The Neuroprotective Effect Of Chrysin And The Underlying Mechanism In Experimental Ischemic Stroke

Objectives: Cerebrovascular disease, cancer, heart disease is the three diseasesin developing countries in the cause of human disability and death. schemicstroke is the most common, accounting for about80%to85%of allcerebrovascular disease. Secondary brain damage following cerebral ischemiacan lead to exacerbations, and affect the prognosis. The mechanism ofSecondary brain damage is very complex, including energy depletion andacidosis, oxygen free radicals, intracellular Ca2+overload, damage ofexcitatory amino acid toxicity, inflammatory cytokines,apoptosis and so on.It’s well known that the inflammatory response and apoptosis plays a key rolein cerebral ischemic pathogenesis.Nuclear factor-kappa B （NF-κB） is a ubiquitously expressedtranscription factor, which regulates hundreds of genes involved in cellsurvival and inflammation. There is ample evidence that NF-κB is activated incerebral ischemia, mainly in neurons. It is known that severalproinflammatory NF-κB target genes （TNF, IL-1α and β, IL-6, iNOS,ICAM-1, COX-2, PLA2and MMP9） are induced in cerebral ischemia,possibly contributing to ischemic damage. Neurons respond to hypoxia causedby ischemia by triggering a dynamic network of signaling pathways includingthe mitogen-activated protein kinases （MAPKs）. The MAPKs which are afamily of related serine/threonine kinases and signal transduction mediatorsincludes the extracellular signal-regulated kinases （ERK） and thestress-activated protein kinases （such as JNK/SAPK1and p38/SAPK2）. ERKsregulate multiple functions that contain cell growth, proliferation,differentiation and apoptosis. The ERK1/2module is primarily activated inresponse to growth factors, oxidative stress, increases of intracellular Ca²⁺levels and also to glutamate receptor stimulation, factors which are activatedby stroke. JNK and p38are also activated by environmental stress andinflammatory cytokines. Activation of p38and JNK plays important roles in transducing stress-related signals involved in cell survival, apoptosis andinflammatory cytokine production. The MAPKs control multitudinousintracellular events in response to a variety of stressors including oxidativestress and anoxia, which finally alter cell function through changed geneexpression or the posttranslational modification of proteins.Recent research found that chrysin observably inhibits expression of thekey pro-inflammatory enzymes （such as inducible nitric oxide synthase,prostaglandin E and cyclooxygenase-2）. It also plays a protective effect onchronic hypoperfusion damage in cerebral by exerting antioxidant andantiapoptosis effects. However, there is still a paucity of data about the exactrole of chrysin on the brain tissue in the acute phase of cerebral ischemia.Thus, we investigated the protective effect of chrysin on cerebral ischemia inthe rat permanent middle cerebral artery occlusion （pMCAO） for the first timeand the potential mechanisms.The purpose of this study is to investigate theneuroprotetion of chrysin in experimental ischemic stroke induced bypermanent middle cerebral artery occlusion （pMCAO） and the effect on theexpression of NF-κB, p-ERK, p-JNK and p-P38MAPK.Methods: Healthy, adult, male Sprague-Dawley rats were subjected tomodified right permanent middle cerebral artery occlusion（pMCAO）, asdescribed by Longa previously. All the animals were randomly divided into5groups: Sham-vehicle group （Sham）: animals received sham operation andequal volume of DMSO; Saline group （Saline）: animals received MCAO andequal volume of NaCl0.9%（saline）; MCAO-vehicle group （DMSO）: animalsreceived MCAO and equal volume of DMSO; and chrysin groups: animalsreceived MCAO and treated with low dose of chrysin10mg/kg （chrysin-L）,and high dose of chrysin40mg/kg （chrysin-H）. The drugs were administratedorally once daily before MCAO surgery. After3days of presurgery treatment,stroke was induced in the rats by MCAO ischemia/reperfusion as previouslydescribed. Rats were treated with chrysin by oral at different doses of10and40mg/kg once daily before MCAO surgery. After3days of presurgery treatment, stroke was induced in the rats by MCAO ischemia/reperfusion aspreviously In the cases of the MCAO and Sham group, equal solvent wasadministered in the same manner. Neurological deficit, infarct volume, andbrain water content were measured at24h after stroke. The expression ofNF-κB were measured by real-time PCR, and Western blot. P38MAPK,extracellular signal-regulated kinase （ERK） and c-jun N-terminal kinase （JNK）were detected by Western blot.Results:1Significant neurological defect of rats in Sham group performed well.Neurological deficit score in Chr-H group was decreased compared withMCAO group（P <0.05）. neurological score in Chr-L group was slightlylower than the MCAO group, but the difference was not statisticallysignificant （P>0.05）. There was no significant difference in the neurologicaldeficit score between MCAO+OIL group and MCAO group（P>0.05）.2ipsilateral hemispheres in MCAO group was significantly higher thansham group （MCAO vs. the sham:85.28%±0.31%vs78.17%±0.42%, P<0.05）. In Chr-H group the brain water content dropped to81.09%±0.52%.compared with the MCAO group difference was statistically significant （P<0.05）.High dose of octreotide reduced the brain water content significantly（Oct-H vs. Vehicle:82.93%±0.51%vs.85.41%±0.27%，P <0.05）. Thebrain water content of Chr-L group was reduced, but there is no significantdifference between Chr-L group and MCAO group （Chr-L vs. of MCAO:84.87%±0.29%vs.85.28%±0.31%, P>0.05）. In MCAO+OIL groups andthe MCAO group infarcted brain tissue water content were no significantchanges （MCAO+OIL vs. MCAO:85.56%±0.35%vs.85.28%±0.31%, P>0.05）.3No lesion was observed in Sham group. High dose of chrysinsignificantly reduced the infarct volume after MCAO（Chr-H vs. MCAO:35.59%±1.02%vs.48.29%±0.94%，P <0.05）. However, the infarctvolume was not significantly decreased in MCAP+OIL group and Chr-Lgroup. 4In Chr-H group the expression of NF-κB and p-P38in protein level wasdecreased significantly （P <0.05）. The protein level of p-ERK in Chr-Hgroup was significantly increased （P <0.05）.5High dose of chrysin decreased the activities of NF-κB in mRNA levelcompared with MCAO group （P <0.05）.Conclusions： This work shows that chrysin could protect the brain againstfocal ischemic injury, improve the neurological deficit, relieve the brainedema and decrease the infarct size, this may be through up-regulationexpression of p-ERK, down-regulation NF-κB and p-P38activities.