Explore The Mechanism Of Wenjing Huayu Zhitong Treatment On Primary Dysmenorrhea With Cold Coagulation And Blood Stasis Syndrome Based On Th1/Th2 Intervening NGF And MAPK/ERK Pathway

Objective:The effectiveness and safety of the Wenjing Huayu Zhitong treatment on PD were evaluated systematically,which provided evidence based medical support for its clinical application.To observe the effect of type 1 helper T cell/2 type helper T cell(Th1/Th2)balance shift on nerve growth factor(NGF)and the effect of Wenjing Huayu Zhitong treatment on NGF and its related mitogen-activated protein kinases/extracellular regulated protein kinases(MAPK/ERK)signal pathway,to further reveal the pathogenesis of primary dysmenorrhea(PD)and the mechanism of Wenjing Huayu Zhitong treatment on PD.Methods:1 Part oneBy searching China National Knowledge Infrastructure(CNKI),VIP,Wanfang database,Pub Med database,Cochrane Library and EMbase database,all randomized controlled trials about the treatment of PD with cold coagulation and blood stasis syndrome were included,in which the intervention was oral administration of Wenjing Huayu Zhitong TCM compound while the control was oral administration of buprofen.The data were screened and sorted out by two researchers,the quality of the included articles was evaluated based on the bias risk assessment tool provided by Cochrane Handbook,and Meta-analysis of the data were performed by Review Manager 5.6.2 Part twoThirty-two female SPF Wistar rats aged 7-8 weeks were randomly divided into blank group,model group,Th1 shift group and Th2 shift group,the cold stimulation combined with estradiol benzoate and oxytocin injection was used to establish the PD rat model with cold coagulation and blood stasis syndrome,besides,the Th1 shift and Th2 shift PD rat models were established by intraperitoneal injection of interferon-?(IFN-?)and interleukin-4(IL-4)respectively.The writhing reaction of rats in each group was observed after oxytocin injection,and Enzyme linked immunosorbent assay(ELISA)was employed to detect the level of prostaglandin F_2?(PGF_2?),prostaglandin E₂(PGE₂),6-keto-prostaglandin F_1?(6-keto-PGF_1?),thromboxane B2(TXB2),cyclooxygenase-2(COX-2),vasopressin(AVP)and oxytocin(OT)in rats'serum,while the protein expression and m RNA transcription level of NGF,Trk A and p75NTR were detected by Western blot and real-time PCR respectively.3 Part threeForty-eight female SPF Wistar rats aged 7-8 weeks were randomly divided into blank group,model group,western medicine group,low dose TCM group,middle dose TCM group and high dose TCM group.The PD rat model with cold coagulation and blood stasis syndrome was established and were gavage with distilled water,buprofen and traditional Chinese medicine from the 7th day of PD modelling.The quantitative scores of symptoms and signs of rats were recorded pre and after treatment,and the writhing reaction of rats after oxytocin injection was observed,the level of PGE₂,PGF_2?,6-Keto-PGF_1?,TXB2,IL-4,IFN-?in rats'serum were detected by ELISA,the expression of NGF,Trk A,MEK1/2,Erk1/2,p-MEK1/2,p-Erk1/2,B-Raf,c PLA2,c-jun,COX-2 were detected by Western blot,and the m RNA transcription levels of NGF,Trk A,MEK1,Mek2,ERK1,ERK2,B-Raf,c PLA2,c-jun and COX-2 were collected by real-time PCR.Results:1 Part oneTwenty-eight studies involving 2510 PD patients with cold coagulation and blood stasis syndrome treated by Wenjing Huayu Zhitong treatment were included in the Meta-analysis.Risk bias assessment showed:There was no high-risk bias study.The clinical effective rate of Wenjing Huayu Zhitong treatment was better than buprofen:OR=3.72,95%CI(2.83,4.89),P<0.00001,the recurrence rate was lower than that of buprofen group after drug withdrawal:OR=0.17,95%CI(0.08,0.36),P<0.00001,the improvement of VAS score was better than that in buprofen group:MD=-1.32,95%CI(-1.86,-0.77),P<0.00001,the improvement of pain score was better than that in buprofen group:MD=-1.52,95%CI(-1.71,-1.34),P<0.00001,the improvement of TCM symptom score was better than that in buprofen group:MD=-2.33,95%CI(-3.59,-1.08),P=0.0003,the level of PGF_2?was lower than that in buprofen group:MD=-12.51,95%CI(-20.80,-4.21),P<0.00001,the level of PGE₂was higher than that in buprofen group:MD=2.80,95%CI(1.95,3.66),P<0.00001.Four studies have evaluated its safety,and only2 patients had mild diarrhea when Wenjing Huayu pain Zhitong treatment was used in PD.2 Part two(1)Writhing reaction:There was no significant difference in writhing latency time between the Th1 shift group,Th2 shift group and model group(P>0.05),but the writhing latency time in the Th1 shift group was longer than that in Th2 shift group(P<0.05).Compared with the model group,the writhing times in the Th1 shift group was significantly decreased(P<0.01),however,the writhing times in the Th2 shift group was significantly increased(P<0.01).(2)ELISA results:Compared with the blank group,the PGF_2??TXB2?COX-2?AVP?OT levels of rats in each group were increased(P<0.05,P<0.01),while the PGE₂and6-Keto-PGF_1?levels were decreased(P<0.01).Compared with the model group,the levels of PGF_2?,TXB2,COX-2 and AVP in the Th1 shift group were decreased(P<0.05,P<0.01)and the levels of PGE₂and 6-keto-PGF_1?were increased(P<0.01),while in the Th2 shift group,the levels of PGF_2?,TXB2,COX-2,AVP and OT were increased(P<0.01)and the levels of PGE₂and 6-keto-PGF _1?were decreased(P<0.01).(3)Western blot and real-time PCR results:Compared with the blank group,the levels of NGF protein,m RNA and Trk A m RNA were increased in each group(P<0.05,P<0.01),and the Trk A protein,p75NTR protein and m RNA level in the model group and Th2 shift group were also increased(P<0.05,P<0.01).Compared with the model group,the levels of NGF protein and m RNA,Trk A protein and m RNA,p75NTR m RNA in the Th1 shift group were decreased(P<0.05,P<0.01),and the levels of NGF,Trk A,p75NTR protein and m RNA in the Th2 shift group were increased(P<0.05,P<0.01).3 Part three(1)Writhing reaction:There was no significant difference in writhing latency(P>0.05).Compared with the model group,the writhing times of each treatment group were significantly reduced(P<0.01).There was no significant difference in writhing times among the western medicine group,middle dose TCM group and high dose TCM group(P>0.05),which were less than low dose TCM group(P<0.05,P<0.01).(2)Quantitative score of symptoms and signs:There was no significant difference in the score of each group(P>0.05)before treatment.And there was no significant difference in scores among the model group,western medicine group and low dose TCM group after treatment(P>0.05),which were higher than those in the middle and high dose TCM groups(P<0.05,P<0.01).(3)ELISA results showed:Compared with the blank group,the levels of PGF_2?,TXB2and IL-4 in the model group were increased(P<0.01),and the levels of PGE₂,6-keto-PGF_1?and IFN-?were decreased(P<0.01).Compared with the model group,the levels of PGF_2?and IL-4 in each treatment group were decreased(P<0.05,P<0.01),the levels of PGE₂,6-keto-PGF_1?and IFN-?in each treatment group were increased(P<0.05,P<0.01),and the levels of TXB2 in the western medicine group,middle and high dose TCM groups were significantly decreased(P<0.01).There was no significant difference in PGF_2?content among the treatment groups(P>0.05),and there was no significant difference in improving the levels of PGE₂and TXB2 between the western medicine group and the middle and high dose TCM groups(P>0.05),while the middle dose TCM group was more efficient than other groups in improving the level of 6-keto-PGF_1?,IFN-?and IL-4(P<0.05,P<0.01).(4)Western blot results:Compared with the blank group,the levels of NGF,Trk A,p-MEK1/2,p-Erk1/2,B-Raf,c PLA2,c-jun and COX-2 in the model group were increased(P<0.05,P<0.01).Compared with the model group,the levels of NGF,Trk A,B-Raf and COX-2 in the treatment groups were decreased(P<0.05,P<0.01),the levels of p-MEK1/2,p-Erk1/2 and c-jun in the western medicine group and the middle and high dose TCM groups were decreased(P<0.05,P<0.01),and the expression of c PLA2 in the middle and high dose TCM groups was decreased(P<0.05,P<0.01).Among the treatment groups,the levels of NGF,p-MEK1/2,p-Erk1/2 in the western medicine group,middle and high dose TCM groups were not significantly different(P>0.05),but were significantly lower than those in the low dose TCM group(P<0.01).The level of Trk A in the high dose TCM group was lower than that in the low dose and middle dose TCM groups(P<0.05,P<0.01),and without significant difference with the western medicine group(P>0.05).There was no significant difference in the levels of B-Raf and c-jun between the middle and high dose TCM groups(P>0.05),which were lower than those in the western medicine group and the low dose TCM group(P<0.05,P<0.01).The level of c PLA2 in the middle dose TCM group was lower than that in the western medicine group and low dose TCM group(P<0.05,P<0.01),and there was no significant difference with high dose TCM group(P>0.05).The level of COX-2 in the high dose TCM group was lower than that in the low dose and middle dose TCM groups(P<0.05,P<0.01),but there was no significant difference with the western medicine group(P>0.05).(5)Real time PCR results showed:Compared with the blank group,the m RNA levels of NGF,Trk A,MEK1,MEK2,Erk1,Erk2,B-Raf,c PLA2,c-jun and COX-2 in the model group were significantly increased(P<0.01).Compared with the model group,the m RNA levels of NGF,Trk A,MEK1,MEK2,Erk1,Erk2,B-Raf,c PLA2 and c-jun in the treatment groups were decreased(P<0.05,P<0.01),and the m RNA levels of COX-2 in the western medicine group,middle and high dose TCM groups were also decreased(P<0.01).Among the treatment groups,there was no significant difference in the m RNA levels of NGF,MEK1,Erk1,Erk2,B-Raf and COX-2 between the western medicine group and the middle and high dose TCM groups(P>0.05),which were lower than those in the low dose TCM group(P<0.01).There was no significant difference in Trk A and MEK2 m RNA levels between the western medicine group and middle dose TCM group(P>0.05).The Trk A m RNA in the western medicine group and MEK2 m RNA in the middle dose TCM group were lower than those in the low and high dose groups(P<0.05,P<0.01)respectively.There was no significant difference in the level of c PLA2 m RNA between the middle and high dose TCM groups(P>0.05),which was lower than that of the low dose TCM group(P<0.05),and there was no significant difference compared with the western medicine groups(P>0.05).There was no significant difference in the c-jun m RNA level between the western medicine group and the high dose TCM group(P>0.05),but the c-jun m RNA level in the western medicine group was lower than that in the low and middle dose TCM groups(P<0.05,P<0.01).Conclusion1 Wenjing Huayu Zhitong treatment is safe and effective in the treatment of PD with cold coagulation and blood stasis syndrome which is superior to buprofen in improving clinical efficiency,reducing VAS score,pain score,TCM symptom score,recurrence rate after drug withdrawal,PGF_2?level and increasing PGE₂level.2 Th1/Th2 balance shift regulates the expression of NGF and its receptor,and then interferes with the synthesis of PGF_2?,which may be considered as one of the pathogenesis of PD.3 The mechanism of Wenjing Huayu Zhitong treatment in PD with cold coagulation and blood stasis syndrome may be involved in its regulation of Th1,Th2 cell related factors,NGF and MAPK/ERK signaling pathway.