芒果苷对大鼠急性脊髓损伤的保护作用及相关机制研究

NEUROPROTECTIVE EFFECTS OF MANGIFERIN ON ACUTE SPINAL CORD INJURY IN RATS AND ITS MECHANISM

目的探讨芒果苷对大鼠急性脊髓损伤（spinal cord injury,SCI）的保护作用并分析其相关机制。方法成年雄性SD大鼠90只,体质量250~300 g,随机分为假手术组（A组）、SCI组（B组）、10 mg/kg芒果苷处理组（C组）、25 mg/kg芒果苷处理组（D组）、50 mg/kg芒果苷处理组（E组）,每组18只。B、C、D、E组采用Allen法（60 g/cm）建立大鼠T9 SCI模型,A组仅切除T8~10椎板;C、D、E组术后每天按照10、25、50 mg/kg剂量腹腔注射芒果苷,共注射30 d;A、B组于对应时间点注射等量生理盐水。术后观察大鼠存活情况,于24、48、72 h采用BBB评分评价大鼠后肢运动功能;72 h时取损伤节段脊髓测量其水含量,ELISA检测氧化应激反应因子丙二醛（malondialdehyde,MDA）、过氧化氢酶（catalase,CAT）、过氧化物歧化酶（superoxide dismutase,SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase,GSH）以及炎性因子NF-κB、TNF-α、IL-1β、IL-6活性;30 d时取损伤节段脊髓采用ELISA法检测Caspase-3、9活性,Western blot检测凋亡蛋白Bax、Bcl-2表达,组织学观察脊髓组织形态,免疫组织化学染色观察Caspase-3蛋白表达。结果术后各组大鼠均存活至实验结束。B、C、D、E组BBB评分均显著低于A组,B、C、D、E组评分呈逐渐增高趋势,组间比较差异均有统计学意义（P〈0.05）。B、C、D、E组脊髓水含量均显著高于A组,B、C、D、E组水含量呈逐渐降低趋势,组间比较差异有统计学意义（P〈0.05）。ELISA检测示,B、C、D、E组MDA、NF-κB、TNF-α、IL-1β、IL-6、Caspase-3、Caspase-9活性均显著高于A组,B、C、D、E组均呈逐渐降低趋势;而CAT、SOD、GSH活性均显著低于A组,B、C、D、E组均呈逐渐增加趋势;以上指标组间比较差异均有统计学意义（P〈0.05）。Western blot示,B、C、D、E组Bax蛋白表达明显高于A组,B、C、D、E组表达呈逐渐降低趋势;Bcl-2蛋白表达明显低于A组,B、C、D、E组表达呈逐渐增高趋势;组间比较差异有统计学意义（P〈0.05）。组织学观察示B组脊髓组织符合SCI病理改变,C、D、E组神经坏死程度较B组好转,并且E组效果优于D组,D组优于C组。免疫组织化学染色观察,B、C、D、E组Caspase-3蛋白表达量显著高于A组,B、C、D、E组呈逐渐降低趋势（P〈0.05）。结论对于大鼠急性SCI,芒果苷可通过减轻脊髓组织水肿、抑制氧化应激反应及炎性反应,调节Bax、Bcl-2蛋白表达,从而发挥神经保护作用。

Objective To investigate the protective effect of mangiferin on acute spinal cord injury（SCI） in rats and its mechanism. Methods Ninety Sprague Dawley rats were randomly divided into 5 groups, 18 rats in each group. SCI was induced by using the Allen＇s method（ 60 g/cm） at T 9 level in the rats of groups B, C, D, and E; laminectomy was performed at T_（8- 10） in group A. The rats were injected intraperitoneally with saline in groups A and B, and with mangiferin in groups C（ 10 mg/kg）, D（ 25 mg/kg）, and E（ 50 mg/kg） every day for 30 days. The survival condition of rats was observed after operation; at 24, 48, and 72 hours after operation, the motor function of the hind limb was evaluated by the Basso, Beattie, Bresnahan（BBB） scores. The spinal cord edema was assessed by measuring the water content in spinal cord tissues at 72 hours. Meanwhile, malondialdehyde（MDA）, catalase（CAT）, superoxide dismutase（SOD）, and glutathione peroxidase（GSH） were detected by ELISA; nuclear factor κB（NF-κB）, tumor necrosis factor α（TNF-α）, interleukin 1 β（IL- 1 β）, and IL- 6 were measured via ELISA at the same time. Caspase- 3 and Caspase- 9 were also detected by ELISA after mangiferin treatment for 30 days. The expressions of Bax and Bcl- 2 proteins were detected by Western blot. Pathological changes of the spinal cord was observed by HE staining. And Caspase- 3 protein expression was detected by immunohistochemical staining. Results All rats survived to the end of experiment. BBB scores of groups B, C, D, and E were significantly less than that of group A（P〈0. 05）, and it showed an increase trend from groups B to E（P〈0. 05）. The content of water of groups B, C, D, and E were significantly greater than that of group A（P〈0. 05）, and it showed a decrease trend from groups B to E（P〈0. 05）. ELISA showed that the activities of MDA, NF-κB, TNF-α, IL- 1 β, IL- 6, Caspase- 3, and Caspase- 9 in groups B, C, D, and E were significantly greater than that in group A（P〈0. 05）, and they showed decrease trends from groups B to E（P〈0. 05）. Meanwhile, the activities of CAT, SOD, and GSH in groups B, C, D, and E were significantly less than that in group A（P〈0. 05）, and they showed increase trends from groups B to E（P〈0. 05）. Western blot showed that the relative expression of Bax protein in groups B, C, D, and E were significantly greater than that in group A（P〈0. 05）, and it showed a decrease trend from groups B to E（P〈0. 05）. Meanwhile, the relative expression of Bcl- 2 protein in groups B, C, D, and E were significantly less than that in group A（P〈0. 05）, and it showed an increase trend from groups B to E（P〈0. 05）. Histological observation showed that the pathological changes in group B were accord with that in SCI, and the degree of necrosis in groups C, D, and E were significantly improved when compared with that in group B, and the effect was better in group E than group D, and group D than group C. Immunohistochemical staining showed that the absorbance（A） value of Caspase- 3 in groups B, C, D, and E were significantly greater than that in group A（P〈0. 05）, and it showed a decrease trend from groups B to E（P〈0. 05）. Conclusion Mangiferin has neuroprotective effects on acute SCI in rats by alleviating edema of spinal cord, inhibiting oxidative stress and inflammation response, and regulating the Bcl- 2 and Bax pathway.