The Role And Mechanism Of Kallistatin In Intrauterine Adhesions

Objective : This experiment is intended to investigate the role and mechanism of kallistatin in endometrial fibrosis at both in vivo and ex vivo levels;it is expected to elucidate the pathogenesis of intrauterine adhesions and to advance the research process of finding targets for prevention and treatment.Methods: 1、In the clinic,the excess endometrial samples from patients with uterine adhesions were collected intraoperatively by hysteroscopy,and the location of kallistatin expression in endometrial cells and the difference in expression between normal(n=22)and IUA(n=21)endometrium were detected by immunofluorescence double-labeling,immunohistochemistry,QPCR and protein immunoblotting experiments,respectively.2、Based on the previous experimental basis,AN3 CA was selected with h ESC cells were selected for in vitro cellular experiments;firstly,TGF-β1 cell growth factor was used to induce cellular fibrosis,and then different concentrations of kallistatin protein were added to co-culture separately,and the difference in expression of the fibrosis marker FN between different subgroups was detected by cellular immunofluorescence assay.3、Human kallistatin knockdown virus was used to transfect AN3 CA cells The knockdown effect of kallistatin gene was verified by QPCR and protein immunoblotting assay;the effects of kallistatin on proliferation,apoptosis and migration of AN3 CA cells were detected by CCK-8,flow cytometry and scratch healing assay;the changes of expression of each signal 4、In vivo experiments were conducted in SD rats by scratching the uterine cavity,and rats with successful modeling were injected with adeno-associated virus under the uterine mucosa to achieve overexpression and knockdown of kallistatin;QPCR and protein immunoblotting assays were performed to detect the differences in expression of fibrosis markers.After males and females were caged together,the rats were followed for later gestation.Results: 1、The expression of kallistatin in the endometrium of the adhesion group was reduced compared to the control group.kallistatin expression was localized in the cytoplasm of AN3 CA cells and h ESC cells.2、The expression of fibronectin(FN)in AN3 CA and h ESC cells increased after treatment with TGF-β1,and the expression of FN in both was reduced after co-culture with kallistatin protein compared to the treatment with TGF-β1.3 、 AN3 CA cells with kallistatin knockdown expression showed reduced proliferation and migration ability and increased apoptosis compared with the control group;FN,TGF-β1,smad3,p-p38 MAPK,and p-Ik Ba expression were increased.4、The endometrium of rats after uterine scratching became thinner and had fewer glands,and TGF-β 1,Smad3,and FN expression were increased,and the number of pregnant embryos was reduced,which proved that the endometrial fibrosis deepened and the uterine adhesion model was successfully constructed;after the expression of kallistatin was changed by viral injection,it was found that kallistatin had a certain improvement effect on uterine adhesion.Conclusion: The present experiment demonstrated that kallistatin in endometrial cells can regulate TGF-β1/Smad3 expression through P38 MAPK pathway and NF-k B pathway,affecting cell activity and migration ability;in vivo experiments in SD rat model,visualized that kallistatin overexpression can improve endometrial fibrosis in uterine adhesions,and knockdown of kallistatin expression can lead to endometrial fibrosis,which in turn affects pregnancy outcome in rats.