| Objective1. To establish endometriosis rat model of combination of disease and syndrome based on theory of traditional Chinese medicine, and then using it to observe the change of appearance and pathomorphism features of ectopic endometrium, and to detected hemorheology and coagulation function of model to confirm the timing of drug intervention.2. To discuss the TCM pathogenesis of EMs that " Hidden cold injures kidney and results in blood stasis damaging collaterals", and further to discuss the EMs pathogenesis joint point of TCM and western medicine that the relationship of "Stasis blocking collaterals " and " angiogenesis ", and then detect the expression level of EphA2, (P)P38MAPK, VEGF and microvessel density (MVD) in nomal and ectopic endometrium. Compared the discrepancies and discuss the relationship between endometriosis and them.3. To detect the change of pathomorphism features, expression level of EphA2, (P)P38MAPK, VEGF and MVD in ectopic endometrium, hemorheology and coagulation function of model after theropy of JINGTONGYUSHU, and discuss the therapeutic mechanism of action of JINGTONGYUSHU for endometriosis.4. To compare the results of preventive intervention and therapeutic intervention of JINGTONGYUSHU for endometriosis, and discuss the impact and mechanism of action of JINGTONGYUSHU preventive intervention.Methods1. Part 1:Female virgin rats were randomly divided into blank control group and model group. There were 4 rats in the former, and 26 rats in the latter. The model group were treated by autologous endometrial transplants and ice water immersion methods. Respectively took out 4 models after 3 days,5 days,4 weeks,6 weeks and 8 weeks of operation randomly and used them to observe the appearance of the rat and pathomorphism features of ectopic endometrium by HE stain, detected hemorheology and coagulation function. All of the results were compared with the results of blank control group.2. Part 2:Female virgin rats were randomly divided into blank control group and model group. There were 12 rats in the former, and 88 rats in the latter. The model group were treated by previous methods. Randomly took out 72 successful models after 4 weeks of operation and divided them into 4 weeks group,8 weeks group, JINGTONGYUSHU group(low-dose, middle-dose, high-dose) and western medicine group with 12 rats in each group. The 4 weeks group were directly executed, and the others were executed after respectively drug intervention of 4 weeks. Took blood when the rats were executed for detecting the hemorheology and coagulation function, and took the ectopic endometrium (normal endometrium for normal rat). HE stain was performed to observe the pathomorphism features. RT-PCR was performed to detect the expression of EphA2mRNA. Western bolt was performed to detect the expression of EphA2 protein. Immunohistochemistry was performed to detect the expression of EphA2, (P)P38MAPK, VEGF and MVD. Determine the best dose of JINGTONGYUSHU.3. Part 3:Female virgin rats were randomly divided into blank control group and model group. There were 12 rats in the former, and 48 rats in the latter. The model group were treated by previous methods. Randomly took out 12 successful models after 5 days of operation to be the prevention group which started to get drug intervention for 4 weeks, and treated by ice water immersion method after 1 week of operation. Randomly took out 24 successful models after 4 weeks of operation and divided them into 4 weeks group and JINGTONGYUSHU (high-dose) group averagely. The 4 weeks group were directly executed, and the others were executed after drug intervention of 4 weeks. All of detect items were same as Part 2.Recults1. Endometriosis model is established by combined method of by autologous endometrial transplants and ice water immersion, and the success rate was 76.9%.3 days and 5 days groups just showed inflammatory reaction, but the 4 weeks,6 weeks and 8 weeks groups showed the mature endometrium tissue. The model group showed the cold-evil appearance, and showed abnormal hemorheology and coagulation function. There was no significant difference of blood index among model groups.2. The success rate of endometriosis modeling is 86.4%. All of the JINGTONGYUSHU groups had a better appearance and mental state after drug intervention, while the western medicine group had no change and got weight gain in addition. The ectopic endometrium volume of all treatment groups obviously shrinked, especially the JINGTONGYUSHU high-dose group and western medicine group. Pathomorphism features of all treatment groups showed varying degrees of growth inhibition, especially the JINGTONGYUSHU high-dose group and western medicine group. All of JINGTONGYUSHU group showed a better hemorheology and coagulation function while the western medicine group had no change.3. RT-PCR and Western blot results showed that the expression of mRNA and protein of EphA2 were higher in each model group and treatment group than blank control group. The expression of mRNA and protein of EphA2 were higher in 8 weeks group while lower in each treatment group than 4 weeks group. Their expression was lower in JINGTONGYUSHU high-doses than in the western medicine group.4. Immunohistochemistry results showed that the expression of EphA2, (P)P38MAPK, VEGF and MVD were higher in model groups and treatment groups than in blank control group. The expression of EphA2, (P)P38MAPK, VEGF and MVD were higher in 8 weeks group while lower in treatment groups than 4 weeks group. Their expression was lower in JINGTONGYUSHU high-dose than in western medicine group. Spearman correlation analysis result showed that the expression of EphA2 was positively correlated with (P)P38MAPK, VEGF and MVD in ectopic endometrium.5. The success rate of endometriosis modeling is 85.4%. The prevention group hadn’t showed the the cold-evil appearance. The ectopic endometrium volume of prevention group and JINGTONGYUSHU (high-dose) group obviously shrinked, and conglutination with surrounding tissue reduced. Pathomorphism features showed that the growth inhibition effect of prevention group was better than it in the JINGTONGYUSHU (high-dose) group. There was no difference of hemorheology and coagulation function between prevetion group and JINGTONGYUSHU (high-dose) group.6. RT-PCR and Western blot results showed that the expression of mRNA and protein of EphA2 were higher in prevention group than in JINGTONGYUSHU (high-dose) group. Immunohistochemistry results showed that the expression of EphA2, (P)P38MAPK and VEGF were higher in prevention group than in JINGTONGYUSHU (high-dose) group. The expression of MVD was lower in prevention group than in JINGTONGYUSHU (high-dose) group. Spearman correlation analysis result showed that the expression of EphA2 was positively correlated with (P)P38MAPK, VEGF and MVD in ectopic endometrium.Conclusion1. The the type of cold coagulation and blood stasis endometriosis model could be successfully established by combined method of autologous endometrial transplants and ice water immersion.2. "Hidden cold injures kidney and results in blood stasis damaging collaterals" was the TCM pathogenesis of EMs. The relationship between "Stasis blocking collaterals" and "angiogenesis" was the EMs pathogenesis joint point of TCM and western medicine.3. The expression of EphA2mRNA and protein were obviously increased in ectopic endometrium, and overexpression of EphA2 was positively correlated with the expression of (P)P38MAPK、VEGF and MVD. These results suggested that EphA2 might play a critical role in the initiation and progression of EMs.4. JINGTONGYUSHU could lead to the obvious inhibition of ectopic endometrium growth and angiogenesis, and it could also decrease the expression of EphA2、(P)PMAPK38、VEGF in ectopic endometrium observably. JINGTONGYUSHU had better effect on improving EMs rat hemorheology and coagulation function, improving cold-evil appearance and without obvious side effect than western medicine danazol.5. In the development progress of EMs, EphA2 may affect the angiogenesis of ectopic endometrium through regulating VEGF via P38MAPK signaling pathway.6. Preventive intervention of JINGTONGYUSHU made a better effect on inhibiting ectopic endometrium growth than therapeutic intervention, supporting the purpose of JINGTONGYUSHU to EMs in prevention before disease onset and prevention of recurrence. |
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