机械通气性牵张致小鼠肺泡上皮细胞损伤时NLRP3表达与线粒体功能的关系

Relationship between NLRP3 expression and mitochondrial function during mechanical stretch-in-duced damage to mouse alveolar epithelial cells

目的评价机械通气性牵张致小鼠肺泡上皮细胞损伤时神经组织NOD样受体热蛋白结构域相关蛋白3（NLRP3）表达与线粒体功能的关系。方法采用随机数字表法，将小鼠肺泡Ⅱ型上皮细胞株MLE-12细胞分为3组（n＝13）：对照组（C组）、机械通气性牵张组（CS组）和机械通气性牵张＋ NLRP3抑制剂TAK-242组（CS＋T组）。CS组给予强度20%，频率0.5 Hz，牵张间歇比1∶1的机械通气性牵张4 h；CS＋T组加入1 μmol/L TAK-242孵育1 h后给予机械通气性牵张4 h，参数同CS组。采用化学荧光法检测细胞活性氧（ROS）含量和线粒体膜电位（ΔΨM），采用ELISA法测定上清液IL-1β浓度，采用Western blot法测定细胞NLRP3表达。结果与C组比较，CS组细胞ΔΨM下降，ROS含量和IL-1β浓度增加，NLRP3表达上调，CS＋T组细胞ΔΨM下降，ROS含量增加，NLRP3表达上调（P〈0.05）；与CS组比较，CS＋T组细胞ΔΨM升高，ROS和IL-1β浓度减少，NLRP3表达下调（P〈0.05）。结论机械通气性牵张可通过上调NLRP3的表达，诱发线粒体损伤，从而导致小鼠肺泡上皮细胞损伤。

ObjectiveTo evaluate the relationship between the expression of Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 （NLRP3） in nerve tissues and mitochondrial function during mechanical stretch-induced damage to mouse alveolar epithelial cells.MethodsAlveolar epithelial type Ⅱ cell line MLE-12 cells were divided into 3 groups （n=13 each） using a random number table： control group （group C）, cyclic stretch group （group CS） and cyclic stretch plus NLRP3 inhibitor TAK-242 group （group CS＋ T）. MLE-12 cells underwent 20% cyclic stretch at a frequency of 0.5 Hz （stretch∶intermittence =1∶1） for 4 h in group CS.In group CS＋ T, after being incubated for 1 h with 1 μmol/L TAK-242, MLE-12 cells underwent 20% cyclic stretch for 4 h, and the parameters were similar to those previously described in group CS.The reactive oxygen species （ROS） content and mitochondrial membrane potential （ΔΨM）were measured using chemiluminescence assay.Enzyme-linked immunosorbent assay was used to determine the concentration of interleukin-1beta （IL-1β） in the supernatant.The expression of NLRP3 in MLE-12 cells was detected using Western blot.ResultsCompared with group C, the ΔΨM of cells was significantly decreased, the ROS content and IL-1β concentration were increased, and the expression of NLRP3 was up-regulated in group CS, and the ΔΨM of cells was significantly decreased, the ROS content was increased, and the expression of NLRP3 was up-regulated in group CS＋ T （P〈0.05）. Compared with group CS, the ΔΨM of cells was significantly increased, the ROS content and IL-1β concentration were decreased, and the expression of NLRP3 was down-regulated in group CS＋ T （P〈0.05）.ConclusionMechanical stretch induces damage to mitochondria through up-regulating the expression of NLRP3, thus leading to damage to mouse alveolar epithelial cells.