| Alcohol consumption is deep-rooted in cultures,social customs and even religions in many countries.However,the global status report on alcohol and health released by the World Health Organization(WHO)states that about 3.3 million individuals die annually due to the abuse of alcohol,accounting for 5.3 % of global deaths.The report also reveals that the per capita alcohol consumption worldwide is declining except in China,where the alcohol consumption keeps increasing;posing a great threat to public health.It also specifies that about 40,000 liver disease patients seek clinical help annually in the Beijing 302 Hospital;recently,however,the distribution of liver disease types among those patients changes a lot,with the proportion of alcoholic liver diseases(ALD)more than doubling.Therefore,prevention of ALD is of great importance.ALD is a form of liver injury induced by alcohol abuse and according to pathomorphology can be termed into 4 forms: alcoholic fatty liver(AFL),alcoholic hepatitis(AH),alcoholic hepatic fibrosis and alcoholic cirrhosis(AC).These forms can coexist or exist separately,and can even further develop into hepatocellular carcinoma(HCC).AFL is the early form of ALD and usually manifested as lipid synthesis abnormity in hepatocytes induced by enzyme activity alteration due to long-term alcohol exposure;if not treated promptly,the AFL can develop into AC withing 10-15 years,which is usually not reversible.Sterol regulatory element-binding proteins(SREBPs)acts as a transcription factor regulating lipid biosynthesis and plays a vital role in lipid metabolism in the liver.The SREBP family consists of 2 members: SREBP1 and SREBP2.SREBP1 can further be subdivided into SREBP1 a and SREBP1c which is the main form in organisms and regulates the fatty acid synthesis,while SREBP2 can regulate the cholesterol metabolism.Therefore,in this study,SREBPs were used as observed indicator variable to investigate the regulation mechanism of AFL,in order to find the effective target and treatment of AFL and block the ALD at the early stage of steatosis.A review of various studies found that β-arrestin 2(Arrb2)contributed to the progression of various diseases such as liver fibrosis and leukemia,and played an important role in metabolic-related diseases such as type 2 diabetes.It is well documented that multifunctional adaptor β-arrestin 2 modulates cell apoptosis and it may have contrary effect in different diseases.For instance,β-arrestin 2 promotes hepatocyte apoptosis in bile duct ligation(BDL)while blocking hepatic stellate cells(HSCs)apoptosis in liver fibrosis.However,the effect of β-arrestin 2 in the AFL has not yet been determined.This study explores the role and mechanism of β-arrestin 2 in regulating lipid metabolism in AFL,in order to find vital targets to treat AFL.This study first adopted the Gao-binge modeling to establish the AFL mice model and extracted primary hepatocytes by perfusion in situ;then immunohistochemistry,RT-q PCR and Western blot were used to analyze the m RNA level and protein level of β-arrestin 2 in the mouse livers and primary hepatocytes.The results showed that the m RNA level and protein level of β-arrestin 2 were upregulated in the Et OH-fed group.In order to explore the effect of β-arrestin 2 in regulating lipid metabolism in AFL,recombinant adeno-associated virus was administrated into mice by intravenous tail injection to silence the β-arrestin 2 expression in the livers.The result showed that the serum levels of ALT,AST,TG and T-CHO all increased in the Et OH-fed group;after the β-arrestin 2 expression was silenced,these biochemical indicators decreased accordingly.The H & E and oil red O staining showed that in the Et OH-fed group,fat vacuoles appeared in the mouse hepatocytes and the number of lipid droplets increased;after the β-arrestin 2 expression was silenced,the severity of liver injury and steatosis both extenuated.The RT-q PCR and Western blot were used to examine the m RNA level and protein level of SREBP1 c and its downstream target gene FASN,SREBP2 and its downstream target gene HMGCR as well as inflammatory factors including TNF-α and IL-6.The result showed that β-arrestin 2 could lead to hepatic lipidosis and resulting in hepatic lipid metabolism disorder.In addition,immortalized mouse AML-12 cell lines were used in vitro for silencing and overexpression of β-arrestin 2,and the results were consistent with the above in vivo studies.Silencing β-arrestin 2 alleviated lipid metabolic disorders in hepatocytes,while overexpression β-arrestin 2 further exacerbated lipid deposition in hepatocytes.To further investigate the molecular mechanism of β-arrestin 2 in regulating lipid metabolism,this study adopted ethanol-exposed AML-12 cell lines in vitro,in which β-arrestin 2 was silenced or overexpressed respectively to detect the expression of AMPKα1 and p-AMPKα1.The result indicated that β-arrestin 2 inhibited AMPK pathway activation.Moreover,GSK621,a specific activator of AMPK pathway,was used in this study to reversely detect β-arrestin 2 and SREBPs levels that reflected lipid synthesis,and the result was consistent with previous result,that is,the activated AMPK signaling pathway could significantly decrease β-arrestin 2 and SREBPs levels.These results revealed that β-arrestin 2 could inhibit AMPK pathway during AFL progression and accelerate fatty acid and cholesterol synthesis,leading to hepatic lipidosis and lipid metabolism disorders.In addition,after silencing β-arrestin 2 with adeno-associated virus in mice,LC-MS technology was used to screen and identify small lipid metabolites in mouse liver tissues;a total of 40 differential metabolites were screened out and their involved metabolic pathways were specifically analyzed.Whether β-arrestin 2 could cause changes in lipids and its metabolites and thereby leaded to metabolic disorder was analyzed,in order to further elucidate the mechanism of β-arrestin 2 in regulating liver lipid metabolism and explore corresponding lipid metabolite changes after being regulated by β-arrestin 2.It was found that β-arrestin 2 could regulate metabolism of multiple lipids,but the lipid metabolites of most significant difference were primary bile acids.The levels of primary bile acids(cholic acid,taurocholic acid and glycocholic acid)in the Et OH-fed group were increased;however,after β-arrestin 2 was silenced,their levels decreased significantly,suggesting that β-arrestin 2 lead to excessive lipidosis in the alcoholic fatty liver,possibly due to abnormal rise of primary bile acids levels.It is worth noting that primary bile acids in the liver were mainly derived from cholesterol metabolism.Therefore,we speculate that β-arrestin 2 could cause excessive cholesterol accumulation in liver cells,which increases the level of primary bile acids;the excessive primary bile acid in turn negatively regulates HMGCR expression,inhibits cholesterol synthesis,and alleviates the lipid metabolism disorder caused by β-arrestin2.In summary,by inhibiting AMPK pathway activation in AFL,β-arrestin 2 on one hand increased the fatty acid synthesis;on the other hand,it increased the cholesterol synthesis,leading to abnormal rise of primary bile acid levels;these two effects together contribute to lipid accumulation in hepatocytes,leading to the hepatic lipid metabolism disorder.In addition,there existed a negative feedback loop: the excessive bile acid in turn negatively regulates HMGCR expression,inhibits cholesterol synthesis,and reduced the lipid metabolism disorder caused by β-arrestin 2. |
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