| ALD(alcoholic liver disease,ALD)refers to a series of liver diseases resulting from long-term and improper alcohol taken.The initial stage of ALD is AFLD(alcoholic fatty liver disease,AFLD),more than 90% of alcoholics show hepatic steatosis.AFLD was previously to be neglected because of its hardly clinical symptoms and can be reversed by alcohol withdrawal.However,increasing studies have found that 20%-40% of patients with AFLD will develop into liver fibrosis,8%-20% of these patients with fibrosis will further develop into cirrhosis,and 3%-10% of these patients with cirrhosis will eventually deteriorate into hepatocellular carcinoma.Meanwhile,some studies have shown that lipid metabolism disorder caused by inappropriate alcohol intake may be closely related to the occurrence and development of tumors,and abnormal lipid metabolism may provide a favorable environment for the occurrence of tumors.CYP450 is a superfamily of proteins with iron porphyrin as its auxiliary group,which is the most important phase ? metabolic enzyme participating in drug biotransformation in liver.CYP450 mediates the oxidation,reduction and hydrolysis of various clinical drugs,environmental substances and endogenous substances,including some drugs may cause hepatotoxicity.The efficacy and toxicity of drugs may be influenced by the alteration of CYP450 activity,which may lead to serious adverse drug reaction.Up to now,there are few researches on the alteration of CYP450 s in AFLD,just some reports about CYP2E1,its regulation and role in AFLD.Therefore,To know the expression and activity regulation of seven major CYP450 subtypes in liver with alcoholic fatty liver,and the role and mechanism of CYP450 in alcohol induced lipid metabolism abnormalities provide experimental basis for exploring the pathological mechanism,treatment and prevention of ALD.There are three parts below in our research: 1.The expression and activity alteration of seven major CYP450 enzymes in liver of rats with alcoholic fatty liver disease.Male Sprague-Dwaley rats were categorized into three groups randomly,named normal control(NC)group,sucrose control(SC)group and alcohol model(AM)group.The AM group was fed with alcoholic beverages containing 18% sucrose and different concentration of ethanol(from 5%-40%)during 12 weeks of ethanol were increased by 5%(v/v)for every next week until the eighth week;The SC group was fed with 18% sucrose for 12 weeks;The NC group was fed with normal potable water.CYP1A2,CYP3A4 and CYP2D6 mRNA levels,protein expression and metabolic activity were increased in AFLD rats.CYP2B6 and CYP2C11 mRNA expression were decreased significantly with AFLD,as same as the protein expression and metabolic activity.CYP2C19 has not changed in mRNA,protein and enzyme activity level in AM group among all the enzymes have been tested.The results suggested that alcohol regulated the major CYP450 isforms differently,the alteration of isforms which involved in the metabolism of clinic drugs or related to drug-induced liver injury deserve special attention,such as CYP1A2,CYP3A4.2.The role of CYP1A2 on alcohol-induced lipid metabolism abnormalities.L02 cells were stimulated by alcohol at different concentrations(0,50,100,200,400,800 mM)for 12,24,48 h,The gene and protein expression of CYP1A2 was determinated by Q-PCR and Western-blot,respectively.The results showed that the gene and protein expression of CYP1A2 elevated significantly after 100 mM Ethanol treated in L02 cells,and the expression of NF-?B and TNF-? was also elevated.In order to research the role of CYP1A2 in alcohol-induced abnormal lipid metabolism,CYP1A2 silenced and fluvoxamine maleate(specific inhibitor of CYP1A2)coincubation were performed before alcoholic treated.Then cells were collected and some biochemical indicators in supernatant and homogenate were detected.Under the conditions that CYP1A2 was silenced or inhibited,the liver injury caused by ethanol metabolism was attenuated to a certain extent.The ALT and TG level of siRNA-CYP1A2 group and fluvoxamine maleate group after ethanol stimulation were significant lower than those of control group.The protein expression of SREBP-1c,PTEN,AKT and p-AKT in L02 cells of each group was analyzed,when the CYP1A2 expression was inhibited,the protein level of SREBP-1c was lower in siRNA-CYP1A2 group and fluvoxamine maleate group than that in normal control group after 100 mM ethanol treated.The expression of PTEN decreased in normal control group after ethanol stimulated,while CYP1A2 expression decreased further reduced PTEN expression in transfected cells and inhibitor incubated cells,which suggest the inhibition of CYP1A2 could down-regulate the expression of SREBP-1c to have an impact on lipid metabolism through the regulation of PTEN.3.Partial mechanism of lipid metabolism regulation by CYP1A2 in alcohol treated liver.To explore the regulatory mechanism of CYP1A2 on lipid metabolism in alcohol-induced liver injury and the relationship among CYP1A2,PTEN and SREBP-1c during this process,PTEN was overexpressed in L02 cells by transfected with pcDNA3.1-PTEN,then the transfected cells were stimulated by 100 mM ethanol for 24 h,the protein levels of PTEN,p-AKT,AKT and SREBP-1c in each group were determined.The expression of p-AKT and SREBP-1c significantly decreased when PTEN was overexpressed.The protein level of PTEN in NC group and transfection group decreased,while the level of p-AKT and SREBP-1c increased significantly,suggesting that PTEN may have regulatory effect on SREBP-1c;In order to confirm the impact of PTEN on p-AKT,AKT and SREBP-1c,siRNA-PTEN and bpv(specific inhibitor of PTEN)was used,protein levels of PTEN,p-AKT,AKT and SREBP-1c in each group were detected as well.The results showed that the expression of p-AKT and SREBP-1c raised significantly when PTEN was silenced or inhibited,the protein level of PTEN in each group decreased after ethanol treatment,while the expression of p-AKT and SREBP-1c increased significantly,which indicated that PTEN/AKT pathway may regulate SREBP-1c in liver lipid metabolism in case of alcohol stimulation. |
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