含中药骨金散血清对大鼠成骨细胞OPG、RANKL mRNA表达的影响

Effects of the expressions of serum with Gujinsan on OPG and RANKL mRNA in cultured osteoblasts of rats

目的:本研究旨在探讨含中药骨金散血清对成骨细胞的增殖及OPG/RANKL系统的影响。方法:6月龄雌性大鼠48只,随机分为假手术组、模型组、骨金散低剂量组和骨金散高剂量组,每组12只,模型组、骨金散低剂量组和骨金散高剂量组大鼠建立去卵巢大鼠骨质疏松的动物模型,4周后低剂量骨金散组和高剂量骨金散组按1.8g/kg和3.6g/kg灌服骨金散,每日一次;模型组和假手术组均每天一次生理盐水灌胃。连续给药8周后处死大鼠,观察骨金散对去卵巢大鼠股骨骨密度的影响,制备含骨金散血清。体外培养成骨细胞,观察含药血清对成骨细胞的增殖和OPG、RANKL mRNA的表达的影响。结果:与假手术组相比,去卵巢大鼠股骨骨密度明显降低(P<0.01),低剂量的骨金散组和高剂量组大鼠股骨骨密度高于去卵巢组(P<0.01)。低剂量的骨金散组和高剂量组血清促进成骨细胞的增殖(P<0.05),升高OPGmRNA表达水平(P<0.01),降低RANKL mRNA的表达水平(P<0.01),使OPGmRNA/RANKLmRNA的表达比值升高(P<0.01)。结论:中药骨金散能增加去势大鼠BMD,促进成骨细胞增殖,增加成骨细胞OPGmRNA表达,降低RANKLmRNA表达,发挥抑制破骨细胞的骨吸收作用,这可能与其治疗骨质疏松的机制有关。

Objective：To establish the animal models of osteoporosis in ovariectomized rats and investigate the effects of the expressions of serum with Gujinsan on proliferation and OPG and RANKL mRNA in cultured osteoblasts of rat. Methods：48 six--month old SD female rats were randomly assigned to two groups, including the ovariectomized group with 36 rats in the group and false operation group with 12 rats in the group. In the ovariectomized group, double ovaries of rats were removed. In the false operation group, some fat around the o- varies on both sides of rats were removed. At 4 weeks after operation, the ovariectomized group were randomly allocated into three groups with 12 rats in each group： the control group, low--dose Gujinsan group, and high --dose Gujinsan group, by drug intervention are as follows. The low--dose Gujinsan group： intragastric ad- ministrated Gujinsan（1.8g/kg per day）. The high--dose Gujinsan group： intragastric administrated Gujinsan （3. 6g/kg per day）. The rest of groups：intragastric administrated the same amount of normal saline. Two months later, BMD of rat was measured by using double functional X--ray digital bone density meter. The Ef- fects of serum with Gujinsan on cultured osteoblasts proliferation was evaluated by MTT assay, and the mRNA expressions of OPG and RANKL in cultured osteoblasts of rat were measured by RT--PCR. Results： In o- variectomized control group,the BMD were significantly lower than that in false operation group （P 〈 0.01）, while compared with ovariectomized control group, BMD of low--dose Gujinsan group and high--dose Gujin- san group increased significantly （P 〈 0. 01）. The serum with Gujinsan of low--dose Gujinsan group and high --dose Gujinsan group can promote the cultured osteoblasts proliferation compared with ovariectomized control group （P 〈0. 01）. The OPG mRNA expression of the cultured osteoblasts in serum with Gujinsan was upregu- lated while the RANKL mRNA expressions of the cultured osteoblasts in serum with Gujinsan was down regu- lated compared with ovariectomized control group （P 〈 0.01）. The serum with Gujinsan increased the expres- sions ratio of OPG mRNA/RANKL mRNA （P 〈 0.01）. Conclusion ：The Gujinsan can increase BMD in O- variectomized rats, promote the proliferation of osteoblasts, increase the osteoblasts OPG mRNA expression, reduce the expression of the osteoblast RANKLmRNA, increase the expressions ratio of OPG mRNA/RANKL mRNA,play the role in inhibition of osteoclasts to absorb bone tissue, which may be related to the mechanism of the Gujinsan in treating osteoporosis.