五环三萜类化合物促进骨形成研究

The stimulating effect of pentacyclic triterpenes on bone formation

目的探讨五环三萜类化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、ELISA试剂盒和荧光实时定量PCR测试五环三萜类化合物对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、含量及mRNA的表达。构建去势大鼠骨质疏松症模型(OVX)25只,并将骨质疏松大鼠随机分为3组不同浓度灌胃给药组[10mg/(kg·d)组、20 mg/(kg·d)组、30 mg/(kg·d)组]、一组空白对照组(OVX组)和一组假手术组(Sham组),给药35 d后采用高效液相色谱测试血清和小肠中血清素水平。采用MTT法测试五环三萜类化合物对成骨细胞促进活性。结果在RBL-2H3细胞中,五环三萜类化合物对于TPH-1的抑制呈剂量依赖性,抑制率为3.6%~76.5%,其中18β-甘草次酸(GA)在30μM浓度下展现出了最高抑制率(76.5%),并能较空白组(219 ng/m L)显著地降低TPH-1含量(34 ng/m L)及mRNA的表达。与Sham组相比,大鼠摘除卵巢(OVX组)后血清和小肠中血清素的水平明显增高,而经过GA给药后的大鼠血清和小肠中血清素的水平却明显降低,在30μM浓度下血清(426 ng/m L)和小肠(304 ng/m L)中血清素的浓度较OVX组(含量分别为1050ng/m L和723 ng/m L)降低了至少2倍;此外,GA能够促进乳小鼠成骨细胞增殖。结论 18β-甘草次酸能够作为促进骨形成的先导化合物,值得深入研究。

Objective To explore the effect of the pentacyclic triterpenes on inhibition of serotonin biosynthesis and increase the bone formation.Methods The inhibition rate and mRNA expression of pentacyclic triterpenes on tryptophan hydroxylase 1（ TPH-1）,which was the principal enzyme in the biosynthesis of serotonin,were tested using HPLC,ELISA kit,and real-time PCR.Osteoporosis model was developed by ovariectomy（ OVX）.The rats were divided randomly into three concentration groups（ 10 mg/kg per day,20 mg/kg per day,or 30 mg/kg per day）,one blank control group（ OVX group）,and one sham operation group（ sham group）.Serum and gut serotonin levels were tested by HPLC method after 35 days of administration.The antiosteoporotic activity of pentacyclic triterpenes was evaluated with MTT assay in osteoblast-like cells isolated from mouse calvariae.Results Pentacyclic triterpenes displayed a dose-dependent suppression of TPH-1 in RBL-2H3 cells,and the inhibition rate was 3.6%-76.5%.Specifically,18β-glycyrrhetinic acid at 30 μMconcentration showed the greatest inhibiting potency in TPH-1,which significantly suppressed TPH-1 protein and mRNA expressions.Compared with those in the sham group,the levels of serum and gut serotonin in OVX group increased,while they significantly decreased in three concentration groups of GA, and in 30 μMconcentration group they reduced by at least 2 times compared with OVX group.Moreover,18β-glycyrrhetinic acid increased the bone formation.Conclusion 18β-glycyrrhetinic acid can be used as a lead compound for promoting bone formation,and it is worth of further study.