| Purpose:1.Through the controlled clinical trial,observe the clinical effect of Tiaoshen Tongluo recipe on the prevention and treatment of postoperative re-adhesion of severe IUA,in order to provide a simple,practical and effective treatment for IUA patients.2.Through the retrospective analysis of endometrium pathological sections in IUA patients,specify the correlation between IUA degree of fibrosis,TGF-?1/Smads signaling pathway and IUA occurrence&development.3.Through in vivo experiments of IUA model for SD rats,analyze the therapeutic mechanism of Tiaoshen Tongluo recipe on the prevention and treatment of postoperative re-adhesion of severe IUA from the perspective of TGF-?1/Smads signaling pathway at the tissue level.4.Through the in vitro experiment of endometrial stromal cells in SD rats,analyze the effect target of Tiaoshen Tongluo recipe on the prevention and treatment of IUA postoperative re-adhesion from the perspective of TGF-?1/Smads signaling pathway at the cell level.Method:1.The clinical efficacy of Tiaoshen Tongluo recipe on the prevention and treatment of IUA postoperative re-adhesion was observed randomly.The patients,who was admitted to the Gynecology Department of the First Affiliated Hospital of Yunnan University of Chinese Medicine(Yunnan Provincial Hospital of Traditional Chinese Medicine)from Apr.2019 to Dec.2020,were chosen and 60 cases were included in the severe IUA patients according to the inclusion criteria.There were divided into the treatment group and control group,respectively(n=30).The treatment group had an average age of 29.87±4.06 and an average course of disease of 3.53±1.70 months;The number of Intrauterine operation was 2.10±0.0.71 and the score of intrauterine adhesion was 15.33±6.78.The control group had an average age of 29.30±3.23 and an average course of disease of 3.73±1.80 months;The number of Intrauterine operation was 1.87±0.68;The score of intrauterine adhesion was 14.93±6.50.The difference between the two groups in the age,course of disease,intrauterine operation time and the score of intrauterine adhesion had no statistical significance(P>0.05).The treatment group was treated with Tiaoshen Tongluo recipe and the control group with Femoston.Both groups were treated for three menstrual cycles and followed up for half a year with the withdrawal during menstruation.The efficacy of each group was evaluated using the following indicators:Intrauterine adhesion score,endometrial thickness on ovulation day,PI,RI,menstrual quantity,color and quality,abdominal pain,back pain,anxiety and other symptoms scores three months after operation.2.Degree of endometrial fibrosis in IUA and correlation between TGF-?1/Smads signaling pathway and IUA.It randomly chose 30 cases with the pathological section of endometrial tissue(10 cases for mild,moderate and severe cases),10 cases with pathological section of non-adhesive endometrium in IUA patients,as well as 10 cases who underwent the hysteroscopic curettage due to endometrial thickness,and was confirmed as endometrial hyperplasia and endometrial polyps from the pathological examination of endometrial tissue.They were admitted to the First Affiliated Hospital of Yunnan University of Chinese Medicine(Yunnan Provincial Hospital of Traditional Chinese Medicine).The glands count shall be carried out by HE dyeing,and the collagen fiber area shall be evaluated according to the Masson dyeing,to clear the fibrosis degree of IUA;Immunohistochemical method was used to detect the expression levels of TGF-?1,Smad2,Smad3 and Smad7 related to TGF-?1/Smads signaling pathway,providing the correlation between TGF-?1/Smads signaling pathway and IUA occurrence&development.3.Animal ExperimentThe double damage method was used to construct a stable IUA animal model for SD rats.The rats were divided into 5 groups randomly(10 uterine specimens):normal control group,model control group,low-dose group with Tiaoshen Tongluo recipe(2.875g/kg TTR),medium-dose group(5.75g/kg TTR)and high-dose group(11.5g/kg TTR).Each group has 5 mice(a total of 10 uterine specimens in each group).The normal control group and model control group were fed regularly,the same as the medication administration group.The same dose of saline was administered in the medication administration group.The low,medium and high-dose groups of Tiaoshen Tongluo recipe received gavage of 2.875g·kg-1,5.75g·kg-1 and 11.5g·kg-1 no-frying particles for 8 weeks.Acquired the endometrial specimens,HE and Masson staining to observe the endometrial morphology,gland count and collagen area of each group,and analyze the impact of Tiaoshen Tongluo recipe on the progress of endometrial fibrosis in SD rats with IUA;Vimentin expression level was tested by immunohistochemical examination to analyze the influence of Tiaoshen Tongluo recipe on endometrial stromal cells of SD rats with IUA;qPCR method was used to test the expression levels of TGF-?1,Smad2,Smad3 and Smad7 mRNA in each group;Western Blot method was used to test the expression levels of TGF-?1,Smad2,p-Smad2,Smad3,p-Smad3 and Smad7 protein.The action mechanism and target of Tiaoshen Tongluo recipe on the prevention and treatment of postoperative re-adhesion of IUA were analyzed from the perspective of TGF-?1/Smads.4.Cell experimentExtracted SD mice endometrial stromal cell and divided them into 5 groups after the cell culture for 48 hours:normal control group,model control group,5%,10%and 20%drug serum groups with Tiaoshen Tongluo recipe.The normal control group was cultured in normal fetal bovine serum.The model control group and Tiaoshen Tongluo recipe groups with drug serum acted on endometrial stromal cells for 48h at 50ng/ml TGF-?1.After that,the model group added the serum of normal feeding rats.The groups of Tiaoshen Tongluo recipe added 5%,10%and 20%drug serum,respectively.Each group was cultured in 37? incubator for 72h to collect the supernatants and cells.qPCR method was used to test the expression levels of Smad2,Smad3 amd Smad7 mRNA;Western Blot method was used to detect the expression levels of Smad2,p-Smad2,Smad3,p-Smad3 and Smad7 protein.The influence of Tiaoshen Tongluo recipe on endometrial stromal cells was analyzed at the cell level from the perspective of TGF-?1/Smads.Results:1.Clinical efficacyAfter the treatment of 3 menstrual cycles,the hysteroscopic adhesion score,endometrial thickness on ovulation date,PI,RI and symptom score in the treatment group were 5.67±3.04,8.64±1.38,0.77±0.14,0.49±0.04 and 32.00±7.93,respectively.The hysteroscopic adhesion score,endometrial thickness on ovulation date,PI,RI and symptom score in the control group were 8.57±3.93,7.08±1.83,0.96±0.14,0.53±0.04 and 38.00±7.85,respectively.The indicators of the two groups were improved after the treatment compared with before treatment,with obvious differences(P<0.05).Compared with the control group,the adhesion score,endometrial thickness on ovulation date,PI,RI and symptom score in the treatment group were improved,with obvious difference(P<0.05).The total effective rate of preventing postoperative re-adhesion of IUA was 83.3%in the treatment group and 53.3%in the control group.The treatment group was better than the control group,and the difference was significant(P<0.05).2.Correlation between IUA fibrosis and TGF-?1/Smads signaling pathway(1)Count of the endometrial glands in each group:HE staining results showed rich non-adhesion group glands in round or oblong shapes,counting 48.13± 6.55.There were few glandes in the adhesion group.The counts of mild,medium,and heavy adhesion glands were 24.8±2.15,18.7±1.34,and 12.1±2.08,respectively.Compared with the control group,the adhesion group significantly increased the Glands count,with significant differences(P<0.001),which was negatively associated with the degree of adhesion.(2)Area ratio of endometrial collagen fibers in each group:Masson staining results showed insignificant blue staining area in the non-adhesion group,with small collagen fiber area and an area ratio of 0.489±0.241.The adhesion group showed an obvious blue staining area and a wide area of collagen fiber.The mild,medium,and heavy adhesion area ratios were 12.92±5.054,25.82±2.636,and 32.5±6.969,respectively.Compared with the control group,the adhesion group significantly increased the collagen fiber area,with significant differences(P<0.001),which was positively associated with the degree of adhesion.(3)Expression of endometrial tissue TGF-?1,Smad2,Smad3 and Smad7 in each group:Immunohistochemistry the results showed that TGF-?1was expressed in the endometrial glandular epithelium and mesenchyme in each group,and it was mainly expressed on the glandular epithelial cell.Relative expression quantity of TGF-?1 in non-adhesion group,mild,moderate and severe adhesion group was 1.37±0.49,2.90±0.31,3.80±0.42 and 4.50±0.42,respectively.Compared with the non-adhesion groups,TGF-?1 expression of the adhesion group was significantly up-regulated and was positively associated with the degree of adhesion,with significant differences(P<0.01).Smad2 was expressed in the endometrial glandular epithelium and mesenchyme in each group.Its relative expression quantity in non-adhesion group,mild,moderate and severe adhesion group was 2.77±0.43,4.80±0.42,5.00±0.47 and 5.00±0.47,respectively.Compared with the non-adhesion group,Smad2 in the adhesion group was significantly up-regulated,with significant differences(P<0.01),which was not significantly associated with the degree of adhesion(P>0.05).Smad3 is mainly expressed on the non-adhesion group.In the adhesion group,it was expressed in glandular epithelium and mesenchyme.Relative expression quantity of Smad3 in non-adhesion group,mild,moderate and severe adhesion group was 1.97±0.56,3.30±0.67,3.70±0.82 and 4.40±0.52,respectively.Compared with the non-adhesion group,the expression of the adhesion group was obviously up-regulated with significant differences(P<0.01),which was positively associated with the degree of adhesion.Smad7 was expressed in each group.The relative expression quantity of Smad7 in the non-cohesion,mild,medium and severe adhesion groups was 3.87±0.86,3.80±0.63m 3.10±0.88 and 1.80±0.42,respectively.Compared with the non-adhesion group,the expression of the adhesion group was obviously down-regulated with significant differences(P<0.01),which was negatively associated with the degree of adhesion.3.Animal Experiment(1)The anatomical morphology of uterus in SD rats:The uterus in vivo of each group presented "Y"-shaped uterus duplex.In the normal control group,the two uteruses were symmetrical with uniform thickness.The uterine wall was smooth and elastic in faint red.The uterus was similar in shape in model control group,low-dose,medium-dose and high-dose group with Tiaoshen Tongluo recipe,but there were different degrees of deformation,stiffness,and adhesion to the surrounding tissue.The surface color of the uterine wall was deeper than the normal group,wherein,the model control group and the low-dose group with Tiaoshen Tongluo recipe were the most obvious ones.The medium and high-dose groups showed good uterus elasticity and lower adhesion degree than the model group and low-dose group.(2)Vimentin immunohistochemical examination of endometrium in SD rats of each group:IHC test results showed a large amount of brown particles in normal control group,high and medium-dose groups with Tiaoshen Tongluo recipe.The relative expression of Vimentin was 21.71±1.88%,17.67±1.06%and 15.39±1.53%,respectively.Less brown particles were found in the model control group and the low-dose group,with a relative expression of 6.11±0.66%and 11.92±1.74%,respectively.Compared with the normal group,the model group and the low-dose group with the Tiaoshen Tongluo recipe showed low expression with significant differences(P<0.01).Compared with the model control group,the expression of high and medium-dose groups with Tiaoshen Tongluo recipe was significantly increased,with obvious differences(P<0.01).(3)SD staining results and glands count of endometrial tissue in SD rats of each group:The normal control group had complete endometrial lines,complete epithelial cells,rich circular or elliptical glands in the endometrial mesenchyme(arranged and clustered).The gland opening was visible on the endometrial surface.Model control group and low,medium and high-dose groups all showed endometrial tissue necrosis,with rare and even missing glands.Among them,the model control group and the low-dose group with Tiaoshen Tongluo recipe were the most serious.Glands counts in normal control group,model control group,and low,medium and high-dose groups with Tiaoshen Tongluo recipe were 54±5/HP,10±2/HP,20±4/HP,34±6/HP and 50±3/HP,respectively.Compared with the normal control group,the gland counts decreased in different degrees(P<0.01).A dose-dependent increase was shown in the gland count of groups with Tiaoshen Tongluo recipe.compared with the model group.However,there was no difference between the low-dose group and the model group(P>0.05).Medium and high-dose groups and model group differed significantly(P<0.01).(4)Masson staining results and fibrosis area of endometrium in SD rats of each group:A small amount of blue collagen fiber was found in the normal control group and high and medium-dose groups with Tiaoshen Tongluo recipe.The fibrosis area ratio was 13.73±1.67%,1 8.56±1.69%and 28.38±2.8%,respectively.Large amounts of blue collagen fibers were seen in the model control group and the low-dose group.The fibrosis area ratio was 53.09±3.46%and 41.82±2.89%,respectively.Compared with the normal control group,the fibrotic area increased in the model group and other groups with Tiaoshen Tongluo recipe with the significant difference(P<0.01).Compared with the model control group,the fibrosis area in the medium and high-dose groups with Tiaoshen Tongluo recipe decreased,with obvious differences(P<0.01).The Tiaoshen Tongluo recipe in the low-dose group and model group has no difference(P>0.05);(5)Expression of TGF-?1,Smad2,Smad3 and Smad7mRNA of endometrium in SD rats of each group:qPCR testing results showed that the relative expression of TGF-?1mRNA in normal control group,model control group,low-dose,medium-dose and high-dose groups was 2.64±0.35,4.61±0.66,4.07±0.38,3.92±0.42 and 3.13±0.41,respectively;The relative expression of Smad2mRNA was 2.96±0.33,3.83±0.55,3.64±0.57,3.28±0.67 and 3.00±0.53,respectively;The relative expression of Smad3mRNA was 3.17±0.22,4.75±0.53,4.14±0.26,3.71±0.28 and 3.27±0.41,respectively;The relative expression of Smad7mRNA was 5.82±0.33,4.03±0.19,4.60±0.62,4.69±0.47 and 5.76±0.27,relatively.Compared with the normal control group,the expression of TGF-?1,Smad2 and Smad3 mRNA was up-regulated in each group and the expression of Smad7 mRNA was lowered,with significant differences(P<0.01).Compared with the model group and control group,TGF-?1,Smad2 and Smad3 mRNA at each group with Tiaoshen Tongluo recipe downregulated expression in dose-dependent manner.Expression of Smad7 mRNA upregulated.Wherein,there was significantly different between the high-dose group and model group(P<0.01),and it had no significant difference in the expression level between the low-dose group and model group(P>0.05).(6)Expression levels of TGF-?1,p-Smad2/3 and Smad2/3/7 protein of endometrium in SD rats of each group:Western blot test results showed that the relative expression of TGF-?1 protein was 0.177±0.08,0.808±0.045,0.584±0.054,0.411±0.164 and 0.206±0.025 in normal control group,model control group,low,medium and high-dose groups,respectively;The relative expression of p-Smad2 protein was 0.126±0.017,0.728±0.370,0.587±0.043,0.281±0.021 and 0.159±0.027,respectively;The relative expression of Smad2 protein was 0.281±0.014,0.780±0.035,0.559±0.057,0.312±0.030 and 0.276±0.047,respectively;The relative expression of p-Smad3 protein was 0110±0.013,0.782±0.133,0.558±0.059,0.290±0.027 and 0.167±0.014,respectively;The relative expression of Smad3 protein was 0.134±0.015,0.570±0.059,0.458±0.032,0.267±0.031 and 0.162±0.018,respectively;The relative expression of Smad7 protein was 0.448±0.030,0.096±0.021,0.131±0.018,0.188±0.019 and 0.198±0.057,respectively.Compared with the normal control group,the expression of TGF-?1,p-Smad2,Smad2,p-Smad3 and Smad3 was up-regulated in each group and Smad7 expression was down-regulated,with obvious differences(P<0.01).Compared with the model group and control group,TGF-(31,Smad2 and Smad3 protein expression at each group with Tiaoshen Tongluo recipe downregulated expression in dose-dependent manner.Expression of Smad7 upregulated.Wherein,there was significantly different between the high-dose group and model group(P<0.01),and it had no significant difference in the expression level between the low-dose group and model group(P>0.05).4.Cell experiment(1)Isolation,culture,and identification of primary endometrial stromal cells in SD rats:The newly separated endometrial stromal cells were in different sizes and shapes,mostly rounded,conical and polyangular.Adherence occurred 0.5 h after the culture.After 24h,the cells were fusiform,triangular and polyangular,which were scattered and arranged without obvious regularity.After 48h,the cells began to gather.After 72h,the cells were gathered obviously in a fusiform shape.The vimentin immunofluorescence was positive in green color,with a positive rate of 90.10±0.15%.(2)Morphology and density of endometrial matrix cells in SD rats:The normal control group and 20%drug serum group showed similar endometrial stromal cells appearance(round or oval).The full cells were arranged in a paving stone shape;The model control group,5%and 10%groups showed long cells and decreased cell density.(3)Expression of Smad2,Smad3 and Smad7mRNA of endometrial stromal cells in SD rats:qPCR results showed that,the relative expression of Smad7mRNA in the normal control group,model control group,and 5%,10%and 20%drug serum groups with Tiaoshen Tongluo recipe was 3.981±0.510,5.040±0.210,4.997±0.323,4.539±0.241 and 4.070±0.265,respectively;The relative expression of Smad3mRNA was 3.539±0.137,5.014±0.321,4.819±0.330,4.31 8±0.368 and 3.792±0.475,respectively;The relative expression of Smad7mRNA was 5.614±0.250,4.508±0.451,4.681±0.074,4.894±0.085 and 5.257±0.370,respectively.Compared with the normal control group,the expression of Smad2 and Smad3 mRNA was up-regulated in the model control group and drug serum groups,while Smad7 expression was down-regulated.Compared with the model group,a dose-dependent down-regulation was presented in Smad2 and Smad3 mRNA expression in drug serum groups,with Smad7 expression up-regulated.The difference was obvious between the medium and high-dose group and model group(p<0.05),while the difference was obvious between the low-dose group and model group(p>0.05).(4)Expression of p-Smad2/3 and Smad2/3/7 protein in endometrial stromal cell of SD rats:Western blot test results showed that:The relative expression of p-Smad2 protein in the normal control group,model control group,5%,10%and 20%drug serum groups with Tiaoshen Tongluo recipe was 0.151±0.016,0.850±0.035,0.730±0.029,0.690±0.024 and 0.621±0.023,respectively.The relative expression of Smad2 protein was 0.190±0.019,0.862±0.028,0.812±0.031,0.681±0.026 and 0.610±0.028,respectively;The relative expression of p-Smad2 protein was 0.130±0.011,0.911±0.027,0.751±0.022,0.660±0.021 and 0.592±0.027,respectively;The relative expression of Smad3 protein was 0.161±0.018,0.841±0.019,0.720±0.025,0.632±0.019 and 0.492±0.018,respectively;The relative expression of Smad7 protein was 0.730±0.035,0.150±0.011,0.261±0.018,0.381±0.029 and 0.550±0.016,respectively.Compared with the normal control group,the expression of p-Smad2,p-Smad3,Smad2 and Smad3 was up-regulated in the model control group and drug serum groups,with Smad7 protein expression down-regulated.Compared with the model group,the p-Smad2,p-Smad3,Smad2 and Smad3 protein expression of the drug serum group with Tiaoshen Tongluo recipe downregulated in the dose-dependent mode.The Smad7 expression up-regulated.Wherein,it had a significant difference between the high-dose group and model group(p<0.05),and had no significant difference between the low-dose group and the model group(p>0.05).Conclusion:1.The application of Tiaoshen Tongluo recipe in postoperative severe IUA surgery can significantly improve the patient's menstrual volume,anxiety,burnout,soreness and weakness of waist&knees and other clinical discomfort;It can increase endometrial thickness,improve endometrial blood flow,promote endometrial repair,and effectively prevent severe IUA postoperative co-adhesion.2.The progress of endometrial tissue fibrosis participates in the occurrence and development of IUA,and the degree of endometrial tissue fibrosis is positively related with adhesion;The activation of TGF-?1/Smads signaling pathway participates in the occurrence and development of IUA.3.From the perspective of TGF-?1/Smads fibrosis signaling pathway,the possible therapeutic mechanism for the application of Tiaoshen Tongluo recipe in postoperative IUA surgery is to:down-regulate the TGF-?1,Smad2 and Smad3 protein and mRNA expression in the intimal tissue,up-regulated the Smad7 protein and mRNA expression;down-regulate the expression of Smad2,Smad3 protein and mRNA expression in the endometrial stromal cell,and up-regulate the expression of Smad7 protein and mRNA;so as to regulate the TGF-?1/Smads signaling pathway and improved the endometrial fibrosis. |
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