sew2871 and Infarction--Middle-Cerebral-Artery

Ischemic stroke is a common and frequent clinical disease. Recent studies have demonstrated that sphingolipid plays an important role in the pathological process of ischemic stroke. PI3K-Akt is a classic protective signaling pathway of cerebral ischemic injury. After acting on the S1P receptor, S1P can activate the downstream PI3K/Akt signaling pathway and play an anti-cerebral ischemia role. Buyang Huanwu Decoction (BHD) is a traditional Chinese medicine formula used to treat ischemic stroke. However, the mechanisms of BHD on ischemic stroke remain unclear based on S1P/S1PR1/PI3K/Akt signaling pathway.. The present study is intended to investigate the action mechanism of BHD on ischemic stroke based on the S1P/S1PR1/PI3K/Akt signaling pathway from multiple perspectives.. The BHD lyophilized product was prepared by vacuum freeze-drying method, of which the chemical composition was determined by UPLC-Q-TOF/MS. The mouse permanent middle cerebral artery occlusion (pMCAO) model was established by the suture-occluded method. Male KM mice were randomly divided into seven groups: sham group, model group, FTY720 (positive control) group, BHD group, BHD + W146 (selective S1PR1 inhibitor) group, SEW2871 (selective S1PR1 agonist) group, and Calycosin group. Each group was administered continuously for 14 days and evaluated with modified neurological severity score (mNSS) and cerebral infarct volume on the 1st, 4th, 7th, and 14th days. The SphK1, SphK2, S1PR1, PI3K, Akt, and p-Akt protein in the prefrontal lobe, hippocampus, and striatum was quantified by Western blot and immunohistochemical (IHC) experiment respectively. The qRT-PCR method was employed to evaluate SphK1, SphK2, and S1PR1 mRNA expression in the above tissue.. BHD and Calycosin both effectively improved mNSS scores with smaller infarct volumes. The SphK1 level in the prefrontal lobe, hippocampus, and striatum of mice in the BHD group was significantly lower, and SphK2, PI3K, and p-Akt in the hippocampus and striatum were significantly higher than those in the model group. BHD significantly decreased SphK1 mRNA expression in the prefrontal lobe, hippocampus, and striatum, and significantly up-regulated SphK2 mRNA and S1PR1 mRNA expression. Additionally, SphK1 protein expression levels of the prefrontal lobe, hippocampus, and striatum in the BHD group was significantly lower than model group, and SphK2, S1PR1, PI3K, Akt, and p-Akt protein expressions levels were increased obviously. Furthermore, SEW2871 can increase S1PR1 and Akt expression, and up-regulate SphK2 and S1PR1 mRNA expression. The effect of BHD on the expression of S1P/S1PR1/PI3K/Akt signaling pathway-related proteins and mRNA were weakened by BHD + W146.. BHD and Calycosin significantly improved the symptoms of neurological deficits in pMCAO mice, reduced the cerebral infarction volume, up-regulated SphK2 and S1PR1 mRNA levels, enhanced SphK2, S1PR1, PI3K, Akt, p-Akt protein expression of the prefrontal lobe, hippocampus and striatum, and down-regulated SphK1 mRNA and protein expression, which may be helpful to clarify the mechanism of BHD through S1P/S1PR1/PI3K/Akt signaling pathway to protect against cerebral ischemic injury.

Topics: Animals; Infarction, Middle Cerebral Artery; Ischemic Stroke; Male; Mice; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Messenger; Signal Transduction

Development of collateral circulation after acute ischemic stroke is triggered by shear stress that occurs in pre-existing arterioles. Recently, sphingosine-1-phosphate receptor 1 (S1P1) on endothelial cells was reported to sense shear stress and transduce its signaling pathways.. BALB/c mice (n = 118) were subjected to permanent middle cerebral artery occlusion (pMCAO) or sham operation. We investigated the effect of an S1P1-selective agonist SEW2871 on leptomeningeal collateral arteries and neurological outcome after pMCAO.. Immunohistochemistry showed that without treatment, the expression of S1P1 on endothelial cells of leptomeningeal arteries and capillaries increased early after pMCAO, peaking at 6 hours, whereas a significant increase in the expression of S1P1 in neurons was seen from 24 hours later. After intraperitoneal administration of SEW2871 for 7 days after pMCAO, the number of leptomeningeal collateral arteries was significantly increased, cerebral blood flow improved, infarct volume was decreased, and neurological outcome improved compared with the controls. Significantly increased phosphorylation of endothelial nitric oxide synthase (eNOS) as early as 6 hours after pMCAO and higher expression of tight junction proteins at postoperative day 3 were observed with SEW2871 treatment as assessed by Western blot. Daily administration of SEW2871 also increased capillary density in peri-infarct regions and promoted monocyte/macrophage mobilization to the surface of ischemic cortex at 7 days after pMCAO.. An S1P1-selective agonist enhanced leptomeningeal collateral circulation via eNOS phosphorylation and promoted postischemic angiogenesis with reinforced blood-brain barrier integrity in a mouse model of acute ischemic stroke, leading to smaller infarct volume and better neurological outcome.

Topics: Animals; Blood-Brain Barrier; Cell Line; Cerebrovascular Circulation; Collateral Circulation; Disease Models, Animal; Endothelial Cells; Infarction, Middle Cerebral Artery; Macrophages; Male; Meninges; Mice, Inbred BALB C; Monocytes; Neovascularization, Physiologic; Nitric Oxide Synthase Type III; Oxadiazoles; Phosphorylation; Receptors, Lysosphingolipid; Recovery of Function; Signal Transduction; Sphingosine-1-Phosphate Receptors; Thiophenes; Tight Junction Proteins; Time Factors

FTY720 is a known sphingosine 1-phosphate receptor agonist. In the present study, we investigated the neuroprotective effect of postischemic administration of FTY720 in rats with 2 hours transient middle cerebral artery occlusion (MCAO).. One hundred eleven male rats were randomly assigned to sham-operated and MCAO treated with vehicle, 0.25 mg/kg and 1 mg/kg of FTY720, another selective sphingosine 1-phosphate receptor-1 agonist SEW2871 (5 mg/kg), or 0.25 mg/kg of FTY720 plus a sphingosine 1-phosphate antagonist, VPC23019 (0.5 mg/kg). Drugs were injected intraperitoneally immediately after reperfusion. Neurological score and infarct volume were assessed at 24 and 72 hours after MCAO. Western blotting, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling were conducted at 24 hours after MCAO.. FTY720 significantly reduced infarct volume and improved neurological score at 24 and 72 hours after MCAO compared with the vehicle group. SEW2871 showed similar neuroprotective effects to FTY720, whereas VPC 20319 abolished the neuroprotective effects of FTY720. FTY720 significantly retained Akt and extracellular signal-regulated kinase phosphorylation and Bcl-2 expression and decreased cleaved caspase-3 expression and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling-positive neurons at 24 hours after MCAO. VPC23019 blocked the antiapoptotic effects of FTY720.. These data suggest that activation of sphingosine 1-phosphate-1 by FTY720 reduces neuronal death after transient MCAO.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Brain; Brain Ischemia; Disease Models, Animal; Drug Administration Schedule; Fingolimod Hydrochloride; Immunosuppressive Agents; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Male; MAP Kinase Signaling System; Nerve Degeneration; Neuroprotective Agents; Oxadiazoles; Propylene Glycols; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sphingosine; Stroke; Thiophenes; Treatment Outcome