The Role And Mechanism Of Melatonin On Cerebral Injury Induced By Intestinal Ischemia-reperfusion In Rats

Objective:The aim of this study was to investigate the role of TLR4(Toll like receptor 4)-MyD88(myeloid differentiation factor 88)signaling pathway in microglia cells on cerebral injury induced by intestinal ischemia-reperfusion;and to confirm whether melatonin can prevent brain injury caused by intestinal ischemia-reperfusion by inhibiting the TLR4/MyD88 signaling pathway in microglia and through anti-inflammatory,anti-oxidative and anti-apoptotic effects.Methods:One hundred healthy male Sprague-Dawley rats were randomly allocated to 5 groups(n=20 each):sham operation group(group Sham),intestinal I/R group(group I/R),low dose of melatonin group(group LMT),high dose of melatonin group(group HMT),and vehicle group(group Veh).Intestinal I/R model was produced by occlusion of the superior mesenteric artery for 90 min followed by reperfusion 2 days.The cognitive function was tested using Morris water maze.The animals were sacrificed after the test and brains were isolated for detection of the morphology by hematoxylin-eosin staining.The apoptosis of frontal cortex and hippocampal CA1 region was detected by TUNEL.The level of TNF-?,IL-6 and IL-1?were detected by ELISA.The reactivity of MDA,SOD and ROS were detected by biochemical method.The expressions of TLR4,MyD88,Cleaved Caspase-3,and NF-?B were detected by Western blot.The activity of microglia and expression of TLR4 and MyD88 were observed by multiple immunofluorescence staining.The experimental data were statistically analyzed by using SPSS 17.0 statistical software.Multiple comparisons of multiple means were performed by one-way analysis of variance.P<0.05 was considered statistically significant.Results:1.The cognitive function of I/R group was obviously damaged than Sham group,and the damage of frontal cortex and hippocampal neurons was more serious than Sham group(P<0.01).Compared with I/R group,the cognitive function and neuronal damage of LMT and HMT group were alleviated,particularly in HMT group(P<0.05 or P<0.01).The results of the Veh group were similar to the I/R group(P>0.05).The number of normal neurons in frontal cortex:[Sham group:(518±72)/mm~2,I/R group:(210±25)/mm~2,LMT group:(307±43)/mm~2,HMT group:(452±48)/mm~2,Veh group:(237±45)/mm~2];The number of normal neurons in hippocampus:[Sham group:(308±37)/mm~2,I/R group:(162±12)/mm~2,LMT group:(203±24)/mm~2,HMT group:(275±28)/mm~2,Veh group:(150±10)/mm~2].2.The results of TUNEL staining showed that the apoptosis of frontal cortex and hippocampal CA1 region of Sham group was lighter,and the apoptosis of other groups was obvious(P<0.01).Compared with I/R group,the percentage of apoptosis in LMT and HMT group decreased(P<0.05 or P<0.01).The percentage of apoptosis in frontal cortex:[Sham group:(5±1)%,I/R group:(86±10)%,LMT group:(63±5)%,HMT group:(54±4)%,Veh group:(88±9)%];The percentage of apoptosis in hippocampal CA1 region:[Sham group:(5±1)%,I/R group:(90±7)%,LMT group:(58±10)%,HMT group:(38±9)%,Veh group:(87±11)%].3.The content of TNF-?,IL-6 and IL-1?in I/R group was significantly higher than Sham group(P<0.05 or P<0.01).Compared with I/R group,the TNF-?,IL-6 and IL-1?were significantly decreased in the HMT(P<0.05)group,and the trend of those are almost same.4.Compared with Sham group,the expression levels of MDA and ROS were significantly increased,whereas SOD activity was significantly lower in I/R,LMT and Veh groups(P<0.05 or P<0.01).The levels of MDA and ROS were significantly decreased whereas SOD activity was significantly higher in HMT group than in the I/R group(P<0.05);5.The expressions of TLR4,MyD88,Cleaved Caspase-3 and NF-?B in the corresponding brain regions of the other groups were higher than those in Sham group(P<0.05 or P<0.01).The expressions of above-mentioned protein in the LMT and HMT group were decreased than in the I/R group(P<0.05 or P<0.01).6.Fluorescence results showed that the changes of TLR4 and MyD88 were consistent with the changes of microglial localization and activity.The expression of NF-?B was consistent with the change of neuron localization and activity.Conclusion:1.TLR4/MyD88 signaling pathway in microglia were involved in cerebral injury induced by intestinal ischemia-reperfusion.2.Melatonin alleviates brain injury in rats subjected to intestinal ischemia-reperfusion via inhibiting TLR4/MyD88 signaling pathway in microglia,and exerts anti-inflammatory,anti-oxidative and anti-apoptotic effects.