Investigating The Mechanisms Of AhR Activation On The Development Of NAFLD Based On Estrogen Signaling Pathway

Nonalcoholic fatty liver disease(NAFLD)is a hepatic complication of the metabolic syndrome.Meanwhile,NAFLD is a continuous liver abnormality from non-alcoholic fatty liver(NAFL)to non-alcoholic steatohepatitis(NASH),which has a variable course may eventually lead to cirrhosis and liver cancer.As NAFLD has an increasing impact on world health,researchers have conducted more in-depth research on NAFL and its more advanced stage,NASH.In 2019,according to statistics,NAFLD affects approximately 25% of the global adult population.This rising disease prevalence will increase the economic burden and be accompanied by both an increasing number of patients with nonalcoholic hepatitis and cirrhosis and an alarming increase in hepatocellular carcinoma.Premenopausal women are protected from the liver metabolic complications of obesity compared with body mass index(BMI)-matched men.This protection may be related to estrogen's ability to limit liver fat accumulation.Aryl hydrocarbon receptor(AhR),a novel regulator of NAFLD,may be an important target for regulating estrogen homeostasis and pathways.The latest research direction indicates that the activation of the AhR pathway is involved in the development of non-alcoholic fatty liver disease.In this study,we used benzo[a]pyrene(BaP),a classic and potent ligand of AhR,to activate AhR pathway causes the expression of important genes involved in hepatic lipid regulation in the estrogen signaling pathway.The steatosis of AhR may be due to the overexpression of the 17?-estradiol(E2)metabolic enzyme CYP1A1,which may cause changes in the body 's estrogen homeostasis,or it may disrupt the estrogen receptor alpha in the estrogen signaling pathway,thereby affecting NAFLD Pathogenesis.In this study,a two-part experiment mainly discusses the role of activated AhR in the pathogenesis of NAFLD and related mechanisms based on estrogen signaling pathway from both in vivo and in vitro.The results of this study may help establish AhR as a novel therapeutic and preventive target for fatty liver disease.1.AhR-dependent induction of CYP450 1A1 enzyme inhibits the effect of estrogen on the development of non-alcoholic fatty liver diseaseObjective: Through reviewing the literature and analyzing the literature,the first part of the experiment was to investigate AhR-dependent induction of CYP450 1A1 enzyme inhibits the effect of estrogen on the development of non-alcoholic fatty liver disease through in vivo and in vitro experiments.Method: In vivo experiments: The NAFLD model of C57BL/6 female mice induced by high-fat diet was the research object.First,the role of estrogen in non-alcoholic fatty liver disease was studied.Bilateral oophorectomy(OVX)was performed on female mice to simulate the course of NAFLD in estrogen deficiency,and 17?-estradiol(ie,17?-estradiol was implanted continuously to verify the role of estradiol in NAFLD.Second,we used benzopyrene(BaP),a classic and effective AhR ligand,to activate the AhR pathway in a high-fat diet to investigate the effects of estrogen in the body on liver lipid regulation.In vitro experiments: To induce excessive lipid accumulation in an in vitro hepatocyte steatosis model,human liver cancer cells(HepG2 cells)were treated with oleic acid(OA)as an in vitro cell model of NAFLD.In this experiment,an E2 bovine serum albumin solution(1 ?M)was first added to the E2 treatment group to verify that estradiol has the effect of reducing lipid accumulation and improving hepatocyte steatosis in NAFLD in vitro.Secondly,BaP(5 ?M)solution was added to another E2 treatment group to observe whether the activation of AhR inhibited the effect of estradiol on steatosis of hepatocytes in vitro.Results: The activation of AhR induces the over-expression of the downstream target gene CYP1A1 and inhibits the protective effect of 17?-estradiol on liver steatosis,which is characterized by the accumulation of triglycerides,and liver injury markers ALT and AST significantly increase.Compared with the control group,the expression of adipogenic genes involved in liver lipid metabolism of sterol regulatory element binding protein-1(SREBP-1c)was increased.In addition,m RNA and protein levels of peroxisome proliferator-activated receptor alpha(PPAR?)were significantly reduced,which is related to fatty acid oxidation.Conclusion: Our results indicate that the steatosis of AhR is caused by the overexpression of the E2 metabolic enzyme CYP1A1,which reduces the level of estrogen,leading to the inhibition of fatty acid oxidation and liver triglyceride output.The results of this study may help establish AhR as a novel therapeutic and preventive target for fatty liver disease.2.Role of AhR-mediated ER? expression in non-alcoholic fatty liver diseaseObjective: The second part of the experiment was to verify the effect of AhR activation on ER? expression in non-alcoholic fatty liver disease through in vivo and in vitro experiments.Method: In vivo experiments: We still use benzopyrene(BaP),a classic and most effective AhR ligand to activate the AhR pathway,and have studied the effect of AhR pathway activation on estrogen receptor alpha in female livers,disrupting participation in nonalcohol estrogen signaling pathway in fatty liver disease.Mice were divided into the following 4 groups(n = 10 / group):(1)LFD group,mice on a Low-fat diet;(2)LFD + BaP group,a Low-fat diet containing BaP(12.5mg / kg / day)Mice;(3)HFD group,high fat diet mice;(4)HFD + BaP,high fat diet mice containing BaP(12.5mg /kg / day).To determine whether the AhR pathway can be activated in the liver,we examined the expression of the classic target gene CYP1A1 of the AhR pathway after exposure to BaP.RT-PCR and western blot analysis showed that CYP1A1 m RNA and protein expression increased in a dose-dependent manner after BaP treatment.Meanwhile,the expression of estrogen receptor alpha protein and m RNA level in the liver were measured.In vitro experiments: RT-PCR and western blot were used to analyze the expression of CYP1A1 and ER? in human liver cancer cells(HepG2) exposed to BaP.Meanwhile,an in vitro model of non-alcoholic fatty liver accumulation was formed in HepG2 treated with oleic acid,which was exposed to BaP and estradiol(E2)and co-exposed to BaP and E2,respectively.To investigate the role of AhR-ER? pathway in non-alcoholic fatty liver in vitro.Results: AhR pathway activation reduces ER? protein levels in liver and HepG2 cells and inhibits the estrogen signaling pathway,thereby promoting lipid accumulation in female rat liver and Hep G2 cells.Our results provide insights into the mechanism and function of the AhR-ER? pathway and its impact on NAFLD progression.Conclusion: AhR activation is involved in steatosis due to reduced levels of the estrogen receptor,which blocks the estrogen signaling pathway.Inhibition of fatty acid oxidation and inhibition of liver triglyceride output.The results of this study further explain that the AhR-ER? pathway is involved in the development of non-alcoholic fatty liver disease.