Neuroprotective Effects Of Shikonin On Focal Cerebral Ischemia/Reperfusion Injury In Mice And Its Mechanism Of Signal Transduction Pathway

Ischemic cerebrovascular disease is the most common type of disease,which is the first leading cause of death and the most frequent cause ofpermanent disability in adults. Ischemic stroke is one of the main diseasesendangering people’s health in our state, have a high incidence, mortality,morbidity, high recurrence rate, and the characteristics of the slow recovery.The worldwide average of one in every400people suffer a stroke in anischemic cerebrovascular disease according to the World Health Organisation.There are250million patients with cerebrovascular disease each year in China,equating to more than75%with ischemic brain disease. Stroke is aslo aleading cause of mortality after heart disease, equating to9%of total deathseach year. Ischaemic stroke accounts for approximately80–85%of all cases,with approximately30%of the former undergoing haemorrhagictransformation.Brain tissue secondary injuries to ischemia often leads to an aggravatedillness after cerebral ischemia/reperfusion. Cerebral ischemia/reperfusioninjury is aslo a complex pathophysiologic process which is not been clarifiednow. With the rapid development of modern immunology and molecularbiology, recent studies found that the injuries caused by cerebral blood flowcessation and reperfusion are a rapid cascade reaction which includesinflammatory damage, oxidative stress, excitatory amino acid toxicity,apoptosis, intracellular calcium overload, free radical damage and so on.These pathophysiologic processes overlap and intercommunicate then form avicious cycle which direct/indirect resulting in blood-brain barrier disruptionand brain edema formation, neurological deficit, as well as cell apoptosis ornecrosis.Nowadays, beyond only a restricted number of hospitalized patients profiting from thrombolytic therapy, the vast majority of patients sufferingfrom stroke resulted in disability even death owing to the limited therapies.Delaying the development of brain damage and protecting neuron areremained. There a large number of agents have been proved to displayanti-inflammation, anti-oxidation properties but never employed in treatingcerebral ischemia. It is a focus in neuroscience that looking for the idealneuroprotective agents that can block ischemic cascade reaction.Shikonin is one of the major naphthoquinone pigment extracted from atraditional herbal medicine Lithospermum erythrorhizon. Evidence has shownthat shikonin possesses a wide range of biological effects, such asantimicrobial, anticancer, antithrombotic, and anti-inflammatory activities totreat thermal edema, arthritis, atherosclerosis. However, the molecular targetsand mechanisms underlying shikonin are not completely characterized, andthe effect of the shikonin in acute stroke is still unknown. In the present study,we demonstrated the unexplored potential of shikonin for the treatment ofcerebral ischemic damage and its potential mechanism.ICR mice were induced into focal cerebral ischemiaby transient middlecerebral artery occlusion (MCAO), and received shikonin treatmentimmediately after MCAO. The present study examined the ability of shikonin,the major constituents of Chinese herb Lithospermum erythrorhizon, to induceexpression of HO-1, p85(PI3-kinase) and AKT phospholation, NF-E2-relatedfactor-2(Nrf2) and analyzed its signaling mechanism in mice brain. Theneurological deficits, brain water content, infarct volume and the expression ofTLR4, p-p38MAPK, NF-κB, TNF-α, MMP-9and claudin-5were measured at24h and72h after cerebral ischemia/reperfusion. The activities of superoxidedismutase (SOD) and malondialdehyde (MDA) content in ischemic corticaltissue were aslo detected to examine the oxidative response at24h and72hafter ischemia.The study was divided into three part list as below.PartⅠAnti-oxidative effect of shikonin on focal cerebralischemia/reperfusion injury in mice and its mechanism of signal transduction pathwayObjective: This study is to evaluate the time course expression regularityof HO-1, Nrf2, p85(PI3-kinase) and p-AKT, and estimate the anti-oxidativeeffects of shikonin on focal cerebral ischemia/reperfusion injury in mice andexplore its possible mechanisms and related signal transduction pathways.Methods: CD-1mice were subjected to transient focal cerebralischemia/reperfusion model was established by middle cerebral arteryocclusion using modified suture occlusion technique. Experiment1: The micewere randomly divided into Sham group(n=30), MCAO group(n=90), Thetime course expression of HO-1in the brain tissue after MCAO. Two groupswere studied, including Sham group, MCAO group. The last two groupincluded3h,6h,24h,48h,72h sub-groups. Experiment2: Shikonin’sneuroprotection against damage from cerebral ischemia/reperfusion. Shikoninwas injected intraperitoneally after MCAO. Mice were reanesthetized andkilled at24h and72h after MCAO. In this part, mice were individed into4groups randomly. Shikonin was injected intraperitoneally immediately afterMCAO. Group1: Sham-operated group (Sham): animals received shamoperation; group2: Vehicle controls (Vehicle): animals received transientMCAO and equal volume PBS including1%DMSO respectively once a dayfor three times before surgery; group3: Shikonin low dose group (L-Shi):animals received transient MCAO and10mg/kg of Shikonin respectively oncea day for three times before surgery; group4: MCAO-Shikonin high dosegroup (H-Shi): animals received transient MCAO and25mg/kg of Shikoninrespectively once a day for three times before surgery. Drug or solvent wasadministered before MCAO, then once daily thereafter. Experiment3:Shikonin was administered before MCAO. Sham-operated group (Sham):animals received sham operation; vehicle controls (Vehicle): animals receivedtransient MCAO and equal volume PBS including1%DMSO; Shikonin lowdose group (L-Shi): animals received transient MCAO and10mg/kg ofShikonin respectively once a day for three times before surgery; MCAO-Shikonin high dose group (H-Shi): animals received transient MCAO and25 mg/kg of Shikonin respectively once a day for three times before surgery;Shikonin+LY-294002group: animals received transient MCAO and25mg/kg of Shikonin respectively once a day for three times before surgery andLY-294002(10μL10mM dissolved in3%DMSO). Infarct volume wasmeasured by TTC staining and morphologic changes were observed by H.E.we measured superoxide dismutase (SOD) and malondialdehyde (MDA) ofcerebral tissue to evaluate antioxidation activitis of shikonin.Immunohistochemistry, RT-qPCR and Western blot were used to analyse theexpression of HO-1, p85(PI3-kinase) and p-AKT and Nrf2.Results: Compared with Sham group, HO-1were upregulated at geneand protein level in ischemia/reperfusion brain, beginning at6h and peakingat24h after MCAO (P＜0.05). Treatment of shikonin for indicated timeperiods increased up-regulation of HO-1in ischemic brain after cerebralischemia. In addition, treatment of various concentrations of shikonin for24halso increased HO-1expression in a concentration-dependent manner.Treatment with shikonin for indicated time periods, or with variousconcentrations of shikonin also increased HO-1mRNA expression in atime-dependent or a concentration dependent manners. Taken together, thesefindings demonstrate that shikonin increases HO-1protein and mRNAexpression. Shikonin high dose (25mg/kg) upregulated Nrf2and HO-1inMCAO-affected brain tissue, The Nrf2CT of Vehicle group, MCAO group,L-Shi group, H-Shi group were (0.22±0.27,0.85±0.10,0.70±0.05,0.53±0.06). Shikonin reduced infarct volume (P＜0.05), and behavioral deficitscaused by MCAO. And also, shikonin significantly increased the activities ofSOD and decreased the production of MDA at24h and72h after ischemia inH-Shi group. Phospho-PI3K and phospho-Akt were examined withimmunohistochemistry, Western blot and RT-qPCR. Few cells were stainedwith phospho-PI3K and phospho-Akt in the cortex in sham group byimmunohistochemistry. In vehicle group, the number of positive cells ofphospho-PI3K and phospho-Akt significantly decreased in the ischemic cortex.In H-Shi group, the number of positive cells of phospho-PI3K and phospho-Akt was significantly increased compared with vehicle group (P <0.05). However, there were no significant differences about the positive cellsbetween vehicle group and L-Shi group. In agreement with the results ofwestern blotting, the mRNA and protein expression of phospho-PI3K andphospho-Akt were down-regulated in Vehicle group compared with Shamgroup (P <0.05). The expression of those factors was significantly increasedin H-Shi group (P<0.05). Whereas L-Shi group did not display changes ofphospho-PI3K and phospho-Akt expression compared with Vehicle group. Tofurther investigate whether the PI3K/Akt pathway mediates theneuroprotection of shikonin in cerebral I/R injury, we treated the mice with aPI3K inhibitor LY-294002(i.c.v.,10μL10mM LY-294002dissolved in3%DMSO) at15min before ischemia. Western blot analysis showed that thePI3K inhibitor LY-294002inhibited the increase of p-Akt level induced byshikonin treatment and almost restored p-Akt level to basal level. In addition,shikonin-induced HO-1expression was antagonized by treatment with PI-3kinase inhibitors LY294002.Conclusions: Nrf2, HO-1were induced at the early stage after MCAO.Shikonin reduce cerebral infarct volume and improve neurologic impairmentand protected the brain from damage caused by MCAO, we found thatshikonin increased HO-1mRNA and protein expression time-dependently. Inaddition, shikonin-induced HO-1expression was attenuated by PI3-kinase(phosphatidylinositol3-kinase) inhibitors LY294002. Treatment of mice withshikonin also induced p85(PI3-kinase) and AKT phospholation. Shikoninalso increased NF-E2-related factor-2(Nrf2) accumulation in the nucleus.Moreover, shikonin-induced increase of Nrf2was reduced by PI3-kinaseinhibitors. Thus, shikonin may be useful as a therapeutic agent for thetreatment of cerebral ischemic-associated disorders. The neuroprotection ofshikonin was accomplished by antioxidation. These findings suggest thatshikonin-increased HO-1expression is mediated by Nrf2activation throughthe PI3-kinase/AKT pathway.PartⅡ Anti-inflammatory effect of shikonin on focal cerebral ischemia/reperfusion injury in mice and its mechanism of signaltransduction pathwayObjective: This study is to evaluate the expression regularity of TLR4,MAPKs, NF-κB, TNF-α and estimate the anti-inflammatory effects ofshikonin on focal cerebral ischemia/reperfusion injury in mice and explore itspossible mechanisms and related signal transduction pathways.Methods: CD-1mice were subjected to transient focal cerebralischemia/reperfusion model in rats was established by middle cerebral arteryocclusion using modified suture occlusion technique. Experiment1: The micewere randomly divided into Sham group(n=30), MCAO group(n=90), Thetime course expression of TLR4, MAPKs, NF-κB, TNF-α in the brain tissueafter MCAO. Two groups were studied, including Sham group, MCAO group.The last two group included3h,6h,24h,48h and72h sub-groups.Experiment2: Shikonin’s neuroprotection against damage from cerebralischemia/reperfusion. In this part, mice were individed into4groups randomly.Shikonin (98%) was purchased from Santa Cruz Biotechnology; it wasdissolved in dimethyl sulfoxide (DMSO) as a10mmol/L stock solution andstored at–20℃. For all experiments the final concentration of the testedcompound was prepared by diluting the stock with PBS including1%DMSO.Shikonin was administered before MCAO. Mice were reanesthetized andkilled at24h and72h after MCAO. In this part, mice were individed into4groups randomly. Group1: Sham-operated group (Sham): animals receivedsham operation; group2: Vehicle controls (Vehicle): animals receivedtransient MCAO and equal volume PBS including1%DMSO respectivelyonce a day for three times before surgery; group3: Shikonin low dose group(L-Shi): animals received transient MCAO and10mg/kg of Shikoninrespectively once a day for three times before surgery; group4: MCAO-Shikonin high dose group (H-Shi): animals received transient MCAO and25mg/kg of Shikonin respectively once a day for three times before surgery.Drug or solvent was administered before MCAO, then once daily thereafter.Immunohistochemistry, RT-qPCR and Western blot were used to analyse the expression of TLR4, MAPKs, NF-κB, TNF-α.Results: TLR4, p-p38MAPK, NF-κB and TNF-α were examined withimmunohistochemistry, Western blot and RT-qPCR. Few cells were stainedwith TLR4, p-p38MAPK, NF-κB and TNF-α in the cortex in sham group byimmunohistochemistry. In vehicle group, the number of positive cells of TLR4,p-p38MAPK, NF-κB and TNF-α significantly increased in the ischemic cortex,among which the location of NF-κB is in nucleus. In H-Shi group, the numberof positive cells of TLR4, p-p38MAPK, NF-κB and TNF-α was significantlydecreased compared with vehicle group (P <0.05). Moreover, positive nucleiof NF-κB were also reduced and lots of cells labeled by NF-κB were stainedonly in cytoplasm (P <0.05). However, there were no significant differencesabout the positive cells between vehicle group and L-Shi group.We found the protein and mRNA levels of TLR4, p-p38MAPK, NF-κB andTNF-α in ischemic tissue were upregulated at24h and72h after MCAO. Wefirst analyzed the protein levels of nuclear NF-κB p65and total TLR4,p-p38MAPK, TNF-α and MMP-9by western blot. The NF-κB p65was rich incytosolic fractions but poor in nuclear extracts in brain tissue of Sham group.In contrast, the protein level of NF-κB in Vehicle group was significantlyenhanced in nuclear fraction and concurrently decreased in cytosol at24h and72h after ischemia, indicating the translocation of these NF-κB subunits fromthe cytosol to the nucleus. High dose of shikonin significantly decreased theexpressions TLR4, p-p38MAPK, NF-κB and TNF-α at24h and72h afterMCAO. However, there were no significant differences in the protein levels ofTLR4, p-p38MAPK, NF-κB and TNF-α between Vehicle group and L-Shigroup. Another parallel set of samples treated with L-Shi and H-Shi werealso evaluated for mRNA expression of TLR4, p-p38MAPK, NF-κB andTNF-α by RT-qPCR. In agreement with the results of western blotting, themRNA expression of TLR4, p-p38MAPK, NF-κB and TNF-α wereup-regulated in Vehicle group compared with Sham group (P <0.05). Theover-expression of those factors was significantly decreased in H-Shi group(P<0.05). Whereas L-Shi group did not display changes of TLR4, p-p38MAPK, NF-κB and TNF-α expression compared with Vehicle group.Conclusions: The expression of TLR4, p-p38MAPK, NF-κB andTNF-α were up-regulated after ischemia. Systemic administration of shikoninis effective which can decrease the expression of TLR4, p-p38MAPK, NF-κBand TNF-α. Therefore, the excessive inflammation of the brain ischemia wasalleviated.Part Ⅲ The permeability of blood-brain barrier after focal cerebralischemia in rats and the protection of shikoninObjective: This study is to evaluate the time course expression ofClaudin-5and MMP-9and explore the underling regulation mechanisms ofshikonin.Methods: CD-1mice were subjected to transient focal cerebralischemia/reperfusion model in rats was established by middle cerebral arteryocclusion using modified suture occlusion technique. Experiment1: The micewere randomly divided into Sham group(n=30), MCAO group(n=90), Thetime course expression of Claudin-5and MMP-9in the brain tissue afterMCAO. Two groups were studied, including Sham group, MCAO group. Thelast two group included3h,6h,24h,48h and72h sub-groups. Experiment2:shikonin’s neuroprotection against damage from cerebral ischemia/reperfusion.shikonin was administered before MCAO. Mice were reanesthetized andkilled at24h and72h after MCAO. In this part, mice were individed into4groups randomly. Group1: Sham-operated group (Sham): animals receivedsham operation; group2: Vehicle controls (Vehicle): animals receivedtransient MCAO and equal volume PBS including1%DMSO respectivelyonce a day for three times before surgery; group3: Shikonin low dose group(L-Shi): animals received transient MCAO and10mg/kg of Shikoninrespectively once a day for three times before surgery; group4: MCAO-Shikonin high dose group (H-Shi): animals received transient MCAO and25mg/kg of Shikonin respectively once a day for three times before surgery.Drug or solvent was administered before MCAO, then once daily thereafter.RT-qPCR and Western blot were used to analyse the expression of Claudin-5 and MMP-9. The loss of BBB integrity was assessed by leakage of Evans bluefrom microvessels after intravenous injection.Results: There was considerable leak of Evans blue into the ischemicbrain after MCAO, showing that the BBB had been disrupted in the ischemichemisphere. Compared with Vehicle group, Evans blue evasion of braintissues was significantly decreased in H-Shi group (P<0.05). Whileclaudin-5’s expression was increased in both L-Shi and H-Shi groups (P<0.01) at gene and protein levels by western blotting and RT-qPCR at24h and72h, but low dose of shikonin did not display significant level. At the sametime, the MMP-9expression was obviously decreased in H-Shi group (P<0.05), relative to that in normal group.Conclusions: The blood-brain barrier was dramatically disrupted at theearly stage of focal cerebral ischemia, which was responsible for the brainedema. Shikonin normalizing Claudin-5by inhibiting MMP-9couldcontribute to the alleviated infarct volume, neurological deficits and brainedema in the study.