PKCα/HO-1信号通路在LPS致大鼠肺泡Ⅱ型上皮细胞损伤中的作用:与线粒体融合的关系

Role of protein kinase Cα/heme oxygenase-1 signaling pathway in LPS-caused damage to typeⅡ alveolar epithelial cells of rats:the relationship with mitochondrial fusion

目的评价蛋白激酶Cα/血红素氧合酶-1(PKCα/HO-1)信号通路在LPS致大鼠肺泡Ⅱ型上皮细胞损伤中的作用及其与线粒体融合的关系。方法肺泡Ⅱ型上皮细胞以2×10^5个/ml密度接种于96孔培养板,采用随机数字表法分为5组(n=40):对照组(C组)、Go6976组(G组)、内毒素组(L组)、内毒素+Go6976组(LG组)和内毒素+二甲基亚砜组(LD组)。LG组加入Go69765μmol/L,LD组加入等容量0.1%二甲基亚砜,30min后L组、LG组和LD组分别加入LPS10μg/ml制备肺泡Ⅱ型上皮细胞损伤模型,G组加入Go6976 5μmol/L,C组加入等容量PBS。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q-PCR法及Westernblot法测定细胞PKCα、HO-1以及线粒体融合蛋白1(Mfn1)、Mfn2和视神经萎缩蛋白1(OPA1)及其mRNA的表达水平。结果与C组比较,L组、LG组和LD组MDA和ROS含量升高,SOD活性降低,PKCα、HO-1及其mRNA的表达上调,Mfn1、Mfn2和OPA及其mRNA的表达下调(P<0.05),G组上述指标差异无统计学意义(P>0.05);与L组比较,LG组MDA和ROS含量升高,SOD活性降低,PKCα、HO-1、Mfn1、Mfn2和OPA1及其mRNA的表达下调(P<0.05),LD组上述指标差异无统计学意义(P>0.05)。结论PKCα/HO-1信号通路激活是LPS致大鼠肺泡Ⅱ型上皮细胞损伤的内源性保护机制,可能与促进线粒体融合有关。

Objective To evaluate the role of protein kinase Cα(PKCα)/heme oxygenase-1(HO-1)signaling pathway in lipopolysaccharide(LPS)-caused damage to typeⅡ alveolar epithelial cells of rats and the relationship with mitochondrial fusion.Methods TypeⅡ alveolar epithelial cells were seeded in 96-well plates at a density of 2×10^5 cells/ml and divided into 5 groups(n=40 each)using a random number table method:control group(group C),Go6976 group(group G),LPS group(group L),LPS plus PKCα inhibitor Go6976 group(group LG)and LPS plus dimethyl sulfoxide(DMSO)group(group LD).Group LG and group LD were pretreated with 5 μmol/L Go6976 and the equal volume of 0.1% DMSO,respectively,for 30 min,lipopolysaccharide(LPS)10 μg/ml was then given to establish the model of type Ⅱ alveolar epithelial cell damage in L,LG and LD groups,Go6976 5 μmol/L was added in group G,and the equal volume of phosphate buffer solution was added in group C.The cells were collected after 24 h of incubation for measurement of malondialdehyde(MDA)and reactive oxygen species(ROS)contents,superoxide dismutase(SOD)activity and expression of PKCα,HO-1,mitochondrial fusion-related proteins 1 and 2(Mfn1,Mfn2),optic atrophy 1(OPA1)protein and mRNA(by fluorescent 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-1 protein and mRNA was up-regulated,and the expression of Mfn1,Mfn2 and OPA1 protein and mRNA was down-regulated in L,LG and LD groups(P<0.05),and no significant change was found in the parameters mentioned above in group G(P>0.05).Compared with group L,MDA and ROS contents were significantly increased,the SOD activity was decreased,and the expression of PKCα,HO-1,Mfn1,Mfn2 and OPA1 protein and mRNA was down-regulated in group LG(P<0.05),and no significant change was found in the parameters mentioned above in group LD(P>0.05).Conclusion Activation of PKCα/HO-1 signaling pathway is the endogenous protective mechanism of LPS-caused damage to typeⅡ alveolar epithelial cells,which may be related to promoting mitochondrial fusion in rats.