慢性睡眠不足诱导小鼠大脑和肝脏氧化应激和炎症反应

Chronic sleep restriction induces oxidative stress and inflammation in mice livers and brains

目的:探讨慢性睡眠不足对小鼠肝脑功能的影响及作用机制。方法:20只4周龄野生雌性C57BL/6J小鼠随机分为正常对照组(n=10)和睡眠剥夺组(n=10),睡眠剥夺组小鼠睡眠剥夺45 d后,观察2组小鼠体质量变化;Morris水迷宫实验分析2组小鼠空间学习记忆能力的变化;测量各组小鼠肝重指数、脾重指数和肾重指数;ELISA法检测血清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)和白细胞介素-1β(interleukin-1β,IL-1β)的含量;酶标比色法检测海马和肝脏超氧化物歧化酶(superoxide dismutase,SOD)活力和还原型谷胱甘肽(reduced glutathione,GSH)含量;Western blot检测海马和肝脏TNF-α、P53、BCL-2相关X蛋白(Bcl-2-associated X protein,BAX)、B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)蛋白相对表达水平;免疫组化SP法检测海马Ki-67的表达水平。结果:睡眠剥夺组小鼠体质量较正常对照组增加缓慢,空间学习记忆能力减退,肝重指数和肾重指数均增高(t=4.274,P=0.000;t=3.629,P=0.002),脾重指数明显降低(t=2.380,P=0.029)。与正常对照组相比,睡眠剥夺组小鼠血清TNF-α[(24.440±4.468)vs.(53.719±4.128)]、IL-6[(14.265±3.718)vs.(31.766±4.535)]和IL-1β[(20.383±3.230)vs.(41.916±4.819)]明显增高(t=10.762,P=0.000;t=6.673,P=0.000;t=8.229,P=0.000),睡眠剥夺组海马和肝脏SOD活力和GSH含量明显降低(t=4.171,P=0.003;t=8.851,P=0.000;t=7.138,P=0.000;t=5.390,P=0.000)。Western blot结果显示,睡眠剥夺组海马和肝脏TNF-α[(0.702±0.088),(0.818±0.074)]、P53[(0.626±0.103),(1.225±0.116)]和BAX[(0.978±0.078),(1.120±0.135)]蛋白相对表达量明显上调(t=13.688,P=0.000;t=11.682,P=0.000;t=6.991,P=0.000;t=10.023,P=0.000;t=13.721,P=0.000;t=10.422,P=0.000),BCL-2蛋白相对表达量[(0.365±0.059),(0.380±0.040)]明显下降(t=15.165,P=0.000;t=12.143,P=0.000),海马齿状回和门区Ki-67蛋白的表达水平明显下降(t=4.171,P=0.003)。结论:慢性睡眠不足可诱导大脑和肝脏的氧化应激,增加血清中炎症因子的浓度,并通过细胞凋亡调节DNA损伤。

Objective：To investigate the effects of chronic sleep restriction （SR） on the function of livers and brains in mice, and to ex- plore the possible mechanism. Methods ：Twenty C57BL/6J female mice at the age of 4 weeks were randomly divided into normal con- trol group（n=10） and SR group（n=10）. Then forty-five days after SR,the mice were observed for the changes of body weight,and the percentage of weight gain was detected in each group. Learning and memorizing abilities were measured by Morris Water Maze test. Liver,spleen and kidney wet weight index were detected in each group. The levels of tumor necrosis factor-α（TNF-α）,inter- leukin-6（IL-6） and interleukin-1β（IL-1β） in the serum were detected by ELISA. The activities of superoxide dismutase（SOD） and contents of reduced glutathione（GSH） were measured by microplate assay. The protein levels of TNF-α, P53, Bcl-2-associated X protein（BAX） and B-cell lymph0ma-2（BCL-2） were measured by Western blot. The expression of Ki-67 was analyzed by immuno- histochemical technique. Results：Compared with the normal control group, the mice in SR had a relatively slow body weight gain, a reduced spatial learning and memorizing capacities, an increased liver and kidney wet weight index（t=4.274,P=0.OOO;t=3.629,P= 0.002） ,and a decreased spleen wet weight index（t=2.380,P=0.029）. The levels of TNF-α,IL=6 and IL-115 were higher in SR group [（53.719 ± 4.128）, （31.766± 4.535）, （41.916±4.819）] than those in the normal control group[（24.440 ± 4.468）, （14.265± 3.718）, （20.383 ± 3.230）]（t=l0.762,P=0.OOO;t=6.673,P--0.OOO;t=8.229,P=0.O00）;the activities of SOD and contents of GSH in hippocampus and livers in SR group lowered significantly, compared with those in the normal control group （t=4.171, P=0.O03 ; t=8.851, P=0.000; t=7.138,P=0.OOO;t=5.390,P=0.000）. Compared with that of normal control group,the expression level of TNF-α[（0.702± 0.088）, （0.818 ± 0.074）],P53[（0.626± 0.103）, （1.225± 0.116）] and BAX[（0.978 ± 0.078）, （1.120 ± 0.135）] in hippocampus and livers in- creased Significantly （t= 13.688, P=0.000; t= 11.682, P=0.000; t=6.991, P=0.000; t= 10.023, P=0.000; t= 13.721, P=0.000; t= 10.422, P=- 0.000） ,while the expression level of BCL-2[（0.365 ±0.059）, （0.380± 0.040）] decreased significantly（t=15.165,P=0.OOO;t=12.143, P=0.000） ,and the positive expression of Ki-67 in hippocampus decreased significantly（t=4.171 ,P=0,003）. Conclusion：SR induces the oxidative stress of brains and livers in mice and elevates the concentration of inflammatory cytokines in the brain and serum, and mediates the DNA damage through cell apoptosis.