Gremlin-1 promotes IL-1ß-stimulated chondrocyte inflammation and extracellular matrix degradation via activation of the MAPK signaling pathway.

The role and mechanism of Gremlin-1 in osteoarthritis (OA) were expected to be probed in this study. Firstly, an in vitro OA model was constructed by stimulating human chondrocyte cell line CHON-001 with IL-1ß. Next, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were utilized for assessing the effect of IL-1ß with different concentrations (5, 10, and 20 ng/mL) on the activity and Gremlin-1 messenger RNA of CHON-001 cells, respectively. Besides, the influence of knocking down/over-expressing Gremlin-1 on the inflammatory factors (IL-6, TNF-α, IL-18 and PGE2), oxidative stress-related substances (malondialdehyde [MDA]; superoxide dismutase [SOD]; lactate dehydrogenase [LDH]), extracellular matrix (ECM) degradation-related proteins, and mitogen-activated protein kinase (MAPK) pathway proteins in IL-1ß-stimulated CHON-001 cells were tested by enzyme-linked immunosorbent assay, related kits, qRT-PCR, and western blot, respectively. IL-1ß inhibited CHON-001 cell proliferation and upregulated Gremlin-1 expression in a concentration-dependent manner. Overexpression of Gremlin-1 increased the IL-6, TNF-α, IL-18, PGE2, and MDA levels, enhanced the LDH activity, and decreased the SOD activity in IL-1ß-induced CHON-001 cells; while the effect of Gremlin-1 knockdown on the above factors was in contrast with that of the overexpression. Furthermore, overexpression of Gremlin-1 upregulated protein expression of matrix metalloproteinase (MMP)-3, MMP-13, and ADAMTS4 while downregulated protein expression of collagen III, aggrecan, and SOX-9 in IL-1ß-stimulated CHON-001 cells. Besides, overexpression of Gremlin-1 increased the p-p38/p38 value while decreased the p-JNK/JNK value in L-1ß-stimulated CHON-001 cells; however, knockdown of Gremlin-1 reversed the above results. Gremlin-1 may promote IL-1ß-stimulated CHON-001 cell inflammation and ECM degradation by activating the MAPK signaling pathway.

Silybin regulates P450s activity by attenuating endoplasmic reticulum stress in mouse nonalcoholic fatty liver disease.

Cytochrome P450s are important phase I metabolic enzymes located on endoplasmic reticulum (ER) involved in the metabolism of endogenous and exogenous substances. Our previous study showed that a hepatoprotective agent silybin restored CYP3A expression in mouse nonalcoholic fatty liver disease (NAFLD). In this study we investigated how silybin regulated P450s activity during NAFLD. C57BL/6 mice were fed a high-fat-diet (HFD) for 8 weeks to induce NAFLD, and were administered silybin (50, 100 mg ·kg-1 ·d-1, i.g.) in the last 4 weeks. We showed that HFD intake induced hepatic steatosis and ER stress, leading to significant inhibition on the activity of five primary P450s including CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP3A in liver microsomes. These changes were dose-dependently reversed by silybin administration. The beneficial effects of silybin were also observed in TG-stimulated HepG2 cells in vitro. To clarify the underlying mechanism, we examined the components involved in the P450 catalytic system, membrane phospholipids and ER membrane fluidity, and found that cytochrome b5 (cyt b5) was significantly downregulated during ER stress, and ER membrane fluidity was also reduced evidenced by DPH polarization and lower polyunsaturated phospholipids levels. The increased ratios of NADP+/NADPH and PC/PE implied Ca2+ release and disruption of cellular Ca2+ homeostasis resulted from mitochondria dysfunction and cytochrome c (cyt c) release. The interaction between cyt c and cyt b5 under ER stress was an important reason for P450s activity inhibition. The effect of silybin throughout the whole course suggested that it regulated P450s activity through its anti-ER stress effect in NAFLD. Our results suggest that ER stress may be crucial for the inhibition of P450s activity in mouse NAFLD and silybin regulates P450s activity by attenuating ER stress.

Selective Photoaffinity Probe for Monitoring Farnesoid X Receptor Expression in Cultured Cells.

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, is a vital ligand-activated transcriptional factor, which is highly expressed in the liver, intestine, and adrenal gland. However, FXR homeostasis is influenced by many factors, such as diet and circadian rhythm, and the expression of FXR differs in diverse organs. Currently, there is no method to monitor the FXR homeostasis in real time, which restricts us from further investigating the function of FXR under physiological and pathological conditions. In this project, classic FXR agonists were selected to be modified to targeting FXR. The photo-cross-linking diazirine group and alkynyl, a click reaction group, were incorporated to the ligands. Through biorthogonal reaction, fluorophore was linked to the ligands to realize the monitoring of FXR expression in cells.

Mechanism-Based Pharmacokinetic Model for the Deglycosylation Kinetics of 20(S)-Ginsenosides Rh2.

Aim: The 20(S)-ginsenoside Rh2 (Rh2) is being developed as a new antitumor drug. However, to date, little is known about the kinetics of its deglycosylation metabolite (protopanoxadiol) (PPD) following Rh2 administration. The aim of this work was to 1) simultaneously characterise the pharmacokinetics of Rh2 and PPD following intravenous and oral Rh2 administration, 2) develop and validate a mechanism-based pharmacokinetic model to describe the deglycosylation kinetics and 3) predict the percentage of Rh2 entering the systemic circulation in PPD form. Methods: Plasma samples were collected from rats after the I.V. or P.O. administration of Rh2. The plasma Rh2 and PPD concentrations were determined using HPLC-MS. The transformation from Rh2 to PPD, its absorption, and elimination were integrated into the mechanism based pharmacokinetic model to describe the pharmacokinetics of Rh2 and PPD simultaneously at 10 mg/kg. The concentration data collected following a 20 mg/kg dose of Rh2 was used for model validation. Results: Following Rh2 administration, PPD exhibited high exposure and atypical double peaks. The model described the abnormal kinetics well and was further validated using external data. A total of 11% of the administered Rh2 was predicted to be transformed into PPD and enter the systemic circulation after I.V. administration, and a total of 20% of Rh2 was predicted to be absorbed into the systemic circulation in PPD form after P.O. administration of Rh2. Conclusion: The developed model provides a useful tool to quantitatively study the deglycosylation kinetics of Rh2 and thus, provides a valuable resource for future pharmacokinetic studies of glycosides with similar deglycosylation metabolism.

LC-MS-based metabolomics reveals metabolic changes in short- and long-term administration of Compound Danshen Dripping Pills against acute myocardial infarction in rats.

BACKGROUND: Mild and systematically improving multiple metabolic disorders was a focused view for Compound Danshen Dripping Pills playing synergistic effects through multiple components and multiple targets. The difference in overall therapeutic effects and endogenous metabolic regulation between short- and long-term administration was still unclear. PURPOSE: This study aimed to explore the difference in endogenous metabolic regulation between short- and long-term Compound Danshen Dripping Pills (CDDP) administration against acute myocardial infarction (AMI). METHODS: The model of AMI was induced by ligating the left anterior descending coronary artery. The cardiac protection effects of CDDP were investigated by echocardiography, 1- or 2-week were defined as short- and long-term based on desirable efficacy variability. The entire metabolic changes between short- and long-term administration of CDDP were profiled by UPLC-Q-TOF-MS. In addition, the metabolic regulatory network of CDDP administration against myocardial infarction rats was also compared with those of a typical chemical drug isosorbide 5-mononitrate (ISMN). RESULTS: After 1- or 2-week continuous oral administration, CDDP could significantly alleviate AMI-induced cardiac dysfunction. By using LC-MS-based metabolomics analyses, we systematically investigated the metabolic profiles of plasma and heart tissue samples at fixed exposure time-points (2 h, 24 h) from AMI rats with CDDP treatment. Most interestingly, global endogenous metabolic changes were observed in cardiac samples collected at different stages post consecutive CDDP administration, fluctuating at 2 and 24 h after 1 week but stabilizing after 2 weeks. The disrupted metabolic pathways such as glycerophospholipid, amino acids, fatty acids, and arachidonic acid metabolism were reconstructed after both short- and long-term CDDP treatment, while taurine and hypotaurine metabolism and purine metabolism contributed to the whole efficacy after long-term CDDP administration. CONCLUSION: Long-term CDDP treatment plays prolonged and stable efficacy against AMI compared with short-term treatment by specifically regulating purine and taurine and hypotaurine metabolism and systematically redressing metabolic disorders.

Exogenous glutathione exerts a therapeutic effect in ischemic stroke rats by interacting with intrastriatal dopamine.

We previously showed that oral administration of exogenous glutathione (GSH) exerted a direct and/or indirect therapeutic effect on ischemic stroke rats, but the underlying mechanisms remain elusive. In the current study, we conducted a quantitative proteomic analysis to explore the pathways mediating the therapeutic effect of GSH in cerebral ischemia/reperfusion (I/R) model rats. Rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion. The rats were treated with GSH (250 mg/kg, ig) or levodopa (L-dopa, 100 mg/kg, ig) plus carbidopa (10 mg/kg, ig). Neurologic deficits were assessed, and the rats were sacrificed at 24 h after cerebral I/R surgery to measure brain infarct sizes. We conducted a proteomic analysis of the lesion side striatum samples and found that tyrosine metabolism and dopaminergic synapse were involved in the occurrence of cerebral stroke and the therapeutic effect of GSH. Western blot assay revealed that tyrosine hydroxylase (TH) mediated the occurrence of I/R-induced ischemic stroke and the therapeutic effect of GSH. We analyzed the regulation of GSH on endogenous small molecule metabolites and showed that exogenous GSH had the most significant effect on intrastriatal dopamine (DA) in I/R model rats by promoting its synthesis and inhibiting its degradation. To further explore whether DA-related alterations were potential targets of GSH, we investigated the therapeutic effect of DA accumulation on ischemic brain injury. The combined administration of the precursor drugs of DA (L-dopa and carbidopa) significantly ameliorated neurological deficits, reduced infarct size, and oxidative stress, and decreased pro-inflammatory cytokines levels in the striatum of I/R injury rats. More interestingly, exogenous L-dopa/carbidopa could also greatly enhance the exposure of intracerebral GSH by upregulating GSH synthetases and enhancing homocysteine (HCY) levels in the striatum. Thus, administration of exogenous GSH exerts a therapeutic effect on ischemic stroke by increasing intrastriatal DA, and the accumulated DA can, in turn, enhance the exposure of GSH and its related substances, thus promoting the therapeutic effect of GSH.

Ginsenoside 20(S)-Rh2 promotes cellular pharmacokinetics and intracellular antibacterial activity of levofloxacin against Staphylococcus aureus through drug efflux inhibition and subcellular stabilization.

Intracellular Staphylococcus aureus (S. aureus) often causes clinical failure and relapse after antibiotic treatment. We previously found that 20(S)-ginsenoside Rh2 [20(S)-Rh2] enhanced the therapeutic effect of quinolones in a mouse model of peritonitis, which we attributed to the increased concentrations of quinolones within bacteria. In this study, we investigated the enhancing effect of 20(S)-Rh2 on levofloxacin (LVF) from a perspective of intracellular bacteria. In S. aureus 25923-infected mice, coadministration of LVF (1.5 mg/kg, i.v.) and 20(S)-Rh2 (25, 50 mg/kg, i.g.) markedly increased the survival rate, and decreased intracellular bacteria counts accompanied by increased accumulation of LVF in peritoneal macrophages. In addition, 20(S)-Rh2 (1, 5, 10 µM) dose-dependently increased the uptake and accumulation of LVF in peritoneal macrophages from infected mice without drug treatment. In a model of S. aureus 25923-infected THP-1 macrophages, we showed that 20(S)-Rh2 (1, 5, 10 µM) dose-dependently enhanced the intracellular antibacterial activity of LVF. At the cellular level, 20(S)-Rh2 increased the intracellular accumulation of LVF by inhibiting P-gp and BCRP. PK-PD modeling revealed that 20(S)-Rh2 altered the properties of the cell but not LVF. At the subcellular level, 20(S)-Rh2 did not increase the distribution of LVF in lysosomes but exhibited a stronger sensitizing effect in acidic environments. Molecular dynamics (MD) simulations showed that 20(S)-Rh2 improved the stability of the DNA gyrase-LVF complex in lysosome-like acidic conditions. In conclusion, 20(S)-Rh2 promotes the cellular pharmacokinetics and intracellular antibacterial activities of LVF against S. aureus through efflux transporter inhibition and subcellular stabilization, which is beneficial for infection treatment.

Silybin alleviates hepatic lipid accumulation in methionine-choline deficient diet-induced nonalcoholic fatty liver disease in mice via peroxisome proliferator-activated receptor α.

Nonalcoholic fatty liver disease (NAFLD) is regarded as the most common liver disease with no approved therapeutic drug currently. Silymarin, an extract from the seeds of Silybum marianum, has been used for centuries for the treatment of various liver diseases. Although the hepatoprotective effect of silybin against NAFLD is widely accepted, the underlying mechanism and therapeutic target remain unclear. In this study, NAFLD mice caused by methionine-choline deficient (MCD) diet were orally administrated with silybin to explore the possible mechanism and target. To clarify the contribution of peroxisome proliferator-activated receptor α (PPARα), PPARα antagonist GW6471 was co-administrated with silybin to NAFLD mice. Since silybin was proven as a PPARα partial agonist, the combined effect of silybin with PPARα agonist, fenofibrate, was then evaluated in NAFLD mice. Serum and liver samples were collected to analyze the pharmacological efficacy and expression of PPARα and its targets. As expected, silybin significantly protected mice from MCD-induced NAFLD. Furthermore, silybin reduced lipid accumulation via activating PPARα, inducing the expression of liver cytosolic fatty acid-binding protein, carnitine palmitoyltransferase (Cpt)-1a, Cpt-2, medium chain acyl-CoA dehydrogenase and stearoyl-CoA desaturase-1, and suppressing fatty acid synthase and acetyl-CoA carboxylase α. GW6471 abolished the effect of silybin on PPARα signal and hepatoprotective effect against NAFLD. Moreover, as a partial agonist for PPARα, silybin impaired the powerful lipid-lowering effect of fenofibrate when used together. Taken together, silybin protected mice against NAFLD via activating PPARα to diminish lipid accumulation and it is not suggested to simultaneously take silybin and classical PPARα agonists for NAFLD therapy.

Metabolic changes in the brain and blood of rats following acoustic trauma, tinnitus and hyperacusis.

It has been increasingly recognized that tinnitus is likely to be generated by complex network changes. Acoustic trauma that causes tinnitus induces significant changes in multiple metabolic pathways in the brain. However, it is not clear whether those metabolic changes in the brain could also be reflected in blood samples and whether metabolic changes could discriminate acoustic trauma, hyperacusis and tinnitus. We analyzed brain and serum metabolic changes in rats following acoustic trauma or a sham procedure using metabolomics. Hearing levels were recorded before and after acoustic trauma and behavioral measures to quantify tinnitus and hyperacusis were conducted at 4 weeks following acoustic trauma. Tissues from 11 different brain regions and serum samples were collected at about 3 months following acoustic trauma. Among the acoustic trauma animals, eight exhibited hyperacusis-like behavior and three exhibited tinnitus-like behavior. Using Gas chromatography-mass spectrometry and multivariate statistical analysis, significant metabolic changes were found in acoustic trauma animals in both the brain and serum samples with a number of metabolic pathways significantly perturbated. Furthermore, metabolic changes in the serum were able to differentiate sham from acoustic trauma animals, as well as sham from hyperacusis animals, with high accuracy. Our results suggest that serum metabolic profiling in combination with machine learning analysis may be a promising approach for identifying biomarkers for acoustic trauma, hyperacusis and potentially, tinnitus.

Cystine supplementation rebalances the redox homeostasis of microenvironment in non-small cell lung cancer cells and reverses their resistance to docetaxel.

Continuous docetaxel (DTX) treatment of non-small cell lung cancer induces development of drug resistance, but the mechanism is poorly understood. In this study we performed metabolomics analysis to characterize the metabolic patterns of sensitive and resistant A549 non-small cell lung cancer cells (A549/DTX cells). We showed that the sensitive and resistant A549 cells exhibited distinct metabolic phenotypes: the resistant cells were characterized by an altered microenvironment of redox homeostasis with reduced glutathione and elevated reactive oxygen species (ROS). DTX induction reprogrammed the metabolic phenotype of the sensitive cells, which acquired a phenotype similar to that of the resistant cells: it reduced cystine influx, inhibited glutathione biosynthesis, increased ROS and decreased glutathione/glutathione disulfide (GSH/GSSG); the genes involved in glutathione biosynthesis were dramatically depressed. Addition of the ROS-inducing agent Rosup (25, 50 µg/mL) significantly increased P-glycoprotein expression and reduced intracellular DTX in the sensitive A549 cells, which ultimately acquired a phenotype similar to that of the resistant cells. Supplementation of cystine (1.0 mM) significantly increased GSH synthesis, rebalanced the redox homeostasis of A549/DTX cells, and reversed DTX-induced upregulation of P-glycoprotein, and it markedly improved the effects of DTX and inhibited the growth of A549/DTX in vitro and in vivo. These results suggest that microenvironmental redox homeostasis plays a key role in the acquired resistance of A549 cancer cells to DTX. The enhancement of GSH synthesis by supplementary cystine is a promising strategy to reverse the resistance of tumor cells and has potential for translation in the clinic.

Technical Report: A New Device Attached to a Smartphone for Objective Vision Screening.

SIGNIFICANCE: A new device attached to a smartphone was created for objective vision screening of young children including infants and newborns. The device is compact, lightweight, portable, cost-effective, and easy to operate. Therefore, it is suitable for screening large numbers of children in clinical settings, schools, and communities. PURPOSE: This article introduces a new device attached to a smartphone for objective vision screening. It can detect and categorize significant refractive errors, anisometropia, strabismus, cloudy ocular media, and ptosis that may cause amblyopia. METHODS: The new device applies the same principles as conventional streak retinoscopy but examines both eyes simultaneously and records the results electronically. The device comprises optical elements that produce a precise streak light beam and move it across a child's both eyes. The smartphone's video camera catches and records the motion of retinal reflex inside the child's pupils. By observing the direction of motion of the retinal reflex relative to the light beam motion, as well as its speed, width, and brightness, the examiner is able to assess the individual and comparative refractive status, ocular alignment, and other conditions. RESULTS: Vision screening with this device does not require any subjective response from children. The examination can be performed and analyzed by nonprofessionals after a short learning period of time. Because the examination results are electronically recorded by the smartphone, they can be stored in the child's files and sent out for professional consultations. CONCLUSIONS: The new device will provide the same functions as conventional streak retinoscopy but examines a child's both eyes simultaneously, so that, in addition to categorizing refractive errors and assessing clarity of refractive media of the eyes, it can also detect anisometropia, strabismus, and anisocoria. In addition to showing the examination results on the smartphone's screen, the device can also store the results electronically.

Feasibility study of 68Ga-labeled CAR T cells for in vivo tracking using micro-positron emission tomography imaging.

Clinical tracking of chimeric antigen receptor (CAR) T cells in vivo by positron emission tomography (PET) imaging is an area of intense interest. But the long-lived positron emitter-labeled CAR T cells stay in the liver and spleen for days or even weeks. Thus, the excessive absorbed effective dose becomes a major biosafety issue leading it difficult for clinical translation. In this study we used 68Ga, a commercially available short-lived positron emitter, to label CAR T cells for noninvasive cell tracking in vivo. CAR T cells could be tracked in vivo by 68Ga-PET imaging for at least 6 h. We showed a significant correlation between the distribution of 89Zr and 68Ga-labeled CAR T cells in the same tissues (lungs, liver, and spleen). The distribution and homing behavior of CAR T cells at the early period is highly correlated with the long-term fate of CAR T cells in vivo. And the effective absorbed dose of 68Ga-labeled CAR T cells is only one twenty-fourth of 89Zr-labeled CAR T cells, which was safe for clinical translation. We conclude the feasibility of 68Ga instead of 89Zr directly labeling CAR T cells for noninvasive tracking of the cells in vivo at an early stage based on PET imaging. This method provides a potential solution to the emerging need for safe and practical PET tracer for cell tracking clinically.

Mechanisms and pharmacokinetic/pharmacodynamic profiles underlying the low nephrotoxicity and ototoxicity of etimicin.

Etimicin (ETM), a fourth-generation aminoglycosides (AGs), is now widely clinically used in China due to its high efficacy and low toxicity. However, the mechanisms underlying its low nephrotoxicity and ototoxicity remain unclear. In the present study we compared the antibacterial and toxicity profiles of etimicin, gentamicin (GM, a second-generation AG), and amikacin (AMK, a third-generation AG), and investigated their pharmacokinetic properties in the toxicity target organs (kidney and inner ear) and subcellular compartments. We first demonstrated that ETM exhibited superior antibacterial activities against clinical isolates to GM and AMK, and it exerted minimal nephrotoxicity and ototoxicity in rats following multi-dose administration. Then, we conducted pharmacokinetic studies in rats, showed that the three AGs accumulated in the kidney and inner ear with ETM being distributed to a lesser degree in the two toxicity target organs as compared with GM and AMK high-dose groups. Furthermore, we conducted in vitro experiments in NRK-52E rat renal tubular epithelial cells and HEI-OC1 cochlear hair cells, and revealed that all the three AGs were distributed predominantly in the mitochondria with ETM showing minimal accumulation; they not only directly inhibited the activity of mitochondrial complexes IV and V but also inhibited mitochondrial function and its related PGC-1α-NRF1-TFAM pathway; ETM caused minimal damage to the mitochondrial complex and mitochondrial biogenesis. Our results demonstrate that the minimal otonephrotoxicity of ETM results from its lesser accumulation in mitochondria of target cells and subsequently lesser inhibition of mitochondrial function. These results provide a new strategy for discovering novel AGs with high efficacy and low toxicity.

Leflunomide increased the renal exposure of acyclovir by inhibiting OAT1/3 and MRP2.

Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (LEF) and the antiviral drug acyclovir to reduce the high risk of infection. Acyclovir is a substrate of organic anion transporter (OAT) 1/3 and multidrug resistance-associated protein (MRP) 2. Considering the extraordinarily long half-life of LEF's active metabolite teriflunomide (TER) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir. Here we used a specific MRP inhibitor MK571 and probenecid (OAT1/3 and MRP2 inhibitor) to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. We showed that LEF and probenecid, but not MK571 significantly increased the plasma concentration of acyclovir. However, kidney and liver exposures of acyclovir were increased when coadministered with LEF, probenecid or MK571. The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid, whereas it was increased to as much as 14.5-fold by MK571. Consistently, these drugs markedly decreased the urinary excretion of acyclovir. TER (0.5-100 µmol/L) dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC50 value of 4.91 µmol/L. TER (5 µmol/L) significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells. These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2, which increased its kidney/plasma ratio and renal injury risk. However, the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells' uptake of acyclovir and increased the plasma concentration.

Rebalancing of the gut flora and microbial metabolism is responsible for the anti-arthritis effect of kaempferol.

Kaempferol is a natural flavonol that possesses various pharmacological activities, including anti-arthritis effects, yet the underlying mechanisms remain controversial. To evaluate the anti-arthritis efficacy and the underlying mechanisms of kaempferol, collagen-induced arthritis (CIA) mice were treated with kaempferol intragastrically (200 mg · kg-1 · d-1) and intraperitoneally (20 mg · kg-1 · d-1). Pharmacodynamic and pharmacokinetic studies showed that the oral administration of kaempferol produced distinct anti-arthritis effects in model mice with arthritis in terms of the spleen index, arthritis index, paw thickness, and inflammatory factors; the bioavailability (1.5%, relative to that of the intraperitoneal injection) and circulatory exposure of kaempferol (Cmax = 0.23 ± 0.06 ng/mL) and its primary metabolite kaempferol-3-O-glucuronide (Cmax = 233.29 ± 89.64 ng/mL) were rather low. In contrast, the intraperitoneal injection of kaempferol caused marginal anti-arthritis effects, although it achieved a much higher in vivo exposure. The much higher kaempferol content in the gut implicated a potential mechanism involved in the gut. Analysis of 16S ribosomal RNA revealed that CIA caused imbalance of 14 types of bacteria at the family level, whereas kaempferol largely rebalanced the intestinal microbiota in CIA mice. A metabolomics study showed that kaempferol treatment significantly reversed the perturbation of metabolites involved in energy production and the tryptophan, fatty acid and secondary bile acid metabolisms in the gut contents of the CIA mice. In conclusion, we demonstrate for the first time that the high level of kaempferol in the gut regulates the intestinal flora and microbiotic metabolism, which are potentially responsible for the anti-arthritis activities of kaempferol.

A hepatoprotection study of Radix Bupleuri on acetaminophen-induced liver injury based on CYP450 inhibition.

We investigated the potential hepatoprotective effect of Radix Bupleuri (RB) by inducing acute liver injury (ALI) in an animal model using acetaminophen (APAP) after pretreatment with RB aqueous extract for three consecutive days. Compared to those of the APAP group, the biochemical and histological results of the RB pretreatment group showed lower serumaspartate transaminase (AST) and alanine transaminase (ALT) levels as well as less liver damage. Pharmacokinetic study of the toxicity related marker acetaminophen-cysteine (APC) revealed a lower exposure level in rats, suggesting that RB alleviated APAP-induced liver damage by preventing glutathione (GSH) depletion. The results of cocktail approach showed significant inhibition of CYP2E1 and CYP3A activity. Further investigation revealed the increasing of CYP2E1 and CYP3A protein was significantly inhibited in pretreatment group, while no obvious effect on gene expression was found. Therefore, this study clearly demonstrates that RB exhibited significant protective action against APAP-induced acute live injury via pretreatment, and which is partly through inhibiting the increase of activity and translation of cytochrome P450 enzymes, rather than gene transcription.

Compound danshen dripping pills normalize a reprogrammed metabolism of myocardial ischemia rats to interpret its time-dependent efficacy in clinic trials: a metabolomic study.

INTRODUCTION: Clinical trials of Compound danshen dripping pills (CDDP) indicated distinct improvement in patients with chronic stable angina. Daily fluctuation of therapeutic effect agreed with a peak-valley PK profile during a 4-week CDDP regimen, but stabilized after 8-week treatment. OBJECTIVES: This article aims to explore the underlying mechanism for the time-dependent drug efficacy of the up-down fluctuation or stabilization in clinic trials. METHODS: A rat model of myocardial ischemia was established via isoproterenol induction. Metabolomics was employed to analyze the energy-related substances both in circulatory system and myocardium in the myocardial ischemia model. RESULTS: CDDP treatment ameliorated myocardial ischemia, reversed the reprogramming of the metabolism induced by ISO and normalized the level of most myocardial substrates and the genes/enzymes associated with those metabolic changes. After 1- or 2-week treatment, CDDP regulated plasma and myocardial metabolome in an analogous, time-dependent way, and modulated metabolic patterns of ischemic rats that perfectly matched with the fluctuated or stabilized effects observed in clinical trials with 4 or 8-week treatment, respectively. CONCLUSION: Metabolic modulation by CDDP contributes to the fluctuated or stabilized therapeutic outcome, and is a potential therapeutic approach for myocardial ischemia diseases.

The Value of the CHADS2 and CHA2DS2-VASc Score for Predicting the Prognosis in Lacunar Stroke with or without Atrial Fibrillation Patients.

BACKGROUND: The CHADS2 and CHA2DS2-VASc scoring systems have been proved efficacy to stratify stroke and thromboembolism risk in patients with atrial fibrillation (AF). Whether CHADS2 and CHA2DS2-VASc score has predictive value for the prognosis in lacunar stroke (LS) patients remains unclear. METHODS: A total of 763 consecutive patients with LS (mean age: 66 ± 12 years; 464 male) were enrolled in this study between January 2013 and December 2014. Patients were divided into LS without AF (LS; n = 679) and LS with AF (LS-AF; n = 84) groups. Measures of performance for the risk scores were evaluated at predicting mortality and restroke in LS-AF and LS without AF patients. All patients were evaluated with respect to clinical features and in-hospital clinical results. RESULTS: During the mean follow-up period of 20 ± 5.8 months, 29 patients (3.8%) experienced all-cause death, 105 patients (13.8%) experienced recurrence of ischemic stroke. Multivariate analysis revealed that CHADS2 and CHA2DS2-VASc score were independently associated with all-cause death (all P < .05). On receiver operating characteristic curve analysis, area under the curve (AUC) for CHADS2 score was .942 with a similar accuracy of the CHA2DS2-VASc score (AUC: .908) in predicting mortality in LS-AF patients. Kaplan-Meier curves were conducted according to the cut-off value of CHA2DS2-VASc score. When CHADS2 score greater than or equal to 4 point or CHA2DS2-VASc score greater than or equal to 5 point, the mortality in LS-AF patients was significantly higher compared with those CHADS2 score less than 4 point or CHA2DS2-VASc score less than 5 point. However, after adjusting for clinical covariates, CHADS2 and CHA2DS2-VASc score could not predict both mortality and restroke in LS without AF patients. CONCLUSIONS: The CHADS2 and CHA2DS2-VASc score have excellent predictive value for mortality in LS-AF patients but could not predict both mortality and restroke in LS without AF patients.

Paeoniflorin inhibits Th1 and Th17 cells in gut-associated lymphoid tissues to produce anti-arthritis activities.

Paeoniflorin shows distinct anti-arthritis and immunoregulatory activities, but its rather low bioavailability via oral administration greatly challenges its known mechanism of in vivo activity. Our data showed that oral administration, instead of intraperitoneal injection, of paeoniflorin significantly reduced the polyarthritis index by 44.4%, reduced paw swelling by 18.4% and delayed the onset of arthritis in collagen-induced arthritis (CIA) mice. Oral paeoniflorin treatment also downregulated the systemic pro-inflammatory cytokines IL-6 (by 52.2%), TNF-α (by 57.7%) and IL-1ß (by 34.1%). A pharmacokinetic study revealed that the maximal plasma concentration of paeoniflorin after oral administration was 4.8 ± 1.9 µM in the CIA mice, much lower than the effective concentration in vitro (30 µM). In contrast, paeoniflorin was highly concentrated in the gut content, intestine and Peyer's patches. T cell analysis showed that paeoniflorin markedly reduced transcription factors of Th1 and Th17, inhibited Th1 by 22.2% and 23.1% and Th17 by 43.2% and 25.4% (p < 0.05) in the mesenteric lymph node and Peyer's patches, respectively. Paeoniflorin did not have a significant impact on Th1 and Th17 in the spleen. For the first time, these data suggest that paeoniflorin accumulates in the intestine and primarily modulates Th1 and Th17 responses in the mesenteric lymph nodes and Peyer's patches, rather than in the spleen, to exert anti-arthritis effects.

Myeloid differentiation protein 1 protected myocardial function against high-fat stimulation induced pathological remodelling.

Myeloid differentiation 1 (MD-1) is a secreted protein that regulates the immune response of B cell through interacting with radioprotective 105 (RP105). Disrupted immune response may contribute to the development of cardiac diseases, while the roles of MD-1 remain elusive. Our studies aimed to explore the functions and molecular mechanisms of MD-1 in obesity-induced cardiomyopathy. H9C2 myocardial cells were treated with free fatty acid (FFA) containing palmitic acid and oleic acid to challenge high-fat stimulation and adenoviruses harbouring human MD-1 coding sequences or shRNA for MD-1 overexpression or knockdown in vitro. MD-1 overexpression or knockdown transgenic mice were generated to assess the effects of MD-1 on high-fat diet (HD) induced cardiomyopathy in vivo. Our results showed that MD-1 was down-regulated in H9C2 cells exposed to FFA stimulation for 48 hours and in obesity mice induced by HD for 20 weeks. Both in vivo and in vitro, silencing of MD-1 accelerated myocardial function injury induced by HD stimulation through increased cardiac hypertrophy and fibrosis, while overexpression of MD-1 alleviated the effects of HD by inhibiting the process of cardiac remodelling. Moreover, the MAPK and NF-κB pathways were overactivated in MD-1 deficient mice and H9C2 cells after high-fat treatment. Inhibition of MAPK and NF-κB pathways played a cardioprotective role against the adverse effects of MD-1 silencing on high-fat stimulation induced pathological remodelling. In conclusion, MD-1 protected myocardial function against high-fat stimulation induced cardiac pathological remodelling through negative regulation for MAPK/NF-κB signalling pathways, providing feasible strategies for obesity cardiomyopathy.