Mechanism of Dahuang Zhechong Pill in Regulating Th17/Treg Balance for Endometriosis with Blood Stasis Syndrome Based on the IL-6/STAT3 Pathway

OBJECTIVE To explore the potential molecular mechanism of Dahuang Zhechong Pill in treating endometriosis(EMs) with blood stasis syndrome by regulating Th17/Treg balance in ectopic endometrial tissue through the IL-6/STAT3 pathway.

METHODS Sixty female SD rats were randomly divided into a sham operation group, a model control group, a positive control group(dienogest), and high, medium, and low dose groups of Dahuang Zhechong Pill according to a random number table method, with 10 rats in each group. A blood stasis syndrome EMs rat model was established using autologous transplantation combined with injection of adrenaline hydrochloride. After 14 d of administration, the general condition of each group of rats was observed, and then the rats were euthanized. The volume of ectopic endometrial tissue was measured, and the blood routine parameters, hemorheological indices, and four coagulation testsprothrombin time(PT), activated partial thromboplatintime(APTT), thrombin time(TT) and fibrinogen(FIB) were determined. Pathological changes in ectopic endometrial tissue were observed, and serum levels of sex hormones, inflammatory factors, p-STAT3 protein expression, IL-6, STAT3, RORγt, and Foxp3 protein and mRNA expression in ectopic endometrial tissue were detected.

RESULTS Compared with the sham operation group, the model control group exhibited dark purple tongue color and disheveled fur, showed significantly enlarged ectopic endometrial tissue volume, and had histopathological findings consistent with the characteristics of ectopic endometrial tissue; both neutrophil lymphocyteratio(NLR) and systemic immune inflammation index(SII) values were significantly elevated(P<0.01); its whole blood viscosity and plasma viscosity were significantly increased(P<0.01); the PT, APTT, and TT values were significantly decreased(P<0.01), while the FIB value was significantly increased(P<0.01); serum E₂, TNF-α, IL-17, and IL-6 levels were significantly increased(P<0.01), while IL-10 and TGF-β1 levels were significantly decreased(P<0.01). The expression of p-STAT3 protein, IL-6 and RORγt protein and mRNA, and STAT3 mRNA in ectopic endometrial tissue were significantly increased(P<0.01), whereas Foxp3 protein and mRNA expression were significantly decreased(P<0.01). Compared with the model control group, the positive control group and the high- and medium-dose Dahuang Zhechong Pill groups showed significant improvement in their condition, with a significant reduction in the volume of ectopic endometrial tissue (P<0.01) and varying degrees of improvement in pathological damage; the NLR and SII values significantly decreased(P<0.01); whole blood viscosity and plasma viscosity were significantly decreased(P<0.01); the PT, APTT, and TT values significantly increased(P<0.01), while the FIB value significantly decreased(P<0.01); serum E₂, TNF-α, IL-17, and IL-6 levels were significantly decreased(P<0.01), while IL-10 and TGF-β1 levels were significantly increased(P<0.01); the expression of p-STAT3 protein, IL-6 and RORγt protein and mRNA, and STAT3 mRNA in ectopic endometrial tissue were significantly reduced(P<0.05 or P<0.01), whereas Foxp3 protein and mRNA expression (except for the positive control group) were significantly increased(P<0.05 or P<0.01).

CONCLUSION Dahuang Zhechong Pill may have a therapeutic effect on endometriosis with blood stasis syndrome by intervening in key proteins of the IL-6/STAT3 signaling pathway, regulating Th17/Treg balance in ectopic endometrial tissue.