褪黑素减轻LPS致大鼠Ⅱ型肺泡上皮细胞损伤的机制:褪黑素受体与线粒体分裂蛋白的关系

Mechanism of melatonin alleviating lipopolysaccharide-induced damage to typeⅡalveolar epithelial cells of rats:relationship between melatonin receptors and mitochondrial fission proteins

目的评价褪黑素受体与线粒体分裂蛋白的关系,明确褪黑素减轻LPS致大鼠Ⅱ型肺泡上皮细胞损伤的机制。方法将大鼠Ⅱ型肺泡上皮细胞以密度2×10^5个/ml接种于6孔板,采用随机数字表法分为5组(n=10):对照组(C组)、LPS组(L组)、LPS+褪黑素组(LM组)、LPS+褪黑素受体阻断剂组(LL组)和LPS+褪黑素+褪黑素受体阻断剂组(LML组)。采用10μg/ml LPS孵育24 h的方法制备LPS致细胞损伤模型;于LPS孵育前12 h时,分别向LM组、LL组和LML组加入褪黑素0.1 mmol/L和/或褪黑素受体阻断剂luzindole 0.2μmol/L。细胞孵育结束后,采用ELISA法测定培养液TNF-α和IL-6浓度。采用GENMED纯化线粒体呼吸控制率(RCR)定量检测试剂盒测定各组RCR。采用Western blot法测定细胞线粒体动力相关蛋白1(Drp1)和线粒体分裂相关蛋白1(Fis1)的表达。结果与C组比较,L组、LM组、LL组和LML组细胞培养液TNF-α和IL-6浓度升高,RCR降低,线粒体Drp1和Fis1表达上调(P<0.05);与L组比较,LM组细胞培养液TNF-α和IL-6浓度降低,RCR升高,线粒体Drp1和Fis1表达下调(P<0.05),LL组上述各指标差异无统计学意义(P>0.05);与LM组比较,LML组细胞培养液TNF-α和IL-6浓度升高,RCR降低,线粒体Drp1和Fis1表达上调(P<0.05)。结论褪黑素减轻LPS致大鼠肺泡Ⅱ型上皮细胞损伤的机制与激活褪黑素受体,抑制线粒体分裂蛋白表达有关。

Objective To evaluate the relationship between melatonin receptors and mitochondrial fission proteins and to clarify the mechanism of melatonin alleviating lipopolysaccharide(LPS)-induced damage to typeⅡalveolar epithelial cells of rats.Methods The rat typeⅡalveolar epithelial cells were seeded in 6-well plates at a density of 2×105 cells/ml and divided into 5 groups(n=10 each)using a random number table method:control group(C group),LPS group(L group),LPS plus melatonin group(LM group),LPS plus melatonin receptor blocking group(LL group),and LPS plus melatonin plus melatonin receptor blocker group(LML group).The model of LPS-induced damage to cells was established by incubating with LPS 10μg/ml for 24 h.Melatonin 0.1 mmol/L and/or melatonin receptor blocker luzindole 0.2μmol/L was added in LM group,LL group and LML group.The concentrations of tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)were measured by enzyme-linked immunosorbent assay after the end of incubation.The mitochondrial respiratory control rate(RCR)was measured by GENMED purified mitochondrial RCR quantitative detection kit in each group.Western blot was used to detect the expression of dynamin-related protein 1(Drp1)and mitochondrial adaptor fission 1(Fis1).Results Compared with C group,the concentrations of TNF-αand IL-6 in culture medium were significantly increased,RCR was decreased,and the expression of Drp1 and Fis1 was up-regulated in L,LM,LL and LML groups(P<0.05).Compared with L group,the concentrations of TNF-αand IL-6 in culture medium were significantly decreased,RCR was increased,and the expression of Drp1 and Fis1 was down-regulated in LM group(P<0.05),and no significant change was found in parameters mentioned above in LL group(P>0.05).Compared with LM group,the concentrations of TNF-αand IL-6 in culture medium were significantly increased,RCR was decreased,and the expression of Drp1 and Fis1 was up-regulated in LML group(P<0.05).Conclusion The mechanism by which melatonin attenuates LPS-induced damage to typeⅡalveolar epithelial cells is related to activating melatonin receptors and inhibiting the expression of mitochondrial fission proteins in rats.