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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 related mechanism. Methods 90 adult male Sprague-Dawley rats weighing 250-300 g were randomly divided into sham operation group (A group), SCI group (B group), 10 mg / kg mangiferin treatment group (C group), 25 mg / kg of mangiferin (group D) and 50 mg / kg of mangiferin (group E), 18 rats in each group. The T9 SCI model was established by Allen’s method (60 g / cm) in group B, C, D and E, and only T8-10 lamina were excised in group A. In group C, D and E, / kg dose of intraperitoneal injection of mangiferin, a total of 30 d injection; A, B group at the corresponding time point injection of normal saline. The survival of rats was observed after operation. BBB score was used to assess the motor function of the hind limbs at 24, 48, and 72 hours. Spinal cord injury was performed at 72 hours, and the levels of malondialdehyde , MDA, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH) and inflammatory factors NF-κB and TNF- α, IL-1β and IL-6. Spinal cord injury was performed on the 30th day, and the activity of Caspase-3 and 9 was detected by ELISA. The expressions of Bax and Bcl-2 were detected by Western blot. Caspase-3 protein expression was observed by immunohistochemistry. Results All rats survived to the end of the experiment. The score of BBB in group B, C, D and E were significantly lower than that of group A, while the score of group B, C, D and E showed a trend of increasing gradually. There was significant difference between two groups (P <0.05). The water content of spinal cord in groups B, C, D and E was significantly higher than that in group A, while the water content in groups B, C, D and E showed a trend of decreasing gradually. There was significant difference between groups (P <0.05). The activity of MDA, NF-κB, TNF-α, IL-1β, IL-6, Caspase-3 and Caspase-9 in group B, C, D and E were significantly higher than those in group A, B, C and D , While the activity of CAT, SOD and GSH in group E were gradually decreased. The activity of CAT, SOD and GSH in group B were significantly lower than that in group A, while the levels in group B, C, D and E increased gradually. The differences among the above groups were statistically significant (P <0.05). Western blot showed that the expression of Bax in B, C, D and E groups was significantly higher than that in A group, while the expression in B, C, D and E groups was gradually decreased; Bcl-2 protein expression was significantly lower than that in A, B, C, D, E group showed a trend of increasing gradually; the difference between the two groups was statistically significant (P <0.05). Histological observation showed that the spinal cord tissue of group B was consistent with the pathological changes of SCI. The degree of neurological necrosis in group C, D and E was better than that of group B, and the effect of group E was better than that of group D and group D was better than group C. Immunohistochemical staining showed that the expression of Caspase-3 in B, C, D and E groups was significantly higher than that in A, B, C, D and E groups (P <0.05). Conclusion For acute SCI in rats, mangiferin can play a neuroprotective role by reducing edema, inhibiting oxidative stress and inflammatory response in spinal cord tissue and regulating Bax and Bcl-2 protein expression.