Effect and related mechanisms of RTA-408 on rat vascular smooth muscle cell calcification induced by advanced glycation end products

Abstract

Objective: To investigate the effect and related mechanisms of RTA-408 on rat vascular smooth muscle cells (VSMCs) calcification induced by advanced glycation end products(AGE). Methods: VSMCs were isolated from the aorta of Sprague Dawley rats and cultured in vitro. The fifth generation of VSMCs were randomly divided into 4 groups with random number table including control group(cells were incubated with normal medium for 2 days, then incubated with bovine serum albumin for 5 days),AGE group (cells were incubated with normal medium for 2 days, then incubated with 200 mg/L AGE for 5 days), experimental group(cells were incubated with 100 nmol/L RTA-408 for 2 days,then incubated with 200 mg/L AGE for 5 days),and RTA group(cells were incubated with 100 nmol/L RTA-408 for 2 days,then incubated with bovine serum albumin for 5 days). Cytosolic calciumin VSMC was measured using arsenazo Ⅲ assay. Von Kossa staining was utilized to detect the calcium deposition.The contents of malondialdehyde(MDA) and superoxide dismutase(SOD) in VSMCs were tested by appropriate kits.The protein expressions of osteopontin (OPN), alkaline phosphatase (ALP), nuclear factor E2 related factor 2(Nrf2), and NAD(P)H: quinone oxidoreductase 1(NQO1) were examined using Western blot. Results: (1) Cytosolic calciumconcentration was significantly higher in AGE group than in control group((2.43±0.15) mmol/L vs. (1.23±0.09) mmol/L, P<0.01), which was significantly reduced in experimental group((1.62±0.18) mmol/L,P<0.01 vs. AGE group). (2) Calcium deposition in VSMCs was significantly upregulated in AGE group than in control group(3.64±0.50 vs. 1.00±0.12, P<0.01), and was downregulated in experimental group (1.56±0.37, P<0.01 vs. AGE group). (3) The MDA contents were higher((3.79±0.27) nmol/mg prot vs.(1.99±0.15) nmol/mg prot, P<0.01), while the SOD activities were lower((308.45±14.28) U/mg prot vs. (428.58±11.00) U/mg prot, P<0.01) in AGE group than in control group. The MDA contents were lower((2.37±0.19) nmol/mg prot vs. (3.79±0.27) nmol/mg prot, P<0.01),while the SOD activities were higher((391.03±22.92) U/mg prot vs. (308.45±14.28) U/mg prot, P<0.05)in experimental group compared with AGE group. (4) The relative expressions of OPN and ALP were higher in AGE group than in control group(3.06±0.21 vs. 1.00±0.07, and 2.89±0.29 vs. 1.00±0.10,both P<0.01), both (OPN(1.15±0.12) and ALP(1.45±0.15)) were downregulated in experimental group (both P<0.01 vs. AGE group). (5) The relative protein expressions of Nrf2 and NQO1 in experimental group were higher than AGE group(2.37±0.17 vs. 1.17±0.09, and 3.91±0.18 vs. 1.05±0.08, both P<0.01). Conclusion: Activation of nrf2/NQO1 signaling pathway by RTA-408 can reduce the AGE-induced VSMC calcification through attenuating oxidative injury.