| ObjectiveAlcoholic liver disease(ALD)was a progressive liver disease caused by excessive ethanol consumption which ranges from alcoholic fatty liver(AFL),alcoholic hepatitis,alcoholic fibrosis,and even hepatocellular carcinoma.AFL was the first stage of alcohol induced liver injury,and has been the focus of ALD investigation due to the reversibility.The mechanism of AFL was complex,which may involve numbers of metabolic activativities and signaling pathways.Oxidative stress has been recognized to play an extremely important role in the pathogenesis of AFL.Cytochrome P450 2E1(CYP2E1)is a member of microsomal ethanol oxidation system(MEOS),and has been demonstrated to be the main source of reactive oxygen species(ROS)induced by ethanol in liver.Mitogen-activated protein kinase(MAPK)is a kind of serine/threonine protein kinase widely existing in eukaryotic cells.MAPK,which can be activated under stress conditions such as ROS-induced oxidative stress,plays important roles in many physiological and pathological activities such as cell proliferation,apoptosis and inflammatory response.Multiple studies had demonstrated that MAPK signaling pathway can regulate lipid metabolism in cell.Then,how does the phosphorylation level of MAPKs change under chronic alcoholic fatty liver model?Are these changes related to CYP2E1?Does MAPK play important roles in the pathogenesis of AFL?These questions are urgently needed to be answered.In this study,chronic AFL mice model was established by feeding mice with Liber-Decarli liquid diet,and the protein level of MAPKs,CYP2E1 and fat metabolizing proteins such as sterol regulatory element binding protein-1c(SREBP-lc)and peroxisomal proliferator activator receptor a(PPAR-a)serially were detected,epidermal growth factor(EGF),was used to investigate whether MAPK activation could alleviate chronic ethanol-induced fatty accumulation in mice liver.In vitro,we measured changes of phoporylation leveal of MAPKs in HepG2 after exposure to ethanol in different concentration and time.Then we used CYP2E1-HepG2 to explore the relationship between CYP2E1 and the changes of phosphorylated MAPKs under chronic ethanol exposure.Finally,we used MKP-1 siRNA interferencing HepG2 to phosphorylate MAPKs,testing protein level of PPAR-a and SREBP-1c.These results may be helpful to reveal the potential roles of MAPKs in the pathogenesis of AFL.Methods1.Animal experiment1.1 The model of chronic AFL:ICR male mice(n=40)were divided into the control group and model group of 1W,2W,3W and 4W randomly.The liquid diet for model group was contained with 5%(m/v)ethanol.At the end of every week 10 mice of each group were sacrificed colleting liver and epididymis fat.Level of liver triglyceride(TG)and protein level of MAPKs,SREBP-1c,PPAR-α were measured.1.2 Intervention of epidermal growth factor(EGF)to chronic AFL:ICR male mice(n=30)were randomly divided into the group of control,model and EGF.Chronic AFL model was constructed with Lieber-DeCarli liquid diet.Beside the chronic alcohol feeding,mice of EGF group were given EGF in a dose of 250μg/kg · bw by intraperitoneal injection everyday from first weekend to the end of feeding.After mice sacrificed,liver were collected for pathological examination and TG determination.2.Cell experiment2.1 Ethanol exposure of HepG2:HepG2 were treated with ethanol in 0mM,100mM and 200mM in 1h,1d or 5d.Cells were collected at the end of experiment for a measurement of MAPKs expression level by western blot.2.2 The effect of CYP2E1 on phosphorylation level of MAPKs in HepG2:CYP2E1-HepG2 and nature control NC-HepG2 were treated with 200mM ethanol for 1d or 5d,the OmM control groups were also established at the same time.Cells were collected at the end of experiment for a measurement of protein level of MAPKs.2.3 The effect of MKP-1 siRNA intervention to HepG2 on lipid metabolism in vitro:After the transfection with MKP-1 siRNA,HepG2 were collected for a measurement of protein level of SREBP-lc,PPAR-a and the level of MKP-1 mRNA by qRT-PCR.Results1.Manifestation of liver in chronic sequentially AFL model:Compared with control group,the mice from model group got higher TG level(P<0.05).In pathological section,alcohol induced liver injury is aggravated with the progress of AFL,manifesting massive fat accumulation in liver.2.Changes of MAPKs phosphorylated level in chronic sequentially AFL model:Compared with control group,p-ERK were decreased respectively in 1W,2W and 4W by 22.31%,48.45%and 21.81%(P<0.05),p-p38 were decreased respectively in 2W and 4W by 28.19%and 42.00%(P<0.05),p-JNK were decreased by 42.60%and 59.74%respectively in 1W and 2W(P<0.05).The level of MAPKs total proteinbetween control and model showed little difference.3.Changes of CYP2E1 in chronic sequentially AFL model:The level of CYP2E1 in model mice was dramatically higher than control group at all time point.CYP2E1 was increased by 110.22%,62.27%,100.85%and 64.93%respectively in 1W,2W,3W and 4W(P<0.05).4.Changes of PPAR-a and SREBP-lc in chronic sequentially AFL model:Protein level of PPAR-a exhibited a remarkable reduction in model mice compared with control group,at the third week it decreased by 64.36%(P<0.05),while SREBP-lc had little change between two groups.5.Pathological changes in chronic AFL mice intervened with MAPK agonist EGF:Liver injury induced by ethanol was eliminated after EGF injection,with the pathological examination showing a normal cell form of hepatocyte with a few lipid drop accumulation.6.Effect of ethanol on MAPKs phosphorylation in HepG2:The phosphorylation level of MAPKs was all changed under ethanol exposure.p-ERK was activated at the early stage and became inhibited later.Level of p-p38 was decreased in short period and increased later.p-JNK reflected as an activated status in HepG2 exposed to ethanol.7.Changes of MAPKs phosphorylation under the intervention of CYP2E1 in HepG2:With the exposure of ethanol for Id,phosphorylation level of ERK and p38 was all increased significantly in both two cells(P<0.05),protein level of p-JNKwas increased significantly in CYP2E1-HepG2(P<0.05).After ethanol exposure for 5d,protein level of p-ERK in two cells was all decreased signicficantly(P<0.05),p38 was activated in both CYP2E1-HepG2 and NC-HepG2(P<0.05),and phosphorylation level of JNK in CYP2E1-HepG2 was decreased(P<0.05).Protein of p-ERK,p-p38 and p-JNK in CYP2E1-HepG2 were all lower than NC-HepG2 under the same ethanol concerntration.8.The effect of changes about MAPKs activation on HepG2 TG level and lipid metabolized protein:TG level in both two cells were all increased after ethanol exposure,and CYP2E1 can promoted the effect of ethanol on TG accumulation.Level of PPAR-a was inhibited with the decreasing MAPK phosphotylation level.SREBP-1c had not obvious changes under the different phosphorylation level of MAPKs.9.Lipid metabolized protein level changes in HepG2 after transfected with MKP-1 siRNA:Compared with control group,Protein level of PPAR-a was increasing significantly in HepG2 transfected with MKP-1 siRNA,showing an elevation by 67.94%or 82.92%compared with control group(P<0.05).The level of SREBP-lc didn’t have remarkable changes.Conclusions1.Chronic ethanol exposure led to the suppression of MAPKs phosphorylation,which may be related with the activation of CYP2E1.2.The declining activity of MAPKs might contribute to ethanol-induced fatty liver through inhibiting PPAR-a in liver。... |
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