抗菌肽LL-37和维生素D受体在呼吸机相关性肺损伤大鼠肺组织中的表达及作用机制

Expression and mechanism of antimicrobial peptide LL-37 and vitamin D receptor in lung tissue of rats with ventilator-associated lung injury

目的探讨抗菌肽LL-37和维生素D受体(VDR)在呼吸机相关性肺损伤(VILI)大鼠肺组织表达及作用机制。方法30只洁净级SD大鼠(雌雄各半)随机分为自主呼吸组,正常潮气量组(VT=6 ml/kg)和大潮气量组(VT=40 ml/kg),每组10只,雌雄各半,3组大鼠经腹腔注射麻醉后行气管切开插管术。其中自主呼吸组保持大鼠自主呼吸,正常潮气量组按6 ml/kg给予机械通气,大潮气量组按40 ml/kg给予机械通气,4 h后经心脏穿刺放血,处死大鼠。采用精密电子秤称量肺湿干比重(W/D),HE染色观察肺形态变化,透射电镜观察Ⅱ型肺泡超微结构,ELISA检测血清和BALF中的白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、髓过氧化物酶(MPO)和人巨噬细胞炎性蛋白2(MIP2)促炎因子的水平,Western-blot检测肺组织中抗菌肽LL-37和维生素D受体蛋白表达。结果大潮气量组W/D明显高于自主呼吸组和正常潮气量组(P<0.05),而正常潮气量组W/D明显高于自主呼吸组(P<0.05);HE染色结果显示大潮气量组有明显的水肿、肺间质变宽,且肺泡充血,同时伴随有大量的炎性细胞浸润,肺泡组织紊乱,大潮气量组病理评分明显高于自主呼吸组和正常潮气量组(P<0.05),而正常潮气量组病理评分明显高于自主呼吸组(P<0.05);大潮气量组AT-Ⅱ细胞的细胞膜、细胞质以及细胞核与自主呼吸组和正常潮气量组相比出现明显改变,细胞膜微绒毛随着潮气量增大而减少,薄膜增厚,细胞质中的板层小体数量减少,分布不均,细胞核形态不一,出现固缩;大潮气量组血清和BALF中IL-6、TNF-α、MPO和MIP2明显高于自主呼吸组和正常潮气量组(P<0.05),而正常潮气量组血清和BALF中IL-6、TNF-α、MPO和MIP2明显高于自主呼吸组(P<0.05);大潮气量组LL-37和VDR明显低于自主呼吸组和正常潮气量组(P<0.05),而正常潮气量组LL-37和VDR明显低于自主呼吸组(P<0.05)。结论机械通气引起的肺损伤会导致抗菌肽LL-37和维生素D受体表达下调,免疫功能紊乱,从而介导或者加重VILI。

Objective To investigate the expression and action mechanism of antibacterial peptide LL-37 and vitamin D receptor(VDR)in lung tissue of rats with ventilator-associated lung injury(VILI).Methods Thirty clean SD rats(half males and half females)were randomly divided into spontaneous breathing group,normal tidal volume group(VT=6ml/kg)and high tidal volume group(VT=40ml/kg),with 10 rats in each group.The rats in the three groups underwent tracheotomy and intubation after intraperitoneal injection anesthesia.The rats in spontaneous breathing group maintained the spontaneous breathing,the rats in normal tidal volume group were given mechanical ventilation at 6ml/kg,and the rats in high tidal volume group were given mechanical ventilation at 40ml/kg.After 4 hours,the rats were sacrificed by heart puncture and bloodletting.The wet-dry specific gravity(W/D)of the lung was measured by a precision electronic scale.The morphological changes of the lung were observed by HE staining.The ultrastructure of typeⅡalveoli was observed by transmission electron microscopy.The serum levels of IL-6,TNF-α,MPO and MIP2 proinflammatory factors and BALF were detected by ELISA.The expression levels of antibacterial peptide LL-37 and vitamin D receptor protein in lung tissue were detected by Western Blot.Results The W/D ratio in high tidal volume group was significantly higher than that in spontaneous breathing group and normal tidal volume group(P<0.05),however,the W/D ratio in normal tidal volume group was significantly higher than that in spontaneous breathing group(P<0.05).HE staining results showed that the high tidal volume group had obvious edema,pulmonary interstitial widening,alveolar hyperemia,accompanied by a large number of inflammatory cell infiltration and alveolar tissue disorder.The pathological scores in high tidal volume group were significantly higher than those in spontaneous breathing group and normal tidal volume group(P<0.05),and the pathological scores in normal tidal volume group were significantly higher than those in spontaneous breathing group(P<0.05).As compared with those in spontaneous breathing group and normal tidal volume group,the cell membrane,cytoplasm and nucleus of AT-Ⅱcells in high tidal volume group had more obvious changes.The microvilli of cell membrane was decreased with the increase of tidal volume,the membrane thickens,the number of lamellar bodies in cytoplasm was decreased,and the distribution was uneven,the shape of nucleus was different,with solid shrinkage.The serum levels of IL-6,TNF-α,MPO and MIP2 and BALF in high tidal volume group were significantly higher than those in spontaneous breathing group and normal tidal volume group(P<0.05),moreover,which in normal tidal volume group were significantly higher than those in spontaneous breathing group(P<0.05).In addition the serum levels of LL-37 and VDR in high tidal volume group were significantly lower than those in spontaneous breathing group and normal tidal volume group(P<0.05),moreover,which in normal tidal volume group were significantly lower than those in spontaneous breathing group(P<0.05).Conclusion The lung injury of rats caused by mechanical ventilation can result in the down-regulation of antibacterial peptide LL-37 and vitamin D receptor expression and immune dysfunction,so as to mediate or aggravate the VILI.