Song Wenjie, Li Ling, Jia Qihua, Cao Nan, Li Li, Ma Ketao, Si Junqiang
Ministry-of-Education Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Department of Physiology, Shihezi University, Shihezi 832000, China.
Department of Traumatology, First Affiliated Hospital, Shihezi University, Shihezi 832000, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2020 Jun;36(6):527-534.
Objective To explore the effects of A549 cells on the proliferation and migration of human pulmonary arterial smooth muscle cells (HPASMCs) and its mechanism. Methods A549 cells and HPASMCs were cultured in vitro. The A549 cells were randomly divided into four groups: control group, dimethylformamide (DMF) solvent group, monocrotaline pyrrole (MCTP) group, MCTP combined with SB431542 group. The cells were assigned into four groups: HPASMC group, A549 and HPASMC co-culture group, MCTP-stimulated A549 and HPASMC co-culture group, MCTP and SB431542-stimulated A549 and HPASMC co-culture group, and IL-6-stimulated HPASMC group. A549 cell viability was detected by CCK-8 assay. The level of IL-6 in the A549 cell culture supernatant was tested by ELISA. The mRNA levels of SMAD2 and SMAD3 in the A549 cells were detected by real-time PCR. The protein levels of TGF-β1, SMAD2, SMAD3 and p-SMAD2, p-SMAD3 in the A549 cells were detected by Western blot analysis. The protein levels of TGF-β1, SMAD2, SMAD3 and p-SMAD2, p-SMAD3 in the A549 cells were examined by Western blot analysis. The protein levels of osteopontin (OPN) and proliferating nuclear antigen (PCNA) in the HPASMCs were determined by Western blot analysis. The migration ability of HPASMCs was measured by wound healing and Transwell assay. Results In the A549 cells, compared with the control group, the cell proliferation ability decreased, the production of IL-6 increased, the mRNA levels of SMAD2 and SMAD3, and the expression of TGF-β1, SMAD2, SMAD3, p-SMAD2, p-SMAD3 proteins significantly increased in the MCTP group. Compared with the MCTP group, the cell proliferation ability increased, the production of IL-6 decreased, the mRNA levels of SMAD 2 and SMAD3, and the expression of TGF-β1, SMAD2, SMAD3, p-SMAD2, p-SMAD3 proteins significantly decreased in the MCTP and SB431542-stimulated group. In the co-culture system, compared with the HPASMC group, the expression of PCNA and OPN proteins and migration ability did not change significantly in the A549 and HPASMC co-cultured group. The expression of PCNA and OPN proteins significantly increased in the MCTP-stimulated A549 and HPASMC co-culture group, and the cell migration ability increased. Compared with the MCTP-stimulated A549 and HPASMC co-culture group, the expression of PCNA and OPN proteins significantly decreased in MCTP and SB431542-stimulated A549 and HPASMC co-culture group, and the cell migration ability decreased. Compared with the HPASMC group, the migration ability of HPASMCs increased and the expression of PCNA and OPN proteins increased in the IL-6 control group. Conclusion Activation of the TGF-β1/SMAD2/SMAD3 signaling pathway in A549 cells induced by MCTP increases IL-6 secretion, thus promoting the proliferation and migration of HPASMCs.
目的 探讨A549细胞对人肺动脉平滑肌细胞（HPASMCs）增殖和迁移的影响及其机制。方法 体外培养A549细胞和HPASMCs。将A549细胞随机分为四组：对照组、二甲基甲酰胺（DMF）溶剂组、野百合碱吡咯（MCTP）组、MCTP联合SB431542组。将细胞分为四组：HPASMC组、A549与HPASMC共培养组、MCTP刺激的A549与HPASMC共培养组、MCTP和SB431542刺激的A549与HPASMC共培养组以及IL-6刺激的HPASMC组。采用CCK-8法检测A549细胞活力。采用ELISA法检测A549细胞培养上清液中IL-6水平。采用实时PCR法检测A549细胞中SMAD2和SMAD3的mRNA水平。采用蛋白质印迹分析法检测A549细胞中TGF-β1、SMAD2、SMAD3以及p-SMAD2、p-SMAD3的蛋白水平。采用蛋白质印迹分析法检测HPASMCs中骨桥蛋白（OPN）和增殖细胞核抗原（PCNA）的蛋白水平。采用划痕愈合实验和Transwell实验检测HPASMCs的迁移能力。结果 在A549细胞中，与对照组相比，MCTP组细胞增殖能力下降，IL-6产生增加，SMAD2和SMAD3的mRNA水平以及TGF-β1、SMAD2、SMAD3、p-SMAD2、p-SMAD3蛋白的表达显著增加。与MCTP组相比，MCTP和SB431542刺激组细胞增殖能力增加，IL-6产生减少，SMAD2和SMAD3的mRNA水平以及TGF-β1、SMAD2、SMAD3、p-SMAD2、p-SMAD3蛋白的表达显著降低。在共培养体系中，与HPASMC组相比，A549与HPASMC共培养组PCNA和OPN蛋白的表达及迁移能力无明显变化。MCTP刺激的A549与HPASMC共培养组PCNA和OPN蛋白的表达显著增加，细胞迁移能力增强。与MCTP刺激的A549与HPASMC共培养组相比，MCTP和SB431542刺激的A549与HPASMC共培养组PCNA和OPN蛋白的表达显著降低，细胞迁移能力减弱。与HPASMC组相比，IL-6刺激组HPASMCs的迁移能力增强，PCNA和OPN蛋白的表达增加。结论 MCTP诱导的A549细胞中TGF-β1/SMAD2/SMAD3信号通路激活增加IL-6分泌，从而促进HPASMCs的增殖和迁移。
