| Objectives 1.Theoretical combing:Interpretation of Professor Liu Yanfeng’s unique understanding of pathogenesis and treatment of ovarian endometriosis.2.To observe the effect of Wenshen Nuangan Xiaozheng Decoction on TCM syndrome,dysmenorrhea,serum CA125 and ectopic mass of patients with ovarian endometriosis,and to explore the effect of Wenshen Nuangan Xiaozheng Decoction on ovarian endometriosis Clinical efficacy.3.By comparing the histological features and molecular biological characteristics of ectopic endometrium,eutopic endometrium in patients with ovarian endometriosis,and endometrium in other patients with benign cysts of ovary,to explore the fibrosis effect of TGF-β1 and Smads in endometriosis,and to reveal the possible mechanism of fibrosis in endometriosis.4.To study the mechanism of TGF-β1/Smads pathway in ectopic follicularization of endometriosis,to explore the therapeutic effect of Wenshen Nuangan Xiaozheng Decoction on endometriosis model mice and its differentiation Effect of lesion fibrosis,and to find the target and biological mechanism of Wenshen Nuangan Xiaozheng Decoction on the treatment of endometriosis cyst fibrosis.Methods 1.From the "kidney yang deficiency,latent cold in liver channel,phlegm and blood stasis" to distinguish the characteristics of ovarian endometriosis,to analyze the Wenshen Nuangan Xiaozheng method,and analyze the drug composition and efficacy.2.Using self contrast test,thirty-five patients with ovarian endometriosis were collected.The patients with yang deficiency cold coagulation syndrome were treated with Wenshen Nuangan Xiaozheng Decoction.Before and after treatment,the patients were observed about the TCM syndrome scores,VAS scores,dysmenorrhea symptom scores,serum CA125 levels,and ectopic cysts.The clinical efficacy of Wenshen Nuangan Xiaozheng Decoction on patients with ovarian endometriosis was analyzed.3.Twenty patients with ovarian endometriosis underwent laparoscopic surgery in the ward,and the ovarian ectopic cystic wall tissue was taken as the ectopic cyst wall group.Nine of them underwent hysteroscopy combined with laparoscopy surgery and we retained their intimal tissue as the eutopic endometrium group.We also retained endometrial tissue from nine patients with other ovarian benign tumors who underwent hysteroscopy combined with laparoscopy surgery as the control group.The expressions of TGF-β1,p-Smad2/3 and Smad7 in three groups were detected by immunohistochemistry,and then were analyzed for differences in expression levels of each group.4.(1)The endometriosis model mice were established by intraperitoneal injection.The histomorphology and fibrosis of the ectopic lesions were analyzed by HE and Masson staining.(2)Animals were divided into blank group,sham group,disease model group,high-dose group,medium-dose group,low-dose group and western medicine group.Masson staining was used to compare the fibrosis of ectopic lesions of each group.The levels of serum TGF-β1 of each group were compared by Elisa detection method.The gene and protein expression of TGF-β1,Smad2/3,Smad7 and Col I in mouse uterus and other ectopic lesions were analyzed by immunohistochemistry,q PCR and Western blot.Results 1.Therapeutic effect of Wenshen Nuangan Xiaozheng Decoction on ovarian endometriosis:(1)TCM sydroms: Wenshen Nuangan Xiaozheng Decoction can significantly alleviate the symptoms of yang deficiency cold coagulation syndrome in patients with ovarian endometriosis(P<0.01).(2)Dysmenorrhea: Wenshen Nuangan Xiaozheng Decoction can significantly relieve degree of dysmenorrhea(P<0.01),and improve symptoms(P<0.01).(3)Serum CA125: Wenshen Nuangan Xiaozheng Decoction can significantly reduce the level of serum CA125(P<0.01).(4)Cyst size: After treatment with Wenshen Nuangan Xiaozheng Decoction,the average size of ectopic cyst size decreased,but there was no significant difference before and after treatment(P>0.05).2.The fibrosis mechanism of ovarian endometriosis cyst:(1)Fibrosis: After Masson staining,fibrosis showed blue staining.A large amount of fibrotic deposition was observed in the ectopic cystic wall tissue.No fibrosis blue staining was observed in the proliferative and secretory phases of the eutopic endometrium group and the control group.(2)The expression of TGF-β1: The expression level of TGF-β1 was the highest in the ectopic cyst wall group,and the expression was statistically different from the control group(P<0.01),but there was no significant difference between the ectopic endometrial group and the eutopic endometrium group(P>0.05).The expression level of TGF-β1 in the eutopic endometrium group was higher than that in the control group,but the difference was not statistically significant(P>0.05).(3)The expression of p-Smad2/3: The expression of p-Smad2/3 was the highest in the ectopic wall group,but there was no statistical difference between the eutopic endometrium group(P>0.05).The expression of p-Smad2/3 was the lowest in the control group,and there was significant difference between the ectopic cystic wall and the eutopic endometrium group(P<0.05).(4)The expression of Smad7: The expression of Smad7 in the ectopic cystic wall group was the lowest,and there was significant difference between the eutopic endometrium group and the control group(P<0.05,P<0.01).The expression level of Smad7 in the eutopic endometrium group was lower than that in the control group,and the difference was statistically significant(P<0.01).3.Experimental study on the effect of Wenshen Nuanggan Xiaozheng Decoction on fibrosis of ectopic lesions in endometriosis model mice:(1)Establishment of the model: 21 days after the model was established,the model was relatively stable.A large number of fibers were stained in the ectopic lesions,mainly in the interstitium and muscular layer.There were a large number of collagen fibers in the ectopic lesions.(2)Ectopic lesion size: Compared with the disease model group,the ectopic lesion size in the high,medium and low dose groups of Chinese medicine and Western medicine groups decreased(P<0.05,P<0.01),and there was no statistical difference among the intervention groups(P>0.05).(3)Fibrosis degree: There was no significant difference in the area ratio of uterine fibrosis between sham group and blank group(P>0.05).The fibrosis area ratio of ectopic lesions in the disease model group was higher than that in the blank group(P<0.01).Compared with the ectopic lesions in the disease model group,the fibrosis area ratio of ectopic lesions in the high and middle dose groups was lower(P<0.05),while the fibrosis area ratio in the low dose group and the western medicine group was lower,but there was no significant difference(P>0.05).(4)Serum TGF-β1 level:There was no significant difference in serum TGF-β1 level between sham group and blank group(P>0.05),and the expression of serum TGF-β1 in disease model group was significantly higher than that in blank group(P< 0.01).Compared with disease model group,the levels of serum TGF-β1 in high,medium,low dose group and western medicine group were significantly lower(P< 0.05,P<0.01).The level of serum TGF-β1 was the lowest in the high-dose group,but there was no significant difference between the high-dose group and the western medicine group(P>0.05).(5)TGF-β1/Smads pathway:(1)m RNA expression : The expressions of TGF-β1,Smad2,Smad3,Smad7 and Col I m RNA were no significant differences between sham operation group and blank group(P>0.05).The expressions of TGF-β1,Smad2,Smad3 and Col I m RNA in the model group were significantly higher than those in the blank group(P<0.01),and the expression of Smad7 was significantly lower(P<0.01).Compared with the model group,the levels of TGF-β1 and Col I m RNA in the high-dose group and the western medicine group were significantly lower(P<0.01),the levels of Smad2 and Smad3 m RNA in the ectopic lesions of the high-dose group,the middle-dose group and the western medicine group were significantly lower(P<0.05,P<0.01),the levels of Smad7 in the ectopic lesions of the high-dose group and the western medicine group were significantly higher(P<0.01).(2)Protein expression:The expressions of TGF-β1,p-Smad2/3,Smad7 and Col I protein levels in uterine between sham group and blank group were no significant differences(P>0.05).The expression of TGF-β1,Smad2,Smad3 and Col I protein in the model group was significantly higher than that in the blank group(P<0.01),and the expression of Smad7 was significantly lower(P<0.01).Compared with the model group,the levels of TGF-β1 and p-Smad2/3 in the ectopic lesions of the high-dose group and the western medicine group were significantly lower(P<0.01),the level of Col I in the ectopic lesions of the high-dose group,the medium-dose group and the western medicine group were lower(P<0.05,P<0.01),and the level of Smad7 in the ectopic lesions of the high-dose group and western medicine group were higher(P<0.05 or P<0.01).Conclusions 1.The results of clinical observation showed that Wenshen Nuangan Xiaozheng Decoction could effectively alleviate the symptoms of yang deficiency cold coagulation syndrome,relieve dysmenorrhea,and reduce the level of serum CA125 and the size of ectopic cysts.2.The mechanism of fibrosis in ovarian endometriotic cysts showed that TGF-β1 and Smad2/3 are highly expressed in endometriosis,which may lead to the formation of endometriosis fibrosis by promoting fibroblast proliferation and collagen deposition.However,the expression of Smad7 as an inhibitory factor in endometriosis is reduced,and the inhibition of Smad-mediated TGF-β1 induced fibrosis is diminished,which also contributes to the progression of endometriosis fibrosis.3.Experimental results show that Wenshen Nuangan Xiaozheng Decoction can slow the growth of ectopic lesions in endometriosis mice and inhibit the formation of ectopic follicular fibrosis.which may be related to its intervention in the fibrogenic effect of TGF-beta 1/Smads pathway.This may play a "Xiao Zheng" therapeutic effect by interfering with the TGF-β1/Smads pathway,which affects the formation and progression of ectopic follicular fibrosis. |
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