不同剂量瘦素对大鼠机械通气所致肺损伤气道黏蛋白MUC5AC的影响

Effects of different doses of leptin on mucin MUC5AC expression of rats with ventilator‑induced lung injury

目的评价不同剂量瘦素对大鼠机械通气肺损伤(ventilator‑induced lung injury,VILI)气道黏蛋白MUC5AC的影响。方法采用随机数字表法将48只6~8周龄健康清洁级SD雄性大鼠分为4组(每组12只):假手术组(A组,气管切开保留自主呼吸)、机械通气模型组(B组)、小剂量瘦素组(C组,10μg/kg)、大剂量瘦素组(D组,50μg/kg)。B组、C组、D组采用机械通气建立VILI模型:潮气量20 ml/kg,通气频率80次/min,吸呼比1∶1,FiO221%,呼气末正压(positive end‑expiratory pressure,PEEP)0 mmHg(1 mmHg=0.133 kPa),通气时间4 h。C组、D组腹腔注射瘦素,A组、B组腹腔注射等容量生理盐水,注射后即刻开始通气。分别于气管插管前即刻(T1)、通气结束后(T2)股动脉取血进行血气分析。造模后收集支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF),测定BALF中TNF‑α、IL‑6、IL‑1β含量,并进行中性粒细胞计数;取肺组织称重,计算肺湿/干重比(wet/dry weight,W/D);观察肺组织病理改变并进行病理评分,H‑E染色观察肺组织形态改变,免疫组织化学法检测气道黏蛋白MUC5AC;检测肺组织研磨液中NF‑κB p65水平及气道黏蛋白MUC5AC蛋白含量。结果C组与D组病理改变较B组明显减轻,其中D组较C组减轻。与T1时比较,B组、C组、D组T2时PaO2降低(P<0.01)。与A组比较,B组、C组、D组T2时PaO2降低(P<0.01)。与A组比较,B组、C组、D组W/D、肺损伤评分、中性粒细胞计数升高(P<0.01)。C组与D组肺损伤评分、BALF中性粒细胞计数均低于B组(P<0.05)。D组肺损伤评分、中性粒细胞计数明显低于C组(P<0.05)。与A组比较,B组、C组、D组BALF中TNF‑α、IL‑6、IL‑1β含量升高(P<0.01)。C组与D组BALF中TNF‑α、IL‑6、IL‑1β含量均低于B组(P<0.05),其中D组均低于C组(P<0.05)。C组与D组气道黏蛋白MUC5AC含量、NF‑κB p65灰度值均低于B组(P<0.05),其中D组均低于C组(P<0.05)。结论瘦素可降低大鼠VILI中炎症因子的含量,降低肺组织气道黏蛋白MUC5AC的水平,减轻肺损伤,较大剂量(50μg/kg)作用明显。

Objective To evaluate the effects of different doses of leptin on mucin MUC5AC in rats with ventilator‑induced lung injury(VILI).Methods According to the random number table method,forty eight healthy adult male SD rats(aged 6 to 8 weeks)were divided into four groups(n=12):a sham‑operated group(group A,with spontaneous breathing after tracheal incision),a VILI group(group B),a small‑dose leptin group(group C,10μg/kg),and a large‑dose group(group D,50μg/kg).Rats in groups B,C and D were mechani‑cally ventilated to establish a model of VILI:tidal volume 20 ml/kg,ventilation frequency 80 bpm,inspiration‑expiration ratio:1∶1,FiO221%,and positive‑end expiratory pressure(PEEP)0 mmHg(1 mmHg=0.133 kPa),ventilation duration 4 h.Groups C and D were intraperitoneally injected with leptin,while groups A and B were intraperitoneally injected with the same volume of normal saline.Ven‑tilation started immediately after injection.Blood samples were collected from the femoral artery immediately before tracheal intubation(T1)and after the end of ventilation(T2).After modeling,bronchoalveolar lavage fluid(BALF)was collected to determine the amounts of tumor necrosis factor‑α(TNF‑α),interleukin‑6(IL‑6),and IL‑1β,and the number of neutrophils was counted.The lung specimens were obtained to determine the wet/dry weight(W/D)ratio.The pathological changes in lung tissues were observed and scored.The morpho‑pathogical changes in the lungs were observed by hematoxylin‑eosin(HE)staining.The expression of mucin MUC5AC was measured by immunohistochemical assay.The levels of nuclear factor‑κB(NF‑κB)p65 and mucin MUC5AC in lung tissue were detected.Results Groups C and D presented relieved pathological changes compared with group B,where declined pathological changes were found in group D compared with group C.Compared with T1,PaO2 decreased in group B,group C and group D at T2(P<0.01).Compared with group A,PaO2 in group B,group C and group D decreased at T2(P<0.01).Compared with group A,W/D,lung injury score and neutrophil count of group B,group C and group D increased(P<0.01).The lung injury score and BALF neutrophil count in group C and D were low‑er than those in group B(P<0.05).The lung injury score and neutrophil count in group D were significantly lower than those in group C(P<0.05).Compared with group A,TNF‑α,IL‑6 and IL‑1βin BALF of group B,C and D were increased(P<0.01).The contents of TNF‑α,IL‑6 and IL‑1βin BALF in group C and D were lower than those in group B(P<0.05),and those in group D were lower than those in group C(P<0.05).The levels of MUC5AC and NF‑κB p65 in group C and D were lower than those in group B(P<0.05),and those in group D were lower than those in group C(P<0.05).Conclusions Leptin can inhibit the expression of inflammatory factors in rats with VILI,reduce the expression of mucin MUC5AC,and relieve pulmonary injury.More obvious effects can be found after injection with a higher dose(50μg/kg).