抑制高迁移率组蛋白1对大鼠急性脊髓损伤后炎性反应的影响

Effect of inhibiting high mobility group box 1 on inflammatory response after acute spinal cord injury in rats

目的探讨抑制高迁移率组蛋白1(HMGB1)对大鼠急性脊髓损伤(ASCI)炎性反应的影响。方法选择健康雄性Wister大鼠32只,随机分为正常组、假手术组、模型组、甘草酸(Gly)组,每组8只。正常组大鼠不予任何刺激,假手术组大鼠只咬除T10椎板,不损伤脊髓,模型组和甘草酸组大鼠采用改良Allen’s法制备T10不完全性急性脊髓损伤模型,甘草酸组给予200mg/kg甘草酸灌胃处理,每天1次,连用3天。术后3天取各组大鼠T10段脊髓,应用ELISA法检测脊髓组织HMGB1、核转录因子κB(NF-κB)、肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、白介素-1β(IL-1β)水平,qRT-PCR法检测脊髓组织HMGB1、NF-κB、TNF-α、IL-6、IL-1βmRNA表达量。结果ELISA结果显示,脊髓损伤后,模型组大鼠脊髓组织HMGB1、NF-κB、TNF-α、IL-6、IL-1β水平较正常组明显升高[(3139.33±68.89)pg/mL比(1977.42±19.08)pg/mL、(2420.16±30.45)pg/mL比(1390.88±15.32)pg/mL;(244.61±5.49)pg/mL比(103.35±2.20)pg/mL、(271.91±3.24)pg/mL比(138.90±3.34)pg/mL、(109.89±3.33)pg/mL比(47.44±1.94)pg/mL,P均<0.01];甘草酸组大鼠脊髓组织HMGB1、NF-κB、TNF-α、IL-6、IL-1β水平较模型组明显降低[(2566.91±23.75)pg/mL比(3139.33±68.89)pg/mL、(1728.55±24.27)pg/mL比(2420.16±30.45)pg/mL、(177.50±4.86)pg/mL比(244.61±5.49)pg/mL、(190.77±1.95)pg/mL比(271.91±3.24)pg/mL、(66.38±3.62)pg/mL比(109.89±3.33)pg/mL,P均<0.01]。RT-qPCR结果表明,模型组大鼠脊髓组织HMGB1、NF-κB、TNF-α、IL-6、IL-1βmRNA水平较正常组明显升高[(1.25±0.08)比(0.57±0.02)、(1.66±0.04)比(0.76±0.02)、(1.26±0.06)比(0.53±0.03)、(1.73±0.08)比(0.94±0.07)、(1.82±0.07)比(1.01±0.06),P均<0.01];甘草酸组大鼠脊髓组织HMGB1、NF-κB、TNF-α、IL-6、IL-1βmRNA水平较模型组明显降低[(0.79±0.03)比(1.25±0.08)、(1.14±0.04)比(1.66±0.04)、(0.75±0.03)比(1.26±0.06)、(1.37±0.04)比(1.73±0.08)、(1.31±0.04)比(1.82±0.07),P均<0.01]。结论 HMGB1至少部分参与介导ASCI炎性反应,并与HMGB1依赖的NF-κB信号通路密切相关,甘草酸可能通过抑制HMGB1/NF-κB通路减轻大鼠急性脊髓损伤后的炎性反应。

Objective To investigate the effect of inhibiting high mobility group box 1(HMGB1) on the inflammatory response of acute spinal cord injury(ASCI) in rats. Methods Thirty-two healthy male Wister rats were randomly divided into normal group, sham operation group, model group, and glycyrrhizic acid(Gly) group, with 8 rats in each group. Rats in the normal group underwent no stimulation. Rats in the sham operation group were only removed the T10 vertebral plates and underwent no damage to the spinal cord. Rats in the model group and the glycyrrhizic acid group were used modified Allen’s method to prepare T10 incomplete acute spinal cord injury models, and Gly was administered at a dose of 200 mg/kg once a day for 3 days. T10 spinal cords of each group of rats were collected 3 days after operation, levels of HMGB1, nuclear transcription factor κB(NF-κB), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and interleukin-1β(IL-1β) were measured by ELISA. mRNA expression of HMGB1, NF-κB, TNF-α, IL-6, IL-1β were detected by qRT-PCR. Results The results of ELISA indicated that levels of HMGB1, NF-κB, TNF-α, IL-6 and IL-1β in model group were significantly higher than those in normal group, HMGB1: [(3139.33±68.89) vs(1977.44±19.08)pg/mL, P<0.01], NF-κB: [(2420.16±30.45) vs(1390.88±15.32)pg/mL, P<0.01], TNF-α: [(244.61±5.49) vs(103.35±2.20)pg/mL, P<0.01];IL-6: [(271.91±3.24)vs(138.90±3.34)pg/mL, P<0.01], IL-1β: [(109.89±3.33) vs(47.44±1.94)pg/mL, P<0.01];while levels of HMGB1,NF-κB, TNF-α, IL-6 and IL-1β in Gly group were significantly lower than those in model group, HMGB1:[(2566.91±23.75) vs(3139.33±68.89)pg/mL, P<0.01], NF-κB: [(1728.52±24.27) vs(2420.16±30.45)pg/mL, P<0.01], TNF-α: [(177.50±4.86) vs(244.61±5.49)pg/mL, P<0.01], IL-6: [(190.77±1.95) vs(271.91±3.24)pg/mL, P<0.01], IL-1β: [(66.38±3.62) vs(109.89±3.33)pg/mL, P<0.01]. The results of RT-qPCR showed that mRNA levels of HMGB1, NF-κB, TNF-α, IL-6 and IL-1β in model group were significantly higher than those in normal group,HMGB1: [(1.25±0.08) vs(0.57±0.02), P<0.01], NF-κB: [(1.66±0.04) vs(0.76±0.02), P<0.01], TNF-α: [(1.26±0.06) vs(0.53±0.03), P<0.01], IL-6: [(1.73±0.08) vs(0.94±0.07), P<0.01], IL-1β: [(1.82±0.07) vs(1.01±0.06),P<0.01];while mRNA levels of HMGB1, NF-κB, TNF-α, IL-6, IL-1β in Gly group were significantly lower than those in model group, HMGB1: [(0.079±0.03) vs(1.25±0.08), P<0.05], NF-κB: [(1.14±0.04) vs(1.66±0.04), P<0.01], TNF-α: [(0.75±0.03) vs(1.26±0.06), P<0.01], IL-6: [(1.37±0.04) vs(1.73±0.08), P<0.01], IL-1β: [(1.31±0.04) vs(1.82±0.07), P<0.01]. Conclusion HMGB1 is at least partly involved in mediating the ASCI inflammatory response and is strongly related to the HMGB1-dependent NF-κB signaling pathway. Gly may reduce the inflammatory response after acute spinal cord injury in rats by inhibiting the HMGB1/NF-κB pathway.