The Neuroprotective Effect Of Luteolin And The Underlying Mechanism In Rat Experimental Stroke

Cerebral ischemia is the most common type of cerebrovascular 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, which has a high incidence, mortality,morbidity, high recurrence rate, and the characteristics of the slow recovery.Brain tissue injuries secondary to ischemia aggravated the illness and inhibitedthe recovery of patients. Despite advances in the understanding of thepathophysiology of cerebral ischemia in recent years, therapeutic optionsremain limited.Brain tissue injuries secondary to ischemia often leads to an aggravatedillness. The pathological mechanisms that trigger ischemic brain damage couldbe related to oxidative stress, inflammation, apoptosis, overproduction ofreactive oxygen species, intracellular acidosis, increased release of excitatoryamion acids, destabilization of intracellular calcium and so on. Thesepathophysiologic processes overlap and intercommunicate then form a viciouscycle which results in a series of pathological cascade. Among them excessiveinflammation, oxidative stress and apoptosis are the main causes of thesecondary injury. Therefore, antioxidant stress, reduce inflammation damage,inhibiting apoptosis treatment become one of the main ways for cerebralinfarction.Luteolin (3′,4′,5,7-tetrahydroxyflavone), an important member of theflavonoid family, is present in various fruits and vegetables. Its molecularformula for: C15H10O6. It exhibits a wide spectrum of pharmacologicalproperties including anti-inflammatory, anti-oxidation, inhibiting apoptosis,anti-allergy, anti-tumor, anti-bacterial, anti-viral and enhancing immunefunction and so on. Besides, it has been proven to have little side effects, andwith good potential clinical application. Male, healthy Sprague-Dawley rats were used and subjected to modifiedpermanent middle cerebral artery occlusion (pMCAO), as described by Longapreviously. The expression of TLR4, TLR5, P38MAPK, ERK1/2, claudin-5,Bcl-2and Bax were detected in the pMCAO; activites of superoxidedismutase (SOD) and catalase (CAT), and malondiadehyde (MDA) contentwere measured in the ipsilateral cortex. Luteolin's neuroprotective effect wasanalyzed. Neurological deficits, brain water content and infarct volume wereevaluated. The expression of TLR4, TLR5, P38MAPK, ERK1/2, claudin-5,Bcl-2and Bax were detected; activites of superoxide dismutase (SOD) andcatalase (CAT), and malondiadehyde (MDA) content were measured.PartⅠAnti-oxidative effect of luteolin and the underlying mechanism inrat experimental strokeObjective: The aim of this study was to evaluate the neuroprotectiveeffects of luteolin and the underlying mechanisms in cerebral ischemia. Brainwater content, infarct volume, the expression of claudin-5, activites ofsuperoxide dismutase (SOD) and catalase (CAT) and malondiadehyde (MDA)content were measured.Methods: Male, healthy Sprague-Dawley rats were used and subjected tomodified permanent middle cerebral artery occlusion (pMCAO). Experiment1was used to analyze luteolin's neuroprotective effect. Neurological deficits,brain water content and infarct volume were evaluated at24h and72h afterpMCAO. Rats were randomly assigned to six groups: Sham-vehicle group(Sham): animals received sham operation and equal volume of DMSO;MCAO group (MCAO): animals received MCAO and equal volume of NaCl0.9%saline; MCAO-vehicle group (MC+DM): animals received MCAO andequal volume of DMSO; and luteolin groups: animals received MCAO andtreated with low dose of luteolin5mg/kg (Luteolin-L), middle dose of luteolin10mg/kg (Luteolin-M) and high dose of luteolin25mg/kg (Luteolin-H).Luteolin was administered immediately after MCAO and then continued with daily injections for2days. Experiment2was used to analyze luteolin'santi-oxidative effect. SOD, CAT activities, and MDA content were measuredat24h and72h after pMCAO. Rats were randomly assigned to five groups:Sham-vehicle group (Sham): animals received sham opera-tion and equalvolume of DMSO; MCAO group (MCAO): animals received MCAO andequal volume of NaCl0.9%; MCAO-vehicle group (MC+DM): animalsreceived MCAO and equal volume of DMSO; and luteolin groups: animalsreceived MCAO and treated with middle dose of luteolin10mg/kg(Luteolin-M) and high dose of luteolin25mg/kg (Luteolin-H). Luteolin wasadministered immediately after MCAO and then continued with dailyinjections for2days. Experiment3was used to detect luteolin's influence onblood-brain barrier (BBB). Rats in this part were assigned to five groups as thesame way of experiment2. Western blotting and RT-qPCR were used to detectthe expression of claudin-5.Results:1Rats in Sham group had no palsy and a neurological score of zero. Ratsin MCAO group, MC+DM group, low dose group, middle dose group andhigh dose group performed a left palsy. Compared with MCAO group andMC+DM group, there was a significant improvement in neurological functionscores in the Luteolin-H group and Luteolin-M group both at24h and72h (P<0.05). By contrast, there was no significant effect in Luteolin-L groupcompared with MCAO and MC+DM groups both at24h and72h (P>0.05for all).2Compared with MCAO group and MC+DM group, Luteolin-H andLuteolin-M group reduced the brain water content significantly both at24hand72h (P <0.05). Luteolin-L group had no effect on brain water contentboth at24h and72h (P>0.05).3Compared with MCAO group and MC+DM group, Luteolin-H andLuteolin-M group reduced the infarct volume significantly both at24h and72h (P <0.05). Luteolin-L group had no effect on infarct volume both at24hand72h (P>0.05). Based on the results above, we demonstrated that luteolin delivered at high dose (25mg/kg) and middle dose (10mg/kg) per day have abetter therapeutic effect after stroke, and therefore we focused on the luteolintreatment at high dose (Luteolin-H) and middle dose (Luteolin-M) per day forsubsequent biochemical and molecular analysis.4Compared with MCAO group and MC+DM group, Luteolin-H andLuteolin-M group significantly increased the activities of SOD and CAT bothat24h and72h (P <0.05), Luteolin-H and Luteolin-M group significantlydecreased the levels of MDA (P <0.05).5Compared with MCAO group and MC+DM group, Luteolin-H groupsignificantly up-regulated the expression of claudin-5both at24h and72h inprotein levels (P <0.05), but only at72h in Luteolin-M group (P <0.05). Inagreement with the results of western blotting, the mRNA expression ofclaudin-5was up-regulated in Luteolin-H group compared with MCAO groupand MC+DM group both at24h and72h (P <0.05), but only at72h inLuteolin-M group (P <0.05).Conclusions: Systemic administration of luteolin is effective which canameliorate the neurological deficit, improve the brain edema, decrease theinfarct size and up-regulate the expression of claudin-5. Luteolin significantlyincreased the activities of SOD, CAT, decreased the levels of MDA. Theunderlying mechanism of this neuroprotection may be involved in increasingthe activities of SOD and CAT, decreasing the levels of MDA andup-regulating claudin-5expression.PartⅡ Anti-inflammatory effect of luteolin and the underlyingmechanism in rat experimental strokeObjective: This study is to evaluate the expression ofTLRs/MAPKs/NF-κB in experimental rats after ischemic injury at differenttime points. The aim of this etudy is to explore anti-inflammatory effect ofluteolin and the underlying mechanism in rat experimental stroke.Methods: Male, healthy Sprague-Dawley rats were used and subjected to modified permanent middle cerebral artery occlusion (pMCAO). Rats wererandomly assigned to five groups: Sham-vehicle group (Sham): animalsreceived sham operation and equal volume of DMSO; MCAO group (MCAO):animals received MCAO and equal volume of NaCl0.9%; MCAO-vehiclegroup (MC+DM): animals received MCAO and equal volume of DMSO; andluteolin groups: animals received MCAO and treated with middle dose ofluteolin10mg/kg (Luteolin-M) and high dose of luteolin25mg/kg(Luteolin-H). Luteolin was administered immediately after MCAO and thencontinued with daily injections for2days. Immunohistochemistry, Westernblotting and RT-qPCR were used to analyse the expression of TLR4, TLR5,NF-κB, p-p38and p-ERK.Results:1In immunohistochemistry, compared with Sham group, TLR4, TLR5,p-p38, NF-κB were upregulated at protein level in ischemic brain, but theexpression of phospho-ERK1/2was significantly increased only at24h andthen decreased at72h (P <0.05).Compared with MCAO and MC+DM groups,luteolin in high dose group dramatically decreased the positive cells of TLR4,TLR5, p-p38, NF-κB and increased the positive cells of p-ERK1/2in theischemic cortex at all time points (P <0.05), while middle dose group onlyshowed effect at72h (P <0.05).2In western blotting, compared with MCAO and MC+DM groups,luteolin in high dose group dramatically decreased the protein level of TLR4,TLR5, p-p38, NF-κB and increased the protein level of p-ERK1/2in theischemic cortex at all time points (P <0.05), while middle dose group onlyshowed effect at72h (P <0.05).3In agreement with the results of immunohistochemistry and westernblotting, compared with MCAO and MC+DM groups, luteolin in high dosegroup dramatically decreased the the mRNA expression of TLR4, TLR5,p-p38, NF-κB and increased the mRNA expression of p-ERK1/2in theischemic cortex at all time points (P <0.05), while middle dose group onlyshowed effect at72h (P <0.05). Conclusions: Systemic administration of luteolin is effective which candecrease the expression of TLR4, TLR5, p-p38, NF-κB and increase theexpression of p-ERK. Therefore, the excessive inflammation of the brainischemia was alleviated.PartⅢ Anti-apoptosis effect of luteolin and the underlying mechanism inrat experimental strokeObjective: This study is to evaluate the expression of Bcl-2/Bax inexperimental rats after ischemic injury at different time points. The aim of thisstudy is to explore anti-apoptosis effect of luteolin and the underlyingmechanism in rat experimental ischemic stroke.Methods: Male, healthy Sprague-Dawley rats were used and subjected tomodified permanent middle cerebral artery occlusion (pMCAO). Rats wererandomly assigned to five groups: Sham-vehicle group (Sham): animalsreceived sham opera-tion and equal volume of DMSO; MCAO group(MCAO): animals received MCAO and equal volume of NaCl0.9%;MCAO-vehicle group (MC+DM): animals received MCAO and equal volumeof DMSO; and luteolin groups: animals received MCAO and treated withmiddle dose of luteolin10mg/kg (Luteolin-M) and high dose of luteolin25mg/kg (Luteolin-H). Luteolin was administered immediately after MCAO andthen continued with daily injections for2days. Immunohistochemistry,Western blotting and RT-qPCR were used to analyse the expression of Bcl-2and Bax.Results:1In immunohistochemistry, compared with Sham group, the expressionof Bcl-2was down-regulated at protein level in ischemic brain, but theexpression of Bax was significantly increased (P <0.05).Compared withMCAO and MC+DM groups, luteolin in high dose group dramaticallyincreased the positive cells of Bcl-2and decreased the positive cells of Bax inthe ischemic cortex both at24h and72h (P <0.05), while middle dose group only showed effect at72h (P <0.05).2In western blotting, compared with MCAO and MC+DM groups,luteolin in high dose group dramatically increased the protein level of Bcl-2and decreased the protein level of Bax in the ischemic cortex both at24h and72h (P <0.05), while middle dose group only showed effect at72h (P <0.05).3In agreement with the results of immunohistochemistry and westernblotting, compared with MCAO and MC+DM groups, luteolin in high dosegroup dramatically increased the the mRNA expression of Bcl-2anddecreased the mRNA expression of Bax in the ischemic cortex both at24hand72h (P <0.05), while middle dose group only showed effect at72h (P <0.05).Conclusions: The expression of Bcl-2was down-regulated afterischemia, but the expression of Bax was up-regulated after ischemia. Systemicadministration of luteolin is effective which can increase the expression ofBcl-2and decrease the expression of Bax. Therefore, the excessive apoptosisof the brain ischemia was alleviated.