| Objective:In this study,JAK2 / STAT3 signaling pathway was introduced into the study of EM rats with kidney deficiency and blood stasis syndrome,and Bushen Wenjing decoction was used to intervene,so as to explore the mechanism of JAK2 / STAT3 signaling pathway in EM rats with kidney deficiency and blood stasis syndrome and the drug target of Bushen Wenjing decoction,Objective to provide a reliable basis for the clinical standard selection of EM prescriptions.Methods: 105 healthy SPF female Sprague Dawley rats were randomly selected as the blank group,and the other 10 rats were randomly selected as the sham operation group after successfully modeled by compound factors method.The remaining 10 rats were randomly selected as the sham operation group,with only laparotomy and no suture of intima,and the other 10 rats were constructed by autologous intima transplantation.Fifty rats were randomly divided into model group,danazol group(63 mg · kg-1)and Bushen Wenjing Decoction low,medium and high dose groups(5,10,20 g · kg-1),10 rats in each group,once a day for 28 days.The length(D1),width(D2)and height(D3)of ectopic foci were measured before and after treatment,and the volume of ectopic foci was calculated(v=0.52×D1×D2×D3).The endometrial tissues were stained with HE to observe the histopathological changes.The levels of IL-10 and IL-17 in serum supernatant were detected by ELISA.IHC was used to detect the expression levels of JAK2,STAT3,p-STAT3,Caspase-8,TNF-α,VEGF,TSP-1,MMP-9,E-cadherin and N-cadherin in the intima of rats;WB was used to detect the expression levels of Caspase-8,TNF-α,VEGF,TSP-1,MMP-9,E-cadherin and N-cadherin.RT-PCR was used to detect the expression levels of caspase-8,TNF-α,MMP-9,E-cadherin and N-cadherin.Results:Before treatment,typical cystic blisters could be seen in the ectopic foci of each model group.Under the microscope,the endometrium structure was complete,showing a uterine cavity or ring closed structure.There were cysts in the endometrium.The epithelium was cubic or columnar epithelium.Most of the epithelial cells had secretion,the stroma was dense,the matrix was a little fibrosis,and the blood vessels proliferated densely.After treatment,the volume of ectopic foci in danazol group and Bushen Wenjing Decoction low,medium and high dose groups was significantly reduced(P<0.01),the endometrial epithelial layer was thinned to varying degrees,and even atrophied in lineararrangement,the cells were not closely arranged,most of the endometrium fell off,the interstitial cells were reduced and showed small and sparse shape,and the blood vessels were significantly reduced.IHC detection: compared with blank group and sham operation group,JAK2,STAT3,p-STAT3 protein expression in eutopic and ectopic endometrial tissue of model group were significantly increased(P<0.05).Compared with the model group,the protein expression of JAK2 in eutopic and ectopic endometrium of Bushen Wenjing Decoction low dose group was significantly decreased(P<0.05)and the protein expression of JAK2,STAT3 and p-STAT3 in eutopic and ectopic endometrium of danazol group and Bushen Wenjing Decoction medium and high dose groups were significantly decreased(P<0.05).ELISA detection: compared with the blank group and sham operation group,the serum IL-10 content in the model group was significantly decreased(P<0.01),and the IL-17 content was significantly increased(P<0.01);compared with the model group,the serum IL-10 content in the danazol group and the low,medium and high dose Bushen Wenjing Decoction groups was significantly increased(P<0.01),and the IL-17 content was significantly decreased(P<0.01).IHC and WB detection:compared with blank group and sham operation group,the protein expressions of caspase-8,TSP-1 and E-cadherin in eutopic and ectopic endometrial tissue of model group were significantly decreased(P<0.01,P<0.05,P<0.01);the protein expressions of TNF-α,VEGF,MMP-9and N-cadherin were significantly increased(P<0.01,P<0.05,P<0.01,P<0.01);compared with model group,the protein expressions of danazol group and Bushen Wenjing Decoction were significantly increased(P<0.01,P<0.05,P<0.01)The expressions of caspase-8,TSP-1 and E-cadherin in eutopic and ectopic endometrium were significantly increased(P<0.01,P<0.05,P<0.01),while the expressions of TNF-α,VEGF,MMP-9 and N-cadherin were significantly decreased(P<0.01,P<0.05,P<0.01,P<0.01).RT-PCR detection: compared with blank group and sham operation group,Caspase-8 and E-cadherin gene expression in eutopic and ectopic endometrium of model group was significantly decreased(P<0.01),while TNF-α,MMP-9 and N-cadherin gene expression was significantly increased(P<0.01).Compared with model group,Caspase-8 and E-cadherin gene expression in eutopic and ectopic endometrium of danazol group and Bushen Wenjing Decoction middle and high-dose group were significantly increased(P<0.01,P<0.05,P<0.01).The expressions of MMP-9 in eutopic and ectopic endometrium in the danazol group and Bushen Wenjing Decoction middle and high dose groups were significantly decreased(P<0.01,P<0.05,P<0.01).The expressions of TNF-αand N-cadherin in eutopic and ectopic endometrium in the danazol group and Bushen Wenjing Decoction middle and high dose groups were significantly decreased(P<0.01,P<0.05,P<0.01).The gene expression of TNF-α and N-cadherin in eutopic and ectopic endometrium of danazol group and Bushen Wenjing Decoction medium and high dose groups were significantly decreased(P<0.05).Conclusion:1.Bushen Wenjing Decoction can inhibit the invasion of ectopic foci in EM rats with kidney deficiency and blood stasis,and its specific mechanism may be related to regulating the immunosuppression mediated by over activation of JAK2 / STAT3 signaling pathway and blocking the function of angiogenesis.2.STAT3 is activated by JAK2,which makes STAT3 hyperphosphorylation,leading to abnormal activation of JAK2 / STAT3 signaling pathway in EM rats with kidney deficiency and blood stasis.3.The abnormal activation of JAK2 / STAT3 signaling pathway was up-regulated the expression levels of IL-17,TNF-α,VEGF,MMP-9 and N-cadherin,down regulated the expression levels of IL-10,Caspase-8,TSP-1 and E-cadherin,resulting in the immune imbalance and a large number of microvascular neovascularization in EM rats with kidney deficiency and blood stasis,leading to the occurrence and development of EM. |
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