[Bioinformatics database and production layout visual analysis platform of medicinal plants:a case study of Dao-di medicinal materials in Sichuan province].

In the new stage of trans-omics and trans-subjects for medicinal plants, it is an urgent need to integrate big data, provide interactive applications, and form a unified and multi-level research system and big data platform. Dao-di medicinal material, as an important source of medicinal plants, is a unique quality concept and comprehensive standard of tranditional Chinese medicine(TCM). Several databases have been developed in China and abroad, such as the Encyclopedia of Traditional Chinese Medicine(ETCM) and the Global Pharmacopoeia Genome Database(GPGD). Yet, most databases do not provide multi-dimensional data, including geographic data, phenotype data, compound data, and genetic data. Sichuan, known as the hometown of TCM therapies and the treasure trove of TCM, is the most representative region of medicinal plant diversity in China. According to the latest data of the fourth national survey of TCM resources, there are more than 8 000 TCM and 86 Dao-di medicinal materials in Sichuan province. Based on resource census data and relevant achievements, this study constructed the bioinformatics database of medicinal plants and the visual analysis platform of production layout by taking the Dao-di medicinal materials in Sichuan province as an example, covering geographic data, phenotype data, compound data, and genetic data. It effectively integrates multi-dimensional data of Dao-di medicinal materials and provides different levels of data interaction applications. The platform is the first large-scale multi-dimensional database and visual platform of Dao-di medicinal materials in Sichuan province, which serves as an essential resource for germplasm resources identification, decomposition of biosynthetic pathways, molecular breeding of varieties and provides medicinal plant resource information and data support for development and utilization of medicinal plants in China and abroad.

[Analysis of WRKY transcription factor family based on full-length transcriptome sequencing in Polygonatum cyrtonema].

WRKY transcription factor family plays an important role in plant growth and development, secondary metabolite synthesis, and biotic and abiotic stress responses. The present study performed full-length transcriptome sequencing of Polygonatum cyrtonema by virtue of the PacBio SMRT high-throughput platform, identified the WRKY family by bioinformatics methods, and analyzed the physicochemical properties, subcellular localization, phylogeny, and conserved motifs. The results showed that 30.69 Gb nucleotide bases and 89 564 transcripts were obtained after redundancy removal. These transcripts had a mean length of 2 060 bp and an N50 value of 3 156 bp. Based on the full-length transcriptome sequencing data, 64 candidate proteins were selected from the WRKY transcription factor family, with the protein size of 92-1 027 aa, the relative molecular mass of 10 377.85-115 779.48 kDa, and the isoelectric point of 4.49-9.84. These WRKY family members were mostly located in the nucleus and belonged to the hydrophobic proteins. According to the phylogenetic analysis of WRKY family in P. cyrtonema and Arabidopsis thaliana, all WRKY family members were clustered into seven subfamilies and WRKY proteins from P. cyrtonema were distributed in different numbers in these seven subgroups. Expression pattern analysis confirmed that 40 WRKY family members had distinct expression patterns in the rhizomes of 1-and 3-year-old P. cyrtonema. Except for PcWRKY39, the expression of 39 WRKY family members was down-regulated in 3-year-old samples. In conclusion, this study provides abundant reference data for genetic research on P. cyrtonema and lays a foundation for the in-depth investigation of the biological functions of the WRKY family.

Dextran sulfate sodium-induced colitis and ginseng intervention altered oral pharmacokinetics of cyclosporine A in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Application of cyclosporine A (CsA) as a rescue treatment in acute severe ulcerative colitis (UC) is limited by its narrow therapeutic window and great interpatient variability. As a substrate of cytochrome P450 3A enzyme (CYP3A) and P-glycoprotein (P-gp), the oral pharmacokinetics of CsA is susceptible to disease status and concomitant medications. Combined treatment with ginseng, a famous medicinal herb frequently prescribed for ameliorating abnormal immune response in many diseases including UC, showed immunologic safety in CsA-based immunosuppression. AIM OF THE STUDY: Since the therapeutic levels of CsA can be achieved within 24 h, this study first assessed the impact of acute colitis and ginseng intervention on the single oral dose pharmacokinetics of CsA and explored the underlying mechanisms in dextran sulfate sodium (DSS)-induced colitis rats and Caco-2 cells. MATERIALS AND METHODS: Rats received drinking water (normal group), 5% DSS (UC group), or 5% DSS plus daily oral ginseng extract (GS+UC group). On day 7, GS+UC group only received an oral dose of CsA (5 mg/kg), while animals of normal or UC group received an oral, intravenous (1.25 mg/kg), or intraperitoneal dose of CsA (1.25 mg/kg), respectively. Blood, liver/intestine tissues and fecal samples were collected for determining CsA and main hydroxylated metabolite HO-CsA or measuring hepatic/intestinal CYP3A activity. Caco-2 cells were incubated with gut microbial culture supernatant (CS) of different groups or ginseng (decoction or polysaccharides), and then CYP3A, P-gp and tight junction (TJ) proteins were determined. RESULTS: Oral CsA exhibited enhanced absorption, systemic exposure and tissue accumulation, and lower fecal excretion, while intravenous or intraperitoneal CsA showed lower systemic exposure and enhanced distribution, in colitis rats. Diminished intestinal and hepatic P-gp expression well explained the changes with DSS-induced colitis. Moreover, blood exposures of HO-CsA in both normal and colitis after oral dosing were significantly higher than intravenous/intraperitoneal dosing, supporting the dominant role of intestinal first-pass metabolism. Interestingly, colitis reduced CYP3A expression in intestine and liver but only potentiated intestinal CYP3A activity, causing higher oral systemic exposure of HO-CsA. Oral ginseng mitigated colitis-induced down-regulation of CYP3A and P-gp expression, facilitated HO-CsA production, biliary excretion and colonic sequestration of CsA, while not affected CsA oral systemic exposure. In Caco-2 cells, gut microbial CS from both colitis and GS+UC group diminished P-gp function, while ginseng polysaccharides directly affected ZO-1 distribution and suppressed TJ proteins expression, explaining unaltered oral CsA systemic exposure. CONCLUSIONS: DSS-induced colitis significantly altered oral CsA disposition through regulating intestinal and hepatic P-gp and CYP3A. One-week ginseng treatment enhanced colonic accumulation while not altered the systemic exposure of CsA after single oral dosing, indicating pharmacokinetic compatibility between the two medications.

[Transcriptome and quality evaluation of Sichuan Dao-di herbs Zanthoxylum bungeanum].

"Huajiao" is dried ripe fruit peel of Zanthoxylum bungeanum or Z. schinifolium, is konwn as geoherbs, especially the "Dahongpao" cultivated in Hanyuan, Maoxian and Jiulong of Sichuan province. However, the genetic basis of Dao-di "Huajiao" is virtually unknown. The transcriptome of the fruit and leaf from Sichuan(Hanyuan, Jiulong, Lixian, Maoxian), Gansu(Wudu) province and Shaanxi(Fengxian) province was sequenced. Trinity de novo assembling resulted in a total of 177 616 unigenes. Through the KEGG, NR, SwissProt, Trembl, KOG/COG, GO, Pfam database comparision 106 644 annotated Unigene finally, 4 574 deferentially expressed genes were found in fruit between Sichuan and other provinces, including 3 740 up-regulated genes and 834 down-regulated genes. Among the up-regulated genes, 27 up-regulated genes were raleted to terpenoids, and 8 up-regulated genes were related to isoquinoline alkaloid bio-synthesis. Furthermore, it was also showed remarkable differences in groups which enrichment ratio of the diffe-rent expressed gene compared. The different expressed genes were annotated by the KEGG database into plant-pathogen interaction, plant hormone signal transduction and phenylpropanoid biosynthesis in fruit and leaf, but isoflavonoid bio-synthesis and betaine bio-synthesis were significantly different in fruit and leaf. The study laid a certain reference basis for comparison of quality and different expressed gene of Z. bungeanum from different groups.

[New method for evaluating pharmacodynamics of traditional Chinese medicine compounds based on its moderate regulation and principle of balanced adjustment of immunity under pan-immunomic].

For the basic research on the traditional Chinese medicine(TCM), objective syndrome of traditional Chinese medicine and evaluation criteria of traditional Chinese medicine compounds are hardly to break though. While, the modern immunology points out that the body is a counterbalance state and immune imbalance is the root of sickness. The thinking mode of treating diseases in traditional Chinese medicine is also "balance", considering disease is the result of bias which present the imbalance of "Yin counters Yang", "exterior counters interior", "cold counters heat" and "weak counters strong". The Chinese herbal compound formula preparation was applied on disease therapy based on theory of Chinese medicine, which was confirmed by long period clinical application. It is composed of multi-compounds and has the characteristic of multi-targeting. Integrative medicine has spawned pan-immunomics, and the evaluation of immune function (immune balance) has become an important basis for diagnosis and treatment models of integrative medicine. In addition, balance is the core idea of whole-systemic conception of traditional Chinese medicine. Therefore, we speculate that immune balance under pan-immunomic can bridge the traditional Chinese medicine and modern integrative medicine and is the important basis for objective syndrome of traditional Chinese medicine and evaluation criteria of traditional Chinese medicine compounds. According to the bridging theory, we attempt to utilize informatics and statistical methods to construct an evaluation system for pharmacodynamics of traditional Chinese medicine based on its moderate regulation and the balanced adjustment of immunity under pan-immunomic, which further reveal the scientific essence of the whole-systemic view of traditional Chinese medicine. This research brings out a new valuable strategy and provides a theoretical basis for accelerating the transformation of traditional Chinese medicine, especially the exploitation of Chinese herbal compound formula, and constructing the new drug innovation and review system for traditional Chinese medicine. Besides as a reference for traditional Chinese medicine objective syndrome and pharmacodynamics of traditional Chinese medicine compounds, the evaluation system can screen the immunity of sub-health population also. With the continuous accumulation of clinical sample and data, the evaluation system will be more accurate and intelligent.

Characteristics and molecular determinants of a highly selective and efficient glycyrrhizin-hydrolyzing ß-glucuronidase from Staphylococcus pasteuri 3I10.

Glycyrrhizin (GL), the principal sweet-tasting bioactive ingredient of licorice (root of Glycyrrhiza glabra), shows poor oral absorption and gut microbial transformation of GL to glycyrrhetinic acid (GA) plays a major role for its multiple pharmacological effects. Co-administration of GL-hydrolyzing bacteria appears to be a feasible strategy to enhance GA exposure. This study reported a gut bacterial strain Staphylococcus pasteuri 3I10 which exhibited moderate p-nitrophenyl-ß-D-glucuronide (PNPG)-hydrolyzing activity but low GL deglucuronidation activity in its crude lysate. The gus gene encoding S. pasteuri 3I10 ß-glucuronidase was successfully cloned and overexpressed in Escherichia coli BL21(DE3). The purified ß-glucuronidase (SpasGUS) was 71 kDa and showed optimal pH and temperature at 6.0 and 50 °C, respectively. Comparing to E. coli ß-glucuronidase (EcoGUS), SpasGUS displayed lower velocity and affinity to PNPG hydrolysis (Vmax 16.1 ± 0.9 vs 140.0 ± 4.1 µmolmin-1 mg-1; Km 469.4 ± 73.4 vs 268.0 ± 25.8 µM), but could selectively convert GL to GA at much higher efficiency (Vmax 0.41 ± 0.011 vs 0.005 ± 0.002 µmolmin-1 mg-1; Km 116.9 ± 15.4 vs 53.4 ± 34.8 µM). Molecular docking studies suggested SpasGUS formed hydrogen bond interactions with the glucuronic acids at Asn414, Glu415 and Leu450, and Val159, Tyr475, Ala368, and Phe367 provided a hydrophobic environment for enhanced activity. Two special substrate interaction loops near the binding pocket of SpasGUS (loop 1 ß-glucuronidase) may account for the selective and efficient bioconversion of GL to GA, predicting that loop 1 ß-glucuronidases show high possibility in processing GL than mini-loop 1 and loop 2 ß-glucuronidases. These findings support potential applications of SpasGUS in cleaving GL to facilitate GA production in vivo or in pharmaceutical industry.

Fecal Microbiota Transplantation in Experimental Ulcerative Colitis Reveals Associated Gut Microbial and Host Metabolic Reprogramming.

Fecal microbiota transplantation (FMT) is gaining attention for the treatment of ulcerative colitis (UC). Data from individual case studies have suggested that FMT may be beneficial for UC, but the detailed microbial and molecular basis remains unknown. Here, we employ 16S rRNA gene sequencing and metabolomics to investigate the influence of FMT on gut microbial community composition and host metabolism in the dextran sulfate sodium-induced UC rat model. The findings from this pilot study suggest that FMT from normal donors to UC recipients could alleviate UC symptoms without close resemblance of donor's gut microbial and metabolic pattern. Meanwhile, FMT from UC donors to normal recipient rats triggered UC symptoms, UC-prone microbial shift, and host metabolic adaption. Gut microbiota under normal conditions could maintain stable species richness and diversity upon FMT intervention, but the disturbed gut microbiota under UC conditions could not maintain such homeostasis. Significant correlations between altered bacterial composition and host metabolism could be assigned to the pathological effects of UC (accounting for 8.0 to 16.2% of total variance) and/or the FMT intervention effects (3.9 to 7.0% of total variance). Overall, our study reveals diverse gut microbial shifts in UC related FMT and their association with host metabolic reprogramming.IMPORTANCE This study combined clinical symptoms measurement, 16S rRNA gene microbial profiling and metabolomics to comprehensively investigate the gut bacterial and host metabolic association and reprogramming in FMT-treated experimental UC. These data can advance our understanding of the effect of FMT on UC and the involvement of gut microbial dysbiosis in the development of UC.

Bacterial Outer Membrane Vesicles from Dextran Sulfate Sodium-Induced Colitis Differentially Regulate Intestinal UDP-Glucuronosyltransferase 1A1 Partially Through Toll-Like Receptor 4/Mitogen-Activated Protein Kinase/Phosphatidylinositol 3-Kinase Pathway.

UDP-glucuronosyltransferase 1A1 (UGT1A1) constitutes an important part of intestinal epithelial barrier and catalyzes glucuronidation of many endogenous compounds and drugs. Downregulation of UGT1A1 in inflammation has been reported, whereas the association with gut dysbiosis is poorly defined. This study verified the involvement of gut microbiota in intestinal UGT1A1 regulation using dextran sulfate sodium (DSS)-induced rat colitis model plus fecal microbiota transplantation (FMT). Generally, both DSS induction and colitis-to-normal FMT suppressed mRNA and protein expressions of UGT1A1 and nuclear xenobiotic receptors (NRs) in colon, but enhanced mRNA and decreased protein of rat UGT1A1/rat NRs in small intestine. Normal-to-colitis FMT alleviated DSS-induced changes. Bacterial outer membrane vesicles (OMVs) from colitis rats and rats receiving colitis feces reduced both mRNA and protein of human UGT1A1 (hUGT1A1)/human NRs (hNRs) in Caco-2 cells. Interestingly, using deoxycholate to reduce lipopolysaccharide, normal OMVs upregulated hUGT1A1/hNRs, whereas colitis OMVs decreased, indicating the involvement of other OMVs components in UGT1A1 regulation. The 10- to 50-kDa fractions from both normal and colitis OMVs downregulated hUGT1A1, human PXR, and human PPAR-γ, whereas >50-kDa fractions from normal rats upregulated hUGT1A1 and human CAR. Additionally, the conditioned medium from OMVs-stimulated rat primary macrophages also reduced hUGT1A1/hNRs expression. Both Toll-like receptor (TLR)2 and TLR4 were activated by DSS, colitis-to-normal FMT, and the opposite, whereas only TLR4 was increased in OMVs-treated cells. TLR4 small interfering RNA blocked hUGT1A1/hNRs downregulation and phosphatidylinositol 3-kinase/Akt, extracellular signal-regulated kinase, and nuclear factor κB phosphorylation evoked by bacterial OMVs. Taken together, this study demonstrated that gut microbiota regulate intestinal UGT1A1 partially through secreting OMVs, which interact with intestinal epithelial cells directly or via activating macrophage.

Ginseng polysaccharides enhanced ginsenoside Rb1 and microbial metabolites exposure through enhancing intestinal absorption and affecting gut microbial metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: Polysaccharides and small molecules commonly co-exist in decoctions of traditional Chinese medicines (TCMs). Our previous study outlined that ginseng polysaccharides (GP) could interact with co-existing ginsenosides to produce synergistic effect in an over-fatigue and acute cold stress model via gut microbiota involved mechanisms. AIM OF THE STUDY: This study aimed to verify the interactions by examining the impact of GP on oral pharmacokinetics of ginsenoside Rb1 (Rb1), the dominant protopanoxadiol (PPD)-type ginsenoside in Ginseng, on a dextran sulphate sodium (DSS) induced experimental colitis model which was characterized by gut dysbiosis, and to delineate the underlying mechanisms in vitro. MATERIALS AND METHODS: Rats received drinking water (normal group), 5% DSS (UC group), or 5% DSS plus daily oral administration of GP (GP group) for 7 days and fecal samples were collected on day -3, 0 and 6. On day 7 all animals received an oral dosage of Rb1 and blood samples were withdrawn for pharmacokinetic study. The in vitro metabolism study of Rb1 in gut microbiota from normal and UC rats and the transport study of Rb1 across Caco-2 cell monolayer were carried out in presence/absence of GP. Rb1 and its bacterial metabolites ginsenoside Rd (Rd), ginsenoside F2 (F2), Compound K (CK) and PPD were determined using LC-MS/MS. Total and target bacteria in fecal samples were determined by using 16S rRNA-based RT-PCR. ß-Glucosidase activity was determined by measuring 4-nitrophenol formed from 4-nitrophenyl-ß-D-glucopyranoside hydrolysis. RESULTS: DSS induction did not alter AUC0-t and Cmax of Rb1, which, however, were doubled together with elevated AUC0-t of the metabolites, in particular Rd and CK, in GP group. GP influenced the microbial composition and showed a prebiotic-like effect. Accordingly, GP treatment could partially restore the ß-glucosidase activity which was reduced by DSS induction. The presence of GP resulted in quicker microbial metabolism of Rb1 and higher Rd formation in first 8 h of incubation, while the impact on F2 and CK formation/conversion became obvious after 8 h. More interestingly, GP slightly stimulated Caco-2 cell growth and facilitated Rb1 transport across the Caco-2 monolayer in both directions, increasing the Papp of Rb1 from 10-7 cm/s to 10-6 cm/s. CONCLUSIONS: GP alleviated DSS-induced colitis-like symptoms and enhanced the systemic exposure of Rb1 through enhancing microbial deglycosylation and intestinal epithelial absorption of Rb1. These findings further demonstrated the important role of gut microbiota in the multifaceted action of polysaccharides in the holistic actions of traditional decoction of TCMs.

[Regulatory mechanisms of gut microbiota on intestinal CYP3A and P-glycoprotein in rats with dextran sulfate sodium-induced colitis].

As important constituents of the first-line of host defense barrier, intestinal cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp) play important roles in disease pathogenesis as well as drug absorption and exposure. Clinical reports and experimental data revealed diminished intestinal CYP3 A and P-gp expression accompanying with gut dysbiosis in inflammatory bowel disease. Yet whether gut dysbiosis is associated with the down-regulation of CYP3A and P-gp and the underlying mechanisms are unclear. In this study, daily administration of fresh feces from normal rats and rats with ulcerative colitis (UC) induced by dextran sulfate sodium to normal rats resulted in alterations of gut bacterial compositions. Intestinal CYP3A2 and P-gp were significantly down-regulated in rats receiving UC feces. Outer-membrane vesicles (OMVs) are nano-scale special buds of the outer membrane which are produced by Gram-negative bacteria and mediate diverse functions including interactions within bacterial communities and communications with host. Expressions of CYP3A4 and P-gp m RNA were diminished in human epithelial colorectal adenocarcinoma cells (Caco-2) treated by OMVs from all different groups with OMVs from UC rats or rats receiving UC feces showing more significant effects. Moreover, the OMVs fractions within 30 000­50 000 Daltons from both normal and UC rats elicited more effects than fractions of other molecular weights. Treatment of Caco-2 cells with toll like receptor 4 (TLR4) inhibitor resatorvid (TAK-242) or TLR4 silence RNA (siRNA) blocked CYP3A4 and P-gp down-regulation induced by bacterial OMVs. Taken together, we proved in this study that gut microbiota can down-regulate intestinal CYP3A and P-gp partially through producing OMVs to activate the TLR4 signaling pathway.