Intervention Study Of TLR4/NF-κB Pathway On Cerebral Ischemia-reperfusion Injury In Mice

Background and ObjectiveCerebral ischemia reperfusion injury(CIRI)is related to inflammatory mechanisms,and TLR4/NF-κB is an important pathway of inflammatory mechanisms.Intermittent fasting has an inhibitory effect on inflammation,but it is not clear whether it can protect CIRI by inhibiting the TLR4/NF-κB pathway.However,intermittent fasting is difficult to be used in clinical practice due to its poor compliance,so it is necessary to find an alternative therapy for intermittent fasting.Metformin has many similarities with intermittent fasting.Metformin has the effect of inhibiting inflammation.It is not clear whether it can protect CIRI by inhibiting the TLR4/NF-κB pathway.In this study,metformin pretreatment and intermittent fasting were used to verify the protective effect of intermittent fasting and metformin on cerebral ischemia-reperfusion injury by inhibiting TLR4/NF-κB pathway,and to identify whether metformin can be an alternative therapy for intermittent fasting,so as to provide theoretical support for alleviating cerebral ischemia-reperfusion injury.MethodsThe mice were randomly divided into five groups: model group(CIRI),intermittent fasting group(IF),metformin group(Met),metformin and intermittent fasting group(Met+IF),sham operation group(Sham).Metformin(10mg/kg)was pretreated by intraperitoneal injection for 14 days.Intermittent fasting was limited daily from 16:00 to 8:00 the next day.The body weight and blood glucose changes of mice in each group were monitored on day 0,day 3,day 7,day 11,and day 14.A transient middle cerebral artery occlusion model was prepared.24 hours after the model was successfully prepared,the Longa score was used to score the mice to determine the neurological damage of the mice.TTC staining was used to understand the cerebral infarction of each group of mice.Brain water was used to understand the situation of brain edema in each group.HE staining was used to understand the damage of the cortex and hippocampus in each group.Immunohistochemistry and Western blot methods were used to detect the protein expression of TLR4 and NF-κB in the ischemic penumbra of each group of mice.Results1.The body weight of the IF group,CIRI group,Met group,Met+IF group and Sham group all increased,but the growth trend was slightly different.Compared with the IF group,the Met group,and the Met+IF group,the CIRI group and the Sham group increased significantly(P<0.05).There was no significant difference in body weight between sham group and CIRI group(P>0.05).Compared with the IF group,there was no significant difference in body weight of mice in Met group(P>0.05).Compared with the IF group,the Met group,the CIRI group,and the Sham group,the growth trend of the Met+IF group was gentle(P<0.05).Compared with the Sham group,CIRI group,IF group,and Met group,the blood glucose level of the Met+IF group was lower(P<0.05).The blood glucose levels of Sham group,CIRI group,IF group and Met group did not change significantly(P>0.05).2.Compared with the Sham group,the Longa score and cerebral infarction volume of the IF group,CIRI group,Met group and Met+IF group changed significantly(P<0.05).Compared with the CIRI group,the Longa score and cerebral infarction volume of the IF group,Met group and Met+IF group were significantly reduced(P<0.05).Compared with the IF group,the Longa score and cerebral infarction volume in the the Met+IF group were significantly reduced(P<0.05).Compared with the IF group,the cerebral infarction volume of the Met group was reduced(P<0.05).3.Compared with the Sham group,the brain water content of the IF group,CIRI group,Met group,and Met+IF group changed significantly(P<0.05).Compared with the CIRI group,the brain water content of the IF group,Met group,and Met +IF group was significantly reduced(P<0.05).Compared with the Met+IF group,the brain water content of the Met group and the IF group increased(P<0.05).Compared with the IF group,there was no significant difference in brain water content in the Met group(P(29)0.05).4.HE staining showed that the morphology of cerebral cortex and hippocampus cells in sham group was normal,and the cells were arranged orderly and completely.The cerebral cortex and hippocampus cells of the CIRI group was arranged disorderly,some of them formed cytoplasmic vacuoles,some neurons were swollen,unevenly distributed,nucleus pyknosis and nucleoli disappeared.The swelling of the cerebral cortex and hippocampal neuron cell bodies in the Met group,the IF group and the Met+IF group were significantly reduced,and the cell morphology were significantly improved.5.Immunohistochemistry and Western blot showed: Compared with the CIRI group,the expression of TLR4 and NF-κB decreased in the IF group,Met group,Met+IF group and Sham group(P<0.05).Compared with IF group,the expression of TLR4 and NF-κB decreased in met group,Met+IF group and sham group(P<0.05).Compared with the Met group,the expression of TLR4 and NF-κB in the Met+IF group and the Sham group were decreased(P<0.05).Compared with the Met group,IF group,and the Sham group,the expression of TLR4 and NF-κB decreased in the Met+IF group(P<0.05).Conclusion1.Intermittent fasting can have neuroprotective effect on CIRI by inhibiting the expression of TLR4 and NF-κB.2.Metformin can inhibit the expression of TLR4 and NF-κB,and has a neuroprotective effect on CIRI.3.The effect of 10mg/kg metformin pretreatment on TLR4 and NF-κB expression was stronger than intermittent fasting.Considering the inflammatory injury after CIRI,metformin may become an excellent alternative to intermittent fasting in the future.4.In this experiment,although metformin combined with intermittent fasting has a strong inhibitory effect on TLR4 and NF-κB and has a strong neuroprotective effect on CIRI,some mice have hypoglycemia.So it is necessary to closely monitor the changes of blood glucose for safety.