TRPV1及其致敏因子与子宫内膜异位症痛经的关系及补肾温阳化瘀方对其影响
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摘要
目的：痛经是子宫内膜异位症患者的主要症状，严重影响患者的生活质量，但其发生机制尚不明确，成为目前研究的热点问题。本研究通过分析TRPV1及其致敏因子NGF、BK/BKB1R、PGF2α在内异症患者血清、在位内膜、异位内膜的变化，以期阐明内异症痛经的部分发病机制；动物实验是研究药物治疗本病作用机制的主要手段，但内异症痛经的动物模型却未见报道，本研究尝试利用近交系BALB/c小鼠建立内异症痛经模型；采用补肾温阳化瘀方对内异症痛经模型动物进行干预，分析治疗前后TRPV1及其致敏因子的变化，进一步探讨补肾温阳化瘀方治疗内异症痛经的作用机制。
方法：
第一部分：TRPV1及其致敏因子与子宫内膜异位症痛经关系的研究
内异症组：选自2012年9月～2013年12月于石家庄市第四医院住院并符合内异症纳入标准的患者34例；对照组来自同期住院的子宫肌瘤无痛经患者16例，所有患者术前抽取空腹肘静脉血，术中取在位及异位内膜组织，采用ELISA法检测血清NGF、BK、PGF2α的含量，采用免疫组化及Western blot方法检测NGF、BKB1R、TRPV1蛋白的表达，采用Real-timePCR方法检测NGFmRNA、BKB1RmRNA、TRPV1mRNA的表达。
第二部分：子宫内膜异位症痛经动物模型的建立
将68只清洁级近交系BALB/c雌性小鼠随机分为6组，分别为假手术组、雌激素+缩宫素组、手术组、手术+雌激素组、手术+缩宫素组、手术+雌激素+缩宫素组，其中假手术组、雌激素+缩宫素组每组10只，其余每组12只。除假手术组、雌激素+缩宫素组外其余各组均采用自体移植法将小鼠自体子宫组织块移植到腹膜，复制子宫内膜异位症模型，术后1~12d采用不同方案诱发小鼠扭体反应，记录扭体潜伏期及扭体次数，并取异位灶行HE染色和病理组织学观察，筛选建立内异症痛经模型的最佳方案。
第三部分：补肾温阳化瘀方对子宫内膜异位症痛经模型小鼠TRPV1及其致敏因子的影响
75只近交系BALB/c雌性小鼠随机分为5组：假手术组、模型组、中药高剂量组、中药低剂量组、西药组，每组15只。除假手术组外，其余各组均采用手术＋雌激素+缩宫素建立小鼠内异症痛经模型，并于术后1-12d同时给药，于第12天诱发扭体反应，断头取血并取子宫及异位灶组织。采用ELISA法、免疫组化法、Western blot方法及Real-time PCR方法检测各组TRPV1及其致敏因子的变化。
结果：
第一部分：TRPV1及其致敏因子与子宫内膜异位症痛经关系的研究
1血清NGF、BK、PGF2α的含量及相关性分析结果
1.1血清NGF、BK、PGF2α的含量
内异症患者痛经组血清NGF、BK、PGF2α水平较对照组及无痛组明显升高，差异有统计学意义(P<0.01或P<0.05)。
1.2血清NGF、BK、PGF2α含量的相关性分析
NGF与PGF2α呈正相关关系(P<0.01)，BK与PGF2α水平亦呈正相关关系(P<0.01)，NGF与BK之间未发现相关关系(P>0.05)。
1.3血清NGF、BK、PGF2α含量与VAS评分的相关性分析
NGF与VAS评分呈正相关(P<0.01)， PGF2α与VAS评分呈正相关(P<0.01)，BK含量与VAS评分呈正相关(P<0.01)。
2免疫组化法检测在位、异位内膜组织中NGF、BKB1R、TRPV1蛋白的表达结果
2.1NGF的表达
在位内膜痛经组NGF表达增强，与对照组及无痛组相比显著升高(P<0.01，P<0.05)；异位内膜痛经组NGF的表达显著高于无痛组(P<0.05)。
2.2BKB1R的表达
在位内膜痛经组BKB1R表达增强，与对照组及无痛组相比显著升高(均为P<0.01);异位内膜痛经组BKB1R的表达显著高于无痛组(P<0.01)。
2.3TRPV1的表达
在位内膜痛经组TRPV1的表达增强，与对照组及无痛组相比显著升高(均为P<0.01)。异位内膜痛经组TRPV1的表达显著高于无痛组(P<0.01)。
3Western blot法检测在位、异位内膜组织中NGF、BKB1R、TRPV1蛋白的表达结果
3.1在位、异位内膜组织中NGF的表达
在位内膜痛经组NGF的表达高于对照组及无痛组(均为P<0.01)；异位内膜痛经组NGF的表达显著高于无痛组(P<0.01)。
3.2在位、异位内膜组织中BKB1R的表达
在位内膜痛经组BKB1R的表达高于对照组及无痛组(均为P<0.01)。异位内膜痛经组显著高于无痛组(P<0.01)。
3.3在位、异位内膜组织中TRPV1的表达
在位内膜痛经组TRPV1的表达高于对照组及无痛组(均为P<0.01)。异位内膜痛经组TRPV1的表达显著高于无痛组(P<0.01)。
3.4NGF、BKB1R、TRPV1蛋白表达之间的相关性分析
在位内膜、异位内膜NGF与BKB1R的蛋白表达呈正相关关系(均为P<0.01)，NGF与TRPV1的表达呈正相关关系(P<0.05，P<0.01)，BKB1R与TRPV1的表达亦呈正相关关系(均为P<0.01)。
4Real-time PCR检测内异症在位、异位内膜组织中NGFmRNA、BKB1RmRNA、TRPV1mRNA的表达结果
4.1在位、异位内膜组织中NGFmRNA的表达
在位内膜痛经组NGFmRNA的表达显著高于对照组及无痛组(均为P<0.01)。异位内膜NGF mRNA的表达：痛经组显著高于无痛组(P<0.01)。
4.2内异症在位、异位内膜组织中BKB1RmRNA的表达
在位内膜痛经组BKB1RmRNA的表达显著高于对照组及无痛组(均为P<0.01)；异位内膜BKB1RmRNA的表达痛经组高于无痛组(P<0.01)。
4.3内异症在位、异位内膜组织中TRPV1mRNA的表达
在位内膜痛经组TRPV1mRNA的表达显著高于对照组及无痛组(均为P<0.01)；异位内膜TRPV1mRNA的表达：痛经组显著高于无痛组，差异有统计学意义(P<0.01)。
4.4NGFmRNA、BKB1RmRNA、TRPV1mRNA表达之间的相关性分析
在位内膜、异位内膜NGFmRNA与BKB1RmRNA的表达呈正相关关系(均为P<0.01)，NGFmRNA与TRPV1mRNA的表达呈正相关关系(均为P<0.01)， BKB1RmRNA与TRPV1mRNA的表达亦呈正相关关系(均为P<0.01)。
4.5NGFmRNA、BKB1RmRNA、TRPV1mRNA表达与VAS评分的相关性分析
在位内膜、异位内膜NGFmRNA、BKB1RmRNA、TRPV1mRNA的表达与VAS评分呈正相关关系(均为P<0.01)。
第二部分：子宫内膜异位症痛经动物模型的建立
与手术+缩宫素组、手术组相比，手术+雌激素+缩宫素组、手术+雌激素组移植物体积明显增大，差异有统计学意义(P<0.01)；手术+雌激素+缩宫素组扭体发生率100%，雌激素+缩宫素组扭体发生率80%，手术+缩宫素组扭体发生率50%，其余组未出现扭体反应，组间差异有统计学意义(P<0.01)；与手术+缩宫素组、雌激素+缩宫素组相比，手术+雌激素+缩宫素组扭体潜伏期明显缩短(P<0.01，P<0.05)，扭体次数明显增多(P<0.01，P<0.05)，差异有统计学意义。
第三部分补肾温阳化瘀方对子宫内膜异位症痛经模型小鼠TRPV1及其致敏因子的影响
1补肾温阳化瘀方对内异症痛经模型小鼠扭体反应的影响与模型组相比，各给药组扭体潜伏期明显延长(均为P<0.01)，扭体次数显著减少(P<0.05或P<0.01)。
2补肾温阳化瘀方对内异症痛经模型小鼠异位灶体积的影响与模型组相比，各给药后异位灶体积明显缩小(均为P<0.01)。
3补肾温阳化瘀方对内异症痛经模型小鼠血清NGF、BK、PGF2α的影响模型组血清NGF、BK、PGF2α含量显著高于假手术组(P<0.01，P<0.01，P<0.05)；与模型组相比，各给药组NGF、BK、PGF2α含量显著下降，差异有统计学意义(P<0.05或P<0.01)
4模型小鼠血清NGF、BK、PGF2α含量与扭体反应的相关性分析血清BK、PGF2α、NGF浓度与扭体次数呈正相关关系(均为P<0.01)；PGF2α浓度与扭体潜伏期呈负相关关系(P<0.05)；NGF、BK与扭体潜伏期之间未发现相关关系(P>0.05)；NGF与BK呈正相关关系(P<0.01)，PGF2α与BK呈正相关关系(P<0.05)，NGF与PGF2α亦呈正相关关系(P<0.01)。
5免疫组化法检测NGF、BKB1R、TRPV1的表达结果模型组子宫NGF、BKB1R、TRPV1表达显著高于假手术组(均为P<0.01)；与模型组相比，各给药组子宫及异位灶NGF、BKB1R、TRPV1表达显著降低，差异有统计学意义(P<0.05或P<0.01)。
6Western blot检测NGF、BKB1R、TRPV1蛋白的表达结果
与假手术组相比，模型组子宫NGF、BKB1R、TRPV1蛋白表达增高(均为P<0.01)；与模型组相比，各给药组子宫及异位灶NGF、BKB1R、TRPV1表达降低(均为P<0.01)，且NGF、BKB1R、TRPV1蛋白的表达存在正相关关系(均为P<0.01)。
7Real-time PCR检测子宫及异位灶NGFmRNA、 BKB1RmRNA、TRPV1mRNA的表达结果
与假手术组相比，模型组子宫NGFmRNA、 BKB1RmRNA、TRPV1mRNA表达增高(均为P<0.01)；与模型组相比，各给药组子宫及异位灶NGFmRNA、BKB1RmRNA、TRPV1mRNA表达不同程度下降(P<0.01或P<0.05)，且NGFmRNA、BKB1RmRNA、TRPV1mRNA的表达存在正相关关系(均为P<0.01)。
结论：
1NGF、BK、PGF2α在内异症患者血清中水平升高，且与痛经程度呈正相关关系，TRPV1、NGF、BKB1R蛋白在内异症患者在位内膜、异位内膜中表达增强，TRPV1mRNA、NGFmRNA、BKB1RmRNA在内异症痛经患者在位内膜及异位内膜中表达增强，且与痛经程度呈正相关。提示TRPV1及其致敏因子NGF、BK/BKB1R、PGF2α可能在内异症痛经的发生过程中起重要作用；
2手术+雌激素+缩宫素组引起的扭体次数最多，扭体潜伏期最短，方法简单易行，可用于内异症痛经发病机制及药物治疗研究，是建立内异症痛经模型的最佳方案；
3补肾温阳化瘀方可以缩小模型小鼠异位灶体积，减少扭体次数，延长扭体潜伏期，降调内异症痛经模型TRPV1及其致敏因子的表达，提示补肾温阳化瘀方可通过影响TRPV1及其致敏因子发挥治疗内异症痛经的作用。
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 dysmenorrhea
Thirty 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 endometriosisdysmenorrhea
A 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 model
A 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 dysmenorrhea
1Concentrations of NGF, BK, and PGF2α in serum by ELISA
1.1Concentrations of NGF, BK, and PGF2α in serum
The 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 serum
There 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 serum
There were positive correlations between NGF and VAS, BK and VASPGF2α and VAS (all P<0.01).
2Expression of NGF、BKB1R、TRPV1by immuno-histochemical staining
2.1The expression of NGF
The 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 BKB1R
The 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 TRPV1
The 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 blot
3.1Expression of NGF by Western blot
The 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 blot
The 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 endometriosis
The 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 PCR
4.1Expression 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,TRPV1mRNA
The 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 andVAS
There 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 endometriosisdysmenorrhea
Except 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 groups
Except 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 groups
Except 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 ELISA
The 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 response
There 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 immunohistochemistry
The 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 blot
The 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 PCR
The 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.
方法：
第一部分：TRPV1及其致敏因子与子宫内膜异位症痛经关系的研究
内异症组：选自2012年9月～2013年12月于石家庄市第四医院住院并符合内异症纳入标准的患者34例；对照组来自同期住院的子宫肌瘤无痛经患者16例，所有患者术前抽取空腹肘静脉血，术中取在位及异位内膜组织，采用ELISA法检测血清NGF、BK、PGF2α的含量，采用免疫组化及Western blot方法检测NGF、BKB1R、TRPV1蛋白的表达，采用Real-timePCR方法检测NGFmRNA、BKB1RmRNA、TRPV1mRNA的表达。
第二部分：子宫内膜异位症痛经动物模型的建立
将68只清洁级近交系BALB/c雌性小鼠随机分为6组，分别为假手术组、雌激素+缩宫素组、手术组、手术+雌激素组、手术+缩宫素组、手术+雌激素+缩宫素组，其中假手术组、雌激素+缩宫素组每组10只，其余每组12只。除假手术组、雌激素+缩宫素组外其余各组均采用自体移植法将小鼠自体子宫组织块移植到腹膜，复制子宫内膜异位症模型，术后1~12d采用不同方案诱发小鼠扭体反应，记录扭体潜伏期及扭体次数，并取异位灶行HE染色和病理组织学观察，筛选建立内异症痛经模型的最佳方案。
第三部分：补肾温阳化瘀方对子宫内膜异位症痛经模型小鼠TRPV1及其致敏因子的影响
75只近交系BALB/c雌性小鼠随机分为5组：假手术组、模型组、中药高剂量组、中药低剂量组、西药组，每组15只。除假手术组外，其余各组均采用手术＋雌激素+缩宫素建立小鼠内异症痛经模型，并于术后1-12d同时给药，于第12天诱发扭体反应，断头取血并取子宫及异位灶组织。采用ELISA法、免疫组化法、Western blot方法及Real-time PCR方法检测各组TRPV1及其致敏因子的变化。
结果：
第一部分：TRPV1及其致敏因子与子宫内膜异位症痛经关系的研究
1血清NGF、BK、PGF2α的含量及相关性分析结果
1.1血清NGF、BK、PGF2α的含量
内异症患者痛经组血清NGF、BK、PGF2α水平较对照组及无痛组明显升高，差异有统计学意义(P<0.01或P<0.05)。
1.2血清NGF、BK、PGF2α含量的相关性分析
NGF与PGF2α呈正相关关系(P<0.01)，BK与PGF2α水平亦呈正相关关系(P<0.01)，NGF与BK之间未发现相关关系(P>0.05)。
1.3血清NGF、BK、PGF2α含量与VAS评分的相关性分析
NGF与VAS评分呈正相关(P<0.01)， PGF2α与VAS评分呈正相关(P<0.01)，BK含量与VAS评分呈正相关(P<0.01)。
2免疫组化法检测在位、异位内膜组织中NGF、BKB1R、TRPV1蛋白的表达结果
2.1NGF的表达
在位内膜痛经组NGF表达增强，与对照组及无痛组相比显著升高(P<0.01，P<0.05)；异位内膜痛经组NGF的表达显著高于无痛组(P<0.05)。
2.2BKB1R的表达
在位内膜痛经组BKB1R表达增强，与对照组及无痛组相比显著升高(均为P<0.01);异位内膜痛经组BKB1R的表达显著高于无痛组(P<0.01)。
2.3TRPV1的表达
在位内膜痛经组TRPV1的表达增强，与对照组及无痛组相比显著升高(均为P<0.01)。异位内膜痛经组TRPV1的表达显著高于无痛组(P<0.01)。
3Western blot法检测在位、异位内膜组织中NGF、BKB1R、TRPV1蛋白的表达结果
3.1在位、异位内膜组织中NGF的表达
在位内膜痛经组NGF的表达高于对照组及无痛组(均为P<0.01)；异位内膜痛经组NGF的表达显著高于无痛组(P<0.01)。
3.2在位、异位内膜组织中BKB1R的表达
在位内膜痛经组BKB1R的表达高于对照组及无痛组(均为P<0.01)。异位内膜痛经组显著高于无痛组(P<0.01)。
3.3在位、异位内膜组织中TRPV1的表达
在位内膜痛经组TRPV1的表达高于对照组及无痛组(均为P<0.01)。异位内膜痛经组TRPV1的表达显著高于无痛组(P<0.01)。
3.4NGF、BKB1R、TRPV1蛋白表达之间的相关性分析
在位内膜、异位内膜NGF与BKB1R的蛋白表达呈正相关关系(均为P<0.01)，NGF与TRPV1的表达呈正相关关系(P<0.05，P<0.01)，BKB1R与TRPV1的表达亦呈正相关关系(均为P<0.01)。
4Real-time PCR检测内异症在位、异位内膜组织中NGFmRNA、BKB1RmRNA、TRPV1mRNA的表达结果
4.1在位、异位内膜组织中NGFmRNA的表达
在位内膜痛经组NGFmRNA的表达显著高于对照组及无痛组(均为P<0.01)。异位内膜NGF mRNA的表达：痛经组显著高于无痛组(P<0.01)。
4.2内异症在位、异位内膜组织中BKB1RmRNA的表达
在位内膜痛经组BKB1RmRNA的表达显著高于对照组及无痛组(均为P<0.01)；异位内膜BKB1RmRNA的表达痛经组高于无痛组(P<0.01)。
4.3内异症在位、异位内膜组织中TRPV1mRNA的表达
在位内膜痛经组TRPV1mRNA的表达显著高于对照组及无痛组(均为P<0.01)；异位内膜TRPV1mRNA的表达：痛经组显著高于无痛组，差异有统计学意义(P<0.01)。
4.4NGFmRNA、BKB1RmRNA、TRPV1mRNA表达之间的相关性分析
在位内膜、异位内膜NGFmRNA与BKB1RmRNA的表达呈正相关关系(均为P<0.01)，NGFmRNA与TRPV1mRNA的表达呈正相关关系(均为P<0.01)， BKB1RmRNA与TRPV1mRNA的表达亦呈正相关关系(均为P<0.01)。
4.5NGFmRNA、BKB1RmRNA、TRPV1mRNA表达与VAS评分的相关性分析
在位内膜、异位内膜NGFmRNA、BKB1RmRNA、TRPV1mRNA的表达与VAS评分呈正相关关系(均为P<0.01)。
第二部分：子宫内膜异位症痛经动物模型的建立
与手术+缩宫素组、手术组相比，手术+雌激素+缩宫素组、手术+雌激素组移植物体积明显增大，差异有统计学意义(P<0.01)；手术+雌激素+缩宫素组扭体发生率100%，雌激素+缩宫素组扭体发生率80%，手术+缩宫素组扭体发生率50%，其余组未出现扭体反应，组间差异有统计学意义(P<0.01)；与手术+缩宫素组、雌激素+缩宫素组相比，手术+雌激素+缩宫素组扭体潜伏期明显缩短(P<0.01，P<0.05)，扭体次数明显增多(P<0.01，P<0.05)，差异有统计学意义。
第三部分补肾温阳化瘀方对子宫内膜异位症痛经模型小鼠TRPV1及其致敏因子的影响
1补肾温阳化瘀方对内异症痛经模型小鼠扭体反应的影响与模型组相比，各给药组扭体潜伏期明显延长(均为P<0.01)，扭体次数显著减少(P<0.05或P<0.01)。
2补肾温阳化瘀方对内异症痛经模型小鼠异位灶体积的影响与模型组相比，各给药后异位灶体积明显缩小(均为P<0.01)。
3补肾温阳化瘀方对内异症痛经模型小鼠血清NGF、BK、PGF2α的影响模型组血清NGF、BK、PGF2α含量显著高于假手术组(P<0.01，P<0.01，P<0.05)；与模型组相比，各给药组NGF、BK、PGF2α含量显著下降，差异有统计学意义(P<0.05或P<0.01)
4模型小鼠血清NGF、BK、PGF2α含量与扭体反应的相关性分析血清BK、PGF2α、NGF浓度与扭体次数呈正相关关系(均为P<0.01)；PGF2α浓度与扭体潜伏期呈负相关关系(P<0.05)；NGF、BK与扭体潜伏期之间未发现相关关系(P>0.05)；NGF与BK呈正相关关系(P<0.01)，PGF2α与BK呈正相关关系(P<0.05)，NGF与PGF2α亦呈正相关关系(P<0.01)。
5免疫组化法检测NGF、BKB1R、TRPV1的表达结果模型组子宫NGF、BKB1R、TRPV1表达显著高于假手术组(均为P<0.01)；与模型组相比，各给药组子宫及异位灶NGF、BKB1R、TRPV1表达显著降低，差异有统计学意义(P<0.05或P<0.01)。
6Western blot检测NGF、BKB1R、TRPV1蛋白的表达结果
与假手术组相比，模型组子宫NGF、BKB1R、TRPV1蛋白表达增高(均为P<0.01)；与模型组相比，各给药组子宫及异位灶NGF、BKB1R、TRPV1表达降低(均为P<0.01)，且NGF、BKB1R、TRPV1蛋白的表达存在正相关关系(均为P<0.01)。
7Real-time PCR检测子宫及异位灶NGFmRNA、 BKB1RmRNA、TRPV1mRNA的表达结果
与假手术组相比，模型组子宫NGFmRNA、 BKB1RmRNA、TRPV1mRNA表达增高(均为P<0.01)；与模型组相比，各给药组子宫及异位灶NGFmRNA、BKB1RmRNA、TRPV1mRNA表达不同程度下降(P<0.01或P<0.05)，且NGFmRNA、BKB1RmRNA、TRPV1mRNA的表达存在正相关关系(均为P<0.01)。
结论：
1NGF、BK、PGF2α在内异症患者血清中水平升高，且与痛经程度呈正相关关系，TRPV1、NGF、BKB1R蛋白在内异症患者在位内膜、异位内膜中表达增强，TRPV1mRNA、NGFmRNA、BKB1RmRNA在内异症痛经患者在位内膜及异位内膜中表达增强，且与痛经程度呈正相关。提示TRPV1及其致敏因子NGF、BK/BKB1R、PGF2α可能在内异症痛经的发生过程中起重要作用；
2手术+雌激素+缩宫素组引起的扭体次数最多，扭体潜伏期最短，方法简单易行，可用于内异症痛经发病机制及药物治疗研究，是建立内异症痛经模型的最佳方案；
3补肾温阳化瘀方可以缩小模型小鼠异位灶体积，减少扭体次数，延长扭体潜伏期，降调内异症痛经模型TRPV1及其致敏因子的表达，提示补肾温阳化瘀方可通过影响TRPV1及其致敏因子发挥治疗内异症痛经的作用。
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 dysmenorrhea
Thirty 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 endometriosisdysmenorrhea
A 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 model
A 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 dysmenorrhea
1Concentrations of NGF, BK, and PGF2α in serum by ELISA
1.1Concentrations of NGF, BK, and PGF2α in serum
The 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 serum
There 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 serum
There were positive correlations between NGF and VAS, BK and VASPGF2α and VAS (all P<0.01).
2Expression of NGF、BKB1R、TRPV1by immuno-histochemical staining
2.1The expression of NGF
The 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 BKB1R
The 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 TRPV1
The 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 blot
3.1Expression of NGF by Western blot
The 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 blot
The 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 endometriosis
The 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 PCR
4.1Expression 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,TRPV1mRNA
The 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 andVAS
There 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 endometriosisdysmenorrhea
Except 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 groups
Except 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 groups
Except 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 ELISA
The 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 response
There 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 immunohistochemistry
The 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 blot
The 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 PCR
The 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.
引文
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