高比例ω-3/ω-6脂肪酸减轻大鼠颅脑外伤后炎症和凋亡

High ω-3/ω-6 ratio fatty acids alleviate inflammation and neuronal apoptosis in a rat model of traumatic brain injury

目的观察高比例ω-3/ω-6脂肪酸对创伤性颅脑损伤(TBI)后炎性反应和神经细胞凋亡的影响.方法 33只雄性Sprague-Dawley大鼠随机分为3组:假手术组、低比例组和高比例组.采用自由落体打击法制作TBI模型,伤后喂食无脂饲料并用相应ω-3/ω-6的混合油灌胃.采用免疫荧光、原位缺口末端标记法(TUNEL)和蛋白质印迹法(Western blot)等方法观察伤后第5天小胶质/巨噬细胞极化、核因子-κB(NF-κB)激活、神经细胞凋亡、B细胞淋巴瘤/白血病-2(bcl-2)和B细胞淋巴瘤/白血病-2相关X蛋白(bax)的表达.计量资料以均值±标准差(Mean±SD)表示,各组样本间的比较采用两独立样本的t检验或单因素方差分析.结果高比例组CD16/32+/离子钙接头蛋白分子-1(Iba-1)+细胞比例[(9.1±0.8)%]显著低于低比例组[(31.8±8.5)%,t=4.720,P<0.01];而高比例组精氨酸酶-1 (Arg-1)+/Iba-1+细胞比例[(28.7±4.9)%]显著高于低比例组[(6.4±2.4)%,t=6.969,P<0.01].高比例组胞核内NF-κB p65的表达显著低于低比例组(t=3.010,P<0.05).同时,高比例组损伤周围皮层和海马CA1区的TUNEL阳性细胞数(49.7 ±6.8、6.3±2.5)均显著低于低比例组(197.7 ±43.7、54.3±14.7)(t=5.801,P<0.01;t=5.559,P<0.01),并伴bcl-2表达增高(t=2.750,P<0.05;t=5.916,P<0.01)和bax表达下降(t=3.754,P<0.01;t=4.238,P<0.01).结论 TBI后高比例ω-3/ω-6脂肪酸有助于减少M1型、增加M2型小胶质/巨噬细胞,这可能与NF-κB通路抑制有关.高比例ω-3/ω-6脂肪酸能抑制伤后神经元凋亡.

Objective To investigate the effects of high ω-3/ω-6 ratio fatty acids on inflammatory response and neuronal apoptosis after traumatic brain injury (TBI). Methods 33 male Sprague-Dawley rats were randomly divided into three groups: sham surgery group, low ratio group and high ratio group. TBI was induced by weigh-dropping method. In addition to a fat-free diet, rats were treated with corresponding ω-3/ω-6 ratio oil mixtures by gavage. Microglia/macrophage polarization, nuclear factor kappa B (NF-κB) activation, neuronal apoptosis and the expression of B-cell lymphoma-2 (bcl-2) and bcl-2 associated X protein (bax) were observed by immunofluorescence, TdT-mediated dUTP nick-end labeling (TUNEL) and Western blotting at day 5 after TBI. The experimental data were expressed as mean±standard deviation(Mean±SD), and two independent-samples t-test or one way ANOVA was used for comparison among groups. Results The percentage of CD16/32+/Ionized calcium binding adapter molecule-1 (Iba-1)+ cells significantly decreased in high ratio group [(9.1±0.8)%] compared to low ratio group [(31.8±8.5)%, t=4.720, P<0.01], and the percentage of Arginase-1(Arg-1)+/Iba-1+ cells significantly increased in high ratio group [(28.7±4.9)%] compared to low ratio group [(6.4±2.4)%, t=6.969, P<0.01]. The nuclear expression of NF-κB p65 in high ratio group was significantly lower than that in low ratio group (t=3.010, P<0.05). Meanwhile, the number of TUNEL-positive cells in the peri-lesion and hippocampus CA1 region decreased in high ratio group (49.7±6.8, 6.3±2.5) compared with low ratio group (197.7±43.7, 54.3±14.7)(t=5.801, P<0.01;t=5.559, P<0.01), which was accompanied by enhanced expression of bcl-2 (t=2.750, P<0.05;t=5.916, P<0.01) and reduced expression of bax protein (t=3.754, P<0.01;t=4.238, P<0.01). Conclusion High ω-3/ω-6 ratio fatty acids could help to decrease M1 and increase M2 type microglia/macrophage cells after TBI, which might be related to the inhibition of NF-κB pathway. High ω-3/ω-6 ratio fatty acids could inhibit posttraumatic neuronal apoptosis.