| Background: Drinking is an indispensable part of the cultural development of many countries,and most people think that a small drink is a pleasure,and a large drink hurts the body.However,in the latest research report,it is shown that there is no so-called safe drinking amount for drinking,and a small amount of alcohol is still harmful to the body.The liver is an important place related to alcohol metabolism,and it is also extremely vulnerable to alcohol attacks,causing alcoholic liver damage.Alcoholic Fatty Liver(AFL)is the early stage of Alcoholic Liver Disease(ALD),and it is also a reversible period,which is of great significance to the prevention and treatment of ALD.Clinically,AFL patients are treated comprehensively,mainly through alcohol withdrawal,dietary regulation,and anti-inflammatory and liver-protecting drugs.In severe cases,glucocorticoids are needed for treatment.Therefore,it is necessary to explore new effective drugs with low side effects to treat alcoholic fatty liver disease.Traditional Chinese medicine has a long history in the treatment of AFL and its curative effect is obvious.The therapeutic effect of Chinese medicine and its active ingredients on AFL and its effect are of great significance to the prevention and treatment of AFL.Hesperetin is the main active ingredient in the fruits of citrus plants in the Rutaceae family.Modern pharmacological studies have shown that hesperetin and its derivatives have various pharmacological effects such as anti-inflammatory,antioxidant and blood lipid regulation.In the preliminary research of our laboratory,it was found that4-fluorobenzyl hesperetin derivatives(4-FH)has strong antioxidant pharmacological activity and small toxic side effects.Objective: This study uses mouse alcoholic fatty liver model and in vitro cell model to explore the protective effect of hesperetin on AFL and its partial mechanism Experimental materials and methods: The research of this subject is mainly divided into the following five parts of experiments:1.The effection of 4-FH on alcohol-induced alcoholic fatty liver injury(1)In vitro : AML-12 mouse hepatocytes were used as experimental objects,and the cells were stimulated by alcohol to simulate an in vitro AFL model.The CCK8 method was used to explore the cytotoxic effects of 4-FH and alcohol on AML-12.(2)In vivo : First,the Gao-Binge modeling method was used to construct a mouse alcoholic fatty liver model,and the administration was performed by oral gavage.C57BL/6J male mice(about 20-22g)were randomly divided into normal group,normal plus high-dose 4-FH group(200 mg/kg),model plus low-dose 4-FH group(50 mg/kg),model The middle-dose 4-FH group(100 mg/kg),the model plus the high-dose 4-FH group(200 mg/kg),and the model plus silybum marianum group(100 mg/kg),each with 6 animals.Through liver-to-body ratio,pathology(HE staining,oil red staining),serology(ALT/AST/TG/TCH content determination),the effect of 4-FH on reducing alcoholic fatty liver injury in mice was detected.2.The effection of 4-FH on alcohol-induced lipid metabolism disorders(1)In vitro : The effects of 4-FH on the expression levels of lipid metabolism-related indexes SREBP1c/SREBP2/FASN and HMGCR were detected by cell oil red,western blotting,and real-time fluorescent quantitative PCR.(2)In vivo : Western bolt and Real-time PCR experiments were used to detect the changes in the expression levels of lipid metabolism-related indicators in each group of mice.3.The effection of 4-FH on the level of alcohol-induced oxidative stress(1)In vitro : Use DCF fluorescent staining to detect intracellular reactive oxygen species(ROS)determination,to detect the effect of 4-FH on alcohol-induced oxidative stress,and to detect superoxide levels by DHE staining for further verification,and LDH and MDA kits were used to detect the level of lipid oxidation in cells.(2)In vivo : GSH/CAT/SOD and MDA kits were used to detect alcohol-induced ROS,oxidative stress levels,and free radical peroxidation in mice in each group.4.Explore the effect of 4-FH on alcohol-induced hepatocyte apoptosis(1)In vitro experiment: Use flow cytometry to detect the effect of 4-FH on alcohol-induced hepatocyte apoptosis,and use Western bolt experiment to detect changes in the expression level of apoptosis-related proteins.(2)In vivo experiment: TUNEL staining was used to detect the apoptosis of liver cells in mouse liver tissues,and Western bolt experiment was used to detect changes in the expression levels of apoptosis-related proteins.5.Explore the influence of 4-FH on the expression of SIRT1(1)In vivo experiment: The expression of SIRT1 in the liver tissue of each group of mice was detected by immunofluorescence staining,and the expression of SIRT1 in the primary hepatocytes of each group of mice was detected by Western blotting and real-time quantitative PCR.(2)In vitro experiment: observe whether SIRT1 affects the efficacy of 4-FH by using si RNA knockdown or plasmid overexpression SIRT1 in vitro6.Explored whether 4-FH is involved in regulating lipid metabolism of alcoholic fatty liver through the SIRT-1-AMPK signaling pathway(1)In vitro : Use Western bolt experiment to explore the expression of phosphorylated AMPK protein in alcoholic fatty liver,and the effect of 4-FH on AMPK phosphorylation level after knocking down or overexpressing SIRT1.(2)In vivo : Western blotting was used to detect the effect of 4-FH on AMPK phosphorylation after knocking down or overexpressing SIRT1.Results:1.4-fluorobenzyl hesperetin derivatives reduces liver damage caused by alcoholic fatty liver: HE and oil red staining results show that compared with the model group,the 4-fluorobenzyl hesperetin derivatives administration model group has fewer fatty vacuoles and lower lipid droplets;liver damage indicators and fat The accumulation index,namely,the level of serological ALT/AST/TG/TCHO was lower than that of the model group.2.4-fluorobenzyl hesperetin reduces the lipid metabolism disorder of alcoholic fatty liver: In the 4-fluorobenzyl hesperetin administration model group,the lipid metabolism index SREBP1c/SREBP2/FASN and the m RNA level and protein level of HMGCR are lower than those in the model group.The results of cell oil red test are consistent with the results of tissue oil red.3.4-fluorobenzyl hesperetin reduces the level of oxidative stress in alcoholic fatty liver:It is found that the content of MDA in the model increases,but after 4-fluorobenzyl hesperetin treatment,it shows a dose-dependent decrease,while the content of SOD is the opposite;The test results also show that 4-fluorobenzyl hesperetin can significantly reduce the level of ROS produced by alcohol.4.4-fluorobenzyl hesperetin reduces alcohol-induced hepatocyte apoptosis: It can be seen from the results of TUNEL staining that after 4-fluorobenzyl hesperetin treatment,hepatocyte apoptosis in the model group is reduced,and the flow cytometry results of AML-12 cells also reflect Apoptosis decreased in 4-fluorobenzyl hesperetin derivatives model administration group;apoptosis-related protein5.4-fluorobenzyl hesperetin may participate in the lipid metabolism process of alcoholic fatty liver through the SIRT1-AMPK signaling pathway:After 4-fluorobenzyl hesperetin treatment,the expression of SIRT1 in mice increased.Overexpression of SIRT1 can enhance the effect of 4-fluorobenzyl hesperetin,while knockdown can weaken it;4-fluorobenzyl hesperetin derivatives can increase AMPK phosphorylation.Conclusion: 4-fluorobenzyl hesperetin reduce oxidative stress levels in alcoholic fatty liver,inhibit hepatocyte apoptosis and reduce lipid metabolism disorders through SIRT1-AMPK signaling pathway... |
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