清热化瘀方调控巨噬细胞极化影响子宫内膜异位症进展的作用机制OA

Objective To investigate the mechanism by which the Heat-Clearing and Stasis-Resolving Formula(HCSRF)affects the progression of endometriosis(EMs)through regulating macrophage polarization.Methods Female SD rats were randomly divided into five groups(n=6 per group):the HCSRF group(granules,3.6 g·kg-1),the Heat-Clearing group(granules,0.72 g·kg-1),the Stasis-Resolving group(granules,1.08 g·kg-1),the gestrinone group(0.45 g·kg-1),and the blank control group,to prepare drug-containing serum or blank serum.Ectopic lesion samples were collected from clinical EMs patients,and primary human ectopic endometrial stromal cells(HESCs)were isolated and extracted.THP-1 cells were induced with 200 ng·mL-1 PMA for 24 hours to differentiate into M0 macrophages.A HESCs-macrophage co-culture model was established.M0 macrophages were induced with 20 ng·mL-1 IL-4+20 ng·mL-1 IL-13 for 48 hours to become M2 macrophages,with simultaneous intervention by drug-containing serum.Cell proliferation activity was detected by the CCK-8 assay.Expression levels of the M1 macrophage surface marker CD80 and the M2 macrophage surface marker CD206 were measured by flow cytometry.mRNA expression levels of TGF-β,TNF-α,IL-10,and IL-6 in macrophages were detected by RT-qPCR.Protein expression of estrogen receptor β(ERβ)in macrophages was detected by Western Blot.Results(1)The inhibitory effects of HCSRF-,Heat-Clearing-,and Stasis-Resolving-drug-containing serum on the proliferation activity of HESCs in the co-culture model showed a certain concentration dependence.The inhibitory effect of 20%HCSRF-containing serum was superior to that of 20%Heat-Clearing-or Stasis-Resolving-containing serum(P＜0.001).Therefore,a 20%concentration of drug-containing serum was used for subsequent experiments.(2)Compared with the 20%blank serum group,the expression proportions of the M1 marker CD80 and the M2 marker CD206 were significantly decreased in the 20%gestrinone-,20%HCSRF-,20%Stasis-Resolving-,and 20%Heat-Clearing-containing serum groups(P＜0.001).The CD206/CD80 ratio was significantly decreased in the 20%HCSRF-and 20%Stasis-Resolving-containing serum groups(P＜0.001).In the 20%HCSRF-containing serum group,mRNA expression levels of the M1-secreted factors IL-6 and TNF-α,as well as the M2-secreted factors IL-10 and TGF-β,were all significantly decreased(P＜0.001).In the 20%gestrinone-,20%Heat-Clearing-,and 20%Stasis-Resolving-containing serum groups,mRNA expression levels of TNF-α,IL-10,and TGF-β in macrophages were significantly decreased(P＜0.05,P＜0.01,P＜0.001).ERβ protein expression in macrophages was significantly downregulated in the 20%HCSRF-and 20%gestrinone-containing serum groups(P＜0.05).(3)Compared with the 20%HCSRF-containing serum group,the expression proportions of CD80 and CD206,as well as the CD206/CD80 ratio,were significantly increased in the 20%gestrinone-,20%Stasis-Resolving-,and 20%Heat-Clearing-containing serum groups(P＜0.001).In the 20%gestrinone-containing serum group,mRNA expression levels of IL-6,IL-10,TNF-α,and TGF-β in macrophages were significantly increased(P＜0.05,P＜0.01,P＜0.001).In the 20%Heat-Clearing-containing serum group,mRNA expression levels of IL-10 and TNF-α in macrophages were significantly increased(P＜0.05).In the 20%Stasis-Resolving-containing serum group,mRNA expression levels of IL-6,TNF-α,and TGF-β in macrophages were significantly increased(P＜0.01,P＜0.001).(4)Compared with the 20%Heat-Clearing-containing serum group,the expression proportions of CD80 and CD206,as well as the CD206/CD80 ratio,were significantly decreased in the 20%Stasis-Resolving-containing serum group(P＜0.001).The mRNA expression level of IL-10 was significantly decreased(P＜0.05),while that of TNF-α was significantly increased(P＜0.001).Conclusion HCSRF-containing serum can inhibit the polarization of macrophages towards the M2 phenotype in the HESCs-macrophage co-culture microenvironment,regulate the M2/M1 cell dynamic balance,thereby inhibiting the progression of EMs caused by HESCs proliferation.This effect may be related to the downregulation of ERβ protein expression.Furthermore,the inhibitory effect of HCSRF-containing serum is superior to that of Heat-Clearing-or Stasis-Resolving-containing serum alone.