Novel Oleanane Triterpenoids Regulate Macrophage Polarization And The Effects On Obesity-associated Metabolic Inflammation

Obesity has become the global healthy challenge,which not only influence the figure and appearance of human but also increase the risk of various chronic disease,including type 2 diabetes,cardiovascular diseases,and multiple forms of cancer.Adipose contains a large amount of innate and adaptive immune cells constituting a complex immune microenvironment.In obesity state,the stress of excess lipid beyond the limit of energy storage in adipocyte induce the changes of gene expression of hypertrophy of adipocytes,which secret a variety of pro-inflammatory cytokines and chemokines.Under the changes of secretion,the immune cells composition and function are remodeled,the classical activated macrophages infiltrate in the adipose,which break the balance of immune system maintaining the homeostasis of adipose tissue.With the apoptosis of a large amount of adipocytes,the inflammation in adipose is aggravated.Aside from persistence of initiating stimuli of lipid,the inflammation in adipose tissue become a type of nonresolving inflammation,which further contribute to the metabolic disorders and inflammation of liver and muscle.The synthetic pentacyclic oleanane triterpenoids(SOs)have uniquely potent ability to control inflammation,oxidative stress and cancer.SOs also have the activity of alleviating metabolic disease.Bardoxolone Methyl(CDDO-Me,trade name:BEACON)is a kind of SOs,which is used to treat diabetic nephropathy.For its excellent activity in preclinical trails,CDDO-Me was be approved to apply to phase III clinical trial.Unfortunately,the trial was terminated,due to the several side effects of CDDO-Me in human beings.Therefore,to discover novel SOs with low side effect and toxicity appear to be very important.Hereby,we screened the anti-inflammatory activity of two novel SOs(SO 1989 and SO1108)which were synthesized by Prof.Sun Hongbing’s team of China Pharmaceutical University with our cooperation.We found that SO1989 has the similar anti-inflammatory activity with CDDO-Me.In addition,We also studied effects of SO1989 on improving obesity-associated metabolic inflammation in mice and regulating the phenotypes of macrophages.The main research contents and results are shown as follows:Ⅰ.Screen and evaluation the anti-inflammation activity of novel oleanane triterpenoids1.Anti-inflammation activity screen in macrophage classical activation model in vitro:RAW264.7 cells were stimulated by LPS and IFN-y with the drugs pretreatment.Then the real-time PCR was used to analyze the expression of pro-inflammatory genes and flow cytometry was applied to detect the ROS content in RAW264.7.Based on the data,we screened SO1989 from the two novel SOs which have the similar anti-inflammation activity with CDDO-Me.2.Assessment of anti-inflammation activity in vivo:BALB/c mice were treated with LPS 12h following SO1989 and CDDO-Me pre-treatment.By detecting the inflammatory markers in serum and the phenotypes changes of peritoneal macrophage,we found that SO1989 had parallel anti-inflammation activity in vivo.Ⅱ.The influences of SO1989 on obesity-associated metabolic inflammation1.The effects of SO1989 on metabolic disorders and inflammation in vivo:C57BL/6J mice were fed with high fat diets containing 60%fat 20 weeks,CDDO-Me and S01989 were treated orally.The glucose tolerance test(GTT)and metabolites detection in serum showed that SO1989 had the similar activity on improving insulin sensitivity and metabolic disorder state.By analyzing the inflammatory markers in serum,we concluded that SO1989 could also improve inflammatory state in whole body 2.The influences of SO1989 on the lipid metabolism and inflammatory cells infiltration in major metabolic organs:Through histological pathology analysis,we found that SO1989 could significantly improve lipid infiltration of liver and skeletal muscle and significantly reduce the infiltration of inflammatory cells in adipose tissue.In addition,using quantitative PCR to detect pro-inflammatory cytokine gene expression of adipose and oxidative metabolism-related gene expression of liver,we found that SO1989 can reduce pro-inflammatory cytokine gene expression of adipose,and up-regulate oxidative metabolism-related gene expression of liver.3.Comparison of side effects of SO1989 and CDDO-Me:Consulted the phase Ⅲclinical literature,CDDO-Me was determined that it had the side effects of lowering body weight,reducing fat content,tamping down appetite,and so on.Through the daily monitoring of food intake and body weight in obese mice,we concluded that SO1989 had no effect on obese mice compared to CDDO-Me,which influenced body weight and appetite.III.Regulation of macrophage polarization by SO19891.Regulation of macrophage polarization by SO1989:By means of the analysis of peritoneal macrophages(PMs)and adipose tissue macrophages(ATMs)phenotype changes in obese mice,we determined that SO1989 was able to reduce the classical activation and increased alternative activation of macrophages,but CDDO-Me can only inhibit the classical activation of macrophages.2.Study the effects of SO1989 on macrophages polarization in vitro:We established adipocytes macrophages co-culture model,together with the IL-4 induced alternative activation of ANA-1 model.Through detecting macrophage phenotype by flow cytometry and quantitative PCR,we found that SO1989 was different from CDDO-Me in regulating macrophage polarization.