[Correlation of gut microbiota and ischemic stroke: a review].

With the widespread application of next-generation sequencing(NGS), especially 16 S rRNA and shotgun sequencing, researchers are no longer troubled with massive data on the gut microbiota, and the correlation between the gut microbiota and the brain(central nervous system) has been gradually revealed. Research on the microbiota-gut-brain axis(MGBA) based on the gut microbiota have provided insights into the exploration of the pathogenesis and risk factors of ischemic stroke(IS), a cerebrovascular disease with high disability and mortality rates, and also facilitate the selection of therapeutic targets of this class of drugs. This study reviewed the application of NGS in the study of gut microbiota and the research progress of MGBA in recent years and systematically collated the research papers on the correlation between IS and gut microbiota. Furthermore, from the bi-directional regulation of MGBA, this study also discussed the high-risk factors of IS under the dysregulation of gut microbiota and the pathophysiological changes of gut microbiota after the occurrence of IS and summarized the related targets to provide a reliable reference for the therapeutic research of IS from the gut microbiota.

Expression of AQP2, AQP4 and AQP 8 in mouse intestine induced by unprocessed and processed Euphorbia lathyris.

The present research was designed to study expression of AQP2, AQP4 and AQP8 in mouse intestines induced by unprocessed and processed Euphorbia lathyris. KM mice were given by different dose lavage of unprocessed and processed Euphorbia lathyris, Euphorbia factor L1, Euphorbia factor L2, Euphorbia factor L3. Samples of mouse intestine were collected for protein levels of AQP2, AQP 4 and AQP 8 which were assessed by immunohistochemical staining and mRNA expression of AQP2, AQP 4 and AQP 8 which were quantified by Real Time-PCR. Comparing to the normal control group, the protein levels of AQP2, AQP 4 and AQP 8 were significantly decreased (P<0.05)by Semen Euphorbiae group and Semen Euphorbiae Pulveratum group (unprocessed and processed Euphorbia lathyris) induced. Protein expression of AQP2, AQP 4 and AQP 8 in the Euphorbia factor L1, Euphorbia factor L2 and Euphorbia factor L3 group were not significantly lower than normal control group. There had no differences on the levels of AQP2 and AQP 8 mRNA expressions between the high-dose group of semen Euphorbiae group, semen Euphorbiae Pulveratum group and positive control group, while significantly lower than normal control group (P<0.05). Expression of AQP4 mRNA in the Semen Euphorbiae group and Semen Euphorbiae Pulveratum group has not significantly decreased. But levels of AQP2, AQP 4 and AQP 8 mRNA in the Euphorbia factor L1 group had no significant differences in normal control group and positive control group. These findings suggest that semen Euphorbiae could regulate expression of AQP2, AQP 4 and AQP 8 protein and mRNA, which may be the possible one reason of semen Euphorbiae induces diarrhea. The semen Euphorbiae group has more significant effects on the levels of AQP2, AQP 4 and AQP 8 protein and mRNA than semen Euphorbiae Pulveratum group, which may be one of the mechanisms of processing attenuation.

[Research and application of quercetin-loaded nano drug delivery system].

Quercetin has a variety of biological activities and pharmacological effects, but its clinical application is limited due to its low water solubility, low bioavailability and poor chemical stability. In order to overcome the above shortcomings of quercetin and achieve better clinical efficacy, many new drug delivery systems have been studied and developed in recent years, of which nano drug delivery system has become a research hotspot. Nano drug delivery system includes nanoparticles, nanocapsules, nanoliposomes, nanomicelles, nanosuspensions and nanoemulsions, with the advantages of reducing particle size, enlarging surface area, increasing drug permeability, improving intracorporeal circulation and distribution of the drug, enhancing drug targeting and so on. This article reviewed the latest progress at home and abroad about the studies and application of quercetin in nano drug delivery system, such as increasing solubility and dissolution, elevating bioavailability, raising drug stability, delaying drug release, enhancing skin permeability, promoting antioxidant capacity and improving therapeutic effect, in the hope of providing references for further research of quercetin preparations.

Synergism and rules of the new combination drug Yiqijiedu formulae (YQJD) on ischemic stroke based on amino acids (AAs) metabolism.

The use of combination drugs is considered to be a promising strategy to control complex diseases such as ischemic stroke. The detection of metabolites has been used as a versatile tool to reveal the potential mechanism of diverse diseases. In this study, the levels of 12 endogenous AAs were simultaneously determined quantitatively in the MCAO rat brain using RRLC-QQQ method. Seven AAs were chosen as the potential biomarkers, and using PLS-DA analysis, the effects of the new combination drug YQJD, which is composed of ginsenosides, berberine, and jasminoidin, on those 7 AAs were evaluated. Four AAs, glutamic acid, homocysteine, methionine, and tryptophan, which changed significantly in the YQJD-treated groups compared to the vehicle groups (P < 0.05), were identified and designated as the AAs to use to further explore the synergism of YQJD. The result of a PCA showed that the combination of these three drugs exhibits the strongest synergistic effect compared to other combination groups and that ginsenosides might play a pivotal role, especially when combined with jasminoidin. We successfully explored the synergetic mechanism of multi-component and provided a new method for evaluating the integrated effects of combination drugs in the treatment of complex diseases.

[Determination of endogenous amino acids in brain tissues after cerebral ischemia by RRLC-QQQ].

OBJECTIVE: To establish a method to determine underivatized endogenous amino acids in brain tissues after cerebral ischemia based on RRLC-QQQ. METHOD: Diamonsil chromatographic column C18 (4.6 mm x 250 mm, 5 microm) was adopted to determine 12 amino acids in 15 min, with acetonitrile-0.1% formic acid for gradient elution. The flow rate was set at 0.5 mL x min(-1). With ESI as the ion source, positive ion scanning mode was adopted for multi-reaction monitoring. RESULT: Each amino acid standard curve (AAs) showed good linear relationship within the detection range (r > 0.996), with the limit of detection of less than 11%, the limit of quantitation of less than 3.09 microg x L(-1). The RSD of intra- and inter-day precisions at high, middle and low concentrations were less than 11%. CONCLUSION: The determination results of actual samples showed that compared with the levels of AAs of the sham operation group, all of the remaining amino acids apart from N-acetyl-aspartate increased in brain tissues. Some amino acids showed significant changes in a time-dependent manner after the operation. The method is so simple, rapid and sensitive that it can be used for finding biological metabolite markers of cerebral ischemia, and exploring cerebral ischemia molecular mechanisms and synergistic mechanism of combined administration of multi-component traditional Chinese medicines.

Differential gene expression profile of the calanoid copepod, Pseudodiaptomus annandalei, in response to nickel exposure.

To better understand the underlying mechanisms of reactions of copepods exposed to elevated level of nickel, the suppression subtractive hybridization (SSH) was used to elucidate the response of the copepod Pseudodiaptomus annandalei to nickel exposure at the gene level. P. annandale is one of a few copepod species that can be cultured relatively easy under laboratory condition, and it is considered to be a potential model species for toxicity study. In the present study, P. annandalei were exposed to nickel at a concentration of 8.86 mgL(-1) for 24h, after which the RNA was prepared for SSH using unexposed P. annandalei as drivers. A total of 474 clones on the middle scale in the SSH library were sequenced. Among these genes, 129 potential functional genes were recognized based on the BLAST searches in NCBI and Uniprot databases. These genes were then categorized into nine groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 129 genes, 127 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains (CD) and proteins using the CD-Search service and BLASTp. Among 129 genes, 119 (92.2%) were annotated to be involved in different biological processes, while 10 genes (7.8%) were classified as an unknown-function gene group. To further confirm the up-regulation of differentially expressed genes, the quantitative real time PCR were performed to test eight randomly selected genes, in which five of them, i.e. α-tubulin, ribosomal protein L13, ferritin, separase and Myohemerythrin-1, exhibited clear up-regulation after nickel exposure. In addition, MnSOD was further studied for the differential expression pattern after nickel exposure and the results showed that MnSOD had a time- and dose-dependent expression pattern in the copepod after nickel exposure. To the best of our knowledge, this is the first attempt to investigate the toxicity effects of nickel on a copepod at molecular level.

[Advance of studies on metabolic fingerprinting analytical techniques and data processing methods].

Metabolomics is an emerging discipline subsequent to genomics, transcriptomics and proteomics, aiming for systematically studying the regularity of changes in metabolite to revealing organism's nature of movement and metabolism. It is especially important in modern pharmacological studies. Metabolic fingerprinting analysis is a method for metabolic analysis on high throughput of all metabolites, studying changes in drugs, organisms and endogenic metabolites caused by drugs and finding out related biomarkers to reflect dynamic changes inside organisms more directly and explain the mechanism of drugs and their effects on diseases. This essay summarizes some new metabolic fingerprint analytical methods and data processing methods used for metabolic fingerprint, elaborates their advantages and disadvantages and looks ahead to their combination with studies on traditional Chinese medicines, providing room for the development of new methods and new approaches for studies on complexity theory system of traditional Chinese medicines.

[High throughput screening for glutathione S-transferase inhibitors].

To identify the inhibitor of glutathione S-transferase (GST), a high-throughput screening method was established in a 384-well microplate with total 35 microL volume, and the absorbance at 340 nm is detected. The concentrations of substrates, CDNB and GST were determined by chromatometry. The optimal enzyme kinetics reaction time and temperature are 2 h and 30 degrees C , respectively. The established model was evaluated by NaOCl, a known GST inhibitor, and the parameter Z' was 0.77, which showed a high feasibility and stability of the assay. A total of 31,098 compounds were screened, of which 4 compounds were shown to inhibit GST activity, high inhibiting activity for their IC50 of GST inhibition was 3.94, 4.05, 74.85, and 77.41 mg x L(-1), separately. The results indicated that the colorimetric method by using CDNB and GSH as substrate is stable, sensitive, reproducible and also suitable for high throughput screening.

Genomic organization and tissue-specific expression analysis of hepcidin-like genes from black porgy (Acanthopagrus schlegelii B).

Hepcidin is an antimicrobial peptide and putative iron regulatory hormone previously described in mice and humans. Dozens of fish hepcidins have been isolated and characterized so far. Here we present seven hepcidin-like cDNA sequences named AS-hepc1-7, amplified from the normal commercially cultured fish (black porgy) by RACE-PCR. Sequence analysis reveals that these seven potential hepcidin peptides have highly conserved sequences with other known hepcidins, but they are different from each other in constitution and characteristics of predicted mature amino acids. Based on the study, it is deduced that AS-hepc1-7 represent different variants of a family of hepcidin genes in black porgy. To understand the organization of these hepcidin-like genes, we sequenced AS-hepc2 DNA, AS-hepc3 DNA, AS-hepc4 DNA, AS-hepc7 DNA and AS-hepc2 upstream region; and all of the four genomic DNAs consisted of two introns and three exons, the same organization as other reported hepcidins. The tissue-specific gene expression of hepcidins in normal black porgy was evaluated using RT-PCR and dot blot approaches. RT-PCR showed that transcripts of hepcidin-like mRNAs were present in each tested tissue of normal juvenile black porgy, including liver, spleen, kidney, heart, brain, stomach, intestine, gill, skin and blood, but abundant hepcidin-like mRNA transcripts were only detected in the liver, kidney, spleen, intestine and stomach by dot blot assay. In addition, using dot blot and Northern blot approach, a significant increase of hepcidin mRNA transcription was observed in the liver within 48 h after immersion in a suspension of live bacteria, which suggested that the expression pattern of hepcidin-like genes in black porgy might be different in the liver from the other tissues as previously reported in several hepcidin studies.