ω-3多不饱和脂肪酸抑制大鼠创伤性脑损伤后小胶质细胞介导的炎症反应

Omega-3 polyunsaturated fatty acids supplementation attenuates microglia-induced inflammation after traumatic brain injury in rats

目的研究ω-3多不饱和脂肪酸对大鼠创伤性脑损伤（TBI）后神经细胞凋亡、脑水肿、小胶质细胞活化、炎症反应及神经功能的影响，以探讨ω-3多不饱和脂肪酸对大鼠TBI的保护及机制。方法采用改良Feeney DM法建立大鼠TBI模型，将90只sD大鼠完全随机分为假手术组（Sham组），TBI组，TBI＋c—Jun氨基末端激酶（JNK）选择性激活剂anisomycin组（TBI＋Aniso组）；TBI＋ω-3多不饱和脂肪酸组（TBI＋ω-3组），TBI＋ω-3多不饱和脂肪酸＋JNK选择性激活剂aniso-mycin组（TBI＋ω-3＋Aniso组），分别于伤后1、3、7d进行神经行为学评分（mNSS）；采用干湿重法测损伤区脑组织脑水含量；TUNEL和免疫荧光等方法测定细胞凋亡和小胶质细胞活化特异标志物IBA-1的表达；PCR、Westerrnblot等检查脑组织炎症因子肿瘤坏死因子仪（TNF—α）、白细胞介素（IL）-1α、IL-1β、IL-6及上游JNK信号通路JNK、p-JNK分子表达水平的变化。结果与同期Sham组比较，其余4组细胞凋亡、脑水肿、神经细胞凋亡和炎症反应明显增高（P〈0．05）；与TBI组比较，TBI＋ω-3组大鼠创伤后脑水含量降低，尤其在脑损伤3d后降低更为明显[（78．14±0．57）％比（82．31±0．81）％，P〈0．01]，神经功能评分相应的改善明显（P〈0．05）。ω-3多不饱和脂肪酸抑制神经细胞凋亡和小胶质细胞的活化；降低大鼠TBI后升高的炎症因子TNF-α、IL-1α、IL-1β、IL-6mRNA及蛋白表达水平；同时抑制了炎症因子上游JNK信号通路的活化。结论ω-3多不饱和脂肪酸明显减轻大鼠TBI后脑水肿的程度，抑制神经细胞凋亡，改善伤后神经行为，其机制可能是通过抑制JNK信号通路和小胶质细胞的活化，减轻小胶质细胞介导的中枢性炎症反应，降低TNF-α、IL—1α、IL-1β、IL-6的表达。

Objective To investigate the effects of omega-3 polyunsaturated fatty acids （ ω-3 PUFA） supplementation on neuron apoptosis, brain edema, activation of microglia, inflammatory response and neural function after traumatic brain injury （TBI） in rats, so as to understand the protection of ω-3 PUFA in rats following TBI and its mechanism. Methods TBI model was established using Feeney＇s method. Ninety SD rats were randomly divided into 5 groups： sham operation group （sham group）, TBI group, TBI ＋ selective activator of c-Jun N-terminal kinase （JNK） anisomycin group （TBI ＋ Aniso group ）, TBI ＋ ω-3 PUFA supplementation group （TBI ＋ ω-3 group）, and TBI ＋ω-3 PUFA supplementation ＋ JNK activation group （TBI ＋ ω-3 ＋ Aniso group）. We measured rat behavioral outcomes by modified neurological severity score （mNSS）on day 1 , 3, and 7 after TBI. Brain water content was measured with wet-dry weight method. The neuron apoptosis and microglial activation （ identified by specific marker IBA-1 ） in TBI cerebral cortex were determined by TUNEL staining and immunofluorescence. Inflammatory cytokines [ tumor necrosis factor-α （TNF-α） , interleukin （IL） -1 α, IL-1β, and IL-6 ] and the JNK signaling pathway （JNK, pJNK） were tested with reverse transcription-polymerase chain reaction and Western blot, respectively. Results Compared with the sham group, the levels of brain cell apoptosis, brain edema, neuron apoptosis, and inflammatory-relatived factors （ TNF-α, IL- 1 α, IL-1β, and IL-6） were significantly increased in the other four groups （ P 〈 0.05 ）. Compared with the TB1 group, ω-3 PUFA supplementation reduced brain water content following TBI, especially on day 3 after TBI [ （ 78. 14 ±0. 57 ） % vs. （ 82. 31 ± 0.81 ） %, P 〈 0. 01], and improved neurological function score （P 〈 0. 05 ）. Meanwhile, ω-3 PUFA supplementation suppressed neuron apoptosis, the activation of microglia, and the mRNA and protein expressions of inflammatory cytokines （ TNF-α, IL- 1α, IL-1 β, IL-6）. The activation of JNK signaling pathway was also inhibited by ω-3 PUFA. Conclusion ω-3 PUFA supplementation may markedly reduce brain edema, suppress neuron apoptosis, and improve neurological outcomes after TBI in rats, possibly mediated by inhibiting JNK signaling pathway and microglial activation, reducing microglia-induced cerebral inflammatory responses, demonstrated as down-regulated expression of TNF-α, IL- 1 α, IL- 1β, and IL- 6.