| To analyze the effects of Wandan Decoction on the methylation and expression of Pik3cd and Wnt8b promoter of immune inflammatory pathway genes in adipose tissue of obese rats with phlegm-dampness syndrome,and to provide more evidence for the mechanism of Wandan decoction’s intervention in obesity phlegm-dampness syndrome.Methods1.High-fat diet to make a model of obesity phlegm-dampness syndrome:SD grade rats were divided into two groups:model group and normal group,and fed high-fat diet and ordinary diet respectively.After 8 weeks,according to body weight and Lee’s index,the model rats were divided into 3 groups:Wendan Decoction high-dose group,Wendan decoction low-dose group and blank control group,and were given high dose of Wendan decoction,low dose of Wendan decoction and pure water.In addition,rats in the normal control group were given pure water intragastric irrigation for 4 weeks.2.Analysis of weight loss results:During the intervention of Wendan Decoction,the weight and length of rats were recorded every week,and Lee’s index was calculated.3.Immunofluorescence staining was used to detect the infiltration of immune cell molecules CD4~+,CD8~+and CD68~+in adipose tissue.4.The promoter methylation expression of Pik3cd and Wnt8b genes in abdominal adipose tissue was detected by Massarray technique.5.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the gene expression of Pik3cd and Wnt8b in abdominal adipose tissue.6.The protein levels of Pik3cd and Wnt8b in rat abdominal adipose tissue were determined by Western blot.7.Data collation and statistics:processing and analysis of test data,statistical analysis of data using SPSS25.00 software.Results1.Body weight and Lee’s results:Compared with the normal group,the body weight of the blank control group was larger,showing a statistical difference;After drug intervention,the weight data of Wwendan Decoction low and high dose groups were compared with the blank control group,P<0.05,which also had statistical difference.At the same time,Lee’s index of the blank control group was compared with the normal group,P<0.05,which also had statistical difference.Lee’s index is down from before.2.Immunofluorescence results:After low dose intervention,the infiltrated CD4~+T cells in adipose tissue decreased;The infiltrated CD8~+T cells in adipose tissue decreased and gradually approached normal adipose tissue.After the intervention of low and high doses of drugs,the infiltrated CD68~+T cells in adipose tissue decreased and gradually approached normal adipose tissue.3.DNA methylation results:Compared with the normal group,the degree of methylation of pik3cd gene in the blank group was increased.After drug intervention,the degree of methylation of pik3cd gene in low and high dose groups decreased slightly compared with blank group.For wnt8b gene,the methylation degree of wnt8b gene in the blank group was also higher than that in the normal group.After intervention,the average methylation degree of WNT8B gene in the low and high dose groups decreased slightly,and the methylation level was very close to that in the blank group,with no significant change.4.mRNA expression:Compared with the normal group,the mRNA expression of pik3cd gene in the blank group was increased;Compared with the blank group,the mRNA expression of genes in the low and high dose groups also decreased.For wnt8b gene,there was no significant difference between blank group,normal group and low-dose group,while mRNA expression in high-dose group decreased significantly(P<0.05).5.Protein expression:the protein expression of pik3cd in the blank group was significantly lower than that in the normal group,while low and high dose drug intervention inhibited it,that is,the protein expression of pik3cd in the dose group was decreased compared with that in the normal group;However,compared with the blank group,it was increased,P<0.05.For wnt8b gene,the protein expression of the blank group was significantly higher than that of the normal group,while the expression of the drug group was increased compared with the blank group.6.Comparative analysis between promoter methylation and RNA and protein expression:In pik3cd,methylation of blank control group increased compared with normal control group.The low dose group had a decreasing trend compared with the blank control group.Compared with the blank control group,the high dose group had a decreasing trend.Compared with the normal control group,the mRNA of the blank control group was increased.The low dose group had a decreasing trend compared with the blank control group.There was a downward trend in the high-dose group compared with the blank control group,but there was no statistical difference.Compared with the normal control group,the protein expression of the blank control group was decreased.Compared with the blank control group,the low-dose group had an upward trend,and the high-dose group also had an upward trend,with statistical difference.In wnt8b,the methylation of blank control group was increased compared with that of normal control group.The low dose group had a decreasing trend compared with the blank control group.Compared with the blank control group,the high dose group had a decreasing trend.Compared with the normal control group,the mRNA of the blank control group was increased.The low dose group had a decreasing trend compared with the blank control group.There was a downward trend in the high-dose group compared with the blank control group,but there was no statistical difference.Compared with the normal control group,the protein expression of the blank control group was increased.The low dose group had a decreasing trend compared with the blank control group.Compared with the blank control group,the high dose group had a decreasing trend,and there was a statistical difference.Conclusion1.For obese rats with phlegm-dampness syndrome,the methylation degree of pik3cd and wnt8b gene promoter,mRNA expression level and wnt8b gene protein expression level were increased in the blank control group compared with the normal group.2.After the intervention of Wendan Decoction,the methylation and protein expression levels of pik3cd were consistent,but wnt8b was not,which verified that Wendan Decoction could interfere with the expression level of pik3cd gene to a certain extent.3.The intervention of Wendan Decoction has certain effects on the methylation degree,mRNA expression and protein expression degree of pik3cd and wnt8b genes,but the relationship between the three is not obvious.Further analysis is needed on the influence of Wendan Decoction on gene methylation degree and whether it stimulates and affects gene expression. |
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