The Role Of PDGFR?/MAPK Signaling Pathway In The Transdifferentiation Mechanism Of Pericytes-myofibroblasts After SAH

Objectives:Subarachnoid hemorrhage(SAH)from a ruptured intracranial aneurysm accounts for 5 % of all strokes and has particularly high morbidity and mortality.Despite a decline in the mortality,survivors are commonly left with permanent neurological disability,cognitive deficits,and mental healthsymptoms,bringing a heavy burden to the family and society.During the prognosis of SAH patients,microinfarction often occurs in the brain parenchyma,remarkably,part of the small infarct is far from the location of the ruptured aneurysm(even in the contralateral hemisphere).Analysis from the perspective of pathological mechanisms,the blood components rapidly enter the glymphatic system following SAH and penetrate into the perivascular parenchyma throughout the brain.Pericytes surround the outer diameter of the microvessels,direct damaged from the blood and its metabolites.Fibrillation factors,including blood metabolites,inflammatory cytokines,products of oxidative stress,provide fibrotic prerequisites in pericytes.Therefore,we speculate here that in the process of diffusion,fibrogenic factors directly or indirectly cause fibrotic changes in pericytes that regulate microcirculation,leading to the formation of microinfarcts in the brain parenchyma.It is proposed to detect the morphological and molecular changes of pericytes in vivo and in vitro SAH models,simultaneously,use the coculture system in vitro to further demonstrate the effect of pericyte fibrosis on the repair of nerve injury.It is important to explore the possible regulation mechanism of PDGFR?/MAPK signaling pathway in the pericyte fibrosis,and lay a solid foundation for the treatment of pericyte fibrosis as a therapeutic target to alleviate early brain injury(EBI)and improve prognosis after SAH.Methods:Experimental SAH in vivo was induced in adult male C57BL/6 mice by prechiasmatic cistern injection.Experimental SAH in vitro were induced in single systems or coculture systems of primary neuron,microglia,pericyte,and astrocyte.First part: Extracted of primary pericytes from adult mouse brain tissue,and passaged to the 5-7th generation by means of staged cultivation.Identified the pericyte purity by flow cytometry and immunofluorescence staining,and then established SAH models in vitro using qualified pericytes.Detected changes in morphological changes,pericyte markers,and myofibroblast markers after SAH.Second part: After detected the PDGFR? changes in vivo by western blot,real time-PCR and IF staining,explored the effects of special inhibitor of SL327 on PDGFR?/ERK signaling pathway in the regulation of pericyte-myofibroblast transdifferentiation and its involved neurological damage repair process.Third part: After detected the PCSK9 changes in vivo or in vitro,explored the effects of special inhibitor of SB203580 on PDGFR?/P38 signaling pathway in the regulation of synthesis and secretion of PCSK9 after SAH.Down-regulation of protein levels of PCSK9 in mouse brain after SAH using lentiviral technology and then contrast nerve damage,inflammatory response changes.Detection of the effects of PCSK9 recombinant protein on neuroprotection and inflammatory response in single system and coculture system of SAH modelResults:First part: Immunofluorescence staining results in the expression of pericyte markers(CD13 and PDGFR?),and flow cytometry double labeling(CD13 and PDGFR?)identification result was up to 89.9%.After SAH,pericyte morphology shrunk significantly,accompanied by the disorder of muscle fibers in the cytoplasm.Meanwhile,smooth muscle contractile protein and collagen expression increased significantly after SAH.Second part: Immunofluorescence suggested that PDGFR? protein was mainly localized to neurons,astrocytes,pericytes.The protein and mRNA levels of PDGFR? were significantly increased,which prompted that the PDGFR? pathway is activated after SAH.SL327 could significantly inhibit PDGFR?/ERK signaling pathway and its downstream of smooth muscle contractile protein and collagen expression,accompanied by nerve damage relief in mice.Inhibiting the PDGFR?/ERK signaling pathway of pericytes in coculture system,nerve damage was alleviated.Third part: The protein and mRNA levels of PCSK9 were significantly increased after SAH,and the secreted PCSK9 level was significantly increased in the supernatant of pericytes after SAH.SB203580 could significantly inhibit PDGFR?/P38 signaling pathway and its downstream of PCSK9,which indicated that PDGFR?/P38 pathway regulated the synthesis and secretion of PCSK9.Lentiviral treatment could significantly down-regulate PCSK9 protein expression in vivo after SAH,accompanied by nerve damage relief in mice.The PCSK9 recombinant protein could significantly inhibit the protective effect of astrocytes on neurons after SAH in the coculture system.The PCSK9 could also significantly increase the release of inflammatory factors after microglia SAH in vitro.Conclusion:Pericytes on the microvascular wall can be transdifferentiated to myofibroblasts after SAH,and the PDGFR?/ERK signaling pathway can regulate transdifferentiation.On the other hand,PDGFR?/P38 pathway regulate the synthesis and secretion of PCSK9 after SAH.Therefore,using PDGFR?/MAPK signaling pathway as a therapeutic target can effectively inhibit transdifferentiation between pericytes and myofibroblasts,alleviate nerve damage and Improve prognosis.