杂环齐墩果酸衍生物-阿司匹林缀合物抑制血清素合成及促骨形成作用

Effects of Heterocyclic Oleanolic Acid Derivatives-Aspirin Conjugates on Inhibition of Serotonin Biosynthesis and Increase of Bone Formation

目的探讨杂环齐墩果酸衍生物-阿司匹林缀合物对血清素合成的抑制活性以及促骨形成活性。方法采用高效液相色谱(HPLC)法、酶联免疫吸附测定(ELISA)试剂盒和荧光实时定量聚合酶链反应(PCR)测试杂环齐墩果酸衍生物-阿司匹林缀合物对RBL-2H3细胞中血清素合成的关键酶色氨酸羟化酶-1(TPH-1)的抑制率、TPH-1蛋白含量及mRNA的表达。构建去势大鼠骨质疏松症模型(OVX),并将骨质疏松大鼠随机分为给药组、甲状旁腺激素组、模型对照组和假手术组,给药35 d后采用HPLC法测定血清和小肠中血清素水平。采用噻唑蓝(MTT)法测定杂环齐墩果酸衍生物-阿司匹林缀合物对成骨细胞促进活性。结果在RBL-2H3细胞中,杂环齐墩果酸衍生物-阿司匹林缀合物对TPH-1的抑制呈剂量依赖性,抑制率为20.4%~92.5%,缀合物7在10μmol·L^(-1)浓度有最高抑制率(92.5%),TPH-1蛋白含量为51 ng·m L^(-1),较对照组(216 ng·m L^(-1))显著降低,TPH-1mRNA表达也降低。与假手术组比较,模型对照组血清和小肠中血清素水平明显增高(P<0.05),给予缀合物后大鼠血清和小肠中血清素水平却明显降低,缀合物7给药后血清和小肠血清素分别为232,155 ng·m L^(-1),较模型对照组1 050和783 ng·m L^(-1)明显降低。缀合物能够促进乳小鼠成骨细胞增殖。结论该研究为开发新型、高效、低毒的具有抗骨质疏松症的齐墩果酸衍生物提供信息和依据。

Objective To explore the effects of the heterocyclic oleanolic acid derivatives-aspirin conjugates on inhibition of serotonin biosynthesis and increase of bone formation. Methods The inhibition rate,content and mRNA expression of heterocyclic oleanolic acid derivatives-aspirin conjugates on tryptophan hydroxylase 1（ TPH-1）,which was the principal enzyme in the biosynthesis of serotonin,were tested by HPLC,ELISA kit and real-time PCR,respectively. Osteoporosis model was established by ovariectomy（ OVX）. The rats were randomly divided into conjugate groups,parathyroid hormone group,model control group and sham operation group. Serum and gut serotonin levels were tested by HPLC after 35 days of administration,and the antiosteoporosis activity of the heterocyclic oleanolic acid derivatives-aspirin conjugates was evaluated by using osteoblast-like cells isolated from murine calvaria by MTT assay. Results The preliminary biological results showed that heterocyclic oleanolic acid derivatives-aspirin conjugates displayed a dose-dependent suppression on TPH-1 in RBL-2H3,and the inhibition rate was 20. 4%-92. 5%. Specifically,conjugate 7 at 10 μmol·L^（-1） concentration showed the greatest inhibition rate on TPH-1（ 92. 5%）. Content of TPH-1 was 51 ng m L^（-1）,significantly lower than that in the control group（ 216 ng·m L^（-1））,as well as the TPH-1 mRNA. Compared with the sham operation group,levels of serum and gut serotonin were increased in OVX group,while they were significanlty decreased in conjugate groups,and reduced significantly in conjugate 7 group（ 232 and 155 ng·m L^（-1）） as compared with OVX group（ 1 050 and 783 ng·m L^（-1））; Moreover,heterocyclic oleanolic acid derivatives-aspirin conjugates could increase the bone formation. Conclusion This study provides information and basis for development of novel,efficient and harmfulless oleanolic acid derivatives with anti-osteoporosis.