The Effects Of Rhubarb In Intracerebral Hemorrhage Through Gut Microbiota-metabolite-brain Axis

Background and purpose: Intracerebral hemorrhage(ICH)is characterized by high morbidity,mortality,and disability.The hematoma and its degradation products are the primary causes of primary and secondary injury in ICH.However,surgery-and single western medicinebased strategies for hematomal clearance have not yielded satisfactory results.According to traditional Chinese medical theory,the core pathogenesis in the acute phase of ICH is "disturbance of qi and blood,blood stasis in collaterals".Rhubarb is the most used traditional Chinese medicine in the acute stage of ICH due to its ability to induce the downward movement of qi and blood and stagnant blood.It has definite clinical efficacy.However,its significant efficacy cannot be explained by the active ingredients entering the brain.Regarding that rhubarb works on the Yang Ming meridian and acts in a "downward" manner,this study was designed to explore the effects and potential targets of rhubarb in improving neurological impairment in the acute phase of ICH from the perspective of gut microbiota-microbial metabolite-brain interplays.Methods: C57BL/6J mice were randomly divided into five groups:sham-operated group,model group,and rhubarb group(low,medium,and high doses).A mouse ICH model was constructed by locally injecting typeⅦ collagenase into brains.The efficacy of rhubarb was assessed using a modified neurological severity score,foot fault test,and body weight change after 3-days drug intervention.Brain histopathological changes,blood-brain barrier permeability,and brain cell mobilization were assessed by hematoxylin-eosin staining,immunoglobulin G immunohistochemical staining and Evans blue leakage assay,and deoxyribonucleotide endtransferase-mediated nick end labeling,respectively.The best dose of rhubarb was determined according to the above results.Based on 16 S r DNA sequencing,untargeted metabolome,and RNA sequencing techniques,we examined the profiles of gut microbiota in cecum contents,metabolites in feces and serum,and m RNAs in the brain,respectively.Subsequently,the gut microbiota-metabolite-brain interaction network in ICH was constructed by comparing the sham and ICH groups.Then the rhubarb-responsive gut microbiota-metabolite-brain histopathological process network was constructed by a combined analysis of the sham,ICH,and rhubarb groups.Then,the effects of core metabolites on post-ICH brain pathophysiology were verified by cellular experiments.Next,animal experiments were designed to validate the effect of rhubarb on microbiotarelated brain pathologies.Finally,the importance of gut microbiota in mediating the therapeutic effects of rhubarb was clarified using an antibiotics-treated pseudo-sterile mouse model.Results: Rhubarb significantly improved neurological function,brain structural disorder,blood-brain barrier damage,and apoptosis in mice in the acute phase of ICH.The high dose was more effective.The integrated microbiome,metabolome,and transcriptome analyses constructed an ICHrelated gut microbiota-metabolite-brain network with PrevotellaceaeUCG-001,Ruminococcus-torques-group,and Desulfovibrio as the major gut microbiota;bilirubin,2-acetyl-3-methylpyrazine,and LPC(20:4)as the core metabolites and inflammatory response as the main related brain histopathological process.At the same time,Prevotellaceae＿UCG＿001,Clostridia＿UCG＿014,Roseburia,Eubacterium＿coprostanoligenes＿group,and Lachnospiraceae＿NK4A136＿group were the main gut microbiota,bilirubin,FAHFA(18:1/22:3)and 2-acetyl-3-methylpyrazine were the core metabolites;and leukocyte activation,differentiation,and migration were the primary microbiota-regulated brain histopathological processes in response to rhubarb treatment.In vitro experiments confirmed that bilirubin significantly promoted microglia inflammatory factor production.Animal experiments showed that rhubarb significantly ameliorated neutrophil-and microglia/macrophage-mediated inflammatory responses in brain tissue and enhanced their ability to phagocytose hematomas.Moreover,the depletion of gut microbiota partially attenuated the efficacy of rhubarb in ICH.Conclusion: 1.The significant alterations of gut microbiota composition,fecal and serum metabolic profiles,and brain tissue transcriptional maps in mice during the acute phase of ICH are regulated by rhubarb.2.Gut microbiota may mediate the neuroinflammation attenuation after ICH through its metabolites.3.Rhubarb ameliorates brain inflammatory injury during the acute phase of ICH probably through regulating gut microbiota and microbial metabolization of metabolites like bilirubin.