加味芍药甘草汤对多囊卵巢综合征模型大鼠的干预作用
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摘要
目的:探究加味芍药甘草汤对多囊卵巢综合征(PCOS)模型大鼠的干预作用及机制。方法:SD雌性大鼠随机分为正常组,模型组,加味芍药甘草汤低、中、高剂量(12.5,25.0,50.0 g·kg-1·d-1)组,达英-35组(0.001 g·kg-1·d-1),每组6只。除正常组给予蒸馏水外,其余各组利用来曲唑(0.001 g·kg-1·d-1)灌胃给药21 d制备PCOS大鼠模型,造模后加味芍药甘草汤及达英-35组分别以相应剂量灌胃给药2周,正常组及模型组灌胃给予等体积的蒸馏水。采用阴道涂片观察动情周期;卵巢组织切片观察形态变化;酶联免疫吸附法(ELISA)检测甾类激素(睾酮,雌二醇,孕酮,促黄体生成素)水平;并用蛋白免疫印迹法(Western blot)测定大鼠卵巢组织中水通道蛋白-9(aquaporin-9,AQP-9)的表达。结果:与正常组比较,模型组大鼠动情周期发生紊乱;体质量与卵巢质量明显增加;卵巢组织中存在大量初级囊性扩张卵泡,颗粒细胞疏松,成熟卵泡内未见卵母细胞,放射冠消失;血清睾酮和促黄体生成素的水平均显著升高(P<0.05),而雌二醇和孕酮水平显著降低(P<0.05)。与模型组大鼠比较,加味芍药甘草汤可以剂量依赖性地减轻体质量和卵巢质量,降低血清中雄激素和促黄体生成素水平,提高血清中雌激素及孕激素水平,并且与卵巢中AQP-9的表达呈相关性。结论:加味芍药甘草汤可能通过增加卵巢颗粒细胞中AQP-9表达干预PCOS大鼠卵巢多囊样变和高雄激素血症。
Objective: To explore the intervention effect and the underlying mechanism of modified Shaoyao Gancaotang on polycystic ovary syndrome( PCOS) in rats. Method: SD female rats were randomly divided into normal group,model group,low,middle and high-dose modified Shaoyao Gancaotang groups( 12. 5,25. 0,50. 0 g·kg-1·d-1) and Diane-35 group( 0. 001 g·kg-1·d-1),with 6 rats in each group. The rats in normal group received distilled water,while all of the other rats were treated with letrozole( 0. 001 g·kg-1·d-1) for 21 days to induce the PCOS model. Diane-35 and different concentrations of modified Shaoyao Gancaotang were given by gavage for two weeks after modeling,and the equal volume of distilled water was given by gavage to normal group and model group. After treatment with drugs,the estrus cycle was determined by vaginal smears,the morphological changes were inspected by ovarian biopsy,the serum hormone level was evaluated by enzyme-linked immunosorbent assay( ELISA),and the protein expression of aquaporin-9( AQP-9) was detected by Western blot technique.Result: Model group showed an irregular estrous cycle with increased body weight and ovarian weight; the ovaries showed plenty of cystic dilating follicles as well as fewer granulosa cells,without oocyte and corona radiating in the follicles; the serum levels of androgen and luteinizing hormone of PCOS rats were significantly increased( P <0. 05),whereas the levels of estrogen and progesterone were significantly decreased( P < 0. 05). Compared with model group,modified Shaoyao Gancaotang groups showed decreased body weight and ovarian weight in a dosedependent manner; serum levels of androgen and luteinizing hormone reduced,whereas the serum levels of estrogen and progesterone were increased. Moreover,modified Shaoyao Gancaotang was associated with AQP-9 expression in ovaries. Conclusion: Modified Shaoyao Gancaotang could intervene polycystic ovaries and hyperandrogenism in PCOS rats,and the underlying mechanism may be correlated with the up-regulation of AQP-9 expression in granulosa cells.
Objective: To explore the intervention effect and the underlying mechanism of modified Shaoyao Gancaotang on polycystic ovary syndrome( PCOS) in rats. Method: SD female rats were randomly divided into normal group,model group,low,middle and high-dose modified Shaoyao Gancaotang groups( 12. 5,25. 0,50. 0 g·kg-1·d-1) and Diane-35 group( 0. 001 g·kg-1·d-1),with 6 rats in each group. The rats in normal group received distilled water,while all of the other rats were treated with letrozole( 0. 001 g·kg-1·d-1) for 21 days to induce the PCOS model. Diane-35 and different concentrations of modified Shaoyao Gancaotang were given by gavage for two weeks after modeling,and the equal volume of distilled water was given by gavage to normal group and model group. After treatment with drugs,the estrus cycle was determined by vaginal smears,the morphological changes were inspected by ovarian biopsy,the serum hormone level was evaluated by enzyme-linked immunosorbent assay( ELISA),and the protein expression of aquaporin-9( AQP-9) was detected by Western blot technique.Result: Model group showed an irregular estrous cycle with increased body weight and ovarian weight; the ovaries showed plenty of cystic dilating follicles as well as fewer granulosa cells,without oocyte and corona radiating in the follicles; the serum levels of androgen and luteinizing hormone of PCOS rats were significantly increased( P <0. 05),whereas the levels of estrogen and progesterone were significantly decreased( P < 0. 05). Compared with model group,modified Shaoyao Gancaotang groups showed decreased body weight and ovarian weight in a dosedependent manner; serum levels of androgen and luteinizing hormone reduced,whereas the serum levels of estrogen and progesterone were increased. Moreover,modified Shaoyao Gancaotang was associated with AQP-9 expression in ovaries. Conclusion: Modified Shaoyao Gancaotang could intervene polycystic ovaries and hyperandrogenism in PCOS rats,and the underlying mechanism may be correlated with the up-regulation of AQP-9 expression in granulosa cells.
引文
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[3]CHEN M J,CHOU C H,CHEN S U,et al.The effect of androgens on ovarian follicle maturation:Dihydrotestosterone suppress FSH-stimulated granulosa cell proliferation by upregulating PPARgammadependent PTEN expression[J].Sci Rep,2015,5(1):18319.
[4]王娜梅.隐丹参酮对多囊卵巢综合征大鼠糖脂代谢及性激素的影响[J].中国妇幼保健,2015,30(20):3490-3493.
[5]Goodman N F,Cobin R H,Futterweit W,et al.American Association of Clinical Endocrinologists,American College of Endocrinology,and Androgen Excess and Pcos Society Disease State Clinical Review:Guide to the Best Practices in the Evaluation and Treatment of Polycystic Ovary Syndrome—Part 1[J].Endocr Pract,2015,21(11):1291-1300.
[6]罗雪.中医药治疗多囊卵巢综合征的系统评价[D].广州:广州中医药大学,2011.
[7]李娟,马红霞,宋金龙,等.多囊卵巢综合征脂代谢异常的国内外研究进展及中医药治疗优势探讨[J].世界中医药,2015,10(7):1112-1116.
[8]Takeuchi T.Effect of shakuyaku-kanzo-to,shakuyaku,kanzo,paeoniflorin,glycyrrhetinic acid and glycyrrhizin on ovarian function in rats[J].Nihon Naibunpi Gakkai Zasshi,1988,64(11):1124-1139.
[9]Armanini D,Castello R,Scaroni C,et al.Treatment of polycystic ovary syndrome with spironolactone plus licorice[J].Eur J Obstet Gynecol Reprod Biol,2007,131(1):61-67.
[10]Takeuchi T,Nishii O,Okamura T,et al.Effect of traditional herbal medicine,shakuyaku-kanzo-to on total and free serum testosterone levels[J].Am J Chin Med,1989,17(1/2):35-44.
[11]刘桢,梁瑞宁,李佩双.多囊卵巢综合征代谢异常特点及中医认识[J].中国中医基础医学杂志,2017,23(5):654-656.
[12]CUI H Z,OH H C,LI X,et al.Ethanol extract of Lycopus lucidus elicits positive inotropic effect via activation of Ca2+entry and Ca2+release in beating rabbit atria[J].J Med Food,2013,16(7):633-640.
[13]REN Q,DING L,SUN S S,et al.Chemical identification and quality evaluation of Lycopus lucidus Turcz by UHPLC-Q-TOF-MS and HPLC-MS/MS and hierarchical clustering analysis[J].Biomed Chromatogr,2017,31(5):3867-3878.
[14]郝琦蓉,张引华.多囊平胶囊治疗多囊卵巢综合征的实验研究[J].山西医科大学学报,2003,34(5):447-450.
[15]Rajan R K,Ss M,Balaji B.Soy isoflavones exert beneficial effects on letrozole-induced rat polycystic ovary syndrome(PCOS)model through anti-androgenic mechanism[J].Pharm Biol,2017,55(1):242-251.
[16]辛卫云,苗明三.泽兰的化学、药理及临床应用[J].中医学报,2015,30(3):418-420.
[17]刘雨娟.芍药苷对大鼠离体脂肪组织影响的实验研究[D].兰州:兰州大学,2009.
[18]Motta A B.The role of obesity in the development of polycystic ovary syndrome[J].Curr Pharm Des,2012,18(17):2482-2491.
[19]尹山兰.多囊卵巢综合征患者卵巢颗粒细胞PPARγ、P450arom表达及与IVF-ET结局关系研究[D].郑州:郑州大学,2013.
[20]Gervasio C G,Bernuci M P,Silva-de-Sa M F,et al.The role of androgen hormones in early follicular development[J].ISRN Obstet Gynecol,2014,doi:10:1155/2014/8/18010.
[21]Filicori M.Roles and novel regimens of luteinizing hormone and follicle-stimulating hormone in ovulation induction[J].J Clin Endocrinol Metab,2001,86(4):1437-1441.
[22]HUANG H F,HE R H,SUN C C,et al.Function of aquaporins in female and male reproductive systems[J].Hum Reprod Update,2006,12(6):785-795.
[23]陆秀娥,钱羽力,黄荷凤.多囊卵巢综合征患者颗粒细胞AQP9的表达[J].浙江大学学报:医学版,2007,36(5):449-453.
[24]QU F,WANG F F,LU X E,et al.Altered aquaporin expression in women with polycystic ovary syndrome:hyperandrogenism in follicular fluid inhibits aquaporin-9in granulosa cells through the phosphatidylinositol 3-kinase pathway[J].Hum Reprod,2010,25(6):1441-1450.
[25]李星,杨菁,尹太郎,等.水通道蛋白9在多囊卵巢综合征大鼠卵巢颗粒细胞的表达[J].中华临床医师杂志(电子版),2012,6(23):7807-7808.
[2]SUN J,JIN C,WU H,et al.Effects of electroacupuncture on ovarian P450arom,P450c17alpha and mRNA expression induced by letrozole in PCOS rats[J].PLo S One,2013,8(11):e79382.
[3]CHEN M J,CHOU C H,CHEN S U,et al.The effect of androgens on ovarian follicle maturation:Dihydrotestosterone suppress FSH-stimulated granulosa cell proliferation by upregulating PPARgammadependent PTEN expression[J].Sci Rep,2015,5(1):18319.
[4]王娜梅.隐丹参酮对多囊卵巢综合征大鼠糖脂代谢及性激素的影响[J].中国妇幼保健,2015,30(20):3490-3493.
[5]Goodman N F,Cobin R H,Futterweit W,et al.American Association of Clinical Endocrinologists,American College of Endocrinology,and Androgen Excess and Pcos Society Disease State Clinical Review:Guide to the Best Practices in the Evaluation and Treatment of Polycystic Ovary Syndrome—Part 1[J].Endocr Pract,2015,21(11):1291-1300.
[6]罗雪.中医药治疗多囊卵巢综合征的系统评价[D].广州:广州中医药大学,2011.
[7]李娟,马红霞,宋金龙,等.多囊卵巢综合征脂代谢异常的国内外研究进展及中医药治疗优势探讨[J].世界中医药,2015,10(7):1112-1116.
[8]Takeuchi T.Effect of shakuyaku-kanzo-to,shakuyaku,kanzo,paeoniflorin,glycyrrhetinic acid and glycyrrhizin on ovarian function in rats[J].Nihon Naibunpi Gakkai Zasshi,1988,64(11):1124-1139.
[9]Armanini D,Castello R,Scaroni C,et al.Treatment of polycystic ovary syndrome with spironolactone plus licorice[J].Eur J Obstet Gynecol Reprod Biol,2007,131(1):61-67.
[10]Takeuchi T,Nishii O,Okamura T,et al.Effect of traditional herbal medicine,shakuyaku-kanzo-to on total and free serum testosterone levels[J].Am J Chin Med,1989,17(1/2):35-44.
[11]刘桢,梁瑞宁,李佩双.多囊卵巢综合征代谢异常特点及中医认识[J].中国中医基础医学杂志,2017,23(5):654-656.
[12]CUI H Z,OH H C,LI X,et al.Ethanol extract of Lycopus lucidus elicits positive inotropic effect via activation of Ca2+entry and Ca2+release in beating rabbit atria[J].J Med Food,2013,16(7):633-640.
[13]REN Q,DING L,SUN S S,et al.Chemical identification and quality evaluation of Lycopus lucidus Turcz by UHPLC-Q-TOF-MS and HPLC-MS/MS and hierarchical clustering analysis[J].Biomed Chromatogr,2017,31(5):3867-3878.
[14]郝琦蓉,张引华.多囊平胶囊治疗多囊卵巢综合征的实验研究[J].山西医科大学学报,2003,34(5):447-450.
[15]Rajan R K,Ss M,Balaji B.Soy isoflavones exert beneficial effects on letrozole-induced rat polycystic ovary syndrome(PCOS)model through anti-androgenic mechanism[J].Pharm Biol,2017,55(1):242-251.
[16]辛卫云,苗明三.泽兰的化学、药理及临床应用[J].中医学报,2015,30(3):418-420.
[17]刘雨娟.芍药苷对大鼠离体脂肪组织影响的实验研究[D].兰州:兰州大学,2009.
[18]Motta A B.The role of obesity in the development of polycystic ovary syndrome[J].Curr Pharm Des,2012,18(17):2482-2491.
[19]尹山兰.多囊卵巢综合征患者卵巢颗粒细胞PPARγ、P450arom表达及与IVF-ET结局关系研究[D].郑州:郑州大学,2013.
[20]Gervasio C G,Bernuci M P,Silva-de-Sa M F,et al.The role of androgen hormones in early follicular development[J].ISRN Obstet Gynecol,2014,doi:10:1155/2014/8/18010.
[21]Filicori M.Roles and novel regimens of luteinizing hormone and follicle-stimulating hormone in ovulation induction[J].J Clin Endocrinol Metab,2001,86(4):1437-1441.
[22]HUANG H F,HE R H,SUN C C,et al.Function of aquaporins in female and male reproductive systems[J].Hum Reprod Update,2006,12(6):785-795.
[23]陆秀娥,钱羽力,黄荷凤.多囊卵巢综合征患者颗粒细胞AQP9的表达[J].浙江大学学报:医学版,2007,36(5):449-453.
[24]QU F,WANG F F,LU X E,et al.Altered aquaporin expression in women with polycystic ovary syndrome:hyperandrogenism in follicular fluid inhibits aquaporin-9in granulosa cells through the phosphatidylinositol 3-kinase pathway[J].Hum Reprod,2010,25(6):1441-1450.
[25]李星,杨菁,尹太郎,等.水通道蛋白9在多囊卵巢综合征大鼠卵巢颗粒细胞的表达[J].中华临床医师杂志(电子版),2012,6(23):7807-7808.