Study On The Effects And Mechanism Of Small GTPase Rab8a Inhibitor Berbamine Hydrochloride In Improving Obesity Of Mice

Objective:In recent years,the incidence of obesity has increased sharply.At present,the main intervention methods for obesity are to increase energy consumption,inhibit fat formation and limit calory intake.Studies have shown that small GTPase Rab8a is an important activator of Fsp27-mediated lipid droplet fusion.Therefore,the present study is aimed to screen small molecule compounds from traditional Chinese medicine that could inhibit the activity of Rab8a GTPase,and study its pharmacological effects and mechanism on metabolic disorders such as obesity,which would provide experimental basis for the R&D of new drugs.Methods:1.The small GTPase Rab8a was purified using eukaryotic cells,and an enzyme activity screening system was established in vitro to screen the small molecule inhibitors of Rab8a from traditional Chinese medicine.2.The inhibitory effect of BBM on the protease activity of wild type and 293TRab8a-/-cells was investigated by colorimetric method.3.The 3T3-L1 differentiation model and the lipid accumulation of L02/Hep G2 cells were established to study the effect and mechanism of BBM on lipid droplet fusion.4.The effect of BBM on lipid metabolism of C.elegans was studied by oil red staining,and investigated its effects on lifespan and diet of C.elegans.5.In DIO mice,body weight,body fat,intake,glucose and lipid metabolism,energy metabolism,liver function and hepatic steatosis were investigated,and q PCR was used to detect the effects of BBM on genes related to glucose and lipid metabolism.In addition,the influence of BBM on the leptin signaling pathway was investigated by the body weight,food intake,blood glucose of db/db mice.6.The DIO mice were treated with BBM by intracerebroventricular(i.c.v.)injection to measure the food intake of mice.The c-Fos expression in hypothalamus neuron was assayed by immunofluorescence.7.The expression of genes or proteins related to leptin pathway,endoplasmic reticulum stress and autophagy in GT1-7 cells and hypothalamy of DIO mice were assayed by Western Blot and q PCR.To explore the mechanism of BBM reducing food intake,autophagy flow was observed by m RFP-GFP-LC3,and the interaction between Rab8a and Jak-Stat3 was explored by immunoprecipitation.Results:1.Rab8a GTPase activity model was established using purified protein from HEK293 cells.By screening more than 700 small molecule compounds from Chinese medicinal herbs,three compounds,Berbamine Hydrochloride,Oleanolic acid and Ursolic acid,that can inhibit the activity of Rab8a GTPase were obtained,and the inhibitory effects of the compounds on Rab8a GTPase activity were in dose-dependent manner.BBM with the strongest inhibitory effects was selected to study its efficacy and mechanism.2.In Rab8a knockout 293 T cells,BBM did not show significant inhibitory effect on protease activity.3.BBM and Rab8a-/-inhibited the differentiation of 3T3-L1,reduced lipid droplets in adipocytes,and inhibited lipid accumulation in L02 and Hep G2 cells induced by oleic acid.4.In c.elegans,BBM reduced the lipid content without affecting the lifespan,brood size and diet of the worms.5.BBM reduced the body weight,weight of abdominal white adipose tissue,size of fat cells and food intake of DIO mice,increased oxygen consumption and carbon dioxide emission,reduced fasting blood glucose,improved glucose tolerance and insulin resistance,lowered the levels of serum ALT,TG,reduced the liver weight and liver TC,TG contents,blocked the liver ballooning change.BBM also reduced body weight and food intake,improved glucose tolerance and adipocyte size in db/db mice.6.BBM reduced food intake and increase the number of C-Fos positive neurons in the paraventricular nucleus(PVH)of DIO mice by i.c.v.injection.7.BBM increased p-Stat3 levels in GT1-7 cells and the hypothalamic tissue of DIO mice,reduced ER stress markers Chop and Grp78 levels and blocked the degradation of autophagosomes.In Rab8a knockout GT1-7 cells,the results are same as the effect of BBM.However,the immunoprecipitation test showed that there was no interaction between Rab8a and Jak2/Stat3.Conclusion:Three Rab8a GTPase inhibitors were obtained by screening of more than 700 compounds from Chinese medicinal herbs.BBM specifically inhibited the activity of Rab8a GTPase,and blocked adipocyte differentiation,lipid droplet fusion and lipid accumulation in hepatocytes.BBM improved obesity,glucose and lipid metabolic disorders in DIO mice and db/db mice,and inhibited feeding behavior by activating hypothalamic feeding neurons,regulating neuronal autophagy,reducing neuronal endoplasmic reticulum stress,increasing leptin pathway sensitivity.Our results suggest that BBM may reduce body weight via the inhibition of Rab8a mediated leptin signaling and feeding behavior.