Chronopharmacology of diuresis via metabolic profiling and key biomarker discovery of the traditional Chinese prescription Ji-Ming-San using tandem mass spectrometry in rat models.

BACKGROUND: Ji-Ming-Shan (JMS) is a traditional prescription used for patients with rheumatism, tendons swelling, relief of foot pain, athlete's foot, diuresis, gout. Although many studies have investigated the active compounds in each herb, the functional mechanism behind its therapeutic effect remains unclear. STUDY DESIGN: Metabolic cages for sample collection. The serum components obtained from the experimental animals were analyzed using LC-MS/MS. Furthermore, cross-analysis using the software MetaboAnalyst and Venn diagrams were used to investigate chronopharmacology of JMS in the animal models. PURPOSE: The aim of this study is to analyze the diuretic effects of JMS and to explore their chronopharmacology involved in organ regulation through four-quarter periods from serum samples of rat models. METHODS: Metabolic cages were used for collecting the urine samples and PocketChem UA PU-4010, Fuji DRI-CHEM 800 were used to examine the urine biochemical parameters. The serum components were identified through ultra-performance liquid chromatography-quadrupole time-of-flight (UPLC-Q-TOF) with a new developed method. Cross analysis, Venn diagram, MetaboAnalyst were used to investigate the key biomarker and major metabolism route with the oral administration of the drug. RESULT: JMS significantly changed the 6 h urine volume with no observed kidney toxicity. Urine pH value ranges from 7.0 to 7.5. The chronopharmacology of JMS diuresis activity were 0-6 and 6-12 groups. UPLC-Q-TOF analyses identified 243 metabolites which were determined in positive mode and negative mode respectively. With cross analysis in the Venn diagram, one key biomarker naringenin-7-O-glucoside has been identified. Major metabolic pathways such as 1: Glycerophospholipid metabolism, 2: Primary bile acid biosynthesis, 3: Sphingolipid metabolism, 4: Riboflavin metabolism, 5: Linoleic acid metabolism, 6: Butanoate metabolism. CONCLUSION: JMS significantly changed the urine output of animals in the 0-6 and 6-12 groups. No change in urine pH was observed and also kidney toxicity. A new UPLC-Q-TOF method was developed for the detection of the metabolites of JMS after oral administration. The cross analysis with Venn diagram and identified the key biomarker of JMS namely naringenin-7-O-glucoside. The results showed that six major pathways are involved in the gastrointestinal system and the liver. This study demonstrated the capability of JMS prescription in the regulation of diuresis and identified a key biomarker that is responsible for its therapeutic effect.

Ginsenoside Rg1 alleviates corticosterone-induced dysfunction of gap junctions in astrocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginsenoside Rg1 (Rg1), one of the major bioactive ingredients of Panax ginseng C. A. Mey, has neuroprotective effects in animal models of depression, but the mechanism underlying these effects is still largely unknown AIM OF THE STUDY: Gap junction intercellular communication (GJIC) dysfunction is a potentially novel pathogenic mechanism for depression. Thus, we investigated that whether antidepressant-like effects of Rg1 were related to GJIC. MATERIALS AND METHODS: Primary rat prefrontal cortical and hippocampal astrocytes cultures were treated with 50µM CORT for 24h to induce gap junction damage. Rg1 (0.1, 1, or 10µM) or fluoxetine (1µM) was added 1h prior to CORT treatment. A scrape loading and dye transfer assay was performed to identify the functional capacity of gap junctions. Western blot was used to detect the expression and phosphorylation of connexin43 (Cx43), the major component of gap junctions. RESULTS: Treatment of primary astrocytes with CORT for 24h inhibited GJIC, decreased total Cx43 expression, and increased the phosphorylation of Cx43 at serine368 in a dose-dependent manner. Pre-treatment with 1µM and 10µM Rg1 significantly improved GJIC in CORT-treated astrocytes from the prefrontal cortex and hippocampus, respectively, and this was accompanied by upregulation of Cx43 expression and downregulation of Cx43 phosphorylation. CONCLUSION: These findings provide the first evidence indicating that Rg1 can alleviate CORT-induced gap junction dysfunction, which may have clinical significance in the treatment of depression.

Alteration of the Intestinal Environment by Lubiprostone Is Associated with Amelioration of Adenine-Induced CKD.

The accumulation of uremic toxins is involved in the progression of CKD. Various uremic toxins are derived from gut microbiota, and an imbalance of gut microbiota or dysbiosis is related to renal failure. However, the pathophysiologic mechanisms underlying the relationship between the gut microbiota and renal failure are still obscure. Using an adenine-induced renal failure mouse model, we evaluated the effects of the ClC-2 chloride channel activator lubiprostone (commonly used for the treatment of constipation) on CKD. Oral administration of lubiprostone (500 µg/kg per day) changed the fecal and intestinal properties in mice with renal failure. Additionally, lubiprostone treatment reduced the elevated BUN and protected against tubulointerstitial damage, renal fibrosis, and inflammation. Gut microbiome analysis of 16S rRNA genes in the renal failure mice showed that lubiprostone treatment altered their microbial composition, especially the recovery of the levels of the Lactobacillaceae family and Prevotella genus, which were significantly reduced in the renal failure mice. Furthermore, capillary electrophoresis-mass spectrometry-based metabolome analysis showed that lubiprostone treatment decreased the plasma level of uremic toxins, such as indoxyl sulfate and hippurate, which are derived from gut microbiota, and a more recently discovered uremic toxin, trans-aconitate. These results suggest that lubiprostone ameliorates the progression of CKD and the accumulation of uremic toxins by improving the gut microbiota and intestinal environment.

Potent activity of nobiletin-rich Citrus reticulata peel extract to facilitate cAMP/PKA/ERK/CREB signaling associated with learning and memory in cultured hippocampal neurons: identification of the substances responsible for the pharmacological action.

cAMP/PKA/ERK/CREB signaling linked to CRE-mediated transcription is crucial for learning and memory. We originally found nobiletin as a natural compound that stimulates this intracellular signaling and exhibits anti-dementia action in animals. Citrus reticulata or C. unshiu peels are employed as "chinpi" and include a small amount of nobiletin. We here provide the first evidence for beneficial pharmacological actions on the cAMP/PKA/ERK/CREB cascade of extracts from nobiletin-rich C.reticulata peels designated as Nchinpi, the nobiletin content of which was 0.83 ± 0.13% of the dry weight or 16-fold higher than that of standard chinpi extracts. Nchinpi extracts potently facilitated CRE-mediated transcription in cultured hippocampal neurons, whereas the standard chinpi extracts showed no such activity. Also, the Nchinpi extract, but not the standard chinpi extract, stimulated PKA/ERK/CREB signaling. Interestingly, treatment with the Nchinpi extract at the concentration corresponding to approximately 5 µM nobiletin more potently facilitated CRE-mediated transcriptional activity than did 30 µM nobiletin alone. Consistently, sinensetin, tangeretin, 6-demethoxynobiletin, and 6-demethoxytangeretin were also identified as bioactive substances in Nchinpi that facilitated the CRE-mediated transcription. Purified sinensetin enhanced the transcription to a greater degree than nobiletin. Furthermore, samples reconstituted with the four purified compounds and nobiletin in the ratio of each constituent's content in the extract showed activity almost equal to that of the Nchinpi extract to stimulate CRE-mediated transcription. These findings suggest that above four compounds and nobiletin in the Nchinpi extract mainly cooperated to facilitate potently CRE-mediated transcription linked to the upstream cAMP/PKA/ERK/CREB pathway in hippocampal neurons.

Characterization of 11-dehydro-thromboxane B2 recombinant antibody obtained by phage display technology.

Recombinant monoclonal antibodies specific for 11-dehydro-thromboxane B(2) (11D-TX) were isolated from the combinatorial libraries on a pComb3 phage-display vector using a magnetic cell sorting (MACS) system. The libraries were constructed from repertories of light and heavy-chains derived from the total RNA of 11D-TX conjugated keyhole limpet haemocyanin-immunized mice. Biotinylation of 11D-TX conjugated bovine serum albumin (BSA) was performed through free thiol groups on BSA using 1-biotinamido-4-[4'-(maleimidomethyl) cyclohexanecarboxamido] butane (Biotin-BMCC). Affinity bio-panning was performed to enrich the phage display libraries against biotinylated 11D-TX conjugated BSA with the MACS system. Results indicated that the selected anti-11D-TX Fab fragments expressed by E. coli exhibited a five-fold higher affinity for BSA conjugated 11D-TX compared to BSA alone and little specificity to other related compounds as determined by the binding assay and inhibition enzyme-linked immunosorbent assay (ELISA). This is the first report of an antibody against prostaglandin produced by phage display technology and also determination of the DNA sequence of this antibody. The MACS system was shown to be a simpler and more efficient method of panning than the conventional ELISA procedure. According to our results, we concluded that the phage display technique combined with the MACS system allowed the selection of the antibody with high affinity and some specificity.