连翘苷对感染性休克小鼠急性肺损伤的作用与机制

Effect and mechanism of Phillyrin on acute lung injury in septic shock mice

目的 探讨连翘苷通过腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)/p70核糖体S6蛋白激酶(p70 S6 kinase,p70S6K)信号通路介导的自噬对感染性休克小鼠急性肺损伤(Acute lung injury,ALI)的影响。方法 随机选择12只小鼠作为对照组,其余小鼠通过腹腔注射20 mg·kg^(-1)脂多糖(Lipopolysaccharide,LPS)构建感染性休克小鼠模型,将感染性休克小鼠随机平分为模型组、低、中、高剂量实验组(5 mg·kg^(-1)、10 mg·kg^(-1)、20 mg·kg^(-1)连翘苷)、高剂量+抑制剂组(20 mg·kg^(-1)连翘苷+20 mg·kg^(-1)AMPK抑制剂compound C),每组均12只小鼠。称量肺干重及湿重,计算W/D比值;ELISA法检测BALF中炎性因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)、白细胞介素6(interleukin-6,IL-6)水平、血清内毒素(endotoxin,ET)含量、肺组织髓过氧化物酶(myeloperoxidase,MPO)活性;HE染色检测肺组织病理变化;Western blot检测自噬蛋白微管相关蛋白-轻链3(microtubule-associated protein-light chain 3,LC3)-II/I、Beclin 1、Ras相关GTP结合蛋白7(Rasassociated GTP binding protein 7,Rab7)、溶酶体关联膜蛋白2(lysosomal associated membrane protein 2,LAMP2)、AMPK/m TOR/p70S6K信号通路蛋白表达。结果对照组、模型组、低、中、高剂量实验组和高剂量+抑制剂组小鼠肺组织LC3-II/I比值分别为1.43±0.14、0.73±0.07、0.81±0.07、1.12±0.10、1.39±0.13、0.76±0.08,Beclin1蛋白水平分别为1.05±0.11、0.43±0.05、0.50±0.05、0.76±0.08、0.98±0.10、0.46±0.05,Rab7蛋白水平分别为1.53±0.17、0.67±0.06、0.70±0.07、1.04±0.10、1.41±0.14、0.69±0.06,LAMP2蛋白水平分别为1.47±0.15、0.72±0.07、0.81±0.08、1.09±0.11、1.35±0.13、0.74±0.07,p-AMPK/AMPK蛋白水平分别为0.95±0.05、0.33±0.03、0.39±0.04、0.68±0.07、0.91±0.09、0.36±0.04,p-m TOR/m TOR蛋白水平分别为0.28±0.02、0.94±0.06、0.88±0.07、0.57±0.05、0.30±0.03、0.87±0.09,p70S6K蛋白水平分别为0.32±0.07、0.96±0.04、0.90±0.07、0.69±0.06、0.38±0.04、0.92±0.06。上述指标:模型组与对照组比较,差异均有统计学意义(均P<0.05);中、高剂量实验组与模型组比较,差异均有统计学意义(均P<0.05);高剂量+抑制剂组与高剂量实验组比较,差异均有统计学意义(均P<0.05)。结论连翘苷可能通过调控AMPK/m TOR/p70S6K信号通路介导的自噬对感染性休克小鼠ALI起到改善作用。

Objective To investigate the impact of phillyrin on acute lung injury (ALI) in septic shock mice through autophagy mediated by adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (m TOR)/p70 S6 kinase(p70S6K) signal pathway.Methods Twelve mice were randomly selected as the control group,and the rest of the mice were injected intraperitoneally with 20mg·kg^(-1)LPS to construct the model of septic shock,the mice with septic shock were randomly divided into a model group,an Experimental-L,-M,-H group (5 mg·kg^(-1),10 mg·kg^(-1),20 mg·kg^(-1)phillyrin),and an Experimental-H+compound C group (20 mg·kg^(-1)phillyrin+20 mg·kg^(-1)AMPK inhibitor compound C),there were 12 mice in each group.The lung dry weight and wet weight were weighed,and the W/D ratio was calculated;the levels of inflammatory factors tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β),interleukin-6 (IL-6) in BALF,serum endotoxin (ET) and myeloperoxidase (MPO) of lung tissue were detected by ELISA;HE staining was applied to detect pathological changes of lung tissue;Western blot was applied to detect the expression of autophagic proteins microtubule-associated protein-light chain 3 (LC3-II/I),Beclin 1,Ras-associated GTP binding protein 7 (Rab7),lysosomal associated membrane protein 2 (LAMP2) and AMPK/m TOR/p70S6K signaling pathway proteins.Results In control group,model group,ExperimentalL,-M,-H group and Experimental-H+compound C group LC3-II/I ratios were 1.43±0.14,0.73±0.07,0.81±0.07,1.12±0.10,1.39±0.13,0.76±0.08,respectively.Beclin1 protein levels were 1.05±0.11,0.43±0.05,0.50±0.05,0.76±0.08,0.98±0.10,0.46±0.05,respectively.Rab7 protein levels were 1.53±0.17,0.67±0.06,0.70±0.07,1.04±0.10,1.41±0.14,0.69±0.06,respectively.LAMP2 protein levels were 1.47±0.15,0.72±0.07,0.81±0.08,1.09±0.11,1.35±0.13,0.74±0.07,respectively.p-AMPK/AMPK protein levels were 0.95±0.05,0.33±0.03,0.39±0.04,0.68±0.07,0.91±0.09,0.36±0.04,respectively.p-m TOR/m TOR protein levels were 0.28±0.02,0.94±0.06,0.88±0.07,0.57±0.05,0.30±0.03,0.87±0.09,respectively.The protein levels of p70S6K were0.32±0.07,0.96±0.04,0.90±0.07,0.69±0.06,0.38±0.04,0.92±0.06,respectively.There were statistical differences between the model group and the control group (all P<0.05).There were statistical differences between Experimental-M,-H group and model group (all P<0.05).There were significant differences between Experimental-H+compound C group and Experimental-H group (all P<0.05).Conclusions Phillyrin may improve ALI in septic shock mice by regulating autophagy mediated by AMPK/m TOR/p70S6K signaling pathway.