| BackgroundStroke is the second leading cause of death worldwide and the leading cause of death in China.Epidemiological data indicate that the morbidity and mortality of stroke are on the rise worldwide due to the aging of the global population and lifestyle changes.Ischemic stroke accounts for 87%of the total incidence of stroke,and as the major type of stroke,it is characterized by high incidence,high fatality,high disability,and high recurrence rates,causing a huge burden to public health,medical systems,and patients and their families.Cerebral ischemia-reperfusion injury may lead to serious neurological complications,such as cerebral edema,cerebral hemorrhage,and neuronal death.Studies have shown that astrocytes and aquaporin 4 play a key role in developing cerebral edema after cerebral ischemia-reperfusion injury.Therefore,it is urgent to develop neuroprotective drugs with good efficacy and low toxic side effects,with broad market prospects and clinical application potential.Pterostilbene is a biologically active trans-stilbene compound,a structural analog of resveratrol.As the main bioactive component in the extract of dragon’s blood,modern pharmacological studies have revealed that Pte plays a key role in the treatment of various diseases.Recent pharmacological studies have also shown that Pte can potentially treat neurological disorders,including degenerative diseases,cognitive disorders,anxiety disorders,and ischemic strokes.Our research group has previously found that Pte has an anti-acute cerebral ischemia-reperfusion injury effect in rats and a protective effect against blood-brain barrier disruption.This study focused on the effect of Pte on cerebral edema in rats with cerebral ischemia-reperfusion injury.It revealed the effects of Pte on metabolites and intestinal flora in rats with cerebral ischemia-reperfusion injury through metabonomics and metagenomic correlation analysis.Part Ⅰ Anti-brain edema after cerebral ischemia-reperfusion injury in rats with pterostilbenePurpose:In this chapter,we establish the middle cerebral artery occlusion rat and the Oxygen-glucose deprivation/reoxygenation astrocyte model to comprehensively investigate the effect of Pte on cerebral edema after cerebral ischemia-reperfusion injury in vitro and in vivo.Methods:SVG p12 cell lines were used at the cellular level and divided into control,model,and Pte groups.MTT assay was used to determine the effects of Pte treatment for 24 and 48 h on the viability of SVG p12 cells and the survival rate under the OGD/R cell model.At the whole organism level,healthy adult male Sprague-Dawley rats with a body weight ranging from 280 to 300 grams were utilized to establish the middle cerebral artery occlusion model.The rats were divided into the Sham,Model,and Pterostilbene(Pte,60 mg/kg)treatment group.The Pte treatment was administered for three durations:1,3,and 7 days.The anti-cerebral edema effect of Pte in MCAO rats was investigated by measuring brain water content,left brain water content,brain index,hemisphere swelling degree,pathological changes of HE in the brain region,ultrastructural changes of brain tissue,GFAP,and AQP4 protein expression.Results:Pte incubated with SVG p12 cells for 24 h or 48 h,the cell viability was enhanced in the range of 1-100 μM or 1-80 μM,with the strongest effect of 80 μM.The IC50 was calculated to be 143.4 μM and 100.3 μM,respectively.Pte 1-40 μM increased the survival of OGD/R cells after 24 h of reoxygenation(P<0.01).Pte 2.5,5,10 μM reduced OGD/R cell swelling in a dose-dependent manner.After 1,3 and 7 days of MCAO model injury,brain water content,left brain water content,brain index and hemispheric swelling of rats were increased compared with Sham group,and the differences were statistically significant(P<0.01),the degree of hemispheric swelling at day 1 after Pte administration was significantly lower than that in the Model group(P<0.01).After Pte administration for 3 and 7 days,cerebral water content,left cerebral water content,cerebral index and hemispheric swelling were all decreased compared with model group,and the difference was statistically significant(P<0.01).After 1,3 and 7 days of Pte administration,the pathological changes of brain tissue in MCAO rats were significantly improved,the pathological score was reduced,the cytotoxicity and vasogenic edema were alleviated,glial cell proliferation was inhibited,and cell necrosis was reduced.Pte can improve the ultrastructure of microvessels and glial cells in MCAO rats,restore the tight junctions and basal membrane of microvascular endothelial cells,and astrocyte morphology to a certain extent,and reduce the blank edema area around cells.The effects of Pte on the contents of GFAP and AQP4 in the ischemic side of MCAO rats were measured by immunofluorescence assay and WB.The results showed that the expressions of GFAP and AQP4 increased in the ischemic side brain tissue of Model rats at 1,3,and 7 days,and Pte could reduce the expressions of GFAP and AQP4 in MCAO rats.Conclusion:Pte has anti-cerebral edema after cerebral ischemia reperfusion injury.Pte can protect astrocytes in vitro,improve cell viability and cell survival rate after OGD/R injury,and reduce cell swelling.Pte can reduce the tissue injury in cerebral ischemia reperfusion rats and reduce the expression of proteins associated with ischemic penumbra edema.Part Ⅱ Effect of Pterostilbene on the Metabolomics of cerebral ischemia-reperfusion RatsPurpose:In this chapter,a MCAO rat model was developed to simulate ischemia stroke.Colonic fecal samples from MCAO rats were analyzed using liquid chromatography-mass spectrometry-based metabolomics.To investigate the impact of Pte on the fecal metabolomic profile of rats subjected to cerebral ischemia-reperfusion injury.Methods:Healthy adult SD rats weighing 280-300 g were employed to establish the MCAO model.The rats were divided into nine groups:Control,Sham,Model,and Pte(30 mg/kg and 60 mg/kg)treatment groups.The Pte treatment was administered for 3,7,and 14 days.The Modified Neurological Severity Scores and laser speckle imaging technique were utilized to assess the success of the MCAO model.LC-MS was employed to analyze the colonic fecal samples of MCAO rats,to investigate the effect of Pte on the metabolomic profile of these rats.Results:A total of 1,303 compounds matching the reference database were identified in 52 samples by non-targeted metabolite detection.Principal Component Analysis dimension reduction results demonstrated a strong correlation between samples within the same group,with no outlier samples and good repeatability in the same group.Differential metabolites exhibited similar trends between the Control and Model groups and between the Sham and Model groups.Many metabolites appeared in the differential metabolites of both groups,exhibiting the same trend of change.The metabolites of different groups were effectively enriched into the Kyoto Encyclopedia of Genes and Genomes metabolic pathways,the results showed that the KEGG metabolic pathways in the top 10 groups had high similarity.A total of 642,973 and 1,154 distinct metabolites were identified between the Model group and the Pte 30 mg/kg 3-day group,Pte 30 mg/kg 7-day group,and Pte 30 mg/kg 14-day group,respectively.Compared with Model group,after 3 and 7 days of Pte 30 mg/kg administration,Deoxyribose 5-phosphate and 1,2-Bis(4-hydroxy 3-methoxyphenyl)ethylene showed significant differences in their upregulated metabolites.After 7 and 14 days of administration,[12]-Gingerol showed significant difference,and the increasing trend was more significant at 14 days;Madasiatic acid was down-regulated,and the difference was most significant at 14 d.A total of 3429,10687 and 10521 distinct metabolites were identified between the Model group and the Pte 60 mg/kg 3-day group,Pte 60 mg/kg 7-day group,and Pte 60 mg/kg 14-day group,respectively.Compared with Model group,p-Cresol sulfate and(R)-Dihydromaleimide were significantly up-regulated 7 and 14 days after Pte administration.4,5-Dimethylthiazole was significantly down-regulated.Prol-histidine continued to be significantly down-regulated during the administration of Pte at 60 mg/kg.KEGG metabolic pathway enriched by differential metabolites between Model group and Pte 30 mg/kg for 7 d and 14 d and Pte 60 mg/kg for 3 d,7 d and 14 d were basically consistent with the results of differential metabolite enrichment in blank control group,Sham group and Model group.Conclusion:Pte can regulate fecal metabolites in MCAO rats in a time-dependent manner.Similar differential metabolites,such as 3-methyladiponic acid and ornithouric acid,between Control group and Model group,Sham group and Model group,may be biomarkers for the identification of pathological changes in cerebral ischemia-reperfusion injury.Compared with the Model group,the significantly upregulated differential metabolites at 3,7 and 14 days after Pte 30 or 60 mg/kg administration were related to the promotion of DNA repair,the regeneration of nerve cells,the inhibition of apoptosis,the anti-oxidative stress,and anti-inflammatory effects.Part Ⅲ Effect of Pterostilbene on intestinal flora of cerebral ischemia-reperfusion RatsPurpose:In this chapter,by establishing a model of MCAO rats to simulate ischemia stroke,metagenomic sequencing was conducted on colon fecal samples of MCAO rats,and the effects of Pte on intestinal microecology of acute cerebral ischemia-reperfusion model rats were discussed.Methods:Healthy adult SD male rats weighing 280-300 g were utilized to establish the MCAO model.The rats were then divided into Control,Model,and Pte(60 mg/kg)groups,with Pte administered for 3,7,and 14 days.The mNSS and laser speckle methods were employed to evaluate the success of the model.DNA extraction and analysis were conducted on the colonic fecal samples of MCAO rats.Subsequently,species and functional annotations of genes were performed to investigate the effects of Pte on the intestinal microecology of MCAO rats.Results:At the kingdom,phylum,class,order,family,genus,and species level,the bacterial abundance of Lactobacillus murinus and Escherichia coli in the Model group decreased.In contrast,the bacterial abundance of Lactobacillus Murinus and Escherichia coli in the Model group increased.The Lactobacillus murinus flora abundance increased gradually with the extension of time after Pte 60 mg/kg intervention,and the Escherichia coli flora abundance decreased 7 and 14 days after administration.The a diversity at the genus level was evaluated by four comparative parameters:Species Richness,Shannon Index,Simpson Index,and Chaol Index.The results showed that compared with the Control group,the species diversity among models was significantly decreased,and the species diversity was significantly increased after intervention of 60 mg/kg Pte for 7 and 14 days.PCA dimension reduction analysis showed the beta diversity results of the five groups.The distribution of Pte 60 mg/kg 3 d group was like that of Model group,while Pte 60 mg/kg 7 d and 14 d group was relatively far away from Model group.The results indicated that Pte 60 mg/kg had a certain effect on the species richness of intestinal flora in MCAO rats after 7 and 14 days of administration.Pte administration of 60 mg/kg can significantly regulate the changes of 20 kinds of intestinal flora.Including the up-regulated of Bacteroides sp.CAG:927,Prevotella sp.CAG:1031 and Prevotella sp.CAG:873,and the down-regulated of Prevotella sp.CAG:891 and Bilophila wadsworthia,Alloprevotella rava,Alloprevotella tannerae,Prevotella sp.CAG:5226,Prevotella sp.CAG:755,Bacteroides nordii and other 17 species of bacteria.The enrichment results of differential KEGG secondary signaling pathway showed that compared with Control group and Model group,the number of differential genes in Model group and Pte 60 mg/kg 3 d group significantly decreased in metabolic pathway and olfactory pathway.On 7 and 14 days of administration,The KEGG pathway and the number of differentially enriched genes in the top 25 pathways were basically the same.Conclusion:Pte administered 60 mg/kg at 3,7 and 14 d can affect the intestinal microbial colony structure of MCAO rats,which can restore the balance of beneficial bacteria and inhibit the growth of potentially harmful bacteria.Improve the species diversity of intestinal flora in cerebral ischemia reperfusion rats and up-regulate the number of differential genes of metabolic pathway and olfactory pathway to protect cerebral ischemia reperfusion rats. |
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