加减知柏地黄丸对特发性中枢性性早熟小鼠ERK1/2、OSX表达的影响

Effect of modified Zhibai Dihuang Pill on the expression of ERK1/2 and OSX in idiopathic central precocious puberty mice

目的探讨加减知柏地黄丸对特发性中枢性性早熟(ICPP)小鼠细胞外信号调控激酶(ERK1/2)、成骨细胞特异性转录因子Osterix(OSX)表达的影响。方法将30只18日龄雌性SPF级C57小鼠随机分为正常组、模型组、加减知柏地黄丸高剂量组、加减知柏地黄丸低剂量组、甲地孕酮组和抑那通组,每组5只。在小鼠20日龄时,正常组给予生理盐水腹腔注射,其余组小鼠腹腔注射瘦素2 mg/kg建立ICPP模型。同时加减知柏地黄丸高、低剂量组分别给予加减知柏地黄丸水溶液3.9 g/mL、1.95 g/mL灌胃,甲地孕酮组给予甲地孕酮水溶液0.13 mg/mL灌胃,正常组和模型组给予等量蒸馏水灌胃,均1次/d,连续7 d;抑那通组分别于20日龄、22日龄和24日龄时依次给予抑那通1.574 mg/kg、1.180 mg/kg、1.180 mg/kg右后肢处肌注。实验结束后,qRT-PCR法检测股骨组织中ERK1、ERK2和OSX mRNA表达情况,Western blot法检测股骨组织中ERK1、ERK2表达情况,ELISA法检测血清骨生化指标[胰岛素样生长因子-1(IGF-1)、碱性磷酸酶(ALP)、鸟氨酸氨基甲酰转移酶(OCT)、N端中段骨钙素(N-MID)]水平。结果模型组股骨组织中ERK1、ERK2、OSX mRNA表达量和ERK1、ERK2蛋白表达量及血清IGF-1、ALP、OCT、N-MID水平均明显高于正常组(P均<0.05),加减知柏地黄丸高剂量组、加减知柏地黄丸低剂量组、甲地孕酮组股骨组织中ERK1、ERK2、OSX mRNA表达量和ERK1、ERK2蛋白表达量及血清IGF-1、ALP、OCT、N-MID水平均明显低于模型组(P均<0.05)。结论加减知柏地黄丸能通过抑制ERK1/2表达,抑制成骨细胞生长和分化成熟,从而延缓ICPP小鼠骨骺提前闭合。

Objective It is to investigate the effect of modified Zhibai Dihuang Pill on the expression of extracellular regulated protein kinases 1/2(ERK1/2)and osteoblast specific transcription factor Osterix(OSX)in mice with idiopathic central precocity puberty(ICPP).Methods Thirty 18 day old female SPF grade C57 mice were randomly divided into normal group,model group,modified Zhibai Dihuang Pill high-dose group,modified Zhibai Dihuang Pill low-dose group,megestrol group and leuprorelin group,with 5 mice in each group.At the age of 20 days,the normal group was given normal saline by intraperitoneal injection,while the mice in the other groups were given leptin 2 mg/kg by intraperitoneal injection to establish ICPP models.At the same time,the high-dose and low-dose groups of Zhibai Dihuang Pill were given 3.9 g/mL and 1.95 g/mL Zhibai Dihuang Pill aqueous solution by gavage,the megestrol group was given 0.13 mg/mL megestrol aqueous solution by gavage,the normal group and model group were given equal amounts of distilled water by gavage,all once a day,continuously treated for 7 days.The leuprorelin group was given leuprorelin 1.574 mg/kg,1.180 mg/kg and 1.180 mg/kg by intramuscular injection on the right hind limb at 20,22,and 24 days of age,respectively.After the experiment,the expressions of ERK1,ERK2 and OSX mRNA in femoral tissue were detected by qRT PCR,the expressions of ERK1,ERK2 in femoral tissue were detected by Western blot,and the levels of serum bone biochemical indicators such as insulin-like growth factor-1(IGF-1),alkaline phosphatase(ALP),ornithine carbamyltransferase(OCT)and N-mid osteocalcin(N-MID)were detected by ELISA.Results The expressions of ERK1,ERK2,OSX mRNA,ERK1,ERK2 protein,and serum IGF-1,ALP,OCT,N-MID levels in the femoral tissue of the model group were significantly higher than those of the normal group(P<0.05).The expression levels of mRNA of ERK1,ERK2,OSX,and protein of ERK1,ERK2 in the femoral tissue,and the levels of serum IGF-1,ALP,OCT,N-MID in the model group were significantly higher than those in the normal group(all P<0.05),the expression levels of mRNA of ERK1,ERK2,OSX,and protein of ERK1,ERK2 in the femoral tissue,and the levels of serum IGF-1,ALP,OCT,N-MID in the high-dose and low-dose groups of Zhibai Dihuang Pill,and megestrol group were significantly lower than those in the model group(all P<0.05).Conclusion Modified Zhibai Dihuang Pill can inhibit the growth and differentiation of osteoblasts by inhibiting the expression of ERK1/2,thus to delay the early closure of the epiphyses in ICPP mice.