| IntroductionSubarachnoid hemorrhage(SAH) is associated with high morbidity and fatality rate at a comparatively young age, resulting in a huge loss to the society. Currently, the pathophysiology of SAH is perceived as referring to the cerebral vascular neural network, which includes all small blood vessels downstream of the major cerebral arteries and cell types associated with these structures. Blood-brain barrier(BBB) is one of the typical components of this vascular neural network. BBB breakdown has close connection with vasogenic brain edema after SAH. Therefore, by targeting BBB breakdown, it may be favorable for the patients with SAH.Artesunate, the water-soluble semi-synthetic derivatives of artemisinin, has been a standard treatment for cerebral malaria and all kinds of other severe malaria. The antimalarial effect of artesunate is commonly recognized as highly efficient, generally safe and well-tolerated. Loads of research pointed out that it had widespread pharmacological activities such as anti-parasites, anti-tumor, anti-inflammation, anti-microbes, etc. As we know, the occurrence and development of neurological disorders usually refer to intricate pathophysiologic mechanisms and multiple etiopathogenesis. Recent progress has also demonstrated that drugs with single mechanism and serious side-effects are not likely the candidates for treatment of the neurological disorders. Therefore, the pluripotent action of artesunate may result in it playing an important role in the prevention and treatment of these neurological disorders. And more importantly, artesunate could be maintained as a higher concentration in the brain. Independent of plasmodium killing, recent studies revealed artesunate can ameliorate BBB breakdown, after experimental malaria infection in mice. In addition, when administered during infection in combination with artesunate, FTY720, the agonist of S1P1, treatment resulted in symptoms against BBB breakdown and thereby increased survival rate to cerebral malaria in mice. Therefore, we have reason to believe that artesunate has a certain therapeutic effect on SAH.S1P1(Sphingosine-1-phosphate receptor-1), the receptor of Sphingosine-1-phosphate(S1P), mainly expresses on vascular endothelial cells. S1P1 couples exclusively to the Gi/o family and plays an important role in maintaining the stability of flow dependent vascular network. Global deletion of S1P1 or deletion of S1P1 in endothelial cells would have serious consequences to the vascular plexus in mice. Previous researches revealed that S1P1 activation could protect injured brain tissue in many stroke models, such as : the study showed that increasing the expression of S1 P could improve the activation of S1P1 and then delay the development of early brain injury after SAH in mice. Nevertheless, it was not clear what the downstream signal pathway was of S1P1 in the protection of injured brain tissue. Previous research showed that S1P1 could activate the PI3K/Akt pathway to inhibit apoptosis, prevent activation of FoxO3 a and then promote PC12 cell survival, enhance adult mouse cardiac myocyte survival during hypoxia, and promote the differentiation of adipose-derived stem cells into endothelial nitric oxide synthase expressing endothelial-like cells, etc. Therefore, PI3 K is a very important downstream effector of S1P1. Taddei, et al. reported that PI3K/Akt activation could inactivate GSK-3β and limit the translocation of β-catenin to the nucleus, which relieved the inhibition and finally increased the expression of Claudin-5. Relevant research also indicated that PI3K/Akt activation could raise the expression of Claudin-3 and Claudin-5 through reduced GSK-3β activation and stabilized β-catenin after ICH in mice. It is well known that tight junction proteins of Claudin-3 and Claudin-5 are very important to BBB integrity. Effectively preserving the expressions of Claudin-3 and Claudin-5 could alleviate BBB breakdown in various central nervous system diseases.Based on these evidences, we purpose that artesunate may be functional via S1P1 activation in the protection of BBB integrity after SAH. By using the rat endovascular perforation model of SAH, the present study sought to investigate the potential protective effects and underlying mechanisms of artesunate on the BBB after SAH.Material and MethodsThis research was conducted in vivo and in vitro experiments. The rat model of SAH was induced by endovascular perforation.1. The 18-point SAH grading scale was employed to evaluate whether the model was successful at 24 and 72 hours after SAH in rats.2. Modified Garcia neurological scores was applied to test the nervous system functions of rats at 24 and 72 hours after SAH in rats.3. The level of cerebral edema was evaluated by brain water content at 24 and 72 hours after SAH in rats.4. The integrity of blood-brain barrier was appraised by Evans blue extravasation, auto-fluorescence of Evans blue, and transmission electron microscope at 24 hours after SAH in rats.5. The expression of S1P1 was detected by immunohistochemistry staining at 24 hours after SAH in rats.6. The expression of S1P1, Sph K1, Sph K2, p-Akt/Akt, p-GSK3β/ GSK3β, p-β-Catenin /β-Catenin, Claudin3 and Claudin5 were assessed by Western blot at 24 or 72 hours after SAH in rats. What’s more, the expression of S1P1 was also detected by Western blot in vitro.7. Cell viability was tested by CCK-8 at 24 hours after administration of artesunate in vitro.8. The inhibitory effect of si RNA was evaluated by RT-PCR in vivo.9. The blood pressure of spontaneously hypertensive rats(SHR) was measured by MRBP system every week after administration of 2% salt solution.10. The therapeutic effect of artesunate was evaluated by neurological score, brain water content, Evans blue extravasation, auto-fluorescence of Evans blue, and transmission electron microscope at 24 or 72 hours after SAH.11. The molecular mechanism of artesunate in protected BBB after SAH was investigated by administration of antagonist(DMS, VPC23019, Wortmannin) and si RNA(S1P1 siRNA and Scr si RNA), respectively.12. The preventive effect of artesunate was evaluated by neurological score, brain water content, the extravasation of Evans blue at 24 hours after SAH in rats.Results1. We found that the relatively high concentration of artesunate(100mg/kg, 200mg/kg) could effectively alleviate neurologic impairment, depress brain edema, and reduce BBB Damage after SAH in Rats.2. Artesunate significantly increased the expression of S1P1 in vivo and in vitro, but not the levels of sphingosine kinase 1(SphK1) and sphingosine kinase 2(Sph K2). And the effect of artesunate could not be reversed by the antagonist of Sph K(N, N-dimethylsphingosine).3. Artesunate(200mg/kg) significantly increased the expression of p-Akt, Claudin-3 and Claudin-5, reduced the ratio of p-GSK-3/GSK-3β and p-β-catenin/β-catenin at 24 h after SAH, and this effect could be reversed by VPC23019, wortmannin and S1P1 siRNA.4. Pretreatment with 200mg/kg artesunate could not improve neurological score, depress brain edema, and reduce the extravasation of Evans blue at 24 h after SAH in SHR. By contrast, pretreatment with artesunate could remarkably alleviate BBB injury by hypertension in SHR.Conclusion1. Artesunate could alleviate SAH-induced brain injury through preserving BBB integrity and thus reduced brain edema. These effects were possible to associate with the improved expression of S1P1 but not with sphingosine kinase by which the sphingosine 1 phosphate(S1P receptor ligand) was produced.2. S1P1 is closely related to the PI3 K signaling pathway. S1P1 stimulation could increase PI3K/Akt activation, then reduce GSK-3β activation and stabilize β-catenin, finally protected BBB by raising the expression of Claudin-3 and Claudin-5 after SAH in rats.3. Hypertension could enhance the permeability of BBB in SHR. Pretreatment with artesunate could remarkably alleviate BBB injury by hypertension in SHR. |
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