N-乙酰半胱氨酸联合泼尼松通过抑制PKCδ的磷酸化缓解大鼠肺间质纤维化的分子机制研究  

Molecular mechanism of N-acetylcysteine combined with prednisone to alleviate pulmonary fibrosis in rats by inhibiting the phosphorylation of PKCδ

作　　者：李阳硕 李德红[1] 刘凡 LI Yang-shuo;LI De-hong;LIU Fan(Department of Respiratory and Critical Care,Daxing District People's Hospital,Beijing 102600,China)

机构地区：[1]北京市大兴区人民医院呼吸与危重症科,北京102600

出　　处：《临床和实验医学杂志》2022年第11期1130-1134,共5页Journal of Clinical and Experimental Medicine

基　　金：首都卫生发展科研专项项目(编号:2018-3-4193)。

摘　　要：目的探究N-乙酰半胱氨酸联合泼尼松通过抑制PKCδ的磷酸化缓解大鼠肺间质纤维化的分子机制。方法40只6~8周龄雌性Wistar大鼠通过博来霉素构建肺损伤大鼠模型,并将其分为博来霉素(BLM)组、N-乙酰半胱氨酸(NAC)组、N-乙酰半胱氨酸和泼尼松(NAC/Ps)组和泼尼松(Ps)组,每组10只,依次进行0.9%氯化钠溶液、80 mg/kg N-乙酰半胱氨酸、40 mg/kg N-乙酰半胱氨酸联合3 mg/kg泼尼松以及6 mg/kg泼尼松干预;另取10只正常鼠为Blank组,仅0.9%氯化钠溶液干预。21 d后对大鼠实施安乐死收集肺样本,其中一部分采用羟脯氨酸测定试剂盒、酸溶性胶原测定试剂盒和Col-Ⅰ酶联免疫吸附试验试剂盒测定羟脯氨酸、酸溶性胶原蛋白和Col-Ⅰ的含量;蛋白质印迹检测Nox4、α-SMA和Col-Ⅰ蛋白表达。另一部分进行细胞转染实验,使用Lipofectamine 2000在NIH-3T3细胞中转染靶向Nox4(Nox4-pEx-3组)和空载体pEx-3质粒(pEx-3组),以未进行任何转染的细胞作为空白对照(Blank组),转染24 h后,免疫荧光染色检测各组大鼠肺细胞NIH-3T3表达水平;蛋白质印迹检测Smad 3和p-PKC-δ蛋白表达。结果(1)BLM组大鼠肺组织中Col-Ⅰ、羟脯氨酸和酸溶性胶原蛋白含量较Blank组升高,NAC组、NAC/Ps组和Ps组大鼠肺组织中Col-Ⅰ、羟脯氨酸和酸溶性胶原蛋白含量较BLM组降低,NAC/Ps组大鼠肺组织中Col-Ⅰ、羟脯氨酸和酸溶性胶原蛋白含量较NAC组和Ps组降低,差异均有统计学意义(P<0.05)。(2)BLM组大鼠肺组织中Nox4、α-SMA和Col-Ⅰ蛋白表达较Blank组升高,NAC组、NAC/Ps组和Ps组大鼠肺组织中Nox4、α-SMA和Col-Ⅰ蛋白较BLM组降低,NAC/Ps组大鼠肺组织中Nox4、α-SMA和Col-Ⅰ蛋白较NAC组和Ps组降低,差异均有统计学意义(P<0.05)。(3)2个pEx-3组和2个Nox4-pEx-3组大鼠肺细胞中NIH-3T3活性较Blank组升高,NAC处理的pEx-3组和Nox4-pEx-3组大鼠肺细胞中NIH-3T3活性较非NAC的pEx-3组和Nox4-pEx-3组降低,2个pEx-3组大鼠�Objective To explore the molecular mechanism of N-acetylcysteine combined with prednisone in alleviating pulmonary interstitial fibrosis in rats by inhibiting the phosphorylation of PKCδ.Methods Forty female Wistar rats aged 6-8 weeks were treated with bleomycin to establish a rat model of lung injury,and they were divided into BLM group,NAC group,NAC/Ps group and Ps group.0.9% sodium chloride solution,80 mg/kg N-acetylcysteine,40 mg/kg N-acetylcysteine combined with 3 mg/kg prednisone and 6 mg/kg prednisone intervention;another 10 normal mice were selected as the Blank group,only physiological Saline intervention.After 21 days,the rats were euthanized to collect lung samples,and some of them were assayed for hydroxyproline,acid-soluble collagen and Col-Ⅰusing hydroxyproline assay kit,acid-soluble collagen assay kit and Col-Ⅰenzyme-linked immunosorbent assay kit The protein expression of Nox4,α-SMA and Col-Ⅰwas detected by Western blotting.The other part performed cell transfection experiments,using Lipofectamine 2000 to transfect targeting Nox4(Nox4-pEx-3 group)and empty vector pEx-3 plasmid(pEx-3 group).The cells without any transfection were used as blank control(Blank group).24 h after transfection,the expression levels of NIH-3T3 in lung cells of rats in each group were detected by immunofluorescence staining;the protein expressions of Smad3 and p-PKC-δ were detected by Western blotting.Results(1)The contents of Col-Ⅰ,hydroxyproline and acid-soluble collagen in the lung tissue of the rats in the BLM group were higher than those in the Blank group.The contents of Col-Ⅰ,hydroxyproline and acid-soluble collagen in the lung tissue of the rats in the NAC group,NAC/Ps group and Ps group were lower than those in the BLM group,and the contents of Col-Ⅰ,hydroxyproline and acid-soluble collagen in the lung tissue of the NAC/Ps group were lower than those in the NAC group and the Ps group,the differences were statistically significant(P<0.05).(2)The protein expressions of Nox4,α-SMA and Col-Ⅰin the lu

关 键 词：N-乙酰半胱氨酸 泼尼松 PKC-δ 磷酸化 肺间质纤维化 

分 类 号：R563[医药卫生—呼吸系统]