目的 觀察積雪草酸對(duì)健康人牙周膜細(xì)胞（human periodontal ligament cells，hPDLCs）增殖、凋亡及成骨分化的作用，探究其作用機(jī)制。方法 采用不同濃度積雪草酸處理健康hPDLCs，CCK8法檢測(cè)不同時(shí)間點(diǎn)積雪草酸對(duì)hPDLCs的增殖能力的影響；流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況；堿性磷酸酶（alkaline phosphatase，ALP）和茜素紅染色檢測(cè)細(xì)胞成骨分化情況，qRT-PCR法檢測(cè)細(xì)胞ALP、Runt相關(guān)轉(zhuǎn)錄因子2（runt-related transcription factor 2，Runx2）、骨鈣素（osteocalcin，OCN）、骨形態(tài)發(fā)生蛋白2（bone morphogenic protein 2，BMP2）和核因子κB受體活化因子配體（receptor activator nuclear factor κB ligand，RANKL）mRNA表達(dá)水平；Western blotting法檢細(xì)胞中B淋巴細(xì)胞瘤-2基因（B-cell lymphoma-2，Bcl-2）、Bcl-2相關(guān)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）蛋白表達(dá)水平。結(jié)果 與對(duì)照組比較，不同濃度積雪草酸對(duì)hPDLCs增殖和凋亡無顯著影響。在成骨誘導(dǎo)條件下，與對(duì)照組比較，不同濃度積雪草酸可提高ALP、Runx2、OCN、BMP2 mRNA表達(dá)，減少RANKL mRNA表達(dá)，上調(diào)PI3K和Akt磷酸化水平，其中50 μg/mL積雪草酸的效果最佳。結(jié)論 100 μg/mL以下積雪草酸對(duì)健康hPDLCs增殖和凋亡無影響，但可促進(jìn)hPDLCs成骨分化能力，其作用機(jī)制可能與激活PI3K/AKT通路有關(guān)。

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.

R965

河南省醫(yī)學(xué)科技攻關(guān)計(jì)劃聯(lián)合共建項(xiàng)目（LHGJ20190198）