Ding L, Xu Y, Yang P, Chen R, Li B, Shao C, Zhong W, Wang Z Q, Yan J C
Department of Cardiology, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2018 Nov 24;46(11):892-900. doi: 10.3760/cma.j.issn.0253-3758.2018.11.014.
To explore whether CD137-CD137L interaction could induce mouse vascular smooth muscle cells(VSMCs) calcification via P38 MAPK signaling. (1) Mouse VSMCs obtained from 8-week old male C57 mice were cultured by using method of tissue piece inoculation.The cells from 3 to 8 passage were divided into 4 groups: control group, agonist-CD137 group(recombinant CD137L protein), anti-P38 group(agonist-CD137 group+P38 inhibitor), single anti-P38 group(P38 inhibitor). The calcification was induced by adding a mixture of 10 mmol/L β-glycerophosphate+10(-8) mol/L dexamethasone+10(-7) mol/L insulin in the culture medium.Immunofluorescence was used to observe the changes of VSMCs markers(α-SMA and OPN).Real time-PCR was used to observe the mRNA expression of OPN and RUNX-2. Western blot was used to observe the protein expression of p-P38, OPN and RUNX-2. The level of cell calcification was observed by detecting alkaline phosphatase activity and calcium concentration. (2) The degeree of local calcium deposition was also tested on Von Kossa staining and Alizarin red staining methods in following 5 mouse VSMCs groups: control group, agonist-CD137 group(recombinant CD137L protein), anti-P38 group (agonist-CD137 group+P38 inhibitor), anti-CD137 group (agonist-CD137 group+CD137 inhibitor),agonist-P38 group(anti-CD137 group+P38 agonist). (1) Compared with the control group, the fluorescence intensity of α-SMA was lower in the agonist-CD137 group(2.79±0.25 vs. 5.42±0.47,0.05), while the fluorescence intensity of OPN was higher(4.91±0.23 vs. 1.63±0.26, 0.05). The fluorescence intensity of α-SMA was partly recovered after adding P38 inhibitor(4.48±0.27 vs. 2.79±0.25,0.05),but it was still lower than the control group (4.48±0.27 vs. 5.42±0.47, 0.05),the fluorescence intensity of OPN decreased(2.66±0.15 vs. 4.91±0.23,0.05),but it was still higher than that in the control group (2.66±0.15 vs. 1.63±0.26,0.05).The fluorescence intensity of α-SMA and OPN(5.32±0.67 vs. 5.42±0.47,1.82±0.30 vs.1.63±0.26,both 0.05) was similar between the control group and single anti-P38 group.(2) Compared with the control group, the protein level of p-P38(4.15±0.24 vs. 3.48±0.26, 0.05), OPN(2.43±0.21 vs. 1.53±0.08, 0.05), RUNX-2(3.20±0.23 vs. 1.13±0.10, 0.05) was significantly increased in agonist-CD137 group,the above effects were blocked by adding specific P38 inhibitor SB203580(1.16±0.12 vs. 4.15±0.24, 0.50±0.02 vs. 2.43±0.21,and 1.74±0.14 vs. 3.20±0.23,all 0.05);the protein level of p-P38(2.93±0.60 vs. 3.48±0.26,0.05),OPN (1.4±0.64 vs. 1.53±0.08,0.05),RUNX-2(1.26±0.26 vs.1.13±0.10, 0.05) was similar between single anti-P38 group and the control group. (3) Compared with the control group, the mRNA level of OPN (1.51±0.34 vs. 1, 0.05) and RUNX-2(2.67±0.19 vs. 1, 0.05) was significantly upregulated in agonist-CD137 group, and these effects were blocked by adding specific P38 inhibitor SB203580(0.33±0.14 vs. 1 and 0.45±0.03 vs. 1,0.05);the mRNA level of OPN (1.05±0.09 vs. 1, 0.05) and RUNX-2(1.18±0.10 vs. 1, 0.05) was similar between the single anti-P38 group and the control group.(4) Compared with the control group,the ALP activity and calcium concentration(2.40±0.25 vs. 1.40±0.21,5.51±0.33 vs. 3.15±0.31,both 0.05) were significantly increased in agonist-CD137 group,while the effects could be blocked by adding specific P38 inhibitor SB203580((1.99±0.07) king unit/gprot vs. (2.40±0.25) king unit/gprot, (3.74±0.20) mmol/gprot vs. (5.51±0.33) mmol/gprot, both 0.05).The ALP activity and calcium concentration was similar between single anti-P38 group and the control group((1.60±0.25) king unit/gprot vs. (1.40±0.21)king unit/gprot, (2.66±0.28) mmol/gprot vs. (3.15±0.31) mmol/gprot, both 0.05). (5) Compared with the control group,the calcification of VSMCs in the agonist-CD137 group was significantly increased,while the calcification in the anti-P38 group was significantly reduced.Compared with the agonist-CD137 group,the level of calcification in the anti-CD137 group was obviously increased,and the calcification in the agonist-P38 group was significantly higher than that in the anti-CD137 group and the control group. These findings suggest that CD137-CD137L signaling may regulate VSMCs calcification via modulating P38 pathway.
探讨CD137-CD137L相互作用是否可通过P38丝裂原活化蛋白激酶（MAPK）信号通路诱导小鼠血管平滑肌细胞（VSMCs）钙化。（1）采用组织块接种法培养8周龄雄性C57小鼠的VSMCs。将第3至8代细胞分为4组：对照组、激动剂-CD137组（重组CD137L蛋白）、抗P38组（激动剂-CD137组+P38抑制剂）、单抗P38组（P38抑制剂）。通过在培养基中添加10 mmol/Lβ-甘油磷酸+10（-8）mol/L地塞米松+10（-7）mol/L胰岛素的混合物诱导钙化。采用免疫荧光法观察VSMCs标志物（α-平滑肌肌动蛋白（α-SMA）和骨桥蛋白（OPN））的变化。采用实时定量聚合酶链反应（Real time-PCR）观察OPN和RUNX-2的mRNA表达。采用蛋白质免疫印迹法（Western blot）观察磷酸化P38（p-P38）、OPN和RUNX-2的蛋白表达。通过检测碱性磷酸酶活性和钙浓度观察细胞钙化水平。（2）采用冯科萨（Von Kossa）染色法和茜素红染色法检测以下5组小鼠VSMCs的局部钙沉积程度：对照组、激动剂-CD137组（重组CD137L蛋白）、抗P38组（激动剂-CD137组+P38抑制剂）、抗CD137组（激动剂-CD137组+CD137抑制剂）、激动剂-P38组（抗CD137组+P38激动剂）。（1）与对照组相比，激动剂-CD137组α-SMA的荧光强度较低（2.79±0.25比5.42±0.47，P<0.05），而OPN的荧光强度较高（4.91±0.23比1.63±0.26，P<0.05）。添加P38抑制剂后，α-SMA的荧光强度部分恢复（4.48±0.27比2.79±0.25，P<0.05），但仍低于对照组（4.48±0.27比5.42±0.47，P<0.05），OPN的荧光强度降低（2.66±0.15比4.91±0.23，P<0.05），但仍高于对照组（2.66±0.15比1.63±0.26，P<0.05）。对照组和单抗P38组之间α-SMA和OPN的荧光强度相似（5.32±0.67比5.42±0.47，1.82±0.30比1.63±0.26，均P>0.05）。（2）与对照组相比，激动剂-CD137组p-P38（4.15±0.24比3.48±0.26，P<0.05）、OPN（2.43±0.21比1.53±0.08，P<0.05）、RUNX-2（3.20±0.23比1.1
