宫粘方通过调控巨噬细胞极化与TGF-β1/CTGF通路改善宫腔粘连大鼠内膜纤维化的机制OA

Objective To investigate the therapeutic effect and mechanism of Gongzhan Formula on intrauterine adhesions(IUA)based on macrophage polarization and the transforming growth factor-β1(TGF-β1)/connective tissue growth factor(CTGF)pathway.Methods A rat model of intrauterine adhesions was established by"mechanical injury plus lipopolysaccharide infection".(1)In vivo experiment:Forty-five female rats were randomly divided equally into sham operation group,model group,and Gongzhan Formula group(all at 14 g·kg-1),and treated by intragastric gavage once daily for 28 days.The above three groups were further randomly divided into 5 subgroups(n=3 each),and rats were sacrificed on days 1,3,5,10,and 28 after modeling to isolate endometrial tissues.Endometrial fibrosis was assessed by Masson staining.TGF-β1 expression in endometrial tissues was detected by immunohistochemistry.(2)In vitro experiment:Six IUA rats at day 1 post-modeling were used to isolate and culture macrophages and stromal cells.Cells were cultured with Gongzhan Formula-containing serum(24 μg·mL-1)and assigned as macrophage+drug-containing serum group and stromal cell+drug-containing serum group,while those cultured with blank serum were assigned as macrophage group and stromal cell group.Immunofluorescence was employed to detect the expression of macrophage polarization markers CD86,CD68,CD163,and CD206.qPCR was used to measure TGF-β1 mRNA expression in macrophages and the mRNA expression of CTGF,COL1A2,FN,and α-SMA in stromal cells.Results(1)Compared with the sham operation group,the model group exhibited significantly enhanced and progressively aggravated endometrial fibrosis from day 1 to 28(P＜0.01),and significantly increased TGF-β1 expression(P＜0.01).Compared with the model group,the Gongzhan Formula group showed significantly reduced endometrial fibrosis(P＜0.05,P＜0.01)and significantly decreased TGF-β1 expression(P＜0.05,P＜0.01)at all time points.(2)In the endometrium of IUA rats,M2(CD163+)macrophage expression was markedly stronger than M1(CD86+),indicating successful model replication.Compared with the macrophage group,the macrophage+drug-containing serum group showed significantly decreased M1(CD86+)macrophages(P＜0.01),with no significant change in M1(CD68+)(P＞0.05),significantly reduced M2(CD163+,CD206+)macrophage expression(P＜0.01),and significantly downregulated TGF-β1 mRNA expression(P＜0.01).Compared with the stromal cell group,the stromal cell+drug-containing serum group exhibited significantly downregulated mRNA expression of fibrosis-related factors CTGF,COL1A2,FN,and α-SMA(P＜0.05,P＜0.01).Conclusion Gongzhan Formula can ameliorate endometrial fibrosis in IUA rats,and the mechanism may be associated with inhibiting excessive polarization of M1 and M2 macrophages,downregulating the TGF-β1/CTGF pathway,reducing myofibroblast transformation,and decreasing extracellular matrix synthesis.