为了研究雷公藤甲素(TP)对Ⅱ型胶原性关节炎(CIA)雌性大鼠生殖系统的毒性作用及其机制,将50只SD大鼠随机分为正常对照组,CIA模型组,和三组以临床等效剂量0接受TP片剂。5、1和2次,分别(TP剂量为3。75、7.5和15μg·kg~(-1)·d~(-1)),每个包括10只大鼠。第一次免疫后的第二天开始胃内给药,一天一次,42天。结果于第21天和第42天计算子宫和卵巢器官指数;在光学显微镜下观察子宫和卵巢的病理和形态学变化;雌二醇(E_2)和细胞色素P450A1(芳香化酶,ELISA法检测卵巢匀浆中CYP19A1)。此外,免疫组化检测转化生长因子β3(TGFβ3)通路相关蛋白的表达水平,母亲在卵巢组织中对抗十截瘫同系物3(Smad3)和类固醇生成因子1(SF-1)。体外,建立了小鼠中国仓鼠卵巢(CHO)细胞系,TP给药24小时后(30、60、120nmol·L~(-1)),用溴化噻唑蓝(MTT)法检测细胞增殖,通过流式细胞术细胞凋亡,用Westernblot方法检测细胞中TGFβ3、Smad3和SF-1蛋白的表达,免疫荧光法检测SF-1的细胞核进入。结果表明,与CIA模型组相比,所有TP给药组子宫腺体数量减少,总卵泡,成熟卵泡,和黄体在给药的第21天和第42天,但是没有统计学差异,在给药42天时,仅给予临床等效剂量的2倍TP可以显着增加闭锁卵泡的数量。TP在3。75μg·kg-1·d-1在给药21天时显着降低了E_2的水平以及卵巢组织中TGFβ3和Smad3因子的表达,但对雌激素合成中的限速酶CYP19A1无明显影响。TP在7。无论给药21天还是给药42天,5和15μg·kg〜(-1)·d〜(-1)均显着降低了SF-1的表达。TP在体外可显著促进卵巢细胞凋亡,细胞凋亡主要集中在给药24小时后的晚期细胞凋亡。此外,60nmol·L~(-1)TP显著降低TGFβ3、Smad3和SF-1蛋白表达,且呈剂量依赖性。总之,以低于临床等效剂量2倍的TP灌胃21天和42天未对CIA大鼠的子宫和卵巢组织造成明显的生殖损伤,只有当2倍的临床等效剂量给药42天时,闭锁卵泡的数量才会发生显着变化。TP通过抑制TGFβ3/Smad3/SF-1通路的表达,在体内对生殖靶器官和体外对卵巢细胞产生生殖毒性。
In order to study the toxic effect and mechanism of
triptolide(TP) on the reproductive system of female rats with Ⅱ type collagen induced arthritis(CIA), 50 SD rats were randomly divided into normal control group, CIA model group, and three groups receiving TP tablets at clinically equivalent doses of 0. 5, 1, and 2 times, respectively(with TP dosages of 3. 75, 7. 5, and 15 μg·kg~(-1)·d~(-1)), each comprising 10 rats. Intragastric administration was started on the day after the first immunization, once a day, for 42 days.The results were taken on the 21st and 42nd days to calculate the uterine and ovarian organ indexes; pathological and morphological changes in uterus and ovaries were observed under a light microscope; and the levels of estradiol(E_2) and cytochrome P450A1(aromatase,CYP19A1) in ovarian homogenate were detected by ELISA. Furthermore, immunohistochemistry was employed to detect the expression levels of transforming growth factor β3( TGFβ3) pathway-related proteins, mothers against decapentaplegic homolog 3(Smad3) and steroidogenic factor-1(SF-1) in ovarian tissues. In vitro, the mouse Chinese hamster ovary(CHO) cell line was established, and after 24 hours of TP administration(30, 60, 120 nmol·L~(-1)), cell proliferation was detected by the thiazolyl blue tetrazolium bromide(MTT) method, apoptosis by the flow cytometry, and TGFβ3, Smad3 and SF-1 protein expression in cells by the Western blot method, and the nuclear entry of SF-1 was detected by immunofluorescence. The results showed that compared with the CIA model group, all TP administration groups showed decreased number of uterine glands, total follicles, mature follicles, and corpus luteum on days 21 and 42 of administration, but there was no statistical difference, and only the administration of 2 times the clinically equivalent dose of TP could significantly increase the number of atretic follicles at 42 days of administration. TP at 3. 75 μg·kg-1·d-1significantly reduced the level of E_2 at 21 days of administration and the expression of TGFβ3 and Smad3 factors in ovarian tissues,but had no significant effect on the rate-limiting enzyme in estrogen synthesis CYP19A1. TP at 7. 5 and 15 μg·kg~(-1)·d~(-1) significantly reduced the expression of SF-1 regardless of administration for 21 days or 42 days. TP can significantly promote ovarian cell apoptosis in vitro, with apoptosis mainly concentrated in the late stage of apoptosis after 24 hours of administration. In addition, 60 nmol·L~(-1) TP significantly reduced the protein expression of TGFβ3, Smad3 and SF-1 in a dose-dependent manner. In summary, intragastric administration of TP at less than 2 times the clinically equivalent dose for 21 days and 42 days did not cause obvious reproductive damage to the uterus and ovarian tissues of CIA rats, and the number of atretic follicles changed significantly only when the 2 times the clinically equivalent dose was administered for 42 days. TP exerted reproductive toxicity in vivo on reproductive target organs and in vitro on ovarian cells by inhibiting the expression of TGFβ3/Smad3/SF-1 pathway.