安石榴苷调节AMPK/SIRT1信号通路对肺炎大鼠氧化应激损伤的影响

Effect of punicalagin on oxidative stress injury in rats with pneumonia by regulating AMPK/SIRT1 signal pathway

目的探讨安石榴苷(Pun)基于AMPK/SIRT1信号通路对肺炎大鼠氧化应激损伤的影响机制。方法利用铜绿假单胞菌建立肺炎大鼠模型,将大鼠分为对照组(NC组)、模型组(M组)、安石榴苷低、中、高剂量组(Pun-L、M、H,10、20、40 mg/kg)、安石榴苷高+AMPK抑制剂Compound C组(Pun-H+CC组,40 mg/kg+0.2 mg/kg)。全自动血气分析仪检测大鼠血气值;HE染色观察大鼠肺组织病理学变化;Wright-Giemsa染色观察肺泡灌洗液中炎性细胞(白细胞)数目;ELISA法测定肺泡灌洗液炎性因子(TNF-α、IL-6、IL-1β)水平;氧化应激因子水平采用试剂盒检测;蛋白表达采用Western Blot法检测。结果与NC组比较,M组大鼠肺损伤严重,PaCO_(2)、炎性因子水平、白细胞数量、MDA含量和p-NF-κB/NF-κB蛋白表达均显著升高,PaO_(2)、SaO_(2)、SOD、GSH-Px、p-AMPK/AMPK、SIRT1、Mn SOD蛋白表达显著降低(P均<0.05);与M组比较,Pun-L、M、H组大鼠肺组织病理学变化好转,PaCO_(2)、炎性因子水平、白细胞数量、MDA含量和p-NF-κB/NF-κB蛋白表达均显著下降,PaO_(2)、SaO_(2)、SOD、GSH-Px、p-AMPK/AMPK、SIRT1、Mn SOD蛋白表达显著上升(P均<0.05);与Pun-H组比较,Pun-H+CC组大鼠肺损伤严重,PaCO_(2)、炎性因子水平、白细胞数量、MDA含量和p-NF-κB/NF-κB蛋白表达均显著升高,PaO_(2)、SaO_(2)、SOD、GSH-Px、p-AMPK/AMPK、SIRT1、Mn SOD蛋白表达显著降低(P均<0.05)。结论Pun可以通过上调AMPK/SIRT1信号通路表达,抑制肺炎大鼠氧化应激损伤,减轻炎症反应。

Objective To investigate the mechanism of the effect of punicalagin(Pun)on oxidative stress injury in rats with pneumonia based on AMPK/SIRT1 signaling pathway.Methods The rat model of pneumonia was established by Pseudomonas aeruginosa.The rats were divided into the control group(the NC group),the model group(the M group),the low-dose,middle-dose and high-dose Pun groups(Pun-L,M,H,10,20,40 mg/kg),and the high-dose Pun+AMPK inhibitor compound C group(Pun-H+CC group,40 mg/kg+0.2 mg/kg).The blood gas value of rats was detected by automatic blood gas analyzer.HE staining was used to observe the pathological changes of lung tissue in rats.Wright-Giemsa staining was used to observe the number of inflammatory cells(leukocytes)in alveolar lavage fluid.The levels of inflammatory factors(TNF-α,IL-6,IL-1β)in alveolar lavage fluid were measured by Elisa method.The level of oxidative stress factor was detected by kit.The protein expression was detected by Western blot.Results Compared with the NC group,lung injury was more severe,the levels of PaCO_(2),inflammatory factor level,leukocyte number,MDA content and p-NF-κB/NF-κB protein expression increased more obviously,and the levels of PaO_(2),SaO_(2),SOD,GSH-Px,p-AMPK/AMPK,SIRT1,and Mn SOD protein expression decreased more obviously in the M group(P<0.05).Compared with the group M,the pathological changes of lung tissue improved more obviously,the levels of PaCO_(2),inflammatory factor level,leukocyte number,MDA content and p-NF-κB/NF-κB protein expression decreased more obviously,and the levels of PaO_(2),SaO_(2),SOD,GSH-Px,p-AMPK/AMPK,SIRT1,and Mn SOD protein expression increased more obviously in the Pun-L,M and H groups(P<0.05).Compared with the Pun-H group,the Pun-H+CC group had more severe lung injury,the higher levels of PaCO_(2),inflammatory factor level,leukocyte number,MDA content and p-NF-κB/NF-κB protein expression,and the lower levels of PaO_(2),SaO_(2),SOD,GSH-Px,p-AMPK/AMPK,SIRT1,and Mn SOD protein expression(P<0.05).Conclusion Pun can inhibit oxidative stress injury and reduce inflammatory reaction in rats with pneumonia by up-regulating the expression of AMPK/SIRT1 signal pathway.