菟丝子总黄酮对多囊卵巢综合征大鼠模型的影响
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摘要
目的:探讨菟丝子总黄酮对脱氢表雄酮(dehydroepiandrosterone,DHEA)联合人绒毛膜促性腺激素(human chorionic gonadotropin,HCG)致大鼠多囊卵巢综合征(olycystic ovary syndrome,PCOS)模型的影响。方法:70只SD大鼠,10只正常组除外,其余大鼠上午颈背部注射DHEA 0. 06 mg·g-1,下午皮下注射1. 5 U HCG/只,连续21 d。造模第16天,阴道涂片,实施动情周期监测。挑选成模大鼠60只,分为模型组,达英-35组(0. 339 2 mg·kg-1),菟丝子总黄酮高、中、低剂量组(0. 2,0. 1,0. 05 g·kg-1),每组10只。分组当天按照组别分别进行给药,正常组和模型组给予等体积溶剂,连续给药21 d。末次给药2 h后,腹主动脉取血,测血清睾酮(testosterone,T),雌二醇(estrogen,E2),促黄体生成素(luteinizing hormone,LH)和卵泡刺激素(follicle stimulating hormone,FSH)的含量;脱颈处死大鼠,取每只大鼠同一部位的卵巢固定于10%甲醛溶液中,进行苏木素-伊红(HE)染色光镜观察卵巢的形态变化;取另一相同部位的卵巢,免疫组化法测量卵巢中雄激素受体(androgen receptor,AR)和凋亡相关蛋白B淋巴细胞瘤-2 (B-cell lymphoma-2,Bcl-2)和Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax),观察相关蛋白在卵巢中的表达情况;取下丘脑和垂体,免疫组化法检测下丘脑中促性腺激素释放激素(gonadotropinreleasing hormone,GnRH),垂体促性腺激素释放激素受体(gonadorelin releasing hormone receptor,GnRHR)的表达情况。结果:PCOS大鼠模型复制成功。与模型组比较,各给药组可以显著降低PCOS大鼠血清T,E2和LH含量,显著升高血清FSH含量(P <0. 01);显著升高卵巢Bcl-2水平(P <0. 01),不同程度升高垂体GnRHR水平(P <0. 05,P <0. 01);不同程度降低卵巢AR和Bax水平(P <0. 05,P <0. 01),显著降低下丘脑中GnRH水平(P <0. 01)和显著改善PCOS大鼠卵巢组织的病理性改变。结论:PCOS大鼠模型复制成功。菟丝子总黄酮可能通过调节雌雄激素分泌、抑制卵巢凋亡蛋白的表达、影响下丘脑-垂体-卵巢轴途径发挥对PCOS模型大鼠的保护作用。
Objective: To investigate the effect of dodder total flavone on polycystic ovary syndrome(PCOS) rat models induced by dehydroepiandrosterone(DHEA) combined with human chorionic gonadotropin(HCG). Method: Except the blank group,the remaining rats were injected with DHEA 0. 06 mg·g-1 in the morning on the nucha and 1. 5 U HCG in the afternoon for 21 consecutive days. On the 16 thday after the modeling,the vaginal smear was performed to monitor the estrus cycle. Sixty rats with successful modeling were selected and divided into model group,dacin-35 group,and high,middle and low-dose dodder total flavonoids groups,with 10 rats in each group. On the day of grouping,drugs were given respectively to the drug treatment groups,and the blank group and the model group were given equal volume solvent. The drugs were given continuously for 21 days.Blood was collected from abdominal aorta 2 h later after the final administration,serum levels of testosterone(T),estrogen(E2),luteinizing hormone(LH) and follicle stimulating hormone(FSH) were measured; rats were put to death,and the ovaries at the same part of each rat were fixed in 10% formalin solution. htoxylin eosin(HE)staining was performed,and the morphological changes in the ovaries were observed by light microscopy; the same part of the ovary was taken,and androgen receptor(AR),B-cell lymphoma-2(Bcl-2) and Bcl-2-associated X protein(Bax) were measured in the ovary by immunohistochemistry to observe the expressions of relevant proteins in the ovary; the hypothalamus and pituitary were taken,and the expressions of gonadotropin-releasing hormone(GnRH) in hypothalamus and gonadorelin releasing hormone receptor(GnRHR) in pituitary were detected by immunohistochemistry. Result: PCOS rat model was successfully replicated. Serum levels of T,E2 and LH were significantly reduced,and FSH level of PCOS was significantly increased in each drug treatment group(P <0. 01). The pituitary GnRHR level was increased to different degrees(P < 0. 05,P < 0. 01); AR and Bax levels of the ovary were decreased to different degrees(P < 0. 05, P < 0. 01); hypothalamus GnRH level was significantly decreased,and pathological changes in ovarian tissue of PCOS rats were obvious(P < 0. 01) in each drug treatment group. Conclusion: The PCOS rat model was successfully replicated. Total flavonoids of dodder may play a protective role in PCOS model rats by regulating the secretion of androgen,inhibiting the expression of ovarian apoptotic protein and impacting the hypothalamic-pituitary-ovary axis pathway.
Objective: To investigate the effect of dodder total flavone on polycystic ovary syndrome(PCOS) rat models induced by dehydroepiandrosterone(DHEA) combined with human chorionic gonadotropin(HCG). Method: Except the blank group,the remaining rats were injected with DHEA 0. 06 mg·g-1 in the morning on the nucha and 1. 5 U HCG in the afternoon for 21 consecutive days. On the 16 thday after the modeling,the vaginal smear was performed to monitor the estrus cycle. Sixty rats with successful modeling were selected and divided into model group,dacin-35 group,and high,middle and low-dose dodder total flavonoids groups,with 10 rats in each group. On the day of grouping,drugs were given respectively to the drug treatment groups,and the blank group and the model group were given equal volume solvent. The drugs were given continuously for 21 days.Blood was collected from abdominal aorta 2 h later after the final administration,serum levels of testosterone(T),estrogen(E2),luteinizing hormone(LH) and follicle stimulating hormone(FSH) were measured; rats were put to death,and the ovaries at the same part of each rat were fixed in 10% formalin solution. htoxylin eosin(HE)staining was performed,and the morphological changes in the ovaries were observed by light microscopy; the same part of the ovary was taken,and androgen receptor(AR),B-cell lymphoma-2(Bcl-2) and Bcl-2-associated X protein(Bax) were measured in the ovary by immunohistochemistry to observe the expressions of relevant proteins in the ovary; the hypothalamus and pituitary were taken,and the expressions of gonadotropin-releasing hormone(GnRH) in hypothalamus and gonadorelin releasing hormone receptor(GnRHR) in pituitary were detected by immunohistochemistry. Result: PCOS rat model was successfully replicated. Serum levels of T,E2 and LH were significantly reduced,and FSH level of PCOS was significantly increased in each drug treatment group(P <0. 01). The pituitary GnRHR level was increased to different degrees(P < 0. 05,P < 0. 01); AR and Bax levels of the ovary were decreased to different degrees(P < 0. 05, P < 0. 01); hypothalamus GnRH level was significantly decreased,and pathological changes in ovarian tissue of PCOS rats were obvious(P < 0. 01) in each drug treatment group. Conclusion: The PCOS rat model was successfully replicated. Total flavonoids of dodder may play a protective role in PCOS model rats by regulating the secretion of androgen,inhibiting the expression of ovarian apoptotic protein and impacting the hypothalamic-pituitary-ovary axis pathway.
引文
[1]周娴颖,周莉,孙祖越.用于治疗多囊卵巢综合征的中药药理学作用机制研究进展[J].中国中药杂志,2016,41(20):3715-3720.
[2] Spritzer P M, Motta A B, et al. Adolescence and polycystic ovary syndrome:current concepts on diagnosis and treatment[J]. Int J Clin Pract,2015,69(11):1236-1246.
[3] SHI X,XIE X,JIA Y,et al. Associations of insulin receptor and insulin receptor substrates genetic polymorphisms with polycystic ovary syndrome:a systematic review and Meta-analysis[J]. J Obstet Gynaecol Res,2016,42(7):844-854.
[4] Ewens K G,Jones M R,Ankener W,et al. FTO and MC4R gene variants are associated with obesity in polycystic ovary syndrome[J]. PLo S One,2011,6(1):e16390.
[5]张美微,侯丽辉,刘颖华.中医药治疗多囊卵巢综合征胰岛素抵抗的研究进展[J].世界中西医结合杂志,2016,11(3):436-439.
[6]谢敏,李丹,赵爽.多囊卵巢综合征的病因病机及治疗研究动态[J].世界中西医结合杂志,2018,13(1):145-148.
[7]刘绍龑,苗明三.基于中药新用的中医药理论创新发展研究[J].中医学报,2012,27(10):1309-1313.
[8] FU Z N,WEI Z Z,MIAO M S. Effects of total flavonoids of raspberry on perimenopausal model in mice[J].Saudi J Biol Sci,2018,25(3):487-492.
[9]辛卫云,白明,苗明三,等.浅析补肾阳中药治疗围绝经期综合征[J].中医学报,2017,32(1):67-70.
[10]王焕江,赵金娟,刘金贤,等.菟丝子的药理作用及其开发前景[J].中医药学报,2012,40(6):123-125.
[11]赵素霞,刘会丽,江红.菟丝子黄酮通过调节Wnt/β-catenin信号通路干预去卵巢大鼠骨代谢的机制研究[J].临床和实验医学杂志,2018,17(1):25-28.
[12]任秀朋,穆文涛,王叶婷,等.补肾活血中药治疗多囊卵巢综合征导致排卵障碍性不孕30例临床观察[J].中国药业,2018,27(17):75-77.
[13]卿朝晖,钟琼兰.观察中药补肾活血方对青春期多囊卵巢综合征内分泌激素的影响[J].中国医药科学,2017,7(8):97-99.
[14]刘玉芳,姚昶,孙海峰.温补脾肾化瘀法治疗多囊卵巢综合症胰岛素抵抗的临床研究[J].中医药学报,2016,44(5):61-63.
[15]姚莉娟,徐晓娟,王婧婧,等.体现中医病因病机的多囊卵巢综合征动物模型评价及筛选[J].世界科学技术—中医药现代化,2014,16(10):2137-2148.
[16]王娜梅.脱氢表雄酮诱导多囊卵巢综合征大鼠模型的表型研究[J].世界中西医结合杂志,2015,10(11):1596-1599.
[17] Andrade V H,Mata A M,Borges R S,et al. Current aspects of polycystic ovary syndrome:a literature review[J]. Rev Assoc Med Bras,2016,62(9):867.
[18]金婧,阮祥燕,华琳,等.多囊卵巢综合征合并卵巢储备功能低下患者的发生现状[J].生殖医学杂志,2018,27(2):103-107.
[19] Enrico C,Franca F,Roger A. Increased anti-mullerian hormone levels and ovarian size in a subgroup of women with functional hypothalamic amenorrhea:further identification of the link between polycystic ovary syndrome and functional hypothalamic amenorrhea[J].Am J Obstet Gynecol,2016,214(6):714-716.
[20] Spalkowska M,Mrozinska S,Galuszka-Bednarczyk A,et al. The PCOS patients differ in lipid profile according to their phenotypes[J]. Exp Clin Endocrinol Diabetes,2018,126(7):437-444.
[21]黄一鸣,康开彪,潘文,等.基于数据挖掘多囊卵巢综合征的中医辨证用药规律分析[J].新中医,2018,50(6):60-64.
[22] MIAO M S,TIAN S,GUO L,et al. The effect of curculigoside on mouse model of perimenopausal depression[J]. Saudi J Biol Sci, 2017,24(8):1894-1902.
[23]闫晓丽,张志强,苗明三.基于中西医临床特征的多囊卵巢综合征模型分析[J].中医学报,2017,32(4):598-601.
[24]罗克燕,杨丹莉,徐敏.菟丝子总黄酮对大鼠排卵障碍的治疗作用及其机制研究[J].现代中西医结合杂志,2013,22(20):2184-2186,2188.
[25]彭申明,陈勤,陈逸青,等.菟丝子总黄酮对内分泌衰退痴呆模型小鼠学习记忆功能的影响及保护作用机制[J].激光生物学报,2014,23(3):218-226.
[26] Mantelli F,Moretti C,Macchi I,et al. Effects of sex hormones on ocular surface epithelia:lessons learned from polycystic ovary syndrome[J]. J Cell Physiol,2016,231(5):971-975.
[27] YANG W,YANG R,YANG S,et al. Infertile polycystic ovary syndrome patients undergoing in vitro fertilization with the gonadotropin-releasing hormone-antagonist protocol:role of hyperandrogen-ism[J]. Gynecol Endocrinol,2018,34(8):715-718.
[28]宋俐.固本祛瘀方对多囊卵巢综合征模型大鼠垂体促性腺激素释放激素受体表达的影响[J].湖北中医杂志,2012,34(2):24-25.
[29]王德军,李路凯,张辉.加味二陈汤对肾虚痰湿型多囊卵巢综合征患者卵巢多囊样改变、内分泌及代谢的影响[J].中国实验方剂学杂志,2017,23(24):190-195.
[30]李俊波,邓芳,李霄汉,等.龙胆泻肝汤对PCOS模型大鼠的影响[J].中国实验方剂学杂志,2018,24(7):121-126.
[31] CHENG X B,Jimenez M,Desai R,et al. Characterizing the neuroendocrine and ovarian defects of androgen receptor-knockout female mice[J]. Am J Physiol Endocrinol Metab,2013,305(6):717-726.
[32]郑艳华,吴婉婷,马红霞,等.低频电针对多囊卵巢综合征大鼠卵巢组织Bax、Bcl-2表达的影响[J].中国中西医结合杂志,2017,37(12):1455-1460.
[33]唐忠庆,吴坚,郑赓唐,等.多囊卵巢综合征与卵巢颗粒细胞凋亡基因蛋白Bcl-2、Bax、P53的相关性研究[J].世界最新医学信息文摘,2016,16(A5):98-99.
[2] Spritzer P M, Motta A B, et al. Adolescence and polycystic ovary syndrome:current concepts on diagnosis and treatment[J]. Int J Clin Pract,2015,69(11):1236-1246.
[3] SHI X,XIE X,JIA Y,et al. Associations of insulin receptor and insulin receptor substrates genetic polymorphisms with polycystic ovary syndrome:a systematic review and Meta-analysis[J]. J Obstet Gynaecol Res,2016,42(7):844-854.
[4] Ewens K G,Jones M R,Ankener W,et al. FTO and MC4R gene variants are associated with obesity in polycystic ovary syndrome[J]. PLo S One,2011,6(1):e16390.
[5]张美微,侯丽辉,刘颖华.中医药治疗多囊卵巢综合征胰岛素抵抗的研究进展[J].世界中西医结合杂志,2016,11(3):436-439.
[6]谢敏,李丹,赵爽.多囊卵巢综合征的病因病机及治疗研究动态[J].世界中西医结合杂志,2018,13(1):145-148.
[7]刘绍龑,苗明三.基于中药新用的中医药理论创新发展研究[J].中医学报,2012,27(10):1309-1313.
[8] FU Z N,WEI Z Z,MIAO M S. Effects of total flavonoids of raspberry on perimenopausal model in mice[J].Saudi J Biol Sci,2018,25(3):487-492.
[9]辛卫云,白明,苗明三,等.浅析补肾阳中药治疗围绝经期综合征[J].中医学报,2017,32(1):67-70.
[10]王焕江,赵金娟,刘金贤,等.菟丝子的药理作用及其开发前景[J].中医药学报,2012,40(6):123-125.
[11]赵素霞,刘会丽,江红.菟丝子黄酮通过调节Wnt/β-catenin信号通路干预去卵巢大鼠骨代谢的机制研究[J].临床和实验医学杂志,2018,17(1):25-28.
[12]任秀朋,穆文涛,王叶婷,等.补肾活血中药治疗多囊卵巢综合征导致排卵障碍性不孕30例临床观察[J].中国药业,2018,27(17):75-77.
[13]卿朝晖,钟琼兰.观察中药补肾活血方对青春期多囊卵巢综合征内分泌激素的影响[J].中国医药科学,2017,7(8):97-99.
[14]刘玉芳,姚昶,孙海峰.温补脾肾化瘀法治疗多囊卵巢综合症胰岛素抵抗的临床研究[J].中医药学报,2016,44(5):61-63.
[15]姚莉娟,徐晓娟,王婧婧,等.体现中医病因病机的多囊卵巢综合征动物模型评价及筛选[J].世界科学技术—中医药现代化,2014,16(10):2137-2148.
[16]王娜梅.脱氢表雄酮诱导多囊卵巢综合征大鼠模型的表型研究[J].世界中西医结合杂志,2015,10(11):1596-1599.
[17] Andrade V H,Mata A M,Borges R S,et al. Current aspects of polycystic ovary syndrome:a literature review[J]. Rev Assoc Med Bras,2016,62(9):867.
[18]金婧,阮祥燕,华琳,等.多囊卵巢综合征合并卵巢储备功能低下患者的发生现状[J].生殖医学杂志,2018,27(2):103-107.
[19] Enrico C,Franca F,Roger A. Increased anti-mullerian hormone levels and ovarian size in a subgroup of women with functional hypothalamic amenorrhea:further identification of the link between polycystic ovary syndrome and functional hypothalamic amenorrhea[J].Am J Obstet Gynecol,2016,214(6):714-716.
[20] Spalkowska M,Mrozinska S,Galuszka-Bednarczyk A,et al. The PCOS patients differ in lipid profile according to their phenotypes[J]. Exp Clin Endocrinol Diabetes,2018,126(7):437-444.
[21]黄一鸣,康开彪,潘文,等.基于数据挖掘多囊卵巢综合征的中医辨证用药规律分析[J].新中医,2018,50(6):60-64.
[22] MIAO M S,TIAN S,GUO L,et al. The effect of curculigoside on mouse model of perimenopausal depression[J]. Saudi J Biol Sci, 2017,24(8):1894-1902.
[23]闫晓丽,张志强,苗明三.基于中西医临床特征的多囊卵巢综合征模型分析[J].中医学报,2017,32(4):598-601.
[24]罗克燕,杨丹莉,徐敏.菟丝子总黄酮对大鼠排卵障碍的治疗作用及其机制研究[J].现代中西医结合杂志,2013,22(20):2184-2186,2188.
[25]彭申明,陈勤,陈逸青,等.菟丝子总黄酮对内分泌衰退痴呆模型小鼠学习记忆功能的影响及保护作用机制[J].激光生物学报,2014,23(3):218-226.
[26] Mantelli F,Moretti C,Macchi I,et al. Effects of sex hormones on ocular surface epithelia:lessons learned from polycystic ovary syndrome[J]. J Cell Physiol,2016,231(5):971-975.
[27] YANG W,YANG R,YANG S,et al. Infertile polycystic ovary syndrome patients undergoing in vitro fertilization with the gonadotropin-releasing hormone-antagonist protocol:role of hyperandrogen-ism[J]. Gynecol Endocrinol,2018,34(8):715-718.
[28]宋俐.固本祛瘀方对多囊卵巢综合征模型大鼠垂体促性腺激素释放激素受体表达的影响[J].湖北中医杂志,2012,34(2):24-25.
[29]王德军,李路凯,张辉.加味二陈汤对肾虚痰湿型多囊卵巢综合征患者卵巢多囊样改变、内分泌及代谢的影响[J].中国实验方剂学杂志,2017,23(24):190-195.
[30]李俊波,邓芳,李霄汉,等.龙胆泻肝汤对PCOS模型大鼠的影响[J].中国实验方剂学杂志,2018,24(7):121-126.
[31] CHENG X B,Jimenez M,Desai R,et al. Characterizing the neuroendocrine and ovarian defects of androgen receptor-knockout female mice[J]. Am J Physiol Endocrinol Metab,2013,305(6):717-726.
[32]郑艳华,吴婉婷,马红霞,等.低频电针对多囊卵巢综合征大鼠卵巢组织Bax、Bcl-2表达的影响[J].中国中西医结合杂志,2017,37(12):1455-1460.
[33]唐忠庆,吴坚,郑赓唐,等.多囊卵巢综合征与卵巢颗粒细胞凋亡基因蛋白Bcl-2、Bax、P53的相关性研究[J].世界最新医学信息文摘,2016,16(A5):98-99.