西红花酸治疗DHT诱导的PCOS小鼠的机制研究

Mechanism of Crocetin on the DHT-induced Polycystic Ovary Syndrome in Mice

目的:观察西红花酸对双氢睾酮(Dihydrotestosterone,DHT)诱导的多囊卵巢综合症(Polycystic ovarian syndrome,PCOS)小鼠的疗效并探讨其作用机制。方法:妊娠15-18天给予孕鼠皮下注射DHT诱导子代雌鼠PCOS模型。待子鼠8周龄后,随机选择一半数量的PCOS小鼠连续4周西红花酸灌胃,作为西红花酸给药组(n=18)。给药期间检测体重和动情周期,待小鼠16周龄左右,通过眼球取血后处死,取出下丘脑、卵巢。采用HE染色观察卵巢组织的病理改变;ELISA试剂盒检测血清中雌二醇(Estradiol,E2)、睾酮(Testosterone,T)、孕酮(Progesterone,P4)、促黄体生成素(Luteinizing hormone,LH)、卵泡刺激素(Follicle-stimulating hormone,FSH);采用免疫组化、Western blot和实时荧光定量PCR法检测下丘脑的前腹侧视旁核(Anteroventral periventricular,AVPV)、弓状核(Arcuate,ARC)的kisspeptin以及视前区(Preoptic area,POA)里Gn RH的表达水平。结果:与对照组相比,PCOS小鼠卵巢与体重的比值上升了22.56%±6.77%,动情周期延长,卵巢内出现大的空泡,闭锁卵泡数量增加了138.74%±33.22%,窦状卵泡、成熟窦状卵泡和黄体数量分别减少了38.80%±4.69%、56.35%±7.32%和63.77%±7.25%,血清中E2、P4和FSH水平分别降低了40.99%±2.69%、56.91%±5.25%、和38.80%±4.69%,而T、LH水平分别升高了43.23%±4.70%和148.46%±28.16%,下丘脑中AVPV中kisspeptin神经元表达减少,ARC中kisspeptin神经元表达增多,POA中Gn RH神经元减少,而以上情况能够被西红花酸改善。结论:西红花酸分别通过促进和抑制下丘脑AVPV核和ARC核团的kisspeptin表达改善PCOS的病理变化。

Objective： To observe the effect of crocetin on polycystic ovary syndrome（PCOS） induced by dihydrotestosterone（DHT） in mice and explore its possible mechanism. Methods： Pregnant dams were subcutaneously（s.c.） injected with DHT from gestation day（GD）16 to GD18, and female offspring were analyzed as the prenatally DHT-treated mice models of PCOS. 50 % of eight-week-old female offspring in PCOS group were treated with crocetin daily for 4 weeks by oral gavage, which were analyzed as crocetin treatment group（n=18）. The body weight and estrous cycle were examined during the treatment. Sixteen weeks later, the mice were sacrificed after getting orbital blood and the hypothalami and ovaries were obtained. HE staining was used to observe the pathological change of ovarian tissue. The levels of serum estradiol（E2）, testosterone（T）, progesterone（P4）, luteinizing hormone（LH） and follicle-stimulating hormone（FSH） were quantified using ELISA kit. Immunohistochemical, western blot and quantitative Real-time PCR methods were used to detect the expression of kisspeptin in anteroventral periventricular（AVPV）, arcuate nucleus（ARC） and the expression of Gn RH in the preoptic area（POA）. Results： Compared with the control group, the ratio of ovary to body weight of in PCOS-mice was increased by 22.56 %±6.77 %. PCOS-mice exhibited an obvious prolongation of diestrus and more atretic cyst-like follicles than the control group. The number of atretic cyst-like follicles was increased by 138.74 %±33.22 %, whereas the numbers of large antral follicles, preovulatory follicles and corpus luteum were reduced by 38.80 %±4.69 %, 56.35 %±7.32 %, 63.77 %±7.25 % respectively. The levels of E2, P4 and FSH in serum were reduced by 40.99 %±2.69 %, 56.91 %±5.25 %, 38.80 %±4.69 % respectively, while the levels of T and LH were increased by 43.23 %±4.70 %, 148.46 %±28.16 % respectively. The expression of hypothalamic kisspeptin in AVPV and Gn RH in POA was reduced, whereas the expression of kisspeptin in ARC was increased. Treatment of crocetin could prevent the prolongation of diestrus and reduction in corpora luteum, recover the levels of Gn RH, FSH, LH, progesterone（P4）, estradiol（E2） and testosterone（T）, and increase the kisspeptin level in AVPV but reduce that in ARC. Conclusion： Crocetin improved the PCOS in mice via increasing AVPV-kisspeptin and reducing ARC-kisspeptin expression.