Synthesis and identification of lithocholic acid 3-sulfate as RORγt ligand to inhibit Th17 cell differentiation.

Primary bile acids (BAs), products of cholesterol metabolism and clearance, are synthesized in the liver and released into the intestine to facilitate the digestion and absorption of lipids. BAs are further converted by gut commensal bacteria into secondary colonic BAs and the metabolism disorder is closely linked to cholestatic liver diseases via regulating immune response. However, the effect and underlying mechanism of these host-microorganism biliary metabolites on T lymphocyte remain unclear. In the current study, we synthesized a sulfated product of lithocholic acid (LCA), lithocholic acid 3-sulfate (LCA-3-S), and investigated the binding affinity of the BAs metabolites on RORγt, the transcription factor of IL-17A. Our results demonstrated that the sulfate of LCA, LCA-3-S, exhibited better effect than its oxidated metabolite, 3-oxo-LCA, binding to RORγt. The results further demonstrated that LCA-3-S selectively suppressed Th17 cell differentiation without influence on Th1, Th2, and Treg cells. Collectively, we synthesized the sulfated biliary metabolite LCA-3-S and demonstrated that LCA-3-S selectively inhibited Th17 cell differentiation by targeting RORγt, indicating that metabolite disorder of BAs resulting in the decrease of LCA-3-S probably contributes to the pathogenesis of cholestatic liver diseases.

Novel ginsenoside derivative 20(S)-Rh2E2 suppresses tumor growth and metastasis in vivo and in vitro via intervention of cancer cell energy metabolism.

Increased energy metabolism is responsible for supporting the abnormally upregulated proliferation and biosynthesis of cancer cells. The key cellular energy sensor AMP-activated protein kinase (AMPK) and the glycolytic enzyme alpha-enolase (α-enolase) have been identified as the targets for active components of ginseng. Accordingly, ginseng or ginsenosides have been demonstrated with their potential values for the treatment and/or prevention of cancer via the regulation of energy balance. Notably, our previous study demonstrated that the R-form derivative of 20(R)-Rh2, 20(R)-Rh2E2 exhibits specific and potent anti-tumor effect via suppression of cancer energy metabolism. However, the uncertain pharmacological effect of S-form derivative, 20(S)-Rh2E2, the by-product during the synthesis of 20(R)-Rh2E2 from parental compound 20(R/S)-Rh2 (with both R- and S-form), retarded the industrialized production, research and development of this novel effective candidate drug. In this study, 20(S)-Rh2E2 was structurally modified from pure 20(S)-Rh2, and this novel compound was directly compared with 20(R)-Rh2E2 for their in vitro and in vivo antitumor efficacy. Results showed that 20(S)-Rh2E2 effectively inhibited tumor growth and metastasis in a lung xenograft mouse model. Most importantly, animal administrated with 20(S)-Rh2E2 up to 320 mg/kg/day survived with no significant body weight lost or observable toxicity upon 7-day treatment. In addition, we revealed that 20(S)-Rh2E2 specifically suppressed cancer cell energy metabolism via the downregulation of metabolic enzyme α-enolase, leading to the reduction of lactate, acetyl-coenzyme (acetyl CoA) and adenosine triphosphate (ATP) production in Lewis lung cancer cells (LLC-1), but not normal cells. These findings are consistent to the results obtained from previous studies using a similar isomer 20(R)-Rh2E2. Collectively, current results suggested that 20(R/S)-Rh2E2 isomers could be the new and safe anti-metabolic agents by acting as the tumor metabolic suppressors, which could be generated from 20(R/S)-Rh2 in industrialized scale with low cost.

Potent Antagonists of RORγt, Cardenolides from Calotropis gigantea, Exhibit Discrepant Effects on the Differentiation of T Lymphocyte Subsets.

RORγt is the master transcription factor of IL-17 cytokine expression and Th17 lymphocyte differentiation, which are responsible for the induction of many autoimmune diseases. Recently, RORγt has become an attractive target for drug development to treat these types of diseases, and the field of RORγt antagonist research is now extremely competitive. In our current study, molecular docking was applied to demonstrate that cardenolides, including uscharin, calactin, and calotropin derived from Calotropis gigantea, probably directly bind to RORγt. Therefore, the inhibitory effect was further validated using a luciferase reporter assay. Because RORγt is the key transcriptional factor for Th17 differentiation, the effects of these compounds on Th17 differentiation were studied by flow cytometry. The results showed that uscharin, calactin, and calotropin inhibited Th17 differentiation from 100 to 500 nM. Furthermore, uscharin had a better effect than digoxin, a well-known inverse agonist of RORγt, in reducing Th17 polarization. Additionally, the effects of the cardenolides on the differentiation of other Th lineages, including Th1, Th2, and Treg, were investigated. Uscharin suppressed Th1, Th2, and Treg cell differentiation, while calactin suppressed the differentiation of Th1 cells, and calotropin did not influence the other T cell subsets, indicating that calactin suppressed Th1 and Th17 differentiation, and calotropin selectively quenched Th17 polarization. Structural analysis of the three compounds showed that the selectivity of uscharin, calactin, and calotropin on the suppression of the different subsets of T cells is correlated to the minor differences in their chemical structures. Collectively, calactin and calotropin have greater potential to be developed as lead compounds than uscharin to treat autoimmune diseases mediated by Th17 and/or Th1 cells.

[Pharmacokinetic study of six aconitine alkaloids in aconiti lateralis radix praeparata in beagle dogs].

OBJECTIVE: To study the pharmacokinetics characteristics of six Aconitum alkaloids aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA) and benzoylhypaconine (BHA) in beagle dogs. METHODS: An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for simultaneous quantitation of six Aconitum alkaloids in beagle dog plasma after oral administration of Aconiti Lateralis Radix Praeparata decoction. UPLC/MS/MS system coupled with an electrospray ionization (ESI) source was performed in multiple-reaction monitoring (MRM) mode. Sample preparation was performed with solid-phase extraction(SPE) on a 3 mL HLB cartridge before the analysis. The separation was applied on a Waters C8 column (100 mm x 2.1 mm, 1.7 microm) and a gradient elution of methanol and 0.2% formic acid-water was used as mobile phase. The pharmacokinetic parameters were calculated by the results of the analysis through the DAS 2. 1 software (Drug and Statistics for Windows). RESULTS: The results showed that the fitting model for the six Aconitum alkaloids was the one-compartment model pharmacokinetics. CONCLUSION: The method is successfully used for the pharmacokinetic evaluation of the six Aconitum alkaloids in beagle dog plasma, it can help monitor the ADME/Tox process when taking Aconiti Lateralis Radix Praeparata by observing the pharmacokinetic process. The results provide a good reference for clinical treatment and safe application of Aconiti Lateralis Radix Praeparata.