基于PI3K/Akt通路探讨积雪草酸对健康人牙周膜细胞增殖、凋亡及成骨分化的作用机制研究

Based on PI3K/Akt pathway, to explore the mechanism of asiatic acid on proliferation, apoptosis and osteogenic differentiation of healthy human periodontal membrane cells

目的观察积雪草酸对健康人牙周膜细胞(human periodontal ligament cells,hPDLCs)增殖、凋亡及成骨分化的作用,探究其作用机制。方法采用不同浓度积雪草酸处理健康hPDLCs,CCK8法检测不同时间点积雪草酸对hPDLCs的增殖能力的影响;流式细胞仪检测细胞凋亡情况;碱性磷酸酶(alkaline phosphatase,ALP)和茜素红染色检测细胞成骨分化情况,qRT-PCR法检测细胞ALP、Runt相关转录因子2(runt-related transcription factor 2,Runx2)、骨钙素(osteocalcin,OCN)、骨形态发生蛋白2(bone morphogenic protein 2,BMP2)和核因子κB受体活化因子配体(receptor activator nuclear factorκB ligand,RANKL)mRNA表达水平;Western blotting法检细胞中B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein gene,Bax)、半胱氨酸蛋白酶-3(caspase-3)、磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(proteinkinase B,Akt)、磷酸化Akt(p-Akt)蛋白表达水平。结果与对照组比较,不同浓度积雪草酸对hPDLCs增殖和凋亡无显著影响。在成骨诱导条件下,与对照组比较,不同浓度积雪草酸可提高ALP、Runx2、OCN、BMP2 mRNA表达,减少RANKL mRNA表达,上调PI3K和Akt磷酸化水平,其中50μg/mL积雪草酸的效果最佳。结论100μg/mL以下积雪草酸对健康h PDLCs增殖和凋亡无影响,但可促进hPDLCs成骨分化能力,其作用机制可能与激活PI3K/AKT通路有关。

Objective To observe the effects of asiatic acid asiatic acid on proliferation, apoptosis, and osteogenic differentiation of healthy human periodontal ligament cells(hPDLCs), and to explore its mechanism. Methods hPDLCs were treated with different concentrations of asiatic acid. CCK8 method was used to detect the effect of asiatic acid on the proliferation of healthy hPDLCs at different time points. Apoptosis was detected by flow cytometry. Osteogenic differentiation was detected by alkaline phosphatase(ALP) and alizarin red staining. The mRNA expression levels of ALP, Runt related transcription factor 2(Runx2), osteocalcin(OCN),bone morphogenetic protein 2(BMP2) and receptor activator nuclear factor ligand(RANKL) were detected by RT-qPCR. Western blot was used to detect B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein gene(Bax), Caspase-3, phosphatidylinositol 3-kinase(PI3K), p-PI3K and protein kinase B(Akt), phosphorylated protein kinase B(p-Akt) protein expression levels. Results Compared with control group, asiatic acid had no significant effect on the proliferation and apoptosis of hPDLCs. Under osteogenic induction conditions, compared with control group, different concentrations of asiatic acid could increase the mRNA expression of ALP, Runx2,OCN and BMP2, decrease the mRNA expression of RANKL, and up regulate the phosphorylation levels of PI3K and Akt. 50 μg/mL asiatic acid had the best effect. Conclusion Asiatic acid below 100 μg/mL has no effect on the proliferation and apoptosis of hPDLCs,but can promote the osteogenic differentiation of healthy hPDLCs, its mechanism may be related to the activation of PI3K/Akt pathway.