Hu Feng, Liu Xin-Xin, Wang Xin, Alashkar Mohammad, Zhang Song, Xu Jun-Tao, Zhong Xue-Lian, He Meng-Wen, Feng Ai-Ping, Chen Hong-Xiang
Department of Dermatology, Wuhan No. 1 Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
J Dermatol Sci. 2015 Jun;78(3):181-8. doi: 10.1016/j.jdermsci.2015.03.009. Epub 2015 Mar 21.
Current in vitro studies show that lipoxin A4 (LXA4) has multiple biological functions including inhibiting cell proliferation and inflammatory cytokine production. Our previous studies showed LXA4 could inhibit the expression of IL-6 and IL-8 in normal human epidermal keratinocytes (NHEKs). However, more specific effects including regulation of cell proliferation and anti-inflammatory mechanisms of LXA4 in NHEKs have not been previously studied.
We proposed to investigate the effects of LXA4 on cell proliferation and inflammatory cytokine/chemokine production in NHEKs, and the possible molecular mechanisms of cell cycle and anti-inflammatory signal transduction pathway.
NHEKs were stimulated with LPS, with or without preincubation with LXA4. Cell proliferation and cell cycle of NHEKs were examined by WST-8, CFSE assay and DNA staining, respectively. The mRNA and protein levels of inflammatory cytokines were quantified by real-time quantitative PCR and ELISA. The expressions of signaling proteins cyclin D1, P16INK4A, ERK1/2 and NF-κB-p65 were analyzed using Western blotting.
Cell proliferation and inflammatory cytokine/chemokine production of NHEKs were suppressed by LXA4, which caused G0/G1 phase cell cycle arrest in NHEKs. The expression of cyclin D1 was down-regulated by LXA4, contrary to the results of P16INK4A. The ERK1/2 phosphorylation and NF-κB-p65 nuclear translocation of NHEKs were both suppressed by LXA4.
Cell growth and inflammatory cytokine/chemokine production of NHEKs were inhibited by LXA4, and the inhibitory effects might be associated with the mechanisms of cyclin D1/P16INK4A, ERK1/2 and NF-κB signal transduction pathway.
目前的体外研究表明，脂氧素A4（LXA4）具有多种生物学功能，包括抑制细胞增殖和炎性细胞因子的产生。我们之前的研究表明，LXA4可抑制正常人表皮角质形成细胞（NHEK）中白细胞介素-6（IL-6）和白细胞介素-8（IL-8）的表达。然而，LXA4在NHEK中对细胞增殖的调控及抗炎机制等更具体的作用此前尚未见研究报道。
研究LXA4对NHEK细胞增殖及炎性细胞因子/趋化因子产生的影响，以及细胞周期和抗炎信号转导通路的可能分子机制。
用脂多糖（LPS）刺激NHEK，LXA4预处理或不预处理。分别采用WST-8法、羧基荧光素二醋酸盐琥珀酰亚胺酯（CFSE）检测法及DNA染色法检测NHEK的细胞增殖和细胞周期。采用实时定量聚合酶链反应（PCR）和酶联免疫吸附测定（ELISA）法检测炎性细胞因子的mRNA和蛋白水平。采用蛋白质印迹法分析细胞周期蛋白D1（cyclin D1）、P16INK4A、细胞外信号调节激酶1/2（ERK1/2）和核因子κB p65（NF-κB-p65）信号蛋白的表达。
LXA4抑制NHEK的细胞增殖及炎性细胞因子/趋化因子的产生，导致NHEK细胞停滞于G0/G1期。LXA4使cyclin D1表达下调，而P16INK4A的结果则相反。LXA4抑制NHEK的ERK1/2磷酸化及NF-κB-p65核转位。
LXA4抑制NHEK的细胞生长及炎性细胞因子/趋化因子的产生，其抑制作用可能与cyclin D1/P16INK4A、ERK1/2及NF-κB信号转导通路机制有关。
