Neiyiting Intervene Th17/Treg Expression Imbalance In Endometriosis Through The PD-1/PD-L1 Signal Pathway

Objective:1.To establish a rat model for endometriosis of endometrium autograft and so detect ectopic endometrium of the model for expression of PD-1 and its ligand,i.e.PD-L1,Th17/Treg cell specific transcription factors,i.e.RoR-?t and Foxp3,and related cytokines secreted by Th17 and Treg cells in peripheral blood,to investigate the presence of PD-1/PD-L1 signal and abnormal expression or distribution of Th17 and Treg cells in this model.2.to observe the effect of Neiyiting(an internal formulation)on PD-1/PD-L1 signal and Th17/Treg expression imbalance,and to preliminarily explore whether Neiyiting may intervene Thl7/Treg expression imbalance through the PD-1/PD-L1 signal pathway,and so have a therapeutic effect,so as to provide a new theory and research foundation for clinical promotion of Neiyiting.Methods:Forty-five female SD rats were randomized to blank control group,endometriosis model group,PD-1 antibody group,Neiyiting group,and low-dose Neiyiting group,9 rats each group.No treatment was given to the blank control group,and all the other groups were performed autotransplantation of endometrium.For each group,one rat was randomly selected and performed laparotomy 28 days after the autotransplantation for observing the growth of ectopic endometrium.Other rats were anesthetized after 2 weeks of continuous administration for blood sampling from the abdominal aorta,and ectopic lesion sampling as well.Westren-blot was used to detect the expression of PD-1,PD-L1,RoR-Y t and Foxp3 in the ectopic endometrium,and ELISA was used to detect the expression of cytokines IL-17A,IL-22,IL-10 and TGF-? in the peripheral blood.Results:1.The SD rat model for endometriosis of endometrium autograft was successfully established.Typical hemispherical cystic vesicles were observed at the graft site upon laparotomy 28 days after autotransplantation.Western-blot results showed that the expression levels of PD-1 and PD-L1 in the ectopic endometrium of rats in the endometriosis model group were significantly higher than in the blank control group,and the expression levels of PD-1 and PD-L1 in the ectopic endometrium of rats in the Neiyiting group and the PD-1 antibody group were significantly lower than in the endometriosis model group.The expression levels of PD-1 and PD-L1 in the ectopic endometrium of rats in the low-dose Neiyiting group showed not obvious change.The difference was not statistically significant.2.As compared with the blank control group,the endometriosis model group had higher expression levels of IL-17A and IL-22 in peripheral blood of the rats and lower expression levels of IL-10 and TGF-?,while the Neiyiting group and low-dose Neiyiting group had lower expression levels of IL-17A and IL-22 and higher expression levels of IL-10 and TGF-?,which were similar to the corresponding conditions of PD-1 antibody group.In the endometriosis model group,the expression level of RoR-?t in the ectopic endometrium was significantly decreased,while the expression level of Foxp3 was significantly increased.In the Neiyiting group,the expression level of RoR-Y t in the ectopic endometrium of rats showed a significant rising trend,which was similar to that in the PD-1 antibody group,and the expression level of Foxp3 in the ectopic endometrium of rats was significantly decreased,which was similar to that in the PD-1 antibody group.Low dose of Neiyiting had no significant effect on the expression level of RoR-?t and Foxp3 in the ectopic endometrium of rats with endometriosis.Conclusion:l.In the ectopic endometrium of rats with endometriosis,there was PD-1/PD-L1 signal activated abnormally,and Neiyiting,a traditional Chinese medicine,had an action similar to PD-1 antibody,i.e.could reduce the expression level of PD-1 and PD-L1.2.Th17/Treg expression and distribution imbalance occurred for endometriosis,blocking PD-1/PD-L1 signal through PD-1 antibody could regulate the Th17/Treg imbalance.3.Neiyiting could inhibit expression of PD-1 and PD-L1 in the ectopic endometrium and regulate the Thl7/Treg imbalance,which was similar to the PD-1 antibody.This indicated that the mechanism of Neiyiting for the treatment of endometriosis might be related to the regulation of Th17/Treg imbalance through blocking the PD-1/PD-L1 signal pathway.The effect of the normal dose group was superior to the low dose group.