p38MAPK信号通路与LPS致大鼠肺泡上皮细胞线粒体分裂的关系:离体实验

Relationship between p38MAPK signaling pathway and lipopolysaccharide-induced mitochondrial fission in alveolar epithelial cells: an in vitro experiment

目的采用离体实验评价p38分裂原激活蛋白激酶(p38MAPK)信号通路与LPS致大鼠肺泡上皮细胞线粒体分裂的关系。方法将传代培养的肺泡上皮细胞以2×10^5个/ml的密度铺于96孔板中,200μl/孔,达到80%融合后,采用随机数字表法分为4组(n=10):对照组(C组)、LPS组、LPS+SB203580组(LPS+SB组)和LPS+二甲基亚砜组(LPS+DMSO组)。LPS组给予LPS10μg/ml孵育24h;LPS+SB组给予p38MAPK抑制剂SB20358010μmol(用二甲基亚砜溶解)孵育1h,再给予LPS10μg/ml孵育24h;LPS+DMSO组给予等容量二甲基亚砜孵育1h,再给予LPS10μg/ml孵育24h。测定MDA含量和SOD活性,采用Westernblot法测定磷酸化p38MAPK(p-p38MAPK)、血红素加氧酶-1(HO-1)、发动蛋白相关蛋白1(DRP1)和分裂相关蛋白1(FIS1)的表达水平。结果与C组比较,LPS组、LPS+SB组和LPS+DMSO组MDA含量升高,SOD活性降低,p-p38MAPK、HO-1、FIS1和DRP1表达上调(P<0.05);与LPS组比较,LPS+SB组MDA含量升高,SOD活性降低,p-p38MAPK和HO-1表达下调,FIS1和DRP1表达上调(P<0.05),LPS+DMSO组上述指标差异无统计学意义(P>0.05)。结论p38MAPK信号通路激活可上调HO-1表达,从而减轻LPS致大鼠肺泡上皮细胞线粒体分裂。

Objective To evaluate the relationship between p38 mitogen-activated protein kinase (p38MAPK) signaling pathway and lipopolysaccharide (LPS)-induced mitochondrial fission in alveolar epithelial cells using an in vitro experiment. Methods The cultured alveolar epithelial cells were subcultured and seeded in 96-well plates at the density of 2 × 105 cells/ml (200 μl/well). The cells were divided into 4 groups (n=10 each) when cell confluence reached 80% using a random number table method: control group (group C), LPS group, LPS+ SB203580 group (group LPS+ SB) and LPS+ dimethyl sulfoxide (DMSO) group.Cells were incubated with LPS 10 μg/ml for 24 h in group LPS.Cells were incubated with p38MAPK inhibitor SB203580 10 μmol (dissolved in DMSO) for 1 h and then with LPS 10 μg/ml for 24 h in group LPS+ SB.Cells were incubated with the equal volume of DMSO for 1 h and then with LPS 10 μg/ml for 24 h in group LPS+ DMSO.Malonaldehyde (MDA) content and superoxide dismutase (SOD) activity were measured.The expression of phosphorylated p38MAPK (p-p38MAPK), heme oxygenase-1 (HO-1), dynamin-related protein 1 (DRP1) and fission protein 1 (FIS1) was determined by Western blot. Results Compared with group C, the MDA content was significantly increased, the SOD activity was decreased, and the expression of p-p38MAPK, HO-1, FIS1 and DRP1 was up-regulated in LPS, LPS+ SB and LPS+ DMSO groups (P<0.05). Compared with group LPS, the MDA content was significantly increased, the SOD activity was decreased, the expression of p-p38MAPK and HO-1 was down-regulated, the expression of FIS1 and DRP1 was up-regulated in group LPS+ SB (P<0.05), and no significant change was found in the parameters mentioned above in group LPS+ DMSO (P>0.05). Conclusion The p38MAPK signaling pathway activation can up-regulate the expression of HO-1, thus reducing LPS-induced mitochondrial fission in alveolar epithelial cells.