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目的 探讨α2A-肾上腺素能受体(α2A-AR)阻断剂BRL-44408 maleate对脂多糖(LPS)诱导小鼠急性肺损伤(ALI)的影响及作用机制.方法 按照随机数字表法将60只雄性C57BL/6小鼠分为假手术组(Sham组)、LPS组、BRL-44408 maleate预处理组(BRL+LPS组),每组20只.采用气管内滴注LPS 5 mg/kg的方法复制ALI小鼠模型;Sham组滴注等量生理盐水(NS).BRL+LPS组于制模前4 h腹腔注射5 mg/kg的BRL-44408 maleate.分别于制模后6 h和24 h各取10只小鼠,其中5只用于检测支气管肺泡灌洗液(BALF)中去甲肾上腺素(NE)、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-6、IL-10)水平〔采用酶联免疫吸附试验(ELISA)〕和总蛋白含量(BCA法);另外5只用于观察肺组织病理学改变,测定肺湿/干重(W/D)比值和丝裂素活化蛋白激酶激酶(MEK)及其下游细胞外信号调节激酶(ERK)表达〔蛋白质免疫印迹试验(Western Blot)〕.结果 与Sham组比较,LPS组制模后6 h肺损伤即明显加重,肺组织病理评分明显增加,肺W/D比值和BALF中总蛋白含量、NE、TNF-α、IL-6、IL-10水平及磷酸化MEK(p-MEK)、磷酸化ERK(p-ERK)表达均明显升高〔肺组织病理评分(分):0.70±0.04比0.14±0.13,肺W/D比值:4.79±0.15比4.35±0.17,总蛋白含量(g/L):1.51±0.36比0.46±0.13,NE(ng/L):85.02±11.28比47.18±10.30,TNF-α(ng/L):186.61±21.93比9.18±2.86, IL-6(ng/L):193.45±26.54比13.58±2.54,IL-10(ng/L):113.46±31.23比25.66±9.41,p-MEK/β-actin:0.246±0.019比0.178±0.030,p-ERK/β-actin:0.257±0.013比0.175±0.014,均1<0.05〕,且随着制模时间延长均呈升高趋势.与LPS组比较,BRL+LPS组制模后6 h即可明显改善肺组织的损伤程度,降低肺组织病理评分(分:0.61±0.05比0.70±0.04),降低肺W/D比值(4.51±0.22比4.79±0.15),抑制BALF中NE、TNF-α、IL-6的表达〔NE(ng/L):55.77±15.86比85.02±11.28,TNF-α(ng/L):54.79±12.68比186.61±21.93,IL-6 (ng/L):67.66±20.08比193.45±26.54〕,抑制肺组织MEK、ERK的活化(p-MEK/β-actin:0.204±0.008比0.246±0.019,p-ERK/β-actin:0.186±0.024比0.257±0.013),差异均有统计学意义(均1<0.05),而BALF中总蛋白含量和IL-10水平差异并无统计学意义.结论 α2A-AR特异性阻断剂BRL-44408 maleate可以显著改善小鼠内源性ALI,其机制可能是通过抑制MEK/ERK信号通路实现的.

Objective To explore the effects and mechanism of α2A-adrenergic receptor (α2A-AR) antagonist BRL-44408 maleate on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Methods Sixty male C57BL/6 mice were randomly divided into three groups (n = 20): sham group, LPS group and BRL-44408 maleate pre-treated group (BRL+LPS group). The model of ALI was replicated by intratracheally administrated of LPS (5 mg/kg), and the mice in the sham group were received an equal volume of saline. Mice in the BRL+LPS group were treated with additionally BRL-44408 maleate (5 mg/kg, i.p) at 4 hours before LPS administration. The mice were sacrificed at 6 hours and 24 hours after LPS administration in each group. Among them, 5 mice were used to collect the bronchoalveolar lavage fluid (BALF) and the other 5 mice were sacrificed for lung tissues. The levels of norepinephrine (NE), tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-10) in BALF were measured by enzyme linked immunosorbent assay (ELISA). The level of protein in BALF was measured by bicinchoninic acid (BCA) method. The histopathological changes and wet/dry (W/D) ratio of lung tissue were observed. The expression of lung phosphorylated mitogen-activated protein kinase kinase (p-MEK) and phosphorylated extracellular regulated protein kinases (p-ERK) were detected by Western Blot. Results Compared with the sham group, the lung histopathological injury was significantly aggravated, and the histopathological injury score was significantly increased, the lung W/D ratio, and total protein content, NE, TNF-α, IL-6, IL-10 in BALF, and p-MEK and p-ERK expressions were significantly increased in LPS group at 6 hours after model setup [the lung histopathological injury score: 0.70±0.04 vs. 0.14±0.13,W/D ratio: 4.79±0.15 vs. 4.35±0.17, protein content (g/L): 1.51±0.36 vs. 0.46±0.13, NE (ng/L): 85.02±11.28 vs.47.18±10.30, TNF-α (ng/L): 186.61±21.93 vs. 9.18±2.86, IL-6 (ng/L): 193.45±26.54 vs. 13.58±2.54, IL-10 (ng/L): 113.46±31.23 vs. 25.66±9.41, p-MEK/β-actin: 0.246±0.019 vs. 0.178±0.030, p-ERK/β-actin:0.257±0.013 vs. 0.175±0.014, all 1 < 0.05], and increase with time after model setup. Compared with the LPS group,BRL-44408 maleate pretreatment for 6 hours could significantly improve the degree of lung injury and reduce the lung histopathological injury score (0.61±0.05 vs. 0.70±0.04), reduce lung W/D weight ratio (4.51±0.22 vs. 4.79±0.15);the expression of NE, TNF-α, IL-6 in BALF were inhibited [NE (ng/L): 55.77±15.86 vs. 85.02±11.28, TNF-α (ng/L): 54.79±12.68 vs. 186.61±21.93, IL-6 (ng/L): 67.66±20.08 vs. 193.45±26.54], in addition, the up-regulation of p-MEK, p-ERK were significantly inhibited (p-MEK/β-actin: 0.204±0.008 vs. 0.246±0.019, p-ERK/β-actin:0.186±0.024 vs. 0.257±0.013), with statistically significant differences (all 1 < 0.05). The protein content and the expression of IL-10 in BALF showed no significant difference. Conclusion α2A-AR blocker BRL-44408 maleate could alleviate endogenous ALI induced by LPS in mice by inhibiting the MEK/ERK pathway.