| The attempt of this article is to increase the yield of Ganoderma lucidum polysaccharide by demethylating DNA,explore the preventive effect and mechanism of G.lucidum polysaccharide on acute alcoholic liver injury in mice.Through pre-experiment,two protoplasts G.lucidum strains YJ16 and YJ20,with the fastest germinating binuclear,were selected by azacitidine induce.The original strain YJCK was used as a control.Comprehensively analyzed the polysaccharide content,preliminary explored the effect of demethylation on the growth and active substances of G.lucidum mycelium.In order to further verify the efficacy of G.lucidum polysaccharides,vivo test was done.Male mice were randomly divided into5 groups,blank group(distilled water),model group(alcohol),GLMPS group(200mg / kg mycelium polysaccharide),GLFPS group(200mg / kg fruiting body polysaccharide)and positive group(300mg / kg bifendate).Mice were gavaged with alcohol to make a model after one-week adaptive feeding.Later,various physiological and biochemical indexes of serum and liver were detected to evaluate the preventive effect of G.lucidum polysaccharide on acute liver injury caused by alcohol.Differentially expressed genes(DEGs)were selected by enrichment analysis of in the transcriptome with the help of GO and KEGG,and RTq PCR was operated to verify the 15 major differential genes in metabolic pathway,which were selected by KEGG,to clearly clarifying the G.lucidum polysaccharides with better liver damage prevention effect and its mechanism on acute alcoholic liver injury in mice.The main findings are as follows:1.In the demethylation treatment,the mycelium morphology of the azacytidine treatment group did not change significantly.But the mycelial growth rate of the YJ16 and YJ20 strains was significantly improved compared with the control;the methylation levels of YJ16 and YJ20 strains were 3.16% and 1.73% respectively,which were 22.22% and 57.40%lower than that of the control.Comparing the hydroxymethylcellulase,hemicellulase and amylase activities of each strain,it showed that the strain YJ20 contained highest enzyme activity compared with the control and strain YJ16 followed.It means that DNA demethylation can effectively improve the enzyme activity of the strain.The assessment of polysaccharide content showed that compared to the control,the polysaccharide content of strain YJ20 was increased by 59.95%,indicating that DNA demethylation may increase polysaccharide content in G.lucidum.Moreover,the stability of the demethylation was tested and the result showed that the methylation level increased by 1.6 times in control strain while that was increased by 0.8 times in strain YJ20 after five times transfer.It means that the DNA methylation level of the strain will spontaneously recover with subculture when no azacytidine environment.2.In terms of liver injury,G.lucidum polysaccharides were used to prevent acute liver injury in mice by gavage,and the physiological and biochemical indicators of serum and liver tissue were detected.The indicators of the model group were significantly different from that of blank group,which proved the success of model of alcoholic liver injury.The levels of ALT,AST,TG,TC,ADH and MDA in serum and liver of mice in GLMPS and GLFPS groups were significantly lower than those in model group,and the contents of CAT and SOD in livers of mice in GLMPS and GLFPS groups were significantly higher than those in model group.Liver pathological slices showed that both the polysaccharides of mycelium and fruiting body of G.lucidum can significantly improve the cell turbidity and hepatic cell disorder caused by alcohol.The above data shows that G.lucidum polysaccharide has the same effect as bifendate in the positive group in preventing liver injury.And the fruiting body polysaccharide was even better than bifendate in terms of increasing CAT and SOD levels.In addition,the effect of fruiting body polysaccharide is better than the mycelium.3.To explore the mechanism of the effect o f G.lucidum polysaccharide on acute alcoholic liver injury in mice by analyzing the transcriptome data.Compared to the blank group,there were 211 genes upregulated and 309 genes down-regulated in model group.In GLFPS group,there were 241 genes up-regulated and 227 genes down-regulated compared to the model group.With the analysis of reported literature,15 significant differential genes were screened and they mainly involved exogenous drug metabolism P450 pathway,cytochrome P450 pathway,chemical carcinogenic pathway,retinol metabolism and steroid metabolism,it indicated that the pathological mechanism of G.lucidum polysaccharide precuring acute liver injury caused by alcohol gavage may related to various biological processes and pathways.4.Further,the 15 screened differential genes were quantitatively detected by q-PCR.The result showed that G.lucidum fruit body polysaccharide intervention can significantly inhibit the expression of CYP450 family Cyp51,Cyp1a1,Cyp2e1 and Aldh3a1,IL-1 and other inflammation-related factors induced by alcohol and significantly improving the Aldh1a2,Aldh1a1,Adh1,Ugt2b38,Egr1,Cyp27b1 and Rdh9 expression level,which were closely related to the exogenous drug metabolism P450 pathway,cytochrome P450 pathway,chemical carcinogenic pathway and retinol metabolism pathway and inhibited expression in the model group.These results were basically consistent with transcriptome analysis,further verifying the accuracy of transcriptome analysis.Compared to the model group,Cyp2e1,IL-1 and Cyp51 were upregulated in the blank group but down-regulated in GLFPS group.Similar,Aldh1a2、Aldh1a1、Adh1、Ugt2b38、Egr1、Cyp27b1 and Rdh9 were down-regulated in the blank group but up-regulated in GLFPS group.It indicated that these genes played a more important role in the G.lucidum polysaccharide prevention effect of acute alcoholic liver injury. |
