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.

Active compounds in RenShenJian decoction ameliorate insulin resistance in vitro

Background:RenShenJian decoction,a combination of Pueraria lobata(Willd.)Ohwi and Panax ginseng C.A.Mey,has been used in China since the Song Dynasty(960-1279 C.E.)to relieve symptoms of diabetes mellitus.However,the key compounds in RenShenJian that ameliorate insulin resistance remain unclear.This study identified the anti-diabetic compounds in RenShenJian by rescuing the decreased function of adenosine 5’-monophosphate-activated protein kinase(AMPK),sirtuin 3(SIRT3),or glucose transporter isoform 4(GLUT4).Methods:After streptozotocin-induced diabetic mice were treated with RenShenJian,fasting blood glucose levels and protein expression of SIRT3,p-AMPK,and AMPK were determined.Compounds from RenShenJian in plasma were monitored using multiple responses by liquid chromatography-mass spectrometry.Additionally,two insulin-resistant cell models were incubated with compounds identified in RenShenJian in the blood.Glucose uptake was determined using the fluorescent analog 2-(N-(7-nitrobenz-2-oxa-1,3-dia-xol-4-yl)amino)-2-deoxyglucose.Protein expression levels of p-AMPK,AMPK,SIRT3,and GLUT4 were detected by western blotting.Results:RenShenJian decreased FBG levels and upregulated SIRT3 expression and AMPK phosphorylation in diabetic mice.Thirteen RenShenJian extracts were identified in the blood,11 of which increased the ratios of 2-(N-(7-nitrobenz-2-oxa-1,3-dia-xol 4-yl)amino)-2-deoxyglucose uptake in two insulin-resistant cell models.Nine extracts increased AMPK phosphorylation,nine increased SIRT3 expression,and six elevated GLUT4 expression in palmitate-induced HepG2 cells.Five extracts-puerarin,puerarin 6″-O-xyloside,genistein,ginsenoside Rb1,and ginsenoside Rd-simultaneously activated AMPK,SIRT3 and GLUT4.Conclusion:A series of compounds in RenShenJian that target AMPK,SIRT3,and/or GLUT4 was confirmed and indicate the chemical material basis of amelioration of insulin resistance by RenShenJian.