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Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin-1β-induced inflammation and apoptosis in vitro
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Kaohsiung Journal of Medical Sciences (2016) xx, 1e7
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ORIGINAL ARTICLE
Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin-1binduced inflammation and apoptosis in vitro Xian-Xiang Xu*, Xiao-Hong Zhang, Yong Diao, Yu-Xiang Huang Department of Pharmacy, Huaqiao University, Quanzhou, China Received 22 September 2016; accepted 16 November 2016
KEYWORDS Achyranthes bidentata saponins; Apoptosis; Chondrocyte; Inflammation; Osteoarthritis
Abstract Achyranthes bidentate Blume (Niuxi) is often employed for treatment of arthritis in Traditional Chinese Medicine and possesses anti-inflammatory properties. Phytochemical and pharmacological studies proved the oleanane-type saponins to be the main bioactive principles. In the present study, protective effects of A. bidentata saponins (ABS) on inflammation and apoptosis in interleukine-1b (IL-1b)-induced chondrocytes were investigated. Rat chondrocytes were pretreated with ABS at 3 mg/mL, 10 mg/mL, and 30 mg/mL, and subsequently stimulated with IL-1b (10 ng/mL). Methylthiazolyldiphenyl-tetrazolium bromide assay and annexin V/propidium iodide dual staining demonstrated that ABS could protect IL-1b-induced chondrocyte injury. ABS suppressed IL-1b-induced apoptosis by suppressing the activation of caspase-3, inhibiting levels of proapoptotic proteins Bax and Bad, decreasing p53 protein phosphorylation, and promoting the expression of antiapoptotic protein Bcl-xL and proliferating cell nuclear antigen. IL-1b-induced inflammation and matrix degradation were also alleviated by ABS through the downregulation of the expressions of matrix metalloproteinases 3 and 9 and cyclooxygenase2. Moreover, ABS inhibited IL-1b-induced nuclear factor kB activation in rat chondrocytes. We demonstrated, for the first time, the protective effects of ABS on IL-1b-stimulated chondrocytes and their molecular mechanisms. Thus, it is suggested that ABS might be a potential drug in the treatment of osteoarthritis. Copyright ª 2016, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Conflicts of interest: All authors declare no conflicts of interest. * Corresponding author. Department of Pharmacy, Huaqiao University, 269 Chenghua North Road, Quanzhou 362021, China. E-mail address: [email protected] (X.-X. Xu). http://dx.doi.org/10.1016/j.kjms.2016.11.004 1607-551X/Copyright ª 2016, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Xu X-X, et al., Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin1b-induced inflammation and apoptosis in vitro, Kaohsiung Journal of Medical Sciences (2016), http://dx.doi.org/10.1016/ j.kjms.2016.11.004
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Introduction Osteoarthritis (OA) is the most common form of joint disease associated with aging. It is characterized by the progressive destruction of articular cartilage [1e3]. Chondrocytes are the only cells present in articular cartilage. The primary function of chondrocytes is to maintain cartilage structure and function, in part through the production of extracellular matrix components [4]. Chondrocyte apoptosis is implicated in cartilage degeneration. Multiple stimuli, including inflammation, affect the development and progression of OA. Proinflammatory cytokines such as interleukin-1b (IL1b) have been shown to be related to chondrocyte apoptosis and degeneration of articular cartilage matrix, which make them prime targets for therapeutic strategies [5e7]. Current therapeutic options such as nonsteroidal antiinflammatory drugs can provide a palliative effect on symptoms but have a limited effect on disease progression. Some drugs have entered into clinical trials, and a few have shown improvements in both pain and structure changes. However, most of them have failed in clinical trials largely due to increased side effects or the failure to identify the right OA phenotype for the right drug in a clinical design. Thus, treatment options for OA remain limited [8]. Achyranthes bidentate Blume (Amaranthaceae) is widely distributed and grown in China and some other Asian countries. Its dried root (“Niuxi” in Chinese, Radix Achyranthes Bidentatae) is an important medicinal herb documented in Chinese Pharmacopeia. The main efficacies of Niuxi include nourishing the liver and kidney, strengthening bones and muscles, invigorating circulation. Radix Achyranthes Bidentatae is usually prescribed for arthritis treatment in Traditional Chinese Medicine. A. bidentata has extensively been described to exhibit anti-inflammatory activities and be useful for the treatment of rheumatoid arthritis [9,10]. Triterpenoid saponins were found in A. bidentata including chikusetsusaponins IVa and V; achyranthosides B, C, D, E, and G; sulfachyranthosides B and D; and betavulgarosides II and IV [11]. A. bidentata saponins (ABS), including about 15 oleanane-type saponins, were found to induce proliferation and differentiation in bone marrow stromal cells and show adjuvant effect [12,13]. In this work, effects of ABS on IL-1b-induced apoptosis and inflammation in rat chondrocytes were investigated.
Materials and methods Chemicals and reagents IL-1b was purchased from PeproTech (Rocky Hill, NJ, USA). Annexin V-FITC/propidium iodide dual staining kit was purchased from MultiSciences Biotech (Hangzhou, China). Rabbit anti-cyclooxygenase-2 (anti-COX-2) polyclonal antibody was purchased from Boster (Wuhan, China). Caspase-3 colorimetric assay kits were purchased from KeyGEN Bio Tech (Najing, China). Hoechst 33258 staining kits, rabbit anti-p53, antiphosphate p65 monoclonal antibody, and mouse anti-b actin monoclonal antibody were provided by Beyotime Biotech (Haimen, China). Anti-Bad, Bax, Bcl-xL, proliferating cell nuclear antigen (PCNA), phospho-p53, matrix metalloproteinase (MMP) 3, MMP 9, IkBa, and p-
X.-X. Xu et al. IkBa monoclonal antibodies were purchased from Epitmics (Burlingame, CA, USA).
Preparation of ABS Radix Achyranthes Bidentatae (roots of A. bidentata) were purchased from a drugstore in Quanzhou, and identified by the pharmacist Jiani Hong at Quanzhou Hospital of Traditional Chinese Medicine according to China Pharmacopoeia. A voucher specimen was deposited in the Department of Pharmacology of Chinese Materia Medica, Huaqiao University. ABS was prepared as follows. Briefly, 3.5-kg crude power of the dried roots was extracted with 70% ethanol three times under reflux for 2 hours and then concentrated in a vacuum to evaporate the solvent. The residue was diluted with water and extracted with n-butyl alcohol until the n-butyl alcohol layer became colorless. The n-butyl alcohol solution was concentrated and dried in vacuum. The dried extract was purified with D101 macroporous absorptive resin, washed with water, and eluted with ethanol. The 50% ethanol-eluted fraction was collected, evaporated in a vacuum, and lyophilized to yield 3.2% yellowish-brown powder (ABS). The total content of saponin was 56.3%, using oleanolic acid as a standard substance and determined by UVevisible colorimetry using a combination of vanillin, acetic acid, and perchloric acid. Chondrocyte isolation and culture Chondrocytes were isolated from the knee joints of 3e4week-old Sprague-Dawley rats (Shanghai SLAC Laboratory Animal Co., Ltd, Shanghai, China). Articular cartilage was isolated from rat femoral grooves and femoral condyles, and then aseptically dissected. After digestion in 0.25% trypsin at 37 C for 30 minutes, the tissue was digested with 0.3% collagenase II for 3 hours at 37 C. The chondrocytes were filtered through a mesh screen to prepare a single-cell suspension, and then centrifuged at 1500g for 10 minutes, transferred into a culture flask, and incubated at 37 C in a 5% CO2 incubator with complete Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/F12) medium (containing 20% newborn calf serum, 100 IU/mL streptomycin, and 100 IU/mL penicillin). Type II collagen immunofluorescence staining was employed to identify the chondrocytes. Two to three passages were used for experimental procedures. Methylthiazolyldiphenyl-tetrazolium bromide assay Cell viability was assessed using methylthiazolyldiphenyltetrazolium bromide (MTT) assay. In brief, chondrocytes were seeded in 96-well plates and cultured for 24 hours. Then the cells were treated with different concentrations of ABS for 1 hour and stimulated with IL-1 b (10 ng/mL) for 48 hours; 20 mL MTT was added. After incubation for 4 hours at 37 C, dimethyl sulfoxide was added and the plates were shaken for 10 minutes until MTT formazan crystals were completely dissolved. Absorbance was measured at a wavelength of 570 nm with a spectrophotometer. Apoptosis analysis After treatments, 1 106 cells were harvested and washed thrice with phosphate-buffered saline, then resuspended in
Please cite this article in press as: Xu X-X, et al., Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin1b-induced inflammation and apoptosis in vitro, Kaohsiung Journal of Medical Sciences (2016), http://dx.doi.org/10.1016/ j.kjms.2016.11.004
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binding buffer followed by annexin V-FITC/propidium iodide staining. The samples were analyzed using a FACScan flow cytometer (Becton Dickinson, CA, USA). The caspase-3 activity was determined with the caspase-3 colorimetric assay kits. An increase in absorbance at 405 nm was used to quantify caspase-3 activation. Western blotting analysis Total proteins from chondrocytes were extracted using extraction kits. Protein concentration was determined according to the bicinchoninic acid method. The proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were preincubated in blocking buffer (5% skimmed milk powder in Tris buffer solution containing 0.1% Tween-20) for 1 hour at room temperature, and then incubated with the primary antibody in overnight at 4 C. The membranes were washed thrice with Tris buffer solution containing 0.1% Tween-20, incubated with the secondary antibody for 1.5 hours, and developed with an enhanced chemoluminescence detection kit. The bands were quantified by densitometry using ‘‘Quantity one’’ software package (Bio-Rad, CA, USA).
Statistical analysis Data were presented as mean standard deviation for at least three experiments. Statistical significance was evaluated by one-way analysis of variance followed by Dunnett’s test. A p value of <0.05 was considered statistically significant.
Results ABS suppressed IL-1b-induced chondrocyte apoptosis ABS at concentrations of 3 mg/mL, 10 mg/mL, and 30 mg/mL prevented IL-1b-induced cell death in a concentrationdependent manner (Figure 1). The following assays were performed in order to confirm that ABS prevented IL-1binduced cell death by blocking apoptosis. IL-1b-induced chondrocyte apoptosis was observed by flow cytometry analysis using annexin V/propidium iodide staining. ABS decreased the rate of apoptotic chondrocytes (Figure 2). Caspase-3 plays an essential role in most types of apoptosis. Caspase-3 was dramatically activated in IL-1b-induced chondrocyte apoptosis. ABS prevented the increase of activation of caspase-3 in a concentration-dependent manner (Figure 3). The expressions of Bcl-xL, Bad, and Bax were investigated to explore whether ABS could influence anti- and proapoptotic genes in IL-1b-induced chondrocyte apoptosis. Western blotting results demonstrated that ABS was able to upregulate the expression of the antiapoptotic protein Bcl-xL, while downregulate the expression of the proapoptotic proteins Bad and Bax (Figure 4).
Figure 1. Effect of Achyranthes bidentate saponins (ABS) on interleukin (IL)-1b-induced chondrocytes vitality. Chondrocytes were treated with 10 ng/mL IL-1b and ABS at different concentrations for 24 hours. Data were expressed as mean standard deviation of triplicates. ** p < 0.01 versus the model control (IL1b alone treatment).
ABS-suppressed proinflammatory and matrixdegraded enzymes in IL-1b-induced chondrocytes Stimulation with IL-1b significantly increased the production of more proinflammatory mediators such as MMPs and COX-2. The effect of ABS on IL-1b-induced MMP 3, MMP 9, and COX-2 is shown in Figure 5. ABS (3 mg/mL, 10 mg/mL, and 30 mg/mL) strikingly suppressed the upregulation of both MMPs and COX-2 in IL-1b-stimulated chondrocytes.
Effects of ABS on IL-1b-induced p53 phosphorylation and PCNA gene expression Stimulation with IL-1b (10 ng/mL) led to p53 phosphorylation without alteration of p53 levels and downregulation of the expression of PCNA. ABS prevented the upregulation of p-p53 and reduction of PCNA in IL-1b-stimulated chondrocytes (Figure 6).
ABS inhibited IL-1b-induced nuclear factor kB activation It is well known that nuclear factor (NF)-kB plays an important role in the regulation of apoptosis and inflammatory mediators. To investigate whether ABS inhibits NFkB activation in IL-1b-stimulated chondrocytes, IkBa and its phosphorylation was detected by Western blotting. The results demonstrated that IL-1b significantly upregulated IkBa degradation and phosphorylation in chondrocytes. However, the upregulation was remarkably inhibited by the treatment of ABS (Figure 7).
Discussion It is well known that inflammatory cytokines are the key elements participating in OA pathogenesis, and IL-1b is thought to be a representative cytokine involved [3].
Please cite this article in press as: Xu X-X, et al., Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin1b-induced inflammation and apoptosis in vitro, Kaohsiung Journal of Medical Sciences (2016), http://dx.doi.org/10.1016/ j.kjms.2016.11.004
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Figure 2. Flow cytometry analysis of annexin V/propidium iodide dual staining of interleukin (IL)-1b-treated chondrocytes. The chondrocytes were treated with 10 ng/mL IL-1b plus Achyranthes bidentate saponins (ABS) at different concentrations for 24 hours. (A) X- and Y-axis indicate V negative/positive and propidium iodide negative/positive, respectively. Numbers in the right upper and right lower quadrants denote percentages of late and early apoptotic chondrocytes (annexin V positive). The small letters indicate the following: a, normal control (medium only); b, IL-1b (10 ng/mL); c, IL-1b (10 ng/mL) þ ABS (3 mg/mL); d, IL-1b (10 ng/mL) þ ABS (10 mg/mL); and e, IL-1b (10 ng/mL) þ ABS (30 mg/mL). (B) Columns, apoptotic rate in each group (late and early apoptotic chondrocytes). n Z 3. * p < 0.05 versus the model control. ** p < 0.01 versus the model control. FL1 Z Fluorescence 1 (Annexin V-FITC); FL2 Z Fluorescence 2 (PI).
Figure 3. Prevention of the activation of caspase-3 by Achyranthes bidentate saponins (ABS). Interleukin (IL)-1bstimulated chondrocytes were treated with ABS (3 mg/mL, 10 mg/mL, and 30 mg/mL) for 24 hours (n Z 3). ** p < 0.01 versus the model control.
Activation of IL-1b triggers a cascade of cartilage damage events such as the production of more proinflammatory cytokines and the release of some inflammatory mediators such as prostaglandin E2 produced by COX-2 [14]. Prostaglandin E2, in turn, amplified the inflammation in chondrocytes [15]. IL-1b is also capable of reducing the production of cartilage-specific macromolecules, including type II collagen, inducing the release of MMPs that are involved in cartilage matrix degradation in OA [16]. Increased IL-1b was detected in synovial fluid of patients with OA. Elevated IL-1b levels could lead to cartilage destruction and pain [17]. Therefore, inhibitor of IL-1b such as IL-1 receptor antagonist may offer a useful therapeutic approach to the management of OA [18]. In the present study, IL-1b promoted the expressions of COX-2, MMP 3, and MMP 9 in IL-1b-stimulated chondrocytes, while ABS remarkably suppressed the increased expressions. To investigate the anti-inflammatory mechanisms of ABS, we focused on the effects of ABS on the transcription factor NF-kB in chondrocytes. IL-1b activates NF-kB via
Please cite this article in press as: Xu X-X, et al., Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin1b-induced inflammation and apoptosis in vitro, Kaohsiung Journal of Medical Sciences (2016), http://dx.doi.org/10.1016/ j.kjms.2016.11.004
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Figure 4. Effects of Achyranthes bidentate saponins (ABS) on the expressions of anti- and proapoptotic proteins in interleukin (IL)-1b-induced chondrocytes. IL-1b-stimulated chondrocytes were treated with ABS (3 mg/mL, 10 mg/mL, and 30 mg/mL) for 24 hours. (A) Expressions of Bad, Bax, and Bcl-xL were detected by Western blotting; b-actin was used as an internal control. (B) Relative protein expression of Bax and Bcl-xL was quantified by densitometry using “Quantity one” software, normalized to b-actin and compared with normal control. n Z 3. * p < 0.05 versus the model control. ** p < 0.01 versus the model control. *** p < 0.05 versus the model control. **** p < 0.01 versus the model control. ***** p < 0.05 versus the model control. ****** p < 0.01 versus the model control.
Figure 5. Achyranthes bidentate saponins (ABS) suppressed proinflammatory and matrix-degraded enzymes in interleukin (IL)1b-induced chondrocytes. IL-1b-stimulated chondrocytes were treated with ABS for 24 hours. (A) Expressions of matrix metalloproteinase (MMP) 3, MMP 9, and cyclooxygenase-2 (COX-2) were detected by Western blotting. b-Actin was as used an internal control. (B) Relative protein expression of MMP 3, MMP 9, and COX-2 was quantified by densitometry using “Quantity one” software, normalized to b-actin and compared with normal control. n Z 3. * p < 0.05 versus the model control. **** p < 0.01 versus the model control. þp < 0.05 versus the model control. þþp < 0.01 versus the model control
Figure 6. Effects of Achyranthes bidentate saponins (ABS) on p53 and proliferating cell nuclear antigen (PCNA) in interleukin (IL)-1b-induced chondrocytes. (A) IL-1b-stimulated chondrocytes were treated with ABS for 24 hours. Western blotting was performed with anti-p53, anti-pp53, and anti-PCNA. b-Actin was used as an internal control. (B) Relative protein expression of p53, pp53 and PCNA was quantified by densitometry using “Quantity one” software, normalized to b-actin and compared with normal control. n Z 3. ** p < 0.01 versus the model control. *** p < 0.05 versus the model control. **** p < 0.01 versus the model control. Please cite this article in press as: Xu X-X, et al., Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin1b-induced inflammation and apoptosis in vitro, Kaohsiung Journal of Medical Sciences (2016), http://dx.doi.org/10.1016/ j.kjms.2016.11.004
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Figure 7. Achyranthes bidentate saponins (ABS) inhibited nuclear factor-kB activation in interleukin (IL)-1b-induced chondrocytes. (A) IL-1b-stimulated chondrocytes were treated with ABS for 24 hours. IkBa and p-IkBa were detected by Western blotting. b-Actin was used as an internal control. (B) Relative protein expression of IkBa and p-IkBa was quantified by densitometry using “Quantity one” software, normalized to b-actin and compared with normal control. n Z 3. ** p < 0.01 versus the model control. **** p < 0.01 versus the model control.
triggering IkBa degradation, contributing to cartilage degradation in OA [19]. Once activated, NF-kB unit p65 dissociates from its inhibitory protein IkBa and translocates from the cytoplasm to the nucleus, and triggers the transcription of specific target genes such as COX-2 [20]. Furthermore, our data also demonstrated that ABS exhibited its anti-inflammatory effects by preventing IL-1binduced IkBa degradation and phosphorylation. Increased IL-1b levels can also trigger apoptosis in chondrocytes, which leads to further degenerative changes in cartilage [21]. In IL-1b-induced chondrocytes, expression of the antiapoptotic protein Bcl-xL was downregulated, while those of the proapoptotic proteins Bad and Bax were upregulated. ABS inhibited both expression changes. Caspase-3 is a crucial biomarker of apoptosis that also acts as an apoptotic executor [22]. ABS markedly alleviated the increase of caspase-3 activity in IL-1b-stimulated chondrocytes. Meanwhile, ABS inhibited the apoptotic rates of IL-1b-indued rat chondrocytes. This study indicated that ABS prevented IL-1b-induced apoptosis by modulating signaling events, such as phosphorylation of p53 and downregulation of PCNA. PCNA is the most representative proliferating marker and plays important roles in DNA replication and repairing. PCNA is regulated by the tumor suppressor gene p53, which can block DNA replication inducing cell apoptosis [23,24]. ABS had no obvious influence on the expression of p53, but could inhibit phosphorylation of p53 and increase the expression of PCNA. OA is a slowly progressing degenerative arthritis. The pharmacological treatment strategies for OA are to decrease symptoms, improve function, and delay time to surgery. Although anti-inflammatory agents such as nonsteroidal anti-inflammatory drugs have good effects on OA, an increased risk of gastrointestinal, cardiovascular, or renal side effects remains one of the problems for their long-term use [25]. There is a strong need to develop an effective treatment regime with fewer side effects. Traditional Chinese Medicine can be an alternative treatment method and is commonly used in China to manage symptoms associated with OA. Recently, a growing body of evidence indicates that active ingredients from
Chinese herbal medicine are noteworthy because of their potential impact on safety and costs. According to the basic theory of Traditional Chinese Medicine, OA is an “impediment disease,” which mainly includes syndromes of qi stagnation and blood stasis, cold dampness, deficiency of Gan (liver) and Shen (kidney), and deficiency of Qi and blood [26]. A. bidentata can strengthen the waist and knee, by eliminating stagnation, removing blood stasis, and nourishing the liver and kidney. Chinese herbal medicine formulas containing A. bidentata have been used in clinical practice for thousands of years, such as Duhuo Jisheng decoction [27] and SiMiaoFang [28]. A. bidentata is used to eliminate inflammation or improve circulation of the knees. Our results show that ABS is effective not only against inflammatory symptoms, but also against apoptosis of IL-1bstimulated chondrocytes in OA. Further pharmacodynamic studies will be performed in a rat model of OA. With both antiapoptotic and anti-inflammatory properties, ABS might help protect against the degeneration of cartilage in patients with OA.
Acknowledgments This work was supported by the Promotion Program for Young and Middle-aged Teacher in Science and Technology Research of Huaqiao University (Number ZQN-PY420), the Key Project of Quanzhou City Science and Technology Program (Number 2011Z8), and the Natural Science Foundation of Fujian Province of China (Number 2015J01364).
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A. bidentata saponins protect chondrocytes [4] Caron MM, Emans PJ, Coolsen MM, Voss L, Surtel DA, Cremers A, et al. Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures. Osteoarthritis Cartilage 2012;20:1170e8. [5] Kapoor M, Martel-Pelletier J, Lajeunesse D, Pelletier JP, Fahmi H. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol 2011;7: 33e42. [6] Goldring MB, Otero M. Inflammation in osteoarthritis. Curr Opin Rheumatol 2011;23:471e8. [7] Bian Q, Wang YJ, Liu SF, Li YP. Osteoarthritis: genetic factors, animal models, mechanisms, and therapies. Front Biosci (Elite Ed) 2012;4:74e100. [8] Wang K, Xu J, Hunter DJ, Ding C. Investigational drugs for the treatment of osteoarthritis. Expert Opin Investig Drugs 2015; 24:1539e56. [9] Vetrichelvan T, Jegadeesan M. Effect of alcoholic extract of Achyranthes bidentata Blume on acute and sub acute inflammation. Indian J Pharmacol 2002;34:115e8. [10] Han SB, Lee CW, Yoon YD, Lee JH, Kang JS, Lee KH, et al. Prevention of arthritic inflammation using an oriental herbal combination BDX-1 isolated from Achyranthes bidentata and Atractylodes japonica. Arch Pharm Res 2005;28:902e8. [11] Kawahara Y, Hoshino T, Morimoto H, Shinizu T, Narukawa Y, Fuchino H, et al. LC-MS-based quantification method for Achyranthes root saponins. J Nat Med 2016;70:102e6. [12] He G, Guo W, Lou Z, Zhang H. Achyranthes bidentata saponins promote osteogenic differentiation of bone marrow stromal cells through the ERK MAPK signaling pathway. Cell Biochem Biophys 2014;70:467e73. [13] Sun HX. Adjuvant effect of Achyranthes bidentata saponins on specific antibody and cellular response to ovalbumin in mice. Vaccine 2006;24:3432e9. [14] van Vulpen LF, Schutgens RE, Coeleveld K, Alsema EC, Roosendaal G, Mastbergen SC, et al. IL-1b, in contrast to TNFa, is pivotal in blood-induced cartilage damage and is a potential target for therapy. Blood 2015;126:2239e46. [15] Dave M, Amin AR. YineYang regulation of prostaglandins and nitric oxide by PGD2 in human arthritis: reversal by celecoxib. Immunol Lett 2013;152:47e54. [16] Rousset F, Hazane-Puch F, Pinosa C, Nguyen MV, Grange L, Soldini A, et al. IL-1beta mediates MMP secretion and IL-1beta neosynthesis via upregulation of p22(phox) and NOX4 activity in human articular chondrocytes. Osteoarthritis Cartilage 2015;23:1972e80. [17] Jotanovic Z, Mihelic R, Sestan B, Dembic Z. Role of interleukin-1 inhibitors in osteoarthritis: an evidence-based review. Drugs Aging 2012;29:343e58.
7 [18] Santangelo KS, Nuovo GJ, Bertone AL. In vivo reduction or blockade of interleukin-1b in primary osteoarthritis influences expression of mediators implicated in pathogenesis. Osteoarthritis Cartilage 2012;20:1610e8. [19] Roman-Blas JA, Jimenez SA. NF-kappaB as a potential therapeutic target in osteoarthritis and rheumatoid arthritis. Osteoarthritis Cartilage 2006;14:839e48. [20] Chen J, Fok KL, Chen H, Zhang XH, Xu WM, Chan HC. Cryptorchidism-induced CFTR down-regulation results in disruption of testicular tight junctions through up-regulation of NFkB/COX-2/PGE2. Hum Reprod 2012;27:2585e97. [21] Fernandes JC, Martel-Pelletier J, Pelletier JP. The role of cytokines in osteoarthritis pathophysiology. Biorheology 2002; 39:237e46. [22] Jiang L, Li L, Geng C, Gong D, Jiang L, Ishikawa N, et al. Monosodium iodoacetate induces apoptosis via the mitochondrial pathway involving ROS production and caspase activation in rat chondrocytes in vitro. J Orthop Res 2013;31: 364e9. [23] Kato K, Kawashiri S, Yoshizawa K, Kitahara H, Okamune A, Sugiura S, et al. Expression form of p53 and PCNA at the invasive front in oral squamous cell carcinoma: correlation with clinicopathological features and prognosis. J Oral Pathol Med 2011;40:693e8. [24] Cvejic D, Selemetjev S, Savin S, Paunovic I, Petrovic I, Tatic S. Apoptosis and proliferation related molecules (Bcl-2, Bax, p53, PCNA) in papillary microcarcinoma versus papillary carcinoma of the thyroid. Pathology 2008;40:475e80. [25] Laev SS, Salakhutdinov NF. Anti-arthritic agents: progress and potential. Bioorg Med Chem 2015;23:3059e80. [26] Chen WH, Liu XX, Tong PJ, Zhan HS, Orthopaedic Professional Committee, Chinese Association of Research and Advancement of Chinese Traditional Medicine, China; Joint Professional Committee, Branch of Orthopaedic of Chinese Association of Integrative Medicine, China. Diagnosis and management of knee osteoarthritis: Chinese medicine expert consensus (2015). Chin J Integr Med 2016;22:150e3. [27] Zhang WM, Wang SQ, Zhang RX, Zhang YY, Li XJ, Lin YP, et al. Evidence of Chinese herbal medicine Duhuo Jisheng decoction for knee osteoarthritis: a systematic review of randomised clinical trials. BMJ Open 2016;6:e008973. [28] Xu Y, Liu Q, Liu ZL, Lim L, Chen WH, Lin N. Treatment with SiMiaoFang, an anti-arthritis Chinese herbal formula, inhibits cartilage matrix degradation in osteoarthritis rat model. Rejuvenation Res 2013;16:364e76.
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Kaohsiung Journal of Medical Sciences (2016) xx, 1e7
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ORIGINAL ARTICLE
Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin-1binduced inflammation and apoptosis in vitro Xian-Xiang Xu*, Xiao-Hong Zhang, Yong Diao, Yu-Xiang Huang Department of Pharmacy, Huaqiao University, Quanzhou, China Received 22 September 2016; accepted 16 November 2016
KEYWORDS Achyranthes bidentata saponins; Apoptosis; Chondrocyte; Inflammation; Osteoarthritis
Abstract Achyranthes bidentate Blume (Niuxi) is often employed for treatment of arthritis in Traditional Chinese Medicine and possesses anti-inflammatory properties. Phytochemical and pharmacological studies proved the oleanane-type saponins to be the main bioactive principles. In the present study, protective effects of A. bidentata saponins (ABS) on inflammation and apoptosis in interleukine-1b (IL-1b)-induced chondrocytes were investigated. Rat chondrocytes were pretreated with ABS at 3 mg/mL, 10 mg/mL, and 30 mg/mL, and subsequently stimulated with IL-1b (10 ng/mL). Methylthiazolyldiphenyl-tetrazolium bromide assay and annexin V/propidium iodide dual staining demonstrated that ABS could protect IL-1b-induced chondrocyte injury. ABS suppressed IL-1b-induced apoptosis by suppressing the activation of caspase-3, inhibiting levels of proapoptotic proteins Bax and Bad, decreasing p53 protein phosphorylation, and promoting the expression of antiapoptotic protein Bcl-xL and proliferating cell nuclear antigen. IL-1b-induced inflammation and matrix degradation were also alleviated by ABS through the downregulation of the expressions of matrix metalloproteinases 3 and 9 and cyclooxygenase2. Moreover, ABS inhibited IL-1b-induced nuclear factor kB activation in rat chondrocytes. We demonstrated, for the first time, the protective effects of ABS on IL-1b-stimulated chondrocytes and their molecular mechanisms. Thus, it is suggested that ABS might be a potential drug in the treatment of osteoarthritis. Copyright ª 2016, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Conflicts of interest: All authors declare no conflicts of interest. * Corresponding author. Department of Pharmacy, Huaqiao University, 269 Chenghua North Road, Quanzhou 362021, China. E-mail address: [email protected] (X.-X. Xu). http://dx.doi.org/10.1016/j.kjms.2016.11.004 1607-551X/Copyright ª 2016, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Xu X-X, et al., Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin1b-induced inflammation and apoptosis in vitro, Kaohsiung Journal of Medical Sciences (2016), http://dx.doi.org/10.1016/ j.kjms.2016.11.004
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Introduction Osteoarthritis (OA) is the most common form of joint disease associated with aging. It is characterized by the progressive destruction of articular cartilage [1e3]. Chondrocytes are the only cells present in articular cartilage. The primary function of chondrocytes is to maintain cartilage structure and function, in part through the production of extracellular matrix components [4]. Chondrocyte apoptosis is implicated in cartilage degeneration. Multiple stimuli, including inflammation, affect the development and progression of OA. Proinflammatory cytokines such as interleukin-1b (IL1b) have been shown to be related to chondrocyte apoptosis and degeneration of articular cartilage matrix, which make them prime targets for therapeutic strategies [5e7]. Current therapeutic options such as nonsteroidal antiinflammatory drugs can provide a palliative effect on symptoms but have a limited effect on disease progression. Some drugs have entered into clinical trials, and a few have shown improvements in both pain and structure changes. However, most of them have failed in clinical trials largely due to increased side effects or the failure to identify the right OA phenotype for the right drug in a clinical design. Thus, treatment options for OA remain limited [8]. Achyranthes bidentate Blume (Amaranthaceae) is widely distributed and grown in China and some other Asian countries. Its dried root (“Niuxi” in Chinese, Radix Achyranthes Bidentatae) is an important medicinal herb documented in Chinese Pharmacopeia. The main efficacies of Niuxi include nourishing the liver and kidney, strengthening bones and muscles, invigorating circulation. Radix Achyranthes Bidentatae is usually prescribed for arthritis treatment in Traditional Chinese Medicine. A. bidentata has extensively been described to exhibit anti-inflammatory activities and be useful for the treatment of rheumatoid arthritis [9,10]. Triterpenoid saponins were found in A. bidentata including chikusetsusaponins IVa and V; achyranthosides B, C, D, E, and G; sulfachyranthosides B and D; and betavulgarosides II and IV [11]. A. bidentata saponins (ABS), including about 15 oleanane-type saponins, were found to induce proliferation and differentiation in bone marrow stromal cells and show adjuvant effect [12,13]. In this work, effects of ABS on IL-1b-induced apoptosis and inflammation in rat chondrocytes were investigated.
Materials and methods Chemicals and reagents IL-1b was purchased from PeproTech (Rocky Hill, NJ, USA). Annexin V-FITC/propidium iodide dual staining kit was purchased from MultiSciences Biotech (Hangzhou, China). Rabbit anti-cyclooxygenase-2 (anti-COX-2) polyclonal antibody was purchased from Boster (Wuhan, China). Caspase-3 colorimetric assay kits were purchased from KeyGEN Bio Tech (Najing, China). Hoechst 33258 staining kits, rabbit anti-p53, antiphosphate p65 monoclonal antibody, and mouse anti-b actin monoclonal antibody were provided by Beyotime Biotech (Haimen, China). Anti-Bad, Bax, Bcl-xL, proliferating cell nuclear antigen (PCNA), phospho-p53, matrix metalloproteinase (MMP) 3, MMP 9, IkBa, and p-
X.-X. Xu et al. IkBa monoclonal antibodies were purchased from Epitmics (Burlingame, CA, USA).
Preparation of ABS Radix Achyranthes Bidentatae (roots of A. bidentata) were purchased from a drugstore in Quanzhou, and identified by the pharmacist Jiani Hong at Quanzhou Hospital of Traditional Chinese Medicine according to China Pharmacopoeia. A voucher specimen was deposited in the Department of Pharmacology of Chinese Materia Medica, Huaqiao University. ABS was prepared as follows. Briefly, 3.5-kg crude power of the dried roots was extracted with 70% ethanol three times under reflux for 2 hours and then concentrated in a vacuum to evaporate the solvent. The residue was diluted with water and extracted with n-butyl alcohol until the n-butyl alcohol layer became colorless. The n-butyl alcohol solution was concentrated and dried in vacuum. The dried extract was purified with D101 macroporous absorptive resin, washed with water, and eluted with ethanol. The 50% ethanol-eluted fraction was collected, evaporated in a vacuum, and lyophilized to yield 3.2% yellowish-brown powder (ABS). The total content of saponin was 56.3%, using oleanolic acid as a standard substance and determined by UVevisible colorimetry using a combination of vanillin, acetic acid, and perchloric acid. Chondrocyte isolation and culture Chondrocytes were isolated from the knee joints of 3e4week-old Sprague-Dawley rats (Shanghai SLAC Laboratory Animal Co., Ltd, Shanghai, China). Articular cartilage was isolated from rat femoral grooves and femoral condyles, and then aseptically dissected. After digestion in 0.25% trypsin at 37 C for 30 minutes, the tissue was digested with 0.3% collagenase II for 3 hours at 37 C. The chondrocytes were filtered through a mesh screen to prepare a single-cell suspension, and then centrifuged at 1500g for 10 minutes, transferred into a culture flask, and incubated at 37 C in a 5% CO2 incubator with complete Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/F12) medium (containing 20% newborn calf serum, 100 IU/mL streptomycin, and 100 IU/mL penicillin). Type II collagen immunofluorescence staining was employed to identify the chondrocytes. Two to three passages were used for experimental procedures. Methylthiazolyldiphenyl-tetrazolium bromide assay Cell viability was assessed using methylthiazolyldiphenyltetrazolium bromide (MTT) assay. In brief, chondrocytes were seeded in 96-well plates and cultured for 24 hours. Then the cells were treated with different concentrations of ABS for 1 hour and stimulated with IL-1 b (10 ng/mL) for 48 hours; 20 mL MTT was added. After incubation for 4 hours at 37 C, dimethyl sulfoxide was added and the plates were shaken for 10 minutes until MTT formazan crystals were completely dissolved. Absorbance was measured at a wavelength of 570 nm with a spectrophotometer. Apoptosis analysis After treatments, 1 106 cells were harvested and washed thrice with phosphate-buffered saline, then resuspended in
Please cite this article in press as: Xu X-X, et al., Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin1b-induced inflammation and apoptosis in vitro, Kaohsiung Journal of Medical Sciences (2016), http://dx.doi.org/10.1016/ j.kjms.2016.11.004
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binding buffer followed by annexin V-FITC/propidium iodide staining. The samples were analyzed using a FACScan flow cytometer (Becton Dickinson, CA, USA). The caspase-3 activity was determined with the caspase-3 colorimetric assay kits. An increase in absorbance at 405 nm was used to quantify caspase-3 activation. Western blotting analysis Total proteins from chondrocytes were extracted using extraction kits. Protein concentration was determined according to the bicinchoninic acid method. The proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were preincubated in blocking buffer (5% skimmed milk powder in Tris buffer solution containing 0.1% Tween-20) for 1 hour at room temperature, and then incubated with the primary antibody in overnight at 4 C. The membranes were washed thrice with Tris buffer solution containing 0.1% Tween-20, incubated with the secondary antibody for 1.5 hours, and developed with an enhanced chemoluminescence detection kit. The bands were quantified by densitometry using ‘‘Quantity one’’ software package (Bio-Rad, CA, USA).
Statistical analysis Data were presented as mean standard deviation for at least three experiments. Statistical significance was evaluated by one-way analysis of variance followed by Dunnett’s test. A p value of <0.05 was considered statistically significant.
Results ABS suppressed IL-1b-induced chondrocyte apoptosis ABS at concentrations of 3 mg/mL, 10 mg/mL, and 30 mg/mL prevented IL-1b-induced cell death in a concentrationdependent manner (Figure 1). The following assays were performed in order to confirm that ABS prevented IL-1binduced cell death by blocking apoptosis. IL-1b-induced chondrocyte apoptosis was observed by flow cytometry analysis using annexin V/propidium iodide staining. ABS decreased the rate of apoptotic chondrocytes (Figure 2). Caspase-3 plays an essential role in most types of apoptosis. Caspase-3 was dramatically activated in IL-1b-induced chondrocyte apoptosis. ABS prevented the increase of activation of caspase-3 in a concentration-dependent manner (Figure 3). The expressions of Bcl-xL, Bad, and Bax were investigated to explore whether ABS could influence anti- and proapoptotic genes in IL-1b-induced chondrocyte apoptosis. Western blotting results demonstrated that ABS was able to upregulate the expression of the antiapoptotic protein Bcl-xL, while downregulate the expression of the proapoptotic proteins Bad and Bax (Figure 4).
Figure 1. Effect of Achyranthes bidentate saponins (ABS) on interleukin (IL)-1b-induced chondrocytes vitality. Chondrocytes were treated with 10 ng/mL IL-1b and ABS at different concentrations for 24 hours. Data were expressed as mean standard deviation of triplicates. ** p < 0.01 versus the model control (IL1b alone treatment).
ABS-suppressed proinflammatory and matrixdegraded enzymes in IL-1b-induced chondrocytes Stimulation with IL-1b significantly increased the production of more proinflammatory mediators such as MMPs and COX-2. The effect of ABS on IL-1b-induced MMP 3, MMP 9, and COX-2 is shown in Figure 5. ABS (3 mg/mL, 10 mg/mL, and 30 mg/mL) strikingly suppressed the upregulation of both MMPs and COX-2 in IL-1b-stimulated chondrocytes.
Effects of ABS on IL-1b-induced p53 phosphorylation and PCNA gene expression Stimulation with IL-1b (10 ng/mL) led to p53 phosphorylation without alteration of p53 levels and downregulation of the expression of PCNA. ABS prevented the upregulation of p-p53 and reduction of PCNA in IL-1b-stimulated chondrocytes (Figure 6).
ABS inhibited IL-1b-induced nuclear factor kB activation It is well known that nuclear factor (NF)-kB plays an important role in the regulation of apoptosis and inflammatory mediators. To investigate whether ABS inhibits NFkB activation in IL-1b-stimulated chondrocytes, IkBa and its phosphorylation was detected by Western blotting. The results demonstrated that IL-1b significantly upregulated IkBa degradation and phosphorylation in chondrocytes. However, the upregulation was remarkably inhibited by the treatment of ABS (Figure 7).
Discussion It is well known that inflammatory cytokines are the key elements participating in OA pathogenesis, and IL-1b is thought to be a representative cytokine involved [3].
Please cite this article in press as: Xu X-X, et al., Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin1b-induced inflammation and apoptosis in vitro, Kaohsiung Journal of Medical Sciences (2016), http://dx.doi.org/10.1016/ j.kjms.2016.11.004
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Figure 2. Flow cytometry analysis of annexin V/propidium iodide dual staining of interleukin (IL)-1b-treated chondrocytes. The chondrocytes were treated with 10 ng/mL IL-1b plus Achyranthes bidentate saponins (ABS) at different concentrations for 24 hours. (A) X- and Y-axis indicate V negative/positive and propidium iodide negative/positive, respectively. Numbers in the right upper and right lower quadrants denote percentages of late and early apoptotic chondrocytes (annexin V positive). The small letters indicate the following: a, normal control (medium only); b, IL-1b (10 ng/mL); c, IL-1b (10 ng/mL) þ ABS (3 mg/mL); d, IL-1b (10 ng/mL) þ ABS (10 mg/mL); and e, IL-1b (10 ng/mL) þ ABS (30 mg/mL). (B) Columns, apoptotic rate in each group (late and early apoptotic chondrocytes). n Z 3. * p < 0.05 versus the model control. ** p < 0.01 versus the model control. FL1 Z Fluorescence 1 (Annexin V-FITC); FL2 Z Fluorescence 2 (PI).
Figure 3. Prevention of the activation of caspase-3 by Achyranthes bidentate saponins (ABS). Interleukin (IL)-1bstimulated chondrocytes were treated with ABS (3 mg/mL, 10 mg/mL, and 30 mg/mL) for 24 hours (n Z 3). ** p < 0.01 versus the model control.
Activation of IL-1b triggers a cascade of cartilage damage events such as the production of more proinflammatory cytokines and the release of some inflammatory mediators such as prostaglandin E2 produced by COX-2 [14]. Prostaglandin E2, in turn, amplified the inflammation in chondrocytes [15]. IL-1b is also capable of reducing the production of cartilage-specific macromolecules, including type II collagen, inducing the release of MMPs that are involved in cartilage matrix degradation in OA [16]. Increased IL-1b was detected in synovial fluid of patients with OA. Elevated IL-1b levels could lead to cartilage destruction and pain [17]. Therefore, inhibitor of IL-1b such as IL-1 receptor antagonist may offer a useful therapeutic approach to the management of OA [18]. In the present study, IL-1b promoted the expressions of COX-2, MMP 3, and MMP 9 in IL-1b-stimulated chondrocytes, while ABS remarkably suppressed the increased expressions. To investigate the anti-inflammatory mechanisms of ABS, we focused on the effects of ABS on the transcription factor NF-kB in chondrocytes. IL-1b activates NF-kB via
Please cite this article in press as: Xu X-X, et al., Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin1b-induced inflammation and apoptosis in vitro, Kaohsiung Journal of Medical Sciences (2016), http://dx.doi.org/10.1016/ j.kjms.2016.11.004
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Figure 4. Effects of Achyranthes bidentate saponins (ABS) on the expressions of anti- and proapoptotic proteins in interleukin (IL)-1b-induced chondrocytes. IL-1b-stimulated chondrocytes were treated with ABS (3 mg/mL, 10 mg/mL, and 30 mg/mL) for 24 hours. (A) Expressions of Bad, Bax, and Bcl-xL were detected by Western blotting; b-actin was used as an internal control. (B) Relative protein expression of Bax and Bcl-xL was quantified by densitometry using “Quantity one” software, normalized to b-actin and compared with normal control. n Z 3. * p < 0.05 versus the model control. ** p < 0.01 versus the model control. *** p < 0.05 versus the model control. **** p < 0.01 versus the model control. ***** p < 0.05 versus the model control. ****** p < 0.01 versus the model control.
Figure 5. Achyranthes bidentate saponins (ABS) suppressed proinflammatory and matrix-degraded enzymes in interleukin (IL)1b-induced chondrocytes. IL-1b-stimulated chondrocytes were treated with ABS for 24 hours. (A) Expressions of matrix metalloproteinase (MMP) 3, MMP 9, and cyclooxygenase-2 (COX-2) were detected by Western blotting. b-Actin was as used an internal control. (B) Relative protein expression of MMP 3, MMP 9, and COX-2 was quantified by densitometry using “Quantity one” software, normalized to b-actin and compared with normal control. n Z 3. * p < 0.05 versus the model control. **** p < 0.01 versus the model control. þp < 0.05 versus the model control. þþp < 0.01 versus the model control
Figure 6. Effects of Achyranthes bidentate saponins (ABS) on p53 and proliferating cell nuclear antigen (PCNA) in interleukin (IL)-1b-induced chondrocytes. (A) IL-1b-stimulated chondrocytes were treated with ABS for 24 hours. Western blotting was performed with anti-p53, anti-pp53, and anti-PCNA. b-Actin was used as an internal control. (B) Relative protein expression of p53, pp53 and PCNA was quantified by densitometry using “Quantity one” software, normalized to b-actin and compared with normal control. n Z 3. ** p < 0.01 versus the model control. *** p < 0.05 versus the model control. **** p < 0.01 versus the model control. Please cite this article in press as: Xu X-X, et al., Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin1b-induced inflammation and apoptosis in vitro, Kaohsiung Journal of Medical Sciences (2016), http://dx.doi.org/10.1016/ j.kjms.2016.11.004
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Figure 7. Achyranthes bidentate saponins (ABS) inhibited nuclear factor-kB activation in interleukin (IL)-1b-induced chondrocytes. (A) IL-1b-stimulated chondrocytes were treated with ABS for 24 hours. IkBa and p-IkBa were detected by Western blotting. b-Actin was used as an internal control. (B) Relative protein expression of IkBa and p-IkBa was quantified by densitometry using “Quantity one” software, normalized to b-actin and compared with normal control. n Z 3. ** p < 0.01 versus the model control. **** p < 0.01 versus the model control.
triggering IkBa degradation, contributing to cartilage degradation in OA [19]. Once activated, NF-kB unit p65 dissociates from its inhibitory protein IkBa and translocates from the cytoplasm to the nucleus, and triggers the transcription of specific target genes such as COX-2 [20]. Furthermore, our data also demonstrated that ABS exhibited its anti-inflammatory effects by preventing IL-1binduced IkBa degradation and phosphorylation. Increased IL-1b levels can also trigger apoptosis in chondrocytes, which leads to further degenerative changes in cartilage [21]. In IL-1b-induced chondrocytes, expression of the antiapoptotic protein Bcl-xL was downregulated, while those of the proapoptotic proteins Bad and Bax were upregulated. ABS inhibited both expression changes. Caspase-3 is a crucial biomarker of apoptosis that also acts as an apoptotic executor [22]. ABS markedly alleviated the increase of caspase-3 activity in IL-1b-stimulated chondrocytes. Meanwhile, ABS inhibited the apoptotic rates of IL-1b-indued rat chondrocytes. This study indicated that ABS prevented IL-1b-induced apoptosis by modulating signaling events, such as phosphorylation of p53 and downregulation of PCNA. PCNA is the most representative proliferating marker and plays important roles in DNA replication and repairing. PCNA is regulated by the tumor suppressor gene p53, which can block DNA replication inducing cell apoptosis [23,24]. ABS had no obvious influence on the expression of p53, but could inhibit phosphorylation of p53 and increase the expression of PCNA. OA is a slowly progressing degenerative arthritis. The pharmacological treatment strategies for OA are to decrease symptoms, improve function, and delay time to surgery. Although anti-inflammatory agents such as nonsteroidal anti-inflammatory drugs have good effects on OA, an increased risk of gastrointestinal, cardiovascular, or renal side effects remains one of the problems for their long-term use [25]. There is a strong need to develop an effective treatment regime with fewer side effects. Traditional Chinese Medicine can be an alternative treatment method and is commonly used in China to manage symptoms associated with OA. Recently, a growing body of evidence indicates that active ingredients from
Chinese herbal medicine are noteworthy because of their potential impact on safety and costs. According to the basic theory of Traditional Chinese Medicine, OA is an “impediment disease,” which mainly includes syndromes of qi stagnation and blood stasis, cold dampness, deficiency of Gan (liver) and Shen (kidney), and deficiency of Qi and blood [26]. A. bidentata can strengthen the waist and knee, by eliminating stagnation, removing blood stasis, and nourishing the liver and kidney. Chinese herbal medicine formulas containing A. bidentata have been used in clinical practice for thousands of years, such as Duhuo Jisheng decoction [27] and SiMiaoFang [28]. A. bidentata is used to eliminate inflammation or improve circulation of the knees. Our results show that ABS is effective not only against inflammatory symptoms, but also against apoptosis of IL-1bstimulated chondrocytes in OA. Further pharmacodynamic studies will be performed in a rat model of OA. With both antiapoptotic and anti-inflammatory properties, ABS might help protect against the degeneration of cartilage in patients with OA.
Acknowledgments This work was supported by the Promotion Program for Young and Middle-aged Teacher in Science and Technology Research of Huaqiao University (Number ZQN-PY420), the Key Project of Quanzhou City Science and Technology Program (Number 2011Z8), and the Natural Science Foundation of Fujian Province of China (Number 2015J01364).
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Please cite this article in press as: Xu X-X, et al., Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin1b-induced inflammation and apoptosis in vitro, Kaohsiung Journal of Medical Sciences (2016), http://dx.doi.org/10.1016/ j.kjms.2016.11.004