| Objective1.To provide the theory basis of Chinese medicine EMs,this paper established a cold coagulation and blood stasis EMs model based on the theory of lurking cold,and then make a comprehensive evaluation on the model.2.To discover the pathway of EMs infertility,PDGFR/JAK2/STAT3 signaling pathway.To discover the mechanism of Tongyushu particles treating EMs rats of cold coagulation and blood stasis type on different doses and to clarify the effects.To compare the effects with Western medicine.3.To provide experimental basis of the etiology and pathogenesis of EMs infertility“lurking cold can cause bruising and damage to the kidney,causing the injury of the uterus”.4.Preventalble intervention of EMs rat model to promote fertility after laparoscope operation.Methods1.Experiment one:30 SPF female SD rats without copulation were randomly divided into a 7-day ice-water group,a 14-day ice-water group and a control group with 10 rats per group.Cold coagulation and blood stasis type model were established by ice-water soaking and epinephrine hydrochloride subcutaneous injection.TCM symptom were observed the next day,blood samples were collected from heart to observe coagulation function and hemorheology to determine establishment of model.2.Experiment two:80 SPF female rats without copulation,10 were randomly picked as the control group and given laparotomy,watered the enterocoelia and sutured napes.The other 70 were given model according to experiment one,and then were given endometrium autotransplantation.1 week after model establishment,soak the models with ice-water once daily for 1 week and pick 6 models 3 days,5 days,1 week,2 weeks,3 weeks,4 weeks,6 weeks,8 weeks and 12 weeks after operation.Compared TCM symptom,focus,endometrium pathologies,coagulation function and hemorheology to determine drug administration time of therapeutic and preventive.3.Experiment three:126 SPF female rats without copulation,18 were picked as a control group and others as model group.Exploratory laparotomy was performed 4 weeks after the operation of the model group,90 models were picked and randomly divided into a 4 weeks group,a 8 weeks group,low dose group,middle dose group,high dose group and western medicine group,with 15 models per group.4 weeks group were executed 4 weeks after operation on implantation window phase,other groups were executed 4 weeks after chronically administered on implantation window phase.All rats were tested gonadal hormone concentrations,coagulation function and hemorheology,model groups taken endometrium and ectopic tissue,control group taken right uterus tissue to do pathomorphism.Immunohistochemical was used to determine HOXA10,PDGFR,p-JAK2,p-STAT3,VEGF expression.Western Blot and RT-PCR to determine HOXA10,PDGFR,p-JAK2,p-STAT3 and to determine best dose of therapeutic usage.4.Experiment four:72 SPF female rats without copulation,18 as control group,establish models as in experiment three.Models were given laparotomy 5 days after operation,15 were picked as prevention group and given best dose of Tongyushu particles,then given a 7-days soaked 1 week after operation.4 weeks after operation,picked 30 models as an 8 weeks group and a best dose group with 15 for each.4 weeks after given drug,models were executed and given the same test as experiment three to determine the effects of preventive usage of Tongyushu particles.Results1.Experiment one:The rat model of congealed blood stasis can be successfully prepared by 14 days ice-water soaking combining with epinephrine hydrochloride subcutaneous injection.2.Experiment two:the success rate of model making was 78.6%.3 days after operation,tissue edema was severe.5 days after operation,compared with 3 days after operation,tissue edema decreased,ectopic tissue did not form completely into endometrial epithelial structure.4 weeks after operation,typical endometrial histology ocurred,and the pathological findings were formed.6 weeks,8 weeks after operation and 12 weeks after operation were similar with 4 weeks after operation.With the exception of 12 weeks after operation,all the rats in the model group showed the symptoms of "cold coagulation and blood stasis'',and the coagulation function and hemorheology changed accordingly.Combined with the pathological results of ectopic endometrium,the therapeutic time point was determined at 4 weeks after operation and the prophylactic use time point on 5 days after operation.3.Experiment three:the success rate of model making in this experiment was 87.03%.The appearance of cold coagulation and blood stasis was improved,the mental state was improved,and the state of the rats in the middle and middle high groups was obviously improved after 4 weeks of administration of Chinese medicine.Compared with the two model groups,thefocus volume of each treatment group was significantly reduced,only a small piece of hyperplastic protuberance was occasionally present,and the pathological changes of ectopic endometrium were obvious,and by contrast the changes of the syndromes in the western medicine group were not obvious.The epithelial cells were arranged incoherently and the number of glands decreased,especially in the high dose group.There was no glandular structure and no inflammatory cell infiltration.In eutopic endometrium,the morphology of endometrium and the internal membrane structure were changed.The number of uterine glands was decreased,the dilatation degree of glandular cavity was reduced,and the loose structure was improved.The changes were more obvious in the high dose group of traditional Chinese medicine,and the cell morphology was close to the normal endometrium.The levels of estradiol in each group were significantly higher than those in the blank control group;in comparison with the model group,the levels of estradiol in each treatment group were significantly decreased,especially in the high dose group;progesterone levels were significantly lower in each treatment group than in the blank control group,but compared with the model group,the treatment group significantly increased.The immunohistochemical results showed that compared with the blank control group,the treatment groups and model groups in PDGFR,p-JAK2,p-STAT3 and VEGF were increased,HOXA10 expression decreased;compared to model group 8 weeks and 4 weeks model group,the expression of the first four were increased and expression of HOXA10 were decreased;comparing the treatment groups with model groups,the expression of the first four were all reduced,the expression of HOXA10 increased,the five expression changes in the high dose group was the most obvious,at the same time,PDGFR,p-JAK2,p-STAT3 and the expression of VEGF is consistent.WB and PCR results show that compared with the blank group,the expression of HOXA10 decreased significantly in treatment groups and model groups,the expression of HOXA10 in each treatment groups was higher than that in model groups,the expression of HOXA10 in high dose group was most obvious.The expression of PDGFR,p-JAK2,p-STAT3 in each treatment groups and model groups was significantly higher than that in control group,the expression of the three mRNA and proteins in the 8 weeks model group was higher than that in the 4 weeks model group,and the expression of the three proteins and mRNA in each treatment groups was lower than that in the model groups,especially in the high dose group.Experiment four:the success rate of this experiment was 88.89%.There was no sign of cold coagulation and blood stasis in the prevention group.Compared with the model 8 weeks group,the ectopic lesion shrank and the adhesion with the surrounding tissue was obviously reduced in the high dose and prevention groups.The pathological changes of ectopic endometriumin the two treatment groups were more significant,and the number of vessels in the prevention group was less than that in the high dose group.The eutopic endometrial pathology of the two treatment groups was close to normal endometrium,and the structure of endometrium in the prevention group was more continuous and intact.There was no significant difference in blood rheology and coagulation function between the two treatment groups,while the sex hormone levels were more obvious in the prevention group to lower estradiol and increase progesterone.The immunohistochemical results showed that both the high dose group and the prevention group could significantly reduce the level of PDGFR,p-JAK2,p-STAT3,VEGF and increased the expression of HOXA10,especially in the prevention group.The results of WB and PCR showed that the expression of PDGFR protein and mRNA in the 8 weeks model group was higher than that in the treatment groups,the expression of the prevention group was significantly lower than that in the high dose group,and the changes of p-JAK2 and p-STAT3 in each groups were consistent with that of PDGFR.The expression of HOXA10 protein and mRNA in the 8 weeks model group was significantly lower than that in the two treatment groups,and the expression in the prevention group was significantly.higher than that in the high dose group.Conclusion1.Ice-water soaking combined with subcutaneous injection and self endometrial transplantation can successful established endometriosis of cold coagulation and blood stasis type.2.It is pointed out that"lurking cold can cause bruising and damage to the kidney,causing the injury of the uterus"is the key to the pathogenesis of EMs with cold coagulation and blood stasis type.3.The expression of PDGFR protein and its mRNA on the endometrium of EMs in the peritoneal implantation window was significantly increased.It is speculated that the abnormal high expression of PDGFR may play an important role in the pathogenesis of endometriosis infertility.Tongyushu particles can effectively inhibit the high expression of PDGFR,it can effectively improve its endometrial receptivity,and the prophylactic application effect is better. |
PDF Full Text Request