| Objective:According to the the cascade trend and the relationship between obesity of pre diabetes,fat deposition in main organs,inflammatory factors releasing and insulin resistance,compound purendan effect ontherapy hyperlipidemia and adiposity,glucolipid metabolism metabolism,serum and adipose tissue inflammation factors and insulin resistance in ob/ob mice were discussed to explore the role of purendan regulation of ob/ob mice epididymal adipose tissue IR and part of its mechanism,so as to provide experimental basis for early prevention of diabetes.Methods:1.Selection of laboratory animals Because of the disease process of model of spontaneous type 2 diabetes mellitus is similar to human,it has high application value in the study of diabetes,drug screening of the pathogenesis.The ob/ob mice have codon mutations Leptin gene genetic defects of in leptin gene,which can lead the defect of expression and normal function of leptin.The defect is linked to obesity,hyperphagia,impaired glucose tolerance,insulin resistance and other symptoms,which showing a pathological change similar to that of human type 2 prediabetes.Therefore,we selected 10 SPF male C57BL/6J mice of 3 weeks old,with a body mass of(9.70 + 0.76)g,50 SPF male ob/ob mice of 3 weeks old,with a body mass of(9.88 + 2.40)g.2.The group and administered of animalsThe ob/ob mice were randomly divided into model group(Model group,M),positive control group metformin(Positive control group,P),Pu Ren Dan high dose group(Purendan high group,G),group of Dan Pu Ren(Purendan medium group,Z),purendan the low dose group(Purendan low group,D),with C57BL/6J mice as control group.The number of experimental animals in each group was 10.Purendan treatment group gavage dose were:low dose(4.5g/kg),middle dose(9.0g/kg),high dose(18g/kg);the positive control drug group intragastric metformin dose of 195mg/kg(the dosage root in human and animal body surface area conversion table);control group and model group mice with the same volume of distilled water;mice were orally once daily and the administration lasted for 8 weeks..3.The detection IR of related indicatorsThe mice were weighed once a week during the administration.After 7 and 8 weeks,glucose tolerance and impaired insulin tolerance were evaluated by oral glucose tolerance test(OGTT)and insulin tolerance test(ITT).After 8 weeks,the mice body weight,body length,body fat and serum biochemical index were determined.The enzyme linked immunosorbent assay(ELISA)was detected for FINS.4.Morphological observationHistopathological changes of the adipose,pancreas,and liver tissue were observed by HE staining.5.Detection of inflammatory factorsELISA was detected for Lep,Retn,Nampt,ADPN,TNF-a and IL-6 content.The fluorescence quantitative RT-PCR were used to determine the level of mRNA of Lep,Retn,Nampt,ADPN,TNF-a and IL-6.6.Detection of associated proteins in JAK/STAT signaling pathwayWestern Blot assay were used to test the level of protein expression and phosphorylation of JAK2,STAT3 and SOCS3 and explored its pathway regulatory mechanisms.Results:1.Purendan effect on glycolipid metabolism and insulin sensitivity in ob/ob miceCompared to the control group,fasting weight,visceral fat index and Lee's index of mice in the model group increased significantly(P<0.01),and the OGTT area under the curve of ITT increased significantly(P<0.01).The levels of TC,LDL-C,FPG and insulin in serum were significantly increased(P<0.01).Compared with the model group,the weight and the Lee's index decreased significantly in purendan group mice(P<0.01).The levels of TC,LDL-C and insulin were significantly reduced in purendan group mice(P<0.05).The levels of FINS were significantly reduced in purendan group mice(P<0.01).Compared with the model group,the Lee's index decreased significantly in Metformin group mice(P<0.01),epididymis adipose tissue and visceral adipose tissue weight was significantly decreased in Metformin group mice(P<0.05).The levels of TC,LDL-C were significantly reduced in Metformin group mice(P<0.01).The levels of FINS were significantly reduced in Metformin group mice(P<0.01).2.The effect of purendan for adipose tissue tissue cell morphology and ectopic fat deposition in ob/ob mice.Compared with the control group,the epididymal adipose cells volume of ob/ob mice increased obviously,and they arranged in loose irregulara.There are a large number of boundary clear vacuoles with different sizes in the liver cells cytoplasm of ob/ob mice increased of the nucleus to one side.The volume of Purendan group and metformin group ob/ob mice adipose cell were decreased,and the number of vacuoles in the cytoplasm of liver cell volume were significantly reduced.3.The effects of Purendan on the cytokines secreted by in adipose tissue of ob/ob miceCompared with the control group,the levels of inflammatory factors TNF-?,IL-6 and Lep in the model group increased significantly in serum(P<0.01),and the levels of Retn,Nampt and ADPN decreased significantly(P<0.01);the level of TNF-? and leptin secreted by adipose tissue increased significantly(P<0.01),and the level of IL-6,Retn,Nampt and ADPN secreted by adipose tissue decreased significantly in adipose tissue of model group ob/ob mice(P<0.01);Compared with the model group,levels of inflammatory factor TNF-?,IL-6 and Lep in serum of ob/ob mice of Purendan group increased significantly(P<0.01);levels of Retn and Nampt were significantly decreased(P<0.01),levelsof ADPN was significantly decreased(P<0.05).Purendan group and metformin group were significantly reduced the level of ADPN,Nampt secreted by adipose tissue(P<0.01).Purendan group and metformin group were significantly reduced the level of TNF-? secreted by adipose tissue4.Purendan effect on the expression of SOCS3 and JAK/STAT signal transduction pathway mediated by inflammatory factor.Compared with the control group,the amount of phosphorylation of JAK2 protein expression in model group decreased in epididymal adipose tissue,and the protein expression and phosphorylation levels of STAT3 were decreased.SOCS3 protein expression decreased,but there is no obvious difference between the indexes(P>0.05).Compared with the model group,the protein expression of JAK2 in epididymal adipose tissue of the high dose group increased slightly but decline of the phosphorylation.The protein expression and phosphorylation level of STAT3 decreased,and SOCS3 protein expression increased slightly,but there is no obvious difference between the indexes(P>0.05).The protein expression levels of JAK2 were not significantly changed in epididymal adipose tissue of Purendan medium dose,phosphorylation levels of JAK2 showed a decreasing trend;and protein expression and phosphorylation level of STAT3 showed a decreasing trend;the protein expression of SOCS3 and increased slightly;the protein expression levels of JAK2 in epididymal adipose tissue of purendan low dose group increased slightly,the phosphorylation level of it were decreased,and levels of protein expression of STAT3 were decreased,levels of phosphorylation of STAT3 were rising,levels of protein expression of SOCS3 were decreased,but there is no obvious difference between the indexes(P>0.05).The levels of the protein expression of JAK2 in epididymal adipose tissue of metformin group increased,The levels of phosphorylation of JAK2 were decreased;The levels of protein expression of STAT3 were decreased;the levels of phosphorylation of STAT3 were increased.The protein expression of SOCS3 was decreased.But there is no obvious difference between the indexes(P>0.05)Conclusion:1.Purendan can effectively control obesity,improve glucolipid metabolism,so as to improve the IR caused by obesity.2.Purendan can effectively reduce the volume of adipose tissue cells and lipid deposition in the liver cells;thereby effectively improve insulin resistance of adipose tissue and liver tissue.3.Purendan also can effectively regulate a variety of inflammatory factors secreted by adipose tissue of ob/ob mice,from the overall maintain secretion imbalance of inflammatory factor in adipose tissue,thereby,improve the chronic inflammatory state and IRof adipose tissue.4.The Purendan can inhibit the JAK2/STAT3 signal pathway by increasing the expression of negative feedback SOCS3 to reduce inflammation and improve insulin resistance of adipose tissue. |
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