The Study Of Mechanism Of Ginsenoside Rg1 Attenuates The Hepatic Ischemia-reperfusion Injury By Inhibiting Mitochondrial Apoptosis And Mitophagy Pathway

Objective:Hepatic ischemia reperfusion injury(HIRI),during the hepatobiliary surgical operations including liver transplantation and hepatectomy,is a common pathological process that the liver function after ischemia injury undergo further aggravated during the subsequent blood-reperfusion process.Therefore,it is essential to find a method to alleviate HIRI.Ischemic preconditioning(IPC)and drug pretreatment are the two pretreatment common methods to prevent or treat HIRI in clinical and basic experimental research.Although IPC has been proved to be an effective treatment method,which drug pretreatment protocol are the best choice for reducing the HIRI has remained unclear.Ginsenoside Rg1 has been proved to reduce various organ I/R injury including heart,brain,liver,et.al,because it posses the multiple pharmacological effects such as anti-oxidation,anti-apoptosis,and improving microcirculation.However,in the HIRI study,the specific mechanism of Rgl in reducing I/R injury is unclear.There are researches suggested that the mitochondrial apoptosis pathway and mitophagy pathway are two important pathways to regulate the pathophysiology of HIRI,and the Cyclophilin D(CypD)protein is associated with mitochondrial structure and function.Therefore,the purpose of this study is to clearly identify the optimal IPC Protocol that can provides the best protection,so as to provides a reference for select the Rgl as the target drug for drug pretreatment.Second,to explore the mechanism of action of Rgl in reducing HIRI through the mitochondrial pathway from the target of CyPD,and provides a certain theoretical basis for traditional Chinese medicine treatment of HIRI in clinical medicine.Methods:① Eastablished a warm ischemia-reperfusion injury model in rat liver(70%),and three ischemic preconditioning were performed:5-10min-IPC,10-10min-IPC,and 3 cycles of 5-5min-IPC.The optimal ischemic preconditioning protocols was optimized through detection of liver function,SOD activity,GSH content,MDA content,histopathological changes,hepatocyte apoptosis.②Compared the Rg1 pretreatment protocol with the optimal IPC protocol,the protective effect of HIRI was evaluated by measuring liver function,SOD activity,GSH content,MDA content,histopathological changes,and hepatocyte apoptosis.It is clearly defined the feasibility and significance of selecting Rgl for subsequent drug pretreatment studies.③To observe the effect of Rg1 pretreatment on rat hepatocyte apoptosis.After the Rg1 pretreatment for rat liver warm ischemia-reperfusion injury model in vivo and OGD/R treated rat BRL-3A cell model in vitro,the apoptosis level was detected by TUNEL method,Annexin V-AF647-PI double staining method to detect the apoptosis levels,and protein expression of apoptosis-related signal molecules(Cyt-c,Caspase9,Caspase3)were detected by western blot.And further to observe the effect of Rg1 pretreatment on mitochondrial apoptosis pathway through measurement the CypD protein expression levels,Mitochondrial membrane potential(MMP),mitochondrial calcium retention capacity(CRC),transmission electron microscope to observe mitochondrial structure.At the same time,evaluate the liver function and histological damage in rats and the BRL-3A Cell damage in vitro to clarify the effectiveness of Rg1.④Using the IRI models in vivo and in vitro,the time points of excessive mitophagy were identified by detecting key proteins of mitophagy(LC3I,LC3II,p62,PINK1 and Parkin)at different reperfusion time points;then,after pretreatment with Rg1,the apoptosis levels were detected by TUNEL method and Annexin V-AF647-PI double staining method,,western blot was used to detect the protein expression level of mitophagy-related signal molecules(LC3Ⅰ,LC3Ⅱ,p62,PINK1 and Parkin),detection of MMP,transmission electron microscopy to observe the number of mitophagosomes,detection of the liver function and histopathological changes in vivo and the proliferation activity of BRL-3A cell in vitro,observation the regulation effect of Rg1 pretreatment on mitophagy pathway,thereby further improving the mechanism of Rg1 protects HIRI through the mitochondrial pathway.Results:① In the studies of rat animal model,it was found that compared with the I/R group,the three IPC protocols showed a significant reduction in liver transaminase activity levels and a reduction in histopathological damage,and an increase in SOD activity and GSH content,the MDA content was reduced;among them,the 5-5min-IPC protocol that implemented the cycle three times was the most obvious,indicating that it have the best effect in reducing I/R injury.② After a comparative study of Rg1 pretreatment and IPC,it was found that the levels of liver transaminase activity,histopathological damage,SOD activity,GSH content,and MDA content of the two showed similar changes.The above of indicators changed was more significantly after the two combined treatment;this shows that the Rg1 pretreatment can achieve the same effect as IPC,both also have an additive effect.③ It was found that the rat HIRI model research in vivo,compared with the I/R group,the Rg1 pretreatment can reduce ALT and AST activity levels,reduce liver pathological damage changes and apoptosis;In the study of the OGD/R model of BRL-3A cells in vitro,40 μM Rg1 pretreatment for 24 hours can provide the best protection,Rg1 significantly enhances cell viability and inhibits apoptosis;in vivo and in vitro models,Rg1 can inhibit CypD expression,stabilize the mitochondrial transmembrane Potential,improve the CRC,and protect the mitochondrial structure and function,limit the mitochondrial apoptotic pathway,reducing hepatocyte apoptosis;and in vitro studies,this beneficial effect is inhibited by H2O2;this indicates that Rg1 pretreatment may reduce HIRI by inhibiting CypD expression and limiting the mitochondrial apoptotic pathway.④In the experiments of animal in vivo and BRL-3A cells in vitro,it was found that the expression level of mitophagy-related protein was highest and the level of liver transaminase activity was highest after 6 hours of reperfusion in vivo,and the expression level of mitophagy-related protein was highest after 6 hours of reoxygenation in vitro;in vivo,compared with the I/R group,pretreatment with Rg1 was significantly reduced the activity levels of ALT and AST,and the changes in liver histological damage were significantly reduced;in vitro,Rg1 pretreatment was significantly enhanced cell proliferation activity;we have found that the expression of the key proteins of mitophagy(LC3I,LC3II,p62,PINK1 and Parkin)decreased,the apoptosis levels were significantly reduced,the MMP was significantly increased,and the number of mitophagosomes was reduced after pretreatment with Rg1 in vivo and in vitro studies;suggesting that the excessive of mitophagy was exacerbates damage during reperfusion,and Rg1 pretreatment may reduce HIRI by inhibiting mitophagy.Conclusion:1.The 5-5min-IPC protocol with 3 cycles provided the optimal protection against hepatic ischemia reperfusion injury among the protocols studied.2.Rg1 pretreatment is able to achieve the same effect as IPC for reduce HIRI in rats;which may be achieved by reducing apoptosis through antioxidant stress injury.3.Rg1 can effectively reduce the expression of CypD protein.4.The pretreatment with Rgl attenuates HIRI in rats,the mechanism was partially attributed to the inhibition of CypD protein expression and regulation of MPTP opening,which ultimately restricted the mitochondrial apoptosis pathway.5.In the reperfusion stage,the excessive mitophagy was increased the liver damage.6.Rg1 pretreatment reduce excessive mitophagy and attenuates HIRI during reperfusion through inhibits the activation of the PINK1/Parkin signaling pathway;the mechanism was partially attributed to the inhibiting the expression of CypD protein and inhibiting the opening of MPTP.