| Objective1.Explore the characteristics of syndromes and syndrome elements in the process of endometrial benign hyperplasia to carcinogenesis,to clarify the evolution of syndromes and syndrome elements in the process of endometrial hyperplasia to carcinogenesis and to guide clinical syndrome differentiation.2.Analyze the correlation between the expression of inflammatory factors(IL-17A,IL-6,TNF-α)and syndromes in various stages of endometrial lesions,and explore closely related syndrome of "inflammatory-cancer" during the process of endometrial hyperplasia to carcinogenesis,to provide ideas and references for the early intervention of Chinese medicine in the prevention of endometrial cancer.3.On the basis of clinical research,the inflammatory factor IL-17A was used in cell experiments to induce human endometrial highly differentiated adenocarcinoma ishikawa cells to mimic the tumor microenvironment status in vivo;and then study the regulation of the environment of genistein and its mechanisms.Methods1.Syndromes and syndrome elements research:a total of 155 patients with endometrial lesions were collected by questionnaire(including 91 patients with endometrial hyperplasia without atypical,ie EHWA patients,41 patients with atypical endometrial hyperplasia,ie AEH patients,23 patients with endometrial adenocarcinoma,ie EC patients)for general demographic data and main symptoms,looking for general characteristics and risk factors of patients with endometrial lesions,and using factor analysis and statistical methods to reduce the dimensionality of main symptoms of patients with endometrial lesions,extracting syndromes Inductive syndrome,combined with professional knowledge to analyze the characteristics of TCM syndromes and syndromes of endometrial lesions,in order to determine the evolution of syndromes and syndromes in the process of endometrial hyperplasia and to guide clinical syndrome differentiation..2.Correlation between inflammatory factors and TCM syndromes:According to the results of the above-mentioned syndrome research,50 patients corresponding to the three syndromes with the highest proportion(ie spleen deficiency and dampness syndrome,spleen dampness heat syndrome,liver qi stagnation syndrome)were selected.,including 18 cases of EHWA(7 cases of liver qi stagnation syndrome,6 cases of spleen deficiency and dampness syndrome,5 cases of spleen deficiency dampness syndrome),18 cases of AEH(5 cases of liver qi stagnation syndrome,spleen deficiency and dampness syndrome 7 Cases,spleen deficiency and dampness syndrome in 6 cases),14 cases of EC(4 cases of liver qi stagnation syndrome,5 cases of spleen deficiency and dampness syndrome,5 cases of spleen deficiency dampness syndrome).Detect the expression of IL-17A,IL-6 and TNF-α protein in endometrial tissue by using immunohistochemical technique,and analyse the correlation between IL-17A,IL-6,TNF-α protein expression with endometrial lesions,TCM syndromes.3.Experimental study(1)Based on the results of clinical research,the inflammatory factor IL-17A was selected to induce human endometrial highly differentiated adenocarcinoma ishikawa cells to mimic the tumor microenvironment status in vivo.CCK-8 method,cell scratch and transwell method,westernblot method were used to detect the effect of IL-17A on cell proliferation,migration and expression of ERa and ERβ protein,and the optimal induction dose of IL-17A in subsequent experiments was determined at the same time.(2)Regulation of genistein on the inflammatory microenvironment of endometrial adenocarcinoma:ishikawa cells were induced with the optimal induction dose of IL-17A,and genistein was added then.Effects of cell proliferation,migration,and expression of ERα,ERβ,AKT/p-AKT,and ERK1/2/p-ERK1/2 proteins were detected by CCK-8 method,cell scratch method and Westernblot method respectively.Results1.Syndromes and syndrome elements research:(1)Distribution characteristics and evolution of syndromes:37 cases of liver qi stagnation syndrome(23.87%),48 cases of spleen deficiency and dampness syndrome(31.61%)41 cases(26.45%)of spleen dampness heat syndrome,16 cases(10.32%)of spleen and kidney yang deficiency syndrome,10 cases(6.45%)of phlegm and blood stasis syndrome,and 2 cases(1.29%)of liver and kidney yin deficiency syndrome of the 155 patients with endometrial disease syndrome.The syndromes of EHWA,AEH and EC patients were mainly liver qi stagnation syndrome,spleen deficiency and dampness syndrome and spleen deficiency dampness syndrome.Among them,EHWA patients had the highest proportion of liver qi stagnation syndrome(32.97%),followed by spleen deficiency and dampness(25.27%);while AEH and EC patients with spleen deficiency and dampness syndrome were accounted for the highest proportion(39.02%,43.48%),followed by spleen dampness heat syndrome(31.71%,34.78%).With the progress of endometrial lesions,the proportion of liver qi stagnation syndrome gradually decreased(P<0.05),while the proportion of spleen deficiency and dampness syndrome and spleen deficiency dampness syndrome increased gradually(P<0.05).(2)Distribution characteristics and evolution of syndromes:cumulative frequency of syndromes of 155 patients with endometrial lesions:spleen(67.10%)>liver(60.00%)>heart(34.84%)>uterine palace(22.58%)>biliary(17.42%)>kidney(10.32%);cumulative frequency of disease syndromes:wet(77.42%)>qi stagnation(39.35%)>sputum(34.19%)>qi deficiency(30.97%)>yang deficiency(29.03%)>hot(26.45%)>blood deficiency(25.81%)>yin deficiency(16.13%)>blood stasis(6.45%)=cold(6.45%)=clood cold(6.45%).Among them,the syndrome elements of EHWA patients mainly were liver(68.13%)and spleen(62.64%),and wet(72.53%),qi stagnation(45.05%)and qi deficiency(36.26%).The syndrome elements of AEH were mainly spleen(73.17%)and liver(51.22%),and wet(85.37%),sputum(43.90%),heat(31.71%),and qi stagnation(31.71%).The syndrome elements of EC patients were mainly spleen(73.91%),liver(43.48%),and wet(82.61%),sputum(39.13%),heat(34.78%).With the progress of endometrial lesions,the syndrome elements changed from"liver to spleen";and the "wet" ran through the whole course,while the proportion of"sputum" and "heat" progresses increased.2.Correlation between inflammatory factors and TCM syndromes:(1)Immunohistochemistry showed that IL-6,IL-17A and TNF-α proteins were mainly expressed in the cytoplasm of glandular epithelial cells of endometrial tissue and the cytoplasm of interstitial cells.The staining was yellow,brownish yellow,and dark brown.(2)Correlation between inflammatory factors and endometrial lesions:IL-6,IL-17A and TNF-α were significantly different between EHWA,AEH and EC(P<0.05).Compared with the EHWA group,IL-6 and TNF-α were significantly increased in the AEH and EC groups(P<0.05),but there was no significant difference between the EC and AEH groups(P>0.05).IL-17A was highest in the AEH group(P=0.024 compared with the EHWA group),but its expression decreased with the lesion progressed to EC.(3)Correlation between inflammatory factors and TCM syndromes of endometrial lesions:Compared with liver qi stagnation syndrome group,IL-6 expression was significantly up-regulated in spleen-deficiency syndrome and spleen-deficiency damp-heat syndrome(P value was 0.000,0.000 and 0.003 respectively),but there was no difference between the two groups in the spleen deficiency and dampness syndrome and spleen dampness heat syndrome(P=0.189).Compared with the liver qi stagnation group,IL-17A was up-regulated in spleen-deficiency syndrome(P=0.026).Although it was up-regulated in spleen-deficiency damp-heat syndrome,it was not statistically significant(P=0.052).There was no difference between the two groups in the spleen deficiency and dampness syndrome and spleen dampness heat syndrome(P=0.776).Compared with the liver qi stagnation group,the expression of TNF-α was significantly up-regulated in the spleen-deficiency damp-heat syndrome(P=0.000).Although the expression was also up-regulated in the spleen-deficiency syndrome,it was not statistically significant(P=0.075).There was no difference between the two groups in the spleen deficiency and dampness syndrome and spleen dampness heat syndrome(P=0.120).(4)Correlation between inflammatory factors and endometrial lesions in patients with spleen deficiency and dampness syndrome and spleen deficiency and dampness syndrome:IL-6 and TNF-α protein showed an increasing trend with the process of endometrial lesions.There was a significant difference in expression between EHWA and EC(P=0.004,0.042,respectively).There was no significant difference of IL-17A expression in each group(P>0.05),except for between AEH and EC group(P=0.000).3.Experimental studies(1)CCK-8 method showed that different doses of IL-17A could promote the proliferation of ishikawa cells(P<0.05),and reached the highest at 50 ng/Ml.Cell scratch test and transwell test showed that IL-17A was dosed-dependently promoted ishikawa cell migration(P<0.05).Western experiment showed that IL-17A up-regulated the expression of ERa protein(P<0.05),but had little effect on ERβ protein expression.(2)Regulation of genistein on the inflammatory microenvironment of tumor:genistein(0.01,0.1 umol/L)could effectively inhibit the proliferation of ishikawa cells induced by IL-17A(P<0.05).To some extent,it inhibited the migration of ishiakwa cells induced by IL-17A,but it was not statistically significant compared with IL-17A group(P>0.05).Western blot showed that ERa protein expression was up-regulated by IL-17A alone(P<0.05),and p-AKT,p-ERK protein expression were also up-regulated(P<0.05),but not on ERβ,AKT,ERK protein expression.But after the combination with genistein,the expression of ERβ protein was up-regulated compared with the IL-17A alone group(P<0.05),while the expression of ERa protein,p-AKT protein and p-ERK protein were down-regulated(P<0.05)at the same time.Conclusions1.Syndromes and syndrome elements research:(1)Syndromes:the syndromes of EHWA patients were mainly liver qi stagnation syndrome,followed by spleen deficiency and dampness syndrome;while AEH and EC patients were spleen deficiency and dampness syndrome and spleen dampness and heat syndrome.In the process of endometrial lesions,the proportion of spleen deficiency and dampness syndrome and spleen dampness and heat syndrome gradually increased.(2)Syndrome elements:"wet" was the core syndrome element of endometrial lesions,and " sputum " and "heat" were the key factors in the progression of endometrial lesions.2.(1)With the deepening of endometrial lesions,the expression of IL-6,IL-17A and TNF-α protein in endometrium increased,indicating that IL-6,IL-17A and TNF-α may be involved in the process of "inflammation-cancer transformation" of endometrial hyperplasia.(2)The expressions of IL-6,IL-17A and TNF-α protein in endometrium of patients with spleen deficiency and dampness syndrome and spleen deficiency dampness syndrome were higher than those of liver qi stagnation syndrome,and the expression showed an increasing trend which indicating that compared with liver qi stagnation syndrome,spleen deficiency and dampness syndrome and spleen dampness heat syndrome might be TCM syndromes related to"inflammation-cancer transformation" of endometrial hyperplasia.3.IL-17A could significantly promote the proliferation and migration of ishikawa cells,which may be closely related to up-regulation of ERα protein expression.Genistein could inhibit the promotion of IL-17A on ishikawa cell proliferation,and its mechanism might was up-regulating the ERβ protein expression,thereby antagonizing the up-regulation of ERa protein and activation of its downstream pathway proteins induced by IL-17A. |
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