槲皮素对钛颗粒诱导的成骨细胞凋亡抑制作用研究

Inhibitory effect of quercetin on titanium particles induced apoptosis in osteoblasts

目的 探讨槲皮素对钛颗粒诱导的成骨细胞凋亡的抑制作用。方法 采用钛颗粒处理小鼠MC3T3-E1细胞系构建细胞凋亡模型,应用槲皮素进行干预并设置对照,噻唑蓝法[3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide,MTT法]法检测细胞增殖活性,流式细胞术检测细胞凋亡,酶联免疫吸附试验(enzyme-linked immunoadsordent assay,ELISA)检测白细胞介素-1β(interleukin-1β,IL-1β)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达,免疫荧光技术检测C/EBP同源蛋白(C/EBP homologous protein,CHOP)、B细胞淋巴瘤因子2(B-cell lymphoma-2,Bcl-2)蛋白的表达,实时定量聚合酶链反应(real-time polymerase chain reaction,RT-PCR)检测CHOP和Bcl-2的mRNA的表达。结果 钛颗粒抑制成骨细胞增殖活性,并呈浓度依赖性。流式细胞术检测结果显示,钛颗粒诱导成骨细胞凋亡。槲皮素预培养能有效缓解钛颗粒对成骨细胞增殖活性的抑制,减少其凋亡。ELISA检测结果显示,槲皮素能减少IL-1β和TNF-α表达。免疫荧光技术显示槲皮素参与调控CHOP蛋白和Bcl-2蛋白的表达,RT-PCR检测结果显示,槲皮素参与调控CHOP和Bcl-2的mRNA的表达。结论 槲皮素能抑制钛颗粒诱导的成骨细胞凋亡,其作用机制可能与内质网应激启动的凋亡途径抑制,调控CHOP、Bcl-2蛋白表达的信号通路有关。

Objective To explore the inhibitory effect of quercetin on titanium particles induced apoptosis in osteoblasts.Methods Mouse MC3T3-E1 cell line was treated with titanium particles to construct apoptosis model,quercetin was applied to intervene and control group was set up.Cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assays(MTT),the apoptosis was analyzed by flow cytometry.The expression levels of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immunoadsordent assay(ELISA).Immunofluorescence technology was used to detect the expression of C/EBP homologous protein(CHOP) and B-cell lymphoma-2(Bcl-2) protein.The protein expression levels of CHOP and Bcl-2 mRNA expression were detected by real-time polymerase chain reaction(RT-PCR).Results Titanium particlesl inhibited osteoblast proliferation in a dose dependent manner.Flow cytometric showed that titanium particles induced osteoblast apoptosis.Quercetin pre-culture can effectively relieve the inhibition of titanium particles on the proliferation of osteoblasts and reduce their apoptosis.ELISA results showed that quercetin could reduce the expression of IL-1β and TNF-α.Immunofluorescence technology showed that quercetin is involved in regulating the expression of CHOP and Bcl-2 proteins,and RT-PCR detection results showed that quercetin is involved in regulating the mRNA expression of CHOP and Bcl-2.Conclusion Quercetin can inhibit the apoptosis of osteoblasts induced by titanium particles,and which may be related to the inhibition of apoptosis pathway initiated by endoplasmic reticulum stress and the signal pathway that regulates the expression of CHOP and Bcl-2 protein.