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.

Optimization of synergistic drug combinations of different sparganins against blood stasis syndrome

Background:Blood stasis syndrome is one of the major syndromes in the development of chronic conditions in traditional Chinese medicine.Blood stasis syndrome is closely related to microcirculation dysfunction and thrombosis.Trigonaceae,as a representative traditional Chinese medicine for promoting blood circulation and removing blood stasis,there are many studies on its anticoagulant,antiplatelet aggregation and antithrombotic effects.Methods:Optimization of sparganin compounds,comprising sparganin A,sparganin B,and sparganin C for the characterization of drug pair effects against blood stasis syndrome was carried out.Ternary quadratic regression orthogonal combination design was carried out in a mouse model of blood stasis syndrome.The concentrations of thromboxane B2,plasminogen activator inhibitor 1,fibrinogen,and endothelin 1 were evaluated by enzyme linked immunosorbent assay.Thymus,hepatic,and spleen indices were also assessed.The outcomes of the evaluations aided in identifying optimum parameter values for the most favorable precipitation against blood stasis syndrome.Subsequently,Cytoscape 3.7.1 was used for network pharmacology to predict the key targets of sparganins and disease.Results:After sparganin A,sparganin B,and sparganin C treatment,hepatic had a downward trend,while spleen indices and thymus had an upward trend.Compared with the model group,thromboxane B2,endothelin 1,plasminogen activator inhibitor 1,and fibrinogen were decreased(P<0.05).The blood stasis syndrome model regression equation was extremely significant,and the correlation coefficient of R was 0.939,indicating that sparganin A,sparganin B and sparganin C were positively correlated at all levels.According to the dosage of these three factors and test results,the equation was fitted and the maximum values of the three sparganin in the corresponding dose range were obtained as follows:A=0.2982 mg/10g,B=0.1438 mg/10g,C=0.0417 mg/10g.Thus,the optimum parameters were found to be 0.298:0.144:0.042 for synergistic drug combinations of sparganin compounds sparganin A,sparganin B,and sparganin C in blood stasis syndrome.The possible key targets of sparganins included matrix metalloproteinase 9,plasminogen,proto-oncogene tyrosine-protein kinase src,and akt serine/threonine kinase 1 in blood stasis syndrome.Conclusion:Our study for the first time found two novel cyclic peptide compounds sparganin B and sparganin C have an anti-blood stasis effect,and speculated sparganins key targets included matrix metalloproteinase 9,plasminogen,proto-oncogene tyrosine-protein kinase src,and akt serine/threonine kinase 1 in blood stasis syndrome.