汉防己甲素干预假体周围磨损颗粒诱导下骨溶解模型的体外实验

In vitro experiment of tetrandrine on the model of osteolysis induced by wear particles around the prosthesis

背景:国内鲜有关于汉防己甲素对磨损颗粒诱导的破骨细胞形成与分化方面的相关研究。目的:探讨汉防己甲素在不同浓度作用下对钛颗粒诱导的小鼠RAW264.7巨噬细胞体外骨溶解模型的积极作用。方法:将小鼠RAW264.7巨噬细胞分为5组培养:空白组,实验组(含0.1 g/L钛颗粒悬液),低、中、高浓度药物组(实验组成分+0.1,0.5,1.0μmol/L汉防己甲素)。CCK-8法检测RAW264.7细胞的增殖活性;抗酒石酸酸性磷酸酶和鬼笔环钛染色法检测成熟破骨细胞的功能和数量;酶联免疫法检测细胞上清液中肿瘤坏死因子α和基质金属蛋白酶9的表达;RT-PCR检测活化T细胞核因子c1、肿瘤坏死因子α和基质金属蛋白酶9的mRNA表达;Western blot法检测核转录因子κBp65、IκBα两者磷酸化蛋白的水平。结果与结论:①与空白组比较,各浓度的汉防己甲素对RAW264.7细胞增殖没有影响(P>0.05);②对照组可观察到较多体积较大、酒红色、F-actin环被荧光绿染的多核破骨细胞,而应用不同浓度的汉防己甲素处理后,破骨的细胞数显著降低(P<0.05),F-actin环大小随着药物浓度的增加而减少;③与空白组比较,实验组的IκBα、核转录因子κBp65及其磷酸化程度,细胞培养上清肿瘤坏死因子α和基质金属蛋白酶9蛋白表达量,活化T细胞核因子c1、肿瘤坏死因子α和基质金属蛋白酶9的mRNA显著升高(P<0.05),汉防己甲素呈浓度依赖性降低上述指标表达;④综上,汉防己甲素通过抑制NF-κB信号通路可以对钛颗粒诱导下小鼠RAW264.7巨噬细胞体外骨溶解模型产生积极作用。

BACKGROUND:There are few domestic studies on the formation and differentiation of osteoclasts induced by wear particles by tetrandrine.OBJECTIVE:To explore the positive effect of tetrandrine on the osteolysis model of mouse RAW264.7 macrophages induced by titanium particles under different concentrations.METHODS:Mouse RAW264.7 macrophages were divided into five groups for culture:blank group,experimental group(0.1 g/L titanium particle suspension),and low,medium,and high concentration drug groups(component+0.1,0.5,and 1.0μmol/L tetrandrine).CCK-8 assay was used to detect the proliferative activity of RAW264.7.The anti-tartaric acid phosphatase staining method and the phalloidin staining method were used to detect the function and number of mature osteoclasts.The enzyme-linked immunosorbent assay was used to detect the cell supernatant expression of tumor necrosis factor-αand matrix metalloproteinase-9.RT-PCR was applied to detect mRNA expression of activated T cell nuclear factor c1,tumor necrosis factor-α,and matrix metalloproteinase 9.Western blot assay was utilized to detect the levels of phosphorylated proteins of nuclear factorκB p65 and IκBα.RESULTS AND CONCLUSION:(1)Compared with the blank group,the various concentrations of tetrandrine had no effect on the proliferation of RAW264.7 cells(P>0.05).(2)In the control group,more bigger,wine red,and F-actin ring stained with fluorescent green multinucleated osteoclasts were observed,and after treatment with different concentrations of tetrandrine,the number of osteoclast cells decreased significantly(P<0.05);and the size of F-actin ring decreased with the increase of drug concentration.(3)Compared with the blank group,IκBαand nuclear factorκB p65 and their phosphorylation levels,tumor necrosis factor-α,matrix metalloproteinase-9 cell culture supernatant protein expression,activated T cell nuclear factor c1 mRNA,tumor necrosis factor-αmRNA,matrix metalloproteinase-9 mRNA expression levels were significantly increased in the experimental group(P<0.05).Tetrandrine reduced the expression of the above indexes in a concentration-dependent manner.(4)In summary,tetrandrine can have a positive effect on the in vitro osteolysis model of mouse RAW264.7 macrophages induced by titanium particles by inhibiting the nuclear factorκB signaling pathway.