补肾调经方和逍遥丸对体外培养小鼠卵泡中ADAM8及ADAMTS-1影响的比较

Comparison of Effects of Bushen Tiaojing Formula and Xiaoyao Pills on Expression of ADAM8 and ADAMTS-1 in Vitro Cultured Follicles of Mice

目的探讨补肾调经方和逍遥丸对体外培养小鼠卵泡中去整合素-金属蛋白酶8(ADAM8)和Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶(ADAMTS-1)表达的影响。方法机械法分离昆明乳鼠窦前卵泡,随机分为空白组、正常组、补肾组、疏肝组,空白组不加血清,正常组加入正常大鼠血清,体外培养12天;补肾组加入补肾调经Ⅱ号方高剂量大鼠含药血清,疏肝组加入逍遥丸高剂量大鼠含药血清体外培养11天。11天时补肾组和疏肝组分别改为补肾调经Ⅲ号方高剂量和逍遥丸低剂量大鼠含药血清体外培养1天。培养12天加入人绒毛膜促性腺激素(hCG)后0、8、12、16 h采用RT-PCR法检测各组卵泡和卵丘卵母细胞复合体(COCs)中ADAM8、ADAMTS-1 mRNA表达,细胞免疫荧光染色法比较各组ADAM8、ADAMTS-1蛋白表达。结果加入h CG后0、8、12、16 h,各组ADAM8、ADAMTS-1 mRNA及蛋白的表达逐渐升高,各时间点空白组与正常组比较,差异无统计学意义(P>0.05)。与本组0、8 h比较,补肾组12、16 h ADAM8、ADAMTS-1 mRNA及蛋白表达升高(P<0.05)。与本组0 h比较,疏肝组8 h ADAM8、ADAMTS-1 mRNA及蛋白表达升高(P<0.05)。与空白组和正常组比较,补肾组与疏肝组8、12、16 h ADAM8、ADAMTS-1 mRNA及蛋白表达升高,8 h疏肝组高于补肾组,12 h补肾组高于疏肝组(均P<0.05)。补肾组ADAM8、ADAMTS-1 mRNA及蛋白的表达高峰高于疏肝组(P<0.05)。结论补肾调经方和逍遥丸均可通过增强ADAM8和ADAMTS-1的蛋白溶解作用,使卵泡壁破裂而诱发排卵,补肾法诱发排卵的作用优于疏肝法。

Objective To observe the effect of Bushen Tiaojing Formula(BSTJF)and Xiaoyao Pills(XYP)on the expression of a disintegrin and metalloprotease 8(ADAM8)and a disintegrin and metalloprotease with thrombospondin motifs-1(ADAMTS-1)in vitro cultured follicles of mice.Methods The preantral follicles of healthy Kunming female mice were separated by microdissection,which were divided into 4 groups:blank group,normal group,BSTJF group,XYP group randomly.The preantral follicles were inoculated by culture medium without medicated serum(blank group),normal rats’serum(normal group),high dose BSTJFⅡ(Bushen group)or high dose XYP containing serums(XYP group)for 12 days.BSTJF group and XYP group were changed to high dose BSTJFⅢand low dose XYP serum respectively in 11 st day.The follicles and excreted cumulus-oocyte complexs(COCs)were collected after the hCG was added after 0,8,12,16 h in 12 nd day.RT-PCR and immunofluorescence staining were used to detect the mRNA and protein expression of ADAM8,ADAMTS-1 in follicles and COCs respectively.Result After the hCG was added,the mRNA and protein expression of ADAM8,ADAMTS-1 increased gradually in each group.There was no significant difference between blank and normal group at each time point(P>0.05).Compared with the same group at 0 and 8 h,m RNA and protein expressions of ADAM8 and ADAMTS-1 were increased at 12 and 16 h in BSTJF group(P<0.05).Compared with the same group at 0 h,the expressions of ADAM8 and ADAMTS-1 mRNA and protein were increased at 8 h in XYP group(P<0.05).Compared with the blank and normal group,the expression of ADAM8,ADAMTS-1 increased in the treatment groups at 8,12 and 16 h.Compared with the BSTJF group,the expression of ADAM8,ADAMTS-1 in XYP group was higher at 8 h(P<0.05)and lower at 12 h(P<0.05).There was no significant difference between the BSTJF group and the XYP group at 16 h(P>0.05).The mRNA and protein expression peak of ADAM8,ADAMTS-1 in BSTJF group was higher than that in XYP group.Conclusions Both BSTJF and XYP can enhance the proteolysis of ADAM8 and ADAMTS-1 to rupture the wall of follicle and induce ovulation.BSTJF is better than XYP in promoting ovulation.