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An Investigation Into The Function And Mechanism Of Lactucopicrin In Reducing Hepatocyte Steatosis By Activating The AMPK Pathway

Posted on:2024-01-05Degree:MasterType:Thesis
Country:ChinaCandidate:H W TanFull Text:PDF
GTID:2544307085973949Subject:Drug Analysis
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Objective:Natural plant development has emerged as a major source of public concern as a result of the prevalence of NAFLD(Nonalcoholic Fatty Liver Disease),which is rising yearly and has become a serious threat to people’s lives and health.This work used FFA(Free Fatty Acid)-induced HepG2 cells to create an in vitro NAFLD model to examine the molecular basis of lactucopicrin’s regulation of lipid metabolism,To provide theoretical support for the development and research of drugs for non-alcoholic fatty liver disease.Methods:(1)The CCK8 method was used to assess the safe dose of lactucopicrin.(2)Lactucopicrin’s ability to reduce intracellular lipid buildup was assessed using Oil Red O staining and TG content testing.(3)By monitoring fatty acid β-oxidase activity,reactive oxygen species level,ATP content,and the level of mitochondrial membrane potential,the improvement effect of lactucopicrin on mitochondrial function was assessed.(4)The regulatory effects of lactucopicrin on coactivator(PGC1α),transcription factors(PPARα 、 PPARγ),and oxidation factors(CPT1A,HADHA)were identified using q RT-PCR and Western Blot.(5)Western Blot was used to detect the effects of Lactucopicrin on P-AMPK and P-m TOR.(6)Dorsomorphin,a specific AMPK inhibitor,was added to verify the changes of AMPK and its downstream proteins by Western Blot.(7)After the addition of Dorsomorphin,oil red O,TG,FAβO,ATP,ROS and JC-1 kits were used to investigate whether the effects of Lactucopicrin on mitochondrial function and lipid accumulation were abolished after AMPK inhibition.Results:(1)0-20μM Lactucopicrin had no significant effect on HepG2 cells.(2)Lactucopicrin significantly reduced the intracellular triglyceride content and decreased intracellular lipid droplets in FFA-induced HepG2 cells.(3)Lactucopicrin significantly increased intracellular fatty acid β-oxidase activity,increased ATP content,decreased reactive oxygen species level and increased mitochondrial membrane potential.(4)Lactucopicrin significantly up-regulated the expression of coactivators,transcription factors,oxidation factors at m RNA level and protein level.(5)Lactucopicrin significantly up-regulated the phosphorylation level of AMPK and inhibited the phosphorylation level of m TOR.(6)The addition of Dorsomorphin inhibited both AMPK and its downstream target proteins and increased the phosphorylation level of m TOR.(7)The protective effect of Lactucopicrin on mitochondria and the amelioration of lipid accumulation were both inhibited by Dorsomorphin.Conclusion:Lactucopicrin are used to improve steatosis and mitochondrial function by activating AMPK and promoting fatty acid beta oxidation.
Keywords/Search Tags:Non-Alcoholic Fatty Liver Disease, Lactucopicrin, HepG2 cells, Fatty acid β-oxidation, AMPK signaling pathway
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