Interface-constrained catalytic hairpin assembly permits highly sensitive SERS signaling of miRNA.

The rapid and precise monitoring of peripheral blood miRNA levels holds paramount importance for disease diagnosis and treatment monitoring. In this study, we propose an innovative research strategy that combines the catalytic hairpin assembly reaction with SERS signal congregation and enhancement. This combination can significantly enhance the stability of SERS detection, enabling stable and efficient detection of miRNA. Specifically, our paper-based SERS detection platform incorporates a streptavidin-modified substrate, biotin-labeled catalytic hairpin assembly reaction probes, 4-ATP, and primer-co-modified gold nanoparticles. In the presence of miRNA, the 4-ATP and primer-co-modified gold nanoparticles can specifically recognize the miRNA and interact with the biotin-labeled CHA probes to initiate an interfacial catalytic hairpin assembly reaction. This enzyme-free high-efficiency catalytic process can accumulate a large amount of biotin on the gold nanoparticles, which then bind to the streptavidin on the substrate with the assistance of the driving liquid, forming red gold nanoparticle stripes. These provide a multitude of hotspots for SERS, enabling enhanced signal detection. This innovative design achieves a low detection limit of 3.47 fM while maintaining excellent stability and repeatability. This conceptually innovative detection platform offers new technological possibilities and solutions for clinical miRNA detection.

Triptolide Reduces Neoplastic Progression in Hepatocellular Carcinoma by Downregulating the Lipid Lipase Signaling Pathway.

Hepatocellular carcinoma (HCC), which is the third leading cause of cancer-related mortality in the world, presents a significant medical challenge. Triptolide (TP) has been identified as an effective therapeutic drug for HCC. However, its precise therapeutic mechanism is still unknown. Understanding the mechanism of action of TP against HCC is crucial for its implementation in the field of HCC treatment. We hypothesize that the anti-HCC actions of TP might be related to its modulation of HCC lipid metabolism given the crucial role that lipid metabolism plays in promoting the progression of HCC. In this work, we first demonstrate that, both in vitro and in vivo, TP significantly reduces lipid accumulation in HCC cells. Additionally, we notice that lipoprotein lipase (LPL) expression is markedly upregulated in HCC, and that its levels are positively connected with the disease's progression. It is interesting to note that TP dramatically reduces LPL activity, which in turn prevents HCC growth and reduces lipid accumulation. Additionally, the effect of TP on LPL is a direct correlation. These results definitely demonstrate that TP protects hepatocytes against abnormal accumulation of lipids by transcriptionally suppressing LPL, which reduces the development of HCC. This newly identified pathway provides insight into the process through which TP exerts its anti-HCC actions.

Corrigendum: Impact of intestinal microbiota on metabolic toxicity and potential detoxification of amygdalin.

[This corrects the article DOI: 10.3389/fmicb.2022.1030516.].

CRISPR/Cas9-based point-of-care lateral flow biosensor with improved performance for rapid and robust detection of Mycoplasma pneumonia.

Screening of acute respiratory infections causes serious challenges in urgent point-of-care scenarios where conventional methods are impractical and alternative techniques suffer from low accuracy, poor robustness, and reliance on sophisticated instruments. As an improvement to this paradigm, we report a point-of-care lateral flow biosensor (LFB) based on the recognition property of clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (Cas9) and apply it to the detection of Mycoplasma pneumoniae (M. pneumoniae). The designed biosensor employs CRISPR/Cas9 for secondary recognition after preamplification of target gene using specific primer set, avoiding false positives caused by nontarget factors. The high amplification efficiency and low applicable temperatures of recombinase polymerase amplification brings the detection limit of the biosensor to 3 copies even at a preamplification temperature of 25 °C. Its practical application is further demonstrated with 100% accuracy by testing with 43 M. pneumoniae-infected specimens and 80 uninfected specimens. Additionally, the entire detection, including pretreatment, preamplification, CRISPR/Cas9 recognition, and visual analysis, can be completed in 30 min. Featured with the combination of CRISPR/Cas9 and LFB, the biosensor we developed herein ensures excellent convenience, accuracy, and robustness, which endows promising point-of-care screening potential for infectious pathogens.

Gut microbiota and host cytochrome P450 characteristics in the pseudo germ-free model: co-contributors to a diverse metabolic landscape.

BACKGROUND: The pseudo germ-free (PGF) model has been widely used to research the role of intestinal microbiota in drug metabolism and efficacy, while the modelling methods and the utilization of the PGF model are still not standardized and unified. A comprehensive and systematic research of the PGF model on the composition and function of the intestinal microbiota, changes in host cytochrome P450 (CYP450) enzymes expression and intestinal mucosal permeability in four different modelling cycles of the PGF groups are provided in this paper. RESULTS: 16S rRNA gene amplicon sequencing was employed to compare and analyze the alpha and beta diversity, taxonomic composition, taxonomic indicators and predicted function of gut microbiota in the control and PGF groups. Bacterial richness and diversity decreased significantly in the PGF group beginning after the first week of establishment of the PGF model with antibiotic exposure. The PGF group exposed to antibiotics for 4-week-modelling possessed the fewest indicator genera. Moreover, increased intestinal mucosal permeability occurred in the second week of PGF model establishment, indicating that one week of antibiotic exposure is an appropriate time to establish the PGF model. The results of immunoblots revealed that CYP1A2, CYP2C19 and CYP2E1 expression was significantly upregulated in the PGF group compared with the control group, implying that the metabolic clearance of related drugs would change accordingly. The abundance of functional pathways predicted in the gut microbiota changed dramatically between the control and PGF groups. CONCLUSIONS: This study provides information concerning the microbial and CYP450 enzyme expression profiles as a reference for evaluating drug metabolism differences co-affected by gut microbiota and host CYP450 enzymes in the PGF model.

Impact of intestinal microbiota on metabolic toxicity and potential detoxification of amygdalin.

Amygdalin (Amy) is metabolized into cyanide in vivo, which may lead to fatal poisoning after oral administration. The defense mechanisms against toxic cyanide have not yet been adequately studied. In this study, comparative toxicokinetics study of Amy was performed in normal and pseudo germ-free rats. The efficiency of cyanide release was significant higher in normal group when given a single oral dose of 440 mg/kg (50% median lethal dose). Thiocyanate, the detoxification metabolite, was firstly detected in feces, caecum, and intestinal microbiota incubation enzymic system. The results suggest intestinal microbiota is involved in bidirectional regulation of toxicity and detoxification of Amy. We further identified the species related to cyanogenesis of Amy with metagenomic sequencing, such as Bifidobacterium pseudolongum, Marvinbryantia formatexigens, and Bacteroides fragilis. Functional analysis of microbiota reveals the detoxification potential of intestinal microbiota for cyanide. Sulfurtransferase superfamily, such as rhodanese, considered as main detoxification enzymes for cyanide, are largely found in Coriobacteriaceae bacterium, Butyricicoccus porcorum, Akkermansia muciniphila, etc. Besides, cyanoamino acid metabolism pathway dominated by Escherichia coli may contribute to the detoxification metabolism of cyanide. In summary, intestinal microbiota may be the first line of defense against the toxicity induced by Amy.

An Open-Label, Randomized Pharmacokinetic Study of 2 Formulations of Eldecalcitol Capsules Following Single Oral Administration in Healthy Chinese Volunteers Under Fasting and Fed Conditions.

Eldecalcitol is an active vitamin D3 derivative that is used for the prevention and treatment of osteoporosis. The objectives of this study were to evaluate the bioequivalence and safety of 2 formulations of eldecalcitol capsule (0.75 µg) in healthy Chinese male and female volunteers, as well as to investigate the food effect on the pharmacokinetics of this drug. An open label, randomized, 3-period, 3-sequence, reference replicated crossover clinical study was performed in 27 healthy Chinese volunteers under fasting conditions, while a 2-way crossover study was carried out in 28 healthy Chinese volunteers under fed conditions. Volunteers were administered a single oral dose of 0.75 µg eldecalcitol after fasting overnight. Blood samples were collected at scheduled time points from 0 to 168 hours after administration of eldecalcitol. The 90%CIs of the test/reference geometric mean ratio (area under the plasma concentration-time curve and maximum plasma concentration) of eldecalcitol after a single-dose administration were within the acceptance criteria based on the average bioequivalence method. The time to maximum concentration of the test and reference formulations were elevated by ≈2.3-fold and 1.7-fold, respectively, after a high-fat meal. Only mild and transient adverse events were reported in this study, and no severe adverse events occurred. These results indicated that the 2 formulations of eldecalcitol were bioequivalent under both fasting and fed conditions. Food intake prolonged the oral absorption of eldecalcitol but did not significantly influence systemic exposure.

Intestinal microbiota-mediated biotransformations alter the pharmacokinetics of the major metabolites of azathioprine in rats after oral administration.

Adverse reactions to azathioprine (AZA) vary greatly among individuals, which is associated with the variable levels of its major metabolites 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). The intestinal microbiota has been proven to contain AZA-metabolizing enzymes, although the explicit role of the intestinal microbiota in AZA metabolism in vivo remains poorly comprehended. In this study, the pharmacokinetic behaviours of 6-TGN and 6-MMP were assessed in the pseudo germ-free (PGF) group and control group following oral administration of AZA. The AUC0-t and Cmax of 6-TGN in the PGF group were significantly decreased by 34.0% and 35.0% (P < 0.05) compared with those in the control group. Additionally, the AUC0-t and Cmax of 6-MMP were reduced by 27.9% and 34.2% in the PGF group, although the differences were not significant. The TPMT and NUDT15 genotypes of rats in the two groups were genetically identical. The expression levels of key AZA-metabolizing enzymes in liver were not different between two groups. Furthermore, the major metabolites of AZA in the incubation system with intestinal microbial enzymes were identified. In summary, shifts in the composition of the intestinal microbiota may regulate the exposure of 6-TGN in vivo by altering the gut microbial metabolism of AZA.

Engineered G-Quadruplex-Embedded Self-Quenching Probes Regulate Single Probe-Based Multiplex Isothermal Amplification to Light Road Lamp Probes for Sensitized Determination of microRNAs.

Design of oligonucleotide probe-based isothermal amplification with the ability to identify miRNA biomarkers is crucial for molecular diagnostics. In this work, we engineered a miRNA-21-responsive G-quadruplex-embedded self-quenching probe (GE-SQP) that can regulate single probe-based multiplex amplifications. The free GE-SQP is tightly locked in a quenching state with no active G-quadruplexes. Introduction of target miRNA to hybridize with GE-SQP would induce a multiplex isothermal amplification to significantly build a lot of one-bulb-contained road lamp probe (OC-RLP) and two-bulb-contained road lamp probe (TC-RLP) using G-quadruplex as the lamp bulb. When lightened by thioflavin T (ThT), beams of fluorescence were emitted to show the presented miRNA-21. Specially, the whole amplification is only a one probe-involved one-step reaction without any wasted species. The mix-to-detection and all-in amplification behavior allows the sensing system a maximally maintained operation simplicity and high assay performance. In such a way, the detection range of miRNA-21 is from 1 fM to 1 nM with a limit of detection of 0.86 fM. The practicability was demonstrated by determining miRNA-21 from serum samples with acceptable results. We expect that this method can open a new avenue for exploring advanced biosensors with improved analytical performances.

Interactions Between Ephedra sinica and Prunus armeniaca: From Stereoselectivity to Deamination as a Metabolic Detoxification Mechanism of Amygdalin.

Mahuang-Xingren (MX, Ephedra sinica Stapf-Prunus armeniaca L.) is a classic herb pair used in traditional Chinese medicine. This combined preparation reduces the toxicity of Xingren through the stereoselective metabolism of its main active ingredient amygdalin. However, whether stereoselectivity is important in the pharmacokinetic properties of amygdalin either in the traditional decoction or in the dispensing granules is unclear. Amygdalin is hydrolyzed to its metabolite, prunasin, which produces hydrogen cyanide by degradation of the cyano group. A comprehensive study of the metabolic pathway of amygdalin is essential to better understand the detoxification process. In this article, the potential detoxification pathway of MX is further discussed with regard to herb interactions. In this study, the pharmacokinetic parameters and metabolism of amygdalin and prunasin were investigated by comparing the traditional decoction and the dispensing granule preparations. In addition, several potential metabolites were characterized in an incubation system with rat liver microsomes or gut microbial enzymes. The combination of Xingren with Mahuang reduces exposure to D-amygdalin in vivo and contributes to its detoxification, a process that can be further facilitated in the traditional decoction. From the in vitro co-incubation model, 15 metabolites were identified and classified into cyanogenesis and non-cyanogenesis metabolic pathways, and of these, 10 metabolites were described for the first time. The level of detoxified metabolites in the MX traditional decoction was higher than that in the dispensing granules. The metabolism of amygdalin by the gut microbial enzymes occurred more rapidly than that by the rat liver microsomes. These results indicated that combined boiling both herbs during the preparation of the traditional decoction may induce several chemical changes that will influence drug metabolism in vivo. The gut microbiota may play a critical role in amygdalin metabolism. In conclusion, detoxification of MX may result 1) during the preparation of the decoction, in the boiling phase, and 2) from the metabolic pathways activated in vivo. Stereoselective pharmacokinetics and deamination metabolism have been proposed as the detoxification pathway underlying the compatibility of MX. Metabolic detoxification of amygdalin was quite different between the two combinations, which indicates that the MX decoctions should not be completely replaced by their dispensing granules.

Evaluation of MTBH, a novel hesperetin derivative, on the activity of hepatic cytochrome P450 isoform in vitro and in vivo using a cocktail method by HPLC-MS/MS.

1. 8-methylene-tert-butylamine-3',5,7-trihydroxy-4'-methoxyflavanone (MTBH), a novel hesperidin derivative, has potential in the prevention of hepatic disease, however, its effects on cytochrome P450 isoforms (CYP450s) remains unexplored. The purpose was to investigate the effects of MTBH on the mRNA, protein levels, and activities of six CYP450s (1A2, 2C11/9, 2D2/6, 3A1/4, 2C13/19, and 2E1) in vitro and in vivo.2. In vitro study, rat and human liver microsomes were adopted to elucidate the inhibitory effect of MTBH on six CYP450s using probe drugs. In vivo study, Sprague-Dawley male rats were treated with MTBH (25, 50, or 100 mg/kg for 28 consecutive days), phenobarbital (80 mg/kg for 12 consecutive days), or 0.5% CMC-Na solution (control group) by intragastric administration, then, the mRNA, protein levels and activities of liver CYP450s were analysed by real-time PCR, western blotting and probe-drug incubation systems, respectively.3. The in vitro study indicated that MTBH inhibits the activities of CYP3A1/4 and CYP2E1 in rat and human liver microsomes. In vivo data showed that MTBH inhibits mRNA, protein levels, and activities of CYP3A1 and CYP2E1 in medium- and high-dose MTBH groups.4. MTBH has the potential to cause drug-drug interactions when co-administered with drugs that are metabolised by CYP3A1/4 and CYP2E1.

Corrigendum: Interactions Between Ephedra sinica and Prunus armeniaca: From Stereoselectivity to Deamination as a Metabolic Detoxification Mechanism of Amygdalin.

[This corrects the article DOI: 10.3389/fphar.2021.744624.].

The permeability characteristics and interaction of main components from Si-Ni-San in a MDCK epithelial cell monolayer model.

1. Si-Ni-San (SNS) possesses extensive therapeutic effects, however, the extent to which main components are absorbed and the mechanisms involved are controversial. 2. In this study, MDCK cell model was used to determine the permeability characteristics and interaction between the major components of Si-Ni-San, including saikosaponin a, paeoniflorin, naringin and glycyrrhizic acid. 3. The transport of the major components was concentration-dependent in both directions. Moreover, the transport of paeoniflorin, naringin and glycyrrhizic acid was significantly reduced at 4 °C or in the presence of NaN3. Additionally, the efflux of paeoniflorin and naringin were apparently reduced in the presence of P-gp inhibitor verapamil. The transport of glycyrrhizic acid was clearly inhibited by the inhibitors of MRP2, indicating that MRP2 may be involved in the transport of glycyrrhizic acid. However, the results indicated that saikosaponin a was absorbed mainly by passive diffusion. Furthermore, the combined incubation of four major components had a powerful sorbefacient effect than a single drug used alone which may be regulated by tight junctions. 4. Taken together, our study provides useful information for pharmacological applications of Si-Ni-San and offers new insights into this ancient decoction for further researches, especially in drug synergism.

Determination of thiorphan, a racecadotril metabolite, in human plasma by LC-MS/MS and its application to a bioequivalence study in Chinese subjects
.

OBJECTIVE: The objective of this study was to use LC-MS/MS to compare the pharmacodynamic properties and bioequivalence of two 200-mg formulations of racecadotril: suspension formulation (test) and granule formulation (reference) in healthy Chinese subjects. MATERIALS AND METHODS: A single-dose, randomized, two-period crossover study was conducted in fasted healthy Chinese subjects, who received a single oral dose of the test or reference formulation, followed by a 7-day washout period and administration of the alternate formulation. RESULTS: The rapid and highly sensitive LC-MS/MS method exhibited a reasonable linearity range (2.324 - 952.000 ng/mL) and high sensitivity (LLOQ of 2.324 ng/mL). The within- and between-run precision, accuracy, and stability results were within the acceptable limits, and no matrix effect was observed. The 90% CI of the ratio of geometric means for AUC0-t, AUC0-∞, and Cmax were 88.1 - 102.3%, 87.9 - 101.5% and 99.5 - 113%, respectively, which met the regulatory criteria for bioequivalence. CONCLUSION: The method is suitable for quantification of thiorphan in human plasma. In addition, the results indicated that the test and reference formulations were bioequivalent in terms of both rate and extent of absorption.

A sensitive LC-MS/MS method to determine ginkgolide B in human plasma and urine: application in a pharmacokinetics and excretion study of healthy Chinese subjects.

1. Ginkgolide B (GB), the most active of the ginkgolides, has been developed as a new drug for the treatment of vascular insufficiency; however, the pharmacokinetics of GB remain unclear. Here, we investigated the pharmacokinetics and urine excretion properties of GB in healthy Chinese subjects administered single- and multiple-dose injectable GB based on a new LC-MS/MS method.2. GB pharmacokinetics were found to be dose-dependent from 20 to 60 mg. GB reached a steady state by day 6 with once-daily dosing at 40 mg. Systemic exposure to GB, as characterised by AUC0-∞, indicated accumulation following repeated once-daily dosing for seven consecutive days. The mean urinary cumulative excretion rate of GB in response to 20, 40, and 60 mg GB was 41.9 ± 18.5%, 32.9 ± 12.2%, and 43.9 ± 8.5%, respectively.3. Dose-proportional pharmacokinetics of GB were observed after intravenous administration in healthy subjects. A gradual reduction in the volume of distribution and slight change in mean resistance time led us to conjecture the limited accumulation of GB based on distribution equilibrium in vivo.4. This comprehensive study of the clinical pharmacokinetics of GB will provide useful information for its application and further development.

Determination of lacidipine in human plasma by LC-MS/MS: Application in a bioequivalence study
.

OBJECTIVE: This study aimed to conduct a sensitive, simple, and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of lacidipine in human plasma. MATERIALS AND METHODS: In this method, the plasma samples were extracted from human plasma using methanol as the precipitant and nisoldipine as internal standard (IS). The analytes were separated on a Phenomenex Luna C18 column (150 mm × 2.0 mm, 3 µm) at 40 °C using isocratic mobile phase consisting of 0.2% formic acid-methanol (13 : 87, v/v) at a flow rate of 0.2 mL/min. The tandem mass detection was constructed on a triple-quadrupole tandem mass spectrometer with an electrospray ionization (ESI) source operating in positive-ion mode. The selected reaction monitoring of transitions was m/z 456.2 → 354.2 for lacidipine and m/z 389.2 → 315.0 for IS, respectively. Then, the established method was applied in a bioequivalence study comparing lacidipine dispersible tablet with commercial tablet in 20 healthy Chinese subjects. RESULTS: The calibration curve exhibited great linearity in the range of 0.10 - 10.00 ng/mL (r2 = 0.999). The intraday and interday precision and accuracy met the acceptance criteria, and no matrix effect was found. In addition, the 90% confidence intervals for the test/reference ratio of Cmax, AUC0-24, and AUC0-∞ fell within the bioequivalence acceptance criteria (80 - 125%). CONCLUSION: The method is suitable for quantification of lacidipine in human plasma. Moreover, the two preparations are bioequivalent.
.

Aspirin suppresses TNF-α-induced MMP-9 expression via NF-κB and MAPK signaling pathways in RAW264.7 cells.

Numerous studies have indicated that the expression of matrix metalloproteinase-9 (MMP-9) contributes to the atherosclerotic plaque hemorrhage and rupture. Aspirin, a non-steroidal anti-inflammation drug, has been known for its anti-platelet effect in the prevention of the vascular complications of atherosclerosis. The present study aimed to investigate the pharmacological effects of aspirin on tumor necrosis factor-α (TNF-α)-induced MMP-9 expression and the underlying molecular mechanisms in murine macrophage RAW264.7 cells. Western blot analysis indicated that the protein level of MMP-9 was reduced by aspirin in a dose-dependent manner. In addition, downregulation of MMP-9 mRNA and activity were detected in aspirin-treated cells using quantitative polymerase chain reaction and a gelatin zymography assay separately. It was also observed that aspirin has a suppressive effect on the activation of nuclear factor (NF)-κB and inhibits the phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases 1/2, p38 and c-Jun N-terminal kinase. Furthermore, subsequent to inhibition of the MAPK pathway by specific inhibitors (PD98059, SB203580 and SP600125), the expression of MMP-9 was reduced, indicating that the inhibitory effect of aspirin on MMP-9 in TNF-α-treated RAW264.7 cells may be, at least in part, through suppression of NF-κB activation and the MAPK pathway. These findings support the notion that aspirin has therapeutic potential application in the prevention and treatment of atherosclerosis disease.

Comparison of tranexamic acid pharmacokinetics after intra-articular and intravenous administration in rabbits.

Tranexamic Acid (TXA) is commonly administered in total knee arthroplasty for reducing blood loss. There has been a growing interest in the topical use of TXA except intravenous use for prevention of bleeding in TKA. The aim of this study was to develop and validate a HPLC-MS method to detect TXA and apply to compare the pharmacokinetic profile of TXA after intravenous (IV) and topical intra-articular (IA) application of TXA at a dose of 20 mg/kg in rabbits. In order to prove intra-articular administration is better than that of intravenous administration from the point of rabbit pharmacokinetic. Two groups of rabbits (n=6/group) respectively received TXA intra-articularly or intravenously. Blood samples were collected at scheduled time. The concentration of TXA in plasma was determined by a validated HPLC-MS method. Excellent linearity was found between 0.015 and 70.0µg/ml with a lower limit of quantitation (LLOQ) of 0.015µg/ml (r>0.99); moreover, all the validation data including accuracy and precision (intra- and inter-day) were all within the required limits. The pharmacokinetic parameters in IA and IV group were: Cmax: 30.65±3.31 VS 54.05± 6.21µg/ml (p<0.01); t1/2: 1.26±0.05 VS 0.68±0.13h (p<0.05); AUC0-t: 42.98±7.73 VS 23.39±4.14µg/ml• h (p<0.01), time above the minimum effective concentration (%T > MEC): 1.5-2.2 VS 0.7-1.2h (p<0.05). HPLC-MS method is suitable for TXA pharmacokinetic studies. The results demonstrated that topical intra-articular application of TXA showed a reduced peak plasma concentration and prolonged therapeutic drug level compared with intravenous TXA from the point of rabbit pharmacokinetic.

Frontline Science: ATF3 is responsible for the inhibition of TNF-α release and the impaired migration of acute ethanol-exposed monocytes and macrophages.

Binge drinking represses host innate immunity and leads to a high risk of infection. Acute EtOH-pretreated macrophages exhibit a decreased production of proinflammatory mediators in response to LPS. ATF3 is induced and counter-regulates the LPS/TLR4 inflammatory cascade. Here, we investigated the potential role of ATF3 in LPS tolerance in acute ethanol-pretreated macrophages. We found that there was an inverse correlation between ATF3 and LPS-induced TNF-α production in acute ethanol-pretreated murine monocytes and macrophages. The knockdown of ATF3 attenuated the inhibitory effects of acute ethanol treatment on LPS-induced TNF-α production. Furthermore, ChIP assays and co-IP demonstrated that ATF3, together with HDAC1, negatively modulated the transcription of TNF-α. In binge-drinking mice challenged with LPS, an up-regulation of ATF3 and HDAC1 and a concomitant decrease in TNF-α were observed. Given that HDAC1 was concomitantly induced in acute ethanol-exposed monocytes and macrophages, we used the HDACi TSA or silenced HDAC1 to explore the role of HDAC1 in acute ethanol-treated macrophages. Our results revealed that TSA treatment and HDAC1 knockdown prevented acute ethanol-induced ATF3 expression and the inhibition of TNF-α transcription. These data indicated a dual role for HDAC1 in acute ethanol-induced LPS tolerance. Furthermore, we showed that the induction of ATF3 led to the impaired migration of BM monocytes and macrophages. Overall, we present a novel role for ATF3 in the inhibition of LPS-induced TNF-α and in the impairment of monocyte and macrophage migration.

Intestinal transport of HDND-7, a novel hesperetin derivative, in in vitro MDCK cell and in situ single-pass intestinal perfusion models.

1. Hesperetin (HDND) possesses extensive bioactivities, however, its poor solubility and low bioavailability limit its application. HDND-7, a derivative of HDND, has better solubility and high bioavailability. In this study, we investigated the intestinal absorption mechanisms of HDND-7. 2. MDCK cells were used to examine the transport mechanisms of HDND-7 in vitro, and a rat in situ intestinal perfusion model was used to characterize the absorption of HDND-7. The concentration of HDND-7 was determined by HPLC. 3. In MDCK cells, HDND-7 was effectively absorbed in a concentration-dependent manner in both directions. Moreover, HDND-7 showed pH-dependent and TEER-independent transport in both directions. The transport of HDND-7 was significantly reduced at 4 °C or in the presence of NaN3. Furthermore, the efflux of HDND-7 was apparently reduced in the presence of MRP2 inhibitors MK-571 or probenecid. However, P-gp inhibitor verapamil had no effect on the transport of HDND-7. The in situ intestinal perfusion study indicated HDND-7 was well-absorbed in four intestinal segments. Furthermore, MRP2 inhibitors may slightly increase the absorption of HDND-7 in jejunum. 4. In summary, all results indicated that HDND-7 might be absorbed mainly by passive diffusion via transcellular pathway, MRP2 but P-gp may participate in the efflux of HDND-7.