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Loganin inhibits the inflammatory response in mouse 3T3L1 adipocytes and mouse model
International Immunopharmacology 36 (2016) 173–179
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Loganin inhibits the inflammatory response in mouse 3T3L1 adipocytes and mouse model☆ Yang Li a,1, Zheng Li a,1, Lei Shi b, Chenxu Zhao a, Bingyu Shen a, Ye Tian a, Haihua Feng a,⁎ a b
Key Laboratory of Zoonosis, Ministry of Education, College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin 130062, PR China Jilin University Library, Changchun, Jilin 130062, PR China
a r t i c l e
i n f o
Article history: Received 15 January 2016 Received in revised form 25 March 2016 Accepted 18 April 2016 Available online 4 May 2016 Keywords: Loganin Atherosclerosis ApoCIII Cytokines NF-κB
a b s t r a c t Atherosclerosis is a chronic inflammatory disease of the vascular walls. ApoCIII is an independent factor which promotes atherosclerotic processes. This study aimed to investigate whether Loganin administration inhibits the inflammatory response in vitro and in vivo. In the apoCIII-induced mouse adipocytes, the levels of cytokines, including TNF-α, MCP-1 and IL-6 were determined by enzyme-linked immunosorbent assay and their gene expressions were measured through RT-PCR. The phosphorylation of nuclear factor-κB (NF-κB) proteins was analyzed by Western blotting. Our results showed that Loganin markedly decreased TNF-α, MCP-1 and IL-6 concentrations as well as their gene expressions. Western blotting analysis indicated that Loganin suppressed the activation of NF-κB signaling. In the Tyloxapol-treated mouse model, Loganin reduced the contents of TC and TG in mouse serum. The results of Oil Red-O Staining showed that Loganin reduced the production of lipid droplets. So it is suggested that Loganin might be a potential therapeutic agent for preventing the inflammation stress in vitro and in vivo. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Atherosclerosis (AS) is a chronic inflammatory process [1,2] which is associated with hypertriglyceridemia, hypercholesterolemia and vascular cell dysfunction. Apolipoprotein CIII (apoCIII), a small protein that resides on the surface of very-low-density lipoprotein (VLDL) and LDL. ApoCIII inhibits clearance from plasma of VLDL and LDL [3–7] and causes accumulation of atherogenic remnant lipoproteins and dense LDL in plasma [5–8]. Overexpression of apoCIII can lead to hyperlipidaemia and can promote atherosclerotic lesion development in mouse models [9]. However, apoCIII deficiency protects against dyslipidaemia and atherogenesis [10]. Recent report demonstrated that apoCIII can promote hyperlipidaemia in human by inhibiting clearance of plasma triglyceride-rich lipoproteins and channeling them to conversion to small, dense LDL [8]. TNF-α, MCP-1 and IL-6 result in inflammation in many types of cells. Their pathophysiological roles in the development of cardiovascular disease, such as atherosclerosis, have been well characterized [11]. Recent clinical studies reported that plasma IL-6 was closely correlated with apoCIII independently of triglyceride, VLDL, and remnant ☆ Authors' contributions: Y.L., Z.L. and H.H.F. contributed to the design. Y.L., B.Y.S. and Y.T. did the data collection. Y.L., Z.L. and C.X.Z. did the analysis. Y.L. did the writing of the article. H.H.F. and L.S did the revisions to the article. ⁎ Corresponding author at: College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin 130062, PR China. E-mail address: [email protected] (H. Feng). 1 The authors Yang Li and Zheng Li contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2016.04.026 1567-5769/© 2016 Elsevier B.V. All rights reserved.
lipoprotein (RLP) cholesterol [12]. MCP-1 plays an important role in the recruitment of macrophages into obese adipose tissue and contributes to the development of obesity [13]. Atherosclerotic processes involve several mediators including adhesion molecules and chemokines, which play a role in different stages of the disease from the initiation of plaque formation to the plaque rupture [14–23]. For example, the recruitment of leucocytes to the vascular intima is a multistep process which depends on a variety of chemokines and adhesion molecules responsible for their chemotaxis, such as MCP-1 and GM-CSF and transendothelial migration including VCAM1, ICAM-1 and E-selectin [24,25]. Activation of endothelial cells and expression of above-mentioned proteins are regulated through NF-κB signaling pathway [26]. The canonical signaling of NF-κB activation controls mediators regulating the thrombotic potential of human atherosclerotic plaques including tissue factor (TF), matrix metalloproteinases (MMPs) and inflammatory cytokines [26]. NF-κB signaling is induced by inflammatory stimuli, such as TNF-α, IL-1 and oxydized LDL. Reactive oxygen species can lead to the ampli cation and long-term maintenance of the vascular inflammatory burden and thus can facilitate atherogenesis. There is no doubt that activation of NF-κB in endothelial cells triggers expression of a variety of adhesion molecules, such as ICAM-1, VCAM-1, IL-1, IL-6, TNF-α and MCP-1. These molecules contribute to the coordination of the invasion, the homing of inflammatory cells into the vascular wall and the migration of smooth muscle cells [27]. These processes are further supported by the factors produced by leucocytes themselves, especially monocytes. What unites these processes in the cells of different origins is the critical
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involvement of IKKα/NF-κB pathway [28]. Together with progressive NF-κB-mediated accumulation and proliferation of smooth muscle cells in intima, these processes warrant progression of atherosclerosis [29]. Herbal medications have been widely used as therapeutic strategies due to their relatively fewer side effects. However, knowledge of their biological actions is limited. Loganin, an iridoid glycoside found in Flos lonicerae, Fruit cornus and Strychnos nux vomica was reported to exhibit immune regulatory activity, anti-inflammatory and anti-shock effects and so on. It also has a variety of biological effects such as antidiabetic, antiapoptotic and neuroprotective [30–32]. Therefore, this study intends to evaluate the potential anti-inflammatory effects of Loganin both in vitro and in vivo. 2. Materials and methods 2.1. Chemicals and reagents Loganin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Jilin, China). Dimethyl sulfoxide (DMSO), Human apoCIII, Triton WR-1339 (Tyloxapol) and CCK8 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), penicillin and streptomycin were obtained from Invitrogen-Gibco (Grand Island, NY). Mouse TNF-α, MCP-1 and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were provided by Biolegend (CA, USA). Rabbit Phospho- specific antibodies for β-actin, NF-κB p65 (Ser536) and IκB were purchased from Cell Signaling Technology Inc (Beverly, MA). HRP-conjugated goat anti-rabbit antibodies were provided by GE Healthcare (Buckinghamshire, UK). TC and TG assay kits were purchased by Jiancheng Co. (Nanjing, China). 2.2. Animals The male BALB/c mice, weighing approximately 18–20 g, were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). These mice were housed for 2–3 days to adapt to the environment before experiments. These mice were housed in microisolator cages and they received food and water ad libitum. The laboratory temperature was 24 ± 1 °C and the relative humidity was 40–80%. All animal experiments were performed in accordance with the guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. 2.3. In vitro study 2.3.1. Cell culture and treatment Mouse 3T3L1 preadipocytes was obtained from the China Cell Line Bank (Beijing, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 3 mM Glutamine, antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) at 37 °C under a humidified atmosphere of 5% CO2. The differentiation of 3T3L1 preadipocytes to adipocytes was described elsewhere [33]. In all vitro experiments, adipocytes were incubated in the presence or absence of various concentrations of Loganin which were always added 1 h prior to apoCIII (100 μg/ml) stimulation. Loganin, was dissolved in DMSO and was stored at −20 °C. The stock solution was diluted to desirable concentrations with DMEM immediately before use. In all experiments, the final DMSO concentration in each sample was b 0.01%, which did not affect cell growth and death. 2.3.2. CCK-8 assay 3T3L1 adipocytes (5 × 104 cells/ml) were mechanically scraped and were plated onto 96-well plates in a 37 °C, 5% CO2 incubator for 1 h. Then the cells were treated with 50 μl Loganin of different concentrations. After 18 h, we added 10 μl CCK-8 to each well and continued to
incubate for 4 h. Then, the optical density was measured at 450 nm on a microplate reader (TECAN, Austria). 2.3.3. Cytokine assay 3T3L1 adipocytes (4 × 105 cells/ml) were plated onto 24-well plates and were incubated in the presence of either apoCIII (100 μg/ml) alone or apoCIII plus Loganin for 18 h. Cell-free supernatants were collected and stored at −20 °C. Then the cytokine levels (TNF-α, MCP-1 and IL6) were determined by using an ELISA kit according to the manufacturer's instructions (BioLegend, Inc, Camino Santa Fe, Suite E, San Diego, CA, USA). 2.3.4. Quantitative real-time PCR 3T3L1 adipocytes (4 × 105 cells/ml) were plated onto six-well plates and were cultured for 1 h. And then they were pretreated with Loganin (32, 64, 128 μM) for 1 h prior to stimulation of 100 μg/ml apoCIII for 24 h in a 37 °C, 5% CO2 Incubator. Total 3T3L1 adipocytes RNA was extracted with TRIzol reagent (TaKaRa Biotechnology Co., Ltd., Tokyo, Japan) according to the supplier's protocol. The RNA concentration was determined by using a K5500 Micro-Spectrophotometer (Beijing Kaiao Technology Development Co., Ltd., Beijing, China). Approximately 5 mg of total RNA was reverse-transcribed to cDNA in 20-ml reactions using PrimeScript Reverse Transcriptase (TaKaRa Biotechnology Co., Ltd., Tokyo, Japan) according to the manufacturer's instructions. The primers were designed using Primer Express software (PE Applied Biosystems, Inc., Foster City, CA, USA). The mRNA expression levels were evaluated by quantitative polymerase chain reaction (qRT-PCR) analysis by using a SYBR Green QuantiTect RT-PCR Kit (Takara Biotechnology Co., Ltd.). qRT-PCR was performed on a 7500 Real-Time PCR System (Applied Biosystems). Mouse MCP-1 primers, forward: 5′-CCACTC ACCTGCTGCTGCTACTCAT-3′, reverse: 5′-TGGTGATCCTCTTGTAGCTC TCC-3′. Mouse IL-6 primers, forward: 5′-ACAACCACGGCCTTCCCTACTT3′, reverse: 5′-CACGATTTCCCAGAGAACATGTG-3′. Mouse TNF-α primers, forward: 5′-AATGAGGCTGGATAAGAT-3′, reverse: 5′-AGAGGT TCAGTGATGTAG-3′. 2.3.5. Determination of NF-κB activity in apoCIII-stimulated mouse adipocytes 3T3L1 adipocytes (4 × 105 cells/ml) were plated onto six-well plates and were cultured for 24 h in a 37 °C, 5% CO2 Incubator, then were pretreated with Loganin (32, 64, 128 μM) for 1 h prior to stimulation of 100 μg/ml apoCIII for 30 min. The cells were collected on ice and were washed with ice-cold PBS twice and lysed in lysis buffer for 30 min. The lysates were centrifuged (12,000 g at 4 °C) for 5 min and the protein concentration was determined by BCA protein assay kit (Beyotime, Haimen, China). The protein samples were separated on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and then electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The samples were incubated overnight with primary antibodies (diluted 1:5000) at 4 °C. Blots were incubated with a peroxidase-conjugated secondary antibody for 1 h and the immunoactive proteins were detected using ECL plus (Thermo, USA). 2.4. In vivo study 2.4.1. Experimental design Mice were randomly divided into six groups, with each group containing three mice: Control, Tyloxapol (300 mg/kg) only, Tyloxapol + Fenofibrate (25 mg/kg), Tyloxapol + Loganin (50, 100, 200 mg/kg). Loganin groups and fenofibrate group received intragastric administration of Loganin and fenofibrate respectively while control group and Tyloxapol received distilled water for consecutively 7 days. On the 6th day of administration, all mice were fasted for 12 h. 1 h after final intragastric administration, Triton WR 1339 was injected intraperitoneally to all mice except for normal control group.
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2.4.2. Measurement of TC and TG After 24 h Tyloxapol treatment, Blood was gained from the eyeballs of mice and contents of TC and TG in serum were measured through using a commercially available assay kit following the manufacturer's recommendation (Jiancheng Company, Nanjing, China). 2.4.3. Oil Red-O Staining Mice livers were excised after 24 h Tyloxapol treatment. Then, the tissues were frozen in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, U.S.A.) and sections (5 μm thick) were made using standard protocol. The production of lipid droplets in liver tissues was observed with a light microscope at ×200 magnification. 2.4.4. Statistical analysis All values were expressed as mean ± SEM. Differences between mean values of normally distributed data were assessed with one-way ANOVA (Dunnett's t-test) and two-tailed Student's t-test. Statistical significance was accepted at P b 0.05 or P b 0.01. 3. Results 3.1. In vitro study 3.1.1. Effect of Loganin on CCK-8 assay After incubating cells for 18 h, we determined the effect of Loganin on cell viability by CCK-8 assay. The result showed that cell viability was not affected by Loganin at the concentrations (8–128 μM) used (Fig. 1). The non-toxic concentrations (32, 64, 128 μM) were used for the following experiment. 3.1.2. Effect of Loganin on the production of cytokines in apoCIII-induced mouse adipocytes The concentrations of TNF-α, MCP-1 and IL-6 in the culture supernatants of mouse adipocytes were measured by sandwich ELISA (Fig. 2). As compared with the control group, treatment of adipocytes with apoCIII alone resulted in a significant increase in cytokines production. However, the concentrations of TNF-α, MCP-1 and IL-6 of apoCIIIinduced mouse adipocytes pretreated with 32, 64 and 128 μM Loganin were significantly reduced compared with apoCIII group. 3.1.3. Effects of Loganin on TNF-α, MCP-1 and IL-6 gene expression Compared with the control group, treatment of adipocytes with apoCIII alone resulted in a significant increase in gene expression. However, the gene expression of TNF-α, MCP-1 and IL-6 of apoCIIIinduced mouse adipocytes pretreated with 32, 64 and 128 μM Loganin were significantly reduced compared with apoCIII group (Fig. 3).
Fig. 1. Effect of Loganin on CCK-8 assay. Cells were cultured with different concentrations of Loganin (8–128 μM) for 18 h. Data are presented as means ± SEM.
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3.1.4. Effect of Loganin on apoCIII-induced NF-κB activation The effect of Loganin on NF-κB activation was examined by western blotting for investigating the anti-inflammatory mechanisms of Loganin. Phosphorylation p65 and IκB in the apoCIII-induced mouse adipocytes increased when compared with control group. Pretreatment with Loganin inhibited apoCIII-induced activation of IκB and p65 in a dose-dependent manner (Fig. 4). 3.2. In vivo study 3.2.1. Effect of Loganin on TC and TG in the Tyloxapol-induced mouse model Serum was harvested for investigating the levels of TC and TG after 24 h Tyloxapol stimulation. The concentrations of TC and TG of Fenofibrate and Loganin groups were markedly reduced compared with Tyloxapol group (Fig. 5). 3.2.2. Effect of Loganin on Oil Red-O Staining in the Tyloxapol-induced mouse model Liver sections of Oil Red-O Staining showed that mice liver was dyed orange and formed red lipid droplets in Tyloxapol group. However, the color of three different doses of Loganin groups (50, 100, 200 mg/kg) and Fenofibrate group lower than Tyloxapol group significantly and lipid droplets obviously reduced compared with Tyloxapol group (Fig. 6). 4. Discussion In this study, cytotoxicity of Loganin on mouse adipocytes was determined by CCK-8 assay. The assay is able to measure mitochondrial activity which is directly correlated to cell viability for both attached and poorly attached cells [34]. The results showed that Loganin didn't have obvious dose-dependent cytotoxic effect even at a higher concentration (128 μM). As the essential mediators of inflammation, cytokines, such as TNFα, MCP-1 and IL-6, play important roles in atherosclerosis. They are found to be involved in the development of atherosclerosis and the related signal transduction pathways such as NF-κB, JNK/AP-1 pathway and JAK/STAT pathway [35]. The present study demonstrated that apoCIII could increase mRNA expression and the release of MCP-1 and IL-6 in mouse 3T3L1 adipocytes. However, Loganin inhibited this trend effectively. NF-κB is a generic name of a series of proteins which can specifically bind with the κB site of multiple gene initiators. NF-κB is composed of five different proteins, including p50, p52, p65/ RelA, RelB and c-Rel. NF-κB is maintained in the cytoplasm in an inactive form bound through inhibitory protein IκBa under normal physiological conditions. Phosphorylated IκBa is degraded by the ubiquitin-pro-teasome pathway after the stimulation. The degradation of IκBa results in p50/p65 dimer phosphorylation and translocation, which leading to the genes transcription [36–38]. The nuclear factor (NF)-κB pathway is significantly related to the inflammatory response in cells. It is involved in the expression of various proinflammatory cytokines and plays a pivotal role in inflammation. The activation of NF-κB is accompanied by expression of a wide range of inflammatory molecules (e.g., IL-6 and TNF-α) and is often triggered by the stimulation of leukocyte receptors involved in innate immunity such as Toll like receptor (TLR) [39–41]. Indeed, pharmacological inhibition of NF-κB dramatically reduces IL production [39]. We tried to study the signaling involved in the effects of apoCIII. Therefore, we measured the phosphorylation p65 and IκBa in apoCIII-induced 3T3L1 adipocytes. In this study, apoCIII activated NF-κB in 3T3L1 adipocytes. Pretreatment with Loganin markedly suppressed the apoCIII-induced p65 and IκBa phosphorylation. These results suggest that the effects of apoCIII were mediated by NF-κB, at least in part.
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Fig. 2. Effect of different concentrations of Loganin on apoCIII-stimulated TNF-α, MCP-1 and IL-6 in mouse 3T3L1 adipocytes. Cells were pretreated with different concentrations (32, 64, 128 μM) of Loganin for 1 h prior to stimulation with 100 μg/ml of apoCIII for 24 h. The values represent mean ± SEMs. ##P b 0.01 indicates significant differences from the control group, *P b 0.05 vs. apoCIII, **P b 0.01 vs. apoCIII.
Fig. 3. Effect of different concentrations of Loganin on apoCIII-stimulated TNF-α, MCP-1 and IL-6 gene expression in mouse 3T3L1 adipocytes. Cells were pretreated with different concentrations (32, 64, 128 μM) of Loganin for 1 h prior to stimulation with 100 μg/ml of apoCIII for 24 h. The values represent mean ± SEMs. ##P b 0.01 indicates significant differences from the control group, *P b 0.05 vs. LPS, **P b 0.01 vs. apoCIII.
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Fig. 4. Effect of Loganin on apoCIII-induced NF-κB in mouse 3T3L1 adipocytes. Cells were pretreated with different concentrations (32, 64, 128 μM) of Loganin for 1 h prior to stimulation with 100 μg/ml of apoCIII for 30 min. Western blotting was used to analyze protein samples with phospho-specific antibodies, β-actin was used as an internal control. Similar results were obtained in three independent experiments expressed as mean ± SEM. ##P b 0.01 indicates significant differences from the unstimulated control group, *P b 0.05 vs. apoCIII, **P b 0.01 vs. apoCIII.
Fig. 5. Effect of Loganin on levels of TC and TG in the serum of mice. Mice received intragastric administration of Loganin (50, 100, 200 mg/kg) and fenofibrate (25 mg/kg) for consecutively 7 days. Then, serum were harvested for measuring TC and TG concentrations. The values presented are the means ± SEMs. ##P b 0.01 vs. Control group; **P b 0.01 vs. Tyloxapol group.
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Fig. 6. Effect of Loganin on Oil Red-O Staining in the Tyloxapol-induced mouse model. Mice received intragastric administration of Loganin (50, 100, 200 mg/kg) and fenofibrate (25 mg/kg) for consecutively 7 days. Then, each group was processed for Oil Red-O Staining. (A) Control group. (B) Tyloxapol group. (C) Fenofibrate group. (D, E and F) Loganin group (50, 100, 200 mg/kg). The panel is magnified 200×.
Triton WR-1339-induced hyperlipidemic rats are a globally accepted model used to evaluate potential hypolipidemic drugs [42]. Triton WR-1339 is a nonionic detergent which prevents catabolism of triacylglycerol-rich lipoproteins by lipo-protein lipase. It is used for the determination of triacylglycerol production, the very low density lipoprotein (VLDL) secretion or clearance rate in vivo commonly [43,44]. The rise of TC levels will accelerate atherosclerosis process and the rise of TG is also a dangerous factor for atherosclerosis. In this study, Pretreatment with Loganin obviously reduced levels of TC and TG in mouse serum. Moreover, Oil red O staining proved that a large quantity of red lipid droplets appeared in liver tissues after WR-1339 stimulating mouse 24 h under light microscope, but lipid droplets ameliorated after Loganin administration. In summary, our results in vitro revealed that Loganin can downregulated the levels of TNF-α, MCP-1 and IL-6 and their gene
expression. Also, it could block the activation of NF-κB signaling markedly in apoCIII-induced mouse adipocytes. Results in vivo also showed that Loganin relieved the inflammation stress. Our experimental results support that Loganin might be a potential therapeutic agent for preventing inflammatory diseases.
Conflict of interest The authors declared that there is no conflict of interest.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (no. 31572347, no. 31372478).
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Loganin inhibits the inflammatory response in mouse 3T3L1 adipocytes and mouse model☆ Yang Li a,1, Zheng Li a,1, Lei Shi b, Chenxu Zhao a, Bingyu Shen a, Ye Tian a, Haihua Feng a,⁎ a b
Key Laboratory of Zoonosis, Ministry of Education, College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin 130062, PR China Jilin University Library, Changchun, Jilin 130062, PR China
a r t i c l e
i n f o
Article history: Received 15 January 2016 Received in revised form 25 March 2016 Accepted 18 April 2016 Available online 4 May 2016 Keywords: Loganin Atherosclerosis ApoCIII Cytokines NF-κB
a b s t r a c t Atherosclerosis is a chronic inflammatory disease of the vascular walls. ApoCIII is an independent factor which promotes atherosclerotic processes. This study aimed to investigate whether Loganin administration inhibits the inflammatory response in vitro and in vivo. In the apoCIII-induced mouse adipocytes, the levels of cytokines, including TNF-α, MCP-1 and IL-6 were determined by enzyme-linked immunosorbent assay and their gene expressions were measured through RT-PCR. The phosphorylation of nuclear factor-κB (NF-κB) proteins was analyzed by Western blotting. Our results showed that Loganin markedly decreased TNF-α, MCP-1 and IL-6 concentrations as well as their gene expressions. Western blotting analysis indicated that Loganin suppressed the activation of NF-κB signaling. In the Tyloxapol-treated mouse model, Loganin reduced the contents of TC and TG in mouse serum. The results of Oil Red-O Staining showed that Loganin reduced the production of lipid droplets. So it is suggested that Loganin might be a potential therapeutic agent for preventing the inflammation stress in vitro and in vivo. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Atherosclerosis (AS) is a chronic inflammatory process [1,2] which is associated with hypertriglyceridemia, hypercholesterolemia and vascular cell dysfunction. Apolipoprotein CIII (apoCIII), a small protein that resides on the surface of very-low-density lipoprotein (VLDL) and LDL. ApoCIII inhibits clearance from plasma of VLDL and LDL [3–7] and causes accumulation of atherogenic remnant lipoproteins and dense LDL in plasma [5–8]. Overexpression of apoCIII can lead to hyperlipidaemia and can promote atherosclerotic lesion development in mouse models [9]. However, apoCIII deficiency protects against dyslipidaemia and atherogenesis [10]. Recent report demonstrated that apoCIII can promote hyperlipidaemia in human by inhibiting clearance of plasma triglyceride-rich lipoproteins and channeling them to conversion to small, dense LDL [8]. TNF-α, MCP-1 and IL-6 result in inflammation in many types of cells. Their pathophysiological roles in the development of cardiovascular disease, such as atherosclerosis, have been well characterized [11]. Recent clinical studies reported that plasma IL-6 was closely correlated with apoCIII independently of triglyceride, VLDL, and remnant ☆ Authors' contributions: Y.L., Z.L. and H.H.F. contributed to the design. Y.L., B.Y.S. and Y.T. did the data collection. Y.L., Z.L. and C.X.Z. did the analysis. Y.L. did the writing of the article. H.H.F. and L.S did the revisions to the article. ⁎ Corresponding author at: College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin 130062, PR China. E-mail address: [email protected] (H. Feng). 1 The authors Yang Li and Zheng Li contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2016.04.026 1567-5769/© 2016 Elsevier B.V. All rights reserved.
lipoprotein (RLP) cholesterol [12]. MCP-1 plays an important role in the recruitment of macrophages into obese adipose tissue and contributes to the development of obesity [13]. Atherosclerotic processes involve several mediators including adhesion molecules and chemokines, which play a role in different stages of the disease from the initiation of plaque formation to the plaque rupture [14–23]. For example, the recruitment of leucocytes to the vascular intima is a multistep process which depends on a variety of chemokines and adhesion molecules responsible for their chemotaxis, such as MCP-1 and GM-CSF and transendothelial migration including VCAM1, ICAM-1 and E-selectin [24,25]. Activation of endothelial cells and expression of above-mentioned proteins are regulated through NF-κB signaling pathway [26]. The canonical signaling of NF-κB activation controls mediators regulating the thrombotic potential of human atherosclerotic plaques including tissue factor (TF), matrix metalloproteinases (MMPs) and inflammatory cytokines [26]. NF-κB signaling is induced by inflammatory stimuli, such as TNF-α, IL-1 and oxydized LDL. Reactive oxygen species can lead to the ampli cation and long-term maintenance of the vascular inflammatory burden and thus can facilitate atherogenesis. There is no doubt that activation of NF-κB in endothelial cells triggers expression of a variety of adhesion molecules, such as ICAM-1, VCAM-1, IL-1, IL-6, TNF-α and MCP-1. These molecules contribute to the coordination of the invasion, the homing of inflammatory cells into the vascular wall and the migration of smooth muscle cells [27]. These processes are further supported by the factors produced by leucocytes themselves, especially monocytes. What unites these processes in the cells of different origins is the critical
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involvement of IKKα/NF-κB pathway [28]. Together with progressive NF-κB-mediated accumulation and proliferation of smooth muscle cells in intima, these processes warrant progression of atherosclerosis [29]. Herbal medications have been widely used as therapeutic strategies due to their relatively fewer side effects. However, knowledge of their biological actions is limited. Loganin, an iridoid glycoside found in Flos lonicerae, Fruit cornus and Strychnos nux vomica was reported to exhibit immune regulatory activity, anti-inflammatory and anti-shock effects and so on. It also has a variety of biological effects such as antidiabetic, antiapoptotic and neuroprotective [30–32]. Therefore, this study intends to evaluate the potential anti-inflammatory effects of Loganin both in vitro and in vivo. 2. Materials and methods 2.1. Chemicals and reagents Loganin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Jilin, China). Dimethyl sulfoxide (DMSO), Human apoCIII, Triton WR-1339 (Tyloxapol) and CCK8 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), penicillin and streptomycin were obtained from Invitrogen-Gibco (Grand Island, NY). Mouse TNF-α, MCP-1 and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were provided by Biolegend (CA, USA). Rabbit Phospho- specific antibodies for β-actin, NF-κB p65 (Ser536) and IκB were purchased from Cell Signaling Technology Inc (Beverly, MA). HRP-conjugated goat anti-rabbit antibodies were provided by GE Healthcare (Buckinghamshire, UK). TC and TG assay kits were purchased by Jiancheng Co. (Nanjing, China). 2.2. Animals The male BALB/c mice, weighing approximately 18–20 g, were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). These mice were housed for 2–3 days to adapt to the environment before experiments. These mice were housed in microisolator cages and they received food and water ad libitum. The laboratory temperature was 24 ± 1 °C and the relative humidity was 40–80%. All animal experiments were performed in accordance with the guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. 2.3. In vitro study 2.3.1. Cell culture and treatment Mouse 3T3L1 preadipocytes was obtained from the China Cell Line Bank (Beijing, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 3 mM Glutamine, antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) at 37 °C under a humidified atmosphere of 5% CO2. The differentiation of 3T3L1 preadipocytes to adipocytes was described elsewhere [33]. In all vitro experiments, adipocytes were incubated in the presence or absence of various concentrations of Loganin which were always added 1 h prior to apoCIII (100 μg/ml) stimulation. Loganin, was dissolved in DMSO and was stored at −20 °C. The stock solution was diluted to desirable concentrations with DMEM immediately before use. In all experiments, the final DMSO concentration in each sample was b 0.01%, which did not affect cell growth and death. 2.3.2. CCK-8 assay 3T3L1 adipocytes (5 × 104 cells/ml) were mechanically scraped and were plated onto 96-well plates in a 37 °C, 5% CO2 incubator for 1 h. Then the cells were treated with 50 μl Loganin of different concentrations. After 18 h, we added 10 μl CCK-8 to each well and continued to
incubate for 4 h. Then, the optical density was measured at 450 nm on a microplate reader (TECAN, Austria). 2.3.3. Cytokine assay 3T3L1 adipocytes (4 × 105 cells/ml) were plated onto 24-well plates and were incubated in the presence of either apoCIII (100 μg/ml) alone or apoCIII plus Loganin for 18 h. Cell-free supernatants were collected and stored at −20 °C. Then the cytokine levels (TNF-α, MCP-1 and IL6) were determined by using an ELISA kit according to the manufacturer's instructions (BioLegend, Inc, Camino Santa Fe, Suite E, San Diego, CA, USA). 2.3.4. Quantitative real-time PCR 3T3L1 adipocytes (4 × 105 cells/ml) were plated onto six-well plates and were cultured for 1 h. And then they were pretreated with Loganin (32, 64, 128 μM) for 1 h prior to stimulation of 100 μg/ml apoCIII for 24 h in a 37 °C, 5% CO2 Incubator. Total 3T3L1 adipocytes RNA was extracted with TRIzol reagent (TaKaRa Biotechnology Co., Ltd., Tokyo, Japan) according to the supplier's protocol. The RNA concentration was determined by using a K5500 Micro-Spectrophotometer (Beijing Kaiao Technology Development Co., Ltd., Beijing, China). Approximately 5 mg of total RNA was reverse-transcribed to cDNA in 20-ml reactions using PrimeScript Reverse Transcriptase (TaKaRa Biotechnology Co., Ltd., Tokyo, Japan) according to the manufacturer's instructions. The primers were designed using Primer Express software (PE Applied Biosystems, Inc., Foster City, CA, USA). The mRNA expression levels were evaluated by quantitative polymerase chain reaction (qRT-PCR) analysis by using a SYBR Green QuantiTect RT-PCR Kit (Takara Biotechnology Co., Ltd.). qRT-PCR was performed on a 7500 Real-Time PCR System (Applied Biosystems). Mouse MCP-1 primers, forward: 5′-CCACTC ACCTGCTGCTGCTACTCAT-3′, reverse: 5′-TGGTGATCCTCTTGTAGCTC TCC-3′. Mouse IL-6 primers, forward: 5′-ACAACCACGGCCTTCCCTACTT3′, reverse: 5′-CACGATTTCCCAGAGAACATGTG-3′. Mouse TNF-α primers, forward: 5′-AATGAGGCTGGATAAGAT-3′, reverse: 5′-AGAGGT TCAGTGATGTAG-3′. 2.3.5. Determination of NF-κB activity in apoCIII-stimulated mouse adipocytes 3T3L1 adipocytes (4 × 105 cells/ml) were plated onto six-well plates and were cultured for 24 h in a 37 °C, 5% CO2 Incubator, then were pretreated with Loganin (32, 64, 128 μM) for 1 h prior to stimulation of 100 μg/ml apoCIII for 30 min. The cells were collected on ice and were washed with ice-cold PBS twice and lysed in lysis buffer for 30 min. The lysates were centrifuged (12,000 g at 4 °C) for 5 min and the protein concentration was determined by BCA protein assay kit (Beyotime, Haimen, China). The protein samples were separated on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and then electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The samples were incubated overnight with primary antibodies (diluted 1:5000) at 4 °C. Blots were incubated with a peroxidase-conjugated secondary antibody for 1 h and the immunoactive proteins were detected using ECL plus (Thermo, USA). 2.4. In vivo study 2.4.1. Experimental design Mice were randomly divided into six groups, with each group containing three mice: Control, Tyloxapol (300 mg/kg) only, Tyloxapol + Fenofibrate (25 mg/kg), Tyloxapol + Loganin (50, 100, 200 mg/kg). Loganin groups and fenofibrate group received intragastric administration of Loganin and fenofibrate respectively while control group and Tyloxapol received distilled water for consecutively 7 days. On the 6th day of administration, all mice were fasted for 12 h. 1 h after final intragastric administration, Triton WR 1339 was injected intraperitoneally to all mice except for normal control group.
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2.4.2. Measurement of TC and TG After 24 h Tyloxapol treatment, Blood was gained from the eyeballs of mice and contents of TC and TG in serum were measured through using a commercially available assay kit following the manufacturer's recommendation (Jiancheng Company, Nanjing, China). 2.4.3. Oil Red-O Staining Mice livers were excised after 24 h Tyloxapol treatment. Then, the tissues were frozen in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, U.S.A.) and sections (5 μm thick) were made using standard protocol. The production of lipid droplets in liver tissues was observed with a light microscope at ×200 magnification. 2.4.4. Statistical analysis All values were expressed as mean ± SEM. Differences between mean values of normally distributed data were assessed with one-way ANOVA (Dunnett's t-test) and two-tailed Student's t-test. Statistical significance was accepted at P b 0.05 or P b 0.01. 3. Results 3.1. In vitro study 3.1.1. Effect of Loganin on CCK-8 assay After incubating cells for 18 h, we determined the effect of Loganin on cell viability by CCK-8 assay. The result showed that cell viability was not affected by Loganin at the concentrations (8–128 μM) used (Fig. 1). The non-toxic concentrations (32, 64, 128 μM) were used for the following experiment. 3.1.2. Effect of Loganin on the production of cytokines in apoCIII-induced mouse adipocytes The concentrations of TNF-α, MCP-1 and IL-6 in the culture supernatants of mouse adipocytes were measured by sandwich ELISA (Fig. 2). As compared with the control group, treatment of adipocytes with apoCIII alone resulted in a significant increase in cytokines production. However, the concentrations of TNF-α, MCP-1 and IL-6 of apoCIIIinduced mouse adipocytes pretreated with 32, 64 and 128 μM Loganin were significantly reduced compared with apoCIII group. 3.1.3. Effects of Loganin on TNF-α, MCP-1 and IL-6 gene expression Compared with the control group, treatment of adipocytes with apoCIII alone resulted in a significant increase in gene expression. However, the gene expression of TNF-α, MCP-1 and IL-6 of apoCIIIinduced mouse adipocytes pretreated with 32, 64 and 128 μM Loganin were significantly reduced compared with apoCIII group (Fig. 3).
Fig. 1. Effect of Loganin on CCK-8 assay. Cells were cultured with different concentrations of Loganin (8–128 μM) for 18 h. Data are presented as means ± SEM.
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3.1.4. Effect of Loganin on apoCIII-induced NF-κB activation The effect of Loganin on NF-κB activation was examined by western blotting for investigating the anti-inflammatory mechanisms of Loganin. Phosphorylation p65 and IκB in the apoCIII-induced mouse adipocytes increased when compared with control group. Pretreatment with Loganin inhibited apoCIII-induced activation of IκB and p65 in a dose-dependent manner (Fig. 4). 3.2. In vivo study 3.2.1. Effect of Loganin on TC and TG in the Tyloxapol-induced mouse model Serum was harvested for investigating the levels of TC and TG after 24 h Tyloxapol stimulation. The concentrations of TC and TG of Fenofibrate and Loganin groups were markedly reduced compared with Tyloxapol group (Fig. 5). 3.2.2. Effect of Loganin on Oil Red-O Staining in the Tyloxapol-induced mouse model Liver sections of Oil Red-O Staining showed that mice liver was dyed orange and formed red lipid droplets in Tyloxapol group. However, the color of three different doses of Loganin groups (50, 100, 200 mg/kg) and Fenofibrate group lower than Tyloxapol group significantly and lipid droplets obviously reduced compared with Tyloxapol group (Fig. 6). 4. Discussion In this study, cytotoxicity of Loganin on mouse adipocytes was determined by CCK-8 assay. The assay is able to measure mitochondrial activity which is directly correlated to cell viability for both attached and poorly attached cells [34]. The results showed that Loganin didn't have obvious dose-dependent cytotoxic effect even at a higher concentration (128 μM). As the essential mediators of inflammation, cytokines, such as TNFα, MCP-1 and IL-6, play important roles in atherosclerosis. They are found to be involved in the development of atherosclerosis and the related signal transduction pathways such as NF-κB, JNK/AP-1 pathway and JAK/STAT pathway [35]. The present study demonstrated that apoCIII could increase mRNA expression and the release of MCP-1 and IL-6 in mouse 3T3L1 adipocytes. However, Loganin inhibited this trend effectively. NF-κB is a generic name of a series of proteins which can specifically bind with the κB site of multiple gene initiators. NF-κB is composed of five different proteins, including p50, p52, p65/ RelA, RelB and c-Rel. NF-κB is maintained in the cytoplasm in an inactive form bound through inhibitory protein IκBa under normal physiological conditions. Phosphorylated IκBa is degraded by the ubiquitin-pro-teasome pathway after the stimulation. The degradation of IκBa results in p50/p65 dimer phosphorylation and translocation, which leading to the genes transcription [36–38]. The nuclear factor (NF)-κB pathway is significantly related to the inflammatory response in cells. It is involved in the expression of various proinflammatory cytokines and plays a pivotal role in inflammation. The activation of NF-κB is accompanied by expression of a wide range of inflammatory molecules (e.g., IL-6 and TNF-α) and is often triggered by the stimulation of leukocyte receptors involved in innate immunity such as Toll like receptor (TLR) [39–41]. Indeed, pharmacological inhibition of NF-κB dramatically reduces IL production [39]. We tried to study the signaling involved in the effects of apoCIII. Therefore, we measured the phosphorylation p65 and IκBa in apoCIII-induced 3T3L1 adipocytes. In this study, apoCIII activated NF-κB in 3T3L1 adipocytes. Pretreatment with Loganin markedly suppressed the apoCIII-induced p65 and IκBa phosphorylation. These results suggest that the effects of apoCIII were mediated by NF-κB, at least in part.
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Fig. 2. Effect of different concentrations of Loganin on apoCIII-stimulated TNF-α, MCP-1 and IL-6 in mouse 3T3L1 adipocytes. Cells were pretreated with different concentrations (32, 64, 128 μM) of Loganin for 1 h prior to stimulation with 100 μg/ml of apoCIII for 24 h. The values represent mean ± SEMs. ##P b 0.01 indicates significant differences from the control group, *P b 0.05 vs. apoCIII, **P b 0.01 vs. apoCIII.
Fig. 3. Effect of different concentrations of Loganin on apoCIII-stimulated TNF-α, MCP-1 and IL-6 gene expression in mouse 3T3L1 adipocytes. Cells were pretreated with different concentrations (32, 64, 128 μM) of Loganin for 1 h prior to stimulation with 100 μg/ml of apoCIII for 24 h. The values represent mean ± SEMs. ##P b 0.01 indicates significant differences from the control group, *P b 0.05 vs. LPS, **P b 0.01 vs. apoCIII.
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Fig. 4. Effect of Loganin on apoCIII-induced NF-κB in mouse 3T3L1 adipocytes. Cells were pretreated with different concentrations (32, 64, 128 μM) of Loganin for 1 h prior to stimulation with 100 μg/ml of apoCIII for 30 min. Western blotting was used to analyze protein samples with phospho-specific antibodies, β-actin was used as an internal control. Similar results were obtained in three independent experiments expressed as mean ± SEM. ##P b 0.01 indicates significant differences from the unstimulated control group, *P b 0.05 vs. apoCIII, **P b 0.01 vs. apoCIII.
Fig. 5. Effect of Loganin on levels of TC and TG in the serum of mice. Mice received intragastric administration of Loganin (50, 100, 200 mg/kg) and fenofibrate (25 mg/kg) for consecutively 7 days. Then, serum were harvested for measuring TC and TG concentrations. The values presented are the means ± SEMs. ##P b 0.01 vs. Control group; **P b 0.01 vs. Tyloxapol group.
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Fig. 6. Effect of Loganin on Oil Red-O Staining in the Tyloxapol-induced mouse model. Mice received intragastric administration of Loganin (50, 100, 200 mg/kg) and fenofibrate (25 mg/kg) for consecutively 7 days. Then, each group was processed for Oil Red-O Staining. (A) Control group. (B) Tyloxapol group. (C) Fenofibrate group. (D, E and F) Loganin group (50, 100, 200 mg/kg). The panel is magnified 200×.
Triton WR-1339-induced hyperlipidemic rats are a globally accepted model used to evaluate potential hypolipidemic drugs [42]. Triton WR-1339 is a nonionic detergent which prevents catabolism of triacylglycerol-rich lipoproteins by lipo-protein lipase. It is used for the determination of triacylglycerol production, the very low density lipoprotein (VLDL) secretion or clearance rate in vivo commonly [43,44]. The rise of TC levels will accelerate atherosclerosis process and the rise of TG is also a dangerous factor for atherosclerosis. In this study, Pretreatment with Loganin obviously reduced levels of TC and TG in mouse serum. Moreover, Oil red O staining proved that a large quantity of red lipid droplets appeared in liver tissues after WR-1339 stimulating mouse 24 h under light microscope, but lipid droplets ameliorated after Loganin administration. In summary, our results in vitro revealed that Loganin can downregulated the levels of TNF-α, MCP-1 and IL-6 and their gene
expression. Also, it could block the activation of NF-κB signaling markedly in apoCIII-induced mouse adipocytes. Results in vivo also showed that Loganin relieved the inflammation stress. Our experimental results support that Loganin might be a potential therapeutic agent for preventing inflammatory diseases.
Conflict of interest The authors declared that there is no conflict of interest.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (no. 31572347, no. 31372478).
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