Integrating Multiomics To Explore The Regulatory Mechanism Of Short Chain Fatty Acids On Secondary Injury After Intracerebral Hemorrhage

BackgroundIntracerebral hemorrhage(ICH)is a common type of brain disease with high morbidity,high disability and high mortality,which brings a heavy burden to the family,society and country.However,the current treatment strategies for secondary injury of ICH are limited,and the treatment effect is not good enough.Therefore,it is of great clinical significance to strengthen the research on the injury mechanism of ICH.Previous studies on ICH based on single omics have enriched our understanding of the secondary injury mechanism after ICH,but it is difficult to fully understand the occurrence and development of ICH by a single omics.Therefore,from a systematic and holistic perspective,we conducted an integrated bioinformatics analysis of the transcriptomics and proteomics expression profile of the brain tissue in ICH mice,so as to find new intervention targets and treatment strategies for the diagnosis and treatment of intracerebral hemorrhage.ObjectiveExplore the changes and characteristics of brain transcriptional expression and protein expression profiles in multiple dimensions after ICH.Find new molecular targets and therapeutic strategies for the prevention and treatment of cerebral hemorrhage by systematically and comprehensively analyzing the pathological mechanism of secondary brain injury after ICH.MethodsBased on type IV collagenase-induced ICH model,the transcriptomic and proteomic analysis were performed on the perihematomal brain tissues to obtain the transcriptomic and proteomic expression profiles of ICH mice.Protein-protein interaction(PPI)analysis,regularized canonical correlation analysis(r CCA)and gene set enrichment analysis(GSEA)were performed to obtain a specific gene-protein related network and to explore mechanisms and potential targets of ICH.The expression of key molecules in pathway and the hub molecules in the m RNA-protein network were verified by RT-q PCR and WB,and the concentration of key molecules in the plasma of ICH patients were detected by ELISA.Results1.H&E staining showed that macrophage infiltration and hemorrhagical foci were significantly increased in the right basal ganglia of mice in ICH group compared with the controls.The modified Garcia score and beam walking tests score in ICH group was significantly decreased compared with the controls.2.A total of 2218 genes(2065 upregulated and 153 downregulated)were identified between the ICH group and control group as DEGs by transcriptomic analysis,and a total of 353 proteins(198 upregulated and 155downregulated)were screened out as DEPs by proteomic analysis.3.Ten candidate hub molecules were screened by co-expression interaction network,namely Lyz2,C3,Serpina3 n,Cfp,Ppbp,Hp,Serping1,Ctss,Itgb2 and Anxa2;two specific ICH-related expression modules were identified r CCA,involving lipid metabolism and immune-inflammatory processes,respectively;three significantly up-regulated gene sets were detected by GSEA analysis,namely inflammasome complex,negative regulation of interleukin-12 production,and pyroptosis.4.The expression of hub molecules(serpina3n,itgb2 and CTSS)in coexpression interaction network were significantly up-regulated,and were significantly correlated with the behavioral score of ICH mice.The expression of related molecules in pyroptosis pathway of NEK7/Caspase-1/GSDMD were significantly up-regulated after ICH.5.The plasma concentrations of Caspase-1,IL-1β,IL-18 and TNF-αwere significantly increased in ICH patients compared with the controls.ConclusionIn this study,we describe the pathological mechanism of brain injury after ICH in a multi-dimensional and systematic manner,and we effectively identified three hub(Itgb2,Ctss and Serpina3n)molecules and revealed ICH-related molecule network in brain of mice.Moreover,Secondary injury after ICH is related to pyroptosis-mediated inflammatory response.Caspase-1 is a potential target for the treatment of ICH.Inhibition of NEK7/Caspase-1/GSDMD-mediated pyroptosis provides a new strategy for the treatment of ICH.BackgroundAt present,there are many studies on local injury in the central system,but the intervention effects are uneven.Therefore,the regulation of peripheral immunity provides a new research perspective for the treatment of intracerebral hemorrhage.The gut microbiota has a powerful immunomodulatory effect on the human gut,peripheral and central systems through the "gut-brain" axis.Short-chain fatty acids(SCFAs)are produced by the fermentation of polysaccharides by gut bacteria and are major players in the interaction between diet,gut microbiota and health.Besides,SCFAs have been widely demonstrated in recent years to regulate nervous system development and microglial differentiation through the "microbiota-gut-brain" axis.Given that the inflammatory cascade mediated by microglia activation is an important initiating link leading to neurological damage after ICH,we hypothesized that SCFAs may have potential neuroprotective effects.However,the current understanding of the relationship between SCFAs and ICH is limited,and further exploratory studies are needed.Objective1.To reveal the correlation and dose-response relationship among ICH,gut microbiota and SCFAs.2.To evaluate the effects of sodium butyrate(Na B)on the central nervous systems,circulation and gut of ICH mice.3.To explore the potential mechanism of the neuroprotective effect of Na B in ICH mice.Methods1.The 16 s r RNA sequencing technology was used to draw a map of the gut microbiome in ICH mice,and to find out the differential microbes after ICH based on the successful establishment of a mouse ICH model.2.The content of total SCFAs and subtypes of SCFAs in feces and serum of mice with cerebral hemorrhage was detected by Gas chromatography-mass spectrometry(GC-MS)and Liquid chromatographyelectrospray ionization-tandem mass spectrometry(LC-ESI-MS/MS).3.Three different concentrations(100m/kg,300mg/kg and 600 mg/kg)of Na B were orally administered to ICH mice.The neurological function of mice at 24 h and 72 h after operation were assessed by the modified Garcia test and the beam walking test.The brain water content was assessed by drywet weight method.The permeability of the blood-brain barrier was evaluated by Evan’s blue extravasation and Western blot.4.Neuronal apoptosis and degeneration were evaluated by TUNEL staining,FJC staining and Western blot.The intestinal mucosal barrier function was assessed by immunofluorescence staining and ELISA.5.The time-related expression characteristics of key molecules(NEK7,GSDMD-N and Caspase-1 p20)were analyzed by Western blot.The cellular localization of NEK7 was analyzed by double immunofluorescence staining.The interaction between NEK7 and NLRP3 was assessed by CO-IP,and the expressions of NEK7,NLRP3,ASC,Caspase-1 p20,GSDMD-N,and IL-1β in the perihematomal area of brain tissue were detected by Western blot.The amount of Caspase-1-positive microglia after ICH was assessed by double immunofluorescence staining.Results1.ICH could lead to changes in the diversity,composition,and abundance of gut microbiota,among which the SCFAs-producing microbiota changes significantly.PICRUSt functional analysis showed that the differential microbiota was mainly related to metabolic function,and four significant differential pathways were screened out,namely " Butanoate metabolism"," Ubiquinone and other terpenoid-quinone biosynthesis"," Secondary bile acid biosynthesis" and " Tetracycline biosynthesis".2.The total amount of SCFA in the feces of ICH mice was significantly decreased.Besides,the contents of acetic acid,propionic acid and butyric acid were significantly decreased,while the content of isobutyric acid,valeric acid,isovaleric acid did not change significantly.3.Compared with ICH + Vehicle group,Administration of Na B(600mg/kg)markedly improved the neurological impairments,reduced brain edema and alleviated the BBB permeability.Besides,compared with sham group,the protein expression of Occludin decreased and MMP-9 increased in ICH + vehicle group,which could be reversed by Na B(600 mg / kg).4.Compared with sham group,the number of TUNEL and FJC positive neurons in ICH + vehicle group significantly increased and the ratio of Bcl-2 to Bax decreased.After Na B treatment,the number of TUNEL + and FJC + neurons decreased significantly,and the ratio of Bcl-2 to Bax increased.Compared with the sham group,the mean fluorescence intensity of the tight junction protein Claudin-1 and mucin Muc2 in the intestinal mucosal barrier in ICH + Vehicle group was significantly decreased,and the serum LBP,DLA and DAO contents were significantly increased.The above trends could be reversed by Na B(600 mg/kg)treatment.5.The expression of NEK7 peaked at 24 h after ICH,gradually decreased at 72 h,and returned to the level similar to that of sham group at 7d.The expression of GSDMD-N and Caspase-1 P20 was consistent with that of NEK7.Double immunofluorescence staining showed that endogenous NEK7 was mainly expressed in microglia and neurons,and the expression increased significantly at 24 h after ICH.6.CO-IP results showed that NEK7 was able to form a NEK7-NLRP3 complex with NLRP3 after ICH.WB results showed that the expressions of NEK7,NLRP3,ASC,Caspase-1 p20,GSDMD-N and IL-1β in the perihematoal tissue were significantly increased at 24 h after ICH,and orally administration of Na B(600 mg/kg)could reversed above trends.Orally administration of Na B(600 mg/kg)decreased the number of Caspase-1-positive microglia in perihematoal tissue at 24 h after ICH.ConclusionICH leads to the dysbiosis of gut microbiota and the disturbance of the metabolic profile of fecal and serum SCFAs,especially the alteration of butyrate metabolic pathway.Na B enhanced the anti-inflammatory defense capacity of perihematomal tissue,peripheral circulation and gut after ICH by reducing the neurological injury and brain edema,inhibiting the microgliamediated pyroptosis,promoting the anti-apoptotic effect of neurons and restoring the intestinal mucosal barrier function.