Study On The Anti Adhesion Effect Of Nei Yi Zhi Tong Decoction On EMs Rat Model And Its Mechanism

Objective:Through the establishment of rat models of endometriosis,combined with pathological and molecular biological methods to study the clinical experience of Neiyi recipe Zhitong decoction is effective in the treatment of endometriosis(EMS)and its molecular mechanism,for the comprehensive evaluation of endometriosis Zhitong decoction is effective and provide scientific basis for clinical application.Method:Histopathological observation after treatment:Through open to observe each rat endometriotic lesions growth and measurement of endometriosis pain before and after the Decoction in the treatment of ectopic lesion volume;by HE staining light microscopy the rats treated with endometrial biopsy.Packet administration:Will make a successful model of the EMS 40 rats,were divided to 4 groups;model group,Chinese high,low dose group,positive drug group,with 10 rats in each group,sham operation group 8(only for abdominal surgery,not for autologous transplantation).Chinese high,low dose treatment group points don't fill the stomach 985.3mg/Kg.d and 245.8mg/Kg.d endometriosis Zhitong decoction and positive drug group irrigation stomach 7.2 mg/Kg.d danazol,model group and sham operation group irrigation stomach 2 ml normal saline,every group were administered once,continuous medication for 4 weeks.Construction of rat EMs model:57 SD rats were used to construct the EMs model.After 4 weeks,the general observation model was constructed successfully,and 5 rats were used to evaluate the histopathological evaluation by HE staining.Pathologic correlation index detection:Using enzyme linked immunosorbent assay(ELISA)rats were treated with serum MMP-9 TIMP-1 expression of E-cadherin levels;by RT-PCR,immunohistochemistry,Western blot method was used to detect treatment of endometriotic lesions of E-cadherin,MMP-9,beta catenin and VEGF gene transcription and protein expression level.Result:Construction of rat EMs model:EMS model construction of 57 SD rats underwent autologous transplantation of endometrium,a total of 45 modeling success(which killed seven,five no abdominal ectopic endometrial tissue),the abdominal wall visible transparent vesicles,visible on the surface of fresh blood vessels and blue purple mass,or see the local lesion and pelvic adhesion,the success rate of about 78%.HE staining microscopically visible EMS model rats ectopic foci epithelial cells more vacuoles,nuclei arranged in disorder,loose stroma,and neovascularization could be observed,glandular structure is incomplete.Normal endometrial stromal cells arranged closely,glandular structure clear epithelial integrity.Histopathological observation after treatment:By measuring the volume of ectopic endometrium cyst size.The results showed that the cyst volume in each treatment group was significantly reduced(P<0.01),and model group cyst volume before and after treatment no significant changes(P>0.05);after treatment between the treatment group with lateral cyst volume comparison,traditional Chinese medicine high dose treatment group cyst volume reduction is obviously better than that of traditional Chinese medicine(TCM)low dose treatment group(P<0.05),its role and positive drug group considerably(P>0.05).After treatment each EMS model rats of ectopic endometrium hematoxylin and eosin(he)staining microscopically visible,and those in the sham operation group of uterine endometrial epithelial cells arranged closely for columnar,glandular structure clear,glandular structures;model group of ectopic endometrium epithelial cells and vacuoles,disordered arrangement of the nuclei,loose stroma,and neovascularization could be observed,glandular structure is incomplete.And traditional Chinese medicine(TCM)high,low dose group and Western medicine group.HE staining ectopic tissue morphology distribution similar to the sham operation group.The changes of pathological correlation index after treatment:Using enzyme linked immunosorbent assay(ELISA)rats were treated with serum MMP-9,TIMP-1,E-cadherin index.The results show that compared with the model group,endometriosis Zhitong decoction in the treatment group significantly decreased the serum MMP-9,elevated TIMP-1,the expression level of E-cadherin and the effect of high dose group was significantly superior to low dose group(P<0.05).Using RT-PCR to detect catenin,E-cadherin,MMP-9 and VEGF gene expression at the transcriptional level show that compared with the model group,endometriosis Zhitong decoction can significantly increase the mRNA in endometriosis model rats and reduce the beta catenin,MMP-9,VEGF mRNA transcription level and high dose of Chinese medicine group the gene transcription level is better than the low dose group,(P<0.01).Using immunohistochemistry,Western blot detected in rats after treatment of ectopic endometrium beta catenin,E-cadherin,MMP-9 and VEGF protein expression.The results show that compared with the model group,the endometriosis Zhitong decoction differences between the three groups can significantly reduce MMP-9,VEGF,beta catenin,elevated E-cadherin protein expression level,and high dose group is better than the low dose group.Conclusion:To sum up the study of ectopic Zhitong decoction has not only inhibit the growth of ectopic endometrium,reducing the volume of the ectopic lesions,improve the role of endometriotic lesions,histopathological changes,with elevated E-cadherin,reduced beta catenin,MMP-9,VEGF of ectopic endometrium gene transcription and protein table role.The stability of endometriosis Zhitong decoction by transcriptional upregulation of E-cadherin or downregulation of-catenin gene and protein expression and to maintain the E-cad/beta-cat complex structure and function,and to strengthen the role of cell adhesion in eutopic endometrium;Neiyi Zhitong decoction may be through increased serum level of TIMP-1 by inhibiting the activity of MMP-9,or by inhibiting-catenin protein the expression of the Wnt/beta-catenin signal pathway is closed,the downstream target genes of MMP-9 enzyme activity is inhibited,thereby reducing the endometrial stromal cells and peritoneal mesothelial cell adhesion,blocking its extracellular matrix to prevent the destruction of ectopic endometrium to distant metastasis and invasion.In conclusion,Neiyi decoction can relieve pain or indirectly influence the formation of EMs adhesion,and achieve the purpose of treatment.