Peptide H11 Targeting Class A Scavenger Receptor Opposites Cerebral Ischemia/reperfusion Injury

Ischemic stroke is one of the major causes of death and disability worldwide,inflammation is the most important pathological process affecting the occurrence,development and prognosis of ischemic stroke.Infiltrating monocyte/macrophages and activated resident microglia are the main participants in the inflammatory response.Researches show that the microenvironment of ischemic cerebral injury induces the microglia/macrophage polarization,and macrophage polarization imbalance reflects inflammatory state of local microenvironment.Macrophage polarization resists in cardiovascular and cerebrovascular diseases,cancer,metabolic diseases and other inflammatory related diseases,Further understanding of molecular mechanisms and intervention targeting on key steps in macrophage polarization may light the way for therapy of a number of inflammation-related diseases.Class A scavenger receptor(SR-A)is a pattern recognition receptor expressed mainly in the monocyte/macrophages,the expression of SR-A closely related to various microglia/macrophages related diseases.Microglia/macrophages play an important role in inflammation due to cerebral ischemia.Different subtypes of microglia/macrophages express class A scavenger receptor are discrepant,therefore,their role in the inflammatory response are often different.Recent studies show,SR-A promotes ischemic brain injury by activating NF-κB,lack of SR-A may mitigate cerebral ischemia/reperfusion injury.H11 is a small peptide previously selected from a phage peptide library,which can inhibit SR-A expression.We constructed H11 transgenic mice,to explore whether H11 inhibit SR-A expression in microglia or macrophages and the potential role in ischemic cerebral injury.The model of mouse middle cerebral artery occlusion(MCAO)was used in our study,60 min after ischemia reperfusion recovery outgoing tie line to establish cerebral ischemia/reperfusion injury model.We used wild-type(WT)and H11 transgenic mice.We confirmed the protein expression level of SR-A in mice brain tissue after MCAO was up-regulated by western blot and immunohistochemistry.1d and 3d after reperfusion,infarct sizes of Tg mice significantly reduced and neurological function score improved significantly.Infarct border zone,TUNEL staining showed neuronal apoptosis significantly reduced.We found cleaved-caspase3 significantly reduced in infarcted brain tissue,suggesting that H11 overexpression can alleviate brain damage caused by MCAO.Then we used immunohistochemistry and flow cytometry to detect microglia/macrophage activation,infiltration and different polar conditions in ischemic brain tissue.The results showed that overexpression of H11 attenuated inflammatory responses of microglia/macrophages after ischemia,reduced the amount of activated microglia/macrophages in the brain,and changed the ratio of M1/M2.At the same time,various types of microglia/macrophage products and markers in ischemic brain tissue were measured by real-time PCR.We found Ml-type markerssuch as tumor necrosis factor-α(TNF-α),interleukin-1p(IL-1p),inducible nitric oxide synthase(iNOS)and monocyte chemoattractant protein 1(MCP-1)in Tg mice were significantly lower than those in WT mice;whereas,the M2-type markers such as IL-10 and CD206 increased significantly than which in WT mice.Further analysis in signaling pathways in brain tissue revealed that the activity of NF-κB was repressed in H11 Tg mice compared with wild-type mice after cerebral ischemia/reperfusion injury.This indicates that the expression of SR-A is suppressed by ischemia/reperfusion-induced activation of microglia/macrophage,cells were shifted to M2 phenotype,and secretion of proinflammatory cytokines were reduced.NF-κB signaling pathway activity was weakened the ischemic brain injury was retarded,subsequently.We treated microglia cells line BV2 cells and mouse peritoneal macrophages under LPS or IL-4 stimulation with or without H11,in vitro.Interestingly,we found that H11 treatment enhanced LPS-induced Ml-type of inflammatory factors in peritoneal macrophages,while weakened M1-type of inflammatory cytokines in BV2 cells;in contrast,H11 enhanced IL-4-induced M2-type inflammatory cytokines in BV2 cells,while reduced M2-type inflammatory factors in peritoneal macrophages.This shows that the role of H11 in centre and peripheral cells may be different.Importantly,when transplanted with H11 transgenic bone marrow cells,wild-type mice,did not show reduced cerebral ischemia/reperfusion injury.So the neuroprotection effect of H11 is mediated through microglia in situ rather than infiltrating macrophages.In summary,our data demonstrate that small peptide H11 alleviates brain tissue damage caused by ischemia/reperfusion by downregulating SR-A expression and inhibition of the inflammation.H11 can inhibit activation of microglia/macrophages induced by brain ischemia,decrease the M1/M2 ratio,which resulting in pro-inflammatory cytokines secretion and act on the brain tissue.Thus,by H11 is expected to become the intervention of cerebral ischemia/reperfusion injury.