The Pharmacological Mechanism Of Wenjing Decoction And Cold Condensate In The Treatment Of Primary Dysmenorrhea Was Preliminarily Studied

Dysmenorrhea(PD)is closely related to infertility and gynecological tumors according to clinical epidemiological investigation with high incidence rate.According to the pathogenesis of TCM,blood stasis due to cold coagulation in Ren channel,an effectiveness as analgesia could be derived from warming meridian and collaterals,Wenjing Decoction(WJD)is the most effective compound prescriptions.Based on the dysfunction of uncoupling proteins 4(UCP4)and relaxin2 being pathogenesis of PD,this work has been focused on the pathogenesis of PD with blood stasis due to cold coagulation in Ren channel in animal models.The results have demonstrated that cold syndromes congealing in Ren channel was the core pathogenesis of PD with blood stasis due to cold coagulation.Pharmacological mechanism of WJD included resistance to the cramps in vitro and increasing the heat production in vivo.1.Relaxant Effect on Isolated Uterus of Rats via PLCp-IP3/DAG pathway Objective:To study the relaxant effect and underlying mechanism of evodiamine on isolated uterus of rats.Methods:PGF2? was used to induce isolated uterus contraction.Relaxin2g effect of evodiamine and influence of capsazepine(antagonist of transient receptor potential cation channel,subfamily V,member 1,TRPV1),U73122(antagonist of phospholipase Cp,PLCp),W-7(antagonist of camodulin,CaM)on effect of evodiamine on uterus were observed respectively.The tensions of isolated uterus were consistently monitored by BL-420F physiological experiment system.The median effective concentration were analyzed with non-linear various slope regression using Prism-5.01 software.Results:evodiamine showed concentration-dependent relaxin2g effects on PGF2?-contracted rat uterus and EC50 was 9.56×10-9mol·L-1.Incubation with capsazepine(6.30×10-11 mol·L-1),U73122(2.57×10-11 mol·L-1)and W-7(5.65×10-13 mol·L-1)markedly increased the relaxant effect of evodiamine.EC50 of evodiamine were lower and dose-effect curves were left shift.EC50 from low to high:W-7-evodiamine(8.88×10-15 mol·L-1)<capsazepine-evodiamine(7.35×10-13 mol·L-1)<U73122-evodiamine(1.95×10-12mol·L-1)?Conclusion:evodiamine can inhibit uterus contraction induced by activating PLCp-IP3/DAG pathway obviously,and the mechanism is probably related to the ability of depression activity of TRPVl,PLC? and CaM.2.Effects of Wenjing Decoction for primary dysmenorrhea with blood stasis due to cold coagulation in Ren channelObjective:To explore the effects of Wenjing Decoction(WJD)on model rats with PD and blood stasis due to cold coagulation in Ren channel.Methods:After the interscapular brown adipose tissue being removed,rats were administrated with high-fat diet at d3-d76 and with diethylstilbestrol lmg/(kg·d)at d2,-d76 to establish the rat model of PD with blood stasis due to cold coagulation in Ren channel.all-trans-Retinoic acid(ATRA)and capsaicine(Cap)were used as positive drugs,Wenjing ecoction(WJD),evodiamine(Evo),Wenjing Decoction evodia rutaecarpa replaced by Fructus corni(SDW),Wenjing Decoction ligusticum wallichii replaced by salviae miltiorrhizae(DDW),were administrated by intragastric administration at d41-d76.The pressure of uterus was observed at d69,and the endometrial volume ratio,the constituent ratio of endometrium,myometrium and cervix,Plasma level of pyruvic acid(PA),total cholesterol(TC),triglyceride(TG),free fatty acid(FFA),prolactin(PROL),follicle-stimulating hormone(FSH),luteinizing hormone(LH),estradiol(E2),progesterone(P)were detected by ELISA method.Control group,model group,WJD group and Evo group were common groups:? pathogenesis of cold coagulation:adding ATRAgroup and SDW group;? pathogenesis of blood stasis:adding Cap group and DDW group;? pathogenesis of(cold coagulation/cold coagulation):(ATRT/SDW)and/or(Cap/DDW)were used to demonstrate efficacy changing after replaced by interior-warming drug but not Ren channel and/or blood-activating drug but not Ren channel.Data were converted into potency,and t-test was used to compare the intergroup difference.Results:? pathogenesis of cold coagulation:compared with ATRA group:WJD,SDW and Evo show uterine organ coefficient decresed,intrauterine pressure decresed,endometrial volume ratio decresed,FFA increased,LH decresed,E2 decresed,P incresed respectively.?pathogenesis of blood stasis:compared with Cap group:WJD,DDW and Evo show uterine organ coefficient decresed,intrauterine pressure decresed,endometrial volume ratio decresed,FFA decreased,LH decresed,E2 decresed,P incresedrespectively.? pathogenesis of(cold coagulation/cold coagulation):ATRT/SDW was higher than Cap/DDW in FFA(P<0.01),intrauterine pressure(P<0.05),myometrium collagen fiber(P<0.05)?cervix collagen fiber(P<0.01),FSH(P<0.05),PROL(P<0.05),E2(P<0.05)?Conclusion:Core pathogenesis of PD with blood stasis due to cold coagulation is cold coagulation in Ren channel,Minor pathogenesis is blood stasis in Ren channel.WJD treats PD though modulating lipid metabolism and sex hormone axis level(warmly resolving cold coagulation),though decreseing intrauterine pressure and endometrial hyperplasia(promoting blood circulation to arrest pain).3.Pharmacological mechanism of Wenjing Decoction for primary dysmenorrhea with blood stasis due to cold coagulation in Ren channelObjective:To explore the pharmacological mechanism of WJD on model rats with primary dysmenorrhea and blood stasis due to cold coagulation in Ren channel.Methods:The style of treatments were same as 2.uterus were obtained at d77 The level of plasma relaxin2 was determined with ELISA.Expression of UCP4 and relaxin2 were determined with immunohistochemical staining,real time RT-PCR and Western blot.The style of statistics were same as 2.Results:? pathogenesis of cold coagulation:compared with ATRA group:WJD,SDW and Evo show UCP4 in situ expression increased,UCP4 RNA expression increased,UCP4 protein expression increased.Relaxin2 plasma level in WJD,SDW and Evo increased.WJD,SDW and Evo show relaxin2 in situ expression increased,relaxin2 RNA expression increased,relaxin2 protein expression increased.? pathogenesis of blood stasis:compared with Cap group:WJD,DDW and Evo show UCP4 in situ expression increased,UCP4 RNA expression increased,UCP4 protein expression increased.Relaxin2 plasma level in WJD,DDW and Evo incresed respectively;WJD,DDW and Evo show relaxin2 in situ expression increased,relaxin2 RNA expression increased,relaxin2 protein expression increased respectively.? pathogenesis of(cold coagulation/cold coagulation):ATRT/SDW was higher than Cap/DDW in UCP4 in situ expression(P<0.05),RNA expression(P<0.05),protein expression(P<0.05);Relaxin2 plasma level(P<0.05),RNA expression(P<0.05),protein expression(P<0.05).Conclusion:Core pathogenesis of PD with blood stasis due to cold coagulation is cold coagulation in Ren channel,Minor pathogenesis is blood stasis in Ren channel.WJD treats PD though upregulating UCP4 compensatory thermogenesis(warmly resolving cold coagulation),though upregulating relaxin2 to accelerate local blood flow(promoting blood circulation to arrest pain).