Lipidomics, which reveals comprehensive characterization of molecular lipids, is a rapidly growing technology used in biomedical research. Lipid extraction is a critical step in lipidomic analysis. However, the effectiveness of different lipid extract solvent systems from cellular samples still remains unclear. In the current study, the protocol of reverse-phase liquid chromatography mass spectrometry (LC/MS)-based lipidomics was optimized for extraction and detection of lipids from human pancreatic cancer cell line PANC-1. Four different extraction methods were compared, including methanol/methyl-tert-butyl ether (MTBE)/H2O, methanol/chloroform, methanol/MTBE/chloroform, and hexane/isopropanol. Data were acquired using high-resolution mass spectrometry in positive and negative ion modes respectively. The number of total detected and identified lipids was assessed with the aid of automated lipid identification software LipidSearch. Results demonstrated that methanol/MTBE/H2O provided a better extraction efficiency for different lipid classes, which was chosen as the optimized extraction solvent system. This validated method enables highly sensitive and reproducible analysis for a variety of cellular lipids, which was further applied to an untargeted lipidomic study on human pancreatic cancer PANC-1 cell lines. Moreover, this optimized extraction solvent system can be further applied to other cancer cell lines with similar chemical and physical properties.

Alcohol abuse leads to alcoholic liver disease and no effective therapy is currently available. Wuzhi Tablet (WZ), a preparation of extract from Schisandra sphenanthera that is a traditional hepato-protective herb, exerted a significant protective effect against acetaminophen-induced liver injury in our recent studies, but whether WZ can alleviate alcohol-induced toxicity remains unclear. This study aimed to investigate the contribution of WZ to alcohol-induced liver injury by using chronic-binge and acute models of alcohol feeding. The activities of ALT and AST in serum were assessed as well as the level of GSH and the activity of SOD in the liver. The expression of CYP2E1 and proteins in the NRF2-ARE signaling pathway including NRF2, GCLC, GCLM, HO-1 were measured, and the effect of WZ on NRF2 transcriptional activity was determined. We found that both models resulted in liver steatosis accompanied by increased transaminase activities, but that liver injury was significantly attenuated by WZ. WZ administration also inhibited CYP2E1 expression induced by alcohol, and elevated the level of GSH and the activity of SOD in the liver. Moreover, the NRF2-ARE signaling pathway was activated by WZ and the target genes were all upregulated. Furthermore, WZ significantly activated NRF2 transcriptional activity. Collectively, our study demonstrates that WZ protected against alcohol-induced liver injury by reducing oxidative stress and improving antioxidant defense, possibly by activating the NRF2-ARE pathway.Wuzhi Tablet, a preparation of extract from Schisandra sphenanthera that is a traditional hepato-protective herb, was demonstrated to protect against alcohol-induced liver injury by reducing oxidative stress and improving antioxidant defense, possibly by activating the NRF2-ARE pathway.Download high-res image (234KB)Download full-size image

Mycoplasma contamination is a common problem in cell culture and can alter cellular functions. Since cell metabolism is either directly or indirectly involved in every aspect of cell function, it is important to detect changes to the cellular metabolome after mycoplasma infection. In this study, liquid chromatography mass spectrometry (LC/MS)-based metabolomics was used to investigate the effect of mycoplasma contamination on the cellular metabolism of human pancreatic carcinoma cells (PANC-1). Multivariate analysis demonstrated that mycoplasma contamination induced significant metabolic changes in PANC-1 cells. Twenty-three metabolites were identified and found to be involved in arginine and purine metabolism and energy supply. This study demonstrates that mycoplasma contamination significantly alters cellular metabolite levels, confirming the compelling need for routine checking of cell cultures for mycoplasma contamination, particularly when used for metabolomics studies.

•Nuclear receptor PXR and CAR regulate a number of genes encoding drug metabolizing enzymes and transporters.•Increasing herbal extract and their active components have been identified as PXR and/or CAR ligands.•PXR and CAR are involved in the herbal effect against multiple human diseases.•Pharmacological targeting of PXR/CAR with herbal compounds may be an effective therapeutic strategy against human diseases.The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are two members of the nuclear receptor superfamily that regulate a broad range of genes involved in drug metabolism and transport. A variety of naturally occurring compounds present in herbal medicines were identified as ligands of PXR and CAR. Recently, accumulative evidences have revealed the PXR- and CAR-mediated herbal effect against multiple human diseases, including inflammatory bowel disease (IBD), cholestatic liver disease, and jaundice. The current review summarized the recent progress in identifying the expanding libraries of herbal medicine as ligands for PXR and CAR. Moreover, the potential for herbal medicines as promising therapeutic agents which were mainly regulated through PXR/CAR signaling pathways was also discussed. The discovery of herbal medicines as modulators of PXR and CAR, and their PXR- and CAR-mediated effect on human diseases will provide a basis for rational drug design, and eventually be explored as a novel therapeutic approach against human diseases. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

The pregnane X receptor (PXR) has been well-established as a critical mediator in regulating important drug metabolizing enzymes and transporter proteins, including cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp). Previous studies identified that PXR served as a molecular target of SUMOylation. However, the impact of SUMOylation of PXR on its transcriptional activity in regulating the expression/activity of the target genes is poorly investigated. In the current study, we established cell-based models of SUMOylated PXR in LS174T cells to investigate the impact of SUMOylation of PXR on regulating the expression/activity of CYP3A4 and P-gp. Our results revealed that rifampicin-induced PXR transactivation of the CYP3A4 and P-gp promoter was suppressed by SUMOylation of PXR in reporter gene assay. The mRNA and protein expression of CYP3A4 and P-gp was also suppressed. Moreover, CYP3A4 enzymatic assay and Rho123 intracellular assay revealed that rifampicin-induced CYP3A4 and P-gp activity was also suppressed by SUMOylated PXR. Our data collectively indicated for the first time that SUMOylation of PXR exerts suppressive effect on rifampicin-induced expression and activity of CYP3A4 and P-gp, which suggest that alteration in the SUMOylation status of PXR will be expected to affect the CYP3A4 mediated drug metabolism and P-gp mediated drug transport.

Ethnopharmacological relevanceHuang-Lian-Jie-Du-Decotion (HLJDD), an important traditional Chinese medicine formula, has been used for various diseases in clinical practice, and thus has high potential to induce cytochrome P450 (CYP) isoenzymes/P-glycoprotein (P-gp) mediated herb–drug interactions (HDIs) with other co-administered drugs. The purpose of this study was to investigate the in vitro effects of multiple extracts including aqueous extracts, total flavonoids, iridoids, alkaloids from HLJDD on the activities of CYPs in rats (CYP1A2, CYP2C6, CYP2D2, CYP2E1 and CYP3A1) and P-gp, and then to predict potential interactions with co-administered drugs.Materials and methodsThe effects of the four extracts from HLJDD on the CYPs activity were evaluated in rat liver microsomes incubation system, and then determined by LC-MS/MS-based CYPs probe substrate assay. Caco-2 cell monolayer was used to investigate the effect of the four extracts on the efflux of Rhodamine 123 to evaluate their influences on P-gp activity.ResultsThe results show that total flavonoids and alkaloids exibited strong inhibition on rat CYP isoenzymes activities. Total flavonoids exhibited different inhibitory effects on CYPs activities with an order of CYP3A1>CYP2C6>CYP2E1>CYP1A2>CYP2D2, and the values of IC50 were 4.24, 8.16, 17.56, 19.03, 29.51 μg/mL, respectively. Total alkaloids possessed similar inhibition on CYPs and could strongly inhibit the activity of CYP2D2 (IC50=2.38 μg/mL), CYP3A1 (IC50=2.61 μg/mL), CYP2E1 (IC50=22.35 μg/mL), CYP1A2 (IC50=23.2 μg/mL) and CYP2C6 (IC50=43.09 μg/mL). Moderate degree of inhibition on CYPs activities was observed in aqueous and total iridoids extracts. Results from transport assay revealed that total flavonoids and alkaloids exhibited significant inhibitory effect on P-gp activity as evidenced by strong inhibition on the efflux of Rhodamine-123 with IC50 of 104.6 and 82.6 μg/mL. But aqueous extract showed weak and iridoids had negligible effect on P-gp activity.ConclusionsThis study clearly demonstrated that total flavonoids and alkaloids from HLJDD can significantly inhibit the activities of CYPs and P-gp, which should be taken into consideration to predict any potential HDIs when HLJDD and its bioactive components are co-administered with other therapeutic drugs metabolized by CYPs or transported by P-gp.Download high-res image (247KB)Download full-size image

Tacrolimus (TAC) is an immunosuppressant that has been widely used alone or in combination with prednisone (PRED) to prevent acute rejection after organ transplantation. Wuzhi tablet (WZ, Schisandra sphenanthera extract) is often prescribed with TAC to prevent drug-induced hepatitis. We recently reported that WZ could significantly increase TAC blood exposure by inhibiting P-gp-mediated efflux and CYP3A-mediated metabolism of TAC. PRED is also a substrate of P-gp and is a weak inducer of CYP3A, and drug-drug interactions within this combination therapy might occur. Therefore, the purpose of this study was to investigate the effect of long-term treatment of WZ and PRED on the pharmacokinetics of TAC in rats. After 14 days of co-administration of WZ and PRED, the AUC0–24 h of oral TAC was increased (from 59.6 ± 37.3 to 95.3 ± 39.4 ng h/ml, p = 0.18) and the clearance was decreased (from 38.4 ± 28.4 to 17.7 ± 6.4 l/h/kg, p = 0.15). When only co-administered with WZ, AUC0–24 h of TAC was demonstrated a significantly increase (from 59.6 ± 37.3 to 135.9 ± 34.8 ng h/ml, p < 0.05). The concomitant administration of PRED resulted in a reduction in the systemic exposure of TAC and an increase in its clearance, though neither was statistically significant. Thus, our study suggested that the presence of WZ and PRED still could increase the systemic exposure of TAC in rats. The drug–drug interactions among this combination therapy should still be taken into consideration in clinical practice.

We recently reported that Wuzhi tablet (WZ), a preparation of the ethanol extract of Wuweizi (Schisandra sphenanthera), had significant effects on blood concentrations of Tacrolimus (FK506) in renal transplant recipients and rats. The active lignans in WZ are schisandrin A, schisandrin B, schisandrin C, schisandrol A, schisandrol B, schisantherin A, and schisantherin B. Until now, whether the pharmacokinetics of these lignans in WZ would be affected by FK506 remained unknown. Therefore, this study aimed to investigate whether and how FK506 affected pharmacokinetics of lignans in WZ in rats and the potential roles of CYP3A and P-gp. After a single oral co-administration of FK506 and WZ, the blood concentration of lignans in WZ was decreased by FK506; furthermore, the AUC of schisantherin A, schisandrin A, schisandrol A and schisandrol B was only 64.5%, 47.2%, 55.1% and 57.4% of that of WZ alone group, respectively. Transport study in Caco-2 cells showed that these lignans were not substrates of P-gp, suggesting decreased blood concentration of lignans by FK506 was not via P-gp pathway. Metabolism study in the human recombinant CYP 3A showed that these lignans had higher affinity to CYP3A than that of FK506, and thus had a stronger CYP3A-mediated metabolism. It was concluded that the blood concentrations of these lignans were decreased and their CYP3A-mediated metabolisms were increased in the presence of FK506 since these lignans had higher affinity to CYP3A.