内毒素诱发大鼠肺泡Ⅱ型上皮细胞的内源性保护机制:与p38MAPK-HO-1-线粒体融合信号通路的关系

Endotoxin-induced endogenous protective mechanism of alveolar type Ⅱ epithelial cells of rats: the relationship with p38MAPK-HO-1-mitochondrial fusion signaling pathway

目的评价内毒素诱发大鼠肺泡Ⅱ型上皮细胞的内源性保护机制:与p38分裂原激活蛋白激酶(p38MAPK)-HO-1-线粒体融合信号通路中的关系。方法将大鼠肺泡Ⅱ型上皮细胞以2×105个/ml的密度接种于6孔板,采用随机数字表法分为5组(n=15):空白对照组(C组)、LPS组(L组)、LPS+p38MAPK抑制剂SB203580组(LS组)、LPS+二甲基亚砜(DMSO)组(LD组)和SB203580组(S组)。C组正常培养,L组、LS组和LD组均给予10 μg/ml LPS制备内毒素诱发模型;LS组和LD组于加入LPS前1 h分别给予SB203580 10 μmol和0.1% DMSO 100 μmol;S组加入SB203580 10 μmol,各组孵育24 h。采用硫代巴比妥酸法测定MDA含量,采用黄嘌呤氧化酶法测定SOD活性,采用Western blot法测定p38MAPK、磷酸化p38MAPK(p-p38MAPK)、血红素加氧酶-1(HO-1)、线粒体融合蛋白1(Mfn1)、Mfn2和视神经萎缩蛋白1(OPA1)的表达水平。结果与C组比较,L组、LS组和LD组MDA含量升高,SOD活性降低,p-p38MAPK和HO-1表达上调,Mfn1、Mfn2和OPA1表达下调(P<0.05);与L组比较,LS组MDA含量升高,SOD活性降低,p-p38MAPK、HO-1、Mfn1、Mfn2 、OPA1表达下调(P<0.05),LD组上述指标差异无统计学意义(P>0.05);各组间p38MAPK表达差异无统计学意义(P>0.05)。结论内毒素诱发大鼠肺泡Ⅱ型上皮细胞的内源性保护机制与p38MAPK-HO-1-线粒体融合信号通路有关。

Objective To evaluate the endotoxin-induced endogenous protective mechanism of alveolar type Ⅱ epithelial cells of rats and the relationship with p38 mitogen-activated protein kinase(p38MAPK)-HO-1-mitochondrial fusion signaling pathway. Methods Rat alveolar type Ⅱepithelial cells were seeded in 6-well plates at a density of 2×105 cells/ml and divided into 5 groups(n=15 each)using a random number table method: control group(group C), lipopolysaccharide(LPS)group(group L), LPS plus p38MAPK inhibitor SB203580 group(group LS), LPS plus dimethyl sulfoxide group(group LD), and SB203580 group(group S). Cells were conventionally cultured in group C. The model of endotoxin-challenged alveolar type Ⅱ epithelial cells was established by giving LPS 10 μg/ml in L, LS and LD groups. SB203580 10 μmol and 0.1% dimethyl sulfoxide 100 μmol were added at 1 h before giving LPS in group LS and group LD, respectively. SB203580 10 μmol was added to the culture medium in group S. All the cells were incubated for 24 h. The malonaldehyde(MDA)content and superoxide dismutase(SOD)activity in the culture medium were determined by thiobarbituric acid assay and xanthine oxidase method, respectively. The expression of p38MAPK, phosphorylated p38MAPK(p-p38MAPK), hemeoxygenase-1(HO-1), mitofusin 1(Mfn1), Mfn2, and optical atrophy-1(OPA1)was measured 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 and HO-1 was up-regulated, and the expression of Mfn1, Mfn2 and OPA1 was down-regulated in L, LS and LD groups(P<0.05). Compared with group L, the MDA content was significantly increased, the SOD activity was decreased, and the expression of p-p38MAPK, HO-1, Mfn1, Mfn2 and OPA1 was down-regulated in group LS(P<0.05), and no significant change was found in the indices mentioned above in group LD(P>0.05). Conclusion The endotoxin-induced endogenous protective mechanism of alveolar type Ⅱ epithelial cells is related to p38MAPK-HO-1-mitochondrial fusion signaling pathway in rats.