To Investigate The Regulation Of Salvianolic Acid A On Mitochondrial Biogenesis And Function Of Vascular Endothelial Cells And Its Related Mechanisms Based On AMPK-PGC-1α Pathway

Objective: Diabetic vasculopathy is one of the complications of diabetes,and also one of the main causes of death and disability in diabetic patients.The decrease of the number of endothelial cells mitochondria,the metabolic dysfunction,and the increase of Reactive oxygen species(ROS)production,which lead to oxidative stress,are the key factors causing diabetic vascular lesions.Salvianolic acid A(SAA)has been shown to have good therapeutic effects on a variety of vascular diseases of Diabetes mellitus(DM),suggesting that it may have the potential to improve the function of vascular endothelial cells.However,the effect of SAA on mitochondrial generation and function of endothelial cells remains unclear.In this study,Human umbilical vein endothelial cells(HUVECs)were used as the model cells to investigate the effect of SAA on mitochondrial biogenesis and the possible related mechanism.On this basis,whether it can improve the oxidative stress state of endothelial cells under high glucose stimulation by promoting mitochondrial biogenesis was investigated..Methods: MTT method was used to determine the cytotoxicity of SAA to HUVECs cells,and the optimal concentration was selected for subsequent experimental exploration.Mitochondrial formation was determined by confocal microscopy and transmission electron microscopy.The expression of Adenosine triphosphate(ATP)was determined by colorimetry and the protein expression of oxidative phosphorylation subunit complex Ш and complex IV was determined by Western blot.The expression of mitochondrial DNA was determined by real-time fluorescence quantitative PCR(RT-q PCR).Western blot and RT-q PCR were used to determine the expression of PGC-1α,NRF1 and TFAM proteins and genes.The expression of PGC-1α,NRF1 and TFAM proteins and the phosphorylation levels of AMP-activated protein kinase(AMPK)and Acetyl Co A Carboxylase(ACC)were detected by Western blot after the cells were treated with Compound C,a specific inhibitor of AMPK.Fluorescence microscope and ROS fluorescence test box were used to measure the production of ROS.Results: Confocal and transmission electron microscopy results showed that SAA increased the number of mitochondria in HUVECs cells after SAA treatment,and RT-q PCR results showed that mitochondrial DNA expression increased in HUVECs cells,suggesting that SAA promoted mitochondrial biogenesis in HUVECs cells.Western blot results showed that after SAA treatment,the expression of oxidative phosphorylation subunit complex Ш and complex IV continued to increase.Compared with the control group,intracellular ATP increased significantly after SAA treatment in a dose-dependent manner,suggesting that SAA enhanced mitochondrial function.Western blot and RT-q PCR showed that the transcription and expression of PGC-1α,NRF1 and TFAM were increased in HUVECs cells treated with SAA.Meanwhile,Western blot and RT-q PCR results after Compound C treatment showed that Compound C eliminated the promotion effect of SAA on PGC-1α and its downstream target proteins,and the promotion effect of SAA on AMPK and ACC phosphorylation levels was also inhibited by Compound C.These results suggest that SAA may promote the generation of mitochondria in endothelial cells by activating the PGC-1α/TFAM signaling pathway mediated by the transcription factor AMPK.The results of fluorescence microscopy and ROS fluorescence method showed that the mitochondrial ROS(mt ROS)and the overall ROS levels in HUVECs cells were decreased under high glucose stimulation after SAA treatment.However,confocal and electron microscopy results showed that mitochondrial production increased.RT-q PCR results showed that mitochondrial DNA expression was also increased,and Western blot and RT-q PCR results showed that the transcription and expression of mitochondrial production-related proteins PGC-1α,NRF1 and TFAM were increased.However,in the presence of Compound C,its role in promoting the expression of mitochondrial production-related proteins and enhancing the phosphorylation levels of AMPK and ACC was inhibited,and SAA’s role in reducing ROS and mt ROS under high glucose stimulation was also inhibited.These results suggest that SAA can improve oxidative stress of endothelial cells under high glucose stimulation,which may be related to the promotion of mitochondrial biogenesis by up-regulating the expressions of PGC-1α,NRF1 and TFAM through activation of AMPK.Conclusions: SAA promotes mitochondrial biogenesis and enhances mitochondrial function of HUVECs cells by activating the PGC-1α/TFAM signaling pathway mediated by the transcription factor AMPK.Moreover,SAA decreased ROS and mt ROS production in HUVECs cells under high glucose stimulation,which was related to the up-regulation of PGC-1α/NRF1/TFAM expression by AMPK activation and the promotion of mitochondrial biogenesis in HUVECs cells.These results provide an important theoretical basis for SAA to improve vascular endothelial function and treat diabetic vascular diseases.