Effects Of Adenosine Preconditioning On HMGB1/TLR4/NF-κB Signaling Pathway During Cerebral Ischemia-reperfusion Injury In Rats

BackgroundCerebral ischemia reperfusion injury（CIRI）is a difficult problem that clinicians often face in the process of diagnosis and treatment.The over-activation of inflammatory response plays an important role in this process.Studies have shown that Toll like receptor4（TLR4）can be used as the receptor of High mobility group box 1（HMGB1）protein in the pathogenesis of various inflammatory diseases.Its combination can stimulate the activation of the Nuclear factor-κB（NF-κB）signaling pathway,which induces the excessive release of a series of inflammatory factors.Adenosine is an active substance involved in energy metabolism in the body.A number of studies have confirmed that adenosine preconditioning can inhibit the release of a series of inflammatory factors and cytokines in the process of inflammation,so as to reduce the damage of inflammation to nerve cells and play its neuroprotective role,but the exact mechanism has not been clearly studied.ObjectiveTo observe the effects of adenosine preconditioning on HMGB1,TLR4 and NF-κB after cerebral ischemia-reperfusion injury in rats,and to explore the neuroprotective mechanism of adenosine preconditioning on cerebral ischemia-reperfusion injury.MethodsThe experimental objects selected in this experiment were 120 healthy adult male Sprague-Dawley rats weighing 220-270 g provided by animal Center of Xinxiang Medical College.They were divided into 3 groups with 40 rats in each group in strict accordance with the random principle.They were labeled as sham operation group（F group）,ischemia-reperfusion group（I/R group）and adenosine preconditioning group（AP group）.According to the different time of post-ischemia reperfusion,they were divided into 4subgroups of 2h,6h,24 h and 48 h,of which 10 rats were randomly divided into 24 h subgroup（3 rats for TTC staining,5 rats for HE staining and 5 rats for Immunohistochemical staining）,and 10 rats were randomly divided into each subgroup（5rats for HE staining and 5 rats for Immunohistochemical staining）.The MCAO model was established by modified thread embolization method,and the neural function of the three groups were quantitatively scored by animal behavior evaluation method.The cell structure and morphology of brain tissue were observed by HE staining,and the ischemic necrosis of brain tissue was observed by Triphenyl Tetrazolium Chloride（TTC）staining,and the volume of ischemic necrosis was calculated by Image J.Immunohistochemical staining was performed to investigate the expression of HMGB1,TLR4 and NF-κB protein in brain tissues of rats in different time periods after cerebral ischemia reperfusion.SPSS26 software was used for statistical analysis of the experimental data.Results（1）All subgroups of I/R group and AP group had different degrees of neurological impairment,and their scores were significantly higher than those of F group,the difference being statistically significant（P<0.05）,while the neurological impairment of AP group was significantly less than that of I/R group,the difference being statistically significant（P<0.05）.HE staining showed that the structure and morphologic pathological changes of brain cells in AP group were less than those in I/R group at different time after reperfusion.（2）TTC staining showed that the volume of pale-brain area（cerebral infarction site）in AP group and I/R group was significantly higher than that in F group,and the difference was statistically significant（P<0.05）.The volume of pale-brain area（cerebral infarction site）in AP group was significantly smaller than that in I/R group,and the difference was statistically significant（P<0.05）.（3）Immunohistochemical staining showed that: A small amount of HMGB1,TLR4 and NF-κB protein were expressed in cerebral cortex of rats in F group,while HMGB1,TLR4 and NF-κB protein were significantly expressed in cerebral cortex of rats in AP group and I/R group compared with rats in F group,the differences were statistically significant（P<0.05）.The protein expression levels of HMGB1,TLR4 and NF-κB in the cerebral cortex of AP group were significantly decreased compared with that in I/R group,and the difference was statistically significant（P<0.05）.Conclusion（1）Adenosine pretreatment can not only reduce the morphological changes of rat nerve cells,reduce the volume of cerebral infarction,but also reduce the neurological function score after cerebral ischemia-reperfusion injury in rats.（2）Adenosine preconditioning may inhibit inflammation by down-regulating the NF-κB signaling pathway mediated by HMGB1-TLR4,thereby alleviating neural injury caused by cerebral ischemia reperfusion in rats and protecting brain cells.