Objective The high post-surgery recurrence rate of endometriosis (EMs) has emerged as a challenge in the long-term manaagement of the condition. This study is aimed at investigating the mechanisms of Neiyiting (NYT) decoction in preventing postoperative recurrence of EMs.

Methods An animal model of EMs postoperative recurrence and a model of endometrial stromal cells (hEM15A) cocultured with macrophages (RAW 264.7 cell line) were established for both in vivo and in vitro experiments. An autotransplantation method was used to establish a rat model of EMs. The rats were divided into 4 groups (6 rats per group) and received the corresponding treatments: a Model group receiving distilled water, a Gestrinone group receiving gestrinone at 0.325 mg/kg, a low-dose NYT (NYT-L) group receiving NYT decoction at 5.04 g/(kg·d), and a high-dose NYT (NYT-H) group receiving NYT decoction at 10.08 g/(kg·d). The treatment was administered for 3 weeks via intragastric gavage. In addition, 6 SD rats were randomly selected for the control group (Control group), and were given distilled water for 3 weeks via intragastric gavage. The sizes and pathological changes of recurrent lesions in EMs rats were observed. Immunohistochemistry and qRT-PCR were performed to assess the expression of M1 macrophage marker CD86 protein and mRNA in vivo. Additionally, immunohistochemistry and qRT-PCR were used to assess the expression of indicator proteins related to the triggering receptor expressed on myeloid cells 1 (TREM1)/Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway and mRNA. The proliferation of hEM15A cells in the coculture experiment was observed. Flow cytometry was performed to determine the polarization of RAW264.7 macrophages, and qRT-PCR was used to determine the expression levels of inducible nitric oxide synthase (iNOS) and interleukin 1β (IL-1β) mRNA. Western blot was performed to determine the expression of signaling pathway-related indicator proteins in vitro. ELISA was performed to determine the levels of inflammatory factors in vitro.

Results Compared with the Model group, the volume of recurrent lesions in the NYT-H group was reduced (P < 0.01). Findings from the macrophage M1 polarization assessment showed that the expression levels of CD86 protein and mRNA in the recurrent lesions of the Model group were higher than those in the control group (P < 0.01). The expression levels of CD86 protein and mRNA in the recurrent lesions of the NYT-H group were lower than those of the Model group (P < 0.01). In addition, the RAW 264.7 cell experiment further verified that NYT decoction could reduce the number of CD86-positive macrophages induced by plasmids overexpressing TREM1 and reduce the expression of IL-1β and iNOS mRNA (P < 0.01). The results of the hEM15A cell proliferation assay showed that NYT decoction down-regulated KI-67 protein expression in hEM15A cells induced by macrophage M1 polarization (P < 0.01). The results of TREM1/TLR4/NF-κB signaling pathway showed that the protein and mRNA expression levels of TREM1, TLR4, and NF-κB in the recurrent lesions of the Model group were higher than those of the control group (P < 0.01). Compared with those in the Model group, the protein and mRNA expression levels of TREM1, TLR4, and NF-κB in the recurrent lesions of the NYT-H group were lower (P < 0.01). In addition, the coculture experiment of RAW264.7 and hEM15A cells further confirmed that NYT decoction reduced the expression of TREM1, TLR4, and P-P65 proteins (P < 0.01).

Conclusion NYT decoction can inhibit macrophage M1 polarization through the TREM1/TLR4/NF-κB signaling pathway, improve the inflammation level, and inhibit the formation of ectopic endometrial lesions, thereby preventing postoperative recurrence of EMs.