TRPV1and Sensitization Factors Relations With Endometriosis Dysmenorrhea And Effects Of Bushenwenyanghuayu Prescription

Objective: Dysmenorrhea, as one of the cardinal symptom ofendometriosis (EM), has a strong impact on patient’ qulity of life. But themechanism of the endometriosis-dysmenorrhea remains unknown, becoming ahot issue at present. The aim of this study is to investigate the expression ofNGF、BKB1R、TRPV1in eutopic and ectopic endometrium of EM and thecontent of NGF、BK、PGF2α in serum, in order to reveal part mechanism ofendometriosis-dysmenorrhea.Take advantage of the BALB/c mice to establishthe model of endometriosis-dysmenorrhea, in order to provide perfect animalmodel for studying the pathogenesis and drug therapy. Applied theBushenwenyanghuayu Prescription (BSWYHYP) to treat the model ofendometriosis-dysmenorrhea, and analyzed the changes of indicators beforeand after treatment inorder to discuss the mechanis of Bushenwenyanghuayuprescription on endometriosis-dysmenorrhea.Methods:Part one: Study the relationships between TRPV1and its’ sensitizationfactors and endometriosis dysmenorrheaThirty four patients who fitted the inclusion criteria of EM in hospital ofthe Fourth Hospital of Shijiazhuang City during September2009toDecemberr2013were collected,16cases with myoma of uterus as controlgroup, before the operation, venous blood were collected, and the eutopic andectopic endometrium of the patients during the operation were collected whichconfirmed by the pathologic detection after operation. Enzyme linkedimmunosorbent assay(ELISA) was used to compare NGF, BK, PGF2αcontents in serum; Western blot and immuno-histochemical stainingtechniques were used to detect the protein expression of NGF、BKB1R andTRPV1in eutopic and ectopic endometrium; Real-time PCR method was used to detect the expression of NGFmRNA, BKB1RmRNA and TRPV1mRNA intissues.Part two: Establishment of an animal model of endometriosisdysmenorrheaA total of sixty eight clean BALB/C female mice were divided randomlyinto6groups, named sham group, estrogen+oxytocin group, operation group,operation+estrogen group, operation+oxytocin group and operation+estrogen+oxytocin group, with10mice in each group. Except for the sham group andestrogen+oxytocin group, the mice in other groups underwent operation,auto-transplantation of uterine tissue into the peritoneum was applied togenerate endometriosis model, Different regimens were applied to treat themice from1th to12th day after operation, observed the writhing response, andcollected tissue samples from the ectopic foci to examine the histopathologicalcharacteristics in order to screen the best regimen.Part three: Effects of Bushenwenyanghuayu Prescription on TRPV1andsensitization factors in Endometriosis Dysmenorrhea modelA total of75BALB/C mice were random divided into five groups, shamgroup, model group, BSWYHYP high dose group, BSWYHYP low dosegroup and gestrinone group, with15in each group. Except for the sham group,all the mice in other groups underwent operation+estrogen+oxytocin toestablish endometriosis dysmenorrhea model. Different drugs were given totreat the mice from1th to12th day after operation, on the12th day, writhingresponse was induced and writhing latency, writhing frequence were orderd.After the observation, the mice were executed and serum, uterus, ectopic fociwere collected. NGF, BK, PGF2α contents in serum were detected by ELISA.Immunohistochemical staining and Western blot techniques were applied todetect the protein expression of NGF, BKB1R, TRPV1and Real-time PCRmethod was used to detect the expression of NGF mRNA, BKB1R mRNA andTRPV1mRNA.Results:Part one: Study the relationships between TRPV1and its’ sensitization factors and endometriosis dysmenorrhea1Concentrations of NGF, BK, and PGF2α in serum by ELISA1.1Concentrations of NGF, BK, and PGF2α in serumThe concentration of NGF, PGF2α in pain group were higher than that incontrol and non-pain groups (P<0.01or P<0.05). The concentration of BK inpain group was higher than that in control group (P<0.01), there was nostatistical significance between pain and non-pain group (P>0.05).1.2The correlation between NGF、BK and PGF2α in serumThere was positive correlation between NGF and PGF2α (P<0.01), therewas positive correlation between BK and PGF2α (P<0.01), there was nocorrelation between NGF and BK (P>0.05).1.3The correlation between NGF、BK PGF2α and VAS in serumThere were positive correlations between NGF and VAS, BK and VASPGF2α and VAS (all P<0.01).2Expression of NGF、BKB1R、TRPV1by immuno-histochemical staining2.1The expression of NGFThe expression of NGF in eutopic and ectopic of pain group enhancedand weakened in non-pain and control group(P<0.01or P<0.05).2.2The expression of BKB1RThe expression of BKB1R in eutopic and ectopic of painl groupenhanced and weakened in non-pain and control group(P<0.01).2.3The expression of TRPV1The expression of TRPV1in eutopic and ectopic of pain group enhancedand weakened in non-pain and control group(P<0.01).3Expression of NGF、BKB1R、TRPV1by Western blot3.1Expression of NGF by Western blotThe eutopic endometrium expression of NGF in pain group was higherthan that control and non-pain groups (P<0.01); The ectopic endometriumexpression of NGF was higher in pain group compared to non-pain group(P<0.01).3.2Expression of BKB1R by Western blot The eutopic endometrium expression of BKB1R in pain group washigher than that control and non-pain groups (P<0.01); The BKB1R in ectopicendometrium was higher in pain group compared to non-pain group (P<0.01).3.3Expression of TRPV1by Western blotThe eutopic endometrium expression of TRPV1in pain group was higherthan control and non-pain groups(P<0.01); The ectopic endometriumexpression of TRPV1was higher in pain group compared to non-pain group(P <0.01).3.4The correlation between NGF、BKB1R、TRPV1in endometriosisThe study result indicated that there was positive correlation betweenNGF and BKB1R in eutopic endometrium、ectopic endometrium (all P<0.01)，there was positive correlation between NGF and TRPV1in eutopicendometrium, ectopic endometrium (P<0.05,P<0.01),there was positivecorrelation between BKB1R and TRPV1in eutopic endometrium、ectopicendometrium (P<0.01).4Expression of NGF mRNA、BKB1R mRNA、TRPV1mRNA in eutopic andectopic endometrium by Real-time PCR4.1Expression of NGF mRNA by Real-time PCR.The eutopic endometrium NGF mRNA expression in pain group wassignificantly higher compared to the control and non-pain groups(P<0.01).The NGF mRNA expression of ectopic endometrium in pain groupwas higher than that in non-pain group (P<0.01).4.2Expression of BKB1R mRNA by Real-time PCR.The eutopic endometrium BKB1R mRNA expression in pain group wassignificantly higher compared to the control and non-pain groups (P<0.01)The BKB1R mRNA expression of ectopic endometrium in pain group washigher than that in non-pain group with significant differences(p<0.01).4.3Expression of TRPV1mRNA by Real-time PCR.The eutopic endometrium TRPV1mRNA expression in pain group wassignificantly higher compared to control and non-pain groups (P<0.01). TheTRPV1mRNA expression of ectopic endometrium in pain group was higher than that in non-pain group with significant differences(P <0.01).4.4The correlation between NGFmRNA,BKB1RmRNA,TRPV1mRNAThe study result indicated that there was positive correlation betweenNGFmRNA and BKB1RmRNA in eutopic endometrium,ectopic endometrium(all P<0.01), there was positive correlation between NGFmRNA andTRPV1mRNA in eutopic endometrium,ectopic endometrium (all P<0.01),there was positive correlation between BKB1RmRNA and TRPV1mRNA ineutopic endometrium、ectopic endometrium (all P<0.01).4.5The correlation between NGFmRNA、BKB1mRNA、TRPV1mRNA andVASThere were positive correlations between NGFmRNA and VAS ineutopic and ectopic endometrium(all P<0.01), there were positive correlationsbetween BKB1RmRNA and VAS in eutopic and ectopic endometrium(allP<0.01), there were positive correlations between TRPV1mRNA and VAS ineutopic and ectopic endometrium(all P<0.01).Part two: Establishment of an animal model of endometriosisdysmenorrheaExcept for the estrogen+oxytocin group and sham operation group, theectopic foci of all the other groups developed well. The lesion volumes of theoperation+estrogen+oxytocin group and operation+estrogen group weresignificantly larger than that in the other groups(P<0.01). The incidence ofwrithing response of the operation+estrogen+oxytocin group was100%,estrogen+oxytocin group was80%and operation+oxytocin group was50%,there were statistically significant differences among different groups(P<0.01). The writhing latency and writhing frequency of the operation+estrogen+oxytocin group were significantly different from that in the othergroups (P<0.01or P<0.05).Part three: Effects of Bushenwenyanghuayu Prescription on TRPV1andsensitization factors in Endometriosis Dysmenorrhea model1Comparison of writhing response among groupsExcept for sham group, the mice in other groups all appeared writhing response. Compared to model group, the writhing latency prolonged and thewrithing frequency reduced in BSWYHYP high dose group, low dose groupand gestrinone group, the differences were statistically significant (P<0.01orP<0.05).2Comparison of ectopic foci volume among groupsExcept for sham group, the ectopic foci developed well in other groups.Compared with model group, the volume of ectopic foci in BSWYHYP highdose group, BSWYHYP low dose group and gestrinone group became smallersignificantly (both P<0.01).3Comparison of serum NGF, BK and PGF2α in groups by ELISAThe level of NGF, BK and PGF2α in model group became higher thanthat of sham (P<0.01，P<0.01，P<0.05), whereas those in the treatment groupswere significantly lower than that of model group (P<0.05,P<0.01,P<0.01).4Correlation analysis among NGF, BK, PGF2α and writhing responseThere were positive correlations between BK, PGF2α, NGF and writhingfrequency(all P<0.01); There was negative correlation between PGF2α andwrithing latency(P<0.05)；There was no correlation between NGF, BK andwrithing latency(P>0.05); There was positive correlation between NGF andBK(P<0.01); There was positive correlation between PGF2α and BK(P<0.05),There was positive correlation between NGF and PGF2α (P<0.01).5Comparison of NGF, BKB1R and TRPV1by immunohistochemistryThe expressions of NGF, BKB1R and TRPV1in uterus of model groupwere higher than that in sham with statistical difference (all P<0.01), andthose in treat groups were significant lower than that of model group not onlyin uterus but also in ectopic foci (all P<0.01).6Comparison of NGF, BKB1R and TRPV1proteins by Western blotThe Western blot analysis showed that NGF, BKB1R and TRPV1proteins in model group increased significantly compared to sham group (allP<0.01), and those in treatment groups decreased significantly compared withthat of model group (all P<0.01), and there were positive correlationsbetween NGF, BKB1R and TRPV1proteins (all P<0.01). 7Comparison of mRNA for NGF, BKB1R and TRPV1by Real-time PCRThe mRNA for NGF, BKB1R and TRPV1in uterus increasedsignificantly in model group compared to sham group (all P<0.01), anddecreased significantly in treat groups compared to model group not only inuterus but also in ectopic foci (P<0.01or P<0.05), and there were positivecorrelations between NGF, BKB1R and TRPV1mRNA with significantdifferences (all P<0.01).Conclusion:1The NGF, BK, PGF2α contents in endometriosis patients increased.TheNGF, BKB1R and TRPV1overexpressed in eutopic and ectopic endometrium,and were positively correlated with the degree of dysmenorrhea. Indicated thatNGF, BK/BKB1R, PGF2α, TRPV1may play an important role in the processof endometriosis-associated dysmenorrhea;2The method of operation+estrogen+oxytocin is the best method toestablish a mouse model of endometriosis dysmenorrhea, and is simple toperform. This animal model can be used to investigate the mechanisms andtreatment of endometriosis dysmenorrheal;3Our studies demonstrated that BSWYHYP can effectively shrink thevolume of ectopic foci, reduce the writhing frequency and prolong writhinglatency, decrease the elevated NGF, BK/BKB1R, PGF2α, TRPV1levels.Bushenwenyanghuayu Prescription may play an important role in preventingthe development of endometriosis dysmenorrhea, with a possible molecularmechanism relating with the inhibition of TRPV1and sensibilization factors.