The Role Of Resvertrol On Microglia Polarization And Postoperative Cognitive Dysfuncti In Mice

Objectivepostoperative cognitive dysfunction(POCD)is a central nervous system complication that usually occurs after surgery,and it is more common in the elderly over 65 years old.Previous epidemiological studies have shown that the incidence of POCD in patients undergoing major cardiac surgery is as high as 20%-79%,but recent statistics have shown that the incidence of POCD in patients undergoing noncardiac surgery is also as high as 4.1%-22.3%.The occurrence of POCD may give rise to the increase of postoperative disability and mortality,the prolongation of hospital stay,the increase of medical costs and the decrease of postoperative quality of life.However,the biological mechanism of POCD is still unclear,and there is no particularly effective drug for the prevention and treatment of POCD in clinic.Resveratrol(RSV)is a natural polyphenol that is rich in plants such as grape,peanut,blueberry and raspberry.Resveratrol has many biological activities such as anti-inflammatory,anti-oxidation,anti-free radical and anti-cancer.Current studies have demonstrated that resveratrol can improve POCD by affecting the activation of microglia in the central nervous system.Nonetheless,the specific molecular mechanisms by which it regulates microglia activation have not been elucidated.The purpose of this research is to investigate the role of CX3CL1/CX3CR1 signaling pathway in resveratrol’s improvement of POCD and reduction of neuroinflammation,in order to provide a molecular mechanism for the clinical application of resveratrol.Methods1.32 ten-month-old C57BL/6 male mice were randomly divided into four groups:Control group(Con group),Resveratrol treatment group(RSV group),Surgery group(Sur group)and Resveratrol + Surgery group(RSV+Sur group).Mice in the Con group did not undergo any treatment.Mice in RSV group were intraperitoneally injected with 100mg/kg resveratrol for 5 consecutive days.Mice in the Sur group underwent exploratory laparotomy under isoflurane inhalation anesthesia.Mice in the RSV+Sur group were intraperitoneally injected with 100mg/kg resveratrol for 5consecutive days before surgery,followed by exploratory laparotomy under isoflurane inhalation anesthesia.The Y-maze,open field test and fear conditioning test were used to evaluate the postoperative cognitive function of the mice.At the end of the behavioral test,the whole brain and hippocampus tissues were extracted after heart perfusion with paraformaldehyde.Western blot was used to detect the protein expression levels of SIRT1,CX3CL1 and CX3CR1 in hippocampus.The expression levels of TNF-α,IL-1β and IL-6 in hippocampus were detected by real-time quantitative immunofluorescence polymerase chain reaction(q RT-PCR).Immunofluorescence was used to detect the expression of microglia activation marker Iba-1 and M1/M2 microglia markers CD86 and CD206 in the hippocampus.2.BV2 microglia were randomly divided into four groups: Control group(Con group),Lipopolysaccharide-stimulation group(LPS group),Lipopolysaccharide +Resveratrol + small interfering RNA group(LPS+RSV+si RNA group)and Lipopolysaccharide + Resveratrol group(LPS+RSV group).Cells in the Con group did not undergo any treatment.Cells in the LPS group were stimulated with 100ng/ml LPS for 6 hours.Cells in the LPS+RSV group were pretreated with 10μM resveratrol for 6 hours before LPS stimulation.Cells in LPS+RSV+si RNA group were treated with 100 n MCX3CR1 small interfering RNA to knock down the CX3CR1 gene before resveratrol pretreatment.The expression levels of inflammatory factors such as TNF-α,IL-1β,IL-6 and M1/M2 microglia markers i NOS and Arg-1 released by the cells were detected by q RT-PCR.Western blot was used to detect the expression levels of SIRT1 and CX3CR1 in the cellular proteins of the extracted cells.Results1.In the behavioral experiments,intraperitoneal injection of resveratrol significantly improved the cognitive dysfunction induced by exploratory laparotomy.2.Surgery significantly reduced the expression of SIRT1,CX3CL1 and CX3CR1 protein in hippocampus,and resveratrol significantly inhibited the surgeryinduced reduction of SIRT1,CX3CL1 and CX3CR1 protein levels.3.Resveratrol significantly reduced the secretion of TNF-α,IL-1β and IL-6 in the hippocampus induced by surgery.4.Immunofluorescence results showed that Iba-1 and CD86 positive cells were significantly increased in the hippocampal sections of the surgery group,while resveratrol significantly inhibited the surgery-induced increase in Iba-1 and CD86 positive cells and significantly increased CD206 positive cells.5.Resveratrol pretreatment significantly reduced the LPS-induced secretion of TNF-α,IL-1β,and IL-6 in BV2 microglia.6.CX3CR1 gene knockout abolished the inhibitory effect of resveratrol pretreatment on LPS induced reduction of SIRT1 and CX3CR1 protein expression levels in BV2 cells.Similarly,CX3CR1 knockdown abolished the reversal effect of resveratrol pretreatment on the increase in i NOS expression and decrease in Arg-1expression induced by LPS stimulation.ConclusionIn the animal experiments,our results demonstrated that resveratrol ameliorated postoperative cognitive dysfunction in mice by inhibiting the activation of M1 phenotype of microglia and promoting the activation of M2 phenotype to reduce neuroinflammation.Meanwhile,resveratrol also increased the synthesis of SIRT1,CXCL1 and CX3CR1 proteins in hippocampus.In cell experiments,we confirmed that CX3CR1 gene knockout inhibited the reduction of inflammatory cytokines release by resveratrol in inflammatory cell model,and also inhibited effect of resveratrol on inhibiting M1 phenotype polarization of microglia and promoting the M2 phenotype polarization.In conclusion,we hypothesize that resveratrol reduces neuroinflammation and improves postoperative cognitive dysfunction by inhibiting microglial M1 phenotype polarization and promoting microglial M2 phenotype polarization through CX3CL1/CX3CR1 signaling pathway.