The Neuroprotective Effects Of Arginine In Cerebral Ischemic Injury

Objective Ischemia stroke causes secondary pathology such as oxidative stress,blood-brain barrier disruption,and inflammatory response.Microglia are the innate immune cells in the brain playing a critical role in the inflammatory response.Activated microglia may present two types of inflammatory and anti-inflammatory phenotypes – M1 type(pro-inflammatory)and M2 type(anti-inflammatory).M1 type microglia release proinflammatory cytokines to exaggerate neuronal damage,while M1 type microglia release anti-inflammatory cytokines and growth factors to promote neuronal survival.Arginine,a conditionally essential amino acid,is also associated with inflammatory response.HIF-1α is a transcript factor that plays a critical role in cell response to hypoxia.This study is aimed to investigate the neuroprotective effects of arginine on cerebral ischemic injury,and effects of arginine in microglial mediated inflammatory response.Methods In a rat model of MCAO and OGD-treated primary culture of microglia and neurons,the effects of arginine on neuronal death,the expression of M1 and M2 type microglial markers,as well as the protein levels of HIF-1α and LDHA were examined.The cerebral infarct volume and neurological function were assessed by TTC staining and neurological behavior test(modified neurological severity score,beam-walking and modified sticky-tape test),respectively.CCK8 and LDH releasing test were used to assay the cell viability of primary neuron cultures.The m RNA levels of M1 type markers(i NOS,TNF-α,and CD32)and M2 type markers(Arg1,YM-1,and CD206)were measured by QPCR.The protein levels of HIF-1α and LDHA were examined by western blotting.To further investigate the role of HIF-1α and LDHA in arginine action,the effects of HIF-1α inhibitor LW6 or LDHA inhibitor FX11 on the expression of M1 type and M2 type microglial markers and cell viability in OGD-treated cultures,as well as on cerebral infarct volume and neurological function in MCAO rats were examined.Results 1)In MCAO rats,arginine reduced cerebral infarct volume and improved functional recovery;arginine decreased the m RNA levels of pro-inflammatory markers i NOS,TNF-α,CD32,and increased m RNA levels of anti-inflammatory factors Arg1,YM-1 and CD206;2)In OGD-treated microglia cells,arginine decreased the m RNA levels of proinflammatory markers i NOS,TNF-α,CD32,while increases the m RNA levels of antiinflammatory factors Arg1,YM-1 and CD206,indicating that arginine promoted the switch of M1 type to M2 type;additionally,arginine pre-treated microglia promoted the neuronal survival in co-culture system;3)In OGD-treated microglia cells,arginine reduced the protein levels of HIF-1α and LDHA,and LDHA expression is positively associated with HIF-1α;4)In OGD-treated microglia cells,LDHA inhibitor FX1 antagonized the effects of arginine on the expression of microglial markers,and promoted the neuronal survival in co-culture system;5)In MCAO rats,HIF-1α inhibitor LW6 or LDHA inhibitor FX11 significantly reduced the cerebral infarct volume and improved neurological function recovery;LDHA inhibitor FX11 blocked the arginine action on expression of microglial functional markers.Conclusion: 1)Arginine promoted the switch of microglial polarization to M2 type via downregulating of HIF-1α/LDHA pathway;2)Arginine may exert neuroprotective effects by regulating the microglial polarization.