Protective Effect Of α-Hederin On CCl₄-Induced Acute Liver Injury And Mechanism Study

Objective:In this study,we used carbon tetrachloride（CCl₄）-induced acute liver injury（ALI）mouse model to explore the the protective effect ofα-hederin on CCl₄-induced acute liver injury model in mice and its mechanism,providing theoretical basis for the clinical application ofα-hederin.Methods:Fifty male ICR mice were randomly divided into five groups:the normal group（Con）,the model group（CCl₄）,theα-hederin-L(40 mg·kg^-1)group,theα-hederin-H(80 mg·kg^-1)group,the BIF positive control group(150 mg·kg^-1).After adaptive feeding,mice were subcutaneously injected with the corresponding dose ofα-hederin for protection.24 hours after the last administration,the mice were received a single intraperitoneal injection of 0.2%CCl₄solution（dissolved in olive oil）at 10 m L·kg^-1to establish the ALI model.Blood was collected and liver was extracted for subsequent experiments after death.The activities of ALT,AST,SOD,GSH-Px and the content of MDA in serum or liver tissues were determined by biochemical method.The histopathological changes of liver were analyzed by HE staining.Inflammatory cytokines IL-1β,TNF-αand IL-6 levels in serum were detected by ELISA.Detecting the expression levels of NLRP3,ASC and caspase-1 proteins in mouse liver by Western Blot.Detecting the expression of NLRP3 and MPO in mouse liver tissue by IHC-P.LPS-induced RAW264.7 cell inflammatory model was constructed in vitro to verify whetherα-hederin play an anti-inflammatory and hepatoprotective role by inhibiting the activation of NLRP3 inflammasome.MTT assay was used to detect the effects of LPS(0,0.5,1.0,2.0,4.0μg·m L^-1)andα-hederin(0,0.2,0.4,0.8,1.6μmol·L^-1)on RAW264.7 macrophage viability.Determine the appropriate range of modeling and dosage concentration.Detecting the secretion of IL-1βand IL-18 in supersuperant of RAW264.7 cells by ELISA,and an appropriate dose was selected to establish LPS induced RAW264.7 macrophage inflammatory model.Detecting the effect ofα-hederin on NLRP3-related protein expression in RAW264.7 inflammatory cells by Western Blot.Results:（1）The serum liver function indexes:In model group,ALT and AST levels were significantly increased（P<0.01）.Indicating that carbon tetrachloride（CCl₄）-induced acute liver injury（ALI）mouse model has been successfully constructed.Inα-hederin or BIF pretreatment group,ALT and AST levels were significantly decreased（P<0.01）in a dose-dependent manner.It was suggested thatα-hederin has protective effect on ALI induced by CCl₄.（2）HE staining showed:The liver tissue structure in the model group was chaotic,the intercellular space was enlarged,and inflammatory cell infiltration appeared around the central hepatic vein.The morphology of liver cells of mice pretreated with low and high doses ofα-hederin or BIF was restored to different degrees,the area of inflammatory infiltration was significantly reduced,and the liver tissue structure gradually became normal.The histopathological analysis results confirmed the protective effect ofα-hederin on liver.（3）Oxidative stress parameters of mouse liver tissue showed that:MDA content in CCl₄group increased significantly,SOD and GSH-Px activity decreased significantly（P<0.01）.However,MDA content in liver tissue of mice pretreated with low-dose and high-doseα-hederin or BIF was decreased significantly,SOD and GSH-Px enzyme activities were recovered（P<0.05,P<0.01）.Suggesting that the resistance ofα-hederin to CCl₄-induced acute liver injury may be related to the alleviation of oxidative stress level during liver injury.（4）The results of inflammatory factors showed that low-dose and high-doseα-hederin or BIF pretreatment significantly inhibited the increase of serum IL-1β,IL-6and TNF-αlevels in a dose-dependent manner in mice with CCl₄-induced liver injury（P<0.05,P<0.01）.These results suggest thatα-hederin resistance to CCl₄induced acute liver injury may be related to the decreased level of inflammatory response during liver injury.（5）Western Blot of liver tissue showed that the expression levels of NLRP3,ASC and Caspase1 proteins in CCl₄-induced acute liver injury mice increased（P<0.05）,while low-dose and high-doseα-hederin pretreatment decreased the expression levels of NLRP3,ASC and Caspase1 proteins（P<0.05,P<0.01）.These results suggest thatα-hederin may play a protective role in liver by inhibiting the activation of NLRP3 inflammasome and alleviating inflammation.（6）The expression of NLRP3 and MPO protein in mouse liver tissue was detected by immunohistochemical staining.The results showed that the expression levels of NLRP3 and MPO were increased in model group.However,low-dose and high-doseα-hederin pretreatment can reduce NLRP3 and MPO expression levels.These results further confirm thatα-hederin can inhibit inflammation by influencing NLRP3 signaling pathway.（7）The results of MTT assay showed that the cell viability was above 90%when LPS was treated with 1μg·m L^-1,so this concentration was selected as the dose to induce inflammation in RAW264.7 mouse macrophage model.When the concentration ofα-hederin was 0.8μmol·L^-1,the cell viability was above 90%.Therefore,the concentration ofα-hederin was set as 0,0.2 and 0.8μmol·L^-1.Western Blot results showed that the NLRP3 and Caspase1 expression levels were increased in RAW264.7inflammatory cells induced by LPS,whileα-hederin treatment decreased the expression levels of these two proteins（P<0.05,P<0.01）.Suggesting thatα-hederin can inhibit inflammation by inhibiting NLRP3/ASC/Caspase1 signaling pathway.Conclusions:（1）α-hederin may play its role in liver protection by alleviating oxidative stress.（2）α-hederin may play its role in liver protection by reducing inflammatory response.（3）α-hederinrelieveinflammationbynegativelyregulating NLRP3/ASC/Caspase1 signaling pathway,thus exerting its protective effect on liver.