Yin Qianran, Jiang Dehua, Li Lei, Yang Yu, Wu Pei, Luo Yuanyuan, Yang Rongli, Li Dongye
Institute of Cardiovascular Disease Research, Xuzhou Medical University, Xuzhou, China.
Department of Cardiology, Xuzhou, China.
Cell Physiol Biochem. 2017;44(6):2189-2200. doi: 10.1159/000486024. Epub 2017 Dec 13.
BACKGROUND/AIMS: Lipopolysaccharide (LPS) is a potent activator of vascular smooth muscle cells (VSMCs) proliferation, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) and Ras-related C3 botulinum toxin substrate 1 (Rac1) expression using small interfering RNA (siRNA) in order to investigate the effects and possible mechanisms of LPS-induced VSMCs proliferation.
VSMCs proliferation was monitored by 5-ethynyl-2'-deoxyuridine staining, and Rac1 activity was measured via Glutathione S-transferase pull-down assay. mRNAs encoding proliferating cell nuclear antigen (PCNA), smooth muscle 22α (SM22α), myosin heavy chain (MYH) and transient receptor potential channel 1 (TRPC1) were detected by qRT-PCR. The expression of total Akt, p-Akt (308), p-Akt (473), SM22α, MYH and TRPC1 protein was analysed by Western blot.
Treatment with TLR4 siRNA (siTLR4) or Rac1 siRNA (siRac1) significantly decreased LPS-induced VSMCs proliferation. Moreover, LPS-induced activation of Rac1 through TLR4 was observed. Western blot analysis revealed that transfection with siTLR4 or siRac1 inhibited LPS-induced Akt phosphorylation. We discovered that LPS stimulated VSMCs proliferation via phenotypic modulation and that this effect was partially inhibited by pre-treatment with siTLR4 or siRac1. Further, TLR4 and Rac1 are involved in LPS-induced activation of TRPC1.
This study suggests that LPS exerts an effect on VSMCs proliferation and that the TLR4/Rac1/Akt signalling pathway mediates this effect.
背景/目的：脂多糖（LPS）是血管平滑肌细胞（VSMCs）增殖的强效激活剂，但其潜在机制仍不清楚。在本研究中，我们使用小干扰RNA（siRNA）敲低Toll样受体4（TLR4）和Ras相关C3肉毒杆菌毒素底物1（Rac1）的表达，以研究LPS诱导的VSMCs增殖的影响及可能机制。
通过5-乙炔基-2'-脱氧尿苷染色监测VSMCs增殖，并通过谷胱甘肽S-转移酶下拉试验测量Rac1活性。通过qRT-PCR检测编码增殖细胞核抗原（PCNA）、平滑肌22α（SM22α）、肌球蛋白重链（MYH）和瞬时受体电位通道1（TRPC1）的mRNA。通过蛋白质印迹分析总Akt、p-Akt（308）、p-Akt（473）、SM22α、MYH和TRPC1蛋白的表达。
用TLR4 siRNA（siTLR4）或Rac1 siRNA（siRac1）处理显著降低了LPS诱导的VSMCs增殖。此外，观察到LPS通过TLR4诱导Rac1激活。蛋白质印迹分析显示，用siTLR4或siRac1转染可抑制LPS诱导的Akt磷酸化。我们发现LPS通过表型调节刺激VSMCs增殖，并且该作用被siTLR4或siRac1预处理部分抑制。此外，TLR4和Rac1参与LPS诱导的TRPC1激活。
本研究表明LPS对VSMCs增殖有影响，并且TLR4/Rac1/Akt信号通路介导了这种影响。
