Dingxin Recipe Ⅲ Promotes White Adipose Tissue Browning In Obese Mice Through The AMPK/SIRT1 Pathway

Background and objective:With the rapid development of the economy,the living standard of residents has improved,and the changes in lifestyle and diet structure have led to a continuous increase in the number of obese people in the world.Therefore,it is indispensable to explore potential novel,safe,and effective avenues to treat obesity for ensuring health and reducing medical burden.Dingxin Recipe Ⅲ(DXR Ⅲ)is used in the treatment of glucose and lipid metabolism disorders such as atherosclerosis and non-alcoholic fatty liver disease clinically.Recent studies have shown that promoting the conversion of white adipose tissue(WAT)to brown adipose tissue(BAT)is beneficial to reduce obesity and obesity-related metabolic disorders.This study aims to investigate whether DXR Ⅲ can promote the browning of WAT and reveal its underlying mechanism,thereby providing theoretical basis for the treatment of adiposity and metabolic disorders associated with obesity.Methods:1.Western diet-fed ApoE-/-mice were used to establish the obesity model.Body weight was monitored monthly.The fasting blood glucose level was measured by tail cutting method.Serum TC,TG,LDL-C and HDL-C,serum ALT and AST,liver TC,TG and NEFA were measured with biochemical kits.HE,Oil red O staining and sirius red staining were used to observe the pathological changes of aortic sinus,liver and adipose tissue.The mRNA expression of IL-6,IL-β,iNOS and Arg-1 in WAT were analyzed by qPCR.Protein levels of lipid synthesis and differentiation regulatory factors SREBP-1c,FAS and CEBP/α in WAT were detected by WB.The expression of brown adipocyte-specific marker UCP1 in WAT was detected by IHC and WB.White adipocyte-specific marker ASCI and browning regulator PPAR-γ in WAT were detected by WB.The expression levels of AMPK,p-AMPK and SIRT1 in WAT were detected by WB.2.In vitro,cocktail-induced 3T3-L1 preadipocytes were used for subsequent experiments.The cytotoxicity of 5%and 10%DXR Ⅲ medicated serum on 3T3-L1 preadipocytes was analyzed by CCK-8 kit.The lipid deposition in adipocytes was detected by Oil red O staining and intracellular TG content.Protein levels of lipid synthesis and differentiation regulatory factors SREBP-lc,FAS,CEBP/α in adipocytes were detected by WB.The expression of brown adipocyte-specific marker UCP1 in adipocytes were detected by IF and WB;White adipocyte-specific marker ASC1,browning regulator PPAR-γ and mitochondrial transcription factors PGC-1α,NRF2 and TFAM were measured by WB;Mitochondrial content and ROS production of adipocytes were analyzed by mitochondrial staining and DCFH-DA staining.The effect of 10%DXR Ⅲ medicated serum on brown-related phenotype and AMPK/SIRT1 signaling pathway was determined after the intervention of AMPK inhibitor Compound C.Results:1.(1)DXR Ⅲ reduced the body weight gain,fasting blood glucose,serum TC,TG,LDL-C and HDL-C levels,inhibited the deposition of TC,TG and NEFA in the liver,and suppressed the activity of ALT and AST in the liver.The pathological changes of aortic and liver caused by western diet were alleviated.(2)DXR Ⅲimproved the pathological morphology of adipose tissue,inhibited the mRNA expression of IL-6,IL-β and iNOS,increased the mRNA expression of Arg-1.DXRⅢ downregulated the protein levels of lipid synthesis and differentiation regulator SREBP-1c,FAS,CEBP/α in WAT.(3)Following DXR Ⅲ administration,the expression of brown adipocyte-specific marker UCP1 and WAT browning regulator PPAR-y were increased and white adipocyte-specific marker ASC1 was decreased.(4)DXR Ⅲ augmented the activation of AMPK/SIRT1 signaling pathway in WAT.2.(1)5%and 10%DXR Ⅲ medicated serum had no toxicity to adipocytes.(2)DXR Ⅲ medicated serum inhibited intracellular lipid accumulation and lipid synthesis factors SREBP-1c,FAS,CEBP/α in adipocytes.(3)The brown adipocyte-specific marker UCP1 and regulators of brown remodeling PPAR-γ were increased and white adipocyte-specific marker ASC1 was decreased after treatment with DXR Ⅲ medicated serum.Mitochondrial content in 3T3-L1 adipocytes and mitochondrial transcription factors PGC-1α,NRF2 and TFAM were upregulated while ROS accumulation decreased following DXR Ⅲ medicated serum treatment.(4)AMPK inhibitor Compound C reversed the effect of 10%DXR Ⅲ medicated serum on browning phenotype and AMPK/SIRT1 signaling pathway.Conclusion:Dingxin Recipe Ⅲ promotes white adipose tissue browning in obese mice through the AMPK/SIRT1 pathway.