白藜芦醇对钛颗粒诱导小鼠巨噬细胞形成的破骨细胞数量及F-actin环面积影响观察

Effect of resveratrol on the number of osteoclasts and F-actin ring area induced by titanium particles in mouse macrophages

目的观察白藜芦醇对钛颗粒诱导小鼠巨噬细胞株RAW264.7形成的破骨细胞数量及F-actin环面积影响,并探讨其机制。方法将小鼠巨噬细胞株RAW264.7分为Sham组、Vehicle组、Low-Res组、Mid-Res组、HighRes组,Sham组加入完全培养基,Vehicle组加入钛颗粒+完全培养基,Low-Res组加入钛颗粒+完全培养基+白藜芦醇(1μmol/L),Mid-Res组加入钛颗粒+完全培养基+白藜芦醇(10μmol/L),High-Res组加入钛颗粒+完全培养基+白藜芦醇(15μmol/L)。各组细胞培养5天后,采用TRAP染色法检测各组破骨细胞数,采用鬼笔环钛染色法检测各组破骨细胞F-actin环面积,采用Realtime-PCR法检测各组细胞中TNF-α、IL-1βmRNA,采用ELISA法检测各组细胞中TNF-α、IL-1β蛋白,采用Western blotting法检测各组细胞p-p65、p-IκB蛋白。结果Vehicle组可观察到较多体积较大、酒红色、内含多个细胞核的破骨细胞,而应用不同浓度白藜芦醇干预后,破骨细胞数随着药物浓度的增加而减少;Sham组、Vehicle组、Low-Res组、Mid-Res组、High-Res组破骨细胞数分别为0、(145.31±7.87)个、(129.63±4.12)个、(85.51±3.65)个、(69.76±5.63)个,其中Vehicle组和Low-Res组破骨细胞数相比,P>0.05,其余组间相比,P均<0.05。Vehicle组可观察到少量较大的F-actin环,Low-Res组、Mid-Res组的F-actin环较Vehicle组略有减少,High-Res组多核细胞明显减少;Sham组、Vehicle组、Low-Res组、Mid-Res组、High-Res组F-actin环面积分别为0、(3642.31±987.01)μm2、(3265.75±871.98)μm2、(2497.45±712.61)μm2和(1741.81±716.07)μm2,组间相比,P均<0.05。Low-Res组、Mid-Res组、High-Res组细胞中TNF-αmRNA、IL-1βmRNA、TNF-α蛋白、IL-1β蛋白、p-p65蛋白、p-IκB蛋白相对表达量或表达水平均低于Vehicle组,且降低程度与白藜芦醇浓度有关。结论白藜芦醇呈浓度依赖性对钛颗粒诱导小鼠巨噬细胞株RAW264.7形成的破骨细胞数量及F-actin环面积产生抑制作用,其抑制作用的机制与NF-κB信号通路有关。

Objective To observe the effect of resveratrol on the number of osteoclasts and F-actin ring area induced by titanium particles in mouse macrophage cell line RAW264.7,and to explore its mechanism.Methods The mouse macrophage cell line RAW264.7 was divided into Sham group,Vehicle group,Low-Res group,Mid-Res group,High-Res group;cells in the Sham group were added with minimal medium,the Vehicle group with titanium particles+minimal medium,Low-Res group with titanium particles+minimal medium+resveratrol(1μmol/L),Mid-Res group with titanium particles+minimal medium+resveratrol(10μmol/L),and High-Res group with titanium particles+minimal medium+resveratrol(15μmol/L),respectively.After 5 days of cell culture in each group,the number of osteoclasts in each group was detected by TRAP staining,and the area of F-actin ring of osteoclasts in each group was detected by phalloidin staining.Real-time PCR was used to detect TNF-αand IL-1βmRNA in each group,ELISA was used to detect TNF-αand IL-1βprotein in each group,and Western blotting was used to detect p-p65 and p-IκB protein.Results In the Vehicle group,more osteoclasts with larger volume,wine red,and multiple nuclei were observed.After intervention with different concentrations of resveratrol,the number of osteoclasts decreased as the drug concentration increased;the number of osteoclasts in the Sham group,Vehicle group,Low-Res group,Mid-Res group,and High-Res group were 0,145.31±7.87,129.63±4.12,85.51±3.65,69.76±5.63,respectively;except between the Vehicle group and the Low-Res group,significant differences were found between the other groups(all P<0.05).A small number of large F-actin rings was observed in the Vehicle group;the F-actin rings in the Low-Res group and the Mid-Res group were slightly reduced as compared with those of the Vehicle group,and the multi-nuclear cells in the High-Res group were significantly reduced;the Factin ring areas of the Sham group,Vehicle group,Low-Res group,Mid-Res group,High-Res group were 0,(3642.31±987.01),(3265.75±871.98),(2497.45±712.61),and(1741.81±716.07)μm2,with statistically significant difference between groups(all P<0.05).The relative expression or expression levels of TNF-αmRNA,IL-1βmRNA,TNF-αprotein,IL-1βprotein,p-p65 protein,and p-IκB protein in cells of the Low-Res group,Mid-Res group,and HighRes group were lower than those of the Vehicle group,and the decreased degree was related to the resveratrol concentration.Conclusion Resveratrol has a concentration-dependent effect on the number of osteoclasts and F-actin ring area induced by titanium particles in the RAW264.7 mouse macrophage cell line,and the mechanism of the inhibition is related to the NF-κB signalling pathway.