The Protective Effect Of Alpinetin On Hepatic Ischemia-Reperfusion Injury And Its Mechanism

Background:Hepatic ischemia-reperfusion injury is inevitable during selective hepatectomy,liver transplantation,traumatic shock,etc.which seriously affect the prognosis of patients.However,the mechanism of liver ischemia-reperfusion injury has not yet been fully elucidated,and interventions are limited.Therefore,there is still an urgent need to find new targets and drugs in order to provide new ideas for the treatment of liver ischemia-reperfusion injury.Hepatic ischemia-reperfusion injury includes two intermediate links of ischemia injury and inflammation related stages regulating reperfusion injury.Excessive cell death and extreme inflammation are two important pathological processes of liver ischemia-reperfusion injury.Under ischemic stress,due to the interruption of oxygen and the depletion of intracellular ATP,hepatocytes will die rapidly,which triggers the inflammatory response,including the infiltration of macrophages and neutrophils and the release of cytokines/chemokines during reperfusion.The inflammatory response breaks out at the stage,which eventually leads to tissue and organ damage.More and more studies have shown that inhibiting cell death and inflammation can effectively alleviate ischemia-reperfusion injury.Alpinetin is a plant flavonoid compound isolated from grass cardamom and turmeric.It has a wide range of beneficial properties,including antioxidant,anti-inflammatory,anti-apoptotic and anti-cancer properties.Studies have shown that alpinetin can improve the inflammatory response of mouse endometritis induced by lipopolysaccharide.Another study found that alpinein can improve non-alcoholic fatty liver induced by high-fat diet by improving oxidative stress,inflammation and lipid metabolism.In addition,it is reported that alpinetin can reduce the apoptosis of rat cardiomyocytes.However,it is still unclear whether alpinetin can protect liver from ischemia-reperfusion injury by regulating inflammation and cell apoptosis.This study will discuss the protective effect of alpinetin on liver ischemiareperfusion injury and its molecular mechanism from the animal level and the cell level,and provide new possible drug targets for the treatment of liver ischemiareperfusion injury.Part 1 The protective effect of alpinetin on in liver ischemiareperfusion injuryObjective:To delve into the protective effect of alpinetin on hepatic I/R injury in vivo and in vitro.Methods:1.To detect the protective effect of alpinetin on hepatic I/R injury in mice1.1 Establish 70%mice model of hepatic I/R injury,and divide them into sham group,Alpinetin pretreated sham group,I/R group and Alpinetin pretreated I/R group.At the corresponding time point,the mice were killed and the samples were taken.The contents of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and lactate dehydrogenase(LDH)in the serum of mice were detected by automatic biochemical analyzer;the dead area and necrosis degree of liver tissue were evaluated by hematoxylin eosin staining(H&E).2.To detect the regulatory effect of alpinetin on the inflammatory response during hepatic I/R injury in vivo and in vitro.2.1 Establish 70%mice model of hepatic I/R injury,and divide them into sham group,Alpinetin pretreatment sham group,I/R group and Alpinetin pretreatment I/R group.At the corresponding time points,the mice were killed and the samples were taken.The infiltration of inflammatory cells in liver tissue were detected by ly6g and CD11b immunofluorescence;the gene expression of inflammatory factors such as TNFa,IL8,IL1B,and MCP-1 in liver tissue were detected by RT-PCR.2.2 The model of hepatocyte H/R was established and divided into control group,Alpinetin pretreatment group,H/R group and Alpinetin pretreatment H/R group.At the corresponding time point,the gene expression of inflammatory factors such as TNFα,IL6,IL8 and MCP-1 were detected by RT-PCR.3.The effect of alpinetin on apoptosis of hepatic ischemia-reperfusion injury in vivo and in vitro.3.1 The 70%mice model of hepatic I/R injury was established and divided into sham group,Alpinetin pretreatment sham group,I/R group and Alpinetin pretreatment I/R group.At the corresponding time points,the mice were killed and the samples were taken.The apoptosis of liver tissue was detected by TUNEL immunofluorescence and Bax immunohistochemistry;the expression of apoptosis related proteins such as Bax,Bcl2 and c-casepase-3 in liver tissue were detected by western blot.3.2 The model of hepatocyte H/R was established and divided into control group,Alpinetin pretreatment group,H/R group and Alpinetin pretreatment H/R group.At the corresponding time points,the expression of Bax,Bcl2 and other apoptosis related genes was detected by RT-PCR;the expression of Bax,Bcl2 and c-caspase-3 in hepatocyte were detected by western blot,apoptosis was also detected by flow cytometry.Results:1.Alpinetin significantly alleviated liver injury after hepatic I/RCompared with the I/R group,the serum transaminase(ALT,AST)and LDH were significantly decreased,and the area and degree of liver necrosis were significantly reduced in the Alpinetin pretreated I/R group.2.Alpinetin significantly inhibited the inflammatory response during hepatic I/R injuryCompared with I/R group,the infiltration of ly6g and CD11b positive inflammatory cells in liver tissue were significantly reduced,and the gene expression of TNFα,IL8,IL1B,and MCP-lin liver tissue was significantly down regulated in Alpinetin pretreated I/R group;Compared with the H/R group,the gene expression of TNFα,IL6,IL8 and MCP-1 were significantly down regulated in Alpinetin pretreated H/R group.3.Alpinetin significantly inhibited the apoptosis of hepatic I/R injuryCompared with I/R group,the number of TUNEL and Bax positive apoptotic cells in I/R group were significantly decreased,the expression of proapoptotic molecules Bax,and c-casepase-3 were significantly down regulated,and the expression of antiapoptotic factor Bcl2 was significantly up-regulated;Compared with H/R group,the expression of proapoptotic factor Bax and antiapoptotic factor Bcl2 were significantly down regulated and up-regulated in Alpinetin pretreated H/R group.At the same time,the proportion of apoptotic cells decreased significantly.Conclusion:Alpinetin significantly inhibit the inflammatory response and apoptosis in the process of hepatic I/R injury,thereby improving hepatic I/R injury.Part 2 The molecular mechanism of Alpinetin improving hepatic I/R injuryObjective:To explore the molecular mechanism of alpinetin improving liver I/R injury.Methods:1.To predict the possible target proteins of kaempferin in regulating liver ischemia-reperfusion injury through databaseTCMSP and BATMAN database were used to predict the target proteins regulated by kaempferin,and CTD2018 update disease database was used to obtain the target proteins of I/R injury.1.To detect the effect of alpinetin on NF-κB pathway during hepatic I/R in vivo and in vitro1.1 Establish 70%mice model of hepatic I/R injury,and divide them into sham group,Alpinetin pretreated sham group,I/R group and Alpinetin pretreated I/R group.The expression of p65,p-p65,IKKβ,p-ikkβ and IκBa in liver tissue was detected by western blot.1.2 The model of hepatocyte H/R was established and divided into control group,Alpinetin pretreatment group,H/R group and Alpinetin pretreatment H/R group.Western blot was used to detect the protein expression of p65,p-p65,IKKp,p-ikkβ and IκBα.2.To detect the effect of alpinetin on MAPK pathway after hepatic I/R in vivo and in vitro2.1 Establish 70%mice model of hepatic I/R injury,and divide them into sham group,Alpinetin pretreatment sham group,I/R group and Alpinetin pretreatment I/R group.The expression of ASK1,p-ASK1,p38,p-p38,JNK and p-JNK in liver tissue was detected by western blot.2.2 The model of hepatocyte H/R was established and divided into control group,Alpinetin pretreatment group,H/R group and Alpinetin pretreatment H/R group.At the corresponding time point,the expression of ASK1,p-ASK1,p38,p-p38,JNK and p-JNK in liver tissue was detected by western blot.3.Whether the protective effect of alpinetin on hepatic I/R injury depends on ASK13.1 The model of hepatocyte H/R was established and divided into H/R group,H/R+flag group,H/R+Alpinetin group and H/R+Alpinetin+flag-ask1 group.At the corresponding time point,the expression of ASK1 and p-ASK1 were detected by Western blot,the expression of TNFα,IL6,IL18 and MCP-1,apoptosis related genes such as BAX,BCL2 were detected by RT-PCR;apoptosis was also detected by flow cytometry.Results:1.NF-κB and MAPK signaling pathway were significantly enriched in the process of kaempferin improving liver I/R injury2.Alpinetin significantly inhibited the activation of NF-κB pathway after hepatic I/R injury in vivo and in vitroCompared with I/R group,the activation of NF-κB pathway was significantly inhibited in Alpinetin pretreatment I/R group.Compared with the H/R group,the activation of NF-κB pathway was significantly inhibited in the Alpinetin pretreatment H/R group.3.Alpinetin significantly inhibited the activation of MAPK pathway after hepatic I/R in vivo and in vitroCompared with I/R group,the activation of MAPK pathway in Alpinetin pretreatment I/R group was significantly inhibited.Compared with the H/R group,the activation of MAPK pathway was significantly inhibited in the Alpinetin pretreatment H/R group.4.The protective effect of Alpinetin on hepatic ischemia-reperfusion injury depends on ASK1-JNK/p38 signal axisCompared with the H/R group,Alpinetin pretreatment significantly inhibited the inflammatory response,apoptosis and activation of ASK1 induced by H/R.Overexpression of ASK1 could counteract the inhibitory effect of Alpinetin pretreatment on inflam matory response,apoptosis and activation of ASK1 after H/R treatment.Conclusion:Alpinetin can inhibits the inflammatory reaction and apoptosis during liver I/R injury through ASK1-JNK/p38 signal axis,thus improving liver I/R injury.