人参皂苷Rb2在体外调控破骨细胞的作用

Effects of ginsenoside Rb2 on osteoclast regulation in vitro

目的探究人参皂苷Rb2在体外对破骨细胞的作用及机制。方法通过培养RAW264.7破骨前体细胞,加入不同浓度Rb2溶液(0.1μM,1μM,10μM),采用CCK-8检测Rb2药物毒性,利用TRAP染色观察破骨细胞形态并计数,使用RT-PCR方法检测TRAP、NFATc1破骨活动特异性基因m RNA的表达,通过Western blot技术检测破骨细胞中LC3 I、LC3 II、m TOR的蛋白表达水平。结果 TRAP阳性计数,与空白对照组相比(168.2±22.6),0.1μM组(131.0±16.3),P=0.018;1μM组(98.8±17.9),P<0.01;10μM组(85.8±17.3),P<0.01,破骨细胞数量明显减少。对照组TRAP、NFATc1破骨分化特异性基因m RNA表达明显下降,与对照组相比各组P<0.01。LC3 II/LC3 I的比值显著增高,m TOR量下降,与对照组相比各组P<0.01。结论 Rb2可能通过自噬途径影响破骨基因的表达,从而减少破骨细胞的形成,m TOR通路在此过程中起重要作用。

Objective To investigate effects and mechanism of ginsenoside Rb2 on osteoclasts in vitro. Methods The mouse osteoclast precursor cells RAW 264.7 were cultivated to induce osteoclasts. Rb2（ 0.1 μM, 1 μM, 10 μM） was respectively added in the culture solution. CCK-8 was used for the detection of drug toxicity. The morphology and count of osteoclasts were observed by TRAP staining. The expression of osteoclast specific gene m RNA, such as TRAP and NFATc1, was detected by RT-PCR. Western blot technique was used to detect the expression levels of LC3 I, LC3 II and m TOR proteins. Results Compared with the control group（ 168.2 ± 22.6）, the number of osteoclasts decreased significantly： grouP〈0.1 μM（ 131.0 ± 16.3）, P = 0.018; group 1 μM（ 98.8 ± 17.9）, P〈0.01; group 10 μM（ 85.8 ± 17.3）, P〈0.01. The expression of TRAP and NFATc1 osteoclast specific gene m RNA decreased significantly, the ratio of LC3, II/LC3 and I increased significantly, and the amount of m TOR decreased, P〈0.01. Conclusions Rb2 may affects the expression of osteoclast genes by autophagy pathway to reduce the osteoclast formation. m TOR pathway plays an important role in this process.