DNA损伤应答通路在高钙磷环境诱导的人主动脉血管平滑肌细胞钙化中的作用

The role of DNA damage response pathway in HVSMC calcification induced by high calcium and phosphorus

目的 明确DNA损伤应答通路在高钙磷环境诱导的人主动脉血管平滑肌细胞(HVSMC)钙化过程中的作用。方法 将HVSMC培养分为对照组、模型组、共济失调毛细血管扩张突变激酶(iATM)组、聚腺苷二磷酸核糖聚合酶(iPARP)组,培养12 d。茜素红-S染色法定性和邻-甲酚酞法定量检测4组细胞钙化情况,彗星实验检测DNA损伤,Western blotting和免疫荧光方法检测组蛋白γH2AX磷酸化水平,酶联免疫吸附试验检测8-羟基-2’-脱氧鸟苷(8-OHDG)水平,NucleoCounterNC-3000^(TM)高级细胞分析仪分析4组细胞的存活率。结果 光学显微镜和茜素红S染色发现第9天开始,与对照组相比,模型组出现细胞内钙质沉积,第12天钙质沉积明显。对照组与模型组分别在第3、6、9、12天培养状态下Ca^(2+)/蛋白比较,结果:(1)不同时间点Ca^(2+)/蛋白有差异(F=168.970,P=0.000);(2)模型组与对照组Ca^(2+)/蛋白有差异(F=203.040,P=0.000),模型组Ca^(2+)/蛋白较高,钙化明显;(3)两组Ca^(2+)/蛋白变化趋势有差异(F=13.213,P=0.000)。培养12 d时,茜素红S染色发现模型组比对照组钙化程度高,iATM组和iPARP组比模型组钙化程度低。σ-甲酚酞试验发现,iATM组和iPARP组Ca^(2+)/蛋白低于模型组(P <0.05)。彗星试验发现,对照组比较,模型组第9天开始出现更多数量的DNA受损细胞。对照组与模型组分别在第3、6、9、12天培养状态下“彗星细胞”比较,结果:(1)不同时间点“彗星细胞”有差异(F=13.141,P=0.000);(2)模型组与对照组“彗星细胞”有差异(F=121.521,P=0.000),模型组“彗星细胞”百分比较高,DNA损伤明显;(3)模型组与对照组“彗星细胞”变化趋势有差异(F=89.290,P=0.000)。模型组γH2AX蛋白相对表达量高于对照组(P <0.05)。对照组、模型组分别在第3和12天免疫荧光显微镜下观察> 3个γH2AX病灶百分比,结果:(1)不同时间点> 3个γH2AX病灶百分比有差异(F=168.970,P=0.000);(2)模型组与对照组> 3个γH2AX病灶百分比有差异(F=203.040,P=0.000),模型组> 3个γH2AX病灶百分比较高,DNA损伤明显;(3)模型组与对照组> 3个γH2AX病灶百分比变化趋势有差异(F=153.410,P=0.000)。模型组8-OHDG水平高于对照组(P <0.05)。模型组细胞存活率低于对照组、iATM组、iPARP组(P <0.05);iATM组、iPARP组与对照组细胞存活率比较,差异无统计学意义(P>0.05)。结论 高Ca^(2+)/P环境激活DNA损伤应答信号通路,诱导HVSMC坏死,进而形成钙化。

Objective To determine the role of the DNA damage response(DDR) pathway in calcification of human vascular smooth muscle cell(HVSMC) induced by high calcium and phosphorus.Methods HVSMCs were divided into the control group,model group,ataxia-telangiectasia mutated inhibitor(iATM) group,and poly(ADPribose) polymerase inhibitor(iPARP) group,and were cultured for 12 days.Qualitative and quantitative analyses of HVSMC calcification in the four groups were performed via Alizarin red S staining and o-Cresolphthalein chromogenic method,respectively.The DNA damage was measured via the comet assay.The phosphorylation level of histone γH2AX was detected by Western blotting and immunofluorescence,while the level of 8-hydroxy-2'-deoxyguanosine(8-OHDG) was detected by enzyme-linked immunosorbent assay(ELISA).Besides,the cell viability of the four groups was analyzed by Nucleo Counter (R) NC-3000^(TM) Advanced Cell Analyzer.Results From the 9th day on,intracellular calcium deposition as shown under the light microscope and by the Alizarin red S staining occurred in the model group in comparison to the control group,and the calcium deposition was obvious on the 12th day.The ratio of the level of calcium to that of proteins on the 3rd,6th,9th,and 12th day was compared between the control group and the model group,which suggested that the ratio of the level of calcium to that of proteins was different among the time points(F = 168.970,P = 0.000) and between the groups(F = 203.040,P =0.000),where the ratio of the level of calcium to that of proteins and the degree of calcification were greater in the model group.In addition,the change trends of the ratio of the level of calcium to that of proteins was different between the two groups(F =13.213,P = 0.000).On the 12th day of culture,the degree of calcification as shown by the Alizarin red S staining was higher in the model group than in the control group,whereas the degree of calcification in the iATM group and the iPARP group was lower than that in the model group.The calcium assay via the o-Cresolphthalein chromogenic method demonstrated that the ratio of the level of calcium to that of proteins was lower in the iATM group and the iPARP group than in the model group(P<0.05).The comet assay found that there were greater numbers of cells bearing DNA damage in the model group than in the control group since the 9th day.The percentage of cells bearing DNA damage on the 3rd,6th,9th,and 12th day of culture was compared between the control group and the model group,and the results exhibited that the percentage of cells bearing DNA damage was different among the time points(F = 13.141,P = 0.000) and between the groups(F = 121.521,P = 0.000).Specifically,the percentage of cells bearing DNA damage was even higher in the model group.Besides,the change trends of the percentage of cells bearing DNA damage were different between the two groups(F =89.290,P =0.000).The relative protein expression of γH2AX in the model group was higher than that in the control group(P<0.05).The comparison of the percentage of 3 γH2AX-positive lesions under the fluorescence microscope on the 3rd and 12th day in the control group and the model group showed that the percentage of >3 γH2AX-positive lesions under the fluorescence microscope was different between the time points(F =168.970,P =0.000) and between the groups(F =203.040,P = =0.000),in which the percentage of >3 γH2AX-positive lesions under the fluorescence microscope was even higher in the model group that suggested obvious DNA damage.The change trends of the percentage of >3 γH2AX-positive lesions under the fluorescence microscope were different between the model group and the control group(F = 153.410,P = 0.000).The level of 8-OHDG in the model group was higher than that of the control group(P<0.05).The cell viability in the model group was lower than that in the iATM group and the iPARP group(P<0.05),while the cell viability in the control group was not different from that in the iATM group and the iPARP group(P>0.05).Conclusions High calcium and phosphorus may activate the DNA damage response signaling pathway and induce necrosis of HVSMC which in turn causes HVSMC calcification.