Protective Effect Of Natural Antioxidants Against Cerebral Ischemia Injury Via The Inhibitory Effect On Nitrosative Stress

Stroke is a major public health problem all over the globe and incident has increased rapidly in developing countries with high rates of morbidity and mortality.The World Health Organization(WHO)has estimated that every year 15 million people experience a stroke episode with 70%individuals suffer from death and disability.Among the aged population,it is considered second deadliest disease with two-thirds deaths worldwide.The ratio of suffering from stroke increased recently due to lifestyle modifications in China and other developing countries.There is an urgent need to understand the underlying mechanisms and development of therapies for cerebral stroke.Nitrosative stress plays an important role during the pathological process of brain ischemia.Considerable evidence suggests that overproduction of highly reactive nitrogen-containing molecules mediates cellular damage.In stroke attack,there is a decreased blood supply to the brain results in oxygen and glucose deprivation and thus,neurons are unable to maintain the ionic gradients necessary for cellular function and homeostasis.The neuronal injury caused by occlusion of cerebral arteries is believed to be mediated by excessive activation of glutamate N-methyl.-D-aspartate(NMDA)receptors and calcium(Ca2+)release.This Ca2+induces the calpain activation and binding of calmodulin(CaM)to the enzyme nitric oxide synthases(NOS)and alters the function of endothelial nitric oxide synthases(eNOS).Uncoupling of eNOS caused increased superoxide(O2-·)production due to activation of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase.Increased generation of nitric oxide(NO)and O2-· results in the formation of peroxynitrite(ONOO-).Nitrosative stress is implicated in a number of disease processes,whereby they immediately damage to cell structures,including lipids,membranes,proteins,and DNA.The cerebral vasculature is a major target of nitrosative stress following a cerebrovascular attack and damaged the blood-brain barrier(BBB).Neuroprotective therapies targeting the nitrosative stress will be beneficial for cerebral stroke treatment.In the present study,we used two natural potent antioxidants,Salvianolic acid A(SAA)and ?-carotene(BC)in two separate experimental designs.We aimed to evaluate their ability to protect the brain from damage during cerebral ischemia.The transient brain ischemia mouse model of middle artery occlusion(tMCAO)used to induce nitrosative stress.SAA is also known as Danshen in Traditional Chinese medicine literature.SAA is a multi-target agent that inhibits distinct ischemic factors triggers multiple intracellular signaling.SAA is obtained from Chinese herb Salviae Miltiorrhizae Bunge(Labiatae),has been reported to have the protective effects against cardiovascular and neurovascular diseases.Slices of roots and rhizomes of Salvia miltiorrhiza analyzed by thin layer liquid(TLC)and purified by column chromatography,high-performance liquid chromatography(HPLC)to obtain the crude product.We tested SAA to investigate the relationship between the effectiveness of SAA against neurovascular injury and its effects on calpain activation and endothelial nitric oxide synthase(eNOS)uncoupling.SAA(1 mg,5 mg per kg)or vehicle(saline)was given by i.p.to C57BL/6 male mice for seven days before the occlusion of the middle cerebral artery for 60 min.High-resolution positron emission tomography scanner(micro-PET)was used for small animal imaging to examine 18F-FDG glucose metabolism.To observe the functional changes in different group rota-rod time and neurological deficit scores were calculated after 23 h of reperfusion.The volume of infarction was determined by Nissl-staining and brain infarct area was evaluated from digital images of Nissl-stained brain sections using Image J software(NIH).The calpain proteolytic activity and eNOS uncoupling and other target proteins were determined using western blot analysis.Our data demonstrated that pretreatment with SAA significantly decreased neurological deficit score compared with that of the vehicle group.The latency to fall in rotarod test was increased in vehicle-treated animals which were significantly reduced by SAA pretreatment.Consistently,infarct areas were significantly reduced in SAA pretreatment groups compared with vehicle treatment.SAA administration increased glucose metabolism,there was no metabolic asymmetry between the hemispheres in sham mice but MCAO decreased the metabolic activity in the affected hemisphere which was protected with SAA pretreatment.The standardized uptake value(SUVs)ratio of contralateral to the ipsilateral region was significantly decreased in vehicle treated group compared to sham.SAA pretreatment preserves significantly contralateral to ipsilateral part SUVs ratio and showed the increased glucose metabolism.SAA pretreatment inhibited calpain proteolytic activity,since western blot analysis shown that the breakdown products of spectrin were significantly preserved by the pretreatment with SAA.In addition,the inhibitory effect of SAA on calpain activation was confirmed by the decreased breakdown products of spectrin and calcineurin(CaN)following brain ischemia.Furthermore,a biochemically active dimeric form of eNOS appeared to be able to generate O2-·,whereas the monomeric form served as a marker for eNOS uncoupling.The SAA pretreatment significantly restored decreased dimer-to-monomer ratio after tMCAO as the dose increased and enhanced the eNOS coupling.Peroxynitrite is a powerful oxidant exhibiting a wide array of tissue-damaging effects.In our western blot analysis,nitrotyrosine levels were significantly increased in penumbra brain region in vehicle treated group compared to sham.The SAA pretreatment significantly reduced nitrotyrosine in ischemic ipsilateral.We observed dephosphorylating of AKT,ERK,and FKHR in the vehicle-treated group but no change in total protein levels.The decreased phosphorylation may be attribution of increased O2-·production as a result of eNOS uncoupling.The SAA pretreatment significantly enhanced phosphorylation of AKT,ERK and FKHR compared to vehicle treated group.Our results suggested that SAA inhibits nitrotyrosine levels which might be associated with neurovascular protective effects.This suggestion is consistent with the inhibitory effect on calpain activation and eNOS uncoupling.Moreover,SAA attenuates glucose metabolism and therefore protects the brain against ischemic/reperfusion injury.?-carotene(BC)is one of the major carotenoids exhibit antioxidative properties.Carotenoids are synthesized by plants and present in various fruits and vegetables.BC has been reported to reduce the risk of myocardial ischemic injury at low doses.BC and other carotenoids have the ability to alter processes result in reduced tissue oxygenation to heart and brain.Nanoparticles have been shown to represent promising drug delivery systems for brain diseases treatment.There are certain brain damage-specific molecules which can be used to target the ischemic region.ONOO-caused severe consequences after ischemic brain damage.Our current drug design strategies predicted that nitrotyrosine antibody might be an appropriate candidate for targeting ONOO-.In this study we develop three different type of BC formulation,?-carotene emulsion(BC-EM)to increase its solubility,BC incorporated in PEGylated lipid nanoparticles(PLNs)and BC incorporated in PEGylated lipid nanoparticles(PLNs)attached with the nitrotyrosine antibody(NT).Mice were divided into seven experimental groups5 sham-operated group,vehicle group,BC-EM-treated group(10 mg/kg),BC-loaded PEGlated nanoparticles-treated group(BC-PLNs,2.5 mg/kg),and BC-loaded-nitrotyrosine-PEGylated nanoparticles-treated groups(BC-NT/PLNs,0.625 mg/kg,1.25mg/kg or 2.5 mg/kg).After the onset of MCAO for 60 mint,drugs or vehicle(saline)were administered intravenously according to the body weight of mice.At the end of 23 h of reperfusion assess the neurodeficit function and take out brain sample for infarct area lesions by Nissl staining.NP3 a peroxynitrite sensitive fluorescent probe used to assess ONOO-.Western blot analysis was used to see the calpain activation,tight junction proteins and phosphorylation of AKT(Ser 473),FKHR(Ser256)and ERK(Thr202/Tyr204).BBB integrity was evaluated by Evans blue extravasations from tissues.We demonstrated that BC-EM has significant effects on the recovery of neurological score compared to the vehicle group and similarly reduced the infarct size.BC-PLNs nano-drug form could enhance neuroprotection at lower dosages versus BC-EM.Functional deficits were significantly reduced by a lower dose of 0.625;1.25 mg/kg BC encapsulated NT/PLNs versus vehicles.Consistently,0.625,1.25 mg/kg BC encapsulated NT/PLNs intravenous treatment effectively reduced infarct area in MCAO mice.NT/BC/PLNs are neuroprotective and beneficial against deficits after brain ischemia,with significantly reduced BC dosages.Our finding demonstrated that BC-EM(10 mg/kg),2.5 mg/kg BC encapsulated in PLNs and 0.625mg/kg NT/PLNs were effective on calpain activation and inhibit the spectrin and CaN breakdown.We found that BC-EM(10 mg/kg)treatment effectively inhibited the upregulation of phospho-P38(Thr180/Tyrl 82)and phospho-JNK(Thr183/Tyr185).The ischemia-induced decrease in phosphorylation of survival protein AKT(Ser 473),FKHR(Ser256)and ERK(Thr202/Tyr204).NT/BC/PLNs(0.625mg/kg)treatment effectively inhibited the downregulation of phospho-AKT(Ser473),phospho-FKHR(Ser256)and phospho-ERK(Thr202/Tyr204).Nitrosative stress causes the breakdown of blood microvessels and damages the neurovascular unit during ischemia.NP3 a peroxynitrite sensitive fluorescent probe showed the great intensity of NP3 emission localized to micro-vessels in the vehicle-treated group as compared to a sham group having no emission of NP3.NT/PEG-nanoconjugate(0.625mg/kg)significantly inhibited this peroxynitrite generation and protects the microvessels from damage.The Evans blue dye easily leaked from the damaged microvessels and reflects the extent of the injury.The amount of Evans blue solution that extravasated from blood vessels into the brain parenchyma was significantly increased at 24 h in the vehicle group.In comparison with vehicle treatment,NT/PEG-nanoconjugate(0.625mg/kg)treatment significantly reduced the Evans blue leakage in the ipsilateral hemisphere.Similarly,NT/PEG-nanoconjugate(0.625mg/kg)treatment significantly blocked the degradation of tight junction proteins zonula occludens-1(ZO-1)and occludin.?-carotene encapsulated PLNs conjugated with nitrotyrosine antibody significant improve the brain injury with the reduced dosages compare to beta carotene emulsion.We reported that NT/BC/PLNs system efficiently delivers beta-carotene at the site of peroxynitrite generation represent suitable for ischemic region-targeted therapy.Moreover,NT/BC/PLNs proved to decrease the generation of ONOO-and inhibition of Evans blue extravasation from microvessels.Furthermore,Calpain activation is inhibited by the NT/BC/PLNs and decreased the breakdown of spectrin or CaN during the ischemia.In summary,nitrosative stress is involved in the pathogenesis of brain injury after ischemia.Salvianolic acid A and ?-carotene have anti-ischemic effects during brain damage,and its molecular mechanism is associated with alleviating eNOS uncoupling and nitrosative stress injury.In addition,we found that the brain-targeted nano-drug delivery system of the coupled nitrotyrosine antibody not only directed the drug to the ischemic brain region but also reduced the dose of the loaded drug to achieve the same therapeutic effect.