壳寡糖对大鼠颅脑冲击伤的保护作用

Protective role of chitosan oligosaccharide on blast brain injury in rats

目的探讨壳寡糖对大鼠颅脑损冲击伤（BBI）的保护作用。方法90只雄性大鼠按随机数字表法分为对照组、BBI组、壳寡糖干预组，每组30只。BBI组采用高压冲击装置制作实验动物模型。壳寡糖干预组在BBI前腹腔注射20mg／kg壳寡糖，1次／d，连续5d。对照组和BB1组给予等量等渗盐水。采用迷宫试验、爬杆试验和反射试验对大鼠进行神经功能缺损评分（NSS）；HE染色观察脑组织病理改变；酶联免疫吸附（ELISA）法检测脑组织不同时相点过氧化氢酶（CAT）、丙二醛（MDA）及活性氧（ROS）含量；Westernblot、免疫荧光检测脑组织肿瘤坏死因子-α（TNF-α）和核因子-κB（NF-κB）蛋白表达；实时荧光定量PCR检测脑组织TNF-α和NF-κBmRNA表达。结果（1）NSS：对照组平均值为0分；BBI组平均值为5．52分；壳寡糖干预组平均值为3．56分（P〈0．05）。（2）HE染色：对照组未见病理改变；BBI组出现不同程度的脑损伤；壳寡糖干预组较BBI组损伤程度减轻。（3）ELISA法检测：与对照组比较，BBI组CAT活性降低，MDA及ROS含量显著增高（P〈0．05）；壳寡糖干预组三项指标均明显改善（P〈0．05）。（4）Westernblot检测：BBI组TNF-α和NF-κB蛋白表达量较对照组显著升高（P〈0．05）；壳寡糖干预组TNF-α和NF-κB蛋白表达量显著降低（P〈0．05）。免疫荧光检测：BBI组TNF-α、NF-κB蛋白表达量高于对照组；壳寡糖干预组TNF-α、NF-κB蛋白表达量低于BBI组。（5）实时荧光定量PCR检测：BBI组TNF-α、NF-κBmRNA表达量较对照组显著升高（P〈0．05）；壳寡糖干预组TNF-αmRNA表达量显著降低（P〈0．05），而NF-κBmRNA表达量与BBI组差异无统计学意义（P〉0．05）。结论壳寡糖能够下调炎症因子TNF-α和NF-κB的表达，减轻氧化应激损伤，对大鼠BBI具有保护作用。

Objective To investigate the protective role of chitosan oligosaccharide on blast brain injury （BBI） in rats. Methods Ninety male SD rats were divided into control group, BBI group and chitosan oligosaccharide group according to the random number table, with 30 rats per group. A model of BBI was induced by high pressure shock wave. Rats in chitosan oligosaccharide group received ehitosan oligosaccharide （20 mg/kg body weight） intraperitoneally once a day for 5 days after the induction of BBI. For control and BBI groups, an equal amount of normal saline was given. Rat neurological severity score（NSS） was evaluated using the maze test, pole test and reflection test. Brain tissue pathological changes were observed using HE staining. Activity of catalase （CAT） and contents of malondialdehyde （MDA） and reactive oxygen species （ROS） in brain tissues were determined using the ELISA method. Protein expressions of tumor necrosis factor α （TNF-α） and nuclear factor-κB （NF-KB） in brain tissues were tested using the Western blot and immunefluorescence method . Expressions of TNF-α and NF-κB mRNA in brain tissues were determined using the Real time PCR. Results Mean NSS was 0 point in control group, 5.52 points in BBI group, and 3.56 points in chitosan oligosaccharide group （ P 〈 0.05 ）. No pathological changes were seen in control group, and the brain damage was less severe in chitosan oligosaccharide group than that in BBI group. BBI group showed significantly attenuated activity of CAT and significantly elevated levels of MDA and ROS compared to control group （ P 〈 0. 01 ）, but marked improvements were observed with the intervention of chitosan oligosaccharide （ P 〈 0. 01 ）. Protein expressions of TNF-α and NF-κB in BBI group were apparently elevated compared to control group （P 〈 0.05 ）, and both expressions were significantly reversed with the intervention of chitosan oligosaccharide （ P 〈 0. 05 ）. Expressions of TNF-α and NF-κB mRNA in BBI group were apparently elevated compared to control group （ P 〈 0. 05 ）, and the expression of TNF-α mRNA were significantly reversed after the intervention with chitosan oligosaccharide. However the expression of NF-κB mRNA was similar between BBI group and chitosan oligosaccharide group （P 〉 0. 05 ）. Conclusion Chitosan oligosaccharide can protect rats against BB1 by down-regulating expressions of TNF-α and NF-κB and reducing oxidative damage.