间歇性禁食及二甲双胍对脑缺血再灌注损伤后Toll样受体4/核转录因子κB通路的影响

Effects of intermittent fasting and metformin on TLR4/NF-κB pathway in mice following cerebral ischemia-reperfusion injury

目的探讨间歇性禁食及二甲双胍通过抑制Toll样受体4(TLR4)/NF-κB通路,从而对脑缺血再灌注的神经保护作用。方法选取雄性KM小鼠180只,随机分为假手术组、模型组、间歇性禁食组、二甲双胍组、二甲双胍+间歇性禁食组(联合组),每组36只。制备模型,采用Longa评分判断小鼠神经功能损伤情况。测定各组脑含水量。采用TTC染色和苏木精-伊红染色判断脑梗死和脑缺血半暗带及海马组织损伤情况。免疫组织化学及Western blot方法检测各组TLR4及NF-κB蛋白表达。结果与假手术组比较,模型组、间歇性禁食组、二甲双胍组、联合组神经功能评分、脑梗死体积、脑含水量、TLR4和NF-κB表达明显增高(P<0.05);与模型组比较,间歇性禁食组、二甲双胍组、联合组神经功能评分,脑梗死体积、脑含水量、TLR4和NF-κB表达明显降低(P<0.05);与间歇性禁食组比较,二甲双胍组脑梗死体积、TLR4和NF-κB表达明显降低,联合组神经功能评分、脑梗死体积、脑含水量、TLR4和NF-κB表达明显降低(P<0.05);与二甲双胍组比较,联合组神经功能评分、脑梗死体积、TLR4和NF-κB表达明显降低[(1.36±0.49)分vs(1.84±0.68)分,(26.25±2.65)mm^(3)vs(32.62±2.44)mm^(3),0.43±0.01 vs 0.59±0.04,0.38±0.02 vs 0.52±0.06,P<0.05]。结论间歇性禁食、二甲双胍可通过抑制TLR4和NF-κB蛋白表达,从而减轻炎性反应,并对脑缺血再灌注损失具有神经保护作用。二甲双胍抑制TLR4、NF-κB效果较间歇性禁食更强,未来可能成为替代间歇性禁食的良好方案。

Objective To investigate whether intermittent fasting and metformin exert neuro-protective effects on cerebral ischemia-reperfusion injury by inhibiting the TLR4/NF-κB pathway.Methods A total of 180 male KM mice were randomly divided into a sham operation group,a model group,an intermittent fasting group,a metformin group,and a metformin+intermittent fasting group(combined group),with 36 mice in each group.The model was prepared,and the neurological impairment was assessed by Longa score.Brain water content was measured in each group.TTC staining and hematoxylin-eosin staining were used to observe cerebral infarction,ischemic penumbra and hippocampal tissue damage.The expression levels of TLR4 and NF-κB were detected by immunohistochemical assay and Western blotting.Results The neurological scores,cerebral infarct volume,brain water content and the protein levels of TLR4 and NF-κB were significantly increased in the model,intermittent fasting,metformin,and combination groups than the sham operation group(P<0.05),and those of the intermittent fasting,metformin and combination groups were obviously reduced than those of the model group(P<0.05).Compared with the intermittent fasting group,the metformin group had notably reduced cerebral infarction volume and the protein levels of TLR4 and NF-κB,while the combination group had lower neurological score,cerebral infarction volume,brain water content and the protein levels of TLR4 and NF-κB(P<0.05).What’s more,the combination group had significantly decreased neurological function score and cerebral infarction volume and the protein levels of TLR4 and NF-κB reduced than the metformin group(1.36±0.49 vs 1.84±0.68,26.25±2.65 mm^(3)vs 32.62±2.44 mm~3,0.43±0.01 vs 0.59±0.04,0.38±0.02 vs 0.52±0.06,P<0.05).Conclusion Intermittent fasting and metformin can reduce the inflammatory response by inhibiting the expression of TLR4 and NF-κB,and excert a neuroprotective effect on rats after cerebral ischemia-reperfusion injury.Metformin shows inhibitory effects on TLR4 and NF-κB more effectively than intermittent fasting,and might become a good alternative to intermittent fasting in the future.