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目的探讨Toll样受体4(Toll-like receptor 4,TLR4)/核转录因子-κB(nuclear transcription factorκB,NF-κB)信号通路对高磷诱导小鼠血管平滑肌细胞(vascular smooth muscle cells,VSMCs)成骨样分化以及血管钙化过程的影响。方法体外培养小鼠血管平滑肌细胞系。首先将实验分为正常对照组和高磷组,观察高磷对VSMCs的影响;其次分为正常对照组、NF-κB抑制剂吡咯烷二硫代氨基甲酸酯(pyrrolidine dithiocarbamate,PDTC)组(PDTC组)、高磷组和高磷+PDTC组,观察PDTC对高磷诱导的VSMCs成骨样分化及钙化的抑制作用;最后分为正常对照组、正常对照+control siRNA组、正常对照+TLR4 siRNA组、高磷组、高磷+control siRNA组、高磷+TLR siRNA组,观察TLR4基因的小分子干扰RNA(siRNA)对高磷诱导的VSMCs成骨样分化及钙化的抑制作用。采用茜素红S染色观察高磷对细胞钙盐沉积的影响,Western blot检测TLR4、p-NF-κB、SM22α、骨形态发生蛋白2(bone morphogenic protein2,BMP2)、Runt相关转录因子2(runt-related transcription factor 2,Runx2)的表达变化。结果与正常对照组相比,高磷处理后,VSMCs的钙盐沉积明显增多[(0.079±0.011)vs(0.193±0.017),P<0.01],TLR4[(0.228±0.008)vs(0.526±0.017),P<0.01]、p-NF-κB[(0.271±0.003)vs(0.454±0.008),P<0.01]蛋白的表达明显升高,与成骨样分化及钙化相关的BMP2、Runx2蛋白表达亦明显升高,而平滑肌细胞标志物SM22α表达则减少;加入PDTC处理后,高磷状态下的VSMCs的钙盐沉积明显降低[(0.121±0.013)vs(0.217±0.031),P<0.01],p-NF-κB[(0.094±0.006)vs(0.380±0.009),P<0.01]表达明显受抑制,BMP2和RUNX2表达明显下降,而SM22α的表达则明显上调;高磷状态下,应用TLR4 siRNA转染VSMCs后,VSMCs的TLR4[(0.117±0.047)vs(0.377±0.012),P<0.01]表达明显受抑制,同样,p-NF-κB[(0.264±0.013)vs(0.601±0.027),P<0.01]的表达亦明显下降,BMP2和Runx2(P<0.01)蛋白的表达明显受抑,而SM22α[(0.321±0.011)vs(0.194±0.004),P<0.01]的表达则明显上升。结论高磷可能通过TLR4/NF-κB信号通路诱导VSMCs成骨样分化及钙化。
Objective To investigate the effects of Toll-like receptor 4 (TLR4) / nuclear transcription factor κB (NF-κB) signaling pathway on vascular smooth muscle cells (VSMCs) Osteogenic differentiation and vascular calcification process. Methods Mouse vascular smooth muscle cell line was cultured in vitro. First, the experiment was divided into normal control group and high-phosphorus group, to observe the effect of high-phosphorus on VSMCs; second, divided into normal control group, pyrrolidine dithiocarbamate (PDTC) PDTC group), high phosphorus group and high phosphorus + PDTC group. The inhibitory effect of PDTC on osteogenic differentiation and calcification of VSMCs induced by high phosphorus was observed. Finally, the cells were divided into normal control group, control + control siRNA group and normal control + TLR4 siRNA group, high phosphorus group, high phosphorus + control siRNA group and high phosphorus + TLR siRNA group. The inhibitory effect of TLR4 siRNA on osteoblastic differentiation and calcification of VSMCs was observed. Alizarin red S staining was used to observe the effect of high P on the deposition of calcium in cells. Western blot was used to detect the expressions of TLR4, p-NF-κB, SM22α, bone morphogenic protein 2 (BMP2), Runt-related transcription factor 2 -related transcription factor 2, Runx2). Results Compared with the normal control group, calcium deposition significantly increased in VSMCs after high phosphorus treatment [(0.079 ± 0.011) vs (0.193 ± 0.017), P <0.01], and TLR4 [(0.228 ± 0.008) vs (0.526 ± 0.017 ), P <0.01]. The protein expression of p-NF-κB [(0.271 ± 0.003) vs (0.454 ± 0.008), P <0.01] was significantly increased. The expression of BMP2 and Runx2 were correlated with osteogenic differentiation and calcification (0.121 ± 0.013) vs (0.217 ± 0.031), P <0.01], and the level of SM22α in VSMCs was significantly decreased after PDTC treatment The expression of p-NF-κB [(0.094 ± 0.006) vs (0.380 ± 0.009, P <0.01] was significantly inhibited, the expression of BMP2 and RUNX2 was significantly decreased, while the expression of SM22α was significantly increased; The expression of TLR4 [(0.117 ± 0.047) vs (0.377 ± 0.012), P <0.01] in VSMCs was significantly inhibited after transfection of VSMCs. Similarly, the expressions of p-NF-κB [(0.264 ± 0.013) vs P <0.01], while the expression of BMP2 and Runx2 (P <0.01) was significantly inhibited. However, the expression of SM22α [(0.321 ± 0.011) vs (0.194 ± 0.004), P <0.01] was significantly increased. Conclusion High phosphorus may induce osteoblastic differentiation and calcification of VSMCs through TLR4 / NF-κB signaling pathway.