基于TLR4/MyD88/NF-κB通路探讨乌司他丁减轻脂多糖诱导的大鼠急性肺损伤的作用

doi:10.3969/j.issn.1006-6233.2019.08.018

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摘要目的:观察乌司他丁对脂多糖诱导的急性肺损伤小鼠保护作用及对TLR4/MyD88/NF-κB信号通路的影响。方法:将48只12~15周龄的SPF级Wistar雄性大鼠随机分为4组,模型组尾部静脉注射0.5mL的脂多糖(LPS)(5mg/kg)构建急性肺损伤(ALI)模型,对照组给予等体积的生理盐水,地塞米松(DXM)干预组和乌司他丁(UTI)干预组则在LPS注射前24h和10min时分别给予腹腔注射1mL的地塞米松(2mg/kg)和1mLUTI溶液(5mg/kg)进行预处理,对照组和模型组则给予等体积的生理盐水代替。计算10h大鼠体重下降比值和肺组织湿干重比(W/D),采用伊文斯兰方法检测肺血管内皮通透性,苏木精-伊红染色法(HE)染色观察大鼠肺部组织病变,并进行评分,ELISA法检测支气管灌洗液(BALF)中TNF-α、IL-6和IL-1β因子水平,同时显微镜下观察并统计总细胞和嗜中性粒细胞的个数,蛋白免疫印迹检测TLR4/MyD88/NF-κB信号通路中的TLR4、MyD88、NF-κB蛋白表达。结果:和对照组相比,模型组,DXM干预组和UTI干预组大鼠体重明显下降(P<0.05),和模型组相比,DXM干预组和UTI干预组大鼠体重明显增加,差异具有统计学意义(P<0.05)。和对照组相比,模型组大鼠在受到LPS刺激后,W/D比值出现明显升高(P<0.05);与模型组相比,DXM干预组和UTI干预组W/D比值比例明显下降(P<0.05)。和对照组相比,模型组BALF中的嗜中性粒细胞和总细胞数量出现明显的增加(P<0.05);和模型组相比,DXM组和UTI组BALF中的嗜中性粒细胞和总细胞数量出现明显的下降(P<0.05)。对照组肺组织结构完整,无充血,肺泡腔清晰,间隔内未发现水肿和炎性细胞浸润。和对照组相比,模型组的肺泡腔明显变小,间隔出现增厚,肺泡壁出现充血水肿,且发现了大量的炎性细胞浸润,HE评分明显高于对照组(P<0.05)。而DXM组、UTI组和模型组相比,肺组织结构接近于正常,炎性细胞浸润得到缓解,DXM组和UTI组HE评分明显低于模型组,差异具有统计学意义(P<0.05)。和对照组相比,模型组大鼠伊文斯兰浓度出现明显的升高(P<0.05),和模型组相比,DXM组和UTI组大鼠伊文斯兰浓度出现明显下降,差异具有统计学意义(P<0.05)。和对照组相比,模型组中IL-1β、IL-6和TNF-α水平出现明显的升高(P<0.05);与模型组相比,DXM组和UTI组大鼠BALF中IL-1β、IL-6和TNF-α水平出现明显的下降,差异具有统计学意义(P<0.05)。和对照组相比,模型组中TLR4、MyD88、NF-κB蛋白表达明显升高(P<0.05);和模型组相比,DXM组和UTI组大鼠肺组织中TLR4、MyD88、NF-κB蛋白表达明显下降,差异具有统计学意义(P<0.05)。结论:乌司他丁可以抑制LPS所致的TLR4/MyD88/NF-κB信号通路的激活,从而减轻炎症反应,发挥对LPS致肺损伤的保护作用。

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	王兮
	母前途
	冯思嘉

关键词 ：乌司他丁, 急性肺损伤, 脂多糖, TLR4, MyD88, NF-κB

Abstract：Objective: To observe the ulinastatin on acute lung injury induced by lipopolysaccharide in mice protection and of TLR4/MyD88/NF-κB signaling pathway. Methods: 48 SPF Wistar male rats aged 12-15 weeks were randomly divided into four groups. The acute lung injury (ALI) model was established by intravenous injection of 0.5mL lipopolysaccharide (LPS) (5mg/kg) into the tail of the model group. The control group was given saline of equal volume. The dexamethasone (DXM) intervention group and ulinastatin (UTI) intervention group were given intraperitoneal injection of 1 mL dexamethasone (2 mg/kg) and 1 mL dexamethasone (1 mL) 24 hours and 10 minutes before LPS injection, respectively. UTI solution (5 mg/kg) was pretreated, while the control group and the model group were given saline of equal volume instead. The weight loss ratio and wet-dry weight ratio (W/D) of lung tissue were calculated for 10 hours. Pulmonary vascular endothelial permeability was measured by Evanslan method. Lung pathological changes were observed by hematoxylin-eosin staining (HE) and scored. The levels of TNF-alpha, IL-6 and IL-1 beta in bronchial lavage fluid (BALF) were detected by ELISA. The number of total cells and neutrophils was observed and counted under microscope. The expression of TLR4, MyD88 and NF-kappa B proteins in TLR4/MyD88/NF-kappa B signaling pathway was detected by Western blot. Results: Compared with control group, model group and DXM intervention group rats weight and UTI intervention group were obviously decreased (P<0.05), compared with model group, DXM intervention group and UTI intervention group obviously increased (P<0.05). Compared with control group, model group rats after by LPS stimulation, W/D ratio increased significantly (P<0.05); Compared with model group, DXM intervention group and UTI intervention group W/D ratio significantly decreased (P<0.05). Compared with control group, model group appeared the number of neutrophil and total cells in BALF significantly increased (P<0.05); Compared with model group, DXM group and ulinastatin group the number of neutrophil and total cells in BALF of obvious declined(P<0.05).Compared with control group, model group rats with Evans blue concentration appear obviously increased (P<0.05), compared with model group and DXM group and UTI group rats Evans blue concentration significantly decline, statistically significant difference (P<0.05). The control structure of lung tissue integrity, no congestion, the alveolar cavity was clear, interval found no edema and inflammatory cell infiltration. Compared with control group, the mo new alveolar cavity decreased significantly, interval appeared thickened, alveolar walls appeared hyperemia and edema, and found a lot of inflammatory cells infiltration, HE score significantly higher than the control group (P<0.05). And DXM group, ulinastatin group compared with model group, was close to normal lung tissue structure, inflammatory cells infiltration, DXM group and ulinastatin group HE scores significantly lower than the model group, the difference was statistically significant (P<0.05). Compared with control group, model group of IL-6 and IL-1,TNF-αlevel appear obviously increased (P<0.05); Compared with model group and DXM group and ulinastatin group rat BALF of IL-6 and IL-1, TNF-αlevel appear obvious dropped, statistically significant difference (P<0.05). Compared with control group, model group of TLR4, MyD88, NF-κB protein expression increased significantly (P<0.05); Compared with model group, TLR4, MyD88, NF-κB protein expression in DXM group and ulinastatin group decreased obviously, statistically significant difference (P<0.05). Conclusion: Ulinastatin can inhibit the activation of TLR4/MyD88/NF-kappa B signaling pathway induced by LPS, thereby alleviating inflammatory response and exerting protective effect on LPS-induced lung injury.

Key words： Ulinastatin Acute lung injury Lipopolysaccharide TLR4 MyD88 NF-κB

通讯作者: 母前途

引用本文:

王兮, 母前途, 冯思嘉. 基于TLR4/MyD88/NF-κB通路探讨乌司他丁减轻脂多糖诱导的大鼠急性肺损伤的作用[J]. 河北医学, 2019, 25(8): 1299-1303.
WANG Xi, MU Qiantu, et al. Effect of Ulinastatin on Lipopolysaccharide-induced Acute Lung Injury in Rats based on TLR4/MyD88/NF-kappa B pathway. HeBei Med, 2019, 25(8): 1299-1303.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2019.08.018 或 http://www.hbyxzzs.cn/CN/Y2019/V25/I8/1299

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