| ObjectiveIn order to provide new ideas and means for the prevention and treatment of phlegm-dampness syndrome of obesity,this study plans to simply analyze the mechanism of action of Wendan decoction in intervening obesity phlegm-damp evidence and the internal connection between promoter methylation and expression of UCP1,and to discuss the correlation between UCP1 methylation and lipid metabolism.Methods1.Establishment of obesity phlegm-dampness syndrome model with high-fat diet:SD rats were divided into model group with high-fat food and normal group with ordinary diet for 8 weeks.According to rouse body weight and lee’s index,The rats that meet the criteria were selected for further experiments.2.Intervention of Wendan Decoction on phlegm and dampness syndrome model of obesity: the rats successfully created were divided into 3 groups: Wendan decoction high-dose group,Wendan decoction low-dose group and blank control group.The rats in the Wendan decoction groups were given the corresponding concentration of Wendan decoction intragastatically for 4 weeks.The other rats in the blank control group and the normal control group were given the intervention of double steaming water for 8 weeks.3.Materials: Blood was taken from the heart of rats after anesthesia to prepare serum;Expose abdominal cavity and take white adipose tissue;Expose the subscapular area and take brown adipose tissue.All samples are stored at-80℃.4.Detection:Serum lipid indexes were detected by enzyme method;The total methylation concentration of UCP1 in adipose tissue was determined by ELISA;The promoter methylation of UCP1 was detected by Massarray technique;Real-time quantitative fluorescence polymerase chain reaction(q RT-PCR)was used to detect UCP1 gene expression in brown adipose tissue;The positive expression of UCP1 protein in brown adipose tissue was detected by immunohistochemistry;The protein expression of UCP1 in brown adipose tissue was detected by Western blotting(WB).5.SPSS 22.00 software statistical analysis data: One-way analysis of variance was used for comparison among groups conforming to normal distribution,LSD method was used for comparison among groups with homogeneity of variances,and Tamhane’s T2 method was used for comparison between groups with uneven variances.Non-parametric Mann-Whitney U test was used between the two groups that did not conform to the normal distribution.Test level α=0.05Results1.Obesity index resultsCompared with the normal group fed with ordinary diet,the body weight of the model group fed with high fat diet was increased(P < 0.05),and the lee’s index of the model group also was increased(P < 0.05),It can be seen that the nutritional obesity model was successfully built.Compared with the normal control group,the body weight of the blank control group was significantly increased(P < 0.01),and the wet weight of white fat was significantly increased(P < 0.01).It can be seen that after modeling,the body weight and white fat of the blank control group were still higher than that of the normal control group.2.Brown adipose tissue resultsCompared with the normal control group,the color of brown fat in the blank control group became lighter,and the volume of brown fat cells increased and the number decreased at a 200-fold microscope.Compared with the blank control group,the color of brown fat in the Wentan decoction group became darker,and the volume of brown fat cells became smaller and the number increased under the 200-fold microscope,which was most obvious in the low-dose group.3.Blood lipid was detected by enzymatic methodCompared with the blank control group,the total cholesterol content in the high-dose group was significantly decreased(P < 0.01),and the total cholesterol content in the low-dose group had a decreasing trend,suggesting that Wentan Decoction could reverse the increase of total cholesterol level caused by high-fat diet.4.The total methylation concentration was determined by ELISACompared with normal control group,the total methylation concentration in blank control group was significantly increased,with statistical significance(P <0.05).Compared with the blank control group,the overall methylation concentration in the low-dose group was significantly decreased,with statistical significance(P <0.05).5.DNA methylation results of UCP1 were detected by Massarray techniqueA total of 13 sites were detected in the promoter region.Compared with the normal control group,the methylation degree of 9 sites in the blank control group was slightly increased,and the overall average methylation degree was slightly increased.Compared with the blank control group,7 sites in the high-dose group and 6 sites in the low-dose group were slightly reduced,and the overall average degree of methylation was slightly reduced.6.Real-time fluorescence quantitative polymerase chain reaction was used to detect UCP1 m RNA expressionThere was no significant difference in m RNA expression between the groups(P > 0.05).Compared with the blank control group,the transcription level of UCP1 gene in the low-dose group showed an upward trend.7.UCP1 protein expression was detected by immunohistochemistry and western blottingImmunohistochemistry showed that UCP1 protein expression in blank control group was significantly decreased compared with normal control group(P < 0.01).Compared with blank control group,UCP1 protein expression in low-dose group was significantly increased(P < 0.01),while UCP1 protein expression in high-dose group was increased,suggesting that Wentan Decoction could promote UCP1 protein expression.Western blotting showed that UCP1 protein expression in blank control group was significantly decreased compared with normal control group(P < 0.01).Compared with blank control group,UCP1 gene expression in low-dose group showed an upward trend,and UCP1 protein expression in drug group was significantly increased(P < 0.01),suggesting that Wentan decoction can promote UCP1 transcription and translation.Conclusion1.Wendan Decoction can reduce the weight of obese rats,reduce the serum total cholesterol content of rats,and has a good effect on the phlegm-dampness syndrome of obesity.2.DNA methylation changes of UCP1 do exist in obese rats with phlegm-dampness syndrome,which may mediate the generation of phlegm-dampness syndrome of obesity.3.Wendan Decoction may play a role in clearing total cholesterol by promoting UCP1 protein expression,thus preventing obesity. |
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