Effects And Mechanism Of SHH Signaling Pathway On Blood-brain Barrier In Mice With Hemorrhagic Brain Injury

Background and Objective: Intracerebral hemorrhage(ICH)is a kind of fatal stroke subtype with high morbidity and mortality.It not only causes serious harm to human health,but also adds heavy economic burden to the society and the patients’ families.So far,there is still a lack of effective therapy for ICH.Therefore,the study and exploration of the pathological mechanism of ICH is the prerequisite for clinical transformation research.Blood-brain Barrier(BBB)damage is an important hallmark of ICH-induced brain injury that leads to the formation of brain edema,infiltration of inflammatory cells and toxic components in brain tissue,and eventually aggravation of the neurological function injury.After ICH,activated astrocytes are essential for the formation and functional regulation of BBB,neurotrophic support,ion balance regulation and neuroinflammatory regulation,etc.Astrocytic Sonic Hedgehog(SHH)signaling pathway has been proved to be closely related to neuronal development,neuroangiogenesis and other important physiological mechanisms of the nervous system.In recent years,the role of SHH signaling pathway in maintaining the integrity of the BBB has attracted much attention,however,its effects on the regulation of BBB function in ICH remain to be clarified.To this end,we used small molecule inhibitors that specifically block the SHH signaling pathway to reveal the effects and mechanism of astrocyte-induced SHH signaling pathway on hemorrhagic brain injury,providing the fresh theoretical basis for the clinical translational reseach of ICH.Methods: C57/BL6 mice(8-10 weeks old)were used for experimental modeling which was established by the method of autologous blood or collagenase injection.Specific inhibitors of SHH signaling pathway(Cyclopamine/LDE-225/GDC-0449)were used for specific intervention in experimental animals and co-cultured cells in vitro.Enzyme-linked immunosorbent assay(ELISA)and immunofluorescence staining were used to detect the expression of SHH in mouse brain tissues;modified Neurological Severity Score(m NSS)and rotarod test were used to assess the neurologic impairment of mice,we also evaluated the hematoma volumes and brain water contents of mice,investigating the effects of SHH signaling pathway inhibition on the ICH-induced brain injury of mice;evans blue(EB)staining was used to assess the BBB permeability of mice;western blot(WB)was used to evaluate the levels of tight junction proteins(TJs)in mouse brain tissue;the immunofluorescence staining was used to evaluate the expressions of SHH signaling pathway-related molecules(Ptch-1,Smo,Gli-1)in mouse brain tissues;primary astrocytes were isolated from mouse brain tissues by magnetic activated cell sorting and co-cultured with mouse brain endothelial cell line(b End3),the immunofluorescence staining and WB were used to evaluate the levels of TJs in endothelial cells.Results: In mice brain tissues,SHH was mainly expressed in astrocyte;compared with the sham group,the expression of SHH in mice brain tissues was significantly increased on day 1 after ICH and reached a peak on day 3 after ICH,it showed a downtrend on day 7 after ICH which was still higher than the sham group.During the acute stage of ICH in mice,specific inhibition of SHH signaling pathway aggravated neurological deficits,hematoma volume and brain edema.During the acute stage of ICH in mice,specific inhibition of SHH signaling pathway increased the EB extravasation,suggesting that the permeability of BBB was significantly increased.During the acute stage of ICH in mice,specific inhibition of SHH signaling pathway downregulated the expressions of ZO-1,Occludin and Claudin-5 in mice brain tissues.During the acute stage of ICH in mice,the expressions of Ptch-1,Smo and Gli-1 were significantly increased,suggesting that the Ptch-1-Gli-1 axis was activated by ICH,however,blockage of SHH signaling pathway suppressed the ICH-induced activation of Ptch-1-Gli-1 axis in brain tissue.Specific inhibition of SHH signaling pathway downregulated the ZO-1 and Claudin-5 expressions of endothelial cells in“astrocyte-endothelial” co-cultured experiment.Conclusions: Astrocytic SHH signaling pathway plays a pivotal role in maintaining the integrity of BBB after ICH,suggesting the potential clinical intervention value of SHH signaling pathway in ICH-induced brain injury.