Study On The Evaluation Of Collateral Circulation And Prognosis Of SICAS Based On Metabonomics

BackgroundStroke is the second leading cause of death and the third leading cause of disability globally.Ischemic stroke（IS）patients accounted for 82.6%of all stroke hospitalizations in China in 2019.The Symptomatic Intracranial Atherosclerotic Stenosis（s ICAS）accounts for 46.6%of the etiology of IS in China.Cerebral collateral reconstruction is clearly associated with the clinical outcome in s ICAS patients.Plasma metabolites can be used to assess the risk,severity,and prognosis of IS,but whether plasma metabolites are associated with ischemic collateral circulation reconstruction remains unclear.In this study,non-targeted metabolomics techniques were used to find plasma metabolites that could evaluate the collateral circulation status of s ICAS.The related molecular mechanism was discussed by basic experiments.MethodsThis study is divided into clinical part and basic experimental part.In the clinical study,s ICAS patients were recruited for a two-stage study.Tan score based on CTA was used to evaluate the collateral circulation,and patients were divided into good and bad collateral circulation groups.In the discovery phase,plasma metabolites were detected by liquid chromatography-high resolution mass spectrometry to find metabolites that could evaluate collateral circulation.At the verification stage,ELISA was used to detect the levels of plasma metabolite sphingosine-1-phosphate（S1P）between the groups.Results（1）In the discovery phase of clinical study,37 different plasma metabolites were initially screened by non-targeted metabolomics method to evaluate the collateral circulation status of s ICAS patients.Through enrichment pathway analysis and KEGG pathway analysis,sphingolipid metabolism was found to be the most relevant pathway for collateral circulation.The expression levels of SA1P and S1P in sphingolipid metabolic pathway were higher in the good collateral circulation group,and the expression levels of SA1P and S1P were significantly correlated with the 90-day functional prognosis.（2）In the validation stage of the clinical part,the expression level of S1P was verified by ELISA method,and it was found that the high level of plasma S1P independently predicted the collateral circulation state well,and the ROC curve area was 0.738.In the validation phase,plasma S1P levels were also found to be positively correlated with functional outcomes at 90 days.（3）At the animal level,compared with wild-type t MCAO model,Sph K2^-/-mice showed more severe neurological defects,higher mortality,and significantly reduced cerebral blood flow on the 7th day after surgery.The results indicate that endogenous Sph K2 may improve stroke function defect symptoms and survival rate by promoting the opening of collateral circulation to increase cerebral blood flow.（4）At the cellular level,the results showed that S1P intervention promoted angiogenesis by increasing the expression of P-stat3 and Cav1 molecules compared to b End.3 cells treated with OGD/R alone.Compared with ABC294640 intervention alone,ABC294640+S1P intervention promoted the cell activity,cell migration and cell tubular formation ability,and the protein expressions of p-stat3 and Cav1 were significantly increased.Stattic intervention decreased cell activity,cell migration and tubular formation ability of b End.3cells treated with OGD/R,while p-stat3 and Cav1 protein expressions were significantly decreased.At the same time,Stattic+S1P intervention showed that the cell activity,cell migration and cell tubular formation ability of b End.3 treated by OGD/R were enhanced again,and the protein expressions of p-stat3 and Cav1were significantly increased.Conclusions（1）37 different plasma metabolites were selected,and sphingomyelin metabolism was the most related metabolic pathway to the collateral circulation.Plasma S1P level can be used as an important predictor of collateral circulation status.（2）Sph K2 promotes the opening of collateral circulation and improves the prognosis of neurological function by increasing cerebral blood flow and the number of new vessels in t MCAO mice.In the molecular mechanism,Sph K2promoted the angiogenesis by promoting the secretion of S1P in OGD/R-treated b End.3 cells,and subsequently increased the expression of P-stat3 and Cav1proteins.These results suggest that Sph K2/S1P/STAT3/Cav1 signaling pathway may be involved in the opening of collateral circulation after cerebral ischemia.