摘要
子宫内膜异位症(endometriosis,以下简称内异症)是育龄妇女最常见的疾病,在一般人群中的发病率为10～15%。内异症的发病机制尚不明确，目前主要的治疗方式是采用手术移除异位病灶和雌激素治疗,但这些治疗通常会有副作用大且易复发的缺点。内异消缓释胶囊是本课题组自行研制的抗子宫内膜异位症的中药新药,由川芎嗪、阿魏酸、延胡索乙素组成,其处方与制备工艺已经申报国家技术发明专利。
目的
本实验主要研究内异消对大鼠子宫内膜异位症模型血管生成与雌激素的影响及相关机制,为将内异消研制成为治疗子宫内膜异位症的中药新药提供试验依据。
方法
1.造模及模型成功判断标准
采用子宫内膜自体移植法建立大鼠子宫内膜异位症模型,以移植物的体积大小、形态及病理检查作为模型成功的依据。以同时符合以下3条标准者为成功模型：①移植物体积增大,被结缔组织或大网膜覆盖并有血管形成,呈隆起透亮的小囊状,出现液体积聚,且积液高度≥2mm；②游标卡尺测量移植物的体积大小,异位组织体积=0.52×长×宽×高,移植物体积≥8mm3；③切取移植物送检,证实移植物中有子宫内膜上皮细胞、腺体及间质的生长。
2.分组及给药
除正常对照组以外,将造模成功的SD大鼠随机分为模型对照组、孕三烯酮0.5 mg·kg-1·d-1组、内异消0.045 g·kg-1·d-1组,0.09 g·kg-1·d-1组,0.18 g·kg-1·d-1组,每组各10只,于模型成功后第4 d开始灌胃给药,每天1次,连续4 wk。
3.内异消抑制子宫内膜异位症大鼠血管生成作用观察
给药4 wk后,择动情期处死大鼠,观察移植物的生长情况,取在位和异位子宫内膜,进行HE染色病理检查观察。免疫组化和Western Blot方法检测异位内膜组织中微血管密度(MVD)、血管内皮生长因子(VEGF)和内皮抑素(endostatin)蛋白的表达,放射免疫分析法(RIA)检测大鼠血清和异位内膜组织中前列腺素E2(PGE2)的含量。
4.内异消对子宫内膜异位症大鼠雌激素影响的研究
参照前面所述方法建立大鼠子宫内膜异位症模型以及进行分组,每组各16只,于模型成功后第4 d开始灌胃给药,每天1次,连续10 wk。分别于给药第4 wk末、10wk末各处死大鼠8只,取大鼠下丘脑、垂体、卵巢、血清、在位和异位子宫内膜。免疫组化检测下丘脑中促性腺激素释放激素(GnRH),垂体中促卵泡激素(FSH)和黄体生成激素(LH),卵巢中雌激素受体(ER)的表达,Western Blot检测异位内膜组织中ER、热休克蛋白90(HSP90)和环氧合酶-2(COX-2)的表达,化学发光免疫分析法(CLIA)和酶联免疫吸附法(Elisa)分别检测大鼠血清中雌二醇(E2)和孕酮(P)的含量。
结果
1.造模结果
造模4 wk后,择动情期开腹观察移植物生长情况,并用游标卡尺测量移植物大小。结果显示,肉眼观察可见移植物被脂肪组织覆盖并有血管生成,呈隆起透亮或黑色的小囊状,出现液体积聚,体积增大,造模成功率为90.91%；HE染色结果显示模型组大鼠异位内膜组织腺体上皮细胞呈高柱状,排列在囊泡内腔面,细胞核大呈多层细胞排列,而正常对照组子宫内膜细胞呈松散排列,上皮细胞薄。
2.内异消抑制子宫内膜异位症大鼠血管生成作用
连续给药4 wk后,比较给药前后移植物体积大小的变化,结果显示内异消3个剂量组给药后移植物体积比给药前明显减小(P<0.01),均能显著抑制异位内膜组织体积的增生；HE染色结果显示内异消组异位内膜腺上皮细胞层明显变薄,细胞呈矮柱状甚至扁平状,单层细胞松散排列。
免疫组化和Western Blot结果显示模型组异位内膜组织中MVD和VEGF蛋白的表达显著高于正常对照组(P<0.01),内异消0.09 g·kg-1、0.18 g·kg-1均能显著降低内异症大鼠MVD和VEGF蛋白的表达(P<0.05,P<0.01)；
模型组异位内膜组织中endostatin蛋白的表达显著低于正常对照组(P<0.01),内异消0.18 g·kg-1能极显著升高内异症大鼠endostatin蛋白的表达(P<0.01)；
RIA结果显示模型组大鼠血清和异位内膜组织中PGE2的含量明显高于正常对照组(P<0.01),内异消0.09 g·kg-1、0.18 g·kg-1均能显著降低内异症大鼠血清和异位内膜组织中PGE2的含量(P<0.05,P<0.01)。
3.内异消对子宫内膜异位症大鼠雌激素的影响
连续给药4 wk后,免疫组化结果显示模型组大鼠下丘脑中GnRH、垂体中FSH和LH、卵巢中ER的表达极显著高于正常对照组(P<0.01)；化学发光法结果显示模型组大鼠血清中E2的含量显著高于正常对照组(P<0.01),内异消0.045g·kg-1、0.09 g·kg-1、0.18 g·kg-1能显著降低大鼠血清中E2的含量(P<0.05,P<0.01)。
连续给药10 wk后,免疫组化结果显示模型组大鼠下丘脑中GnRH、垂体中FSH和LH、卵巢中ER的表达极显著高于正常对照组(P<0.01),内异消0.09 g·kg-1、0.18 g·kg-1均能显著降低下丘脑中GnRH的表达、垂体中FSH和LH的表达、卵巢中ER的表达(P<0.05,P<0.01)；
酶联免疫法结果显示模型组大鼠血清中P的含量极显著低于正常对照组(P<0.01),内异消0.09 g·kg-1、0.18 g·kg-1均能显著增高大鼠血清中P的含量(P<0.05,P<0.01)：
免疫组化和Western Blot结果显示模型组大鼠异位内膜组织中ER, HSP90和COX-2的表达极显著高于正常对照组(P<0.01),内异消0.045 g·kg-1、0.09 g·kg-1、0.18 g·kg-1均能显著降低ER和HSP90蛋白的表达(P<0.05,P<0.01),内异消0.09g·kg-1、0.18 g·kg-1能显著降低COX-2蛋白的表达(P<0.05,P<0.01)。
结论
1.内异消具有抗子宫内膜异位症大鼠异位组织血管生成作用,其作用机制与抑制VEGF蛋白表达和促进endostatin蛋白的表达有关。
2.内异消能够抑制子宫内膜异位症大鼠下丘脑-垂体-卵巢轴中与激素分泌相关的蛋白的表达,显著降低子宫内膜异位症大鼠血清中E2的含量,显著升高P的含量,在异位病灶处抑制雌激素的表达,从而达到降低雌激素生成。
Endometriosis was a common disease in women and its incidence rate was 10～15% in crowd. The pathogenesy of endometriosis was not definite until now. The therapeutics method of endometriosis were operation and estrin treatment,but its defect were adverse effect and relapse. Neiyixiao was a new traditional chinese drug developed by our team which can treat the endometriosis,it was composed of ligustrazine, ferulic acid and tetrahydropulmatine. The prescription and the preparation technology were submissioned to national technical invention patent.
Objective
To explore the effect and the mechanism of Neiyixiao on angiogenesis and estrogen of endometriosis in rats, and it can provide experiments background to clinical.
Methods
1. The standard of succeed rat model of endometriosis
The rat model of endometriosis was established by surgical implant of the endometrial tissue which belong to its body.The standard of succeed model were based on the volumes,forms and pathology sections of the transplants. It will be the succeed model when it consistent the three standards.①The grafts increased in size,much fluid accumulated to form the transparent saccule appearance,being covered by connective tissue or colic omentum and vascularized in it.and the height of hydrops was more than two millimeter.②We mearsured the volume of grafts.The calculation formula was 0.52×length×width×height,and the volume was more than eight cubic millimeter.③There were endometrial endothelial cells.glandular organ and mesenchymal in grafts being observed with microscope.
2.The grouping and administration of the rat model
The succeed rats model were grouping by random.it were composed of Model group,Gestrinone 0.5 mg·kg-1·d-1 group,Neiyixiao 0.045 g·kg-1·-d-1 group, Neiyixiao 0.09 g·kg-1·d-1 group, Neiyixiao 0.18 g·kg-1·d-1 group,and every group has ten rats include the Control group.The rats were treated by intragastric administration for 4 weeks,one time every day.
3.The effect of Neiyixiao on angiogenesis of endometriosis in rats model
After 4 weeks,the rats were sacrificed on its estrus time.We observed the growing condition of the transplants and surgical ablation the transplants.Using the method of hematoxylin and eosin stain to observed the pathology of the transplants. We explored microvessels density (MVD) by the immunohistochemical test as well as the expression of vascular endothelial growth factor (VEGF) protein and endostatin protein in ectopic endometriotic tissue by the immunohistochemical test and western blot. The level of secretion of prostaglandin E2 (PGE2) in the blood serum and ectopic endometriotic tissue was measured by radioimmunoassay (RIA).
4.The effect of Neiyixiao on estrogen of endometriosis in rats model
The rats model of endometriosis and the grouping were in accordance with the method we referred before.every group has sixteen rats.The rats were treated by intragastric administration for 10 weeks.one time every day.After 4 weeks and 10 weeks,the rats were sacrificed on its estrus time,eight rats every time.We surgical ablation the hypothalamus. pituitary, ovary and ectopic endometriotic tissue. We explored the expression of gonadotrop in hormone-releasing hormone (GnRH) in hypothalamus、follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in pituitary、estrogen receptor (ER) in ovary by the immunohistochemical test as well as the expression of ER、heat shock protein 90 (HSP90) and cyclooxygenase-2 (COX-2) in ectopic endometriotic tissue by western blot.The level of estradiol (E2) and progesterone (P) in the blood serum were measured by chemiluminescent immunoassay (CLIA) and enzyme linked immunosorbent assay (Elisa).
Results
1.The result of the endometriosis rat model
After the rats were autotransplanted with endometrial for 4 weeks,we measured the siza of graft using vernier caliper. The result display that the graft was vascularized in it,and much fluid accumulated to form the transparent saccule appearance,the color was clearing or black.The achievement ratio of model was 90.91%. The result of hematoxylin and eosin stain display that in the model group that the epithelial cells submit high prismatical and the nucleus display multilayer cells,but in normal group that epithelial cells submit low prismatical and the nucleus display monolayer cells.
2.The effect of Neiyixiao on angiogenesis of endometriosis in rats model
After the rats were administer for 4 weeks,we observed the volums of the transplants. Neiyixiao 0.045,0.09,0.18g·kg-1 markedly inhibited the volume of ectopic endometriotic tissue.The results of hematoxylin and eosin stain indicated that that epithelial cells submit low prismatical and the nucleus display monolayer cells.
The results of immunohistochemisty and western blot display that the expression of MVD and VEGF protein in model group were significant more than normal group(P<0.01). Neiyixiao 0.09,0.18g·kg-1 reduced MVD and the expression of VEGF protein(P<0.05,P<0.01).The expression of endostatin protein in model group were significant fewer than normal group(P<0.01), Neiyixiao 0.18g·kg-1 advanced the expression of endostatin protein(P<0.01).
The results of radioimmunoassay display that the level of secretion of PGE2 in blood serum and ectopia endometrium in model group were significant more than normal group(P<0.01). Neiyixiao 0.09,0.18g·kg-1 reduced the level of secretion of PGE2(P<0.05, P<0.01).
3.The effect of Neiyixiao on estrogen of endometriosis in rats model
After the rats were administer for 4 weeks, the results of immunohistochemisty display that in the model groups the expression of the GnRH in hypothalamus,the FSH and LH in pituitary,the ER in ovary were significant more than normal group(P<0.01).The results of chemiluminescent immunoassay display that the level of E2 in model group were ignificant more than normal group(P<0.01), Neiyixiao 0.045, 0.09,0.18g·kg-1 reduced the level of secretion of E2(P<0.05, P<0.01).
After the rats were administer for 10 weeks, the results of immunohistochemisty display that in the model groups the expression of the GnRH in hypothalamus,the FSH and LH in pituitary,the ER in ovary were significant more than normal group(P<0.01), Neiyixiao 0.09,0.18g·kg-1 significant reduced the expression of the GnRH in hypothalamus,the FSH and LH in pituitary,the ER in ovary(P<0.05, P<0.01).
The results of enzyme linked immunosorbent assay display that the level of P in model group were significant lower than normal group, Neiyixiao 0.09,0.18g·kg-1 significant advanced the level of P(P<0.05, P<0.01).
The results of immunohistochemisty and western blot display that the expression of ER,HSP90 and COX-2 protein in model group were significant more than normal group(P<0.01). Neiyixiao 0.09.0.18g·kg-1 reduced the expression of ER,HSP90 and COX-2 protein(P<0.05, P<0.01).
Conclusion
1.Neiyixiao can reduce the degree of angiogenesis of endometriosis in the animal models,and its mechanisms might be associated with the regulation of estrogen and the expression of VEGF and endostatin.
2. Neiyixiao can reduce the degree of estrogen of endometriosis in the animal models, and its mechanisms might be associated with the regulation of hypothalamic-pituitary-ovarian axis.
引文
[1]Becker CM, D'Amato RJ. Angiogenesis and antiangiogenic therapy in endometriosis [J]. Microvascular Research,2007,74(2-3):121-130.
[2]Kodati VL, Govindan S, Mowa S, et al. Role of shigella infection in endometriosis:A novel Hypothesis[J]. Medical Hypotheses,2008,70(2):2392-43.
[3]Lebovic D, Kir M, Casey C. Peroxisome proliferator-activated receptor-gamma Induces regression of endometrial explants in a rat model of endometriosis [J]. Fertil Steril,2004, 82(3):1009-1013.
[4]曹立幸.活血.化瘀、软坚散结法对子宫内膜异位症神经-内分泌-免疫网络的整体调作用-对免疫功能影响的实验研究[D].天津：天津中医学院,2002.
[5]徐晓玉，严鹏科,陈刚.等.川芎嗪对小鼠肺癌血管生长和VEGF表达的抑制[J].中国药理学通报.2004,20(2)：151-154.
[6]陈刚,徐晓玉，叶兰，等.川芎嗪对大鼠胶原性关节炎滑膜VEGF及VEGF mRNA表达的影响[J].中国药理学通报,2006，22(10)：1199-1202.
[7]徐晓玉,陈伟海,叶兰.等.阿魏酸钠和白芍总苷对小鼠H22肿瘤生长与血管内皮细胞生长因子及增殖细胞核抗原表达的影响[J].中草药,2007，38(4)：570-573.
[8]徐晓玉.祛因治病药[M]//徐晓玉.中药药理学.北京：中国中医药出版社,2010：288-91.
[9]冯彬彬,张建海,徐晓玉，等.川芎嗪、阿魏酸及其配伍药代动力学比较研究[J].中国中药杂志，2010,35(7)：900-903.
[10]Tai-Ping Fan, Ju-Ching Yeh, Kar Wah Leung, et al. Angiogenesis:from plants to blood vessels[J]. Trends in Pharmaclolgical Sciences.2006,27(6):297-309.
[11]吴家明,陆茵,张伟伟.等.血管生成实验模型研究进展[J].中国药理学通报，2008,24(1)：11-14.
[12]陈怡，徐晓玉，叶兰,等.三棱丸抑制大鼠子宫内膜异位症血管生成及VEGF,TNF-α表达的研究[J].中国中药杂志,2008，33(3)：303-307.
[13]Donnez J, Smoes P. Gillerot S, et al.Vascular endothelial growth factor(VEGF) in内异症[J].Hum Reprod,1998.13(6):1686-1690.
[14]黄学应,陈匕虎,张晓明,等.内抑素抗血管生成及抗类风湿性关节炎治疗[J].中国药理学通报，2009,25(3)：287-9.
[15]李傲.加味三棱丸对子宫内膜异位症雌激素生成的影响及机制研究[D].重庆：重庆医科大学，2008.
[16]陈怡.加味三棱丸主要成分抑制子宫内膜异位症效果观察及相关机制研究[D].重庆：重庆医科大学,2006.
[17]Nikos F. Vlahos. Odysseas Gregoriou, Aikaterini Deliveliotou, et al. Effect of pentoxifylline on vascular endothelial growth factor C and flk-1 expression on endometrial implantsin the rat endometriosis model[J]. Fertility and Sterility,2009,93(4):1316-23.
[18]叶兰.加味三棱丸主要成分抗大鼠子宫内膜异位症血管生成与侵袭及其作用机制研究[D].重庆：重庆医科大学,2006.
[19]Hawighorst H, Knapstein PQ, Knopp MV, et al. Uterine cervical cervical carcinorma:comparison of standard and pharmacokinetic analysis of time-intensity curve for assessment of tumor angiogenesis and patient survival[J]. Cancer Res,1998,58(6):3598-3602.
[20]J F. what is the evidence that tumors are angeogenesis depedent[J]. Natl Cancer Inst.1990, 82(65):4-6.
[21]任旭,何援利.彭冬先.缺氧诱导因子-1α在子宫内膜异位症中的表达与微血管密度的关系[J].广东医学，2007，28(2)：229-231.
[22]Velasca I RJ. Acien P. Aromatase expression in endometriotic tissues and cell cultures of patients with endometriosis [J].Mol Hum Reprod,2006,12(6):377-381.
[23]Dizerga GS, Barber DL, Hodgen GD. Endometriosis:role of ovarian steroids in initiation. maintenance and suppression[J]. Fertil Steril,1980,33(6):649-653.
[24]于祖茹,章明放，李玉玮,等.去势大鼠腺垂体促卵泡素细胞形态结构研究[J].天津医药，2004，32(12)：758-759.
[25]Acconcia F,Kumar R.Signaling regulation of genomic and nongenomic functions of estrogen receptors[J]. Cancer Let,2006,238(1):1-14
[26]赵晓民，徐小明.雌激素受体及其作用机制[J].西北农林科技大学学报(自然科学版).2004,32(12)：154-158.
[27]镇澜,刘义.刘娜.等.17β-雌二醇对子宫内膜异位症患者在位子宫内膜间质细胞PI3K/Akt信号转导通路活化的影响[J].华中科技大学学报(医学版)，2010，39(3)：321-326.
[28]霍海如,谭余庆，姜廷良.等.桂枝汤有效部位A对下丘脑热休克蛋白含量的影响[J].中国中药杂志.2000,25(10)：619-621.
[29]Rochelle Bagatell.Luke Whitesell. Altered Hsp90 function in cancer.A unique therapeutic opportunity[J]. Mol Cancer Ther.2004.3(8):1021-1030.
[30]Wataba K.Saito T,Fukunaka K.et al. Overexpression of heat shock proteins in the carcinogenic endometrium[J]. Int J Cancer,2001.91(4):448-456.
[31]白晓霞，王文栋，陈亚琼.子宫内膜热休克蛋白70和90表达与ER表达的关系[J].第四军医大学学报,2002，23(14)：1337-1340.
[32]陈坤华,方军.莫启忠,等.覆盆子水提取液对大鼠下丘脑-垂体-性腺轴功能的作用[J].中国中药杂志,1996，21(9)：560-562.
[33]孙艳红，李吉，郭秋平.更年乐颗粒剂对12月龄大鼠下丘脑-垂体-卵巢轴的调节作用[J].中成药，2007，29(1)：44-47.
[34]李勤操,郑喜邦，丁丽华,等.雌激素受体α真核表达载体的构建及其转录活性[J].生 物技术通讯,2008，19(4)：529-531.
[35]贵春山，沈建华，蒋华良,等.雌激素受体和选择性雌激素受体调节剂的研究进展[J].中国药物化学杂志，2005，15(5)：313-320.
[36]Shao W,Brown M. Advances in estrogen receptor biology:prospects for improvements in targeted breast cancer therapy[J]. Breast Cancer Res,2004,6(1):39-52.
[37]吕秋军,温利青.雌激素受体配体组织选择性作用机制的研究[J].中国药学杂志，2000，35(1)：1-3.
[38]Witkin SS. Heat shock protein expression and immunity:Relevance to gynecolgic oncology[J]. Eur J Gynaecol Oncol,2001,22(4):249-256.
[39]Bulun SE.Zeitoun KM.Takayama K,et al. Estrogen biosynthesis in ensometriosis:molecular basis and clinical relevance[J]. J Mol Endocrinol,2000,25():35-42.
[40]Mestre JR,Mackrell PJ.Rivadeneira DE. Redundancy in the signaling pathways and promoter elements regulating cyclooxygenase-2 gene expression in endotoxin-treated macrophage/monocytic cells[J]. J Biol Chem,2001.276(6):3977-3982.
[41]Tjandrawinata RR,Dahiya R,Hughes Fulford M. Induction of cyclooxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells[J].1997,75(8):1111-1118.
[1]Christian M.Becker RJDA.Angiogenesis and antiangiogenic therapy in endometriosis [J]. Microvascular Research.2007,74:121-130.
[2]Lebovic D KM. Casey C.Peroxisome proliferator-activated receptor-gamma Induces regression of endometrial explants in a rat model of endometriosis[J].Fertil Steril,2004,82:1009-1013.
[3]司徒仪,沈碧琼,莫莉莉.等.子宫内膜异位症瘀证本质及活血化瘀疗效机理探讨[J].北京中医，1998，3：11-13.
[4]李秀英,刘爽.马中夫治疗子宫内膜异位症的三三理论[J].2003,30(2)：1051.
[5]汪素卿，王惠珍.中医治疗子宫内膜异位症的研究进展[J].陕西中医学院学报，2009，32(2)：68-70.
[6]曹立幸.活血化瘀、软坚散结法对子宫内膜异位症神经-内分泌-免疫网络的整体调节作用-对免疫功能影响的实验研究[D].天津.天津中医学院,2002.
[7]季秋梅.贾晓航.中医中药治疗子宫内膜异位症的药效机理探讨[J].辽宁中医药大学学报，2008,10(3)：33-34.
[8]何红兵.活血化瘀法治疗子宫内膜异位症心得[J].中国医药指南,2008.6(6)：131-132.
[9]孙洪胜.活血化瘀法治疗子宫内膜异位症[J].山东医药,2003,43(29).
[10]黄彩霞.活血化瘀法治疗子宫内膜异位症46例[J].基层医学论坛,2006，10(5)：475.
[11]何子强，黄崇巧，牙延艺,等.活血化瘀法治疗子宫内膜异位症43例[J].医学理论与实 践,1995,8(10).
[12]韩冰,李同玺，常暖，等.活血化癖、软坚散结法治疗子宫内膜异位症药效学研究[J].中国医药学报,1995,10(4)：196-197.
[13]许丽芬,韩冰,李同玺.活血化瘀、软坚散结法(妇痛宁)对子宫内膜异位症神经内分泌影响的实验研究[J].天津中医,2002,19(2)：61-62.
[14]曹立幸,韩冰,马平仲,等.活血化瘀软坚散结法对子宫内膜异位症细胞免疫和体液免疫功能影响的实验研究[J].天津中医药,2003,20(4)：25-28.
[15]王艳丽,李艳.活血化瘀法治疗子宫内膜异位症的临床观察[J].中国实用医药杂志,2007,2(14)：76-77.
[16]刘冠春.活血化瘀法治疗子宫内膜异位症48例临床观察[J].现代中医药.2005,25(5)：37.
[17]陈怡,叶兰，徐晓玉.等.三棱丸抑制大鼠子宫内膜异位症血.管生成及VEGF,TNF-α表达的研究[J].中国中药杂志,2008,33：303-307.
[18]叶兰，徐晓玉,陈怡，等.三棱、莪术对大鼠皮下移植人工海绵新生血.管的影响研究[J].中国药房,2008,21.
[19]李傲,董伟,徐晓玉，等.三棱丸抑制大鼠异位子宫内膜芳香化酶及环氧合酶-2表达的研究[J].中国中药杂志,2008，11.
[20]李傲，陈刚,徐晓玉.等.加味三棱丸抗子宫内膜异位症内膜细胞雌二醇生成机制的研究[J].中国中药杂志.2008.33：687-690.
[21]李卓恒,陈刚,徐晓玉，等.加味三棱丸对子宫内膜异位症黏附分子ICAM-1,CD44表达的影响[J].中国中药杂志,2009,34：1285-1289.