Yang Yaru, Wang He, Lv Xiongwen, Wang Qi, Zhao Han, Yang Feng, Yang Yan, Li Jun
School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province, China; Department of Pharmacy, The Second Hospital of Anhui Medical University, Hefei, Anhui Province, China.
School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province, China; Institute for Liver Disease of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province, China.
Biochimie. 2015 Aug;115:59-70. doi: 10.1016/j.biochi.2015.04.019. Epub 2015 May 6.
The present study was undertaken to investigate the mechanism by which adenosine receptors (ARs)-mediated the cAMP/PKA/CREB signal pathway regulates the activation of acetaldehyde-induced hepatic stellate cells (HSCs). Primary HSCs were isolated from SD rats, cultured in vitro, and activated with different concentrations of acetaldehyde at different time points. Quantitative real-time PCR and Western blotting were used to quantify both protein and mRNA levels of the four AR (A1R, A2AR, A2BR, and A3R) in rat HSCs. Selective inhibitors of PDEs and the Gi/o protein pathway, general AR agonists, and AR subtype specific agents were used to study the AR signaling. The level of cAMP was measured by radio-immunoassay, and the expression of α-SMA, collagen type I and III, PKA and p-CREB were also detected by Western blotting. Acetaldehyde could significantly promote HSC proliferation, with a maximum stimulatory effect observed at 48 h after exposure to 200 μM acetaldehyde. All four AR subtypes could be present in rat HSCs, and the mRNA and protein expression levels for A2AR and A1R in much greater abundance than those for A2BR and A3R. The expression of A2AR and A1R was significantly increased in acetaldehyde-induced HSCs as compared with that of control group, whereas the expression of A2BR and A3R remained unaffected by the addition of acetaldehyde. Curiously, there is coupling of A2AR to the Gs-AC signaling, as well as coupling of A1R to the Gi/o-AC signaling pathway in acetaldehyde-induced HSCs. Both the A2AR and A1R antagonists could suppress the activation of HSC, although they have opposing effects on cAMP signal transduction. These results suggested that a combination of cAMP/PKA/CREB signals via A2AR and A1R likely mediate the activation of acetaldehyde-induced HSCs, and A1R coupled to the Gi/o-AC signaling pathway may be masked by the more predominant A2AR that coupled to the Gs-AC signaling pathway.
本研究旨在探讨腺苷受体（ARs）介导的cAMP/PKA/CREB信号通路调节乙醛诱导的肝星状细胞（HSCs）激活的机制。从SD大鼠中分离出原代HSCs，进行体外培养，并在不同时间点用不同浓度的乙醛进行激活。采用定量实时PCR和蛋白质印迹法对大鼠HSCs中四种AR（A1R、A2AR、A2BR和A3R）的蛋白质和mRNA水平进行定量。使用磷酸二酯酶（PDEs）和Gi/o蛋白途径的选择性抑制剂、一般AR激动剂以及AR亚型特异性试剂来研究AR信号传导。通过放射免疫测定法测量cAMP水平，并通过蛋白质印迹法检测α-SMA、I型和III型胶原蛋白、PKA和p-CREB的表达。乙醛可显著促进HSC增殖，在暴露于200μM乙醛后48小时观察到最大刺激作用。大鼠HSCs中可存在所有四种AR亚型，且A2AR和A1R的mRNA和蛋白质表达水平远高于A2BR和A3R。与对照组相比，乙醛诱导的HSCs中A2AR和A1R的表达显著增加，而添加乙醛后A2BR和A3R的表达未受影响。奇怪的是，在乙醛诱导的HSCs中，A2AR与Gs-AC信号偶联，A1R与Gi/o-AC信号通路偶联。A2AR和A1R拮抗剂均可抑制HSC的激活，尽管它们对cAMP信号转导具有相反的作用。这些结果表明，通过A2AR和A1R的cAMP/PKA/CREB信号组合可能介导乙醛诱导的HSCs的激活，并且与Gi/o-AC信号通路偶联的A1R可能被与Gs-AC信号通路偶联的更主要的A2AR所掩盖。
