| Ischemic stroke(IS),also referred to as stroke in Chinese medicine,is the second most common cause of death and disability in the world.It is brought on by the abrupt loss of blood flow to a portion of the brain due to thrombotic or embolic cerebral artery occlusion or narrowing,which results in the loss of corresponding neurological function.According to recent research,the microecology of the gut is intrinsically related to the pathophysiology and prognosis of IS.Currently,thrombolysis and interventional surgery are the primary methods of treating IS,but due to their restricted temporal window and high risk of bleeding transformation,their clinical utility is significantly constrained.Xingnaojing injection(XNJ)has demonstrated good efficacy in the treatment of IS after years of clinical use.Recent research has demonstrated that XNJ significantly protects the intestinal mucosal barrier.Its mechanism of action is thought to be connected to the control of intestinal flora.This work suggests the notion XNJ may indirectly exhibit cerebral protective effects through regulating intestinal microecology,in addition to directly entering the brain to produce neuroprotective benefits.Clinical exploration,mechanism prediction,and experimental validation make up the three sections of this study.Firstly,a retrospective clinical investigation employing frequency of feces as an indicator of intestinal microecological state was carried out to observe the impact of XNJ on defecation in patients with IS.Secondly,a network pharmacological study was conducted to investigate the effect of XNJ on inflammatory bowel disease(IBD)and constipation following IS.Finally,an experimental research and the detection of intestinal microbiota confirmed the prediction mechanism.Objective:To explore the mechanism of XNJ in the treatment of IS based on intestinal microecology.(1)A retrospective study was conducted to observe the relationship between XNJ and defecation of IS patients,and to preliminarily investigate the effect of XNJ on intestinal microecology in the clinical treatment of IS.(2)By using an ultra-high performance liquid chromatography-MS/MS(UHPLC-MS/MS)technique to analyze the chemical makeup of XNJ.A network pharmacological analysis was conducted to investigate potential mechanisms of the effects of XNJ on intestinal microecology when treating the target diseases of IS,constipation,and IBD.(3)Experimental animal research and intestinal flora testing supported a potential mechanism for the impact on intestinal microecology during XNJ treatment of IS.Methods:(1)Medical records of IS patients admitted to the Department of Encephalopathy of Dongzhimen Hospital of Beijing University of Chinese Medicine between January 1,2011 and June 30,2021 were collected to compare the NIHSS,ADL,mRS scores and frequency of defecation within 14 days of hospitalization between the exposed and non-exposed groups,with the application of XNJ as the exposure factor.(2)UHPLC-MS/MS technology was used to identify and evaluate the chemical makeup of XNJ.The TCMSP,SwissTargetPrediction,ETCM,and SEA databases were used to search for and predict the target relating to the chemical composition of XNJ.GeneCard and DisGeNet provided the disease targets for IS,IBD,and constipation.The medications and disease targets were then intersected for protein-protein interaction networks(PPI),gene ontology(GO),functional enrichment,and kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analyses.(3)Specific pathogen Free(SPF)grade male C57BL/6 mice were randomly separated into 7 groups,which were normal group(NG),sham-surgery group(SG),control group(CG),experiment group(EG),sham germ-free normal group(SGFNG),sham germ-free control group(SGFCG),and sham germ-free experiment group(SGFEG).A thread occlusion method was used to create a transient middle cerebral artery occlusion(tMCAO)model,and 2,3,5Triphenyltetrazolium chloride(TTC),hematoxylin-eosin(HE),real-time quantitative PCR(RTqPCR),Westernblot,and enzyme linked immunosorbent assay(ELISA)were used for model evaluation and indexes of inflammation and brain and intestinal(TLR4、My D88、NF-κ BP65、Occludin、MMP-9、ZO-1).High-throughput 16s RNA gene sequencing technology was used to assess the levels of mouse intestinal flora and short-chain fatty acids(SCFAs)in the feces.Results:(1)A total of 431 IS patients were enrolled,including 390 in the exposed group and 41 in the non-exposed group.Patients in the exposed group made of 60.5%men and 39.5%women.63.4%of the patients in the non-exposed group were men and 36.6%were women.Patients in the non-exposed group ranged in age from 39 to 88.Ages of the patients in the exposed group ranged from 34 to 97.Patients who used laxatives and had a high frequency of bowel movements on the day of admission had a higher average number of defecations during hospitalization(P<0.05).There was a trend toward lower NIHSS scores following therapy in the non-exposed patients(P>0.05),while patients in the exposed group had significantly lower NIHSS scores than those in the non-exposed group(P<0.001).There was no significant difference in mRS scores between the two groups before and after treatment(P>0.05),and ADL scores of the two groups were significantly increased after treatment(P<0.05).(2)XNJ was found to have 36 different chemical components,including 23 Yujin,8 Musk,1 Bingqi,and 4 Gardenia.XNJ was found to have 36 different chemical components,including 23 Radix Curcumae,8 Moschus,1 Borneolum Syntheticum,and 4 Fructus gardeniae.The analysis of XNJ improved IS showed a total of 25 drug components and 228 common targets related to IS.The biological processes including cellular response to hypoxia,response to lipopolysaccharide,negative regulation of apoptotic process,regulation of blood pressure,negative regulation of inflammatory response,platelet activation,negative regulation of neuronal apoptosis,positive regulation of angiogenesis,positive regulation of endothelial cell proliferation,and it is mainly related to PI3K-Akt signaling pathway,apoptosis,toll-like receptor signaling pathway,VEGF signaling pathway,Ras signaling pathway,JAK-STAT signaling pathway,etc.(3)The analysis of XNJ to improve IBD showed a total of 32 chemical components and 263 common targets related to IBD.The biological processes including regulation of inflammatory response,regulation of apoptosis and migration,and regulation of endothelial cell proliferation,and it is mainly related to PI3K-Akt signaling pathway,toll-like receptor signaling pathway,cGMP-PKG signaling pathway,FoxO signaling pathway,and Shigella production.(4)The analysis of XNJ for constipation showed a total of 36 components and 185 common targets related to constipation,The biological processes including negative regulation of apoptotic processes,positive regulation of MAP kinase activity,positive regulation of smooth muscle cell proliferation,positive regulation of inflammatory response,positive regulation of NF-κB transcription factor activity,epithelial cell proliferation,5hydroxytryptamine binding,neurotransmitter receptor activity etc.,and it is mainly in related to serotonin-activated synapses,FoxO signaling pathway,PI3K-Akt signaling pathway,Tolllike receptor signaling pathway,VEGF signaling pathway,apoptosis,dopaminergic synapses,and Shigella and Salmonella.(5)There are 36 components and 103 related targets in the analysis of the co-action mechanism of anti-IS,IBD and constipation.The biological processes involved include response to lipopolysaccharide,negative regulation of apoptosis,positive regulation of vascular smooth muscle cell proliferation,positive regulation of endothelial cell proliferation,regulation of blood pressure,positive regulation of cell migration,positive and negative regulation of inflammatory response,platelet activation,etc.It is mainly related to atherosclerosis,PI3K-Akt signaling pathway,VEGF signaling pathway,Toll-like receptor signaling pathway,MAPK signaling pathway,serotonin-activated synapses,NF-Kappa B signaling pathway,gap junction and bacterial invasion of epithelial cells.(6)① The neurological function scores of mice in CG and SGFCG were higher than those in SG(P<0.01).② TTC staining revealed that the area of cerebral infarction was substantially smaller in the EG mice compared to the CG(P<0.01),however,the difference between the SGFCG and the SGFEG mice was not statistically significant(P>0.05).③HE staining revealed considerable improvements in neuronal morphology in the EG and SGFEG but severe damage to the cerebral cortex in the CG and SGFCG on the ischemic side.④ Brain immune function:In the ischemic cortex of EG,TLR4 and MyD88 mRNA expression were considerably reduced as compared to CG(P<0.01)and SGFCG(P<0.01),respectively.⑤In terms of BBB permeability,Occludin protein expression was trending greater in EG compared to CG(P>0.05),while MMP9 protein expression was considerably higher in SGFEG compared to SGFCG(P<0.05).When compared to the corresponding model groups,EB staining revealed considerably reduced levels of EB in EG(P<0.01)and SGFEG(P<0.05).⑥ Regarding intestinal immune function,mice in the EG tended to exhibit decreased expression of TLR4,MyD88,and NF-Bp65 mRNA proteins in the colon compared to CG(P>0.05),whereas mice in the SGFEG exhibited significantly increased expression of TLR4 mRNA compared to SGFCG(P<0.01).There was a decreasing trend in plasma LPS levels in EG and SGFEG compared with the corresponding model groups(P>0.05).⑦ In terms of intestinal barrier function,EG mice had significantly higher mRNA levels of Occludin(P<0.01)and ZO-1(P<0.01)compared to CG mice.Occludin(P<0.05)and ZO-1(P<0.01)mRNA levels in the colon of SGFEG mice were substantially higher than those of SGFCG,whereas the expression of Occludin tended to decrease(P>0.05),⑧For intestinal motility,the results of the powder propulsion experiment showed a significantly lower toner propulsion ratio in the CG(P<0.01)and SGFCG(P<0.05)compared to the SG.The toner propulsion ratio in the EG was significantly larger than those in the CG(P<0.05),however there was no significant difference between SGFEG and SGFCG(P>0.05).(7)① In terms of the diversity and richness of the intestinal flora,there was a significant difference between the sham germ-free group and the other groups(P<0.05),but not between the CG and EG(P>0.05).The SGFCG had a higher diversity than the SGFEG(P<0.05).Candidatus Saccharibacteria and Deferribacteres were considerably reduced at the phylum level in the EG compared to the CG(P<0.05),and Deferribacteres in SGFEG decreased significantly compared with SGFCG(P<0.05).In the EG compared to the CG,Enterobacteriaceae and Peptostreptococcaceae increased significantly,while Flavobacteriaceae and Deferribacteraceae decreased dramatically(P<0.05).When compared to the SGFCG,Sutterellaceae were substantially more prevalent in the SGFEG(P<0.05).In the SGFEG as compared to the SGFCG,Morganella was decreased dramatically and Parasutterella was increased significantly(P<0.05).When comparing the EG to the CG,Akkermansia was considerably higher and Morganella was significantly lower(P<0.05).②In terms of SCFA levels,the sham germ-free group had lower levels than the other groups(P<0.05).When compared to the SGFCG,the levels of PA,VA,IB A,and IVA were higher in the SGFEG(P<0.05),but there was no discernible change in the levels of SCFAs between the CG and EG(P>0.05).Conclusion:XNJ is incredibly effective at treating IS.Since it not only has a directly protective effect on the brain,but has the ability to regulate intestinal microbiology to improve the prognosis for the IS indirectly.Specifically,XNJ reduced the area of cerebral infarction,protected the BBB,inhibited the ischemic inflammatory response in mice,and improved the neurological function and capacity for daily activities of patients.On the other hand,XNJ improved post-stroke constipation by reducing intestinal inflammation,mending the intestinal mucosal barrier,and speeding up intestinal peristalsis.It also restored the homeostasis of the intestinal environment by increasing the level of probiotics and decreasing the level of pathogenic bacteria in the intestine,which indirectly improved the prognosis of IS. |
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