IL-38拮抗RhoA/ROCK信号抑制PDGF-BB诱导的血管平滑肌细胞增殖和迁移

IL-38 antagonizes RhoA/ROCK signaling to inhibit PDGF-BB-induced proliferation and migration of vascular smooth muscle cells

目的探究白细胞介素(IL)-38对血小板源性生长因子-BB(PDGF-BB)诱导的血管平滑肌细胞增殖和迁移的作用和机制。方法将人主动脉血管平滑肌细胞(HA-VSMC)随机分成4组:对照组(不进行任何处理)、PDGF-BB组(加入20 ng/ml重组人PDGF-BB蛋白处理24 h)、PDGF-BB+空质粒组(转染pcDNA3.1空质粒48 h后加入20 ng/ml重组人PDGF-BB蛋白处理24 h)和PDGF-BB+IL-38组(转染pcDNA3.1-IL-38质粒48 h后加入20 ng/ml重组人PDGF-BB蛋白刺激24 h)。MTT法检测细胞活力、Transwell检测细胞迁移、Western blot检测增殖细胞核抗原(PCNA)、RhoA、Rho相关蛋白激酶(ROCK)1、ROCK2、肌球蛋白磷酸酶靶标亚基1(MYPT1)和磷酸化MYPT(p-MYPT)蛋白水平的表达。结果与对照组相比,PDGF-BB组中细胞活力,迁移细胞数目,PCNA、RhoA、ROCK1、ROCK2的表达和p-MYPT/MYPT1的值均增加,差异均具有统计学意义(均P<0.05);与PDGFBB组相比,PDGF-BB+空质粒组中这些指标变化无统计学意义(均P>0.05);与PDGF-BB+空质粒组相比,PDGF-BB+IL-38组中细胞活力,迁移细胞数目,PCNA、RhoA、ROCK1、ROCK2的表达和p-MYPT/MYPT1的值均降低,差异均具有统计学意义(均P<0.05)。结论IL-38能够减弱PDGF-BB诱导的血管平滑肌细胞增殖和迁移,这种作用可能是通过抑制RhoA/ROCK通路活化发挥作用的。

This study was aimed to investigate the role and mechanism of interleukin(IL)-38 in the proliferation and migration of vascular smooth muscle cells induced by platelet-derived growth factor-BB(PDGFBB). Human aorta vascular smooth muscle cells(HA-VSMC) were randomly divided into 4 groups: the control group(without any treatment), PDGF-BB group(treatment with 20 ng/ml PDGF-BB recombinant human protein for 24 h),PDGF-BB + empty group(transfection with pcDNA3.1 plasmid for 48 h, followed by stimulation with 20 ng/ml PDGF-BB recombinant human protein for 24 h), and PDGF-BB+IL-38 group(transfection with pcDNA3.1-IL-38 plasmid for 48 h, followed by stimulation with 20 ng/ml PDGF-BB recombinant human protein for 24 h). The cell viability was determined by MTT assay. Transwell analysis was used to measure cell migration;Western blot was used to analyze the protein levels of proliferating cell nuclear antigen(PCNA), RhoA, Rho-associated protein kinase (ROCK)1, ROCK2, myosin phosphatase target subunit1(MYPT1), and phospho-MYPT1(p-MYPT1). Data showed that compared with the control group, PDGFBB group demonstrated significantly elevation of cell viability, migrated cell numbers, the expression of PCNA, RhoA, ROCK1 and ROCK2, and p-MYPT/MYPT1(all P<0.05), which did not different with those in the PDGF-BB + empty group(all P>0.05). Compared with the PDGF-BB+ empty group, cell viability, migrated cell numbers, the expression of PCNA, RhoA, ROCK1 and ROCK2, and pMYPT/MYPT1 were all markedly reduced in the PDGF-BB+IL-38 group(all P<0.05). In conclusion, IL-38 can attenuate PDGF-BB-induced proliferation and migration of VSMC cells, which may relate with the inactivation of RhoA/ROCK signaling pathway.