| Objective:Centella asiatica is a medicine and food herb.It has been reported that C.asiatica has a positive effect on obesity.However,it has not yet been fully elucidated whether C.asiatica inhibits weight gain via a mechanism involving the hypothalamic-sympathetic-brown adipose tissue(BAT)axis.Therefore,in this study,we evaluated the effect of C.asiatica on the hypothalamic-sympathetic-BAT pathway in obese diabetic mice,which was based on transcriptomics.In addition,the main triterpene component of CentellaAsiatica-madecassoside has been validated for its efficacy and mechanism of action.Methods:1.Sixteen six-week-old SPF male db/db mice.After adapted for one week,The randome blood glucose of mice were be measured,and 8 mice with random blood glucose>11.1 mmol/L were randomly selected as the model group(db/db group),8 male mice in the Centella asiatica group(db/db+C group)and 10 male mice in the normal group(db/m group).All the mice were fed a regular chow diet,and had free access to water.The food was only removed during the experiment.The mice in the normal group were given deionized water by gavage.The mice in the Centella asiatica group were given 100 mg/kg/day by gavage,and body weight of mice were measured everyday.After 6 weeks of intervention,all mice were subjected to oral glucose tolerance test(OGTT).After the mice were anesthetized with isoflurane,blood was taken from the abdominal aorta and all mice were sacrificed.The liver,brown adipose tissue,and epididymal fat of the mice were quickly separated,weighed,and the corresponding organ weight index was calculated.Quickly collected mice hypothalamic tissue and put them in 4%paraformaldehyde solution for subsequent analysis.The serum cholesterol(CHO),triglyceride(TG),high-density lipoprotein(HDL),low-density lipoprotein(LDL),aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels were measured using a special kit according to the manufacturer's instructions.Oil red O staining and HE staining were performed on liver,and HE staining was performed on brown adipose tissue.The differentially expressed genes in hypothalamus and brown adipose tissue of these mice were analyzed by transcriptomics,and the mechanism of Centella asiatica inhibiting weight gain in db/db mice were explored.2.A total of 16 male KKay/TaJcl obese diabetic mice weighing 35-40g were randomly divided into two groups,wherein the mice in the control group(n=8)were administered with water,and those in MA group(n=8)were administered with MA(40mg/kg/day)via gavage.At the end of the 8th week,all mice were subjected to an oral glucose tolerance test(OGTT),and then the body mass index of each mouse was measured by bioelectrical impedance analysis(BIA).The blood samples were collected through abdominal aorta,and the organs(epididymal fat,mesenteric fat,brown fat,abdominal fat,brain,heart,etc.)from the mice were separated to measure their respective weights immediately.The serum cholesterol(CHO),triglycerides(TG),aspartate aminotransferase(AST),alanine aminotransferase(ALT)and blood glucose levels were detected using a special kit according to the manufacturer's instructions.Results:1.The results of the 6-week intervention experiment showed that compared with the model group,Centella asiatica water extract can effectively reduce body weight of db/db mice,reduce the weight of brown adipose tissue,liver and epididymal adipose tissue.It also has positive effect for the level of cholesterol,triglycerides,high-density lipoprotein and low-density lipoprotein,moreover,it has no obvious damage to liver function.The results of adipose tissue morphology indicated that Centella asiatica had no significant effect on epididymal fat and mesenteric fat in db/db mice,but it could effectively improve the morphology of brown adipose tissue in mice.The above results indicate that Centella asiatica has a positive effect on the fat metabolism of db/db mice,which possibly by affecting the function of brown adipose tissue.But the treatment with C.asiatica did not modify pathoglycemia,therefore,we abandoned any research into the hypoglycemic mechanism.2.Centella asiatica can significantly promote the expression of UCP1,Cidea,Cox8b,Cox7a and PPAR-? transcription factors in brown adipose tissue,which relates closely to the function of BAT thermogenesis.In addition,the mRNA expression of adiponectin receptor 1,ACO and MCAD in BAT also increased significantly after 6 weeks of intervention with C.asiatica,which relates closely to ?-oxidation.Western blotting results showed that the expression of UCP1 and TH protein in brown adipose tissue of db/db group mice was significantly lower than that of db/m group,but after 6 weeks of intervention by Centella asiatica,UCP1 and TH protein in brown adipose tissue of db/db group mice were significantly increased.Transcriptomics results showed that after 6 weeks of intervention by Centella asiatica,the transcriptome results of brown adipose tissue in db/db group mice changed significantly.Compared with mice in the db/db group,111 differential genes in the db/db+C group mice were up-regulated and 431 down-regulated.The functional enrichment analysis of these differential genes showed that Centella asiatica can significantly regulate metabolism-related functions.Taken together,combining our transcriptome data and experimental results,we found that C.asiatica has positive effects on the function of BAT thermogenesis via the activation of sympathetic nerves.3.Transcriptome analysis of hypothalamic tissue showed that after 6 weeks of intervention by Centella asiatica,compared with db/db group mice,there were 131 transcription factors up-regulated and 51 transcription factors down-regulated in hypothalamic tissue.And these differentially expressed genes are all related to the biological process of metabolic diseases.Enrichment analysis of the KEGG signaling pathway showed that the differentially expressed genes in the hypothalamus were enriched in biological processes such as endocrine and metabolic system disease signaling pathways,lipid metabolism signaling pathways,and energy metabolism signaling pathways.The bubble chart of the differential genes showed that they were mainly distributed in dopaminergic synapses.These results indicate that Centella asiatica has a significant effect on the metabolism-related signal pathways in the hypothalamus,which may act by affecting the hormone-related receptors in the hypothalamus.4.The joint transcriptomics analysis of hypothalamus and brown adipose tissue showed that Centella asiatica can significantly affect hormone-related signaling pathways in hypothalamic.The functional enrichment analysis of the KEGG signaling pathway in brown adipose tissue showed that Centella asiatica can significantly affect energy metabolism-related signaling pathways.The immunohistochemical results of P-AMPK in hypothalamus also showed that Centella asiatica can significantly inhibit the expression of P-AMPK.Collectively,these findings may indicate that Centella asiatica can promote the expression of a variety of hormone-related transcription factors in the hypothalamus,and trigger the thermogenic function of brown adipose tissue by activating sympathetic nerve activity.Thus,it may be beneficial in the treatment of obesity.5.Centella asiatica has no significant burden on the liver of db/db mice.Pathological staining results showed that Centella asiatica has a positive effect in reversing liver lipid deposition of db/db mice,PCR results showed that Centella Asiatica could significantly promote the expression of MCAD and PPARa transcription factors in the liver of db/db mice.Therefore,these also indicated that Centella asiatica can improve liver lipid metabolism disorders by affecting liver fatty acid oxidation,thus,it has a protective effect on the liver of db/db mice.6.Madecassoside can significantly promote the expression of SIRT1,P-AMPK and P-ACC in mesenteric fat(P<0.05),activating SIRT1-AMPK-HSL signaling pathway in epididymal fat,and it can significantly enhance the expression of following three transcription factors in epididymal fat(PGC-1?,PPARa and CPT-1a).We also found that madecassoside can significantly promote the expression of UCP-1 in brown adipose tissue and mesenteric adipose tissue,moreover,it also has a possitive role in promoting the expression of genes related to mitochondrial function(Cidea,Cox7al and Cox8b).Conclusions:1.Centella asiatica has a positive effect on inhibiting the weight gain of db/db mice.The results of LC-MS showed that the triterpene components were main component.2.Centella asiatica can significantly promote the expression of UCP1,Cidea,Cox8b,Cox7a and PPAR-a transcription factors in brown adipose tissue in db/db mice,and Western blotting results showed that Centella asiatica can significantly promote the expression of UCP1 and TH protein in brown adipose tissue of db/db mice,thus,it possibly by activating the expression of hormone-related transcription factors in the hypothalamus,activating sympathetic nerve activity,and promoting thermogenesis of brown adipose tissue.3.Centella asiatica has a positive effect in reversing liver lipid deposition and the possible mechanism involved in activating fatty acid oxidation in the liver.4.Madecassoside has a significant effect on inhibiting weight gain in KKay mice.The mechanism of action was similar to that of Centella asiatica,both of them can active the function of BAT and it may be one of the effective components of Centella asiatica. |
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