探索建立雷公藤多苷致早发性卵巢功能不全肾虚血瘀证的小鼠模型
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摘要
为研究补肾促卵方治疗早发性卵巢功能不全的作用机制,采用雷公藤多苷灌胃建立早发性卵巢功能不全肾虚血瘀证小鼠模型并进行卵巢功能和生育力评价。60只SPF级动情周期规则的雌性Blab/c小鼠随机分为空白1组、空白2组、空白生育组、模型组、模型恢复组、模型生育组,每组10只。模型组予40 mg·kg-1雷公藤多苷混悬液灌胃,空白组予等量生理盐水灌胃,每日2次,连续14 d。第15天空白1组和模型组解剖取材;其余4组终止雷公藤灌胃恢复正常喂养,空白2组和模型恢复组连续观察动情周期变化14 d后解剖取材,观察各组一般表征、动情周期、血清性激素、性腺指数、卵巢形态及卵泡计数、卵巢血管内皮生长因子(VEFG)和内皮抑制素(ES)的变化。空白生育组和模型生育组以雌雄2∶1进行合笼,记录雌鼠受孕率、产仔数、仔鼠存活数及雌雄比例。造模期间模型组和模型生育组小鼠体质量下降(P<0. 05)。与空白1组相比,模型组小鼠动情周期紊乱,卵巢及子宫指数下降,原始及生长卵泡数量减少、闭锁卵泡数量增多,VEGF表达下降、ES表达升高(P<0. 05)。与空白2组相比,模型恢复组小鼠动情周期仍紊乱,血清卵泡刺激素(FSH)升高,卵巢指数未恢复,原始及生长卵泡数量仍减少、闭锁卵泡数量仍增加,VEGF表达仍下降(P<0. 05)。与空白生育组相比,模型生育组小鼠产仔数和仔鼠存活数下降(P<0. 05)。40 mg·kg-1雷公藤多苷每日2次,连续灌胃14 d可成功建立早发性卵巢功能不全肾虚血瘀证小鼠模型,停药14 d后模型小鼠卵巢功能尚未恢复,为后续补肾促卵方的干预性实验治疗时间提供证据。
To establish a mouse model of premature ovarian insufficiency( POI) with kidney deficiency and blood stasis pattern by Tripterygium wilfordii polyglycoside( TWP) gavage,and to evaluate the ovarian function and fertility of the model,in order to find Bushen Culuan Decoction therapeutic mechanism. 60 SPF level Blab/c female mice with normal estrous cycle were randomly divided into 6 groups of 10 each: blank group 1( BG1),blank group 2( BG2),blank fertility group( BFG),model group( MG),model recovery group( MRG) and model fertility group( MFG). The mice in three model groups were treated by gastric gavage with TWP suspension 40 mg·kg-1 twice a day for 14 days,while the mice in three blank groups were treated by gastric gavage with same volume normal saline for 14 days. The mice in BG1 and MG were sacrificed and dissected on day 15. The mice in BG2,BFG,MRG and MFG were returned normal feeding from day 15 and were sacrificed and dissected on day 29. The mice in BFG and MFG were cohabited with male mice with a ratio of 2 ∶1( female ∶male) from day 15. The general situation and estrous cycles of all mice were observed every day. Serum sex hormone levels,ovarian index,uterine index,ovarian morphology,follicle count,ovarian VEGF and ES index were observed within the mice in BG1,BG2,MG and MRG. Pregnancy rate,litter size,survival number of newborn mice and male-female proportion were reported within the mice in BFG and MFG. In model establishing stage,the body weight of mice significantly decreased( P <0. 05) in MG and MFG. Compared with BG1,the mice in model group had irregular estrous cycle,decreased ovarian and uterine indexes,less primordial and developing follicles,more atretic follicles,increased VEGF expression and decreased ES expression( P <0. 05). Compared with blank group 2,the mice in model recovery group had irregular estrous cycle,increased FSH level,decreased ovarian indexes,less primordial and developing follicles,more atretic follicles,increased VEGF expression( P<0. 05). Compared with blank fertility group,the mice in model fertility group had smaller litter size and newborn mice survival count( P<0. 05). Gastric gavage with TWP 40 mg·kg-1 twice a day for 14 days is a feasible way to establish a POI kidney deficiency and blood stasis pattern mouse model. The mice ovarian functions didn't recovery on day 14 after stopping TWP intervening,which could suggest the effectiveness of subsequent therapeutic intervention.
To establish a mouse model of premature ovarian insufficiency( POI) with kidney deficiency and blood stasis pattern by Tripterygium wilfordii polyglycoside( TWP) gavage,and to evaluate the ovarian function and fertility of the model,in order to find Bushen Culuan Decoction therapeutic mechanism. 60 SPF level Blab/c female mice with normal estrous cycle were randomly divided into 6 groups of 10 each: blank group 1( BG1),blank group 2( BG2),blank fertility group( BFG),model group( MG),model recovery group( MRG) and model fertility group( MFG). The mice in three model groups were treated by gastric gavage with TWP suspension 40 mg·kg-1 twice a day for 14 days,while the mice in three blank groups were treated by gastric gavage with same volume normal saline for 14 days. The mice in BG1 and MG were sacrificed and dissected on day 15. The mice in BG2,BFG,MRG and MFG were returned normal feeding from day 15 and were sacrificed and dissected on day 29. The mice in BFG and MFG were cohabited with male mice with a ratio of 2 ∶1( female ∶male) from day 15. The general situation and estrous cycles of all mice were observed every day. Serum sex hormone levels,ovarian index,uterine index,ovarian morphology,follicle count,ovarian VEGF and ES index were observed within the mice in BG1,BG2,MG and MRG. Pregnancy rate,litter size,survival number of newborn mice and male-female proportion were reported within the mice in BFG and MFG. In model establishing stage,the body weight of mice significantly decreased( P <0. 05) in MG and MFG. Compared with BG1,the mice in model group had irregular estrous cycle,decreased ovarian and uterine indexes,less primordial and developing follicles,more atretic follicles,increased VEGF expression and decreased ES expression( P <0. 05). Compared with blank group 2,the mice in model recovery group had irregular estrous cycle,increased FSH level,decreased ovarian indexes,less primordial and developing follicles,more atretic follicles,increased VEGF expression( P<0. 05). Compared with blank fertility group,the mice in model fertility group had smaller litter size and newborn mice survival count( P<0. 05). Gastric gavage with TWP 40 mg·kg-1 twice a day for 14 days is a feasible way to establish a POI kidney deficiency and blood stasis pattern mouse model. The mice ovarian functions didn't recovery on day 14 after stopping TWP intervening,which could suggest the effectiveness of subsequent therapeutic intervention.
引文
[1]陈子江,田秦杰,乔杰,等.早发性卵巢功能不全的临床诊疗中国专家共识[J].中华妇产科杂志,2017(9):577.
[2]杨冬梅,刘俊.雷公藤多苷临床应用及不良反应的研究进展[J].中国医院药学杂志,2018(10):1.
[3]曹艳,运乃茹,邹爱英.雷公藤多苷片致不良反应的Meta分析[J].中国药房,2018,29(1):125.
[4]郭健敏,黄远铿,雷夏凌,等.雷公藤多苷对雄性SD大鼠生殖系统损伤的动态变化及其可能机制[J].中国药理学与毒理学杂志,2018,32(6):469.
[5]马堃,刘雁峰,何军琴,等.补肾活血方案治疗排卵障碍性不孕不育的多中心随机双盲对照临床研究[J].中国中药杂志,2015,40(20):3911.
[6]冯桂玲,李静,周小琳.补肾健脾方对免疫性卵巢功能衰退小鼠性激素及卵子质量的影响[J].中医杂志,2016,57(16):1416.
[7]孟玥,任艳玲,孙月娇,等.左归丸、右归丸及其拆方对去卵巢骨质疏松症模型大鼠肾脏碱性磷酸酶、骨钙素表达的影响[J].中医杂志,2016,57(5):423.
[8]秦忠,李文,管连城,等.冬虫夏草VDR沉默大鼠卵巢颗粒细胞CYP19表达的影响[J].中国民族民间医药,2018,27(9):21.
[9]马堃,佟雅婧.从肾虚血瘀论治排卵障碍性不孕[J].中国中药杂志,2017,42(23):4451.
[10]神农本草经[M].吴普述,孙星衍,孙冯翼辑.沈阳:辽宁科学技术出版社,1997.
[11]高学敏.中药学[M].北京:中国中医药出版社,2002.
[12]贾微,吴晓君,岑妍慧,等.雷公藤多苷治疗肾脏疾病的研究进展[J].中华中医药学刊,2018,36(4):866.
[13]刘素晓,闫凤娜,王幼平.雷公藤多苷的抗炎作用与临床应用进展[J].中医临床研究,2017,9(32):131.
[14]刘莹,仲青香,邱辉辉,等.基于肝毒性的雷公藤中药复方配伍减毒的研究进展[J].中国中药杂志,2017,42(16):3044.
[15]冯雪,方赛男,高雨鑫,等.雷公藤制剂安全性的循证评价研究[J].中国中药杂志,2018,43(3):425.
[16]孙凤,杨兴华,马冬梅,等.雷公藤用药者生殖毒性发生率的Meta分析[J].中国药物警戒,2014,11(2):94.
[17]蔡竞,吴克明,黄丽,等.雷公藤多苷通过PI3K/AKT信号通路影响大鼠卵泡发育的实验研究[J].陕西中医,2014,35(7):923.
[18]付雨,吴克明.雷公藤多苷对大鼠卵巢血管生成及血供的影响[J].四川中医,2011,29(6):50.
[19]杨威.雷公藤多苷对消化道的毒性研究[C].武汉:中国毒理学会第七次全国毒理学大会暨第八届湖北科技论坛,2015.
[20]Ji Y,Tang B,Traub R J. The visceromotor response to colorectal distention fluctuates with the estrous cycle in rats[J]. Neuroscience,2008,154(4):1562.
[21]何国珍,杨美春,谢桂珍,等.雷公藤多苷诱导大鼠卵巢功能低下模型的实验研究[J].广西中医药大学学报,2017,20(4):9.
[22]Marcondes F K,Bianchi F J,Tanno A P. Determination of the estrous cycle phases of rats:some helpful consideration[J]. Braz J Biol,2002,62(4a):609.
[23]严大为,周莉,孙祖越.卵巢功能早衰动物模型的建立及其评价指标的研究进展[J].中国药理学与毒理学杂志,2015,29(3):486.
[24]梁爽,田舸,田燕,等.雷公藤多苷致小鼠卵巢早衰后的自然修复时间探讨[J].大连医科大学学报,2010,32(2):146.
[25]史志明.中成药雷公藤多苷片对儿童成年后生殖能力影响的临床随访观察与分析[D].郑州:河南中医学院,2014.
[26]徐文君,高慧,李杨,等.和颜坤泰胶囊对卵巢早衰大鼠性激素水平和VEGF,b FGF mRNA表达的影响[J].重庆医学,2017,46(6):738.
[27]杜江,李楠,王和鸣.肾虚模型造模方法及相关指标[J].中国组织工程研究与临床康复,2010,14(50):9433.
[28]邹桂林,方芳,何爱先,等.分析探讨更年期卵巢衰退动物模型与肾虚动物模型[J].中华中医药杂志,2015,30(4):1171.
[29]钟素琴.滋肾活血方治疗肾虚血瘀型卵巢早衰的临床研究[J].中国中医药现代远程教育,2019,17(1):76.
[30]沈江平.补肾活血方联合克龄蒙治疗卵巢储备功能下降肾虚血瘀证临床观察[J].新中医,2017,49(11):84.
[31]罗晶婧,李花.电针配合补肾活血方治疗肾虚血瘀型卵巢早衰30例临床观察[J].中医药导报,2015,21(6):46.
[32]邓高丕,骆欢欢,赵颖.活血化瘀对血瘀证模型大鼠子宫卵巢组织形态学的影响[J].辽宁中医药杂志,2007,34(6):851.
[2]杨冬梅,刘俊.雷公藤多苷临床应用及不良反应的研究进展[J].中国医院药学杂志,2018(10):1.
[3]曹艳,运乃茹,邹爱英.雷公藤多苷片致不良反应的Meta分析[J].中国药房,2018,29(1):125.
[4]郭健敏,黄远铿,雷夏凌,等.雷公藤多苷对雄性SD大鼠生殖系统损伤的动态变化及其可能机制[J].中国药理学与毒理学杂志,2018,32(6):469.
[5]马堃,刘雁峰,何军琴,等.补肾活血方案治疗排卵障碍性不孕不育的多中心随机双盲对照临床研究[J].中国中药杂志,2015,40(20):3911.
[6]冯桂玲,李静,周小琳.补肾健脾方对免疫性卵巢功能衰退小鼠性激素及卵子质量的影响[J].中医杂志,2016,57(16):1416.
[7]孟玥,任艳玲,孙月娇,等.左归丸、右归丸及其拆方对去卵巢骨质疏松症模型大鼠肾脏碱性磷酸酶、骨钙素表达的影响[J].中医杂志,2016,57(5):423.
[8]秦忠,李文,管连城,等.冬虫夏草VDR沉默大鼠卵巢颗粒细胞CYP19表达的影响[J].中国民族民间医药,2018,27(9):21.
[9]马堃,佟雅婧.从肾虚血瘀论治排卵障碍性不孕[J].中国中药杂志,2017,42(23):4451.
[10]神农本草经[M].吴普述,孙星衍,孙冯翼辑.沈阳:辽宁科学技术出版社,1997.
[11]高学敏.中药学[M].北京:中国中医药出版社,2002.
[12]贾微,吴晓君,岑妍慧,等.雷公藤多苷治疗肾脏疾病的研究进展[J].中华中医药学刊,2018,36(4):866.
[13]刘素晓,闫凤娜,王幼平.雷公藤多苷的抗炎作用与临床应用进展[J].中医临床研究,2017,9(32):131.
[14]刘莹,仲青香,邱辉辉,等.基于肝毒性的雷公藤中药复方配伍减毒的研究进展[J].中国中药杂志,2017,42(16):3044.
[15]冯雪,方赛男,高雨鑫,等.雷公藤制剂安全性的循证评价研究[J].中国中药杂志,2018,43(3):425.
[16]孙凤,杨兴华,马冬梅,等.雷公藤用药者生殖毒性发生率的Meta分析[J].中国药物警戒,2014,11(2):94.
[17]蔡竞,吴克明,黄丽,等.雷公藤多苷通过PI3K/AKT信号通路影响大鼠卵泡发育的实验研究[J].陕西中医,2014,35(7):923.
[18]付雨,吴克明.雷公藤多苷对大鼠卵巢血管生成及血供的影响[J].四川中医,2011,29(6):50.
[19]杨威.雷公藤多苷对消化道的毒性研究[C].武汉:中国毒理学会第七次全国毒理学大会暨第八届湖北科技论坛,2015.
[20]Ji Y,Tang B,Traub R J. The visceromotor response to colorectal distention fluctuates with the estrous cycle in rats[J]. Neuroscience,2008,154(4):1562.
[21]何国珍,杨美春,谢桂珍,等.雷公藤多苷诱导大鼠卵巢功能低下模型的实验研究[J].广西中医药大学学报,2017,20(4):9.
[22]Marcondes F K,Bianchi F J,Tanno A P. Determination of the estrous cycle phases of rats:some helpful consideration[J]. Braz J Biol,2002,62(4a):609.
[23]严大为,周莉,孙祖越.卵巢功能早衰动物模型的建立及其评价指标的研究进展[J].中国药理学与毒理学杂志,2015,29(3):486.
[24]梁爽,田舸,田燕,等.雷公藤多苷致小鼠卵巢早衰后的自然修复时间探讨[J].大连医科大学学报,2010,32(2):146.
[25]史志明.中成药雷公藤多苷片对儿童成年后生殖能力影响的临床随访观察与分析[D].郑州:河南中医学院,2014.
[26]徐文君,高慧,李杨,等.和颜坤泰胶囊对卵巢早衰大鼠性激素水平和VEGF,b FGF mRNA表达的影响[J].重庆医学,2017,46(6):738.
[27]杜江,李楠,王和鸣.肾虚模型造模方法及相关指标[J].中国组织工程研究与临床康复,2010,14(50):9433.
[28]邹桂林,方芳,何爱先,等.分析探讨更年期卵巢衰退动物模型与肾虚动物模型[J].中华中医药杂志,2015,30(4):1171.
[29]钟素琴.滋肾活血方治疗肾虚血瘀型卵巢早衰的临床研究[J].中国中医药现代远程教育,2019,17(1):76.
[30]沈江平.补肾活血方联合克龄蒙治疗卵巢储备功能下降肾虚血瘀证临床观察[J].新中医,2017,49(11):84.
[31]罗晶婧,李花.电针配合补肾活血方治疗肾虚血瘀型卵巢早衰30例临床观察[J].中医药导报,2015,21(6):46.
[32]邓高丕,骆欢欢,赵颖.活血化瘀对血瘀证模型大鼠子宫卵巢组织形态学的影响[J].辽宁中医药杂志,2007,34(6):851.