Icaritin Regulates Glial Cell Polarization Via GPER-NF-κB To Promote Neurological Recovery After Cerebral Ischemia

Background:Ischemic stroke is a syndrome in which neurological deficits occur due to impaired blood supply to the brain and ischemic and hypoxic necrosis of nerve cells caused by various cerebrovascular pathologies.It is the third leading cause of human death and disability worldwide and is a serious threat to human life expectancy.Ischemic stroke can lead to a variety of sequelae,such as motor function,sensory function,speech and swallowing function,and psychosomatic impairment.Drugs that can effectively improve the sequelae of ischemic stroke are special scarce,and therefore it is clinically important to research and develop drugs to treat the sequelae of ischemic stroke.Microglia and astrocytes are neuroglia in the central nervous system that can be activated and polarized into M1/M2 and A1/A2 phenotypes after cerebral ischemia,to exerting neurodamaging effects and neuroprotective effects and affect the prognosis of cerebral ischemia.Icaritin(ICT)is a phytoestrogen extracted from the traditional Chinese medicine Epimedium,which has various pharmacological effects such as anti-inflammatory and anti-tumor.Our previous study found that ICT can impact on G protein-coupled estrogen receptor(GPER)to exert cerebral protective effects in acute cerebral ischemia,but it is unclear whether ICT improves neurological function during recovery period of ischemic stroke.Therefore,the aim of this study was to investigate the regulation and possible mechanism of ICT on microglia and astrocytes during the recovery period of cerebral ischemia,to provide an experimental basis for further revealing the treatment of stroke sequelae by ICT.Objective:Observe the effects of ICT on the improve neurological function in rats recovering from cerebral ischemia/reperfusion injury;to clarify the regulatory effects of ICT on the activation and polarization of microglia and astrocytes;and to illustrate the regulatory effects of ICT on the activation and polarization of microglia and astrocytes may achieved through the GPER-NF-κB signaling pathway.Method:SPF-grade,healthy male SD rats,weighing 210-230 g.The transient middle cerebral artery occlusion(t MCAO)reperfusion surgery was operated by the wire embolization method and treated with 0.5 mg/kg of ICT administered intraperitoneally for 28 days.The survival rate,body weight,and Garcia JH neurological function score were measured to assess the neurological function of the rats during the rehabilitation period;the motor ability of the rats was examined by the rotarod test and gait test;the learning and memory ability of the rats during the rehabilitation period was examined by the Morris water maze test and Y-maze test;the number of neurons in the ischemic cortex were observed by Neu N immunofluorescence staining;the content of IL-1β and TNF-α in the ischemic cortex was measured by ELISA.GFAP,C3,GBP2,Nrf2,CD11 b,CD40,CD206,BDNF,Trk B,TNF-α,IL-1β,IL-6,IL-10,Doublecortin,Synapsin-1,PSD-95,Occludin,Claudin-5,GPER and NF-κB protein expression were detected by Western blot.Tissue immunofluorescence for Iba-1 and CD68,CD206;GFAP and GBP2,S100A10,Brd U and Neu N co-labeling respectively.The above indices were retested after intervention with the GPER-specific inhibitor G15.Results:1.ICT long-term treatment improved body weight,neurological function and reduced the degree of right brain deficit in t MCAO rats during the rehabilitation period;increased the time on rod and rotating speed in rotarod test,and improved gait;shortened the escape latency,improved learning memory function and promoted neuronal survival in the ischemic cortex in the water maze experiment.2.Long-term treatment with ICT downregulated the expression levels of CD11 b and M1 phenotype microglia marker CD40 protein,elevated the expression levels of M2 phenotype marker CD206 protein in t MCAO rats during recovery;immunofluorescence results showed that ICT reduced the number of CD68 microglia and increased the number of CD206 microglia.3.Long-term treatment with ICT downregulated the expression levels of GFAP and A1 phenotype astrocytes marker C3 and GBP2 protein,increased the expression levels of A2 phenotype astrocytes marker Nrf2 protein in t MCAO rats during recovery;immunofluorescence results showed that ICT reduced the number of GBP2 astrocytes and increased the number of S100A10 astrocytes.4.ICT long-term treatment significantly reduced the levels of TNF-α and IL-1β,increased BDNF/Trk B protein expression in the ischemic cortex during the recovery period in t MCAO rats,promoted the blood-brain barrier tight junction proteins expression of Occludin and Claudin-5,promoted neural regeneration in the DG region of the hippocampus,and increased the protein expression of Doublecortin and promoted expression of synapsin-1 and PSD-95.5.Long-term treatment with ICT increased the expression level of GPER protein and inhibited NF-κB protein phosphorylation in the ischemic cortex of t MCAO rats during recovery.6.Intraperitoneal administration of G15 abolished the therapeutic effect of ICT on t MCAO rats during the recovery period,reversed the effect of ICT in inhibiting M1 phenotype microglia and A1 phenotype astrocytes,and promoting M2 phenotype microglia and A2 phenotype astrocytes,and flipped the effect of ICT in inhibiting NF-κB protein phosphorylation.Conclusion:1.ICT has neuroprotective,improve motor,gait,and learning memory function on t MCAO rats during the rehabilitation period;2.ICT can promote neuronal regeneration in the DG region of hippocampus and maintain normal synaptic function and BBB integrity;3.ICT regulates microglia and astrocytes activation and polarization may through GPER-NF-κB signaling in t MCAO rats during the recovery period,to promoting neurological recovery after cerebral ischemia.