| Aim: Metabolic inflammation is closely related to the occurrence of obesity,and can also lead to the occurrence of many metabolic diseases which is related to obesity.During the development of obesity,the expansion of adipose tissue is accompanied by the infiltration and activation of macrophages as the key immune cells,which leads to the occurrence of acute and chronic inflammation in the tissue.Many studies have shown that the natural drug berberine(BBR)has the effect of lowering blood glucose,lowering blood lipids and improving the balance of intestinal flora.Therefore,the purpose of this study is to study the therapeutic effect of berberine on obesity and of which related metabolic diseases and related molecular mechanisms.Methods:1.In vivo experiment: In this study,4-week-old C57BL/6J mice were fed with highfat diet for 16 weeks,and then berberine(200 mg/kg)was given for 6 weeks to observe the improvement effect of berberine on glycolipid metabolism and tissue inflammation in mice.First,monitor the overall general situation of mice under the condition of highfat diet,such as body weight and food intake,and then detect various biochemical metabolic indicators such as blood glucose,triglyceride and total cholesterol;At the same time,combined with insulin tolerance test(ITT),glucose tolerance test(GTT)and glucose stimulated insulin secretion test(GSIs),the changes of glucose homeostasis in the two groups of mice were evaluated under the condition of high-fat feeding.Then,in order to further explore the effect of berberine on adipose tissue of obese mice,the infiltration of berberine on adipose tissue inflammation of obese mice was analyzed by pathological section staining,laser confocal immunofluorescence,ELISA and QPCR.2.In vitro experiment: We extracted and isolated the stromal vascular fraction(SVF)in visceral fat and subcutaneous fat of two groups of mice under high-fat diet,and used flow cytometry and laser confocal analysis to analyze the effect of berberine on the type and proportion of macrophages in adipose tissue of obese mice.At the same time,we used QPCR and ELISA to analyze the inflammatory gene expression level of SVF in mice adipose tissue under high-fat diet and the secretion of inflammatory factors in primary adipose tissue culture.Finally,we extracted the primary peritoneal macrophages of C57BL/6J mice,stimulated the activation of macrophages in the simulated tissue by LPS,and detected the effect of berberine on the activation of primary macrophages and chronic inflammation by QPCR.3.Molecular mechanism: Extract and isolate SVF in visceral fat and subcutaneous fat of the two groups of mice under high-fat diet,and analyze the difference of gene expression profile by RNA sequencing;The target molecules interacting with berberine were screened by molecular docking,and then the overlap analysis was carried out with the sequenced differential genes.The validation experiment was carried out after the candidate genes were obtained by combining the sequencing analysis of clinical samples and animal model validation.Results:1.In vivo experiment: First of all,at the overall animal level.Compared with the control group under the high-fat diet,the weight of mice in the berberine treatment group decreased significantly,and the physiological indexes such as blood glucose,triglyceride and total cholesterol decreased significantly.The results of GTT,ITT and GSIS showed that the glucose metabolism of mice in the berberine group was significantly improved.Secondly,from the level of adipose tissue.Compared with the control group under highfat diet,HE staining showed that the number of CLS in adipose tissue of mice treated with berberine decreased significantly,immunofluorescence showed that the expression of CD11 c,a classic anti-inflammatory marker of macrophages,in adipose tissue of mice treated with berberine decreased significantly;and the results of QPCR and ELISA showed that the expression of inflammatory genes and secretion of inflammatory factors in adipose tissue of mice treated with berberine decreased significantly.2.In vitro experiment: from the cell level.Compared with the control group under high-fat diet,the QPCR results of SVF in adipose tissue showed that the expression of SVF pro-inflammatory gene in adipose tissue of mice treated with berberine was significantly decreased,and the expression of anti-inflammatory gene was significantly increased.The results of flow cytometry also showed that the number of M1 antiinflammatory macrophages in mice treated with berberine decreased.In vitro,the expression of LPS-stimulated pro-inflammatory genes was significantly reduced after berberine treatment in peritoneal macrophages by QPCR analysis.3.Molecular mechanism: FKBP5,Chit1,Mmp7,P2rx3 and Mgam genes were obtained by SVF RNA-seq and docking overlap analysis,and FKBP5 was initially screened by macrophage LPS stimulation model.Finally,we found that the expression of FKBP5 gene was positively correlated with the BMI of the clinical population(r=0.3648,P=0.0436)through RNA-seq of clinical preperitoneal and subcutaneous adipose tissue;The expression of FKBP5 the SVF in adipose tissue of spontaneously obese diabetes db/db mice was significantly increased.Therefore,we infer that berberine may regulate macrophages by down-regulating the expression of FKBP5 to improve tissue inflammation and metabolic homeostasis.Conclusions: This topic focuses on the study of gene expression related to glycolipid metabolism and inflammatory response in adipose tissue of obese mice,in order to further clarify the effect of berberine on macrophages,adipose tissue and systemic inflammation and metabolic homeostasis.It was found that berberine could significantly improve the disorder of glucose and lipid metabolism and systemic metabolic inflammation in obese mice induced by high-fat diet;It was further found that berberine may reduce the inflammatory effect of obese mice by targeting FKBP5 gene to regulate the activation of macrophages in adipose tissue. |
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