右美托咪定对呼吸机相关性肺损伤大鼠肺组织内质网应激的影响及PI3K/Akt 信号通路的关系

Effect of dexmedetomidine on endoplasmic reticulum stress in lung tissue of rats with ventilation-associated lung injury and its relationship with PI3K/Akt signaling pathway

目的探讨右美托咪定(DEX)对大鼠呼吸机相关性肺损伤(ventilator associated lung injury,VALI)时内质网应激(ERS)诱导的细胞凋亡及磷脂酰肌醇3-激酶/丝氨酸苏氨酸蛋白激酶(PI3K/Akt)信号通路的影响。方法将80只SD大鼠采用随机数字表法分为四组(n=20):对照组(C组)、机械通气组(V组)、右美托咪定组(D组)和右美托咪定+PI3K/Akt信号转导通路抑制剂(LY294002)组(DL组)。C组不行机械通气,自主呼吸空气;V组、D组、DL组机械通气4h,机械通气前20minD组静脉输注右美托咪定5.0μg/kg,机械通气期间以5.0μg/(kg·h)的速率静脉输注;DL组在给予右美托咪定前5min静脉输注LY2940020.3mg/kg;V组给予等容量生理盐水。机械通气4h时处死大鼠,测定肺通透指数(LPI)与肺湿/干质量(W/D)比值。采用蛋白质印迹(Westernblot)法检测Akt、磷酸化Akt(P-Akt)、葡萄糖调节蛋白78(GRP78)、CAAT增强子结合蛋白同源蛋白(CHOP)及半胱氨酸天冬氨酸蛋白酶12(caspase-12)的表达水平。光镜下观察肺组织病理学结果,测定肺泡损伤率(IAR),采用原位末端标记技术(TUNEL)法观察肺组织细胞凋亡情况并计算凋亡指数(AI)。结果与C组比较,V组与DL组W/D比、LPI、IAR(%)、AI(%)明显升高,GRP78、CHOP、caspase-12表达升高(C组:0.35±0.02、0.28±0.02、0.51±0.03;V组:1.27±0.11、1.09±0.13、1.37±0.15;DL组:0.97±0.08、0.66±0.07、1.01±0.09;P<0.05),P-Akt表达降低(C组:0.81±0.04;V组:0.23±0.01;DL组:0.44±0.02;P<0.05);与V组比较,DL组与D组W/D比、LPI、IAR(%)、AI(%)明显降低,GRP78、CHOP、caspase-12表达降低(V组:1.27±0.11、1.09±0.13、1.37±0.15;DL组:0.97±0.08、0.66±0.07、1.01±0.09;D组:0.62±0.04、0.43±0.05、0.79±0.07;P<0.05),P-Akt表达升高(V组:0.23±0.01;DL组:0.44±0.02;D组:0.65±0.03;P<0.05)且肺组织病理损伤减轻;与D组比较,DL组W/D比、LPI、IAR(%)、AI(%)明显升高,GRP78、CHOP、caspase-12表达升高(D组:0.62±0.04、0.43±0.05、0.79±0.07;DL组:0.97±0.08、0.66±0.07、1.01±0.09;P<0.05),P-Akt表达降低(D组:0.65±0.03;DL组:0.44±0.02;P<0.05),肺组织病理损伤较重。结论右美托咪定可减轻大鼠VALI,与其激活PI3K/Akt信号通路,抑制ERS,减少肺组织细胞凋亡有关。

Objective To investigate the effects of dexmedetomidine on endoplasmic retieulum stress-induced apoptosis and PI3K/Akt signaling pathway in rats with ventilation -associated lung injury.Methods Eighty Spragne -Dawly (SD)rats were randomly (random number)divided into 4 groups (n =20 each):the control group (Group C),Mechanical ventilation Group (Group V), Dexmedetomidine groups (Group D)and Dexmedetomidine +LY294002 group (group DL).Group C, no mechanical ventilation,and breathed independently;Mechanical ventilation in group V,D and DL for 4 h,dexmedetomidine 5.0 μg/(kg·h )were infused intravenously,20 min before mechanical ventilation,5.0 μg/(kg·h)intravenous infusion during mechanical ventilation.In the Group DL, LY294002 0.3 mg/kg was infused intravenously 5 min prior to dexmedetomidine.Group V was given normal saline.Rats were sacrificed at 4 hours after mechanical ventilation and the lung permeability index (LPI)and lung wet/dry weight (W/D)ratio were measured.Western blot was used to detect the level of Akt,phosphorylated Akt (P -Akt),glucose -regulated protein 78 (GRP78),CAAT enhancer binding protein homologous protein (CHOP),and caspase -12.The pathological changes of lung tissues were observed under light microscope,and the alveolar injury rate (IAR)was determined.The lung cell apoptosis was observed and apoptosis index was calculated by TUNEL method.Results Compared with the group C,ratio of W/D,LPI,IAR (%),AI (%)in group V and group DL increased,and the expressions of GRP78,CHOP,caspase -12 increased (group C:0.35 ±0.02,0.28 ±0.02,0.51 ±0.03;group V:1.27 ±0.11,1.09 ±0.13,1.37 ±0.15;group DL:0.97±0.08,0.66 ±0.07, 1.01 ±0.09;P <0.05),the expression of P-Akt decreased (group C:0.81 ± 0.04;group V: 0.23 ±0.01;group DL :0.44 ±0.02;P <0.05).Compared with the group V,group D and group DL Ratio of W/D,LPI,IAR(%),Al(%)decreased,and the expressions of GRP78,CHOP,caspase -12 decreased (group V :1.27 ± 0.11,1.09 ± 0.13,1.37 ±0.15;group DL:0.97 ± 0.08,0.66 ± 0.07, 1.01±0.09;group D:0.62 ±0.04,0.43 ±0.05,0.79 ±0.07;P <0.05),the expression of P-Akt increased (group V:0.23±0.01;group DL:0.44 ±0.02;group D:0.65 ±0.03;P <0.05),the pathological changes of lung tissues were significantly attenuated;Compared with the group D,ratio of W/D,LPI,IAR (%),AI (%)in group DL increased,and the expressions of GRP78,CHOP, caspase-12 increased (group D:0.62±0.04,0.43±0.05,0.79 +0.07;group DL:0.97 ±0.08, 0.66 ±0.07,1.01 ± 0.09;P <0.05 ),the expression of P -Akt decreased (group D :0.65 ± 0.03;group DL:0.44 ± 0.02;P <0.05 ),and the lung tissue pathological damage was heavier.Conclusion Dextomidine can alleviate ventilation -associated lung injury in rats,which is related to activation of PI3K/Akt signaling pathway,inhibition of endoplasmie reticulum stress,and reduction of apoptosis in lung tissue.