Inhibition Of CCR5/Akt/IKK/NF-κB Signaling Pathway Promotes Axonal Growth And Neurological Recovery In The Chronic Phase Of Intracerebral Hemorrhage

Objective: Cerebral hemorrhage refers to hypertension,cerebral arteriosclerosis and other factors caused by cerebrovascular non-traumatic rupture bleeding,is a common subtype of stroke.The annual incidence of cerebral hemorrhage accounts for about 20% of the total incidence of stroke.Cerebral hemorrhage can lead to a series of severe pathological changes in the brain rapidly,including the compression effect of hematoma,edema of brain tissue,inflammatory response and neuronal apoptosis.Cerebral hemorrhage has a high disability rate and fatality rate,which seriously damages the neurological function of patients.At present,the clinical treatment of cerebral hemorrhage is very limited,mainly absolute bed rest,reduce intracranial pressure and surgical treatment,but these treatment methods are only suitable for the acute phase of cerebral hemorrhage.But the brain ’s self-repair process continues,not limited to the acute phase,and this feature may provide a relatively appropriate treatment opportunity for the chronic recovery phase of intracerebral hemorrhage.Many studies have shown that after intracranial hemorrhage or ischemic injury,motor neuron axons in the undamaged cortex on the lesion side and the motor neuron axons in the unaffected cerebral cortex will appear budding to a certain extent,and the extended branches innervate the damaged area and re-establish structural and functional connections with the cervical enlargement area,which is known as axonal regeneration of neurons,and this feature provides an anatomical basis for the recovery of neurological function.Therefore,it is very important to find the control mechanism of axonal growth induced by intracerebral hemorrhage and intervene it to promote the recovery of neurological function in patients with intracerebral hemorrhage.Chemokine receptor 5 is a seven-transmembrane member of the G protein-coupled receptor superfamily.Stroke has now been found to trigger high CCR5 expression in the brain,so it has received attention from researchers.There are many studies on the role of CCR5 after stroke,and it has been found in animal experiments that inhibition of CCR5 can promote motor recovery in the acute phase of hemorrhagic stroke and various stages after ischemic stroke,and in addition,a CCR5 antagonist,Maraviroc(MVC),has been applied in some experiments of hemorrhagic and ischemic stroke,which has led the FDA to approve drugs that can be used to treat AIDS,which gives us the possibility of the CCR5 antagonist,Maraviroc,in the treatment of stroke.In addition,it has been found that motor function recovery after cerebral ischemia is better in patients with CCR5 gene deletion than in ordinary patients.These results point to a positive effect of CCR5 inhibition on the prognosis of stroke.However,in the chronic phase of intracerebral hemorrhage,whether CCR5 can also affect neurological function,and through which way to affect neurological function,it needs further exploration.In summary,we will use the CRISPR/Cas9 system to construct a CCR5 knockout rat model to first explore the phenomenon to verify the conjecture,and then use the CCR5 inhibitor Maraviroc to observe whether the application of the drug on axonal growth and neurological recovery is consistent with gene knockout,and to explore what mechanism the beneficial effect of Maraviroc on inhibiting CCR5 is produced.Methods: 1.In this experiment,we first divided SD rats into sham operation group(sham WT group)and intracerebral hemorrhage group(ICH WT group),and detected the changes of CCR5 expression in the peri-hemorrhagic tissues using RT-qPCR technique;and observed the expression of CCR5 on various cells using immunofluorescence co-localization.2.CCR5 knockout rats were constructed using the CRISPR/Cas9 system and divided into the following four groups: wild-type sham group(sham WT group),CCR5-mutant sham group(sham CCR5-Mut group),wild-type intracerebral hemorrhage group(ICH WT group)and CCR5-mutant intracerebral hemorrhage group(ICH CCR5-Mut group).The neurological recovery in chronic phase was evaluated by balance beam test and cylinder test by observing the behavioral performance of rats in each group.BDA anterograde labeling and immunofluorescence were used to evaluate axonal growth in the spinal cord after intracerebral hemorrhage in rats.3.Subsequently,we applied Maraviroc(Maraviroc,MVC)to inhibit CCR5.Rats were divided into the following three groups: sham operation group(Sham WT group),intracerebral hemorrhage group(ICH WT group)and intracerebral hemorrhage + MVC intervention group(ICH + MVC group).RT-qPCR was used to detect the expression of CCR5 in the cortex around the hemorrhage after Maraviroc,behavioral experiments were used to detect the recovery of motor function in each group,immunofluorescence was used to detect the axonal growth in the spinal cord of each group,and Western blot was used to detect the expression of axon-related parameters in each group.4.In order to explore the possible molecular mechanism by which inhibition of CCR5 promotes axonal growth and neurological recovery,we performed transcriptomic sequencing of cortical tissues around intracerebral hemorrhage in the three groups,and GSEA enrichment analysis was performed on the sequencing results to select differential pathways.5.Afterwards,we examined the expression levels of major kinases downstream of G protein-coupled receptors,and key transcription factors and their downstream indicators on selected differential pathways.6.Finally,the key indicators on the pathway were given corresponding interventions to verify the role of the examined pathway in CCR5-regulated axonal growth.To more comprehensively confirm the important role CCR5 and its downstream signaling pathways may play in axonal growth after intracerebral hemorrhage.Results: 1.The CCR5 expression was significantly higher in the perihematomal tissue around the ICH by RT-qPCR;immunofluorescence co-localization revealed that CCR5 was present in intracranial neurons,microglia,and astrocytes in the brains of wild-type rats;and CCR5 was significantly up-regulated mainly in neurons,upper microglia,and astrocytes in the basal cortex around the ICH at 28 days after ICH.2.In the rat behavioral test experiment,rats in the ICH CCR5-Mut group showed significant neurological recovery compared with the ICH WT group.It is mainly characterized by lower footstep error rates in both forelimbs and hindlimbs in the balance beam walking experiment,as well as more frequent spontaneous forelimb use.These behavioral results suggest that inhibition of CCR5 improves neurological function to some extent in the chronic phase of intracerebral hemorrhage.Moreover,the results of anterograde BDA labeling and immunofluorescence showed that rats in the ICH CCR5-Mut group showed significant axonal growth in the spinal cord compared with other groups.3.After CCR5 inhibition with maraviroc(MVC),we found that the behavior of ICH + MVC group was also significantly recovered,and significant axonal growth was also observed in the spinal cord of ICH + MVC group rats,and Western blot detection of related indicators also showed that CCR5 inhibition with maraviroc significantly increased intracranial axonal plasticity in the chronic phase of intracerebral hemorrhage.4.The results of transcriptomic sequencing and subsequent GSEA enrichment analysis showed that NF-κB signaling pathway was significantly up-regulated after intracerebral hemorrhage,and NF-κB signaling pathway was significantly down-regulated after inhibiting CCR5.5.Examination of key kinases downstream of CCR5 revealed that intervention with Maraviroc resulted in a significant decrease in the expression of phosphorylation levels of protein kinase B(Akt),but no difference in the expression levels of protein kinase A(PKA)and protein kinase C(PKC).The important transcription factor p NF-κB on NF-κB signaling pathway was down-regulated,and the phosphorylation level expression of IKK-β,a key hub connecting Akt and NF-κB signaling pathway,was also found to be decreased.This links the CCR5-Akt-IKK-NF-κB signaling pathway.6.Subsequent administration of the Akt agonist SC79 reversed the beneficial effects of Maraviroc on the chronic phase of intracerebral hemorrhage.Combined with the above experimental results,we hypothesized that inhibition of CCR5 promotes axonal growth and neurological recovery may be achieved by inhibiting the CCR5-Akt-IKK-NF-κB signaling pathway.Conclusion: 1.Cerebral hemorrhage leads to increased CCR5 expression in neurons,microglia,and astrocytes in the basal cortex surrounding hemorrhage during the chronic phase.2.Inhibition of CCR5 can promote axonal growth in the chronic phase of intracerebral hemorrhage and thus promote neurological recovery after intracerebral hemorrhage.3.Inhibition of CCR5 promotes axonal growth and neurological recovery in the chronic phase of intracerebral hemorrhage may be achieved by inhibiting the CCR5-Akt-IKK-NF-κB signaling pathway.