甘草酸二铵对大鼠脊髓缺血再灌注损伤后NF-κB表达及神经元凋亡影响的研究

RESEARCH ON THE INFLUENCE OF DIAMMONIUM GLYCYRRHIZINATE ON THE EXPRESSION OF NF-κB AND NEURON APOPTOSIS AFTER SPINAL CORD ISCHEMIA-REPERFUSION INJURY IN RATS

目的探讨甘草酸二铵(diammonium glycyrrhizinate,DG)对大鼠脊髓缺血再灌注损伤后NF-κB的表达及神经元凋亡的影响。方法取健康雄性SD大鼠48只,体重220～270g,随机分为实验组和对照组,每组24只。实验组于缺血开始前10min舌下静脉注射DG(20mg/kg),对照组注射等量生理盐水。夹闭左右肾动脉之间腹主动脉制备脊髓缺血再灌注损伤模型。两组于缺血再灌注3、24、72、168h取腰段脊髓组织,切片,行HE染色、免疫组织化学染色观察及TUNEL法凋亡细胞检测,并进行相关性分析。结果HE染色示对照组缺血再灌注3h时,可见脊髓组织水肿,神经细胞形态基本正常;24h和72h时组织病理改变进一步加重;168h时灰质前角中大量空泡形成,尚残存多个结构清楚的运动神经元。实验组各时间点明显好于对照组。免疫组织化学染色示两组缺血再灌注3h表达NF-κB p65增强,24h达高峰,随后缓慢下降。实验组缺血再灌注各时间点吸光度(A)值分别为0.3060±0.0244、0.3964±0.0227、0.2966±0.0211和0.2679±0.0153;对照组分别为0.3611±0.0177、0.4966±0.0201、0.3563±0.0210和0.3014±0.0181(P<0.05)。DG对各时间点NF-κB p65表达的抑制率分别是15.40%、20.17%、19.28%和11.11%。细胞凋亡检测示两组于缺血再灌注后神经元凋亡表达增强。实验组各时间点A值分别为0.1710±0.0291、0.1755±0.0311、0.1751±0.0279和0.1832±0.0237;对照组分别为0.2368±0.0636、0.2412±0.0426、0.2015±0.0498和0.2501±0.0484(P<0.05)。DG各时间点对神经元凋亡表达的抑制率分别为27.79%、27.23%、13.08%和26.74%。实验组和对照组NF-κB表达和神经元凋亡表达成正相关(r=0.838,P<0.01)。结论脊髓缺血再灌注可导致NF-κB表达及凋亡神经元增多,DG可抑制脊髓神经元NF-κB的表达,减少神经元凋亡。

Objective To investigate the influence of diammonium glycyrrhizinate （DG） on the expression of NF-κB and neuron apoptosis after spinal cord ischemia-reperfusion injury in rats. Methods Fourty-eight healthy SD male rats, weighing 220-270 g, were randomly divided into the experimental group and the control group, with 24 rats in each group. A model of spinal cord ischemia-reperfusion injury was completed by intercepting the rats＇ abdominal aorta between right and left renal arteries for 30 minunts. In the experimental group, each rat was injected 20 mg/kg DG via sublingual vein 10 minutes before ischemia occurred. Equal qualities of physiological saline were injected into the rats in the control group. The two groups were observed at 3, 24, 72 and 168 hours after ischemia-reperfusion, respectively. Lumbar myeloid tissues were prepared at the different times, respectively. The expression of NF-κB p65 in lumbar myeloid tissues was analyzed by immunohistochemistry and the apoptosis of neurons was examined by TUNEL reaction. Meanwhile, histological changes of spinal cord were observed by HE staining. Then the correlation between NF-κBand neuron apoptosis was analyzed. Results HE staining showed obvious histological changes of spinal cord of the two groups. In the control group, myeloid tissue edema and normal neurons were observed at 3 hours; there were more histological changes at 24 hours and 72 hours; vacuolus in gray matters and some survived neurons were seen at 168 hours. The histological changes at each time in the experimental group were fewer than those in the control group. The immunohistochemical staining showed that the expression of NF-κB p65 was observed. After ischemia-reperfusion, the expression strengthened at 3 hours, reached the peak at 24 hours and then weakened slowly. At 3, 24, 72 and 168 hours after ischemia-reperfusion, the absorbency （A）value of NF-κB p65 in the experimental group was 0.306 0± 0.0244, 0.396 4 ± 0.022 7, 0.296 6 ± 0.021 1 and 0.267 9 ± 0.015 3, respectively, and that in the control group was 0.361 1 ± 0.017 7, 0.496 6± 0.020 1, 0.356 3 ± 0.0210 and 0.3014 ± 0.018 1, respectively. There were significant differences between the two groups （P 〈 0.05）. The inhabitation ratio of NF- κB p65 expression by DG was 15.40%, 20.17%, 19.28% and 11.11% at 3, 24, 72 and 168 hours after ischemia-reperfusion, respectively. Neuron apoptosis was observed, which strengthened at 3 hours and was the most serious at 24 and 168 hours after ischemia-reperfusion. At 3, 24, 72 and 168 hours after ischemia-reperfusion, the A value of neuron apoptosis in the experimental group was 0.171 0 ± 0.029 1, 0.175 5 ± 0.031 1, 0.175 1 ± 0.027 9 and 0.183 2 ±0.023 7, respectively, and that in the control group was 0.236 8 ± 0.063 6, 0.241 2± 0.042 6, 0.201 5± 0.049 8 and 0.250 1 ± 0.048 4, respectively. There were significant differences between the two groups （P 〈 0.05）. The inhabitation ratio of neuron apoptosis by DG was 27.79%, 27.23%, 13.08% and 26.74% at 3, 24, 72 and 168 hours after ischemia-reperfusion, respectively. The expression of NF-κB in myeloid tissues was positively correlated with neurons apoptosis in the two groups （r = 0.838, P 〈 0.01）. Conclusion Spinal cord ischemia-reperfusion injury may cause a marked expression of NF-κB and notable evidence of neurons apoptosis. DG can reduce neurons apoptosis by inhibiting the expression of NF-κB.