ObjectiveTo investigate the mechanism by which Luoshi Neiyi prescription treats endometriosis （EMs） through the Toll-like receptor 4 （TLR4）/nuclear factor-κB （NF-κB） signaling pathway. MethodsAnimal experiments were conducted with 50 female SD rats， which were randomized into a sham operation group （10 rats） and a modeling group （40 rats）. An autologous endometrial transplantation method was used for the modeling of EMs. The 36 successfully modeled rats were randomly allocated into four groups （n=9 each）： The model group， the low-dose （7.87 g·kg^-1） Luoshi Neiyi prescription group， the high-dose （15.74 g·kg^-1） Luoshi Neiyi prescription group， and the dienogest （0.20 mg·kg^-1） group. The physiological status and ectopic lesion volumes of rats in each group were observed. Hematoxylin-eosin （HE） staining was used to observe the morphology of the eutopic endometrial tissue. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of inflammatory factors including interleukin-1β （IL-1β） and prostaglandin E₂ （PGE₂） in the serum of EMs rats. Immunohistochemistry was used to detect the expression of matrix metalloproteinase-9 （MMP-9） and vascular endothelial growth factor A （VEGFA） in the eutopic endometrial tissue. Western blot was adopted to determine the protein levels of TLR4， myeloid differentiation factor 88 （MyD88）， phosphorylated nuclear factor-κB （p-NF-κB）/NF-κB， MMP-9， and VEGFA in the eutopic endometrial tissue. In the cell experiments， the cell-counting kit-8 （CCK-8） assay was employed to screen the optimal concentration of Luoshi Neiyi prescription-containing serum， and a scratch assay was performed to assess the migration ability of iheESCs cells. Interventions with Luoshi Neiyi prescription-containing serum， resatorvid （TAK-242， a TLR4 inhibitor）， and lipopolysaccharides （LPS， a TLR4 agonist） were conducted， and Western blot was used to detect the expression of proteins related to the TLR4/NF-κB signaling pathway. ResultsThis experiment successfully replicated 36 EMs rat models. Compared with the sham operation group， the model group exhibited visible ectopic lesions on the abdominal wall and an increase in the writhing response score （P<0.01）. Furthermore， HE staining revealed the model group exhibited a thickened eutopic epithelium， stromal cell disarrangement， and evident infiltration of inflammatory cells. In addition， the model group showed elevated serum levels of IL-1β and PGE₂ （P<0.05， P<0.01）， increased positive expression of MMP-9 and VEGFA in the ectopic endometrial tissue （P<0.01）， and up-regulated protein levels of TLR4， MyD88， p-NF-κB/NF-κB， MMP-9， and VEGFA in the ectopic endometrial tissue （P<0.05， P<0.01）. Compared with the model group， all treatment groups exhibited a reduction in the ectopic lesion volume （P<0.01）. Furthermore， the writhing response scores were decreased in the high-dose Luoshi Neiyi prescription group and the dienogest group （P<0.05， P<0.01）. The pathological state of the ectopic endometrial tissue was alleviated to varying degrees in the treatment groups. Low-dose Luoshi Neiyi prescription reduced serum PGE₂ levels， and high-dose Luoshi Neiyi prescription and dienogest decreased serum IL-1β and PGE₂ levels in EMs rats （P<0.05， P<0.01）. The treatment groups showed decreased positive expression of MMP-9 and VEGFA in the ectopic endometrial tissue （P<0.05， P<0.01） and down-regulated protein levels of TLR4， MyD88， p-NF-κB/NF-κB， MMP-9， and VEGFA in the ectopic endometrial tissue （P<0.05， P<0.01）. The cell experiments showed that 5%， 10%， and 20% Luoshi Neiyi prescription-containing sera significantly reduced the viability and inhibited the migration of iheESCs. Compared with the control group， 5%， 10%， and 20% Luoshi Neiyi prescription-containing serum groups and the TAK-242 group showed reduced protein levels of TLR4， MyD88， p-NF-κB/NF-κB， MMP-9， and VEGFA （P<0.01）. Compared with the control group， the LPS group showed increased expression of the above proteins （P<0.01）. Compared with the LPS group， the LPS+5%， 10%， and 20% Luoshi Neiyi prescription-containing serum groups showed reduced expression of the above proteins （P<0.05， P<0.01）. ConclusionLuoshi Neiyi prescription may modulate the TLR4/NF-κB signaling pathway to reduce the inflammatory response and histopathological damage in the eutopic endometrium and suppress the adhesive， invasive， and angiogenic biological processes in the ectopic endometrial tissue， thereby exerting its therapeutic effect on EMs.

Objective To investigate the intervention effect and mechanism of LUO's Neiyi Prescription on the proliferation and angiogenesis of eutopic endometrium in rats with endometriosis(EMs)based on hypoxia-inducible factor 1A(HIF1A)/enhancer of zeste homolog 2(EZH2)/anthrax toxin receptor 2(ANTXR2)signaling pathway.Methods SD rats were randomly divided into sham operation group,model group,Danazol group(4.2 g·kg-1),low-dose LUO's Neiyi Prescription group and high-dose LUO's Neiyi Prescription group(15.74,31.48 g·kg-1),eight rats in each group.The rat model of EMs with qi stagnation and blood stasis syndrome was constructed by autologous endometrial transplantation combined with multi-factor intervention.Intragastric administration was given once a day for 28 consecutive days.The pathological changes of endometrial tissue were observed by HE staining.The expression levels of proliferating cell protein 67(Ki67)and platelet endothelial cell adhesion molecule 1(CD31)in endometrial tissue were detected by immunohistochemistry.The mRNA and protein expression levels of HIF1A,EZH2 and ANTXR2 in endometrial tissues were detected by qRT-PCR and Western Blot.The expression levels of YAP1,CD44 and β-catenin in endometrial tissues were detected by Western Blot.Results Compared with the sham operation group,the epithelial cells of the eutopic endometrium of the model group were thickened,the interstitial cells were arranged disorderly,and the inflammatory cells increased.The expression levels of Ki67 and CD31 in endometrial tissues were significantly increased(P＜0.01),the mRNA and protein expression levels of HIF1A and ANTXR2 were significantly increased(P＜0.01),while the mRNA and protein expression levels of EZH2 were significantly decreased(P＜0.01),and the protein expression levels of YAP1,CD44 and β-catenin were significantly increased(P＜0.01).Compared with the model group,the epithelial layer of the eutopic endometrial tissue of the rats in each administration group became thinner,the interstitial disorder and inflammatory infiltration were improved,and the levels of Ki67 and CD31 in the eutopic endometrial tissue were significantly decreased(P＜0.01).The mRNA and protein expression levels of HIF1A and ANTXR2 in the endometrium of rats in the Danazol group and the high-dose LUO's Neiyi Prescription group were significantly decreased(P＜0.05,P＜0.01),and the mRNA and protein expression levels of EZH2 were significantly increased(P＜0.05,P＜0.01).The protein expression levels of YAP1,CD44 and β-catenin in endometrial tissue of rats in each administration group were significantly decreased(P＜0.05,P＜0.01).Conclusion LUO's Neiyi Prescription can play a role in the treatment of EMs by inhibiting the proliferation and angiogenesis of eutopic endometrial cells,and its mechanism may be related to the inhibition of HIF1A/EZH2/ANTXR2 signaling pathway.