Role Of Maresin1 And Its Receptor RORα In Cognitive Impairment Caused By Chronic Cerebral Hypoperfusion

Objective The aging population leads to increased global burden of senile dementia.Alzheimer’s Disease(AD)and Vascular dementia(VaD)are the most common types of senile dementia.Chronic cerebral hypoperfusion(CCH)is one of the early common pathological mechanisms of AD and VaD,resulting in a gradual decline in cognition.The neuroinflammatory cascade occurs and persists in the early stage of CCH,induces the release of proinflammatory cytokines and toxic activation of glial cells,leads to neuronal death and demyelination,which ultimately evolves into brain damage and cognitive impairment.Neuroinflammation also promotes the hydrolysis of tight junctions(TJs),leads to an increase in the permeability of the blood-brain barrier(BBB),which disrupts the homeostasis of the brain microenvironment.CCH-induced neuroinflammation and BBB damage play an important role in the development of cognitive impairment.Macrophage mediator in resolving inflammation 1(Maresin1,hereinafter referred to as MaR1)is a member of the family of specialized pro-resolving lipid mediators(SPMs),which has biological activities promoting the resolution of inflammation at doses of nanograms or picograms.MaR1 has been confirmed to have a certain inhibitory effect on neuroinflammation,but the role and mechanism of MaR1 on CCH-induced neuroinflammation and BBB injury have not yet been elucidated.Retinoic acid-related orphan receptor α(RORα)is an endogenous receptor of MaR1,which forms a positive feedback pathway with MaR1 to inhibit the inflammatory response.Studies have found that RORα is widely expressed in neurons and astrocytes,and regulates inflammatory pathways within astrocytes through nuclear factor kappa B(NF-κB).However,the effect and mechanism of RORα on CCH-induced neuroinflammation and BBB injury have not been elucidated.In this study,we injected MaR1 into the CCH rats’ brain through an intrathecal catheter to explore its function in inhibiting cognitive impairment and neuroinflammation caused by CCH,and to evaluate its protective effect on BBB.In addition,we overexpressed RORα in CCH rats’ hippocampus via adeno-associated virus(AAV),and then evaluated cognitive function,neuroinflammation and BBB damage in CCH rats to further elaborate the possible mechanism of MaR1’s pro-inflammatory effect in CCH.It is hoped that these will provide a new intervention target for the prevention and treatment of CCH-related cognitive impairment.Methods 1.2-Vessel Occlusion(2VO)surgery was used to induce CCH model.Rats were randomly divided into 3 groups: Sham operation+PBS administration group,2VO operation+PBS administration group,2VO operation+MaR1 administration group(hereinafter referred to as Sham group,2VO group,2VO+MaR1 group respectively).The rats in the respective groups received either 2VO or Sham surgery,and received lumbar intrathecal catheterization.Then,the rats in 2VO+MaR1 group received MaR1 administration,and the rats in Sham group and 2VO group received PBS administration.The Morris water maze(MWM)was used to evaluate the cognitive function of rats 4 weeks after administration.The expressions of NF-κB,TNF-α and IL-1β were detected by enzyme-linked immunosorbent assay(ELSIA)and Western blot.The expressions of microglia marker Iba-1,astrocyte marker GFAP and myelin basic protein(MBP)in rats’ hippocampus were observed by immunofluorescence staining.2.Rats were randomly divided into 3 groups: Sham group,2VO group,2VO+MaR1 group.14 days after administration of MaR1 or PBS,the ultrastructure of BBB was observed by transmission electron microscope(TEM),the content of Evans blue(EB)in rat hippocampus was measured by chemical colorimetry.The expressions of tight junction proteins(ZO-1,Claudin-5)and MMP-9 were detected by WB and ELISA.The expressions of NF-κB were detected by enzyme-linked immunosorbent assay(ELSIA)and Western blot.3.Rats were randomly divided into 3 groups: Control-AAV+Sham group,Control-AAV+2VO group,RORα-AAV+2VO group.The adeno-associated virus(AAV2/9-CMV-r-RORα-3xflag-GFP virus)overexpressing RORα was injected into the bilateral hippocampus of rats in RORα-AAV+2VO group,and the empty virus(AAV2/9-CMV-GFP virus)was injected into the bilateral hippocampus of rats in Control-AAV+Sham group and Control-AAV+2VO group.Sham and 2VO surgery were performed 14 days after stereotaxic injection.14 days after 2VO/Sham,the content of EB in the hippocampus of rats was detected by chemical colorimetry.28 days after 2VO/Sham,the cognitive function of rats was assessed using MWM test.the expressions of NF-κB and IL-6 in the hippocampus of rats were detected by WB.Immunofluorescence staining was used to observe the co-staining of C3d/GFAP or S100A10/GFAP in the hippocampus,and Neu N immunohistochemical staining was used to observe the survival of hippocampal neurons.Results 1.2VO surgery reduced hippocampal blood flow in rats,induced the decline in spatial learning and memory,increased expression of NF-κB,IL-1β and TNF-α in hippocampus and white matter,increased activation of microglia and astrocytes and damage of white matter.MaR1 administration reduced the expression of pro-inflammatory factors,inhibited glial cell activation,reduced demyelination lesions,and improved spatial learning and memory in CCH rats.2.CCH increased the EB leakage of BBB,decreased expression of tight junction proteins(ZO-1,Claudin-5)and increased expression of MMP-9.Ultrastructural destruction of BBB was observed in TEM result.MaR1 administration inhibited the NF-κB pathway,reduced BBB leakage,suppressed MMP-9 expression and tight junction proteins disruption,and restored BBB Ultrastructure.3.The rats in the Control-AAV+2VO group had decreased spatial learning and memory,increased EB leakage of BBB,increased number of A1 astrocytes activation,decreased surviving neurons,and increased expression levels of NF-κB and IL-6 compared with the Control-AAV+Sham group.RORα overexpression improved cognitive function,attenuated BBB leakage,prevented A1 astrocyte activation,promoted neuronal survival,and inhibited the NF-κB/IL-6 pathway in the hippocampus of CCH rats.Conclusions 2VO surgery could successfully induce CCH rat model,resulting in neuroinflammation,BBB damage,and cognitive impairment in rats.Administration of the pro-resolving lipid mediator MaR1 or overexpression of the MaR1 receptor RORα could improve cognitive function,inhibit neuroinflammation,and protect BBB function in the CCH rats.RORα may be the target of MaR1 to inhibit CCH-induced neuroinflammation.MaR1/RORα may inhibit neuroinflammation and BBB injury,and improve cognitive impairment by inhibiting NF-κB/IL-6 pathway and activation of A1 astrocytes in CCH model.MaR1 and its receptor RORα may be a new target and idea for the treatment of CCH-related cognitive impairment.