| BackgroundIschemic stroke is an acute clinical syndrome caused by a disturbance of the blood supply to the brain,which may eventually develop into devastating or irreversible brain damage.Although evidence-based medicine research confirmed that intravenous thrombolysis and endovascular intervention are the first-line treatment directed at early reperfusion of tissue,less than 10%of all ischemic stroke patients are available to these effective therapies safely in most centers,due to the limitation of 4.5 hours of time window.As resident immune cells in the central nervous system,microglia are the first to respond to dramatic changes in brain homeostasis,and activated into a pro-inflammatory M1 phenotype to amplify cellular damage or an anti-inflammatory M2 phenotype to remove the damaged tissue.To identify new potential targets is critically urgent for addressing the lack of stroke therapeutic strategies.Microglia-mediated inflammation and disruption of the blood-brain barrier(BBB)play pivotal roles in ischemia-reperfusion injuries.Recent findings have clarified that gut microbiota are closely implicated in microglial maturation and activation in disorders of the brain,and are expected to be a novel prevention for patients with ischemic stroke.Acorus tararinowii oils(AT oils)are considered as the active ingredient with promising pharmacological activities of Acorus tararinowii,which is a principal aromatic herb for herbal formula in the treatment of Alzheimer disease,epilepsy,depression,stroke and other cognitive disorders.However,it remains to be clarified that whether the potential effects of AT essential oils are relevant to microglial maturation and activation,as well as gut microbiota.The role of intestinal flora dysbiosis on the anti-inflammatory effect of AT essential oils is still unknown.ObjectiveIn the present study,we present evidence that AT essential oils could relieve cerebral ischemia-reperfusion injury via modulating microglia polarization and BBB permeability,in which gut microbiota might play a vital role.MethodsSixty rats weighing 250-270 g were subjected to 2 h of middle cerebral artery occlusion(MCAO)following by reperfusion for 7 days.Neurological deficit was assessed at 1 and 7 days after MCAO or sham operation using modified neurological severity score(mNSS)scale.1.Preparation and Gas chromatography-mass spectrometry of AT oilsThe air-dried plant materials were acquired from GuangDong Zihetang Pharmacy Co.Ltd.and authenticated as roots and rhizomes of Acorus tatarinowii(AT)by Professor Wei Li(School of Chinese Materia Medica,Guangzhou University of Chinese Medicine).Gas chromatography-mass spectrometry(GC-MS)was used for identifying the compositions of AT oils.2.AT oils alleviates cerebral infarct volume and neurological recovery in ischemic stroke ratsSD rats were randomly divided into sham group,model group(MCAO),low dose,medium dose and high dose(5mg/kg/day,10mg/kg/day,20mg/kg/day)of AT oils treatment groups.The drug was administered by oral gavage in AT oils groups at designed dosages(5,10 and 20 mg/kg/day)for 7 days similarly as previously described.Equal volume of saline with 3%Tween-80 was given to the sham and MCAO-reperfusion(MCAO/R)groups.Neurological deficit was assessed at 1 and 7 days after MCAO or sham operation using modified neurological severity score(mNSS)scale.We investigated cerebral infarct size by 2,3,5-triphenyltetrazolium chloride(TTC)staining.Furthermore,we employed a TUNEL assay for detecting the neuroprotective effects of AT oils against cell apoptosis.To investigate the effects of AT oils on microglial phenotype and neuroinflammation in the prefrontal cortex after ischxemic stroke,we examined the microglial polarization using double staining of the co-incubation of Iba-1 with CD16/32 or CD206,and we also tested the serum levels of inflammatory cytokines,including TNF-α,IL-17,IL-6 and IFN-y by using ELISA.Western Blot was used to detect the effects of AT oils on the levels of JNK,p-JNK,ERK,p-ERK,iNOS and the expression of brain apoptosis-related proteins Bc12 and Bax in MCAO rats.3.Analysis on AT oils of the diversity and composition of the gut microbiota in MCAO/R rat modelFecal samples from rats were collected and preserved in the dry sterilized centrifuge tubes at-80℃ until analyzed.The V3-V4 region of the 16S rRNA amplicon sequencing was carried out for analyzing bacteria taxa in each fecal sample to compare the differences in microbiological investigation between groups.4.Gut microbiota mediates gut-brain barrier function and microglial phenotype of AT oils after strokeTo further confirm the relationship between the neurological protection of AT oils and gut microbiota,the FMT experiments were performed as removing intestinal flora by antibiotic cocktails with 1%(w/v)amoxicillin and 0.1%(w/v)clavulanic acid solution in the drinking bottles for 14 days(50 ml solution/rat/day)before MCAO operation and then transferring the feces of the rats in the I/R group into the recipient rats.The infarct volume,neurological recovery and double immunofluorescence staining were tested to demonstrate the key role of AT oils in neuroinflammation induced by MCAO.Results1.Preparation and Gas chromatography-mass spectrometry of AT oilsIt is reported that AT oils are the main bioactive ingredient of Acorus tatarinowii.GCMS was conducted to detect the chemical compounds in AT oils.The representative GC-MS chromatogram of the scan mode showed that 32 compounds were identified in the AT oils.The main components contained Elemicin,Methyleugenol and β-asarone.2.AT oils alleviates cerebral infarct volume and neurological recovery in ischemic stroke ratsCompared with the I/R group,no significant differences were observed in the low dose of AT oils on neurological deficit test,while it showed a prominent reduction in the groups with AT oils treatment similar to the sham group.As determined by 2,3,5triphenyltetrazolium chloride(TTC)staining on the 7th day after MCAO treatment,the ischemia-reperfusion(I/R)rats exhibited cerebral ischemic injury.In the TTC staining,the infarct volume was much larger in the I/R group than in the sham-control,whereas AT oils showed a decreased percentage of infarct volume in a dose dependent manner.After exposed to 2 h of MCAO and 7 days of reperfusion,rats displayed a high increase on apoptotic cell death as TUNEL-positive puncta accumulated around the infarct area of the frontal cortex,and the treatment of AT oils remarkably decreased the accumulation of TUNEL-positive staining in MCAO rats,suggesting that the essential oils of AT could remarkably reduce the apoptosis rate.The co-incubation of Iba-1 with CD16/32 or CD206 proved that the population of Iba-1 was remarkably increased in the groups with MCAO and reperfusion,besides,we observed an increased number of Iba-1+/CD16/32+cells and a growing population of Iba-1+/CD206+cells in I/R group.Meanwhile,AT oils(20 mg/kg/day)treatment inhibited the increase of Iba-1+/CD16/32+-positive cells obviously,and promoted the growth of Iba-1+/CD206+-positive cells with certainty.The co-immunofluorescence visualization of Iba-1/CD 16/32 and Iba-1/CD206 confirmed that AT oils could maintained the number of M2 microglia instead of M1 microglia,compared with the MCAO/R group.The expression levels of TNF-α,IL-17,IL-6 and IFN-y were abundantly increased after MCAO/R operation,and they were down-regulated by the treatment of AT oils with gradient concentrations at day 7 of reperfusion compared with MCAO/R-operated rats.Westen blot showed that AT oils inhibited the phosphorylation of JNK and ERK,decreased the expression of iNOS,and increase the protein expression ratio of Bcl2 to Bax.3.Analysis on AT oils of the diversity and composition of the gut microbiota in MCAO/R rat modelThe result shown that AT oils altered the diversity of gut microbiota following the ischemic stroke.We could observe that the relative abundance of Verrucomicrobiae,Verrucomicrobiaceae,Akkermansia,Verrucomicrobiales,Tenericutes,Verrucomicrobia and Akkermansia_muciniphil was substantially exacerbated and the relative abundance of Tenericutes and Prevotella_copri was inhibited within the I/R group,while AT oils therapy sharply reversed variations in Verrucomicrobia and Tenericutes.4.Gut microbiota mediates gut-brain barrier function and microglial phenotype of AT oils after strokeTo further confirm the relationship between the neurological protection of AT oils and gut microbiota,the FMT experiments were performed as removing intestinal flora by antibiotic cocktails and transferring the feces of the rats in the I/R group into the recipient rats.As expected,the reduced infarct volume and improved neurological recovery by AT oils were withdrawn by the decrease of bacterial load,while FMT treatment displayed statistical significance on reducing infarct volume and neurological deficit scores.he immunofluorescence staining indicated that the colocalization of Occludin with ZO-1 was markedly decreased in the frontal cortex after MCAO.Nevertheless,AT oils group receiving microbiota from I/R rats showed better performance in the fluorescence intensity of Occludin and ZO-1 than AT oils group without FMT treatment in the cerebral cortex.Similar changes were observed in immunofluorescence staining of Occludin and ZO-1 in the ileum tissues.Collectively,these findings indicated that gut microbiota could reduce the permeability of brain and gut.ConclusionIn conclusion,the results of our study manifested that AT oils served as a neuroprotective and anti-inflammatory polarizing agent accompanied by altering the composition of gut microbiota and enhancing constant remodeling of tight junctions in the intestine and the brain.These findings offered evidences that AT oils could be a novel therapeutic/preventive approach for patients with ischemic stroke by harnessing the microbiota. |
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