The Prevention And Treatment Effect Of Jujube And Puerarin Against Alcoholic Liver Disease And Its Molecular Mechanism

Objectives：Alcoholic liver disease(ALD) is a liver damage resulted froma long-term heavy drinking. Early ALD showed only fatty liver, and thus candevelop into alcoholic hepatitis, alcoholic liver fibrosis and (alcoholic liver)cirrhosis. The disease is the second-largest liver in China following viralhepatitis.Studies have shown that CYP2E1, TNF-α and Sirt1participate in themetabolic pathways of the body: inflammation, oxidative stress and energyfeelings in three different ways. It has confirmed that CYP2E1can induceincreasing metabolic activity in the long-term heavy drinking, on one hand, itaccelerate the removal of alcohol, and on the other, it generates a large numberof Reactive oxygen species that damages the liver. The serum levels ofTNF-αwere detected increase in animal models and people who abuse alcohol,and TNF-α participate in the deposition of collagen fibers and cirrhosis, this isthe molecular basis of alcoholic cirrhosis. Sirt1is closely related to the energymetabolism of the liver cells, and it is an "energy feeling" element, which canfeel the body and cellular energy metabolism. Its expression level is closelyrelated to the ratios of the NAD+/NADH and lactate/pyruvate, when the tworatios increased, the expression of Sirt1increased, and vice versa. Whathappens with Sirt1in alcoholic liver disease?Jujube contains flavonoids, and the application of ethanol extract ofjujube can significantly increase the vitality of the endogenous antioxidantenzymes and the amount of glutathione in liver tissue. Studies have shown thatjujube has a hepatoprotective effect, and significantly reduces the oxidationand nitro-oxidative stress and inflammation of liver, thus reduces liver damage.The Pueraria is a traditional Chinese medicine, which contains flavonoidshaving antioxidant, drug hangover effects. The experiments showed thatPueraria flavonoids could reduce the increase of ethanol-induced intestinal permeability, and alcohol-induced liver steatosis.The purpose of this experiment was to observe the role of jujube andpuerarin on the prevention and treatment of ALD. Contrary to thehepatoprotective drug bifendate, it was verified that jujube and puerarin haveeffects of anti-alcoholic liver injury from pathological changes, and the otherpurpose was to explore the effect of alcohol liver injury molecule changesafter administration.Methods：1.1Animal114Kunming mice with2-3weeks old in clean grade were selected.According to the weight, they were randomly divided into two groups, controlgroup (n=24) and model group (n=90). The model mice intragastriced for4weeks, eight mice were randomly killed in both control and model group toget specimens and were detected the changes of the indicators. Model groupwere randomly divided into4groups according to weight:1)model group(n=22)were intragastriced with alcoholic drinking8ml/kg the next day, and treatedwith distilled water0.01ml/kg as a substitute for drugs,2) jujube group(n=20)was intragastriced with jujube extract0.01ml/kg (equivalent to jujube0.02g/kg) before intragastricing with alcoholic drinking8ml/kg the next day,3)puerarin group(n=20) was intragastriced with pueraria extract0.01ml/kg(equivalent to pueraria raw herbs0.01g/kg) before intragastricingwith54degrees Red Star Erguotou8ml/kg the next day,4) bifendategroup(n=20) was intragastriced with bifendate0.01ml/kg/day(equivalent tobifendate drugs7.5mg/kg) before intragastricing with alcoholic drinking8ml/kg the next day. They were sacrificed at8weeks or12weeks.1.2SampleThe mice were executed by the mean of cervical dislocation after takingthe femoral vein blood, fasting for12hours before death. Then the liver wasimmediately removed, the left lateral lobe was fixed by4%paraformaldehyde,and the other part of the specimens was immediately put in liquid nitrogen. Itwas subpacked for molecular biochemical indicators, and was put in-80 degrees for long-term preservation. The blood samples were used to determinethe biochemical indicators.1.3Western-blotting detected the CYP2E1and Sirt1Total liver protein was extracted and determined using Lowry; its proteincontent was expressed in mg per milliliter of sample (mg/ml). Relativeexpression level of CYP2E1and Sirt1were both detected by Western-blottingand β-actin was used as the internal reference.1.4Detected the TNF-αand the level of the ALT、AST.Total liver protein was extracted and determined using Lowry; TheTNF-αand the level of the ALT、AST were both detected by kit.1.5Statistical analysisAll data are compared by mean using SPSS16.0statistical software, andall values are expressed as mean±standard deviation （±S）, statisticalevaluation of the results was performed by One-way ANOVA. According totesting the homogeneity of variance, LSD multiple comparison test wasselected, A probability value of0.05or0.01was considered statisticallysignificant.Result：1In generalThe mice with ethanol liquid gavage early appeared to lethargy, poorappetite, eating less, slow response, short ataxia, irritability, superficial lossand so on. After4weeks, the above symptoms were gradually improved. Bycontrary, the control mice were without these changes.2weightAfter4weeks and8weeks, there were no significant difference withweight in each group (P>0.05). After12weeks, contrary to pathology group,jujube group and puerarin group, the weights of the control mice were heavier,which was statistically significant (P>0.05).3The serum AST and ALT level3.1ASTIn the4th week, there was no significant difference with the serum level of AST between the control and pathological group (P>0.05). In the8th week,the serum level of AST of the pathological group was significantly highercompared with the control group and the treatment group, which wassignifieantly different (P<0.01). In the12th week, the serum level of AST ofpathological group was still higher than that of other groups, which wassignifieantly different (P <0.05).3.2ALTIn the4th week, there was no significant difference with the serum ALTlevel between pathological group and control group (P>0.05). In the8th week,the serum ALT levels of pathological group was significantly higher comparedto the control and other groups, which was signifieantly different (P <0.05). Inthe12th week, the serum ALT levels of the pathological group wassignificantly higher compared with other groups, which was signifieantlydifferent (P <0.05). There was no significant difference with the serum ALTlevels among bifendate group, the control group and the treat groups (P>0.05).4Histological changes of live tissueLver tissue were stained by hematoxylin-eosin (HE) staining in thecontrol mice (4th,8th, and12th week) showed that liver cells were arrangedradially to the central vein, outline of the hepatic lobule was clear and livercable was neat, intracellular lipid droplets were rarely seen, and there was notfiber formation with Masson staining. HE staining in pathological group (4thweek) showed that there was blood stasis in a portal area, hepatic sinusoidsand around the central vein, and various degrees of hydropic degeneration insome cells. Serious cells became swollen or into a spherical, cytoplasm wastransparent, sinusoidal was pressed, liver cable disordered, a little of lipiddroplets gathered in hepatic lobule, and there was a little of necrosis points,and there was not fiber formation with Masson staining. HE staining inpathological group (8th week) showed that the liver tissue around the centralvein appeared blood stasis, which significantly aggraved compared to one ofthe4th weeks, liver cells showed vacuolar degeneration, most cells swelled into spherical, cytoplasm was transparent, steatosis significantly increasedthan that of the4th weeks, different sizes of lipid droplets were in thecytoplasm, hepatocyte necrosis significantly increased than that of modelgroup in the4th week, accompanied by invasion of inflammatory cell, andthere was not fiber formation with Masson staining. The above-mentionedpathological changes of liver tissue in jujube group (8th week), puerarin group(8th week), and bifendate group (8th week) were all appeared, but which weremuch lighter than those of pathological group. Cells had a little of fattydegeneration, but were dramatically reduced compared with the control. HEstaining in pathological group (12th week) showed that there was a lot ofblood stasis in the liver portal area, hepatic lobule around the central vein andsinusoidal, vacuolar degeneration of the liver cells increased, more amount ofliver cells were swelled to spherical, cytoplasm was transparent, hepatic lobuleshowed increasing necrosis points and spots, inflammatory cells appearedinfiltration, and some of the necrotic area found proliferation of fiber cell.Masson staining showed blood in portal area and central venous wallthickening, vessel wall was colored light green, and there was no other fibrosis.These changes in jujube group (12th week), puerarin group (12th week), andbifendate group (12th week) were lighter than that of pathological group, andfatty of cells decreased.5The level of TNF-α in liver tissueIn the4th week, the TNF-α level of pathological group was significantlyhigher compared with concrol group(P<0.05). In the8th week, the TNF-αlevel of pathological group was significantly higher compared with controlgroup (P<0.01), puerarin group and bifendate group (P<0.05), but it did nothave any difference with jujube group. In the12th week, the TNF-α level ofpathological group was significantly higher than those of control group(P<0.01) and puerarin group (P<0.05), but they had no difference with thoseof jujube group and bifendate group.6the expression of protein in liver tissue6.1SIRT1 In the4th week, SIRT1level of of pathological group was significantlyhigher compared with the control one (P<0.01). In the8th week, SIRT1levelof pathological group was significantly higher than those of the control andbifendate group, but lower than that of puerarin one, the results werestatistically significant (P<0.01). The protein level of Sirt1of bifendate groupwas significantly lower compared with pueraria, jujube, and pathologicalgroup (P<0.01), but it showed no difference compared with the control one(P>0.05). In the12th week, the results of pathology group and bifendate groupwere statistically significant (P<0.01), while pathology group showed nosignificant differences compared with jujube group and puerarin group (P>0.05).6.2CYP2E1In the4th week, the relative expression level of protein CYP2E1ofpathological group elevated significantly compared with control group, whichwas statistically significant (P<0.01). In the8th week, the relative expressionlevel of CYP2E1of pathological group increased significantly in comparisonwith other groups, the results have statistical difference (P<0.01), there wereno significance compared with jujube group, puerarin group and bifendategroup (P>0.05),while CYP2E1level of puerarin group was much higher thanthat of bifendate group (P<0.05). In the12th week, the protein level ofCYP2E1of pathological group significantly increased compared with othergroups, and the results were statistically significant (P<0.01). There were notsignificant difference among jujube group, puerarin group and bifendate group(P>0.05). CYP2E1protein levels of pueraria group is still higher than that ofbifendate group, and the results were statistically significant (P<0.05).Conclusion:In mice models with alcoholic liver disease, liver tissues appearedpathological damage, and the level of Sirt1, CYP2E1and TNF-α in the livertissue significantly increased with the increase of drinking time. Aftertreatment with jujube, puerarin and bifendate, pathological damage reduced invarious degrees, and the level of CYP2E1, TNF-α also appeared to reduce. Bifendate could partly fight against the increasing of alcohol-induced Sirt1,while jujube, puerarin have no antagonism effect on the increasing ofalcohol-induced Sirt1.Thus the molecular mechanisms of the three drugs aredifferent.