Study On The Mechanism Of Apigenin Alleviates Liver Injury Induced By High Fat Diet Through Activating Autophagy

With the improvement of living standards,people’s dietary patterns are developing in the direction of low fat and low calories.Studies have shown that long-term excessive intake of high-fat diet（HFD）could damage human health and induce a series of liver injuries such as hepatocyte lipid accumulation,steatosis,inflammation and fibrosis.Liver injury induced by HFD involves multiple death modes,including pyroptosis and ferroptosis.Pyroptosis is a form of inflammatory cell death in which HFD activates the NOD-like receptor family pyrin domain containing 3（NLRP3）inflammasome to cause the release of cellular contents,ultimately leading to liver injury.Ferroptosis is a newly discovered mode of cell death characterized by iron-dependent accumulation of lipid peroxidation to lethal levels.HFD increases lipid accumulation and lipid peroxidation,further accelerating cellular ferroptosis,and ultimately leading to liver injury.Therefore,it is of great significance to explore intervention methods for liver injury caused by HFD based on hepatocyte pyroptosis and ferroptosis.Autophagy is a lysosome-dependent degradation pathway that is generally considered a pro-survival mechanism of cells in response to various stresses and is involved in the process of hepatocyte injury and death.Apigenin is a natural flavonoid found in fruits and vegetables,has been shown to have a protective effect on the liver.However,there are no studies investigating the protective effect of apigenin on HFD-induced hepatocyte pyroptosis and ferroptosis.Therefore,this thesis uses HFD feeding to construct a high-fat mouse model,uses palmitic acid（PA）to treat AML12 cells and Hep G2 cells to construct a high-fat cell model,and studies the role and mechanisms of apigenin alleviates HFD-induced liver injury by activating autophagy.The main study contents and results are as follows:（1）Apigenin alleviated HFD-induced hepatocyte lipid accumulation and injury through activation of autophagy（1）To study the effect of apigenin on liver lipid accumulation and injury induced by HFD in C57BL/6 mice,histopathological（H&E）staining,Oil red O staining,Masson staining,triglyceride（TG）,total cholesterol（TC）,aspartate aminotransferase（AST）and alanine aminotransferase（ALT）levels of mice were detected.The results showed that the livers of mice in the HFD group showed significant lipid accumulation and typical liver injury such as inflammation and fibrosis.At the same time,the levels of TC,TG,AST and ALT in the liver tissue of mice also significantly increased.However,after apigenin intervention,the above-mentioned liver lipid accumulation and injury phenomena were significantly alleviated,and the levels of TC,TG,AST and ALT in liver tissue were also significantly reduced.The above results indicated that apigenin alleviated liver lipid accumulation and injury in HFD-induced mice.（2）To study the effect of apigenin-activated autophagy on lipid accumulation and injury in HFD-induced mice,we first observed the number of autophagosomes and also detected the protein expression of LC3 and P62 in C57BL/6 mice.The results showed that the HFD group showed an increase in the number of autophagosomes and the protein levels of LC3 and P62,indicating autophagy inhibition in the hepatocytes of HFD-fed mice.However,apigenin intervention further increased the protein expression level of LC3,reduced the protein expression level of P62,and activated hepatocyte autophagy inhibited by HFD.Next,lipid accumulation and injury in mouse livers were detected after adding the autophagy inhibitor chloroquine（CQ）.The results showed that CQ treatment reversed the effect of apigenin in alleviating lipid accumulation and liver injury,increasing the number of lipid droplets and the levels of TC and TG in mice.At the same time,H&E staining results showed typical liver injury conditions such as large amounts of inflammatory cell infiltration and a small amount of nuclear shrinkage,accompanied by increased AST and ALT levels.The above results indicated that apigenin alleviated hepatic lipid accumulation and injury in HFD-induced mice by activating autophagy.（3）To study the effect of apigenin-activated autophagy on lipid accumulation and injury in PA-treated AML12 cells and Hep G2 cells,we first observed the number of autophagosomes and the protein expression of LC3 and P62 in AML12 cells and Hep G2 cells.The results showed that the number of autophagosomes and the protein expression levels of LC3 and P62 were increased in PA-treated AML12 cells and Hep G2 cells.However,the intervention of apigenin further increased the protein expression level of LC3 and reduced the protein expression level of P62,thus activating PA-inhibited autophagy.We next added CQ to detect lipid accumulation in AML12 cells and Hep G2 cells.The results showed that CQ treatment reversed the lipid-lowering effect of apigenin.Through Oil Red O staining,it was observed that the number of intracellular lipid droplets increased,and the levels of TC and TG increased after CQ treatment.The above results indicated that apigenin alleviated lipid accumulation and injury in PA-induced hepatocytes by activating autophagy.（2）The effect and mechanism of apigenin in alleviating HFD-induced hepatocyte pyroptosis（1）To study the effect of apigenin on HFD-induced hepatocyte pyroptosis,the release of lactate dehydrogenase（LDH）,interleukin 1β（IL-1β）and interleukin 18（IL-18）,the protein expression levels of NLRP3,cleaved-caspase-1,N-terminal domain of GSDMD（GSDMD-N）,IL-1βand IL-18 were detected.The results showed that the release of LDH,IL-1βand IL-18 were increased,and the expression levels of pyroptosis marker proteins were also increased in HFD group and PA-treated AML12cells.However,apigenin intervention significantly improved the above abnormal indicators,reduced the release of LDH,IL-1βand IL-18,and reduced the expression levels of pyroptosis marker proteins and m RNA.The above results indicated that apigenin could alleviate HFD-induced hepatocyte pyroptosis.（2）To study the mechanism of apigenin on HFD-induced hepatocyte pyroptosis,the effect of apigenin on the mitophagy-reactive oxygen species（ROS）-lysosomal membrane permeabilization（LMP）pathway was first examined.Next,the protein levels of PTEN induced putative kinase1（PINK1）,Parkinson’s disease gene 2（Parkin）,lysosomal associated membrane protein 1（LAMP1）and cathepsin B（CTSB）in C57BL/6 mice and AML12 cells were analyzed.The accumulation of total ROS and mitochondrial ROS（mt ROS）were observed along with acridine orange（AO）staining and Lyso Tracker Red staining.The results showed that in HFD group and PA group of AML12 cells the protein expression levels of PINK1,Parkin and LAMP1were decreased,the release of ROS,mt ROS and CTSB was increased,the fluorescence intensity of AO and Lyso Tracker Red were decreased.However,the above abnormal changes were significantly improved after apigenin intervention.Subsequently,we used LC3-si RNA,mitophagy inhibitor cyclosporin A（Cs A）,ROS scavenger N-Acetyl-L-cysteine ethyl ester（NAC）,CTSB inhibitor CA-074 methyl ester（CA-074 Me）and NLRP3 inhibitor MCC950 to detect pyroptosis indicators and NLRP3 and CTSB co-localization staining.The results showed that LC3-si RNA and Cs A weakened the effect of apigenin in alleviating hepatocyte pyroptosis to a certain extent,while NAC,CA-074 Me and MCC950 promoted the effect of apigenin in alleviating hepatocyte pyroptosis.The above results indicated that apigenin alleviated HFD-induced hepatocyte pyroptosis through the mitophagy-ROS-LMP pathway.（3）The effect and mechanism of apigenin on HFD-induced hepatocyte ferroptosis（1）To study the effect of apigenin on HFD-induced hepatocyte ferroptosis,we examined the Lillie staining,mitochondrial changes,intracellular iron content,the levels of malondialdehyde（MDA）,glutathione（GSH）and the protein levels of glutathione peroxidase 4（GPX4）,ferritin heavy chain（FTH）,ferroportin（FPN）,longchain acyl-coa synthetase 4（ACSL4）and transferrin receptor 1（Tf R1）in C57BL/6 mice and AML12 cells.The results showed that in HFD group and PA-treated AML12 cells,the levels of MDA,ROS,iron content and the protein levels of ACSL4 and Tf R1 were elevated,the levels of GSH and the protein levels of GPX4,FTH and FPN were decreased,while apigenin intervention significantly alleviated the above abnormal indicators.The above results indicated that apigenin could alleviate HFD-induced hepatocyte ferroptosis.（2）To study the mechanism of apigenin on HFD-induced hepatocyte ferroptosis,we detected the ferroptosis index and PINK1 and FTH co-localization by adding LC3-si RNA,Cs A,NAC,iron ion chelator DFO and NH₄Cl.The results showed that LC3-si RNA and Cs A could weaken the effect of apigenin in alleviating hepatocyte ferroptosis,while NAC,DFO and NH₄Cl further promoted the alleviating effect of apigenin on hepatocyte ferroptosis.The above results indicated that apigenin alleviated HFD-induced hepatocyte ferroptosis through the mitophagy-ROS-LMP pathway.In summary,this thesis illustrates the role of apigenin in activating autophagy to alleviate HFD-induced liver injury.The specific mechanism is that apigenin alleviates HFD-induced hepatocyte lipid accumulation,pyroptosis and ferroptosis through activating the mitophagy-ROS-LMP pathway,and thus alleviates liver injury.Our study provides new ideas for the study of hepatoprotective effects of apigenin,which is of great significance for safeguarding human health.