- Email: [email protected]
Nifedipine inhibits oxidative stress and ameliorates osteoarthritis by activating the nuclear factor erythroid-2-related factor 2 pathway
Journal Pre-proof Nifedipine inhibits oxidative stress and ameliorates osteoarthritis by activating the nuclear factor erythroid-2-related factor 2 pathway
Jun Yao, Huiping Long, Jianping Zhao, Gang Zhong, Jia Li PII:
S0024-3205(20)30039-4
DOI:
https://doi.org/10.1016/j.lfs.2020.117292
Reference:
LFS 117292
To appear in:
Life Sciences
Received date:
13 October 2019
Revised date:
23 December 2019
Accepted date:
7 January 2020
Please cite this article as: J. Yao, H. Long, J. Zhao, et al., Nifedipine inhibits oxidative stress and ameliorates osteoarthritis by activating the nuclear factor erythroid-2-related factor 2 pathway, Life Sciences(2020), https://doi.org/10.1016/j.lfs.2020.117292
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier.
Journal Pre-proof
Nifedipine inhibits oxidative stress and ameliorates osteoarthritis by activating the nuclear factor erythroid-2-related factor 2 pathway
Jun Yao#1, 2,3, Huiping Long#3, Jianping Zhao1,3, Gang Zhong*1, 2,3 and Jia Li*3,4
Author information Department of Bone and Joint Surgery, The First Affiliated Hospital of Guangxi Medical University, 530021, Nanning, China
2
Guangxi Collaborative Innovation Center for Biomedicine, The First Affiliated Hospital of Guangxi Medical University, 530021, Nanning, China
3
The First Affiliated Hospital of Guangxi Medical University, 530021, Nanning, China
3
Department of Pathology, First Affiliated Hospital of Guangxi Medical University, 530021, Nanning, China
oo
f
1
pr
Abstract:
e-
Nifedipine is a voltage-gated calcium channel inhibitor widely used in the treatment of hypertension. Nifedipine
Pr
has been reported to have antioxidant and anti-apoptotic effects and promotes cell proliferation. However, the effects
al
of nifedipine on oxidative stress and apoptosis in osteoarthritic (OA) chondrocytes are still unclear. In this study, we
Jo ur n
sought to investigate whether nifedipine alleviates oxidative stress and apoptosis in OA through nuclear factor erythroid-2-related factor 2 (Nrf2) activation. The cytotoxicity of nifedipine against human chondrocytes was detected using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit, whereas mRNA and protein expression levels were measured using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The oxidative stress level was analyzed by measuring reactive oxygen species (ROS), glutathione peroxidase (GSH-px), catalase (CAT) and superoxide dismutase (SOD) activities. The role of Nrf2 in the effect of nifedipine on OA was analyzed using an Nrf2 inhibitor brusatol (BR). The result showed that nifedipine inhibited the expression of matrix metalloprotein(MMP)-13, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, inducible nitric oxide (NO) synthase (iNOS), and prostaglandin E2 (PGE2), as well as reduced ROS production in human OA chondrocytes, which was partially reversed by BR. Nifedipine prevented cartilage degeneration and contributed to the expression of Nrf-2 in chondrocytes. These results indicate that nifedipine inhibited inflammation and oxidative stress in chondrocytes via activation of Nrf-2/HO-1 signaling.
Keywords: Osteoarthritis; nifedipine; oxidative stress; inflammation; Nrf2/HO-1 pathway
Journal Pre-proof
1. Introduction Osteoarthritis (OA) is the most common degenerative disease that causes joint deformity [1]. It is widely endemic in middle-aged and elderly individuals [2]. The incidence of OA is more than 60% in people over 65 years old [3]. Molecularly, massive loss of cartilage is caused by multiple factors including oxidative stress, mitochondrial dysfunction, and intracellular metabolic disorders in chondrocytes, the only cells in cartilage tissue [4-6]. Oxidative stress in chondrocytes is mainly due to production and accumulation of a large number of reactive oxygen species (ROS) in cells by exogenous factors including trauma, overload, synovial inflammation, and local intra-articular lesions [7]. Recent studies have shown that oxidative stress caused by elevated ROS levels creates a poor cell microenvironment for chondrocytes. ROS regulate multiple signaling pathways, activate inflammation, exacerbate metabolic disorders, promote apoptosis, and eventually lead to OA. Moreover, the loss of cartilage matrix is
oo
f
positively correlated with the apoptosis of chondrocytes caused by endogenous and exogenous inflammatory factors [8]. It is well known that oxidative stress, apoptosis, and inflammation are integral factors in the entire spectrum of
e-
very promising approach to remission of OA is reasonable.
pr
OA pathogenesis. Therefore, a hypothesis that pharmacological regulation of related signaling pathways may be a
Pr
Nrf2 is a redox-related transcription factor and a major regulator of the antioxidant response and the expression
al
of genes related to the cellular oxidative defense system [9]. Nrf2 plays a crucial role in regulating the production of
Jo ur n
oxygen radicals, which control the protein expression of heme oxygenase1 (HO-1), and antioxidases including superoxide dismutase (SOD), glutathione peroxidase (GSH-px), and catalase (CAT) [10-12]. A recent study revealed that Nrf2 adjusts heme oxygenase 1(HO-1) expression and relieves inflammation and cartilage degradation in chondrocytes during OA [13].
Currently, the mainstream of OA treatments involves physical measures, drug therapy, and surgery[14] . Physical therapy is a simple, everyday adjunctive treatment, including spa, moderate exercises, massage, and acupuncture, which are beneficial but lack of evidence-based medical evidence to support efficacy[15]. Surgical treatment is used cautiously, only considered in serious cases, when conservative therapy is ineffective [16]. Pharmacological treatment is the most widely used treatment strategy, including non steroidal anti-inflammatory drugs, paracetamol, opioid analgesics and emerging therapeutic agents such as artemisinin[17] and phloretin[18]. Anti-inflammatory and analgesic as the main strategy for early drug relief symptoms, and non-steroidal anti-inflammatory drugs (NSAIDs) are used most frequently clinically [19]. However, long-term evidence-based medicine indicates that the prolonged use of NSAIDs is associated with serious side effects including peptic ulcer, nervous system dysfunction, and bleeding diseases[20, 21]. The safety of alternative pharmacological treatment drugs has not been clinically proven. Therefore, safe and effective treatment for OA are urgently needed to reverse the pathological processes.
Journal Pre-proof
Nifedipine, a dihydropyridine calcium blocker that has been widely used clinically in the treatment of hypertension and angina pectoris, has potent pharmacological properties and negligible side effects [22, 23]. Recent studies have found that nifedipine inhibits chronic inflammation of the nervous system and peptic ulceration by neutralizing over-oxidative environments [24]. Previous studies suggest that nifedipine protects cartilage in the OA microenvironment, but its specific mechanism in relieving oxygen free radical-mediated OA by effects on mitochondrial dysfunction, NRF2 signaling, or both are unknown. In this study, we investigated the effects of nifedipine on human OA cells, and its underlying mechanisms. Our results suggest that nifedipine attenuated human OA cells by activating Nrf2/HO-1 signaling. 2. Materials and methods 2.1 Chemicals and reagents
f
Nifedipine (purity > 98 %), brusatol (BR), Safranin-O/Fast Green, collagenase type II, and dimethylsulfoxide
horseradish
peroxidase
conjugates
were
purchased
from
Boster
(Wuhan,
China).
The
pr
anti-rabbit
oo
(DMSO) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Goat anti-mouse and goat
e-
immunohistochemistry kit was purchased from Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China), and
Pr
penicillin/streptomycin was purchased from Gibco (Carlsbad, CA, USA). TRIzol reagent, Hipure, total RNA mini kit were from Magen (China) and the live-dead viability assay kit was purchased from Invitrogen (CA, USA).
al
QuantiTect reverse transcription (RT) kit was purchased from Qiagen (Valencia, CA, USA). SYBR Green Master
Jo ur n
Mix was purchased from Bio-Rad Laboratories (CA, USA). Griess reagent was purchased from Beyotime Institute of Biotechnology (Shanghai, China). All other chemicals were of reagent grade. 2.2 Primary human chondrocytes isolation and culture Human cartilage tissues were provided by 15 donors (6 males (50-70kg), 9 females (40-60kg), 60-75 years old) from the First Affiliated Hospital of Guangxi Medical University (Nanning, China) from Jun. 29 th, 2017 to Jan. 5th, 2018. The diagnosis of OA is based on the US OA diagnostic criteria (Knee Treatment Guidelines). American Osteoarthritis, 2008). The collection and use of human samples were agreed in writing by the donor, and all procedures were approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Approval No.: 2017 (KY-E-040)). Cartilage tissue was obtained from patients with OA undergoing total knee arthroplasty. Primary human chondrocyte was obtained from each volunteer and primary chondrocytes were isolated from the articular cartilage. Briefly, cartilage pieces were digested with 2 mg/mL collagenase type II (Gibco, USA) in alpha-modified Eagle’s medium (α-MEM; Gibco, USA) for 4 h at 37°C, centrifuged at 1000 rpm for 5 min, and then the supernatant was discarded. The inner chondrocytes were obtained, cultured in Dulbecco's MEM (DMEM) High Glucose (Gibco, USA) containing 10% (v/v) fetal bovine serum (FBS; Zhejiang Tianhang Biotechnology Ltd., China) and 1% (v/v) penicillin/streptomycin (Solarbio, China), and the cells were finally incubated under a humidified
Journal Pre-proof
atmosphere of 5% CO2 at 37°C. The medium was changed every 2-3 days and the cells were passaged every 5 days using 0.25% trypsin (Solabio, China). The passage 3 cells were used for further research. 2.3 chondrocytes treatment After chondrocytes were raised to the third passage, all the OA cells were divided into the following four groups and were treated as indicated: (1) OA group, untreated and (2-4) 2, 4, and 8 μM groups, incubated with 2, 4, and 8 μmol/L nifedipine, respectively. All the groups of cells were incubated for 24 h for the subsequent experiments. 2.4 Cytotoxicity assay The effect of nifedipine on the activity of the chondrocytes was detected using the MTT kit (Gibco, USA) in strict accordance with the manufacturer’s instructions. Briefly, human chondrocytes were seeded in 96-well plates at a density of 5 × 103 cells per well for 24 h, treated with various concentrations (0, 0.5, 1, 2, 4, 8, 16, and 32 μM) of
f
nifedipine for 24 h, followed by incubation with 20 μL MTT for 4 h, and then 200 μL DMSO was added to each well
oo
after aspirating all the liquid. The absorbance of the resultant reaction solution was measured using a microplate
pr
reader at a wavelength of 450 nm and all experiments were repeated three times using the above described procedure.
e-
2.5 Cell viability assay
Pr
Cell viability was detected using a live-dead viability assay. Briefly, chondrocytes were washed three times with
al
phosphate buffer saline (PBS), incubated with 1 μM each of calcein-AM and propidium iodide (PI, Invitrogen, USA),
Jo ur n
and then incubated at 37°C for 5 min in the dark. After rinsing with PBS, the images were captured using a fluorescence imaging microscope (Olympus BX53, Japan) and Quantitative analysis of the ratio of living to dead cells was performed through Image J (USA,National Institutes of Health, NIH). All analyses were repeated three times. 2.6 Safranin O staining The synthesis of glycosaminoglycans (GAGs) in chondrocytes was evaluated using Safranin O staining. Chondrocytes were fixed in 4% (w/v) paraformaldehyde for 15 min and then incubated with 0.1% safranin O (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. The chondrocytes were washed three times with PBS, sealed with a neutral gum, and then photographed using an inverted phase contrast microscope (Olympus BX53, Japan) and all analyses were repeated three times. 2.7 Quantification of intracellular GAG secretion Intracellular GAG secretion was evaluated using 1,9-dimethylmethylene blue (DMMB; Sigma-Aldrich, St. Louis, MO, USA) dye, and the DNA content was quantified using Hoechst 33258 dye (Sigma-Aldrich, St. Louis, MO, USA). A spectrophotometer was used to detect the absorbance of the reaction solution at a wavelength of 46 nm using the calf thymus DNA content as a quantitative criterion. The secretion of GAGs in the chondrocytes was
Journal Pre-proof
quantified using a spectrophotometer at a wavelength of 525 nm using chondroitin sulfate as the analytical standard. Finally, the intracellular GAG content was normalized to that of intracellular DNA. All analyses were repeated three times 2.8 Measurement of, IL-6 and TNF-α The production of NO in the culture medium was detected using Greiss reagent kit (Sigma, USA) as previously described[25]. The concentration of PGE2, IL-6 and TNF-α in the medium supernatants was detected using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA) in strict accordance with the manufacturer’s instructions, repeating all tests three times. 2.9 Measurement of ROS levels Chondrocyte ROS levels were assessed using the fluorogenic probe 2,7-dichlorodi-hydrofluorescein diacetate
f
(Beyotime Biotechnology, China). Briefly, human chondrocytes were stained with DCFH 2 -DA (10 μM) probes for
oo
0.5 h at 37°C and ROS levels were estimated by measuring the fluorescence at emission and emission wavelengths
pr
of 488 and 525 nm respectively, using a microplate reader (Thermo Fisher Scientific, USA) and all analyses were
e-
repeated three times.
Pr
2.10 SOD, CAT and GSH-Px activity assay
al
After 24h of treatment with three different concentrations of nifedipine (2 μM, 4 μM and 8 μM), the cultured
Jo ur n
medium of cells were collected and subjected to detection of antioxidant capacity by using Superoxide dismutase (SOD, Beyotime Biotechnology, China, Catalog #S1017) and Catalase (CAT, Beyotime Biotechnology, China, Catalog #P3541) and Glutathione peroxidase (GSH-Px, Beyotime Biotechnology, China, Catalog # S0052) according to the instructions. Then the absorbance at 450 nm was recorded using a Fluorescence microplate reader (Bio-Tek Instruments, USA). 2.11 Measurement of mitochondrial O2- using MitoSOX To measure the cellular mitochondrial O2-, the different groups of OA chondrocytes were loaded with MitoSOX TM Red (red, Eugene, USA, 5 μM) for 15 min at 37°C and O2- levels were evaluated by measuring the fluorescence at 510 and 580 nm excitation and emission wavelengths, respectively as previously described [26] and all analyses were repeated three times. 2.12 Quantitative polymerase chain reaction (qPCR) Total RNA was extracted from human chondrocytes using the Hipure Total RNA Mini kit and cDNA was synthesized from 1000 ng total RNA using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara, China). The quantitative polymerase chain reaction (qPCR) reaction was performed using the FastStart Universal SYBR Green Master Mix (Roche, Germany) under the following conditions: 10 min at 95°C, 15 s at 95°C, and 1 min at 60°C.
Journal Pre-proof
Glyceraldehyde 3-phosphate dehydrogenase (GADPH, Abcam, USA) level was used as the internal control while the 2−ΔΔCT method was used for data analysis. Each gene analysis was repeated three times and the primer sequences of the targeted genes are listed in Table 1. 2.13 Western blotting For the Western blotting, proteins were extracted from human chondrocytes using a total protein extraction kit (BOSTER, China), Fifty μg protein was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE), and then transferred onto a polyvinylidene difluoride membrane (Bio-Rad, USA). After incubation with 5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T), the membrane was further incubated with primary antibodies against IL-1β (BOSTER, Wuhan, China. Catalog #BA3711 1:200), IL-6 (BOSTER, Wuhan, China. Catalog #A00102-1, 1:200), Collagen 2A1 (BOSTER, Wuhan, China. Catalog #A00002, 1:200), aggrecan (BOSTER, Wuhan, China. Catalog #BA3711 1:200), IL-6 (BOSTER, Wuhan,
oo
f
China. Catalog #A00102-1, 1:200), MMP13 (BOSTER, Wuhan, China. Catalog #BA0574, 1:200), iNOS (BOSTER,
pr
Wuhan, China. Catalog #A00368, 1:200), COX-2 (BOSTER, Wuhan, China. Catalog #BM4419, 1:200), Nrf-2
e-
(Abcam, USA, Catalog # ab76026, 1:5000), HO-1(BOSTER, Wuhan, China. Catalog #PB9212, 1:200), quinone dehydrogenase 1 (NQO1, BOSTER, Wuhan, China. Catalog #PB0526, 1:200), glutamate-cysteine ligase modifier
Pr
subunit (GCLM, BOSTER, Wuhan, China. Catalog #BM5483, 1:200 ), and SOD2 (BOSTER, Wuhan, China.
al
Catalog #BA4566, 1:200) followed by secondary antibodies. GAPDH ((BOSTER, Wuhan, China. Catalog #A00227,
three times 2.14 Statistical analysis
Jo ur n
1:1000)) was used as the standard vertebralization control for protein expression levels. All analyses were repeated
All tests are performed at least triplicate. Statistical analysis of all data (mean ± S.D., n = biological replicates) was analyzed by SPSS 64.0 (SPSS Inc., Chicago, Illinois, USA). Comparison between OA and treatment groups was examined using the Independent-Samples t-tests. The level of significance was set to p < 0.05.
3. Results 3.1Preliminary Screening of nifedipine. The chemical structure of nifedipine is shown in Fig. 1a. The effect of different concentrations nifedipine on chondrocyte viability was detected using an MTT assay. As shown in Fig. 1b, the cell viability was increased gradually when the chondrocytes treated by nifedipine from 0 µM to 8 µM and reached its peak at the concentration of 8 μM. Then, the viability of chondrocyte decreased significantly when the chondrocytes treated by nifedipine from 8 µM to 32 µM. When the concentration of nifedipine was 32μM, the viability of chondrocyte was even lower than
Journal Pre-proof
that of OA group (the concentration of nifedipine was 0 μM). Therefore, 2, 4, and 8 μ m were considered to be the optimal concentrations for nifedipine in the treatment of chondrocytes and were selected for further experiments. 3.2 Effects of nifedipine on chondrocyte viability. The live/dead cells assay by Calcein-AM/PI staining revealed that the nifedipine group had more viable cells (green) and less dead cells (red) than those in the OA group (Fig. 1c). This shows that nifedipine effectively protected the chondrocytes from OA microenvironment, confirming the results of the MTT assay. 3.3 Effect of nifedipine on osteoarthritis-mediated GAG loss To evaluate the effect of nifedipine on GAG secretion and degenerative change in human OA chondrocytes, GAG cintent, aggrecan and collagen 2A1 in OA chondrocytes were detected by DMMB assay, qPCR and Western blotting analysis. As shown in Fig.2a, OA chondrocytes showed less intense positive staining with safranin O, which
oo
f
became more positive after treatment with nifedipine, suggesting that the secretion of GAG increased after the treatment with nifedipine. Similar conclusions were drawn from DMMB analysis (Fig. 2b), nifedipine rescued the
pr
loss of GAG in OA chondrocyte. As evidence, it significantly increased the secretion of GAG in nifedipine-treated
e-
human chondrocyte compared with OA group. And treated with 8 μM nifedipine, the GAG content in human OA
Pr
chondrocytes was increased by 4.56 times compared with the OA group. In addition, the transcription and translation levels of aggragen and collagen2A1 are in a trough in the microenvironment of OA, whereas the treatment of
Jo ur n
al
nifedipine significantly enhances its expression (Fig. 2c, 2d). 3.4 Effects of nifedipine on the expression of MMP-13, ADAMTS-5, SOX-9, NO, and PGE2 by human OA chondrocytes
To evaluate the effect of nifedipine on extracellular matrix (ECM) degradation in human chondrocytes, the expression of MMP-13, a disintegrin and metalloprotease with thrombospondinmotifs DAMTS)-, SOX-9 and PGE2 was analyzed using qPCR. PGE2 was analyzed using ELISA. NO was analyzed using Griess reagent, As illustrated in Fig. 3, MMP-13 and ADAMTS-5 levels were reduced and that of SOX-9 increased significantly in chondrocytes treated with nifedipine for 24 h in a concentration-dependent manner as shown in the qPCR analysis (p < 0.05). The OA group showed the content of NO and PEG2, which were downregulated by nifedipine treatment in a dose-dependent manner. These results indicated that nifedipine significantly inhibited OA-mediated ECM degradation. 3.5 Nifedipine inhibits expression of IL-1β, IL-6, TNF-α, iNOS, and COX-2 of human OA chondrocytes To evaluate the effect of nifedipine on secretion of inflammatory factors in human chondrocytes, the expression levels of IL-1β, IL-6, TNF-α, iNOS, and COX-2 was performed using qPCR analysis. As illustrated in Fig. 4, the expression of IL-1β, IL-6, TNF-α, iNOS and COX-2 reduced significantly in nifedipine-treated chondrocytes
Journal Pre-proof
compared with levels of the OA group in a dose-dependent manner (p < 0.05), according to the qPCR analysis. Corresponding results were also observed in the Western blotting and ELISA analysis. These results indicated that nifedipine significantly inhibited OA-mediated secretion of inflammatory factors. 3.6 Nifedipine inhibits oxidative stress in OA chondrocytes We monitored both the basal and induced oxidative stress by measuring the ROS levels using the oxidation-sensitive dye DCFH-DA. Furthermore, the antioxidant enzyme (SOD, CAT and GSH-px) was determined using SOD kit, CAT kit and GSH-px kit, respectively. and mitochondrial O2- levels were determined using MitoSOX. The levels of oxidative stress were explored in OA chondrocytes treated with different concentrations of nifedipine and Fig. 5a shows that the ROS levels of nifedipine-treated OA chondrocytes were significantly and dose-dependently reduced compared to those of the untreated OA group were. Furthermore, nifedipine 8 μM treatment induced the lowest ROS level (p < 0.05, Fig. 5a) and Fig. 5b shows a clear difference in the SOD
oo
f
concentrations among the four groups of human OA chondrocytes. Moreover, the SOD levels in the nifedipine
pr
groups were significantly higher than that in the OA group was (p < 0.05) after 24 h nifedipine treatment. A similar
e-
pattern was also observed with CAT and GSH-px activity (p < 0.05, Fig.5c, d). The CAT and GSH-px concentration of human OA chondrocytes also differed significantly among the four groups (p < 0.05, Fig. 5c, d). The expression
Pr
of CAT and GSH-px was significantly higher in nifedipine treated groups (2, 4, and 8 μM groups) than it was in the
al
OA group. Furthermore, O2-, is a direct cellular oxidative damage index, and as illustrated in Fig. 5e, its levels were
Jo ur n
significantly inhibited in human OA chondrocytes treated with nifedipine in a dose-dependent manner, consistent with the levels of ROS, SOD, CAT, and GSH-px (p < 0.05, Fig. 5a-d). These results indicate that nifedipine significantly inhibited OA-mediated elevation of oxidative stress. 3.7 Effect of nifedipine on Nrf-2/HO-1 signaling of OA chondrocytes To clarify the role of the Nrf-2/HO-1 signaling pathway in nifedipine, the gene and protein expression levels of Nrf2, HO-1, NQO1, GCLM and SOD2 were analyzed using qPCR and Western blotting analysis, respectively. Fig. 4a shows low expression levels of all the proteins in the untreated control, whereas nifedipine treatment significantly increased the expression in a dose-dependent manner (p < 0.05, Fig. 6a-c). Gene expression levels showed a similar pattern in the qPCR analysis, and these results indicate that nifedipine significantly upregulated OA-mediated inhibition of the Nrf-2/HO-1 signaling pathway. 3.8 Involvement of Nrf2/HO-1 signaling pathway in anti-inflammatory and antioxidant effects of nifedipine on OA chondrocytes To further elucidate the anti-inflammatory and antioxidant mechanism of nifedipine in chondrocytes, the Nrf2/HO-1 signaling pathway was analyzed using qPCR and Western blotting analysis. The Nrf2/HO-1 pathway inhibitor brusatol (BR, USA, Sigma-Aldrich, 0.3μg/mL) was used to investigate the role of Nrf-2 in the effect of
Journal Pre-proof
nifedipine on oxidative stress and inflammation. Fig. 6a and Fig. 6b shows that the expression of Nrf2 and HO-1 in chondrocytes was more significantly increased in the nifedipine groups than it was in the untreated OA group. However, treatment with BR reversed this result, with a significant decrease in Nrf2 and HO-1 in the nifedipine plus BR group. Moreover, we examined the expression of inflammation-related factors (IL-1β, IL-6, and MMP-13) and found that BR abolished the decline in nifedipine-mediated inflammatory and oxidative stress markers (P < 0.001, Fig. 6c). In summary, the above results indicated that positive role of Nrf2/HO-1 signaling in nifedipine-induced effects on human OA chondrocytes.
4 Discussion The present treatment modality for OA mainly involves relieving the symptoms of patients including joint pain, swelling, and muscle tension to improve the quality of life. Currently, the most commonly used pharmacological
oo
f
clinical treatments for OA such as NSAIDs require long-term use, which causes numerous serious side effects and a
pr
huge economic burden to patients[19]. Thus, the search for safer and more effective OA therapeutic strategies
e-
continues. Nifedipine is a commonly used, safe drug for the treatment of hypertension, which has excellent L-type calcium channel inhibitory properties[27]. The recent accumulation of evidence suggests that nifedipine also has
Pr
excellent anti-inflammatory properties, particularly with the discovery of almost no serious associated side
al
effects[24]. In our study, nifedipine significantly increased the viability of OA chondrocytes without showing
Jo ur n
cytotoxicity in the range of 0-8 μM. Furthermore, nifedipine remarkably inhibited the expression of inflammatory responses in OA chondrocytes, as evidence by the downregulation of expression levels of the inflammatory factors IL-1β, IL-6, TNF-α, iNOS, COX-2, MMP-13, and PGE2, as well as collagen 2A1 and aggrecan. More importantly, we found that nifedipine greatly reduced oxidative stress in the OA microenvironment. Further studies have shown that nifedipine activated the Nrf2 pathway, which is inhibited in human OA cells and might mediate the mechanism underlying the OA relieving effects. The synthesis of MMPs as well as loss of type II collagen and GAGs are the main pathological features of OA, and inflammatory factors are the mediators of OA, affecting its pathological progression [28]. The most direct evidence is the large increase in the synthesis of iNOS and PEG2 in chondrocytes in the pathological environment of OA, which are thought to increase the production of MMPs and accelerate the loss of type II collagen [29]. In the ensuing chain reaction, iNOS efficiently catalyzes the production of NO, and PEG2 promotes the production of COX-2, which are also important mediators of MMP production and degradation of the extracellular matrix (ECM) in OA [30,31]. Studies have shown that inhibition of iNOS and PEG2 gene expression relieves OA [8,32]. In our study, we found that the expression of iNOS and PEG2 was significantly increased in OA chondrocytes, and nifedipine reverse this trend at both the gene and protein levels. IL-1β, IL-6, and TNF-α the most common inflammatory factors, also exhibited a similar pattern to the above results. Therefore, based on the effects of
Journal Pre-proof
nifedipine on the expression of the above inflammatory factors, we concluded that nifedipine has good anti-inflammatory activity. Degradation of the ECM, which is synthesized and secreted by chondrocytes and provides a microenvironment for the growth of chondrocytes, is another important cause of OA. [33]. In OA, damage to type II collagen and loss of
GAGs
mechanically
weakens
the
cartilage
tissue
rendering
it
insufficient
to
balance
the
mechanical wear and corros caused by joint motion [34]. These processes lead to progressive degeneration of joint tissue, with almost irreversible OA. MMP-13 is a key gene for chondrocyte hypertrophy and is significantly elevated in the OA environment [35]. Nifedipine significantly reduced the expression of MMP-13 in OA cells. In the ECM, SOX-9 is a key anabolic gene while MMPs and ADAMTS are key catabolic genes [36], and promoting the synthesis and inhibiting the degradation of the ECM is a basic proposed treatment strategy for OA. In our study, nifedipine not only promoted the translation of SOX-9 and synthesis of the ECM, but it also inhibited the expression of MMP-13
oo
f
and ADAMTS-5. Thus, nifedipine could inhibit ECM metabolism and eventually maintain its metabolic balance in human the body.
pr
Recently, increasing evidence has shown that oxidative stress, which is the imbalance of free radical scavenging
e-
mechanism, is an important mechanism for the onset of OA [37]. Oxidative stress not only destroys cartilage, but
Pr
also participates in the process of secretion and degradation of inflammatory mediators and promotes clinically asymptomatic cartilage breaks down, leading to a significant OA transition [38]. ROS are the mediators of oxidative
al
stress and there are many sources in the cell including the mitochondria and NADPH oxidase, uncoupled NOS, and
Jo ur n
xanthine oxidase [39]. In addition to the cell dysfunction, apoptosis, and necrosis that lead to dysfunction, the well-known ROS also control some of the specific post-translational modifications of the mRNA, leading to functional changes in important proteins and cellular signaling pathways. The Nrf2 signaling pathway is mainly responsible for cellular defense against oxidative stress and physiologically maintaining the cellular redox balance [40]. Some studies have indicated that Nrf2 signaling plays a critical role in regulating the inflammatory response, and its dysfunction has been shown to increase susceptibility to inflammatory diseases [41]. In our study, we found that Nrf2 was significantly inhibited in OA chondrocytes, and nifedipine relieved the inhibition of Nrf2 by oxidative stress microenvironment. Thus, we hypothesized that nifedipine-mediated activation of Nrf2 is a key component in the inhibition of oxidative stress and inflammation leading to cartilage protection. We obtained chondrocytes from patients with OA to demonstrate the chondroprotective effects of nifedipine-mediated Nrf2 activation and further elucidate the mechanism of nifedipine's activation of Nrf2 in the pathological OA environment. Nifedipine-mediated decrease in SOD, CAT, GSH-px, and ROS levels was shown to be associated with Nrf2 activation and increased translation of Nrf2-dependent genes including Nrf2, HO-1, NQO1, SOD-2, and GCLM in OA chondrocytes. Nrf2 activation has been proven to induce the expression of numerous key signals related to inflammation. For
Journal Pre-proof
instance, Abusarah et al. showed that protandim and 6-gingerol-mediated activation of Nrf2 significantly inhibited the OA-induced elevation of NO, PEG2, iNOS, and COX-2, leading to cartilage protection. Nifedipine treatment of OA chondrocytes under the oxidative stress microenvironment significantly suppressed the expression of the inflammatory cytokines, IL-1β, IL-6, TNF-α, iNOS, and COX-2 in OA cells (Fig. 4a, b). We proved our hypothesis that Nrf2 activation is responsible for the anti-inflammatory and antioxidative effects of nifedipine using brusatol, which significantly abrogated the nifedipine-mediated suppression of expression of inflammatory cytokine in OA cells that contributes to the associated antioxidant effects associated with the Nrf2 pathway (Fig. 6a-c). In conclusion, our study demonstrated the cartilage protective effects of nifedipine by inhibiting mitochondrial damage and dysfunction in the pathological OA microenvironment. The study also revealed that nifedipine-mediated reduction of ROS, which modulated oxidative stress in OA chondrocytes, was mediated by the Nrf2 pathway, which contributed to the anti-inflammatory properties of nifedipine in OA. Furthermore, we also highlighted the
oo
f
considerable potential of antioxidants and ROS scavenger as anti-inflammatory and chondrocyte-protective drugs. Our research creates new awareness of the potential usefulness of Nrf2 as a candidate for the development of a novel
pr
and effective treatment for OA.
Pr
e-
Acknowledgement
This study was supported by grants from the National Natural Science Foundation of China (Grant No. 81760390)
al
and the National Natural Science Foundation of China (Grant No. 81760402). This study was also received support
Jo ur n
from grants from the Innovation and Entrepreneurship base of Guangxi Medical University (Grant No. 02404217017C) and Innovation and Entrepreneurship Base (Grant No. GCICB-IE-2017011). Conflicts of Interest
We declare that we have no conflict of interest. Author Contributions
Gang Zhong developed the study design; Xiaohan Zhang collected the clinical cartilage tissue from patients with OA; Huiping Long conducted the Western blotting; Jianjun Wu performed the cell staining and analyzed the data; Jun Yao wrote the manuscript; and Jia Li revised and polished the article. ORCID Gang Zhong https://orcid.org/0000-0002-9098-0449
References 1.
Nelson, A.E., Osteoarthritis year in review 2017: clinical. Osteoarthritis Cartilage, 2018. 26(3): p. 319-325.
2.
Glyn-Jones, S., et al., Osteoarthritis. The Lancet, 2015. 386(9991): p. 376-387.
3.
Varady, N.H. and A.J. Grodzinsky, Osteoarthritis year in review 2015: mechanics. Osteoarthritis Cartilage, 2016. 24(1): p. 27-35.
4.
Minguzzi, M., et al., Emerging Players at the Intersection of Chondrocyte Loss of Maturational Arrest, Oxidative Stress, Senescence and Low-Grade Inflammation in Osteoarthritis. Oxid Med Cell Longev, 2018. 2018: p. 3075293.
Journal Pre-proof 5.
Blanco, F.J., I. Rego, and C. Ruiz-Romero, The role of mitochondria in osteoarthritis. Nat Rev Rheumatol, 2011. 7(3): p. 161-9.
6.
Hosseinzadeh, A., et al., Apoptosis signaling pathways in osteoarthritis and possible protective role of melatonin. J Pineal Res, 2016. 61(4): p. 411-425.
7.
Blanco, F.J., A.M. Valdes, and I. Rego-Perez, Mitochondrial DNA variation and the pathogenesis of osteoarthritis phenotypes. Nat Rev Rheumatol, 2018. 14(6): p. 327-340.
8.
Rahmati, M., et al., Aging and osteoarthritis: Central role of the extracellular matrix. Ageing Res Rev, 2017. 40: p. 20-30.
9.
Ferrandiz, M.L., J. Nacher-Juan, and M.J. Alcaraz, Nrf2 as a therapeutic target for rheumatic diseases. Biochem Pharmacol, 2018. 152: p. 338-346.
10.
Na, H.K. and Y.J. Surh, Oncogenic potential of Nrf2 and its principal target protein heme oxygenase-1. Free Radic Biol Med, 2014. 67: p. 353-65.
11.
Done, A.J. and T. Traustadottir, Nrf2 mediates redox adaptations to exercise. Redox Biol, 2016. 10: p. 191-199.
12.
Liu, X.F., et al., Nrf2 as a target for prevention of age-related and diabetic cataracts by against oxidative stress. Aging Cell, 2017. 16(5): p. 934-942.
13.
Khan, N.M., I. Ahmad, and T.M. Haqqi, Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis. Free Radic Biol Med, 2018. 116: p. 159-171. 8.
Rahmati, M., et al., Aging and osteoarthritis: Central role
of the extracellular matrix. Ageing Res Rev, 2017. 40: p. 20-30. 14.
Zhang, W., et al., OARSI recommendations for the management of hip and knee osteoarthritis, Part II: OARSI evidence-based, expert consensus guidelines. Osteoarthritis Cartilage, 2008. 16(2): p. 137-62.
15.
Wang, C., Complementary and Alternative Medicine and Osteoarthritis. Int J Integr Med, 2013. 1.
16.
Laupattarakasem, W., et al., Arthroscopic debridement for knee osteoarthritis. Cochrane Database Syst Rev, 2008(1): p. CD005118.
17.
Zhong, G., et al., Artemisinin Ameliorates Osteoarthritis by Inhibiting the Wnt/beta-Catenin Signaling Pathway. Cell Physiol Biochem, 2018. 51(6): p. 2575-2590.
18.
Zheng, W., et al., The protective effect of phloretin in osteoarthritis: an in vitro and in vivo study. Food Funct, 2018. 9(1): p. 263-278.
19.
Nakata, K., et al., Disease-modifying effects of COX-2 selective inhibitors and non-selective NSAIDs in osteoarthritis: a systematic review. Osteoarthritis Cartilage, 2018.
20.
Wongrakpanich, S., et al., A Comprehensive Review of Non-Steroidal Anti-Inflammatory Drug Use in The Elderly. Aging Dis, 2018. 9(1): p. 143-150.
21.
Bjarnason, I., et al., Mechanisms of Damage to the Gastrointestinal Tract From Nonsteroidal Anti-Inflammatory Drugs. Gastroenterology, 2018. 154(3): p. Ghamami N, C.S., Dormuth C, Wright JM,
oo
22.
f
500-514. CD010052[PMIDT25173808].pdf>. Cochrane Database of Systematic Reviews, 2014.
Chaumais, M.C., E.A. Macari, and O. Sitbon, Calcium-channel blockers in pulmonary arterial hypertension. Handb Exp Pharmacol, 2013. 218: p. 161-75.
24.
Kang, M., G.R. Ross, and H.I. Akbarali, The effect of tyrosine nitration of L-type Ca2+ channels on excitation-transcription coupling in colonic inflammation.
pr
23.
Br J Pharmacol, 2010. 159(6): p. 1226-35.
Au, R.Y., et al., Avocado soybean unsaponifiables (ASU) suppress TNF-alpha, IL-1beta, COX-2, iNOS gene expression, and prostaglandin E2 and nitric oxide
e-
25.
production in articular chondrocytes and monocyte/macrophages. Osteoarthritis Cartilage, 2007. 15(11): p. 1249-55. Li, Y., et al., Inhibition of autophagy protects against PAMAM dendrimers-induced hepatotoxicity. Nanotoxicology, 2015. 9(3): p. 344-55.
27.
Taira, N.,. Drugs 2006.
28.
Goldring, M.B. and M. Otero, Inflammation in osteoarthritis. Curr Opin Rheumatol, 2011. 23(5): p. 471-8.
29.
Leonidou, A., et al., Inducible nitric oxide synthase as a target for osteoarthritis treatment. Expert Opin Ther Targets, 2018. 22(4): p. 299-318.
30.
Avendano, M.S., et al., Role of COX-2-derived PGE2 on vascular stiffness and function in hypertension. Br J Pharmacol, 2016. 173(9): p. 1541-55.
31.
Hardy, M.M., et al., Cyclooxygenase 2-dependent prostaglandin E2 modulates cartilage proteoglycan degradation in human osteoarthritis explants.
Jo ur n
al
Pr
26.
Arthritis Rheum, 2002. 46(7): p. 1789-803. 32.
Loeser, R.F., J.A. Collins, and B.O. Diekman, Ageing and the pathogenesis of osteoarthritis. Nat Rev Rheumatol, 2016. 12(7): p. 412-20.
33.
Guilak, F., et al., Osteoarthritis as a disease of the cartilage pericellular matrix. Matrix Biol, 2018.
34.
Hochberg, M., et al., Symptom and structure modification in osteoarthritis with pharmaceutical-grade chondroitin sulfate: what's the evidence? Curr Med Res Opin, 2013. 29(3): p. 259-67.
35.
Ma, C.H., et al., PKR activation causes inflammation and MMP-13 secretion in human degenerated articular chondrocytes. Redox Biol, 2018. 14: p. 72-81.
36.
Yang, C.Y., A. Chanalaris, and L. Troeberg, ADAMTS and ADAM metalloproteinases in osteoarthritis - looking beyond the 'usual suspects'. Osteoarthritis Cartilage, 2017. 25(7): p. 1000-1009.
37.
Ansari, M.Y., et al., Parkin clearance of dysfunctional mitochondria regulates ROS levels and increases survival of human chondrocytes. Osteoarthritis and Cartilage, 2017.
38.
Wauchope, O.R., et al., Oxidative stress increases M1dG, a major peroxidation-derived DNA adduct, in mitochondrial DNA. Nucleic Acids Res, 2018. 46(7): p. 3458-3467.
39.
Moon, Y., et al., Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms. Antioxid Redox Signal, 2017. 26(17): p. 966-985.
40.
Vomund, S., et al., Nrf2, the Master Regulator of Anti-Oxidative Responses. Int J Mol Sci, 2017. 18(12).
41.
Anuranjani and M. Bala, Concerted action of Nrf2-ARE pathway, MRN complex, HMGB1 and inflammatory cytokines - implication in modification of radiation damage. Redox Biol, 2014. 2: p. 832-46.
Journal Pre-proof
Figure Legend
Figure 1 Nifedipine increase the viability of human OA chondrocytes. (a) Chemical structure of nifedipine. (b) The cytotoxic effect of nifedipine on chondrocytes was determined at various concentrations for 24 h using a MTT assay. (c) Calcein-AM/PI stained for cell viability. (d) Quantitative analysis of the ratio of living cells to dead cells in live-dead viability assay by Image J. All experiments were performed three times. Values are the means ± SD, n=6, *, p < 0.05; **, p < 0.01; ***, p < 0.001, relative to the OA group; #, p < 0.05;
Jo ur n
al
Pr
e-
pr
oo
f
##, p < 0.01; ###, p < 0.001, relative to the 2 μM group. Scale bar, 40 µm.
Journal Pre-proof
Figure 2 Nifedipine maintains phenotype for human OA chondrocytes. Human OA chondrocytes were stimulated or not stimulated with various concentrations of nifedipine (2 μM, 4 μM, 8 μM) for 24 h. (a) Safranin O stained for GAG production. (b) Quantification of intracellular production of GAG. (c)The mRNA expression levels of collagen 2A1 and aggrecan were assayed by qPCR. (d)The protein expression levels of collagen 2A1 and aggrecan were determined by Western blot and quantification analysis. OA (OA chondrocytes were not treated with nifedipine); 2 μM (OA chondrocyte treated with 2 μM nifedipine); 4 μM (OA chondrocyte treated with 4 μM nifedipine); 8 μM (OA chondrocyte treated with 8 μM nifedipine). All experiments were performed three times. Values are the means ± SD, n=6, *, p < 0.05; **, p < 0.01; ***, p < 0.001, relative to the OA group; #, p < 0.05; ##, p < 0.01; ###, p < 0.001, relative to the 2
Jo ur n
al
Pr
e-
pr
oo
f
μM group. Scale bar, 40 µm.
Journal Pre-proof
Figure 3 Nifedipine inhibits the expression of MMP-13, ADAMTS-5, SOX-9, NO and PGE2 from human OA chondrocytes. Human OA chondrocytes were stimulated or not stimulated with various concentrations of nifedipine (2 μM, 4 μM, 8 μM) for 24 h. (a, b, c) The levels of MMP-13, ADAMTS-5, SOX-9 were determined using qPCR. (d) The nitrite levels in the culture medium were assessed by Griess reaction. (e) PGE2 was determined using ELISA. OA (OA chondrocytes were not treated with nifedipine); 2 μM (OA chondrocyte treated with 2 μM nifedipine); 4 μM (OA chondrocyte treated with 4 μM nifedipine); 8 μM (OA chondrocyte treated with 8 μM nifedipine). All experiments were performed three times. Values are the means ± SD, n=6, *, p < 0.05; **, p < 0.01; ***, p < 0.001,
Jo ur n
al
Pr
e-
pr
oo
f
relative to the OA group; #, p < 0.05; ##, p < 0.01; ###, p < 0.001, relative to the 2 μM group.
Journal Pre-proof
Figure 4 Nifedipine inhibits the expression of IL-1β, IL-6, TNF-α, iNOS and COX-2 from human OA chondrocytes Human OA chondrocytes were stimulated or not stimulated with various concentrations of nifedipine (2 μM, 4 μM, 8 μM) for 24 h. (a)The mRNA expression levels of IL-1β, IL-6, TNF-α, iNOS and COX-2 were assayed by qPCR. (b)The protein expression levels of IL-6 and TNF-α were determined by ELISA. (c)The protein expression levels of IL-1β, IL-6, TNF-α, iNOS and COX-2 were determined by Western blot and quantification analysis. OA (OA chondrocytes were not treated with nifedipine); 2 μM (OA chondrocyte treated with 2μM nifedipine); 4 μM (OA chondrocyte treated with 4 μM nifedipine); 8 μM (OA chondrocyte treated with 8 μM nifedipine). All experiments were performed three times. Values are the means ± SD, n=6, *, p < 0.05; **, p < 0.01; ***, p < 0.001, relative to the OA
Jo ur n
al
Pr
e-
pr
oo
f
group; #, p < 0.05; ##, p < 0.01; ###, p < 0.001, relative to the 2 μM group.
Journal Pre-proof
Figure 5 Nifedipine inhibits oxidative stress from OA chondrocytes Human OA chondrocytes were stimulated or not stimulated with various concentrations of nifedipine (2 μM, 4 μM, 8 μM) for 24 h. The levels of intracellular ROS in different groups were analyzed by fluorogenic probes DCFH-DA. (b) The levels of SOD equivalents were detected by assay kit. (c) The levels of CAT equivalents were detected by assay kit. (d) The levels of GSH-px equivalents were detected by assay kit. (e)The level of O2- in human OA chondrocyte was stained with MitoSox Red dye and analyzed by confocal microscopy. OA (OA chondrocytes were not treated with nifedipine); 2 μM (OA chondrocyte treated with 2 μM nifedipine); 4 μM (OA chondrocyte treated with 4 μM nifedipine); 8 μM (OA chondrocyte treated with 8 μM nifedipine). All experiments were performed three times. Values are the means ± SD, n=6, *, p < 0.05; **, p < 0.01; ***, p < 0.001, relative to the OA group; #, p < 0.05; ##, p < 0.01; ###, p <
Jo ur n
al
Pr
e-
pr
oo
f
0.001, relative to the 2 μM group. Scale bar, 100 µm.
Journal Pre-proof
Figure 6 Nifedipine inhibits oxidative stress and ameliorates OA by activating Nrf2 pathway. Human OA chondrocytes were stimulated or not stimulated with nifedipine (8 μM) or stimulated with nifedipine (8 μM) and BR(0.3μg/ml) for 24 h. (a)The mRNA expression of Nrf2, HO-1 and GCLM were measured by qPCR. (b) Oxidative stress related protein Nrf2, HO-1, NQO1, GCLM and SOD2 expression was detected by Western blot. (c) Inflammation-related proteins IL-1β, IL-6 and MMP-13 expression detected by Western blot. OA (OA chondrocytes were not treated with nifedipine); Nifedipine (OA chondrocyte treated with 8 μM nifedipine); Nifedipine+BR (OA chondrocyte treated with 8 μM nifedipine and 0.3μg/ml BR). All experiments were performed three times. Values are the means ± SD, n=6, *, p < 0.05; **, p < 0.01; ***, p < 0.001, relative to the OA group; #, p < 0.05;
Jo ur n
al
Pr
e-
pr
oo
f
##, p < 0.01; ###, p < 0.001, relative to the Nifedipine group.
Journal Pre-proof
Table 1. Primer Sequences Used in qPCR Experiments (human) Gene name
Forward primer
Size
Reverse primer
Size
5’-AGGGCCCTGACAACTCTTTT-3’
20bp
5’-AGGGGTCTACATGGCAACTG-3’
20bp
IL-1β
5’-TGAGCTCGCCAGTGAAATGA-3’
21bp
5’-CATGGCCACAACAACTGACG-3’
20bp
MMP-13
5’-GCCATTACCAGTCTCCGAGG-3’
20bp
5’-TACGGTTGGGAAGTTCTGGC-3’
20bp
TNF-α
5’-TCAGAGGGCCTGTACCTCAT-3’
20bp
5’-GGAGGTTGACCTTGGTCTGG-3’
20bp
IL-6
5’-TCTGCGCAGCTTTAAGGAGT-3’
20bp
5’-CCCAGTGGACAGGTTTCTGA-3’
20bp
Aggrecan
5’-CCAGAAGGGTCAGGAGAAAC-3’
20bp
5’-AAAGTGTCCAAGGCATCCAC-3’
20bp
Collaggen Ⅱ
5’-GGCTCCCAGAACATCACCTA-3’
20bp
5’-GCCCTCATCTCCACATCATT-3’
20bp
ADAMTS-5
5’-TAGGCGTCTTTGGCTTCAGT-3’
20bp
5-GCTTTCAGGCTGACCAGTTC-3’
20bp
oo
f
GAPDH
e-
Pr
Osteoarthritic Nuclear factor erythroid-2-related factor 2 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Reverse transcription-polymerase chain reaction Reactive oxygen species Glutathione peroxidase Catalase Superoxide dismutase Brusatol Interleukin Tumor necrosis factor α Cyclooxygenase2 Nitric oxide Inducible nitric oxide synthase Prostaglandin E2 Heme oxygenase 1 Non-steroidal anti-inflammatory drugs Dimethylsulfoxide propidium iodide Fetal bovine serum Glycosaminoglycans 1,9-dimethylmethylene blue Tris-buffered saline sulfate-polyacrylamide gel electrophoresis a disintegrin and metalloprotease with thrombospondinmotifs NAD(P)H dehydrogenase (quinone)1 Glutamate-cysteine ligase
al
OA Nrf2 MTT RT-PCR ROS GSH-px CAT SOD BR IL TNF-α COX-2 NO iNOS PGE2 HO-1 NSAIDs DMSO PI FBS GAG DMMB TBS SDS PAGE ADAMTS-5 NQO1 GCLM
Full name
Jo ur n
Abbreviation
pr
Table2. Abbreviation
Journal Pre-proof
Highlights
Research highlights Nifedipine has anti-inflammatory effect on OA chondrocytes. Nifedipine has chondro-protective effect on OA chondrocytes.
Jo ur n
al
Pr
e-
pr
oo
f
Nifedipine inhibits oxidative stress through Nrf2/HO-1 signaling pathway in OA chondrocytes
Jun Yao, Huiping Long, Jianping Zhao, Gang Zhong, Jia Li PII:
S0024-3205(20)30039-4
DOI:
https://doi.org/10.1016/j.lfs.2020.117292
Reference:
LFS 117292
To appear in:
Life Sciences
Received date:
13 October 2019
Revised date:
23 December 2019
Accepted date:
7 January 2020
Please cite this article as: J. Yao, H. Long, J. Zhao, et al., Nifedipine inhibits oxidative stress and ameliorates osteoarthritis by activating the nuclear factor erythroid-2-related factor 2 pathway, Life Sciences(2020), https://doi.org/10.1016/j.lfs.2020.117292
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier.
Journal Pre-proof
Nifedipine inhibits oxidative stress and ameliorates osteoarthritis by activating the nuclear factor erythroid-2-related factor 2 pathway
Jun Yao#1, 2,3, Huiping Long#3, Jianping Zhao1,3, Gang Zhong*1, 2,3 and Jia Li*3,4
Author information Department of Bone and Joint Surgery, The First Affiliated Hospital of Guangxi Medical University, 530021, Nanning, China
2
Guangxi Collaborative Innovation Center for Biomedicine, The First Affiliated Hospital of Guangxi Medical University, 530021, Nanning, China
3
The First Affiliated Hospital of Guangxi Medical University, 530021, Nanning, China
3
Department of Pathology, First Affiliated Hospital of Guangxi Medical University, 530021, Nanning, China
oo
f
1
pr
Abstract:
e-
Nifedipine is a voltage-gated calcium channel inhibitor widely used in the treatment of hypertension. Nifedipine
Pr
has been reported to have antioxidant and anti-apoptotic effects and promotes cell proliferation. However, the effects
al
of nifedipine on oxidative stress and apoptosis in osteoarthritic (OA) chondrocytes are still unclear. In this study, we
Jo ur n
sought to investigate whether nifedipine alleviates oxidative stress and apoptosis in OA through nuclear factor erythroid-2-related factor 2 (Nrf2) activation. The cytotoxicity of nifedipine against human chondrocytes was detected using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit, whereas mRNA and protein expression levels were measured using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The oxidative stress level was analyzed by measuring reactive oxygen species (ROS), glutathione peroxidase (GSH-px), catalase (CAT) and superoxide dismutase (SOD) activities. The role of Nrf2 in the effect of nifedipine on OA was analyzed using an Nrf2 inhibitor brusatol (BR). The result showed that nifedipine inhibited the expression of matrix metalloprotein(MMP)-13, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, inducible nitric oxide (NO) synthase (iNOS), and prostaglandin E2 (PGE2), as well as reduced ROS production in human OA chondrocytes, which was partially reversed by BR. Nifedipine prevented cartilage degeneration and contributed to the expression of Nrf-2 in chondrocytes. These results indicate that nifedipine inhibited inflammation and oxidative stress in chondrocytes via activation of Nrf-2/HO-1 signaling.
Keywords: Osteoarthritis; nifedipine; oxidative stress; inflammation; Nrf2/HO-1 pathway
Journal Pre-proof
1. Introduction Osteoarthritis (OA) is the most common degenerative disease that causes joint deformity [1]. It is widely endemic in middle-aged and elderly individuals [2]. The incidence of OA is more than 60% in people over 65 years old [3]. Molecularly, massive loss of cartilage is caused by multiple factors including oxidative stress, mitochondrial dysfunction, and intracellular metabolic disorders in chondrocytes, the only cells in cartilage tissue [4-6]. Oxidative stress in chondrocytes is mainly due to production and accumulation of a large number of reactive oxygen species (ROS) in cells by exogenous factors including trauma, overload, synovial inflammation, and local intra-articular lesions [7]. Recent studies have shown that oxidative stress caused by elevated ROS levels creates a poor cell microenvironment for chondrocytes. ROS regulate multiple signaling pathways, activate inflammation, exacerbate metabolic disorders, promote apoptosis, and eventually lead to OA. Moreover, the loss of cartilage matrix is
oo
f
positively correlated with the apoptosis of chondrocytes caused by endogenous and exogenous inflammatory factors [8]. It is well known that oxidative stress, apoptosis, and inflammation are integral factors in the entire spectrum of
e-
very promising approach to remission of OA is reasonable.
pr
OA pathogenesis. Therefore, a hypothesis that pharmacological regulation of related signaling pathways may be a
Pr
Nrf2 is a redox-related transcription factor and a major regulator of the antioxidant response and the expression
al
of genes related to the cellular oxidative defense system [9]. Nrf2 plays a crucial role in regulating the production of
Jo ur n
oxygen radicals, which control the protein expression of heme oxygenase1 (HO-1), and antioxidases including superoxide dismutase (SOD), glutathione peroxidase (GSH-px), and catalase (CAT) [10-12]. A recent study revealed that Nrf2 adjusts heme oxygenase 1(HO-1) expression and relieves inflammation and cartilage degradation in chondrocytes during OA [13].
Currently, the mainstream of OA treatments involves physical measures, drug therapy, and surgery[14] . Physical therapy is a simple, everyday adjunctive treatment, including spa, moderate exercises, massage, and acupuncture, which are beneficial but lack of evidence-based medical evidence to support efficacy[15]. Surgical treatment is used cautiously, only considered in serious cases, when conservative therapy is ineffective [16]. Pharmacological treatment is the most widely used treatment strategy, including non steroidal anti-inflammatory drugs, paracetamol, opioid analgesics and emerging therapeutic agents such as artemisinin[17] and phloretin[18]. Anti-inflammatory and analgesic as the main strategy for early drug relief symptoms, and non-steroidal anti-inflammatory drugs (NSAIDs) are used most frequently clinically [19]. However, long-term evidence-based medicine indicates that the prolonged use of NSAIDs is associated with serious side effects including peptic ulcer, nervous system dysfunction, and bleeding diseases[20, 21]. The safety of alternative pharmacological treatment drugs has not been clinically proven. Therefore, safe and effective treatment for OA are urgently needed to reverse the pathological processes.
Journal Pre-proof
Nifedipine, a dihydropyridine calcium blocker that has been widely used clinically in the treatment of hypertension and angina pectoris, has potent pharmacological properties and negligible side effects [22, 23]. Recent studies have found that nifedipine inhibits chronic inflammation of the nervous system and peptic ulceration by neutralizing over-oxidative environments [24]. Previous studies suggest that nifedipine protects cartilage in the OA microenvironment, but its specific mechanism in relieving oxygen free radical-mediated OA by effects on mitochondrial dysfunction, NRF2 signaling, or both are unknown. In this study, we investigated the effects of nifedipine on human OA cells, and its underlying mechanisms. Our results suggest that nifedipine attenuated human OA cells by activating Nrf2/HO-1 signaling. 2. Materials and methods 2.1 Chemicals and reagents
f
Nifedipine (purity > 98 %), brusatol (BR), Safranin-O/Fast Green, collagenase type II, and dimethylsulfoxide
horseradish
peroxidase
conjugates
were
purchased
from
Boster
(Wuhan,
China).
The
pr
anti-rabbit
oo
(DMSO) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Goat anti-mouse and goat
e-
immunohistochemistry kit was purchased from Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China), and
Pr
penicillin/streptomycin was purchased from Gibco (Carlsbad, CA, USA). TRIzol reagent, Hipure, total RNA mini kit were from Magen (China) and the live-dead viability assay kit was purchased from Invitrogen (CA, USA).
al
QuantiTect reverse transcription (RT) kit was purchased from Qiagen (Valencia, CA, USA). SYBR Green Master
Jo ur n
Mix was purchased from Bio-Rad Laboratories (CA, USA). Griess reagent was purchased from Beyotime Institute of Biotechnology (Shanghai, China). All other chemicals were of reagent grade. 2.2 Primary human chondrocytes isolation and culture Human cartilage tissues were provided by 15 donors (6 males (50-70kg), 9 females (40-60kg), 60-75 years old) from the First Affiliated Hospital of Guangxi Medical University (Nanning, China) from Jun. 29 th, 2017 to Jan. 5th, 2018. The diagnosis of OA is based on the US OA diagnostic criteria (Knee Treatment Guidelines). American Osteoarthritis, 2008). The collection and use of human samples were agreed in writing by the donor, and all procedures were approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Approval No.: 2017 (KY-E-040)). Cartilage tissue was obtained from patients with OA undergoing total knee arthroplasty. Primary human chondrocyte was obtained from each volunteer and primary chondrocytes were isolated from the articular cartilage. Briefly, cartilage pieces were digested with 2 mg/mL collagenase type II (Gibco, USA) in alpha-modified Eagle’s medium (α-MEM; Gibco, USA) for 4 h at 37°C, centrifuged at 1000 rpm for 5 min, and then the supernatant was discarded. The inner chondrocytes were obtained, cultured in Dulbecco's MEM (DMEM) High Glucose (Gibco, USA) containing 10% (v/v) fetal bovine serum (FBS; Zhejiang Tianhang Biotechnology Ltd., China) and 1% (v/v) penicillin/streptomycin (Solarbio, China), and the cells were finally incubated under a humidified
Journal Pre-proof
atmosphere of 5% CO2 at 37°C. The medium was changed every 2-3 days and the cells were passaged every 5 days using 0.25% trypsin (Solabio, China). The passage 3 cells were used for further research. 2.3 chondrocytes treatment After chondrocytes were raised to the third passage, all the OA cells were divided into the following four groups and were treated as indicated: (1) OA group, untreated and (2-4) 2, 4, and 8 μM groups, incubated with 2, 4, and 8 μmol/L nifedipine, respectively. All the groups of cells were incubated for 24 h for the subsequent experiments. 2.4 Cytotoxicity assay The effect of nifedipine on the activity of the chondrocytes was detected using the MTT kit (Gibco, USA) in strict accordance with the manufacturer’s instructions. Briefly, human chondrocytes were seeded in 96-well plates at a density of 5 × 103 cells per well for 24 h, treated with various concentrations (0, 0.5, 1, 2, 4, 8, 16, and 32 μM) of
f
nifedipine for 24 h, followed by incubation with 20 μL MTT for 4 h, and then 200 μL DMSO was added to each well
oo
after aspirating all the liquid. The absorbance of the resultant reaction solution was measured using a microplate
pr
reader at a wavelength of 450 nm and all experiments were repeated three times using the above described procedure.
e-
2.5 Cell viability assay
Pr
Cell viability was detected using a live-dead viability assay. Briefly, chondrocytes were washed three times with
al
phosphate buffer saline (PBS), incubated with 1 μM each of calcein-AM and propidium iodide (PI, Invitrogen, USA),
Jo ur n
and then incubated at 37°C for 5 min in the dark. After rinsing with PBS, the images were captured using a fluorescence imaging microscope (Olympus BX53, Japan) and Quantitative analysis of the ratio of living to dead cells was performed through Image J (USA,National Institutes of Health, NIH). All analyses were repeated three times. 2.6 Safranin O staining The synthesis of glycosaminoglycans (GAGs) in chondrocytes was evaluated using Safranin O staining. Chondrocytes were fixed in 4% (w/v) paraformaldehyde for 15 min and then incubated with 0.1% safranin O (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. The chondrocytes were washed three times with PBS, sealed with a neutral gum, and then photographed using an inverted phase contrast microscope (Olympus BX53, Japan) and all analyses were repeated three times. 2.7 Quantification of intracellular GAG secretion Intracellular GAG secretion was evaluated using 1,9-dimethylmethylene blue (DMMB; Sigma-Aldrich, St. Louis, MO, USA) dye, and the DNA content was quantified using Hoechst 33258 dye (Sigma-Aldrich, St. Louis, MO, USA). A spectrophotometer was used to detect the absorbance of the reaction solution at a wavelength of 46 nm using the calf thymus DNA content as a quantitative criterion. The secretion of GAGs in the chondrocytes was
Journal Pre-proof
quantified using a spectrophotometer at a wavelength of 525 nm using chondroitin sulfate as the analytical standard. Finally, the intracellular GAG content was normalized to that of intracellular DNA. All analyses were repeated three times 2.8 Measurement of, IL-6 and TNF-α The production of NO in the culture medium was detected using Greiss reagent kit (Sigma, USA) as previously described[25]. The concentration of PGE2, IL-6 and TNF-α in the medium supernatants was detected using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA) in strict accordance with the manufacturer’s instructions, repeating all tests three times. 2.9 Measurement of ROS levels Chondrocyte ROS levels were assessed using the fluorogenic probe 2,7-dichlorodi-hydrofluorescein diacetate
f
(Beyotime Biotechnology, China). Briefly, human chondrocytes were stained with DCFH 2 -DA (10 μM) probes for
oo
0.5 h at 37°C and ROS levels were estimated by measuring the fluorescence at emission and emission wavelengths
pr
of 488 and 525 nm respectively, using a microplate reader (Thermo Fisher Scientific, USA) and all analyses were
e-
repeated three times.
Pr
2.10 SOD, CAT and GSH-Px activity assay
al
After 24h of treatment with three different concentrations of nifedipine (2 μM, 4 μM and 8 μM), the cultured
Jo ur n
medium of cells were collected and subjected to detection of antioxidant capacity by using Superoxide dismutase (SOD, Beyotime Biotechnology, China, Catalog #S1017) and Catalase (CAT, Beyotime Biotechnology, China, Catalog #P3541) and Glutathione peroxidase (GSH-Px, Beyotime Biotechnology, China, Catalog # S0052) according to the instructions. Then the absorbance at 450 nm was recorded using a Fluorescence microplate reader (Bio-Tek Instruments, USA). 2.11 Measurement of mitochondrial O2- using MitoSOX To measure the cellular mitochondrial O2-, the different groups of OA chondrocytes were loaded with MitoSOX TM Red (red, Eugene, USA, 5 μM) for 15 min at 37°C and O2- levels were evaluated by measuring the fluorescence at 510 and 580 nm excitation and emission wavelengths, respectively as previously described [26] and all analyses were repeated three times. 2.12 Quantitative polymerase chain reaction (qPCR) Total RNA was extracted from human chondrocytes using the Hipure Total RNA Mini kit and cDNA was synthesized from 1000 ng total RNA using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara, China). The quantitative polymerase chain reaction (qPCR) reaction was performed using the FastStart Universal SYBR Green Master Mix (Roche, Germany) under the following conditions: 10 min at 95°C, 15 s at 95°C, and 1 min at 60°C.
Journal Pre-proof
Glyceraldehyde 3-phosphate dehydrogenase (GADPH, Abcam, USA) level was used as the internal control while the 2−ΔΔCT method was used for data analysis. Each gene analysis was repeated three times and the primer sequences of the targeted genes are listed in Table 1. 2.13 Western blotting For the Western blotting, proteins were extracted from human chondrocytes using a total protein extraction kit (BOSTER, China), Fifty μg protein was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE), and then transferred onto a polyvinylidene difluoride membrane (Bio-Rad, USA). After incubation with 5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T), the membrane was further incubated with primary antibodies against IL-1β (BOSTER, Wuhan, China. Catalog #BA3711 1:200), IL-6 (BOSTER, Wuhan, China. Catalog #A00102-1, 1:200), Collagen 2A1 (BOSTER, Wuhan, China. Catalog #A00002, 1:200), aggrecan (BOSTER, Wuhan, China. Catalog #BA3711 1:200), IL-6 (BOSTER, Wuhan,
oo
f
China. Catalog #A00102-1, 1:200), MMP13 (BOSTER, Wuhan, China. Catalog #BA0574, 1:200), iNOS (BOSTER,
pr
Wuhan, China. Catalog #A00368, 1:200), COX-2 (BOSTER, Wuhan, China. Catalog #BM4419, 1:200), Nrf-2
e-
(Abcam, USA, Catalog # ab76026, 1:5000), HO-1(BOSTER, Wuhan, China. Catalog #PB9212, 1:200), quinone dehydrogenase 1 (NQO1, BOSTER, Wuhan, China. Catalog #PB0526, 1:200), glutamate-cysteine ligase modifier
Pr
subunit (GCLM, BOSTER, Wuhan, China. Catalog #BM5483, 1:200 ), and SOD2 (BOSTER, Wuhan, China.
al
Catalog #BA4566, 1:200) followed by secondary antibodies. GAPDH ((BOSTER, Wuhan, China. Catalog #A00227,
three times 2.14 Statistical analysis
Jo ur n
1:1000)) was used as the standard vertebralization control for protein expression levels. All analyses were repeated
All tests are performed at least triplicate. Statistical analysis of all data (mean ± S.D., n = biological replicates) was analyzed by SPSS 64.0 (SPSS Inc., Chicago, Illinois, USA). Comparison between OA and treatment groups was examined using the Independent-Samples t-tests. The level of significance was set to p < 0.05.
3. Results 3.1Preliminary Screening of nifedipine. The chemical structure of nifedipine is shown in Fig. 1a. The effect of different concentrations nifedipine on chondrocyte viability was detected using an MTT assay. As shown in Fig. 1b, the cell viability was increased gradually when the chondrocytes treated by nifedipine from 0 µM to 8 µM and reached its peak at the concentration of 8 μM. Then, the viability of chondrocyte decreased significantly when the chondrocytes treated by nifedipine from 8 µM to 32 µM. When the concentration of nifedipine was 32μM, the viability of chondrocyte was even lower than
Journal Pre-proof
that of OA group (the concentration of nifedipine was 0 μM). Therefore, 2, 4, and 8 μ m were considered to be the optimal concentrations for nifedipine in the treatment of chondrocytes and were selected for further experiments. 3.2 Effects of nifedipine on chondrocyte viability. The live/dead cells assay by Calcein-AM/PI staining revealed that the nifedipine group had more viable cells (green) and less dead cells (red) than those in the OA group (Fig. 1c). This shows that nifedipine effectively protected the chondrocytes from OA microenvironment, confirming the results of the MTT assay. 3.3 Effect of nifedipine on osteoarthritis-mediated GAG loss To evaluate the effect of nifedipine on GAG secretion and degenerative change in human OA chondrocytes, GAG cintent, aggrecan and collagen 2A1 in OA chondrocytes were detected by DMMB assay, qPCR and Western blotting analysis. As shown in Fig.2a, OA chondrocytes showed less intense positive staining with safranin O, which
oo
f
became more positive after treatment with nifedipine, suggesting that the secretion of GAG increased after the treatment with nifedipine. Similar conclusions were drawn from DMMB analysis (Fig. 2b), nifedipine rescued the
pr
loss of GAG in OA chondrocyte. As evidence, it significantly increased the secretion of GAG in nifedipine-treated
e-
human chondrocyte compared with OA group. And treated with 8 μM nifedipine, the GAG content in human OA
Pr
chondrocytes was increased by 4.56 times compared with the OA group. In addition, the transcription and translation levels of aggragen and collagen2A1 are in a trough in the microenvironment of OA, whereas the treatment of
Jo ur n
al
nifedipine significantly enhances its expression (Fig. 2c, 2d). 3.4 Effects of nifedipine on the expression of MMP-13, ADAMTS-5, SOX-9, NO, and PGE2 by human OA chondrocytes
To evaluate the effect of nifedipine on extracellular matrix (ECM) degradation in human chondrocytes, the expression of MMP-13, a disintegrin and metalloprotease with thrombospondinmotifs DAMTS)-, SOX-9 and PGE2 was analyzed using qPCR. PGE2 was analyzed using ELISA. NO was analyzed using Griess reagent, As illustrated in Fig. 3, MMP-13 and ADAMTS-5 levels were reduced and that of SOX-9 increased significantly in chondrocytes treated with nifedipine for 24 h in a concentration-dependent manner as shown in the qPCR analysis (p < 0.05). The OA group showed the content of NO and PEG2, which were downregulated by nifedipine treatment in a dose-dependent manner. These results indicated that nifedipine significantly inhibited OA-mediated ECM degradation. 3.5 Nifedipine inhibits expression of IL-1β, IL-6, TNF-α, iNOS, and COX-2 of human OA chondrocytes To evaluate the effect of nifedipine on secretion of inflammatory factors in human chondrocytes, the expression levels of IL-1β, IL-6, TNF-α, iNOS, and COX-2 was performed using qPCR analysis. As illustrated in Fig. 4, the expression of IL-1β, IL-6, TNF-α, iNOS and COX-2 reduced significantly in nifedipine-treated chondrocytes
Journal Pre-proof
compared with levels of the OA group in a dose-dependent manner (p < 0.05), according to the qPCR analysis. Corresponding results were also observed in the Western blotting and ELISA analysis. These results indicated that nifedipine significantly inhibited OA-mediated secretion of inflammatory factors. 3.6 Nifedipine inhibits oxidative stress in OA chondrocytes We monitored both the basal and induced oxidative stress by measuring the ROS levels using the oxidation-sensitive dye DCFH-DA. Furthermore, the antioxidant enzyme (SOD, CAT and GSH-px) was determined using SOD kit, CAT kit and GSH-px kit, respectively. and mitochondrial O2- levels were determined using MitoSOX. The levels of oxidative stress were explored in OA chondrocytes treated with different concentrations of nifedipine and Fig. 5a shows that the ROS levels of nifedipine-treated OA chondrocytes were significantly and dose-dependently reduced compared to those of the untreated OA group were. Furthermore, nifedipine 8 μM treatment induced the lowest ROS level (p < 0.05, Fig. 5a) and Fig. 5b shows a clear difference in the SOD
oo
f
concentrations among the four groups of human OA chondrocytes. Moreover, the SOD levels in the nifedipine
pr
groups were significantly higher than that in the OA group was (p < 0.05) after 24 h nifedipine treatment. A similar
e-
pattern was also observed with CAT and GSH-px activity (p < 0.05, Fig.5c, d). The CAT and GSH-px concentration of human OA chondrocytes also differed significantly among the four groups (p < 0.05, Fig. 5c, d). The expression
Pr
of CAT and GSH-px was significantly higher in nifedipine treated groups (2, 4, and 8 μM groups) than it was in the
al
OA group. Furthermore, O2-, is a direct cellular oxidative damage index, and as illustrated in Fig. 5e, its levels were
Jo ur n
significantly inhibited in human OA chondrocytes treated with nifedipine in a dose-dependent manner, consistent with the levels of ROS, SOD, CAT, and GSH-px (p < 0.05, Fig. 5a-d). These results indicate that nifedipine significantly inhibited OA-mediated elevation of oxidative stress. 3.7 Effect of nifedipine on Nrf-2/HO-1 signaling of OA chondrocytes To clarify the role of the Nrf-2/HO-1 signaling pathway in nifedipine, the gene and protein expression levels of Nrf2, HO-1, NQO1, GCLM and SOD2 were analyzed using qPCR and Western blotting analysis, respectively. Fig. 4a shows low expression levels of all the proteins in the untreated control, whereas nifedipine treatment significantly increased the expression in a dose-dependent manner (p < 0.05, Fig. 6a-c). Gene expression levels showed a similar pattern in the qPCR analysis, and these results indicate that nifedipine significantly upregulated OA-mediated inhibition of the Nrf-2/HO-1 signaling pathway. 3.8 Involvement of Nrf2/HO-1 signaling pathway in anti-inflammatory and antioxidant effects of nifedipine on OA chondrocytes To further elucidate the anti-inflammatory and antioxidant mechanism of nifedipine in chondrocytes, the Nrf2/HO-1 signaling pathway was analyzed using qPCR and Western blotting analysis. The Nrf2/HO-1 pathway inhibitor brusatol (BR, USA, Sigma-Aldrich, 0.3μg/mL) was used to investigate the role of Nrf-2 in the effect of
Journal Pre-proof
nifedipine on oxidative stress and inflammation. Fig. 6a and Fig. 6b shows that the expression of Nrf2 and HO-1 in chondrocytes was more significantly increased in the nifedipine groups than it was in the untreated OA group. However, treatment with BR reversed this result, with a significant decrease in Nrf2 and HO-1 in the nifedipine plus BR group. Moreover, we examined the expression of inflammation-related factors (IL-1β, IL-6, and MMP-13) and found that BR abolished the decline in nifedipine-mediated inflammatory and oxidative stress markers (P < 0.001, Fig. 6c). In summary, the above results indicated that positive role of Nrf2/HO-1 signaling in nifedipine-induced effects on human OA chondrocytes.
4 Discussion The present treatment modality for OA mainly involves relieving the symptoms of patients including joint pain, swelling, and muscle tension to improve the quality of life. Currently, the most commonly used pharmacological
oo
f
clinical treatments for OA such as NSAIDs require long-term use, which causes numerous serious side effects and a
pr
huge economic burden to patients[19]. Thus, the search for safer and more effective OA therapeutic strategies
e-
continues. Nifedipine is a commonly used, safe drug for the treatment of hypertension, which has excellent L-type calcium channel inhibitory properties[27]. The recent accumulation of evidence suggests that nifedipine also has
Pr
excellent anti-inflammatory properties, particularly with the discovery of almost no serious associated side
al
effects[24]. In our study, nifedipine significantly increased the viability of OA chondrocytes without showing
Jo ur n
cytotoxicity in the range of 0-8 μM. Furthermore, nifedipine remarkably inhibited the expression of inflammatory responses in OA chondrocytes, as evidence by the downregulation of expression levels of the inflammatory factors IL-1β, IL-6, TNF-α, iNOS, COX-2, MMP-13, and PGE2, as well as collagen 2A1 and aggrecan. More importantly, we found that nifedipine greatly reduced oxidative stress in the OA microenvironment. Further studies have shown that nifedipine activated the Nrf2 pathway, which is inhibited in human OA cells and might mediate the mechanism underlying the OA relieving effects. The synthesis of MMPs as well as loss of type II collagen and GAGs are the main pathological features of OA, and inflammatory factors are the mediators of OA, affecting its pathological progression [28]. The most direct evidence is the large increase in the synthesis of iNOS and PEG2 in chondrocytes in the pathological environment of OA, which are thought to increase the production of MMPs and accelerate the loss of type II collagen [29]. In the ensuing chain reaction, iNOS efficiently catalyzes the production of NO, and PEG2 promotes the production of COX-2, which are also important mediators of MMP production and degradation of the extracellular matrix (ECM) in OA [30,31]. Studies have shown that inhibition of iNOS and PEG2 gene expression relieves OA [8,32]. In our study, we found that the expression of iNOS and PEG2 was significantly increased in OA chondrocytes, and nifedipine reverse this trend at both the gene and protein levels. IL-1β, IL-6, and TNF-α the most common inflammatory factors, also exhibited a similar pattern to the above results. Therefore, based on the effects of
Journal Pre-proof
nifedipine on the expression of the above inflammatory factors, we concluded that nifedipine has good anti-inflammatory activity. Degradation of the ECM, which is synthesized and secreted by chondrocytes and provides a microenvironment for the growth of chondrocytes, is another important cause of OA. [33]. In OA, damage to type II collagen and loss of
GAGs
mechanically
weakens
the
cartilage
tissue
rendering
it
insufficient
to
balance
the
mechanical wear and corros caused by joint motion [34]. These processes lead to progressive degeneration of joint tissue, with almost irreversible OA. MMP-13 is a key gene for chondrocyte hypertrophy and is significantly elevated in the OA environment [35]. Nifedipine significantly reduced the expression of MMP-13 in OA cells. In the ECM, SOX-9 is a key anabolic gene while MMPs and ADAMTS are key catabolic genes [36], and promoting the synthesis and inhibiting the degradation of the ECM is a basic proposed treatment strategy for OA. In our study, nifedipine not only promoted the translation of SOX-9 and synthesis of the ECM, but it also inhibited the expression of MMP-13
oo
f
and ADAMTS-5. Thus, nifedipine could inhibit ECM metabolism and eventually maintain its metabolic balance in human the body.
pr
Recently, increasing evidence has shown that oxidative stress, which is the imbalance of free radical scavenging
e-
mechanism, is an important mechanism for the onset of OA [37]. Oxidative stress not only destroys cartilage, but
Pr
also participates in the process of secretion and degradation of inflammatory mediators and promotes clinically asymptomatic cartilage breaks down, leading to a significant OA transition [38]. ROS are the mediators of oxidative
al
stress and there are many sources in the cell including the mitochondria and NADPH oxidase, uncoupled NOS, and
Jo ur n
xanthine oxidase [39]. In addition to the cell dysfunction, apoptosis, and necrosis that lead to dysfunction, the well-known ROS also control some of the specific post-translational modifications of the mRNA, leading to functional changes in important proteins and cellular signaling pathways. The Nrf2 signaling pathway is mainly responsible for cellular defense against oxidative stress and physiologically maintaining the cellular redox balance [40]. Some studies have indicated that Nrf2 signaling plays a critical role in regulating the inflammatory response, and its dysfunction has been shown to increase susceptibility to inflammatory diseases [41]. In our study, we found that Nrf2 was significantly inhibited in OA chondrocytes, and nifedipine relieved the inhibition of Nrf2 by oxidative stress microenvironment. Thus, we hypothesized that nifedipine-mediated activation of Nrf2 is a key component in the inhibition of oxidative stress and inflammation leading to cartilage protection. We obtained chondrocytes from patients with OA to demonstrate the chondroprotective effects of nifedipine-mediated Nrf2 activation and further elucidate the mechanism of nifedipine's activation of Nrf2 in the pathological OA environment. Nifedipine-mediated decrease in SOD, CAT, GSH-px, and ROS levels was shown to be associated with Nrf2 activation and increased translation of Nrf2-dependent genes including Nrf2, HO-1, NQO1, SOD-2, and GCLM in OA chondrocytes. Nrf2 activation has been proven to induce the expression of numerous key signals related to inflammation. For
Journal Pre-proof
instance, Abusarah et al. showed that protandim and 6-gingerol-mediated activation of Nrf2 significantly inhibited the OA-induced elevation of NO, PEG2, iNOS, and COX-2, leading to cartilage protection. Nifedipine treatment of OA chondrocytes under the oxidative stress microenvironment significantly suppressed the expression of the inflammatory cytokines, IL-1β, IL-6, TNF-α, iNOS, and COX-2 in OA cells (Fig. 4a, b). We proved our hypothesis that Nrf2 activation is responsible for the anti-inflammatory and antioxidative effects of nifedipine using brusatol, which significantly abrogated the nifedipine-mediated suppression of expression of inflammatory cytokine in OA cells that contributes to the associated antioxidant effects associated with the Nrf2 pathway (Fig. 6a-c). In conclusion, our study demonstrated the cartilage protective effects of nifedipine by inhibiting mitochondrial damage and dysfunction in the pathological OA microenvironment. The study also revealed that nifedipine-mediated reduction of ROS, which modulated oxidative stress in OA chondrocytes, was mediated by the Nrf2 pathway, which contributed to the anti-inflammatory properties of nifedipine in OA. Furthermore, we also highlighted the
oo
f
considerable potential of antioxidants and ROS scavenger as anti-inflammatory and chondrocyte-protective drugs. Our research creates new awareness of the potential usefulness of Nrf2 as a candidate for the development of a novel
pr
and effective treatment for OA.
Pr
e-
Acknowledgement
This study was supported by grants from the National Natural Science Foundation of China (Grant No. 81760390)
al
and the National Natural Science Foundation of China (Grant No. 81760402). This study was also received support
Jo ur n
from grants from the Innovation and Entrepreneurship base of Guangxi Medical University (Grant No. 02404217017C) and Innovation and Entrepreneurship Base (Grant No. GCICB-IE-2017011). Conflicts of Interest
We declare that we have no conflict of interest. Author Contributions
Gang Zhong developed the study design; Xiaohan Zhang collected the clinical cartilage tissue from patients with OA; Huiping Long conducted the Western blotting; Jianjun Wu performed the cell staining and analyzed the data; Jun Yao wrote the manuscript; and Jia Li revised and polished the article. ORCID Gang Zhong https://orcid.org/0000-0002-9098-0449
References 1.
Nelson, A.E., Osteoarthritis year in review 2017: clinical. Osteoarthritis Cartilage, 2018. 26(3): p. 319-325.
2.
Glyn-Jones, S., et al., Osteoarthritis. The Lancet, 2015. 386(9991): p. 376-387.
3.
Varady, N.H. and A.J. Grodzinsky, Osteoarthritis year in review 2015: mechanics. Osteoarthritis Cartilage, 2016. 24(1): p. 27-35.
4.
Minguzzi, M., et al., Emerging Players at the Intersection of Chondrocyte Loss of Maturational Arrest, Oxidative Stress, Senescence and Low-Grade Inflammation in Osteoarthritis. Oxid Med Cell Longev, 2018. 2018: p. 3075293.
Journal Pre-proof 5.
Blanco, F.J., I. Rego, and C. Ruiz-Romero, The role of mitochondria in osteoarthritis. Nat Rev Rheumatol, 2011. 7(3): p. 161-9.
6.
Hosseinzadeh, A., et al., Apoptosis signaling pathways in osteoarthritis and possible protective role of melatonin. J Pineal Res, 2016. 61(4): p. 411-425.
7.
Blanco, F.J., A.M. Valdes, and I. Rego-Perez, Mitochondrial DNA variation and the pathogenesis of osteoarthritis phenotypes. Nat Rev Rheumatol, 2018. 14(6): p. 327-340.
8.
Rahmati, M., et al., Aging and osteoarthritis: Central role of the extracellular matrix. Ageing Res Rev, 2017. 40: p. 20-30.
9.
Ferrandiz, M.L., J. Nacher-Juan, and M.J. Alcaraz, Nrf2 as a therapeutic target for rheumatic diseases. Biochem Pharmacol, 2018. 152: p. 338-346.
10.
Na, H.K. and Y.J. Surh, Oncogenic potential of Nrf2 and its principal target protein heme oxygenase-1. Free Radic Biol Med, 2014. 67: p. 353-65.
11.
Done, A.J. and T. Traustadottir, Nrf2 mediates redox adaptations to exercise. Redox Biol, 2016. 10: p. 191-199.
12.
Liu, X.F., et al., Nrf2 as a target for prevention of age-related and diabetic cataracts by against oxidative stress. Aging Cell, 2017. 16(5): p. 934-942.
13.
Khan, N.M., I. Ahmad, and T.M. Haqqi, Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis. Free Radic Biol Med, 2018. 116: p. 159-171. 8.
Rahmati, M., et al., Aging and osteoarthritis: Central role
of the extracellular matrix. Ageing Res Rev, 2017. 40: p. 20-30. 14.
Zhang, W., et al., OARSI recommendations for the management of hip and knee osteoarthritis, Part II: OARSI evidence-based, expert consensus guidelines. Osteoarthritis Cartilage, 2008. 16(2): p. 137-62.
15.
Wang, C., Complementary and Alternative Medicine and Osteoarthritis. Int J Integr Med, 2013. 1.
16.
Laupattarakasem, W., et al., Arthroscopic debridement for knee osteoarthritis. Cochrane Database Syst Rev, 2008(1): p. CD005118.
17.
Zhong, G., et al., Artemisinin Ameliorates Osteoarthritis by Inhibiting the Wnt/beta-Catenin Signaling Pathway. Cell Physiol Biochem, 2018. 51(6): p. 2575-2590.
18.
Zheng, W., et al., The protective effect of phloretin in osteoarthritis: an in vitro and in vivo study. Food Funct, 2018. 9(1): p. 263-278.
19.
Nakata, K., et al., Disease-modifying effects of COX-2 selective inhibitors and non-selective NSAIDs in osteoarthritis: a systematic review. Osteoarthritis Cartilage, 2018.
20.
Wongrakpanich, S., et al., A Comprehensive Review of Non-Steroidal Anti-Inflammatory Drug Use in The Elderly. Aging Dis, 2018. 9(1): p. 143-150.
21.
Bjarnason, I., et al., Mechanisms of Damage to the Gastrointestinal Tract From Nonsteroidal Anti-Inflammatory Drugs. Gastroenterology, 2018. 154(3): p. Ghamami N, C.S., Dormuth C, Wright JM,
oo
22.
f
500-514. CD010052[PMIDT25173808].pdf>. Cochrane Database of Systematic Reviews, 2014.
Chaumais, M.C., E.A. Macari, and O. Sitbon, Calcium-channel blockers in pulmonary arterial hypertension. Handb Exp Pharmacol, 2013. 218: p. 161-75.
24.
Kang, M., G.R. Ross, and H.I. Akbarali, The effect of tyrosine nitration of L-type Ca2+ channels on excitation-transcription coupling in colonic inflammation.
pr
23.
Br J Pharmacol, 2010. 159(6): p. 1226-35.
Au, R.Y., et al., Avocado soybean unsaponifiables (ASU) suppress TNF-alpha, IL-1beta, COX-2, iNOS gene expression, and prostaglandin E2 and nitric oxide
e-
25.
production in articular chondrocytes and monocyte/macrophages. Osteoarthritis Cartilage, 2007. 15(11): p. 1249-55. Li, Y., et al., Inhibition of autophagy protects against PAMAM dendrimers-induced hepatotoxicity. Nanotoxicology, 2015. 9(3): p. 344-55.
27.
Taira, N.,
28.
Goldring, M.B. and M. Otero, Inflammation in osteoarthritis. Curr Opin Rheumatol, 2011. 23(5): p. 471-8.
29.
Leonidou, A., et al., Inducible nitric oxide synthase as a target for osteoarthritis treatment. Expert Opin Ther Targets, 2018. 22(4): p. 299-318.
30.
Avendano, M.S., et al., Role of COX-2-derived PGE2 on vascular stiffness and function in hypertension. Br J Pharmacol, 2016. 173(9): p. 1541-55.
31.
Hardy, M.M., et al., Cyclooxygenase 2-dependent prostaglandin E2 modulates cartilage proteoglycan degradation in human osteoarthritis explants.
Jo ur n
al
Pr
26.
Arthritis Rheum, 2002. 46(7): p. 1789-803. 32.
Loeser, R.F., J.A. Collins, and B.O. Diekman, Ageing and the pathogenesis of osteoarthritis. Nat Rev Rheumatol, 2016. 12(7): p. 412-20.
33.
Guilak, F., et al., Osteoarthritis as a disease of the cartilage pericellular matrix. Matrix Biol, 2018.
34.
Hochberg, M., et al., Symptom and structure modification in osteoarthritis with pharmaceutical-grade chondroitin sulfate: what's the evidence? Curr Med Res Opin, 2013. 29(3): p. 259-67.
35.
Ma, C.H., et al., PKR activation causes inflammation and MMP-13 secretion in human degenerated articular chondrocytes. Redox Biol, 2018. 14: p. 72-81.
36.
Yang, C.Y., A. Chanalaris, and L. Troeberg, ADAMTS and ADAM metalloproteinases in osteoarthritis - looking beyond the 'usual suspects'. Osteoarthritis Cartilage, 2017. 25(7): p. 1000-1009.
37.
Ansari, M.Y., et al., Parkin clearance of dysfunctional mitochondria regulates ROS levels and increases survival of human chondrocytes. Osteoarthritis and Cartilage, 2017.
38.
Wauchope, O.R., et al., Oxidative stress increases M1dG, a major peroxidation-derived DNA adduct, in mitochondrial DNA. Nucleic Acids Res, 2018. 46(7): p. 3458-3467.
39.
Moon, Y., et al., Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms. Antioxid Redox Signal, 2017. 26(17): p. 966-985.
40.
Vomund, S., et al., Nrf2, the Master Regulator of Anti-Oxidative Responses. Int J Mol Sci, 2017. 18(12).
41.
Anuranjani and M. Bala, Concerted action of Nrf2-ARE pathway, MRN complex, HMGB1 and inflammatory cytokines - implication in modification of radiation damage. Redox Biol, 2014. 2: p. 832-46.
Journal Pre-proof
Figure Legend
Figure 1 Nifedipine increase the viability of human OA chondrocytes. (a) Chemical structure of nifedipine. (b) The cytotoxic effect of nifedipine on chondrocytes was determined at various concentrations for 24 h using a MTT assay. (c) Calcein-AM/PI stained for cell viability. (d) Quantitative analysis of the ratio of living cells to dead cells in live-dead viability assay by Image J. All experiments were performed three times. Values are the means ± SD, n=6, *, p < 0.05; **, p < 0.01; ***, p < 0.001, relative to the OA group; #, p < 0.05;
Jo ur n
al
Pr
e-
pr
oo
f
##, p < 0.01; ###, p < 0.001, relative to the 2 μM group. Scale bar, 40 µm.
Journal Pre-proof
Figure 2 Nifedipine maintains phenotype for human OA chondrocytes. Human OA chondrocytes were stimulated or not stimulated with various concentrations of nifedipine (2 μM, 4 μM, 8 μM) for 24 h. (a) Safranin O stained for GAG production. (b) Quantification of intracellular production of GAG. (c)The mRNA expression levels of collagen 2A1 and aggrecan were assayed by qPCR. (d)The protein expression levels of collagen 2A1 and aggrecan were determined by Western blot and quantification analysis. OA (OA chondrocytes were not treated with nifedipine); 2 μM (OA chondrocyte treated with 2 μM nifedipine); 4 μM (OA chondrocyte treated with 4 μM nifedipine); 8 μM (OA chondrocyte treated with 8 μM nifedipine). All experiments were performed three times. Values are the means ± SD, n=6, *, p < 0.05; **, p < 0.01; ***, p < 0.001, relative to the OA group; #, p < 0.05; ##, p < 0.01; ###, p < 0.001, relative to the 2
Jo ur n
al
Pr
e-
pr
oo
f
μM group. Scale bar, 40 µm.
Journal Pre-proof
Figure 3 Nifedipine inhibits the expression of MMP-13, ADAMTS-5, SOX-9, NO and PGE2 from human OA chondrocytes. Human OA chondrocytes were stimulated or not stimulated with various concentrations of nifedipine (2 μM, 4 μM, 8 μM) for 24 h. (a, b, c) The levels of MMP-13, ADAMTS-5, SOX-9 were determined using qPCR. (d) The nitrite levels in the culture medium were assessed by Griess reaction. (e) PGE2 was determined using ELISA. OA (OA chondrocytes were not treated with nifedipine); 2 μM (OA chondrocyte treated with 2 μM nifedipine); 4 μM (OA chondrocyte treated with 4 μM nifedipine); 8 μM (OA chondrocyte treated with 8 μM nifedipine). All experiments were performed three times. Values are the means ± SD, n=6, *, p < 0.05; **, p < 0.01; ***, p < 0.001,
Jo ur n
al
Pr
e-
pr
oo
f
relative to the OA group; #, p < 0.05; ##, p < 0.01; ###, p < 0.001, relative to the 2 μM group.
Journal Pre-proof
Figure 4 Nifedipine inhibits the expression of IL-1β, IL-6, TNF-α, iNOS and COX-2 from human OA chondrocytes Human OA chondrocytes were stimulated or not stimulated with various concentrations of nifedipine (2 μM, 4 μM, 8 μM) for 24 h. (a)The mRNA expression levels of IL-1β, IL-6, TNF-α, iNOS and COX-2 were assayed by qPCR. (b)The protein expression levels of IL-6 and TNF-α were determined by ELISA. (c)The protein expression levels of IL-1β, IL-6, TNF-α, iNOS and COX-2 were determined by Western blot and quantification analysis. OA (OA chondrocytes were not treated with nifedipine); 2 μM (OA chondrocyte treated with 2μM nifedipine); 4 μM (OA chondrocyte treated with 4 μM nifedipine); 8 μM (OA chondrocyte treated with 8 μM nifedipine). All experiments were performed three times. Values are the means ± SD, n=6, *, p < 0.05; **, p < 0.01; ***, p < 0.001, relative to the OA
Jo ur n
al
Pr
e-
pr
oo
f
group; #, p < 0.05; ##, p < 0.01; ###, p < 0.001, relative to the 2 μM group.
Journal Pre-proof
Figure 5 Nifedipine inhibits oxidative stress from OA chondrocytes Human OA chondrocytes were stimulated or not stimulated with various concentrations of nifedipine (2 μM, 4 μM, 8 μM) for 24 h. The levels of intracellular ROS in different groups were analyzed by fluorogenic probes DCFH-DA. (b) The levels of SOD equivalents were detected by assay kit. (c) The levels of CAT equivalents were detected by assay kit. (d) The levels of GSH-px equivalents were detected by assay kit. (e)The level of O2- in human OA chondrocyte was stained with MitoSox Red dye and analyzed by confocal microscopy. OA (OA chondrocytes were not treated with nifedipine); 2 μM (OA chondrocyte treated with 2 μM nifedipine); 4 μM (OA chondrocyte treated with 4 μM nifedipine); 8 μM (OA chondrocyte treated with 8 μM nifedipine). All experiments were performed three times. Values are the means ± SD, n=6, *, p < 0.05; **, p < 0.01; ***, p < 0.001, relative to the OA group; #, p < 0.05; ##, p < 0.01; ###, p <
Jo ur n
al
Pr
e-
pr
oo
f
0.001, relative to the 2 μM group. Scale bar, 100 µm.
Journal Pre-proof
Figure 6 Nifedipine inhibits oxidative stress and ameliorates OA by activating Nrf2 pathway. Human OA chondrocytes were stimulated or not stimulated with nifedipine (8 μM) or stimulated with nifedipine (8 μM) and BR(0.3μg/ml) for 24 h. (a)The mRNA expression of Nrf2, HO-1 and GCLM were measured by qPCR. (b) Oxidative stress related protein Nrf2, HO-1, NQO1, GCLM and SOD2 expression was detected by Western blot. (c) Inflammation-related proteins IL-1β, IL-6 and MMP-13 expression detected by Western blot. OA (OA chondrocytes were not treated with nifedipine); Nifedipine (OA chondrocyte treated with 8 μM nifedipine); Nifedipine+BR (OA chondrocyte treated with 8 μM nifedipine and 0.3μg/ml BR). All experiments were performed three times. Values are the means ± SD, n=6, *, p < 0.05; **, p < 0.01; ***, p < 0.001, relative to the OA group; #, p < 0.05;
Jo ur n
al
Pr
e-
pr
oo
f
##, p < 0.01; ###, p < 0.001, relative to the Nifedipine group.
Journal Pre-proof
Table 1. Primer Sequences Used in qPCR Experiments (human) Gene name
Forward primer
Size
Reverse primer
Size
5’-AGGGCCCTGACAACTCTTTT-3’
20bp
5’-AGGGGTCTACATGGCAACTG-3’
20bp
IL-1β
5’-TGAGCTCGCCAGTGAAATGA-3’
21bp
5’-CATGGCCACAACAACTGACG-3’
20bp
MMP-13
5’-GCCATTACCAGTCTCCGAGG-3’
20bp
5’-TACGGTTGGGAAGTTCTGGC-3’
20bp
TNF-α
5’-TCAGAGGGCCTGTACCTCAT-3’
20bp
5’-GGAGGTTGACCTTGGTCTGG-3’
20bp
IL-6
5’-TCTGCGCAGCTTTAAGGAGT-3’
20bp
5’-CCCAGTGGACAGGTTTCTGA-3’
20bp
Aggrecan
5’-CCAGAAGGGTCAGGAGAAAC-3’
20bp
5’-AAAGTGTCCAAGGCATCCAC-3’
20bp
Collaggen Ⅱ
5’-GGCTCCCAGAACATCACCTA-3’
20bp
5’-GCCCTCATCTCCACATCATT-3’
20bp
ADAMTS-5
5’-TAGGCGTCTTTGGCTTCAGT-3’
20bp
5-GCTTTCAGGCTGACCAGTTC-3’
20bp
oo
f
GAPDH
e-
Pr
Osteoarthritic Nuclear factor erythroid-2-related factor 2 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Reverse transcription-polymerase chain reaction Reactive oxygen species Glutathione peroxidase Catalase Superoxide dismutase Brusatol Interleukin Tumor necrosis factor α Cyclooxygenase2 Nitric oxide Inducible nitric oxide synthase Prostaglandin E2 Heme oxygenase 1 Non-steroidal anti-inflammatory drugs Dimethylsulfoxide propidium iodide Fetal bovine serum Glycosaminoglycans 1,9-dimethylmethylene blue Tris-buffered saline sulfate-polyacrylamide gel electrophoresis a disintegrin and metalloprotease with thrombospondinmotifs NAD(P)H dehydrogenase (quinone)1 Glutamate-cysteine ligase
al
OA Nrf2 MTT RT-PCR ROS GSH-px CAT SOD BR IL TNF-α COX-2 NO iNOS PGE2 HO-1 NSAIDs DMSO PI FBS GAG DMMB TBS SDS PAGE ADAMTS-5 NQO1 GCLM
Full name
Jo ur n
Abbreviation
pr
Table2. Abbreviation
Journal Pre-proof
Highlights
Research highlights Nifedipine has anti-inflammatory effect on OA chondrocytes. Nifedipine has chondro-protective effect on OA chondrocytes.
Jo ur n
al
Pr
e-
pr
oo
f
Nifedipine inhibits oxidative stress through Nrf2/HO-1 signaling pathway in OA chondrocytes