Characterization of Chinese medicine celastrol and celastrol-loaded biodegradable nanoparticles towards the therapy of obesity and non-alcoholic fatty liver disease

Abstract

Obesity and non-alcoholic fatty liver disease (NAFLD) share the common pathological hallmarks including aberrant lipid deposition, sustained chronic inflammation and metabolic dysfunctions. Celastrol is a pentacyclic triterpene isolated from herbal medicine Tripterygium wilfordii. Previous studies suggest that celastrol could be a lipid-lowering and anti-inflammatory drug. However, the poor solubility, limited oral bioavailability and possible side effects largely hamper its clinical application. Thus, this thesis was designed to pursue two major aims: 1) to identify the primary targets of celastrol and then characterize the molecular mechanisms of celastrol-protein covalent conjugates for regulating proinflammatory response and lipid metabolism against NAFLD; 2) to develop biodegradable nanoparticles for improving the bioavailability and liver-targeting ability of celastrol towards the therapy of obesity and NAFLD. Firstly, we investigated whether celastrol formed covalent conjugates with cellular proteins to ameliorate inflammation via skewing macrophage M1/M2 polarization.We identified pyruvate kinase M2 (PKM2) as the primary celastrol-bound protein and identified the residue Cys31 as the covalent binding site for celastrol. Celastrol inhibited the enzymatic activity and nuclear translocation of PKM2 and abolished the binding of PKM2 with HIF-1α. Mechanistic studies demonstrated that celastrol downregulated the expression of glycolysis-related proteins, suppressed the Warburg effects and skewed macrophage M1/M2 polarization both in vitro and in vivo. Collectively, celastrol ameliorated hepatic steatosis, inflammation and fibrosis in NAFLD mice by skewing hepatic macrophage M1/M2 polarization via covalent inhibition of PKM2 to suppress glycolysis and PKM2-HIF-1α signaling pathway in the induction of glycolytic enzymes and inflammatory mediators. Secondly, we prepared and evaluated the celastrol-loaded bovine serum albumin nanoparticles (BSA/Cela) for improving the intestinal absorption, oral bioavailability and therapeutic efficacy of celastrol in obese mice. BSA/Cela displayed better water-solubility, intestinal absorption and oral bioavailability than free celastrol. In animal experiments, compared with free celastrol, BSA/Cela not only more effectively reduced lipid accumulation and inflammation, but also outperformed in improving glucose metabolism and insulin resistance in obese mice. Thus, BSA/Cela may be an effective clinical drug candidate against obesity. Thirdly, we developed the celastrol-loaded lactosylated bovine serum albumin nanoparticles (Lac-BSA/Cela) for delivering celastrol into hepatocytes and evaluated the therapeutic potential in NAFLD mice. Compared with free celastrol and BSA/Cela, Lac-BSA/Cela showed best hepatocyte uptake and most effectively decreased lipid deposition in hepatocytes. Consistently, animal experiment results demonstrated that Lac-BSA/Cela was the best in hepatic deposition, reduction ofhepatic steatosis, and improvement of metabolic homeostasis as well as liver function. Mechanistic studies showed that Lac-BSA/Cela outperformed free celastrol and BSA/Cela in downregulating lipogenesis and lipid transportation while upregulating lipolysis. Western blot analysis confirmed that Lac-BSA/Cela most effectively activated the AMPK and SIRT1 and subsequently reduced the expression levels of FASN and SREBP1c to inhibit lipogenesis. Collectively, LacBSA/Cela achieved higher hepatocyte uptake and better liver-targeting ability, thus enhancing the lipid-lowering efficacy of celastrol against NAFLD. In conclusion, covalent inhibition of PKM2 by celastrol may reprogram the metabolic and inflammatory pathways in hepatic macrophages against NAFLD. In addition, the results of this thesis may help translate celastrol-loaded albumin nanoparticles into potential clinical drugs against obesity and NAFLD.