β1受体阻滞剂通过TLR4/NF-κB信号通路抑制脓毒症心肌炎症反应

β1 receptor blocker decreases the myocardial inflammation in the sepsis adult rats through inhibition of TLR4/NF-κB signaling pathway

目的探讨β1受体阻滞剂是否通过Toll样受体4?/核转录因子-κB (TLR4/NF-κB)信号通路抑制脓毒症大鼠心肌炎症反应。方法按随机数字表法将3月龄250~300 g清洁级雄性Wistar大鼠分配至假手术组(S组)、脓毒症模型组(CLP组)、β1受体阻滞剂艾司洛尔干预组(ES组)、TLR4受体拮抗剂干预组(E5564组),每组15只。采用盲肠结扎穿孔术(CLP)制备脓毒症动物模型;S组仅剖腹,不进行盲肠结扎、穿孔。S组、CLP组、E5564组关腹后皮下注射0.9%氯化钠(NaCl)2.0 mL/kg补液;除皮下补液外,ES组经尾静脉持续泵入艾司洛尔15 mg·kg-1·h-1,E5564组于术前1 h经尾静脉持续泵入E5564 0.3 mg·kg-1·h-1。各组于制模后6 h采集标本,采用PU电传导心电监护仪监测平均动脉压(MAP)及心排血指数(CI),采用酶联免疫吸附试验(ELISA)检测血清心肌肌钙蛋白I(cTnI)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)水平,采用蛋白质免疫印迹试验(Western Blot)检测心肌组织TLR4、NF-κB p65、IL-1β、TNF-α蛋白表达。结果各组大鼠MAP比较差异均无统计学意义。与S组相比,CLP组CI明显降低,血清cTnI、IL-1β、TNF-α水平明显升高,心肌组织TLR4、NF-κB p65、IL-1β、TNF-α蛋白表达水平明显上调。与CLP组比较,ES组和E5564组CI均明显升高(mL·s-1·m-2:58.6±4.3、58.9±4.4比41.2±3.9,均P<0.01),血清cTnI、IL-1β、TNF-α水平明显降低〔cTnI(μg/L):1113.81±26.64、1?115.74±25.90比1975.96±42.74,IL-1β(ng/L):39.6±4.3、38.9±4.4比61.2±3.9,TNF-α(ng/L):43.1±2.8、48.7±2.6比81.3±4.4,均P<0.01〕,心肌组织NF-κB p65、IL-1β、TNF-α蛋白表达水平显著下调(NF-κB p65/β-actin:0.31±0.03、0.43±0.04比0.85±0.08,IL-1β/β-actin:0.28±0.05、0.32±0.03比0.71±0.06,TNF-α/β-actin:0.18±0.04、0.28±0.03比0.78±0.07,均P<0.01),而TLR4蛋白表达水平差异无统计学意义(TLR4/β-actin:0.89±0.07、0.87±0.09比0.95±0.09,均P>0.05)。ES组与E5564组CI和血清cTnI、IL-1β、TNF-α水平以及心肌组织TLR4、NF-κB p65、IL-1β、TNF-α蛋白表达水平比较差异均无统计学意义(均P>0.05)。结论β1受体阻滞剂艾司洛尔可能通过TLR4/NF-κB信号通路抑制脓毒症大鼠心肌炎症反应,从而减轻脓毒症心肌损伤。

Objective To explore whether β1 receptor blocker could decrease the myocardial inflammation through the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling pathway in the sepsis adult rats. Methods Sixty male Wistar rats (250-300 g) aged 3 months old were allocated to four groups by random number table (n = 15): sham operation group (S group), sepsis model group (CLP group),β1 receptor blocker esmolol intervention group (ES group), and inhibitor of the TLR4 E5564 intervention group (E5564 group). The rat sepsis model was established by cecal ligation and puncture (CLP);S group of rats underwent only an incision. Rats in S group, CLP group and E5564 group were subcutaneous injected with 0.9% sodium chloride (NaCl) 2.0 mL/kg. Besides, the rats in ES group were injected with esmolol (15 mg·kg-1·h-1) by micro pump through the caudal vein. The rats in E5564 group were injected with E5564 (0.3 mg·kg-1·h-1) by micro pump through the caudal vein 1 hour before the CLP surgery. Samples were collected 6 hours after the modelling in each group. The average arterial pressure (MAP) and cardiac output index (CI) were monitored by PU electrical conduction ECG monitor. The levels of serum cardiac troponin I (cTnI), interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were detected by enzyme linked immunosorbent assay (ELISA). The expressions of TLR4, NF-κB p65, IL-1β, TNF-α in myocardial tissue was detected by Western Blot. Results There was no significant difference in MAP in each group. Compared with the S group, the CI in the CLP group was significantly decreased, the levels of serum cTnI, IL-1β, TNF-α were significantly increased, the protein expressions of myocardial tissue TLR4, NF-κB p65, IL-1β and TNF-α were significantly increased. Compared with the CLP group, the CI in the ES group and E5564 group were significantly increased (mL·s-1·m-2: 58.6±4.3, 58.9±4.4 vs. 41.2±3.9, both P < 0.01), the levels of serum cTnI, IL-1β and TNF-α were significantly decreased [cTnI (μg/L): 1113.81±26.64, 1?115.74±25.90 vs. 1975.96±42.74;IL-1β(ng/L): 39.6±4.3, 38.9±4.4 vs. 61.2±3.9;TNF-α(ng/L): 43.1±2.8, 48.7±2.6 vs. 81.3±4.4, all P < 0.01], the protein expressions of myocardial tissue NF-κB p65, IL-1β, TNF-αwere significantly decreased (NF-κB p65/β-actin: 0.31±0.03, 0.43±0.04 vs. 0.85±0.08;IL-1β/β-actin: 0.28±0.05, 0.32±0.03 vs. 0.71±0.06;TNF-α/β-actin: 0.18±0.04, 0.28±0.03 vs. 0.78±0.07, all P < 0.01), but there was no significant difference in protein expression of TLR4 (TLR4/β-actin: 0.89±0.07, 0.87±0.09 vs. 0.95±0.09, both P > 0.05). There was no significant difference in CI, the levels of serum cTnI, IL-1β, TNF-α, and the protein expressions of myocardial tissue TLR4, NF-κB p65, IL-1β, TNF-αbetween ES group and E5564 group (all P > 0.05). Conclusion β1 receptor blocker esmolol may inhibit myocardial inflammatory response in sepsis adult rats through TLR4/NF-κB signaling pathway, thereby alleviating sepsis-induced myocardial injury.