Integrated gut microbiota,plasma metabolomics and network pharmacology to reveal the anti-ischemic stroke effect of cooked rhubarb

Background:Cooked rhubarb(CR)is obtained by steaming raw rhubarb(Rheum palmatum L.,Rheum officinale Baill.,or Rheum tanguticum Maxim.ex Balf.)with millet wine.It is used as a traditional Chinese medicine for treating cerebral stroke,where patients often face severe constipation.This study explored the pharmacological effects and mechanisms of CR against ischemic stroke(IS)in rats.We used integrated analysis of gut microbiota,metabolomics,and network pharmacology.Methods:The compounds in CR were identified using LC-MS/MS.The impact of CR on rat middle cerebral artery occlusion/reperfusion(MCAO/R)-induced cerebral infarct size and brain tissue pathology was assessed through 2,3,5-triphenyl tetrazolium chloride and HE staining.Changes in hemorheology were measured by evaluating blood viscosity,and serum levels of pro-inflammatory cytokine were also determined.Gut microbiota composition was analyzed through 16S rRNA gene sequencing.Serum metabolites were examined using untargeted metabolomics and Spearman correlation analysis.The pseudo-germ-free test was used to determine whether tumour necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)improvement in IS are dependent on gut microbiota.Results:In vivo studies showed CR enhances neurobehavioral function in MCAO/R rats and reduces cerebral infarction area.CR also ameliorates brain and intestinal barrier damage caused by stroke.It also decreased inflammation and oxidative stress in IS rats.Furthermore,CR promotes the growth of Akkermansia and Verrucomicrobia,aiding in intestinal barrier repair.Notably,CR primarily influences the primary bile acid biosynthesis pathway.The pseudo-germ-free experiment and network pharmacology confirmed TNF-αand IL-1βas potential targets.CR relies on gut microbiota for its anti-inflammatory effects to improve IS.Conclusion:Cooked rhubarb offers neuroprotective benefits by enhancing beneficial bacteria abundance and regulating bile acid metabolism.It emerges as a potential therapeutic agent for IS.