Angiotensin-(1-7) protects H9c2 cardiac cells against high glucose-in- duced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis / 中国病理生理杂志

ABSTRACT

AIM:To investigate whether angiotensin-(1-7) [Ang-(1-7)] protects H9c2 cardiac cells against high glucose (HG)-induced injury and inflammation by inhibiting the interaction between Toll-like receptor 4 (TLR4) acti-vation and necroptosis .METHODS:The expression levels of receptor-interacting protein 3 ( RIP3;an indicator of necrop-tosis) and TLR4 were determined by Western blot .Cell viability was measured by CCK-8 assay.The activity of lactate de-hydrogenase ( LDH) in the culture medium was measured with a commercial kit .The releases of interleukin-1β( IL-1β) and tumor necrosis factor-α( TNF-α) were measured by ELISA .The intracellular level of reactive oxygen species ( ROS) was analyzed by 2 ’ , 7 ’-dichlorfluorescein-diacetate ( DCFH-DA ) stating followed by photofluorography .Mitochondrial membrane potential ( MMP) was examined by rhodamine 123 staining followed by photofluorography .RESULTS:After the H9c2 cardiac cells were treated with HG (35 mmol/L glucose) for 24 h, the expression of RIP3 was obviously increased . Co-treatment of the cells with 30μmol/L TAK-242 (an inhibitor of TLR4) attenuated the up-regulation of RIP3 induced by HG.Furthermore, the expression of TLR4 was significantly increased after the cells were exposed to HG for 24 h, and co-treatment of the cells with 100μmol/L necrostatin-1 ( Nec-1;a specific inhibitor of necroptosis ) and HG for 24 h attenua-ted the up-regulation of TLR4 expression induced by HG .Moreover, 1μmol/L Ang-(1-7) simultaneously blocked the up-regulation of the RIP3 and TLR4 induced by HG.On the other hand, co-treatment of the cells with 1μmol/L Ang-(1-7), 30 μmol/L TAK-242 or 100 μmol/L Nec-1 and HG for 24 h attenuated HG-induced injuries and inflammatory response , leading to the increase in the cell viability , and the decreases in the activity of LDH , ROS generation , MMP loss as well as the releases of IL-1βand TNF-α.CONCLUSION:Ang-(1-7) protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the interaction between TLR 4 activation and necroptosis .