The Effect Of Celastrol On Polarization Of Mouse Peritoneal Macrophage And The Initial Exploration Of Its Controlling Mechanism

Currently,metabolic diseases are the most common non-contagious diseases which are a major threat to people's health.Although the exact mechanism of these metabolic diseases is still unclear,an increasing evidences suggest that overactivation of inflammation is involved.Inflammatory infiltration of macrophages and other immune cells is a major feature of the inflammation environment.Phenotypic/functional plasticity and heterogeneity are two main characteristics of macrophages.Therefore,dependent on the corresponding extracellular environmental stimulations,macrophages can be grouped as classically activated M1 macrophages,which have proinflammatory function,and alternatively activated M2 macrophages with anti-inflammatory capabilities.Differential regulation of macrophage phenotypes has a profound impact on the development of these metabolic diseases.Celastrol is a natural quinine methide pentacyclic triterpenoid isolated from the root bark of Tripterygium wilfordii,a perennial creeping plant indigenous to areas in southern China.Celastrol has shown anti-inflammatory effects in several animal research models,and thus it is generally used to treat chronic inflammation,autoimmune diseases and neurodegenerative diseases.Whether celastrol can exert anti-inflammatory effect by regulating the polarization of mouse peritoneal primary macrophages still needs to be further investigated.Sirtl(Silent mating type information regulation 2 homolog 1)is an evolutionally highly conserved NAD+ dependent protein deacetylase that regulates metabolism.Recent findings have identified the critical roles of Sirtl in glucose-lipid metabolism in multiple tissues.Previous studies in our laboratory have shown that celastrol ameliorates mouse liver metabolic damages such as inflammation caused by a high-fat diet through over expression of Sirtl.Heme oxygenase is an essential enzyme in heme catabolism and it has three types of isozymes:HO-1(oxidative stress inducible type),HO-2(constitutive type)and HO-3(not yet clear).It has been proved that HO-1 has important functions in antioxidant defence,anti-inflammatory and anti-apoptotic aspects.Previous studies in our laboratory have shown that overexpression of Sirtl can induce the expression of HO-1,thereby exerting an antioxidant effect.It is still a question whether celastrol can exert anti-inflammatory effect to ameliorate metabolic damage by regulating the polarization of macrophages through Sirtl and HO-1.The following experiments were carried out to address this issue.Firstly,we use the remaining freezing liver tissues of WT(ND/HFD;con/cel)and Sirt1-/-(ND/HFD;con/cel)mice to conduct a preliminary experiment.After extracting RNA from the above liver tissues,the transcriptional levels of related genes for macrophage polarization were detected by qRT-PCR technique.The results show that celastrol can enhance the mRNA levels of related biomarkers of M2 macrophages vastly in the liver tissues of WT mice,but it decreases the mRNA expressions of related biomarkers of M2 macrophages in the liver tissues of Sirt-/-mice.This result indicates that celastrol has effects on macrophage polarization and the mechanism may be related to Sirtl gene.Then we take mouse peritoneal macrophages isolated by intraperitoneal injection of broth(stimulation method)as the main research object.Flow cytometry and qRT-PCR technique were used to detect related markers'expressions of M1 and M2 macrophages in con,cel,IFN-y+LPS and IFN-y+LPS+cel group.The results of qRT-PCR experiments show that celastrol can decrease the expressions of revelant markers CD11c and iNOS of M1 macrophages and can enhance the expressions of revelant marker Arg-1 of M2 macrophages.The results of flow cytometry show that celastrol can reduce the proportions of F4/80+CDllc+ and CD11c+CD206-Ml macrophages.These results suggest that celastrol can suppress the M1 macrophage polarization of mouse peritoneal macrophages.The subsequent results show that EX-527,the Sirtl inhibitor,can decrease the mRNA expressions of related biomarkers of M2 macrophages,and the proportion of Ml macrophages in cel+EX-527 group increases.Besides,the results of Western blot experiments show that celastrol can enhance the expressions of Sirtl protein and HO-1 protein in both mouse peritoneal macrophages and Raw264.7 cells.In the adipose tissue inflammation model,which we build by mature 3T3-L1 adipocytes and Raw264.7 cells,we find that celastrol can also enhance the protein expressions of Sirtl and HO-1,and the protein expressions of Sirtl and HO-1 decrease after EX-527 treatment.All these results indicate that celastrol can regulate mouse peritoneal macrophages polarization through Sirtl and HO-1.This may have significance for people to find a way to ameliorate macrophage infiltrated inflammatory environment.