PKCα／HO-1信号通路在内毒素诱发大鼠肺泡巨噬细胞损伤中的作用：与Mfn1的关系

Role of PKCα/HO-1 signaling pathway in endotoxin-induced damage to alveolar macrophages of rats： the relationship with mitofusin-1

目的评价蛋白激酶Cα（PKCα）／血红素加氧酶-1（HO-1）信号通路在内毒素诱发大鼠肺泡巨噬细胞损伤中的作用及其与线粒体融合蛋白-1（Mfnl）的关系。方法将体外培养的大鼠肺泡巨噬细胞NR8383细胞以1×104个／ml密度接种于96孔板中。采用随机数字表法分为5组（n=15）：对照组（C组）、内毒素攻击模型组（E组）、PKCα抑制剂G06976组（G组）、PKCα激动剂PMA组（P组）和二甲基亚砜组（D组）。采用给予10μg／mlLPS刺激NR8383细胞的方法制备肺泡巨噬细胞内毒素攻击模型。G组、P组和D组于LPS刺激前30min分别给予5txmol／LG06976、100nmol／LPMA、0．1％二甲基亚砜预处理30min，再给予10μg／mlLPS，各组均孵育24h后收集细胞，检测丙二醛（MDA）、活性氧（ROS）的含量和超氧化物歧化酶（SOD）活性，采用Westernblot法及q-PCR法检测PKCα、HO-1和Mfnl的表达。结果与C组比较，E组、G组、P组和D组MDA和ROS含量升高，SOD活性降低，PKCα、HO-1及其mRNA表达上调，Mfnl及其mRNA表达下调（P〈0．05）；与E组比较，G组MDA和ROS含量升高，SOD活性降低，PKCα、HO-1、Mfnl及其mRNA表达下调，P组MDA和ROS含量降低，SOD活性升高，PKCα、HO-1、Mfnl及其mRNA表达上调（P〈0．05），D组上述指标差异无统计学意义（P〉O．05）。结论PKCαHO-1信号通路激活后促进Mfnl表达是内毒素诱发大鼠肺泡巨噬细胞损伤时的内源性保护机制。

Objective To evaluate the role of protein kinase Cct （PKCct）/heine oxygenase-1 （HO-1） signaling pathway in endotoxin-induced damage to alveolar macrophages and the relationship with mitofusin-1 （Mfnl） in rats. Methods Rat alveolar macrophagcs NR8383 cells cultured in vitro were see- ded in 96-well plates at a density of 1× 104 cells/ml. NR8383 cells were divided into 5 groups （rt = 15 each） using a random number table： control group （group C） , endotoxin challenge model group （group E） , PKCαinhibitor Go6976 group （group G）, PKCα agonist PMA group （group P） and dimethyl sulfoxide group （group D）. NR8383 cells were stimulated with 10 μg/ml lipopolysaecharide （LPS） to establish the model of endotoxin challenge in alveolar macrophages. In G, P and D groups, cells were pretreated with 5 μmol/L Go6976, 100 nmol/L PMA and 0.1% dimethyl sulfoxide, respectively, for 30 rain starting from 30 min before stimulation with LPS, and 10 μg/ml LPS was then given. The cells were collected after 24 h of incubation tor measurement of malondialdehyde （MDA） and reactive oxygen species （ROS） contents, superoxide dismutase （SOD） aetiv, ity and expression of PKCα, HO-1 and Mfnl protein and mRNA （by flu- orescent quantitative polymerase chain reaction or Western blot）. Results Compared with group C, MDA and ROS contents were significantly increased, the SOD activity was decreased, the expression of PKCα and HO-I protein and mRNA was up-regulated, and the expression of Mfnl protein and mRNA was down- regulated in E, G, P and I） groups （P〈0.05）. Compared with group E, MDA and ROS contents were significantly increased, the SOl） activity was decreased, and the expression of PKCα, HO-1 and Mfnl protein and mRNA was down-regulated in group G, MDA and ROS contents were significantly decreased, the SOD activity was increased, and the expression of PKCα, HO-1 and Mfnl protein and mRNA was up- regulated in group P （ P〈0. 05） , and no significant change was found in the parameters mentioned above in group D （P〉0.05）. Conclusion Promotion of Mfnl expression following PKCα/HO-1 signaling pathway activation is the endogenous protective mechanism of endotoxin-induced damage to alveolar macrophages of rats.