氯胺酮调控Akt/mTOR信号通路对大鼠肺缺血再灌注损伤的作用机制研究

Effect of ketamine on lung ischemia-reperfusion injury in rats through regulation on Akt/mTOR signaling pathway

目的:探讨氯胺酮对大鼠肺缺血再灌注损伤(LIRI)的保护作用及其机制。方法:将60只SD大鼠随机分为5组:假手术组、模型组、氯胺酮低剂量组(2.5 mg/kg)、氯胺酮中剂量组(5 mg/kg)和氯胺酮高剂量组(10 mg/kg),每组12只。除假手术组外,其他4组均复制LIRI大鼠模型。比较各组大鼠肺组织湿重/干重比值(W/D),采用酶联免疫吸附测定(ELISA)法检测血清中白细胞介素(IL)-6、IL-1β和肿瘤坏死因子(TNF)-α的含量,比色法检测肺组织丙二醛(MDA)含量及髓过氧化物酶(MPO)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性,苏木素-伊红(HE)染色法检测肺组织的病理学变化,TUNEL法检测细胞凋亡率,Western blotting法检测磷酸化丝氨酸-苏氨酸蛋白激酶(p-Akt)、p-哺乳动物雷帕霉素靶蛋白(p-mTOR)、p-核因子κB(pNF-κB)p65蛋白表达。结果:与假手术组比较,模型组大鼠肺组织结构损伤严重,肺间质充血水肿,肺间隔变厚,大量炎性细胞浸润和红细胞渗出,W/D比值、细胞凋亡率、MPO活性及IL-6、IL-1β、TNF-α、MDA含量显著升高,SOD、CAT活性显著降低(均P<0.05);氯胺酮处理后肺组织损伤程度明显减轻,仅有少量炎性细胞浸润,肺组织W/D比值、细胞凋亡率、MPO活性及IL-6、IL-1β、TNF-α、MDA含量显著降低,SOD、CAT活性显著升高,且呈剂量依赖性(均P<0.05)。此外,与模型组比较,氯胺酮各剂量组p-NF-κB p65蛋白水平显著降低,p-Akt、p-mTOR蛋白水平显著升高(均P<0.05)。结论:氯胺酮可能通过激活Akt/mTOR信号通路减轻炎症反应及氧化应激,保护大鼠LIRI。

Objective:To investigate the protective effect and mechanism of ketamine on lung ischemia reperfusion injury(LIRI)in rats.Methods:A total of 60 SD rats were randomly divided into 5 groups:sham-operation group,model group,low-dose ketamine group(2.5 mg/kg),middle-dose ketamine group(5 mg/kg)and high-dose ketamine group(10 mg/kg),with 12 rats in each group.Except for the sham-operation group,the LIRI rat models were established in other 4 groups.The ratio of wet/dry lung weight(W/D)was calculated.The levels of IL-6,IL-1βand TNF-αin serum were detected by enzyme-linked immunosorbent assay(ELISA).The contents of malondialdehyde(MDA)and myeloperoxidase(MPO),the activities of superoxide dismutase(SOD)and catalase(CAT)in lung tissue were detected by colorimetric test.Hematoxylin-eosin staining was used to observe the pathological changes of lung tissue.TUNEL method was used to detect the apoptosis of lung tissue.The protein expression levels of p-Akt,p-mTOR and p-NF-κB p65 were detected byWestern blotting.Results:Compared with the shamoperation group,the lung tissue structure of the model group was seriously damaged,the pulmonary interstitium was congested and edematous,the pulmonary septum became thicker,a large number of inflammatory cells infiltrated and red blood cells exuded,W/D ratio,apoptosis index,the contents of IL-6,IL-1β,TNF-α,MDA and MPO as well as the protein level of p-NF-κB p65 were significantly increased,whereas the contents of SOD and CAT,the protein levels of p-Akt and p-mTOR were decreased(P<0.05).Compared with the model group,the degree of lung injury in ketamine-treated groups was significantly reduced,a small amount of inflammatory cell infiltration was observed,the W/D ratio,apoptosis index,the contents of IL-6,IL-1βand TNF-αin serum,the contents of MDA and MPO in lung tissues and protein level of p-NF-κB p65 were significantly decreased,while the contents of SOD and CAT,the protein levels of p-Akt and p-mTOR were significantly increased(P<0.05).Conclusion:Ketamine may protect LIRI in rats,reduce inflammation and oxidative stress by activating Akt/mTOR signaling pathway.