Role Of Sigma-1 Receptor In Pericyte Survival After Acute Ischemic Stroke

Objective:The present study was aimed to investigate whether sigma-1 receptor was involved in the regulation of the pericyte survival in the context of ischemic stroke.Methods:For in vivo study,acute ischemia stroke was precessed in C57BL/6J as well as in sigma-1R KO male mice(six to eight weeks old).After permanent middle cerebral artery occlusion(pMCAO)24 h,infarct volume was observed by using magnetic resonance imaging(MRI)and 2,3,5-triphenyltetrazolium chloride(TTC)staining.Neurological score was used to evaluate the neurological deficits.The blood-brain barrier(BBB)permeability was assessed by using the Evans blue extravasation.The integrity of microvessels was observed by the transmission electron microscopy and the immunofluorescence staining.Western Blot was used to evaluate tight junction proteins,pericyte makers,apopotosis proteins and autophagy-related proteins.For in vitro study,C3H/10T1/2(C3H)cells and primary mouse pericytes were cultured and were subjected to oxygen glucose deprivation(OGD)for 0 h,1 h,3 h,6 h,12 h and 24 h.The cell viability was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT);Western Blot analysis was used to examine the expression of pericytes makers,apopotosis proteins and autophagy proteins.The autophagy inhibitor 3-MA and the autophagy inducer rapamycin were added for exploration the role of autophagy on the OGD-induced pericyte apopotosis.Moreover,the sigma-1R inhibitor BD1047 and the sigma-1R agonist PRE084 were used to explore the role of sigma-1R in the OGD-induced pericyte apopotosis and autophagy.MTT assay was used to screen the compounds which have a high affinity with sigma-1R in pericytes after OGD treatment,in vivo the therapeutic relevance study was explored by using TTC,MRI,Evans blue extravasation and Western Blot in both pMCAO and transient middle cerebral artery occlusion(tMCAO)model.Results:Our in vivo study demonstrated that knockout of sigma-1R aggravated infarction and neurological deficits in pMCAO mice,exacerbated the disruption of BBB and aggravated the autophagy and apoptosis in ischemic core area of pMCAO mice.These findings were further confirmed in in vitro study as evidenced by the fact that OGD treatment increased the autophagy and apoptosis of C3H cells and primary pericytes in a time-dependent manner.Morove,pretreatment of C3H cells and primary mouse pericytes with the autophagy inhibitor 3-MA significantly attenuated the OGD-induced apopotosis,while pretreatment of C3H cells and primary pericytes with the autophagy inducer rapamycin for 1 h augmented the OGD-induced apopotosis.Next,we further examined the role of sigma-1 receptor in the pericity survival.Pretreatment with sigma-1R agonists PRE084 significantly decreased the expression of apoptosis proteins,however,sigma-1R inhibitor BD1047 enhanced the expression of apoptosis proteins in both C3H cells and primary mouse pericytes.In terms of the expression of autophagy-related proteins,sigma-1R inhibitor BD1047 significantly increased the expression of autophagy proteins but sigma-1R agonists PRE084 decreased the expression of autophagy proteins in both C3H cells and primary pericytes.Finally,according the role of sigma-1 receptor in the pericyte survival,our finding indicated that Compound M1 significantly decreased the ischemia outcomes induced by pMCAO or tMCAO induced,suggesting M1 as a potential therapeutic strategy for acute ischemic stroke.Conclusions:Sigma-1R was involved in regulating pericyte survival via inhibiting autophagy and protected BBB integrity after acute ischemia stroke.The synthesized sigma-1 receptor agonist-Ml compound exerted protection against ischemic injury.Our findings indicated that sigma-1R is a promising therapeutic target for acute ischemic stroke.