| 1 ObjectiveIn view of this situation,the aim is to find the distribution characteristics of TCM Syndromes of female Wilson disease combined with reproductive system damaged.To study the clinical observation on the damage to Wilson’s reproductive system by Invigorating the kidney Eliminating phlegm Resolving stasis recipe.The mechanism for improving the damage of female Wilson disease and reproductive system was further discussed by animal experiment.2 Methods2.1 Clinical investigationFrom January to November 2017,the patients with Wilson disease who were admitted to the first affiliated hospital of anhui university of traditional Chinese medicine(central hospital of anhui province)were investigated.A total of 143 patients with Wilson disease(WD)were screened,and 105 cases were associated with reproductive system damage.Patients who met the criteria for inclusion in the group were able to collect the symptoms and signs of traditional Chinese medicine.2.2 Clinical researchFrom January 2017 to November 2017,a total of 50 patients with Wilson’s disease were treated in the first affiliated hospital of anhui university of traditional Chinese medicine(anhui provincial hospital).They were randomly divided into treatment and control group,each group have 25 people.The control group was treated with western medicine and the treatment group was treated with western medicine and Invigorating the kidney Eliminating phlegm Resolving stasis recipe.They were treated for 90 days.To observe the changes of syndrome differentiation and the improvement of the reproductive system.2.3 Experimental research120 CK57 female mice were fed for 1 weeks were randomly divided into six groups:blank group,copper load model group,DMSA group,GDL group Invigorating the kidney Eliminating phlegm Resolving stasis recipe group,Invigorating the kidney Eliminating phlegm Resolving stasis recipe+DMSA group,20rats in each group.The dosage of the mice was changed according to the body surface area,and the ratio was about 1:9 according to the daily dosage of adult.All the animals in each group were gavaged in the morning,and the dosage of 0.2ml solution was 10g,one times a day.The blank group and the copper load model group were given equal volume of physiological saline.The mice were given by the copper concentration of 100 mg/kg body weight daily lavage of copper solution,lavage volume:0.2 ml/10 g weight of liquids.Gavage dose when these mice taking for 15 days,until to the end of the 28th day.HE staining was used to observe the pathological changes of the cells in mice;in fallopian tube of periampullary remove eggs from the ovary,computation is the number of groups of mice ovulation differences and by immunofluorescence staining to detect egg dome complex granulosa cells in mice to observe granulosa cell proliferation Ki67 expression level.3 Results3.1 TCM syndrome about Wilson disease combined with reproductive abnormality in female patients3.1.1 The distribution of TCM syndromes in two groups of subjectsThe distribution rate of phlegm,blood stasis,kidney Yin deficiency and kidney Yang deficiency in reproductive system damage group was significantly higher than that in WD group(P<0.05).The distribution rate of heat,qi deficiency and yangon in WD group was significantly higher than that in reproductive system damage group(P<0.05 or P<0.01).There was no significant difference in the distribution rreproductive system damage ate of qi depression,blood deficiency,liver Yin deficiency and spleen Yang deficiency(P>0.05).3.1.2 Comparison of TCM syndromes in two groups of subjectsThe syndromes of phlegm,blood stasis,kidney Yin deficiency and kidney Yang deficiency in reproductive system damage group were significantly higher than that in WD group(P<0.01).The syndromes of WD group and liver Yin deficiency were significantly higher than that in reproductive system damage group(P<0.01).There was no significant difference in TCM syndrome differentiation between qi depression,qi deficiency,blood deficiency,Yang kang and spleen Yang deficiency(P>0.05).3.1.3 Comparison of the distribution of composite syndromes in the two groupsThe proportion of multiple syndromes in reproductive system damage group was greater than that in WD group(P<0.01).The proportion of single syndrome in WD group was greater than that in reproductive system damage group(P<0.01),and there was no significant difference in the proportion of multiple syndromes between the two syndromes of groups(P>0.05).3.1.4 The research on the risk degree of TCM syndromes in combination with reproductive system damage groupResearch various syndromes by Logistic regression analysis for the risk of liver degeneration reproductive abnormalities,phlegm dampness,blood stasis,kidney Yin deficiency,kidney Yang deficiency regression coefficient was 8.629,4.496,3.829 and 8.629,respectively,and were statistically significant(P<0.05 or P<0.01).3.2 Patients syndrome differentiation and reproductive system damage symptom improvementCompared with before treatment,both groups of patients were significantly reduced after treatment of TCM syndromes(P<0.01);Compared with the two groups after treatment,the comparison of TCM syndromes in the treatment group was more obvious than that in the control group(P<0.01);The was significant difference between the treatment group and the control group(P<0.01).3.3 Experimental results3.3.1 Effect of tonifying phlegm and removing blood stasis on the ovulation of the copper loaded mice Compared with the normal control group,the difference is significant and the number of eggs in the row was decreased(P<0.01);compared with the copper load group and GDL group,the difference is significant and the number of eggs excreted was increased(P<0.01);Compared with the DMSA group,the number of eggs excreted by Invigorating the kidney Eliminating phlegm Resolving stasis recipe and DMSA the difference is significant and the number of eggs in the row was increased(P<0.01).3.3.2 Effect of Invigorating the kidney Eliminating phlegm Resolving stasis recipe on follicular development in mice with copper loadThe number of normal secondary follicles and normal sinus follicles in the mice of the copper load model group decreased significantly than the normal control group(P<0.01);Compared with the copper load model group,there was significantly increased by Invigorating the kidney Eliminating phlegm Resolving stasis recipe(P<0.01);Compared with the GDL group,the number of normal sinus follicles and normal secondary follicles was significantly increased by Invigorating the kidney Eliminating phlegm Resolving stasis recipe(P<0.05);Compared with the DMSA group,the number of normal sinus follicles and normal secondary follicles was significantly increased by Invigorating the kidney Eliminating phlegm Resolving stasis recipe and DMSA(P<0.05).3.3.3 Effect of Invigorating the kidney Eliminating phlegm Resolving stasis recipe on the ultrastructure of the egg mound complex in the copper load miceThe normal mitochondria’s number decreased significantly in the copper load model group than the normal control group(P<0.01);The number of mitochondrial abnormalities was significantly increased(P<0.01);Compared with the copper load model group,it can significantly increase the number of normal mitochondria and reduce the number of abnormal mitochondria(P<0.01);Compared with the GDL group,the number of normal mitochondria could be increased by Invigorating the kidney Eliminating phlegm Resolving stasis recipe(P<0.01);it can significantly increase the number of normal mitochondria and reduce the number of abnormal mitochondrial by Invigorating the kidney Eliminating phlegm Resolving stasis recipe and DMSA than the DMSA group(P<0.01).3.3.4 Effect of Invigorating the kidney Eliminating phlegm Resolving stasis recipe on the proliferation of granulocytes in the copper loaded miceCompared with the normal control group,the copper load model group expression level of Ki67 protein was significantly reduced(P<0.01);the expression level of Ki67 protein in the mice with Invigorating the kidney Eliminating phlegm Resolving stasis recipe was increased obviously than the copper load model group(P<0.01);The expression level of Ki67 protein was increased in the mice with Invigorating the kidney Eliminating phlegm Resolving stasis recipe than the GDL group(P<0.01);Compared with the DMSA group,it can significantly increase the expression level of Ki67 protein in Invigorating the kidney Eliminating phlegm Resolving stasis recipe and DMSA(P<0.01).4.Conclusion4.1 The main syndromes of WD with abnormal reproductive are phlegm-dampness,blood stasis,kidney Yin deficiency and kidney Yang deficiency;4.2 The main risk factors for the WD with abnormal reproductive are phlegm-dampness,blood stasis,kidney Yin deficiency and kidney Yang deficiency;4.3 Invigorating the kidney Eliminating phlegm Resolving stasis recipe can obviously improve the syndrome differentiation of female Wilson disease with reproductive system and significantly improve the clinical symptoms of patients;4.4 The development of oocyte and follicle and mature harvest in copper loading mice were disturbed;and the development and maturation of the follicles in the copper load mice and enhance the development potential of oocyte can improved by Invigoratingthe kidney Eliminating phlegm Resolving stasis recipe;4.5The copper loading canbring about the apoptosis and the proliferation ability of the granule cells;and invigorating the kidney Eliminating phlegm Resolving stasis recipe can improve the proliferation ability of the granule cells of the oocyte complex,while inhibiting the apoptosis of the granulocytes in the copper loading mice. |
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