The Longitudinal Expression Of SphK2 In Acute Cerebral Ischemia In Rats And The Neuroprotective Effect Of Oxymatrine

Objective: Ischemia with high mortality and serious disability is the most common type of cerebral vascular disease. The pathological mechanism for ischemic injury was a perplexing cascade reaction, the perturbation of calcium homeostasis plays an important role in cerebral ischemic pathogenesis. The intracellular calcium overload activated kinds of enzymes regulated by Ca2+, induced membrane phospholipid resolved and brokedown cytoskeleton with subsequent cell death. Sphingosine kinase (SphK) is a critical enzyme that catalyzes the phosphorylation of sphingosine to sphingosine-1-phosphate. The catalytic activity and specific subcellular localization also contributes to the pro-apoptotic effect of SphK2, which affect the intracellular Ca2+. Oxymatrine (OMT) is an alkaloid extracted from Sophora flavescens Ait and its molecular formula is C15H24N2O2, with a tetracyclic quinolizine structure. In both preclinical and clinical studies, it has been confirmed to possess a variety of biological activities including anti-inflammatory, anti-oxidant, anti-cancer and anti-anaphylaxis properties. In recent years, there are many studies conducted on OMT, most of them are focused on anti-hepatitis and anti-carcinoma, but there are rare reports on the effects of OMT on cerebralvascular diseases. This study tested the dynamic expression of SphK2 in ischemic rats brain tissues using Western Blot, RT-PCR and immunohistochemistry technology; What's more we studied the influence of OMT on brain water content and infarction volume to testify the neuroprotective effect of OMT on cerebral ischemia and the underlying mechanism.Methods: Male, healthy Sprague-Dawley rats were subjected to permanent focal cerebral ischemia by the middle cerebral artery occlusion (MCAO). Experiment 1 was used to evaluate the longitudinal expression of SphK2 in the cerebral ischemia. Two groups were studied, including normal-control group and the MCAO group. The MCAO group included 3 h, 6 h, 12 h, 24 h, 48 h and 72 h sub-groups. We tested the dynamic expression of SphK2 in ischemic rat brain tissues using Western Blot, RT-PCR and immunohistochemistry technology. Experiment 2 was used to detect OMT's influence on SphK2 and claudin-5 expression. The rats were randomly divided into 3 groups: sham operated group that received equal volume 0.9% NaCl (Sham); MCAO group that received equal volume 0.9% NaCl after MCAO (MCAO); OMT group received OMT at 120 mg/kg after MCAO (OMT). OMT were administered immediately through intraperitoneal injection after the MCAO. For Sham and MCAO group, equal volume saline was administered in the same manner. Neurological behavior was evaluated at 24 h after operation then rats were sacrificed. Brain water content was measured by wet-dry method ; Infarct volume was analyzed with 2, 3, 5- triphenyltetrazolium chloride (TTC) staining. SphK2 and claudin-5 expression were measured by immunohistochemistry, Western Blot and RT-PCR.Results:1 The expression of SphK2 was upregulated compared with normal-control (P < 0.05), beginning at 12 h, and peaking at 72 h after MCAO.2 OMT ameliorated neurologic deficits, reduced brain water content and infarct volume. Neurological deficit score in OMT group was decreased compared with MCAO group (P < 0.05); No infarction was observed in Sham group. Compared with MCAO group, the infarct volume was significantly reduced in OMT high dose group (MCAO vs. OMT: 46.66±3.36 vs. 26.01±6.10, P < 0.01); Compared with MCAO group, the water content was significantly reduced in OMT high dose group (MCAO vs. OMT: 83.08±0.56 vs. 81.00±0.43, P < 0.01).3 OMT downregulated the expression of SphK2. The number of positive cells, the protein level and the mRNA level were increased after MCAO, and the overexpression of SphK2 was downregulated with administration of OMT (Western Blot: MCAO vs. OMT: 2.21±0.34 vs. 1.74±0.06, P < 0.05; RT-PCR: MCAO vs. OMT: 0.45±0.01 vs. 0.34±0.02, P < 0.05).4 OMT ameliorated BBB permeability. RT-PCR and Western Blot showed that OMT upregulated the expression of claudin-5 in the cerebral ischemia (RT-PCR: MCAO vs. OMT: 0.47±0.04 vs. 0.55±0.01, P < 0.05; Western Blot: MCAO vs. OMT: 0.95±0.04 vs. 1.25±0.14, P < 0.05).Conclusion: SphK2 increased in the delayed injury processes after cerebral ischemia. Systemic administration of OMT is effective which leads to decreased neurologic impairment and tissue injury and increased survival under cerebral ischemic conditions. Those effects may be through down-regulation of SphK2, inhibiting apoptosis; up-regulation of claudin-5, ameliorating BBB permeability.