Zhang Xin-cai, Chen Jin-qin, Li Bo
*Department of Cardiology, Weifang People's Hospital, Weifang, People's Republic of China; †Department of Cardiology, Central Hospital of Zibo, Zibo, People's Republic of China; and ‡Shandong University, Jinan, People's Republic of China.
J Cardiovasc Pharmacol. 2014 Oct;64(4):318-25. doi: 10.1097/FJC.0000000000000117.
The CC chemokine ligand-20 (CCL-20)/macrophage inflammatory protein-3α has been seen as one of the most important chemokines and played a key role in atherogenesis, but the mechanism that underlies the regulation of CCL-20 has not been established clearly yet. The aim of this study was to investigate the influence of salvianolic acid A (SAA) on the expression of CCL-20 in macrophages and ApoE-deficient (ApoE) mice.
The expression of CCL-20 was detected both at protein and messenger RNA levels in RAW264.7 cells. We validated the result in ApoE mice that were intraperitoneally injected with SAA. Phosphorylation of p38 mitogen-activated protein kinase was detected with Western blot, and inhibitor of p38 was used to investigate the mechanism of regulation of CCL-20. Hematoxylin and eosin and Oil-Red-O staining were used to evaluate the atherosclerotic lesions and lipid accumulation in ApoE mice. Immunohistochemical analysis was used to detect the expressions of CCL-20 and CCR6 in the atherosclerotic lesions. Immunofluorescent analysis was used to certify the origination of CCL-20.
Recombinant tumor necrosis factor-α (TNF-α) upregulated CCL-20 production in dose- and time-dependent manners in RAW264.7 cells. The activity of TNF-α-induced CCL-20 production seemed to be significantly suppressed by SAA. Using p38 mitogen-activated protein kinase inhibitor, we found that p38 mediated the effects of TNF-α- and SAA-induced CCL-20 expression changes. In addition, immunohistochemical analysis of aortic root of ApoE mice also demonstrated that the expressions of CCL-20 and CCR6 were both downregulated significantly with SAA treatment. Furthermore, treatment of SAA inhibited the progression of the atherosclerotic plaques and lipid accumulation.
These results demonstrate that TNF-α increased but SAA suppressed CCL-20 production significantly via a novel mechanism.
CC趋化因子配体20（CCL-20）/巨噬细胞炎性蛋白-3α被视为最重要的趋化因子之一，在动脉粥样硬化形成中起关键作用，但CCL-20的调控机制尚未完全明确。本研究旨在探讨丹酚酸A（SAA）对巨噬细胞和载脂蛋白E缺陷（ApoE）小鼠中CCL-20表达的影响。
在RAW264.7细胞中检测CCL-20在蛋白质和信使RNA水平的表达。我们在腹腔注射SAA的ApoE小鼠中验证了该结果。用蛋白质印迹法检测p38丝裂原活化蛋白激酶的磷酸化，并使用p38抑制剂研究CCL-20的调控机制。用苏木精和伊红染色以及油红O染色评估ApoE小鼠的动脉粥样硬化病变和脂质蓄积。用免疫组织化学分析检测动脉粥样硬化病变中CCL-20和CCR6的表达。用免疫荧光分析确定CCL-20的来源。
重组肿瘤坏死因子-α（TNF-α）以剂量和时间依赖性方式上调RAW264.7细胞中CCL-20的产生。SAA似乎能显著抑制TNF-α诱导的CCL-20产生活性。使用p38丝裂原活化蛋白激酶抑制剂，我们发现p38介导了TNF-α和SAA诱导的CCL-20表达变化的效应。此外，对ApoE小鼠主动脉根部的免疫组织化学分析也表明，SAA处理后CCL-20和CCR6的表达均显著下调。此外，SAA治疗可抑制动脉粥样硬化斑块的进展和脂质蓄积。
这些结果表明，TNF-α通过一种新机制显著增加CCL-20的产生，而SAA则抑制其产生。
