重楼皂苷I通过抑制自噬减轻磷酸三钙磨损颗粒诱导成骨细胞损伤的实验研究

Polyphyllin I alleviates osteoblasts injuries induced by TCP wear particles in vitro via inhibition of autophagy

目的观察重楼皂苷I(PPI)对磷酸三钙(TCP)磨损颗粒诱导的成骨细胞损伤的影响,并探讨其调控机制。方法通过消化法从SD大鼠颅骨中获得原代成骨细胞,应用碱性磷酸酶(ALP)染色鉴定成骨细胞。TCP磨损颗粒(0.1 mg/m L)与成骨细胞共孵育48 h制备成骨细胞损伤模型,实验随机分为对照组、模型组和PPI(30、100μg/m L)组。CCK-8和化学比色法分别测定成骨细胞活力和上清液中乳酸脱氢酶(LDH)水平;实时荧光定量PCR法检测成骨细胞中ALP、I型胶原(Collagen I)和Runt相关转录因子2(RUNX2)m RNA水平;Annexin-V/PI染色经流式细胞术定量分析成骨细胞凋亡情况;应用茜素红S染色观察成骨细胞矿化结节形成;Western blotting法检测成骨细胞中Bcl-2、Bax、cleaved Caspase-3、Atg5、p62和微管相关蛋白1轻链3(LC-3)蛋白的表达。结果与对照组比较,模型组成骨细胞活性降低,特征蛋白ALP、Collagen I、RUNX2m RNA水平及矿化结节形成显著减少,LDH释放量、细胞凋亡和Bax、cleavedCaspase-3、Atg5和LC-3等蛋白表达及LC-3II/LC-3I值明显增加,而Bcl-2和p62蛋白表达显著减少;与模型组比较,PPI各组成骨细胞活性升高,ALP、Collagen I和RUNX2 m RNA水平和矿化结节形成明显增加,LDH释放和细胞凋亡显著减少,Bax、cleaved Caspase-3、Atg5和LC-3蛋白表达及LC-3II/LC-3I值也明显减少,而Bcl-2和p62蛋白表达显著增加。结论PPI可通过抑制自噬的活化而减轻TCP磨损颗粒诱导的成骨细胞损伤。

Objective To observe the effect of polyphyllin I(PPI)on osteoblasts injuries induced by tricalcium phosphate(TCP)wear particles in vitro,and explain its regulation mechanisms.Methods Primary osteoblasts obtained from the calvaria of neonatal SD rat by the series of digestion were identified with ALP staining.The osteoblasts were treated with TCP wear particles(TCP,0.1 mg/mL)for 48 h to establish an in vitro injuries model of the calvarial osteoblasts.The experiment was randomly divided into control group,model(TCP,0.1 mg/mL)group,PPI(30μg/mL)group and PPI(100μg/m L)group.CCK-8 and chemical colorimetry were used to examine cell viability and lactic dehydrogenase(LDH)content in culture media;Real-time PCR was performed to detect mRNA levels of ALP,Collagen I and RUNX2 in osteoblasts;The flow cytometry was used to examine apoptosis of osteoblasts using Annexin V/PI double staining;When the osteoblasts were treated for 14 d,mineral nodules formation was observed with alizarin S staining;Western blot was applied to examine proteins expression of Bax,Bcl-2,cleaved Caspase-3,Atg5,p62,and microtubule associated protein 1 light chain3(LC-3).Results Compared with control group,model group showed that the cell viability of osteoblasts,m RNA levels of ALP,Collagen I and RUNX2,and mineral nodules formation were significantly decreased;LDH content,percentage of apoptosis and proteins expression of Bax,cleaved Caspase-3,Atg5,LC-3 and the ratio of LC-3 II/LC-3 I were obviously increased in calvarial osteoblasts,whereas proteins expression of Bcl-2 and p62 was remarkably decreased.Compared with model group,PPI groups indicated that cell viability of osteoblasts,m RNA levels of ALP,Collagen I and RUNX2,and mineral nodules formation were dramatically increased;LDH content,percentage of apoptosis,protein expressions of Bax,cleaved Caspase-3,Atg5,and LC-3 and the ratio of LC-3 II/LC-3 I were obviously decreased,but Bcl-2 and p62 expression were obviously increased.Conclusion PPI alleviates osteoblasts injuries induced by TCP wear particles via inhibition of autophagy.