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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.
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Objective To investigate the effect of Bortezomib on renal tubular epithelialmesenchymal transition (EMT),and to determine whether Smad ubiquitin regulatory factor 2 (Smurf2) expression induced by tumor necrosis factor-α (TNF-α) can be reversed by Bortezomib in human renal tubular epithelial cells (HK-2).Methods The HK-2 cells were divided into control group (cultured with fetal bovine serum),TNF-α group (cultured with TNF-α),Bortezomib group (cultured with Bortezomib) and experimental group (treated with Bortezomib and TNF-α together).Each group of cells was observed under an inverted microscope,and then the cells of each group were collected.RT-PCR and Western blotting were performed to detect the expression levels of fibronectin (FN),Smurf2,E-cadherin and α-smooth muscle actin (α-SMA).Results As compared with the TNF-α-treated group,the morphology of HK-2 cells was still cobblestone-like after intervention with bortezomib;however,the cellular morphology did not change significantly in the bortezomib-treated group as compared with the control group.As compared with the control group,the expression of FN mRNA and protein in the TNF-α group was significantly increased (P＜0.05).As compared with the TNF-α group,bortezomib inhibited the expression of FN induced by TNF-α (P＜0.05) There was no significant difference in the expression of FN between bortezomib-treated group and control group (P ＞0.05).As compared with the control group,E cadherin was significantly decreased in HK-2 cellsafter treatment with TNF-α,and α-SMA was significantly increased (P＜0.05).As compared with TNFα group,co-stimulation with bortezomib reversed the expression of E cadherin and α-SMA induced by TNF-α (P ＜ 0.05),but the expression of E-cadherin and α-SMA did not change significantly in the bortezomib-treated group as compared with the control group (P ＞ 0.05).As compared with the control group,TNF-α could increase the expression of Smurf2 mRNA and protein (P＜0.05),and bortezomib could inhibit the increase in Smurf2 induced by TNFα (P＜0.05).As compared with the control group,the expression of Smurf2 did not change significantly in the bortezomib-treated group (P＞0.05).Conclusion Bortezomib can antagonize the expression of Smurf2 and EMT induced by TNF-α in HK-2 cells.
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Objective To explore the role and mechanism of tumor necrosis factor-α (TNF-α) in the formation of chronic transplant renal interstitial fibrosis and to study Smurf2 expression change and role played in this process.Methods We collected 26 cases of normal renal tissues and 26 cases of renal allograft specimens from chronic allograft dysfunction patients to observe the degree of renal interstitial fibrosis by Hematoxylin and eosin (HE) staining and Masson staining.Immunohistochemical staining was applied to detect the expression and distribution of E-cadherin,α-smooth muscle actin (α-SMA) and TNF-α,Smad ubiquitination regulatory factor 2 (Smurf2).Furthermore,The HK2 cells were divided into five groups depending on different concentrations of TNF-α (0,10,20,50,and 100 ng/mL).After 48 h,we collected the cells to analyze the expression changes of Smurf2,E-cad,α-SMA by Western blotting.Results As compared with normal group,the degree of renal interstitial fibrosis in CAD group was aggravated according to the results of HE and Masson staining (P＜0.01).Immunohistochemical staining results showed that the positive expression of E-cadherin was reduced,and that of α-SMA,TNF-α and Smurf2 increased greatly in CAD group as compared with normal group (P＜0.01).The results of Western blotting also revealed that after treatment with different concentrations of TNF-α for 48 h,the expression of E-cadherin was downregulated,and that of α-SMA and smurf2 was up-regulated in HK2 cells (P＜0.05).Conclusion TNF-α may promote the formation of allograft renal interstitial fibrosis via transdifferentiation of human tubular epithelial cells to mesenchymal cells by up-regulating the expression of Smurf2.