The Role And Mechanism Of β2-Adrenergic Receptor In Sepsis-Related Encephalopathy

Objective Sepsis associated encephalopathy(SAE)is an acute diffuse cognitive dysfunction caused by peripheral infection.The specific mechanism of SAE is still unclear.Beta-2-adrenoceptor(β2-AR)is a G protein-coupled receptor,which regulates immune response and synaptic plasticity.The abnormal β2-AR may lead to a variety of neurodegenerative diseases.However,the role of beta 2-AR in SAE has rarely been reported.Therefore,we hypothesize that the dysfunction of β2-AR is involved in the cognitive impairment caused by sepsis.In this study,sepsis-related encephalopathy model was established by cecal ligation and perforation.β2-AR was used as the starting point,and animal behavior and molecular biology were used to study the possible pathophysiological mechanism of SAE occurrence,providing a new idea for the prevention and treatment of sepsis-related encephalopathy.Methods 220 male C57BL/6 mice,aged 3-4 months and weighing 22-26 g,were used to establish SAE animal models by cecal ligation and puncture(CLP).The first group of mice were executed at 6h,12h,24h,7d and 16 d after sepsis.The hippocampus was abtained to detect the changes of inflammatory response and synaptic plasticity-related proteins.The second batch of mice were randomly divided into five groups:sham group,CLP group,CLP+0.02mg/kg group,CLP+0.1mg/kg group and CLP+0.5mg/kg group.The mice in CLP+0.02mg/kg group,CLP+0.1mg/kg group,CLP+0.5mg/kg group were injected with clenbuterol 0.02mg/kg,0.1mg/kg and 0.5mg/kg intraperitoneally immediately after CLP,and then executed 16 days after operation.Sham group and CLP group mice were injected with the same volume of saline.Open field test(OF)and conditioning test(FC)were used to detect the behavioral changes of mice 13,14 and 15 days after operation,and the effects of clenbuterol on SAE were detected.The third group of mice were randomly divided into four groups:sham group,sham+clenbuterol group,CLP group,CLP+clenbuterol group,to explore the intervention effect of immediate activation of β2AR on SAE.The mice in sham+clenbuterol group and CLP+clenbuterol group were intraperitoneally injected with clenbuterol 0.5 mg/kg immediately after CLP,once a day and stopped every five days for two days until they were executed 16 d after operation.The mice in sham group and CLP group were injected with the same volume of saline.Three mice in each group were executed 24 hours after CLP to detect the changes of biochemical indexes in the acute phase of sepsis.The rest of the mice were reared to 16 days after operation.Open-field and conditional fear tests were carried out on the 13d,14d and 15d after operation to detect the motor and learning and memory abilities of the experimental mice.The mice were executed on the next day after behavioral studies.The changes of biochemical indicators during recovery period of sepsis were measured.The four group of mice were randomly divided into sham group,sham+clenbuterol group,CLP group,CLP+clenbuterol group,in order to explore the intervention effect of activating β2-AR on SAE during the recovery period of SAE.The mice in sham+clenbuterol group and CLP+clenbuterol group were intraperitoneally injected with clenbuterol 0.5 mg/kg on the 8d after operation(the same administration method as above).The mice in sham group and CLP group were injected with corresponding saline of equal volume.The mice in each group were fed until 16 days after operation.The exercise and learning and memory abilities of the mice were tested by open field and conditioned fear test at 13d,14d and 15d after operation.The mice were executed on the next day after behavioral study to detect the changes of biochemical indicators related to the recovery period of sepsis.Results In the first batch of experiments,compared with sham group,the expression level of β2-AR began to decrease gradually at 12h after CLP and lasted until 16th day after operation.At the same time,the expression levels of CD11b,inflammatory factors(IL-1b,TNF-a and IL-6)increased in varying degrees,while the expression levels of synaptic plasticity-related proteins pCREB,BDNF,PSD95 and GluN2b decreased in varying degrees.In the second group of experiments,there was no significant difference in the total distance of movement and the time in the central region between groups in open field experiments.In the context test of fear conditioning test,compared with CLP group,clenbuterol treatment immediately after operation significantly increased the freezing time of mice,significantly reduced the expression levels of CD68,CD 16,IL-1b and TNF-a,and increased the expression of M2 microglia markers(CD206 and arginase-1).The levels of M1 and M2 microglia markers and inflammatory factors on the 16th day after operation showed no significant difference among the groups.At the same time,compared with CLP group,clenbuterol immediately after operation significantly increased the expression levels of synaptic plasticity-related proteins pCREB,BDNF,PSD95 and GluN2b on the 24th and 16th day after operation.In the third batch of experiments,compared with CLP group,delayed clenbuterol treatment(at the recovery stage of sepsis)also significantly increased the freezing time of CLP group mice,and significantly reduced the expression levels of synaptic plasticity-related proteins pCREB,BDNF,PSD95 and GluN2b 16 days after CLP.Conclusion 1.In SAE model,the expression level of beta 2-AR in hippocampus is significantly reduced.2.Early clenbuterol-activated beta 2-AR can improve the cognitive impairment of SAE by promoting the change of microglia from M1 to M2,reducing the production of inflammatory factors,up-regulating the CREB/BDNF signaling pathway and increasing the expression levels of synaptic plasticity-related proteins.3.Even activate β2-AR during the recovery period of SAE,it can also promote the recovery of cognitive function of SAE by increasing the level of synaptic plasticity-related proteins.This study shows that β2-AR is closely related to cognitive impairment of SAE.