甘草酸苷对大鼠脑创伤后高迁移族蛋白B1表达的影响

发布时间：2018-06-25 07:32

  本文选题：高迁移率族蛋白B1 + 创伤性脑损伤　； 参考：《南京医科大学》2016年博士论文

【摘要】：第一部分急性创伤性脑损伤患者血浆中HMGB1的表达及临床意义目的：探讨急性创伤性脑损伤患者血浆中高迁移率簇蛋白B1 (High-mobility group box 1,HMGB1)的表达以及临床意义。方法：选取2012年1月至2012年6月住院的急性创伤性脑损伤患者142例,轻度脑创伤组68例(GCS 13～15分)；中度脑创伤组48例(GCS 9-12分)；重度脑创伤组26例(GCS 3-8分)。所有患者均于伤后4-6小时内采集上肢静脉血,并采用酶联免疫吸附测定法(Enzyme Linked ImmunoSorbent Assay, ELISA)测定血浆中HMGB1的表达。并且选取同期体检健康正常者15例作为对照组。比较脑创伤组和对照组两者HMGB1的表达水平,并分析急性创伤性脑损伤患者伤后1个月内死亡和6个月内功能预后不良的危险因素。结果：急性创伤性脑损伤患者血浆中HMGB1水平均比对照组明显增高(P0.01)；血浆中HMGB1水平与入院时GCS评分呈显著负相关(P0.01)。单因素Logistic回归分析显示,血浆中HMGBl水平是急性创伤性脑损伤患者伤后1个月内死亡和6个月内功能预后不良的危险因素。ROC曲线分析显示血浆中HMGB1水平对急性创伤性脑损伤患者伤后1个月内死亡和6个月内功能预后不良有显著预测价值。结论：急性创伤性脑损伤患者血浆中HMGB1表达水平增高,且其增高的程度与脑伤情的轻重呈正相关,血浆中HMGB1的水平可作为评估脑创伤患者预后的一项重要观察指标。第二部分HMGB1在实验性大鼠急性创伤性脑损伤中的表达目的：探讨实验性大鼠急性创伤性脑损伤后HMGB1在血浆和脑组织中的表达情况,以及脑水肿和细胞凋亡情况。方法：大鼠随机分为7组：假手术组、脑创伤2h组、脑创伤6h组、脑创伤12h组、脑创伤24h组、脑创伤48h组、脑创伤72h组(每组12只),改良Feeney重物自由落体法建立大鼠脑创伤模型,ELISA检测血浆中HMGB1的表达,免疫组化染色观察HMGB1阳性细胞的表达,湿/干法测定脑水含量,TUNEL染色检测细胞凋亡。结果：血浆中HMGB1在假手术组为0.83±0.15ng/ml,伤后2h为3.50±0.57ng/m1(与假手术组相比P0.01),伤后6h为7.87±0.60ng/ml(与伤后2h相比尸0.01),伤后12h为7.85±0.59ng/ml,伤后24h为7.6l±0.66ng/ml(脑创伤6h组、脑创伤12h组、脑创伤24h组各组间相比尸0.05),伤后48h为4.63±0.68ng/ml(与伤后24h相比P0.01),伤后72h为3.11±0.66ng/ml(与伤后48h相比P0.01);HMGB1阳性细胞在假手术组为7.49±1.76%,伤后2h为36.54±5.92%(与假手术组相比P0.01),伤后6h为65.58±5.46%(与伤后2h相比尸0.01),伤后12h为65.18±4.83%,伤后24h为64.68±4.09%(脑创伤6h组、脑创伤12h组、脑创伤24h组各组间相比P0.05),伤后48h为43.17±4.48%(与伤后24h相比P0.01),伤后72h为36.50±4.92%(与伤后48h相比P0.01)；伤侧脑组织含水量在假手术组为79.21±0.96%,伤后2h为79.27±0.92%(与假手术组相比尸0.05),伤后6h为81.00±0.85%(与伤后2h组相比P0.01),伤后12h为82.26±0.95%(与伤后6h组相比P0.05),伤后24h为83.54±0.93%(与伤后12h组相比P0.05),伤后48h为82.31±0.50%(与伤后24h组相比P0.05),伤后72h为81.22±0.88%(与伤后48h组相比P0.05)；细胞凋亡在假手术组为6.62±1.38%,伤后2h为8.73±1.52%(与假手术组相比P0.05),伤后6h为35.11±4.72%(与伤后2h组相比P0.01),伤后12h为43.23±4.61%(与伤后6h组相比P0.05),伤后24h为52.29±4.76%(与脑创伤12h组相比尸0.01),伤后48h为39.91±4.66%(与脑创伤24h组相比P0.01),伤后72h为32.56±4.85%(与脑创伤48h组相比P0.05)。结论：大鼠脑创伤后血浆中HMGB1在伤后2h开始升高,伤后6h达到峰值,持续24h,伤后48h开始明显下降；大鼠脑创伤后2h HMGB1由细胞核向细胞质分布,伤后6h达到峰值,持续24h,伤后48h开始明显下降；大鼠脑创伤后6h伤侧脑组织含水量开始升高,至伤后24h达高峰,伤后48h开始有所缓解；大鼠脑创伤后伤后6h凋亡细胞逐步增多,伤后24h达到最多,伤后48h逐步减少。第三部分甘草酸苷对大鼠脑创伤后HMGB1表达的影响及其作用机制探讨目的：探讨甘草酸苷对大鼠创伤性脑损伤的神经保护作用和对高迁移率族蛋白B1 (HMGB1)的影响。方法：雄性SD大鼠随机分为三组：假手术组、脑创伤组、甘草酸苷组(每组36只)。采用改良Feeney法建立大鼠脑创伤模型。在甘草酸苷组,伤后30分钟静脉注射10mg/kg甘草酸苷。伤后24小时行运动功能、脑组织含水量测定,同时在损伤部位周围的脑组织采样,用RT-PCR、Western blot、 EMSA和ELISA方法检测HMGB1/HMGB1的受体(TLR4和RAGE)/NF-κB的信号通路和炎性细胞因子,并且分析HMGB1、RAGE和TLR4免疫组化和细胞凋亡的情况。结果：脑创伤组明显引起运动功能障碍(与假手术组相比P0.01),甘草酸苷组运动功能受损明显减轻(与脑创伤组比较,P0.01)；损伤侧脑组织含水量在假手术组为79.97±0.82%,在脑创伤组为82.94±0.65%(与假手术组相比,P0.01),在甘草酸苷组为80.97±0.49%(与脑创伤组相比,P0.01)；创伤性脑损伤区周围脑组织HMGB1/HMGB1受体(TLR.4和RAGE) /NF-κB信号通路表达显著增加(与假手术组相比,各指标P0.01)。甘草酸苷治疗后,HMGB1/HMGB1受体(TLR.4和RAGE)/NF-κB信号通路表达受到抑制(与脑创伤组相比,各指标P0.05)；假手术组大鼠脑组织IL-1β、TNF-α和IL-6的含量分别为39.48±5.67pg/mg、10.52±1.53pg/mg、5.38±0.82pg/mg,脑创伤组大鼠脑组织IL-1β、TNF-α和IL-6的含量分别为79.57±5.17pg/mg、 18.94±1.45pg/mg、16.08±1.06pg/mg(与假手术组相比,各指标P0.01),甘草酸苷组大鼠脑组织IL-1β、TNF-α和IL-6的含量为62.02±5.54pg/mg、15.25 ±1.52pg/mg、11.60±0.90pg/mg(与脑创伤组相比,各指标P0.01)；.在假手术组HMGB1、RAGE和TLR4阳性细胞及凋亡细胞分别为7.98±1.44%、5.60±1.12%、7.60±1.29%、8.19±1.46%,在脑创伤组HMGB1、RAGE和TLR4阳性细胞及凋亡细胞分别为58.37±5.06%、54.15±4.65%、65.50±4.83%、52.02±4.63%(与假手术组相比,各指标P0.01),在甘草酸苷组分别为39.99±4.99%,34.87±5.02%,43.33±4.54%,37.84±5.16%(与脑创伤组相比,各指标P0.01)。结论：甘草酸苷下调大鼠创伤周围脑组织HMGB1/HMGB1受体(TLR4和RAGE)/NF-κB介导的炎症反应,减轻继发性脑损伤,改善大鼠脑创伤的预后。我们的研究表明甘草酸苷对大鼠创伤性脑损伤具有神经保护作用。
[Abstract]:The expression and clinical significance of HMGB1 in the first part of the patients with acute traumatic brain injury: To explore the expression and clinical significance of high mobility cluster protein B1 (High-mobility group box 1, HMGB1) in patients with acute traumatic brain injury and its clinical significance. Methods: 1 patients with acute traumatic brain injury from January 2012 to June 2012 were selected. 42 cases, 68 cases of mild brain trauma (GCS 13~15), 48 cases of moderate brain trauma (GCS 9-12) and 26 cases of severe brain trauma (GCS 3-8). All patients collected upper limb venous blood within 4-6 hours after injury, and the expression of HMGB1 in plasma was determined by enzyme linked immunosorbent assay (Enzyme Linked ImmunoSorbent Assay, ELISA). 15 cases of healthy and normal physical examination were taken as control group. The expression level of HMGB1 in both brain trauma group and control group was compared, and the risk factors of poor functional prognosis within 1 months after acute traumatic brain injury and 6 months after injury were analyzed. Results: the plasma levels of HMGB1 in patients with acute traumatic brain injury were significantly higher than those in the control group. High (P0.01); HMGB1 level in plasma was negatively correlated with GCS score at admission (P0.01). Single factor Logistic regression analysis showed that HMGBl level in plasma was a risk factor for acute traumatic brain injury within 1 months of injury and a risk factor for poor functional prognosis within 1 months of injury, and.ROC curve analysis showed that the level of HMGB1 in plasma was acute to acute traumatic injury. Conclusion: the level of HMGB1 expression in the plasma of patients with acute traumatic brain injury is higher, and the degree of the increase is positively correlated with the severity of brain injury. The level of HMGB1 in plasma can be an important observation to evaluate the prognosis of patients with brain injury. Index. Second part HMGB1 expression in experimental rat acute traumatic brain injury. Objective: To investigate the expression of HMGB1 in plasma and brain tissue after acute traumatic brain injury in experimental rats, as well as brain edema and apoptosis. Methods: rats were randomly divided into 7 groups: sham operation group, brain trauma 2H group, brain trauma 6h group, brain Group 12h, group 24h of brain trauma, group 48h of brain trauma, group of brain trauma, group 72h (each group of 12), modified Feeney weight free falling body method to establish rat brain trauma model, ELISA to detect the expression of HMGB1 in plasma, immunohistochemical staining to observe the expression of HMGB1 positive cells, wet / dry assay of brain water content, TUNEL staining to detect cell apoptosis. Results: H in the plasma. MGB1 was 0.83 + 0.15ng/ml in the sham operation group and 3.50 + 0.57ng/m1 after injury (compared with that of the sham group) (compared with the sham operation group), and the 6h was 7.87 + 0.60ng/ml (0.01) after injury (compared with 2h after injury), and the 12h was 7.85 + 0.59ng/ml after injury. The 24h was 7.6l + 0.05 after injury (group of brain trauma, brain trauma group, 0.05 of all groups of brain trauma groups), and 4.63 + 0.05 after injury. (compared with 24h after injury P0.01), 72h was 3.11 + 0.66ng/ml after injury (compared with 48h after injury); HMGB1 positive cells were 7.49 + 1.76% in sham operation group and 36.54 + 5.92% after injury (P0.01 compared to sham operation group), 6h was 65.58 + 5.46% after injury (0.01), 65.18 + 4.83% after injury, and after injury, it was 64.68 + 4.09% (brain trauma group, brain creation) 12h group was injured in group 12h and group 24h was compared with P0.05 in group 24h (compared with 24h P0.01 after injury), and 72h was 36.50 + 4.92% (P0.01 compared with 48h after injury). The water content of the injured brain tissue was 79.21 + 0.96% in sham operation group and 79.27 + 0.92% after injury (0.05 in sham operation group) and 81 + 0.85% after injury. 12h was 82.26 + (P0.05) after injury (compared with group 6h after injury), 24h was 83.54 + 0.93% (P0.05 compared with group 12h) and 48h was 82.31 + 0.50% (compared with group 24h after injury), and 81.22 + 0.88% (compared with 48h group after injury); apoptosis was 6.62 + 1.38% in sham operation group and 8.73 + 1.52% (and artificial hand) after injury. Compared with P0.05, 6h was 35.11 + 4.72% (P0.01 compared with group 2H), 12h was 43.23 + 4.61% (P0.05 compared with group 6h), 24h was 52.29 + 4.76% after injury (0.01), and 48h was 39.91 + 4.66% (compared to 24h group of brain trauma) after injury, and 32.56 + (compared with brain trauma group). After traumatic brain injury, the plasma HMGB1 began to rise in 2h after injury, and the 6h reached its peak after injury. After the injury, the 48h began to decrease obviously after injury. After the injury, the 2H HMGB1 was distributed to the cytoplasm of the nucleus to the cytoplasm, and the 6h reached the peak after injury. The 48h began to decrease obviously after injury, and the water content in the 6h injured brain tissue began to rise after the traumatic brain injury. After injury, 24h reached a peak, and 48h began to remission after injury; 6h apoptotic cells gradually increased after traumatic brain injury in rats, the 24h reached most after injury, and 48h gradually decreased after injury. The effect and mechanism of third part of glycyrrhizin on HMGB1 expression after traumatic brain injury in rats and its mechanism of action were discussed. Methods: the effect of protective action and high mobility group protein B1 (HMGB1). Methods: male SD rats were randomly divided into three groups: sham operation group, brain trauma group and glycyrrhizin group (36 rats in each group). The rat model of brain trauma was established by improved Feeney method. In the glycyrrhizin group, 10mg/kg glycyrrhizin was injected into the vein 30 minutes after injury. The exercise function was performed after 24 hours after injury. The water content of brain tissue was measured, and the brain tissue around the injured part was sampled. RT-PCR, Western blot, EMSA and ELISA methods were used to detect the signaling pathway and inflammatory cytokines of HMGB1/HMGB1 receptor (TLR4 and RAGE) /NF- kappa B, and HMGB1, RAGE and immunohistochemical and apoptosis were analyzed. The motor dysfunction in the glycyrrhizin group was significantly reduced (compared with the brain trauma group, P0.01), and the water content of the injured side brain tissue was 79.97 + 0.82% in the sham operation group and 82.94 + 0.65% in the brain trauma group (compared with the sham group, P0.01) and 80.97 + 0.49% in the glycyrrhizin group (compared with the brain trauma group, P0.0, compared with the brain trauma group, P0.0). 1): the expression of HMGB1/HMGB1 receptor (TLR.4 and RAGE) /NF- kappa B signaling pathway in the peripheral brain tissue of the traumatic brain injury area was significantly increased (compared with the sham operation group, P0.01). After glycyrrhizin treatment, the expression of HMGB1/HMGB1 receptor (TLR.4 and RAGE) /NF- kappa B signaling pathway was suppressed (compared with the brain trauma group, each index P0.05); sham operation group rats. The content of IL-1 beta, TNF- alpha and IL-6 in brain tissue was 39.48 + 5.67pg/mg, 10.52 + 1.53pg/mg and 5.38 + 0.82pg/mg respectively. The content of IL-1 beta, TNF- A and IL-6 in brain tissue of brain trauma rats were 79.57 + 5.17pg/mg, 18.94 + 1.45pg/mg, 16.08 + 1.06pg/mg (compared with sham operation group). The content of L-6 was 62.02 + 5.54pg/mg, 15.25 + 1.52pg/mg and 11.60 + 0.90pg/mg (P0.01), respectively. In the sham operation group HMGB1, RAGE and TLR4 positive cells and apoptotic cells were 7.98 + 1.44%, 5.60 + 1.12%, 7.60 + 1.29% and 8.19 +, respectively, in the brain trauma group, and RAGE and TLR4 positive cells and apoptotic cells were 58.37. 5.06%, 54.15 + 4.65%, 65.50 + 4.83%, 52.02 + 4.63% (compared with the sham operation group, each index P0.01), in the glycyrrhizin group were 39.99 + 4.99%, 34.87 + 5.02%, 43.33 + 4.54%, 37.84 +, respectively (compared with the brain trauma group, each index P0.01). Conclusion: glycyrrhizin downregulated the HMGB1/HMGB1 receptor (TLR4 and RAGE) of /NF- kappa B in the brain tissue around the trauma of rats The inflammatory response alleviates secondary brain damage and improves the prognosis of brain trauma in rats. Our study shows that glycyrrhizin has a neuroprotective effect on traumatic brain injury in rats.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R651.15

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