Inhibitory effect of irisin on vasular smooth muscle cell inflammation and proliferation by regulating autophagy through adenosine monophosphate-actived protein kinase/mammalian target of rapamycin pathway

Abstract

Objective To explore the underlining mechanisms of irisin inhibiting inflammation and proliferation of vascular smooth muscle cells (VSMCs). Methods The cultured primary rat aortic VSMCs were pre-incubated with irisin and then treated with platelet derived growth factor-BB (PDGF-BB). Enzyme linked immunosorbent assay (ELISA) and methyl thiazol tetrazolium (MTT) assay were used to measure the VSMCs inflammation and proliferation, respectively. The protein expression and phosphorylation of adenosine monophosphate-actived protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway related molecule AMPK/p-AMPK (Thr172), acetyl-CoA carboxylase (ACC)/phosphorylated (p)-ACC (Thr308), mTOR/p-mTOR (Ser2448), ribosomal protein S6 kinase (p70SK6)/p-70SK6 (Thr389), and autophay related molecule microtubule-associated protein 1 light chain 3 (LC3) Ⅰ, LC3Ⅱ and nucleoporin p62 (P62) were detected by Western blotting. Results After treatment with PDGF-BB, VSMCs proliferation dramatically increased, and the inflammatory cytokines IL-1β (37.9±8.5 vs. 10.8±2.9, t=18.620, P<0.01), IL-6 (57.4±14.1 vs. 32.4±8.7, t=6.250, P<0.01), MCP-1 (158.3±39.1 vs. 57.2±12.5, t=14.280, P<0.01), ICAM-1 (371.8±77.3 vs. 160.4±39.8, t=10.850, P<0.01), tumor necrosis factor-α (TNF-α) (16.3±3.8 vs. 7.8±2.4, t=7.390, P<0.01) in the cell supernatant were significantly up-regulated. The protein expression levels of AMPK (0.39±0.11 vs. 0.83±0.18, t=14.250, P<0.01), p-AMPK (0.25±0.03 vs. 0.49±0.12, t=12.370, P<0.01), ACC (0.54±0.16 vs. 1.07±0.24, t=10.670, P<0.01), p-ACC (0.38±0.07 vs. 0.97±0.30, t=18.130, P<0.01), LC3Ⅰ (0.67±0.21 vs. 0.67±0.21, t=5.780, P<0.01) and LC3Ⅱ (0.42±0.13 vs. 0.93±0.38, t=6.820, P<0.01) were down-regulated, mTOR (2.57±0.53 vs. 1.23±0.25, t=21.350, P<0.01), and p-mTOR (1.35±0.0.37 vs. 0.57±0.12, t=8.870, P<0.01), p70SK6 (1.85±0.39 vs. 0.81±0.19, t=7.170, P<0.01), p-70SK6 (0.69±0.22 vs. 0.38±0.11, t=9.260, P<0.01) and P62 (1.24±0.38 vs. 0.58±0.14, t=5.270, P<0.01) were up-regulated. However, pre-incubation with irisin and then stimulation with PDGF-BB could significantly decrease VSMCs proliferation [(122.14±9.42)%, t=4.970, P<0.01], and significantly down-regulate the inflammatory cytokines IL-1β (17.38±4.10, t=6.740, P<0.01), IL-6 (34.9±8.9, t=2.760, P<0.05), MCP-1 (47.28±9.20, t=16.270, P<0.01), ICAM-1 (169.2±55.8, t=7.780, P<0.01) and TNF-α (6.9±2.0, t=8.180, P<0.01) in the cell supernatant. The protein expression levels of AMPK (0.72±0.26, t=2.670, P<0.05), p-AMPK (0.46±0.13, t=3.280, P<0.05), ACC (0.95±0.28, t=5.870, P<0.01), p-ACC (0.78±0.25, t=3.140, P<0.01), LC3Ⅰ (1.34±0.35, t=1.960, P<0.05) and LC3Ⅱ (1.03±0.19, t=7.260, P<0.01) were up-regulated, and mTOR (1.16±0.33, t=6.910, P<0.01), p-mTOR (0.66±0.18, t=6.830, P<0.01), p70SK6 (1.12±0.34, t=2.360, P<0.05), p-70SK6 (0.37±0.12, t=3.120, P<0.01) and P62 (0.68±0.18, t=3.360, P<0.05) were down-regulated. Conclusion Irisin could inhibit the inflammation and proliferation of VSMCs possibly by up-regulating autophagy through activation of the AMPK/mTOR signaling pathway. Key words: Irisin; Adenosine monophosphate-actived protein kinase/mammalian target of rapamycin signaling pathway; Autophagy; Vascualar smooth muscle cell proliferation; Vein graft stenosis