Effect Of Triptolide On Repolarization Of Mouse Peritoneal Macrophages

Purpose:Through the mouse macrophage model in vitro,we observed the regulation of triptolide（TP）on macrophage polarization and repolarization,so as to provide a new idea for the study of triptolide immunotherapy with macrophages as the target.Methods:1.Mice were intraperitoneally injected with 0.25ml of 3%sodium mercaptoacetate to stimulate the production and extraction of peritoneal macrophages.2.Grouping:according to the added reagent,the cultured cells were divided into control group（including blank group,LPS group and IL-4 group）,TP group(treated with triptolide of different concentrations(100 ng/ml,50 ng/ml,25 ng/ml,including TP₁₀₀,TP₅₀,TP₂₅ groups)and RP group (macrophages were induced by IL-4 for 24 hours and then induced by Triptolide at different concentrations（100 ng/ml,50 ng/ml and 25 ng/ml）for 48 hours,including RP₁₀₀,RP₅₀ and RP₂₅groups).3.The morphological changes of macrophages in each group before and after culture were observed by inverted microscope.4.Collect the cells of each group,and analyze the proportion of macrophage polarization markers M1（CD68⁺CD86⁺）and M2（CD68⁺CD206⁺）by flow cytometry.5.Collect the supernatant of macrophages in each group,and detect and analyze the contents of IL-12 and IL-10 in the macrophage supernatant of each group by ELISA.Results:1.The results of microscopic morphological observation showed that after co culture with LPS,IL-4 and TP stimulation of mouse peritoneal macrophages for 72 hours,most of the cells gradually extended pseudopodia from the initial round and oval to irregular polygonal or long spindle cells.2.The results of flow cytometry showed that the proportion of M2 macrophages（CD68⁺CD206⁺）in IL-4 group was significantly higher than that in blank group,LPS group,TP group and RP group（P<0.05）;The proportion of M1 macrophages（CD68⁺CD86⁺）in LPS group,TP group and RP group was significantly higher than that in blank group and IL-4 group（P<0.05）.When the TP concentration was 100 ng/ml,the proportion of M2 macrophages（CD68⁺CD206⁺）in TP group and RP group was higher than that in blank group（P<0.05）,but lower than that in M1 macrophage（P<0.05）.3.The results of ELISA showed that the content of IL-10 in the supernatant of IL-4 group was significantly higher than that of blank control group,IL-4 group and LPS group（P<0.05）;The content of IL-12 in LPS group,TP group and RP group was higher than that in blank control group and IL-4 group（P<0.05）.The content of IL-12 in macrophage supernatant of LPS group,TP group and RP group was significantly higher than that of IL-10（P<0.05）.Conclusions:TP can induce macrophages to polarize more towards M1 macrophages;At the same time,it can make IL-4-induced M2 macrophages repolarize more to M1 macrophages.