Clinical And Experimental Study On Wenjing Huayu Zhitong Treatment On Primary Dysmenorrhea With Cold Coagulation And Stasis Syndrome Based On NGF

Objective:To observe the clinical efficacy of Wenjing Huayu Zhitong Treatment on primary dysmenorrhea(PD)with cold coagulation and stasis syndrome.Reveal the pathogenesis of PD furtherly based on nerve growth factor(NGF)and its receptors,and explore the mechanisms of Wen Jing Huayu Zhitong Treatment on PD.Our study will provide scientific basis for Traditional Chinese Medicine(TCM)to treat PD.Methods:This study is divided into two parts:clinical trials and animal experiments.1 Clinical study:The clinical study was a randomized,double-blind,parallel controlled design with positive and placebo.A total of 108 patients of PD with cold coagulation and stasis syndromes were collected and divided into TCM group,ibuprofen group and placebo group according to the random number table method.There were 36 cases in each group.All patients started to be administered 3 days before menstrual cramps,continuous for 6 days.The TCM group was treated with TCM of free decoction granules and ibuprofen sustained-release capsules simulator.The ibuprofen group was treated with ibuprofen sustained-release capsules and TCM simulator of free decoction granules.The placebo group was treated with TCM simulator of free decoction granules and ibuprofen sustained-release capsules simulator.One menstrual cycle is one course of treatment,and all the patients were treated continuously for 3 courses.After treatment,the TCM group and ibuprofen group were followed up for three menstrual cycles.The changes of VAS scores,cumulative time of abdominal pain,and TCM symptoms during baseline,treatment,and follow-up periods,and prostaglandin F_2?(PGF_2?),NGF,tyrosine kinase receptor A(Trk A)and p75 neurotrophin receptor(p75NTR)levels in peripheral blood.At the same time,the blood routine,liver and kidney function,and electrocardiogram of patients were monitored before and after treatment.We evaluated the clinical efficacy and safety of Wenjing Huayu Zhitong Treatment on PD with cold coagulation and stasis syndromes.2 Experimental study:Forty-eight female SPF-grade Wistar rats were randomly divided into blank group,model group,western medicine group,TCM low dose group,middle dose group and high dose group.There were eight rats in each group.In addition to the blank group,dysmenorrhea rat model of cold coagulation and stasis syndrome was established by cold stimulation combined with estradiol benzoate and oxytocin.All rats were treated with TCM,ibuprofen and distilled water once a day from 7th day of modeling,and for 6 days consecutively.Observe the writhing latency,the number of writhing times and the writhing response score of rats within 30 min after oxytocin injection.PGE₂,PGF_2?,COX-2,and vasopressin(AVP)in serum were detected by enzyme-linked immunosorbent assay(ELISA).Trk A and p75NTR protein expression in uterine tissues were detected by Western Blot.NGF,Trk A and p75NTR m RNA expression in uterine tissues were detected by Real-time PCR.Results:1 Clinical research:(1)VAS score and cumulative time of abdominal pain:During the treatment period,with the extension of the treatment time,the VAS score and cumulative time of abdominal pain in the TCM group and ibuprofen group showed a downward trend.Compared with the baseline period at each time point,and the differences were significant statistically(P<0.01).The TCM group was similar to that of the ibuprofen group(P>0.05),and both were better than the placebo group(P<0.01).During follow-up period,compared with the baseline period,the VAS score and cumulative time of abdominal pain in the TCM group and ibuprofen group were significantly reduced(P<0.01).Compared with the 3 courses of treatment,the VAS score and cumulative pain of ibuprofen group at each time point were significantly increased significantly(P<0.01),but there was no significant change in the TCM group(P>0.05).The comparisons among the three groups showed that the VAS score and the cumulative time of abdominal pain at each time point during the follow-up period of the TCM group were lower than those of the ibuprofen group and placebo group(P<0.01).This demonstrates that the analgesic effect of the TCM group in short-term is comparable to the ibuprofen group,but the long-term effect is better than the ibuprofen group.(2)TCM symptom scores:The total effective rate of the TCM group was 91.43%,the ibuprofen group was 66.67%,and the placebo group was 30.30%.The TCM group was superior to the ibuprofen group and the placebo group(P<0.01).During the treatment period,the TCM symptom scores of the three groups were significantly lower than the baseline period at each time point(P<0.01),and the TCM group was lower than the ibuprofen and placebo groups(P<0.05,P<0.01).During the follow-up period,compared with the baseline period,the TCM symptom scores of the TCM group and ibuprofen group were significantly reduced at each time point(P<0.05,P<0.01).Compared with the 3 courses of treatment,the TCM symptom scores of each time point of the ibuprofen group were increased significantly(P<0.01),but there was no significant change in the TCM group(P>0.05).Comparing among the three groups,the TCM symptom score of the TCM group was significantly lower than that of the ibuprofen group and the placebo group(P<0.01).(3)Symptoms of TCM:After three courses of treatment,the abdominal pain scores of the three groups were significantly lower than the baseline period(P<0.05,P<0.01).The efficacy of the TCM group was similar to the ibuprofen group and better than placebo group(P>0.05,P<0.01).In terms of dark menstruation,low menstrual flow,cold and chills in the limbs,diarrhea,and anal slump,the efficacy of the TCM group was significantly better than that of the ibuprofen and placebo groups(P<0.05,P<0.01).In terms of pale complexion and nausea and vomiting,the TCM group was better than the placebo group(P<0.01),and the difference was not statistically significant compared with the ibuprofen group(P>0.05).(4)Test results of laboratory indicators:After treatment,the contents of PGF_2?,NGF,Trk A,and p75NTR in the TCM group and ibuprofen group were all lower than before treatment(P<0.01),and there was no significant difference between the two groups(P>0.05),and both were lower than the placebo group(P<0.05,P<0.01).2 Experimental study:(1)Writhing response:Compared with the model group,the writhing latency of rats in each treatment group was prolonged,but the difference was not statistically significant(P>0.05).The writhing frequency and writhing response scores in each treatment group were significantly reduced compared with the model group(P<0.01).The western medicine group and TCM medium and high dose groups were better than the TCM low dose group in reducing the frequency of writhing(P<0.01,P<0.05).There was no significant difference in writhing response scores among all treatment groups(P>0.05).(2)ELISA test results:Compared with the blank group,the content of PGE₂ decreased,and the PGF_2?,COX-2 increased in the model group and each treatment group(P<0.01),The content of AVP increased in the model group,the TCM low and medium dose groups(P<0.01,P<0.05).Compared with the model group,the content of PGE₂ increased,the PGF_2?and COX-2 decreased in each treatment group(P<0.05,P<0.01).The content of AVP decreased in the western medicine group,the TCM medium and high dose groups(P<0.01).Comparing the treatment groups,the content of PGE₂ in the western medicine group was higher than that in the low dose group of TCM.The content of COX-2 in the western medicine group,the TCM medium and high dose groups were lower than that in the TCM low dose group.The content of AVP in the western medicine group and TCM high dose group were lower than that in the TCM low dose group.The differences were statistically significant(P<0.05,P<0.01).There was no significant difference in reducing PGF_2?between the treatment groups(P>0.05).(3)Western blot test results:Compared with the blank group,the levels of NGF and Trk A in the model group and each treatment group were significantly increased,and the levels of p75NTR in the model group and the three TCM groups were increased(P<0.05,P<0.01).Compared with the model group,the levels of NGF,Trk A,and p75NTR in each treatment group were significantly reduced(P<0.01).Comparing among the treatment groups,the levels of NGF in the western medicine group and the TCM high dose group were significantly lower than those in the TCM low dose group(P<0.01).The levels of Trk A in the western medicine group were lower than that in TCM low and medium dose groups(P<0.01),and the high dose group and medium dose group were significantly lower than the low dose group(P<0.05,P<0.01),and the high dose group was lower than the medium dose group(P<0.05).The levels of p75NTR in western medicine group and the TCM high dose group were lower than that of the low dose group(P<0.05,P<0.01).(4)Real-time PCR test results:Compared with the blank group,the expressions of NGF,Trk A,and p75NTR m RNA in the model group and each treatment group were significantly increased(P<0.01).Compared with the model group,the expression of NGF and Trk A m RNA decreased in each treatment group(P<0.01),and the expression of p75NTR m RNA in the western medicine group and the TCM high dose group decreased(P<0.01).Comparing the treatment groups,the expressions of NGF and p75NTR m RNA in the western medicine group,the TCM medium and high dose groups were lower than those in the TCM low dose group(P<0.05,P<0.01).The differences of Trk A m RNA expression among all treatment groups were no statistical significance(P>0.05).Conclusion:(1)Clinical studies have shown that Wenjing Huayu Zhitong Treatment is comparable to ibuprofen in terms of its recent analgesic effect and reducing the levels of PGF_2?,NGF and Trk A,p75NTR in patients'peripheral blood,but significantly better than ibuprofen in improving TCM symptoms and long-term efficacy.(2)Experimental study indicated that Wenjing Huayu Zhitong Treatment can inhibit the writhing reaction of rats effectively,and increase the level of PGE₂ in serum,and reduce the levels of PGF_2?,COX-2 and AVP.The dysmenorrhea of cold coagulation and stasis syndromes model rats had abnormal expression of NGF,Trk A,and p75NTR,and the Wenjing Huayu Zhitong Treatment could regulate the abnormal expression of NGF,Trk A,and p75NTR,indicating that the Wenjing Huayu Zhitong Treatment may affect the PGF_2?level by regulating the expression of NGF,Trk A and p75NTR,and thus achieve the purpose of treating PD.