2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside Protects TNF-α Induced Damage Of Human Brain Microvascular Endothelial Cells By Inhibiting NF-ΚB Signaling Pathway

Objective: To observe the protection of 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside(TSG)on TNF-α induced damage of human brain microvascular endothelial cells(HBMECs),and then discuss the function of NF-κB signaling pathway on protection.Thus to provides theoretical basis for the prevention and treatment of clinical stroke.Methods: HBMECs were cultured in vitro,and the cells were directly treated with TSG of different concentrations to detect the toxicity of TSG by CCK-8 kit.Subsequently,HBMECs were treated with the concentration of 2.5 μmol/L,5.0 μmol/L,10.0 μmol/L,20.0 μmol/L of TSG for 1h,and then stimulated for 24 h with 60 ng/ml TNF-α to detected Cell viability by CCK-8 kit;Western-blot was used to detect the expression levels of NF-κB p65,caspase-3,I-κB,MMP-2,MMP-9 and iNOS.ELISA was used to detect the secretion levels of MMP-2 and MMP-9 in HBMECs;After 1h pretreatment with a different concentration(20 mmol /L,40 mmol/L,80 mmol/L,and 160 mmol /L)of NF-κB inhibitor PDTC,the HBMECs were stimulated by 60 ng/ml TNF-α for 24 h to detected cell viability by CCK-8 kit.Results: 1.The results of cytotoxicity test showed that 0~20.0 μmol/L TSG had no cytotoxicity to human brain microvascular endothelial cells(P>0.05).2.TNF-α can induced the cell damage of HBMECs,while TSG in different concentrations can significantly inhibit TNF-α induced cell damage(P<0.05),however,there was no significant difference in cell viability between the TSG concentration of 10.0 μmol/L and 20.0 μmol/L(P>0.05).Therefore,we can use the concentration of 10.0 μmol/L for subsequent experiments.3.Western-blot results showed that TNF-α could induce a significant increased expression levels of NF-κB p65 and caspase-3 in HBMECs,however,which were significantly decreased after the pretreatment of TSG with a concentration of 10.0 μmol/L(P<0.05).4.The results of NF-κB inhibition experiment showed that there was no significant difference in cell viability when PDTC treatment concentration was 20 μmol/L and 40 μmol/L,and the cell activity increased significantly compared to control when the concentration was 80 μmol/L and 160 μmol/L(P<0.05).5.The results of ELISA showed that TNF-α could induce the increased secretion levels of MMP-2 and MMP-9 in HBMECs,while the secretion levels of MMP-2 and MMP-9 were significantly decreased after the TSG pretreatment with 2.5 μmol/L,5.0 μmol/L and 10.0 μmol/L,respectively(P<0.05).6.Western-blot results showed that TSG with 2.5 μmol/L,5.0 μmol/L and 10.0 μmol/L could significantly reduce the expression level of MMP-2,MMP-9 and iNOS in HBMECs.Conclusions: 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside could provide a protection on TNF-α induced damage of Human brain microvascular endothelial cells by inhibiting NF-κB signaling pathway.