1.Protective Effects Of Guattegaumerine Against Hydrogen Peroxide Concomitant With Serum Deprivation Incuced Injury In Primary Cultured Cortical Neurons 2.Betulinic Acid Protects Against Cerebral Ischemia/Reperfusion Injury In ApoE Knock Out Mice

First Part Protective Effects of Guattegaumerine Against HydrogenPeroxide Concomitant with Serum Deprivation Incuced Injury inPrimary Cultured Cortical NeuronsAims: To establish the convenient and effective model of ischemia induced injury inprimary cultured neurons, and evaluate the neuroprotective effects of guattegaumerine onrat primary cultured cortical neurons. Methods: Research on primary cultured corticalneurons of Wistar rats, different concentrations of H₂O₂ and Glutamate (Glu) expoured tocells in serum free and low glucose medium for 24h and cell viability determined by3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and lactatedehydrogenase (LDH) leakage were detected. 1.25μmol/L and 2.5μmol/L guattegaumerinewere added 30 min before cells exposure to 6.25μmol/L H₂O₂ in serum free and lowglucose medium for 24h, then cell viability,LDH leakage,malondialhehyde (MDA) andtotal antioxidative capacity (TAC) were observred. Cells apoptosis and necrosis weremeasured by Annexin V/Propidium iodide double staining assay using flow cytometry, andBax, Bcl-2 protein expression were detected by immunohistochemistry. The calciumfluorescent label fura-2 AM was used to measure the intracellular calcium concentrationinduced by H₂O₂ and KC1, and the effects of guattegaumerine on the increased intracellularcalcium stimulated by H₂O₂ and KC1. Results: 6.25μmol/L～200μmol/L H₂O₂ and12.5μmol/L～50μmol/L Glu could decreased cell viability and increased leakage of LDH.Preincubation of guattegaumerine dramatically improved the cell viability and inhibitedLDH release. Preincubation of guattegaumerine also dramatically inhibited MDAproduction and elevated TAC in cells. Results of flow cytometry andimmunohistochemistry showed that preaddition of guattegaumerine interrupted theapoptosis of the neurons, reversed the up regulation of the pro-apoptotic gene (Bax) and thedown regulation of the anti-apoptotic gene (Bcl-2). Furthermore, guattegaumerinesuppressed the increase of intracellular calcium concentration ([Ca²⁺]_i) stimulated by eitherH₂O₂ or KC1 in Ca²⁺-containing extracellular solution, and high concentration of guattegaumerine also suppressed the increase of [Ca²⁺]_i induced by H₂O₂ in Ca²⁺-freesolution. Conclusions: 6.25μmol/L～200μmol/L H₂O₂ and 12.5μmol/L～50μmol/L Glu aretwo convenient and effective models to induce ischemic injury in neurons.Guattegaumerine protects cultured cortical neurons against 6.25μmol/L H₂O₂ concomitantwith serum deprivation incuced injury, which may relate to its inhibition of lipidperoxidation, regulation of apoptotic related gene expression and prevention of intracellularcalcium elevation by blocking the selective and non-selective calcium channels on the cellmembrane, and the release of calcium from ER.Second Part Betulinic Acid Protects against CerebralIschemia/Reperfusion Injury in ApoE Knock Out icemAims: To establish focal cerebral ischemia-reperfusion injury by middle cerebral arteryocclusion in ApoE knock out (KO) mice, and investigate the neuroprotective potential ofbetulinic acid against brain ischemia-reperfusion. Methods: ApoE KO mice were treatedwith 50mg/kg betulinic acid via gavage per day for 7days, then subjected to 2h of the leftmiddle cerebral artery occlusion (MCAO) followed by 22h of reperfusion. Brain infarctionwas indicated by TTC staining. NADPH oxidase (NOX) subunits (NOX1,NOX2,NOX4and p22phox) and nitric oxide synthase (NOS) subunits (nNOS,iNOS and eNOS) mRNAexpression were measured by qRT-PCR, and NOS subunits (nNOS and eNOS) andnitrotyrosine protein expression were measured by western-blot. Results: Inatherosclerosis-prone apolipoprotein E knockout (ApoE-KO) mice, 2h of middle cerebralartery occlusion (MCAO) followed by 22h of reperfusion led to an enhanced expression ofseveral NADPH oxidase subunits (NOX2, NOX4 and p22phox) and an up-regulation ofNOS isoforms (nNOS and iNOS) in the ischemic hemisphere. This was associated withelevated levels of 3-nitrotyrosine, an indicator of peroxynitrite-mediated oxidative proteinmodification. Pretreatment of the mice with betulinic acid (50 mg/kg/day for 7 days via gavage) prior to MCAO reduced infarct volume. In addition, treatment of betulinic acidresulted in a reduction of the ischemia/reperfusion-induced up-regulation of NOX2, nNOSand iNOS. Moreover, treatment with betulinic acid enhanced the expression of endothelialNOS (eNOS), both in the ischemic and non-ischemic hemispheres. In parallel, betulinicacid reduced the levels of 3-nitrotyrosine. Conclusions: these results suggest theneuroprotective potential of BA in cerebral ischemia-reperfusion injury, the mechanismsmay involve inhibiting of both superoxide and NO·production via down-regulated NOX2,iNOS and nNOS gene expression, leading to decreased oxidative stress injury duringcerebral ischemia-reperfusion.