Mechanism Of Gastrodin Regulating P38 MAPK/Nrf2/HO-1 Pathway To Alleviate Oxidative Damage Of Liver Sinus Endothelial Cells In Mouse

Objective:Hepatic ischemia reperfusion injury refers to the phenomenon that hepatic injury is further aggravated after temporary blocking of hepatic blood supply in the process of liver resection and liver transplantation and the subsequent restoration of blood supply.As the first line of defense in hepatic ischemia reperfusion injury,liver sinus endothelial cells are the earliest damaged cells and play a key role in hepatic oxidative stress.Oxidative stress is mainly caused by the combined action of reactive oxygen species(ROS)accumulation and inflammatory reactions.Therefore,it is beneficial to protect HIRI if it can effectively protect the function of liver sinus endothelial cells and reduce oxidative stress.At present,there are no clinical drugs for the treatment of oxidative damage in HIRI,so the development of effective drugs for the treatment of oxidative damage in HIRI has become a problem to be solved.As an effective antioxidant and oxygen free radical scavenger,Gastrodin (GSTD)is mainly used in the treatment of nervous system and cardiovascular diseases,and has the function of liver protection.Therefore,the purpose of this study is to explore the protective effect of gastrodin(GSTD)on the oxidative damage of LSECs and its molecular mechanism,so as to provide a theoretical basis for the prevention and treatment of clinical HIRI.Methods:Firstly,we used 1mM H2O2 to construct the oxidative stress model of LSECs,and treated cells with 1,10 and 50 M GSTD at the same time.By detecting the content of ROS and MDA,we evaluated the alleviating effect of GSTD in the oxidative stress process of LSECs.The effect of GSTD treatment on apoptosis induced by H2O2 was detected by TUNEL and Annexin V-FITC-PI double staining,and the expression of apoptosis-related protein was detected by western blot.Secondly,we pretreated cells with SB 203580 of lOmM and Znpp of ImM,respectively,and detected the protein changes of p38MAPK/Nrf2/HO-1,a pathway related to oxidative stress,by western blot and index of correlation in oxidative stress and apoptosis were detected.Cell models of Nrf2 overexpression and siRNA knockout were constructed to further verify the role of GSTD in regulating Nrf2/HO-1 in the antioxidant process through p38MAPK.In addition,the model of HIRI in mice was established after intraperitoneal perfusion of GSTD,and the protective effect of GSTD on liver injury was observed by detecting the level of AST,ALT in serum and the staining of HE in liver tissue.Apoptosis was detected by TUNEL assay.The expression of mRNA and protein in Nrf2,HO-1,IL-6 and TNF-α were examined by RT-PCR and western blot in liver tissue respectively.To further study the protective effect of GSTD on HIRI in mice.Results:Firstly,we found that the content of ROS and MDA in the LSECs pretreated by GSTD decreased in the oxidative stress model induced by H2O2,suggesting that GSTD could inhibit the oxidative stress induced by H2O2.The apoptosis decreased by TUNEL assay and Annexin V_FITC-PI double staining assay after treatment of GSTD,suggesting that GSTD has an inhibitory effect on H2O2-induced apoptosis.Secondly,it is suggested that GSTD inhibits H2O2-induced apoptosis and oxidative stress by promoting the phosphorylation of p38 MAPK and activating the Nrf2/HO-1 pathway.After GSTD treatment,the phosphorylation level of p38MAPK increased significantly,and high concentration of GSTD could offset the effect of p38MAPK inhibitor.Meanwhile,GSTD can significantly up-regulate Nrf2 and HO-1 expression.After knockdown of Nrf2 expression with siRNA,the expression of HO-1 was decreased,while the percentage of TUNEL positive cells was increased,and the contents of ROS and MDA were significantly increased.The results of nucleosome separation and immunoprecipitation showed that the main pathway by which GSTD regulated Nrf2 under oxidative stress was to induce nuclear translocation of Nrf2.In addition,the results showed that the oxidative stress,inflammation,apoptosis and necrosis of liver were caused by HIRI and GSTD pretreatment can reduce level of AST,ALT in the serum and decrease MDA level,increase the SOD activity,raise the expression of Nrf2 and HO-1 and lower the expression of TNF-α and IL-6.In addition,GSTD pretreatment can prompt anti-inflammatory,antioxidant,antiapoptotic effect the abilities in HIRI.Conclusion:1.GSTD has antioxidant and anti-apoptotic effects in H2O2-induced oxidative stress;2.GSTD inhibits LSECs oxidative damage and apoptosis induced by H2O2 by activating p38MAPK/Nrf2/HO-1 pathway;3.GSTD can reduce hepatic ischemia reperfusion injury in mice by anti-inflammation,anti-oxidation and anti-apoptosis.