姜黄素对大鼠离体肺缺血再灌注损伤时mTOR信号通路的影响

Effect of cuecumin on mammalian target of rapamyein signaling pathway during ischemia-reperfusion injury in isolated rat lungs

目的评价姜黄素对大鼠离体肺缺血再灌注损伤时mTOR信号通路的影响。方法清洁级健康雄性SD大鼠64只,月龄3~4月,体重250~320g,采用随机数字表法分为4组(n=16):假手术组(S组)、缺血再灌注组(I/R组)、低剂量姜黄素组(LC组)和高剂量姜黄素组(HC组)。S组仅离体灌流不缺血;I/R组通过停止灌流60min再恢复75min的方法建立肺缺血再灌注损伤模型;LC组和HC组分别于复灌开始时从灌流液中加入5和10μmol/L姜黄素。于初灌10min(T0)、复灌15、45和75min(T1-3)时记录肺气道压力(Res)、肺顺应性、灌注流量(Flow)及肺静脉氧分压(PaO2);再灌注末时确定肺组织湿/干重(W/D)比值;分别于光镜和电镜下观察形态学及超微结构;采用RT-PCR法检测mTOR、Tau蛋白、NF-κB及TNF-αmRNA的表达;采用Westernblot法检测mTOR、磷酸化Tau蛋白(pS396Tau蛋白)、NF-κB及TNF-α的表达。结果与S组比较,I/R组、LC组和HC组T1-3时Res和T3时W/D比值升高,T1-3时肺顺应性、Flow和PaO2降低,T3时mTOR、NF-κB和TNF-αmRNA及其蛋白、Tau蛋白mRNA及pS396Tau蛋白表达上调(P<0.05);与I/R组比较,LC组和HC组T1-3时Res和T3时W/D比值降低,T1-3时肺顺应性、Flow和PaO2升高,T3时mTOR、NF-κB和TNF-αmRNA及其蛋白、Tau蛋白mRNA及pS396Tau蛋白表达下调(P<0.05);与LC组比较,HC组T1-3时Res和T3时W/D比值降低,T1-3时肺顺应性、Flow和PaO2升高,T3时mTOR、NF-κB和TNF-αmRNA及其蛋白、Tau蛋白mRNA及pS396Tau蛋白表达下调(P<0.05)。光镜及电镜显示LC组和HC组肺组织损伤程度较I/R组减轻。结论姜黄素减轻大鼠离体肺缺血再灌注损伤的机制与其抑制mTOR信号通路有关。

Objective To evaluate the effect of curcumin on the mammalian target of rapamyein(mTOR)signaling pathway during ischemia-reperfusion(I/R)injury in isolated rat lungs. Methods Sixty-four clean-grade healthy male Sprague-Dawley rats, aged 3-4 months, weighing 250-320 g, were divided into 4 groups(n=16 each)using a random number table method: sham operation group(S group), I/R group, low-dose curcumin group(LC group)and high-dose curcumin group(HC group). The rats only received in vitro perfusion without ischemia in S group.Isolated rat lungs were subjected to 60 min of ischemia followed by 75-min reperfusion to establish the lung I/R injury model in I/R group.Curcumin 5 and 10 μmol/L were added to perfusion fluid from the beginning of reperfusion in LC and HC groups, respectively.Airway resistance(Res), lung compliance, perfusion flow(Flow)and pulmonary venous partial pressure of oxygen(PaO2)were recorded at 10 min of first perfusion(T0)and 15, 45 and 75 min of reperfusion(T1-3). Wet/dry lung weight ratio(W/D ratio)was measured at the end of reperfusion.The morphological structure and ultrastructure of lung tissues were observed by using a light microscope and a transmission electron microscope, respectively.The expression of mTOR, Tau protein, nuclear factor kappa B(NF-κB)and tumor necrosis factor-alpha(TNF-α)mRNA in lung tissues was detected by real-time polymerase chain reaction.The expression of mTOR, phosphorylated Tau protein(pS396 Tau protein), NF-κB and TNF-α protein in lung tissues was determined by Western blot. Results Compared with S group, Res at T1-3 and W/D ratio at T3 were significantly increased, lung compliance, Flow and PaO2 were decreased at T1-3, and the expression of mTOR, NF-κB and TNF-α protein and mRNA, Tau protein mRNA and pS396 Tau protein was up-regulated at T3 in I/R, LC and HC groups(P<0.05). Compared with I/R group, Res at T1-3 and W/D ratio at T3 were significantly decreased, lung compliance, Flow and PaO2 were increased at T1-3, and the expression of mTOR, NF-κB and TNF-α protein and mRNA, Tau protein mRNA and pS396 Tau protein was down-regulated at T3 in LC and HC groups(P<0.05). Compared with LC group, Res at T1-3 and W/D ratio at T3 were significantly decreased, lung compliance, Flow and PaO2 were increased at T1-3, and the expression of mTOR, NF-κB and TNF-α protein and mRNA, Tau protein mRNA and pS396 Tau protein was down-regulated at T3 in HC group(P<0.05). The microscopic examination showed that the injury to lung tissues was significantly attenuated in LC and HC groups as compared with I/R group. ConclusionThe mechanism by which curcumin reduces I/R injury in isolated rat lungs is related to inhibiting mTOR signaling pathway.