Common bacterial metabolite indole directly activates nociceptive neuron through transient receptor potential ankyrin 1 channel.

ABSTRACT: Nociceptors are known to directly recognize bacterial cell wall components or secreted toxins, thereby leading to pain induced by bacterial infection. However, direct activation of nociceptors by bacterial metabolites remains unclear although bacteria produce numerous metabolites related to health and disease. In this study, we investigated whether and how a common bacterial metabolite, indole, which is produced by normal microflora of the gastrointestinal tract and oral cavity, can directly activate nociceptive sensory neurons. We found that indole elicits calcium response and evokes inward currents in subsets of dorsal root ganglia (DRG) neurons. Intraplantar (i.pl.) injection of indole produced nocifensive behaviors in adult mice, which were enhanced in complete Freund's adjuvant-induced chronic inflammatory condition. Indole increased calcitonin gene-related peptide release in DRG neurons, and i.pl. injection of indole increased hind paw thickness, suggesting its role in generation of neurogenic inflammation. These in vitro and in vivo indole-induced responses were pharmacologically blocked by transient receptor potential ankyrin 1 (TRPA1) antagonist, HC-030031, and significantly abolished in TRPA1 knockout (KO) mice, indicating that indole targets TRPA1 for its action in DRG neurons. Nocifensive licking behavior induced by the injection of live Escherichia coli was significantly decreased in tryptophanase mutant (TnaA KO) E. coli- injected mice that lack indole production, further supporting the idea that bacteria-derived indole can induce pain during infection. Identifying the mechanism of action of indole through TRPA1 provides insights into bacteria-neuron interactions and the role of bacterial metabolites in pain signaling, especially in inflammation-accompanied bacterial infection.

The analgesic effect of refeeding on acute and chronic inflammatory pain.

Pain is susceptible to various cognitive factors. Suppression of pain by hunger is well known, but the effect of food intake after fasting (i.e. refeeding) on pain remains unknown. In the present study, we examined whether inflammatory pain behavior is affected by 24 h fasting and 2 h refeeding. In formalin-induced acute inflammatory pain model, fasting suppressed pain behavior only in the second phase and the analgesic effect was also observed after refeeding. Furthermore, in Complete Freund's adjuvant-induced chronic inflammatory pain model, both fasting and refeeding reduced spontaneous pain response. Refeeding with non-calorie agar produced an analgesic effect. Besides, intraperitoneal (i.p.) administration of glucose after fasting, which mimics calorie recovery following refeeding, induced analgesic effect. Administration of opioid receptor antagonist (naloxone, i.p.) and cannabinoid receptor antagonist (SR 141716, i.p.) reversed fasting-induced analgesia, but did not affect refeeding-induced analgesia in acute inflammatory pain model. Taken together, our results show that refeeding produce analgesia in inflammatory pain condition, which is associated with eating behavior and calorie recovery effect.

Identification and characterization of in vitro inhibitors against UDP-glucuronosyltransferase 1A1 in uva-ursi extracts and evaluation of in vivo uva-ursi-drug interactions.

Uva-ursi leaf is widely used to treat symptoms of lower urinary tract infections. Here, we evaluated the in vitro inhibitory effects of uva-ursi extracts on 10 major human UDP-glucuronosyltransferases (UGT) isoforms. Of the 10 tested UGT isoforms, uva-ursi extracts exerted the strongest inhibitory effect on UGT1A1-mediated ß-estradiol 3-glucuronidation with the lowest IC50 value of 8.45 ±â€¯1.56 µg/mL. To identify the components of uva-ursi extracts showing strong inhibitory effects against UGT1A1, the inhibitory effects of nine major constituents of the extracts were assessed. Among the tested compounds, gallotannin exerted the most potent inhibition on UGT1A1, followed by 1,2,3,6-tetragalloylglucose; both demonstrated competitive inhibition, with Ki values of 1.68 ±â€¯0.150 µM and 3.55 ±â€¯0.418 µM. We found that gallotannin and 1,2,3,6-tetragalloylglucose also inhibited another UGT1A1-specific biotransformation, SN-38-glucuronidation, showing the same order of inhibition. Thus, in vitro UGT1A1 inhibitory potentials of uva-ursi extracts might primarily result from the inhibitory activities of gallotannin and 1,2,3,6-tetragalloylglucose present in the extracts. However, in rats, co-administration with uva-ursi extracts did not alter the in vivo marker for UGT1A1 activity, expressed as the molar ratio of AUCSN-38 glucuronide/AUCSN-38, because plasma concentrations of gallotannin and 1,2,3,6-tetragalloylglucose may be too low to inhibit the UGT1A1-mediated metabolism of SN-38 in vivo. The poor oral absorption of gallotannin and 1,2,3,6-tetragalloylglucose in uva-ursi extracts might cause the poor in vitro-in vivo correlation. These findings will be helpful for the safe and effective use of uva-ursi extracts in clinical practice.

In Vitro Inhibition of Human UDP-Glucuronosyl-Transferase (UGT) Isoforms by Astaxanthin, ß-Cryptoxanthin, Canthaxanthin, Lutein, and Zeaxanthin: Prediction of in Vivo Dietary Supplement-Drug Interactions.

Despite the widespread use of the five major xanthophylls astaxanthin, ß-cryptoxanthin, canthaxanthin, lutein, and zeaxanthin as dietary supplements, there have been no studies regarding their inhibitory effects on hepatic UDP-glucuronosyltransferases (UGTs). Here, we evaluated the inhibitory potential of these xanthophylls on the seven major human hepatic UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7 and UGT2B15) in vitro by LC-MS/MS using specific marker reactions in human liver microsomes (except UGT2B15) or recombinant supersomes (UGT2B15). We also predicted potential dietary supplement-drug interactions for ß-cryptoxanthin via UGT1A1 inhibition. We demonstrated that astaxanthin and zeaxanthin showed no apparent inhibition, while the remaining xanthophylls showed only weak inhibitory effects on the seven UGTs. ß-Cryptoxanthin mildly inhibited UGT1A1, UGT1A3, and UGT1A4, with IC50 values of 18.8 ± 2.07, 28.3 ± 4.40 and 34.9 ± 5.98 µM, respectively. Canthaxanthin weakly inhibited UGT1A1 and UGT1A3, with IC50 values of 38.5 ± 4.65 and 41.2 ± 3.14 µM, respectively; and lutein inhibited UGT1A1 and UGT1A4, with IC50 values of 45.5 ± 4.01 and 28.7 ± 3.79 µM, respectively. Among the tested xanthophyll-UGT pairs, ß-cryptoxanthin showed the strongest competitive inhibition of UGT1A1 (Ki, 12.2 ± 0.985 µM). In addition, we predicted the risk of UGT1A1 inhibition in vivo using the reported maximum plasma concentration after oral administration of ß-cryptoxanthin in humans. Our data suggests that these xanthophylls are unlikely to cause dietary supplement-drug interactions mediated by inhibition of the hepatic UGTs. These findings provide useful information for the safe clinical use of the tested xanthophylls.

In vitro stereoselective inhibition of ginsenosides toward UDP-glucuronosyltransferase (UGT) isoforms.

We evaluated in vitro, the potential of the six pairs of ginsenoside isomers, stereoisomers at the chiral carbon on position 20, to inhibit the enzymatic activity of several UDP-glucuronosyltransferase (UGT) isoenzymes, major players in the human phase II drug metabolism. The results show that the tested six pairs of ginsenoside isomers exhibited stereoselective inhibitory effects of varying degrees on the ten UGT isoenzymes explored. Of the tested twelve stereoselective ginsenosides, 20(R)-Rg3 had the strongest inhibitory effect on the UGT1A8 isoform with the lowest IC50 value of 5.66±1.04µM. On the other hand, the (S)-isomers of Rg3 and Rh2 also exerted remarkable inhibition on UGT1A8, with IC50 values of 6.89±0.812µM and 5.85±0.821µM, respectively. Although the inhibitory effect was low, both 20(R)-PPT and 20(S)-PPT also inhibited UGT1A8 activity. Considering 1) that the relative contents of 20(R)-Rg3 in processed ginseng are high, 2) that higher exposure to (R)-isomers of ginsenosides occur in the intestine compared to that in the liver, and 3) the inhibitory effects of other ginsenosides on enzymatic activity [20(S)-Rg3, 20(S)-Rh2, 20(R)- and 20(S)-PPT], there may be a potential for herb-drug interactions between processed ginseng and UGT1A8 substrates when concomitantly administered.

Pharmacokinetic properties of trifolirhizin, (-)-maackiain, (-)-sophoranone and 2-(2,4-dihydroxyphenyl)-5,6-methylenedioxybenzofuran after intravenous and oral administration of Sophora tonkinensis extract in rats.

1. SKI3301, a standardized dried 50% ethanolic extracts of Sophora tonkinensis, contains four marker compounds (trifolirhizin, TF; (-)-maackiain, Maack; (-)-sophoranone, SPN, and (2-(2,4-dihydroxyphenyl)-5,6-methylenedioxybenzofuran, ABF), is being developed as an herbal medicine for the treatment of asthma in Korea. This study investigates the pharmacokinetic properties of SKI3301 extract in rats. 2. The dose-proportional AUCs suggest linear pharmacokinetics of TF, Maack, SPN and ABF in the SKI3301 extract intravenous dose range of 5-20 mg/kg. After the oral administration of 200-1000 mg/kg of the extract, TF and Maack exhibited non-linearity due to the saturation of gastrointestinal absorption. However, linear pharmacokinetics of SPN and ABF were observed. 3. The absorptions of TF, Maack, SPN and ABF in the extract were increased relative to those of the respective pure forms due to the increased solubility and/or the decreased metabolism by other components in the SKI3301 extract. 4. No accumulation was observed after multiple dosing, and the steady-state pharmacokinetics of TF, Maack, SPN and ABF were not significantly different from those after a single oral administration of the extract. 5. The pharmacokinetics of TF, SPN and ABF were not significantly different between male and female rats after oral administration of the extract, but a significant gender difference in the pharmacokinetics of Maack in rats was observed. 6. Our findings may help to comprehensively elucidate the pharmacokinetic characteristics of TF, Maack, SPN and ABF and provide useful information for the clinical application of SKI3301 extract.

Nardostachys jatamansi protects against cerulein-induced acute pancreatitis.

OBJECTIVES: Nardostachys jatamansi belonging to the family Valerianaceae has been used as a remedy for stomach and skin ailments in Korea. The effect of N. jatamansi on acute pancreatitis (AP) has not been defined. Therefore, we investigated the effect of N. jatamansi on cerulein-induced AP. METHODS: In the pretreatment group, N. jatamansi was administered orally to mice at 10 and 20 mg/kg for 5 days, and the mice were intraperitoneally injected with the stable cholecystokinin analogue cerulein hourly for 6 hours. In the posttreatment group, cerulein was injected hourly for 6 hours, and N. jatamansi was administered at the indicated time (1, 3, and 5 hours after the first cerulein injection) and dose (10 and 20 mg/kg) during the cerulein injection. Blood samples were taken 6 hours later to determine the serum amylase, the lipase, and the cytokine levels. The pancreas and the lung were rapidly removed for morphologic examination, myeloperoxidase assay, and real-time reverse transcription polymerase chain reaction. RESULTS: Nardostachys jatamansi treatment attenuated the AP, as shown by the histological examination results of the pancreas and the lung, reductions in pancreatic edema, neutrophil infiltration, serum amylase and lipase levels, serum cytokine levels, and messenger RNA expressions of inflammatory mediators. CONCLUSIONS: These results suggest that N. jatamansi attenuates the severity of AP and pancreatitis-associated lung injury.

Gardenia jasminoides protects against cerulein-induced acute pancreatitis.

AIM: To investigate the effect of Gardenia jasminoides (GJ) on cerulein-induced acute pancreatitis (AP) in mice. METHODS: C57BL/6 mice weighing 18-20 g were divided into three groups. (1) Normal saline-treated group, (2) treatment with GJ at a dose of 0.1 g/kg, (3) treatment with GJ at a dose of 1 g/kg. GJ was administered orally (n = 6 per group) for 1 wk. Three hours later, the mice were given an intraperitoneal injection of cerulein (50 microg/kg), a stable cholecystokinin (CCK) analogue, every hour for a total of 6 h as described previously. The mice were sacrificed at 6 h after completion of cerulein injections. Blood samples were obtained to determine serum amylase, lipase and cytokine levels. The pancreas was rapidly removed for morphologic examination and scoring. A portion of pancreas was stored at -70 degree and prepared for the measurement of tissue myeloperoxidase (MPO) activity, an indicator of neutrophil sequestration, and for reverse-transcriptase PCR (RT-PCR) and real-time PCR measurements. RESULTS: Treatment with GJ decreased significantly the severity of pancreatitis and pancreatitis-associated lung injury. Treatment with GJ attenuated the severity of AP compared with saline-treated mice, as shown by reduction in pancreatic edema, neutrophil infiltration, serum amylase and lipase levels, serum cytokine levels, and mRNA expression of multiple inflammatory mediators. CONCLUSION: These results suggest that GJ attenuated the severity of AP as well as pancreatitis-associated lung injury.