Young Yum pill inhibits inflammatory mediators and nuclear factor-kappa B signaling in lipopolysaccharide-stimulated RAW 264.7 macrophages

OBJECTIVE: To investigate the effect of Young Yum pill (YYP) on inflammatory mediators in cultured RAW 264.7 cells and elucidate the nuclear factor- kappa B (NF-κB)-related mechanism behind the action. METHODS: YYP was extracted with 95% ethanol Lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were used to evaluate the effect of YYP on inflammatory mediators. Production of nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess test and enzyme-linked immunosorbent assay, respectively. The levels of genes and proteins involved in the generation of inflammatory mediators were examined using real-time polymerase chain reaction and Western blotting, respectively. RESULTS: YYP dose-dependently suppressed LPS-induced production of NO, PGE2 and tumor necrosis factor-α(TNF-α), and elevation of mRNA and protein levels of inducible NO synthase and cyclooxygenase- 2 in RAW 264.7 macrophages. These observations were associated with decreased NF-κB p65 phosphorylation and nuclear localization, enhanced Akt (protein kinase B) phosphorylation, as well as reduced inhibitor of κB (IκB)α degradation and IκB kinase α/β phosphorylation. CONCLUSION: The present study demonstrated an inhibitory effect of YYP on the NF-κB-regulated inflammatory mediators NO, PGE2 and TNF-α in LPS-stimulated RAW 264.7 macrophages, providing a pharmacological basis for the use of YYP in treating inflammatory disorders.

Total Ginsenoside Extract from Panax ginseng Enhances Neural Stem Cell Proliferation and Neuronal Differentiation by Inactivating GSK-3β

Objective:To study the effects of total ginsenosides(TG)extract from Panax ginseng on neural stem cell(NSC)proliferation and differentiation and their underlying mechanisms.Methods:The migration of NSCs after treatment with various concentrations of TG extract(50,100,or 200μg/mL)were monitored.The proliferation of NSCs was examined by a combination of cell counting kit-8 and neurosphere assays.NSC differentiation mediated by TG extract was evaluated by Western blotting and immunofluorescence staining to monitor the expression of nestin and microtubule associated protein 2(MAP2).The GSK-3β/β-catenin pathway in TG-treated NSCs was examined by Western blot assay.The NSCs with constitutively active GSK-3βmutant were made by adenovirus-mediated gene transfection,then the proliferation and differentiation of NSCs mediated by TG were further verified.Results:TG treatment significantly enhanced NSC migration(P<0.01 or P<0.05)and increased the proliferation of NSCs(P<0.01 or P<0.05).TG mediation also significantly upregulated MAP2 expression but downregulated nestin expression(P<0.01 or P<0.05).TG extract also significantly induced GSK-3βphosphorylation at Ser9,leading to GSK-3βinactivation and,consequently,the activation of the GSK-3β/β-catenin pathway(P<0.01 or P<0.05).In addition,constitutive activation of GSK-3βin NSCs by the transfection of GSK-3βS9 A mutant was found to significantly suppress TG-mediated NSC proliferation and differentiation(P<0.01 or P<0.05).Conclusion:TG promoted NSC proliferation and neuronal differentiation by inactivating GSK-3β.

Evaluation of the Pharmacokinetics and Renal Excretion of Ma-Zi-Ren-Wan in Health Subjects

Background: Ma-Zi-Ren-Wan(MZRW) is a classic Chinese formula for treating human constipation. It is comprised of six herbs.Our previous studies have shown its great therapeutic effect. The absorbed compounds had been studied in rat, while there was no study about its components in human body.Objectives: To observe the components of MZRW absorbed in health subjects and study the pharmacokinetics of major compounds. At the same time, to observe the renal excretion of MZRW in health subjects based on the quantification of major compounds.Methods: Health adults were randomly assigned to three dose groups(5 g, 7.5 g and 10 g q.d.) of MZRW. Blood samples were collected from the medial cubital vein just before and at 0.25, 0.5, 1, 2, 4, 8 and 12 h after administration. Urine samples were collected at 0 to 3 h, 3 to 6 h, 6 to 9 h and 9 to 12 h after MZRW administration, with the urine volume recorded for each time segment. Plasma and urine samples were analyzed by optimized LC-MSMS(Liquid chromatography-tandem mass spectrometry) method for pharmacokinetics and renal excretion study of MZRW.Results: Ten compounds of MZRW were observed in 23 health subjects. Due to the low concentration in plasma at the current dose, only four compounds(Albiflorin, paeoniflorin, magnolol and rhein) were quantified in the plasma sample. Honokiol, aloe emodin and emodin could only meet the LLOQ at some time points of the high dose group. Hesperidin, naringin and amygdalin could not be detected in plasma sample. While seven compounds(Amygdalin, albiflorin, paeoniflorin, magnolol, honokiol, rhein and aloe emodin) could be quantified in urine, the renal excretion was well studied.Conclusion: MZRW was safe and well tolerated in this clinical study. Albiflorin, paeoniflorin, magnolol and rhein was well quantified in plasma. The renal excretion of paeoniflorin, albiflorin and rhein were dose dependent for doses ranging between 5 and 10 g.

An ethanolic extract of Bailian(Radix Ampelopsis Japonicae):demonstration of colorectal cancer treatment efficacy via inhibition of β-catenin signaling in vitro

OBJECTIVE:To investigate the underlying mechanism of Bailian (Radix Ampelopsis Japonicae,BL) extract action on colorectal cancer (CRC).METHODS:We explored the involvement of β-catenin signaling on the anti-CRC effects of an BL ethanolic extract (BLE) in cell models by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,immunofluorescent staining,luciferase assay,Western blot analysis and real-time quantitative polymerase chain reaction analysis.Anti-CRC compounds were quantified by high performance liquid chromatography.RESULTS:The contents of gallic acid,catechin,and epicatechin in the BLE were 0.23,1.25,and 0.18 g/kg,respectively.BLE-mediated cytotoxic and apoptotic effects were accompanied by lowered β-catenin/Tcf transcriptional activity,reduced β-catenin nuclear localization,and downregulated protein and mRNA levels of both β-catenin and molecules regulated by β-catenin.CONCLUSION:The mechanism underpinning the anti-CRC effects of BLE may involve inhibition of β-catenin signaling.Further studies are necessary to establish the role of β-catenin signaling in the action of BLE-mediated anti-CRC effects.

An integrated quantitative proteomics strategy reveals the dual mechanisms of celastrol against acute inflammation

Inflammation is a defense mechanism associated with a wide range of diseases.Celastrol is a small molecule isolated from traditional Chinese medicine with potent anti-inflammation activity.In this study,we established an integrated quantitative proteomics strategy to investigate the acute response to celastrol treatment in a rat macrophage cell line challenged with lipopolysaccharide(LPS).Both stableisotopic based non-targeted quantitative profiling and PRM-based targeted quantitation methods were employed.Dimethyl-labeling based non-targeted profiling revealed 28 and 52 proteins that significantly up-and down-regulated by celastrol.Bioinformatics analysis pinpoint key signaling pathways affected.Seven proteins were selected for examining their time-dependent regulatory pattern in response to celastrol using targeted PRM quantitation.The abundance of mRNA at multiple time-points of selected proteins was also examined.Celastrol induced an acute response of selected key transcriptional factors in terms of mRNA or protein abundance within one hour.Interestingly,regulatory trend of mRNA and protein abundance suggested a novel dual mechanism of celastrol in the terms of acute antiinflammation.The integrated quantitative proteomic strategy established in this study constitutes an efficient workflow to characterize key components and their time-dependent regulatory pattern for monitoring drug response.