Transcriptomic analysis of the HPG axis-related tissues reveals potential candidate genes and regulatory pathways associated with testicular size in Hu sheep.

Testicular size is an excellent proxy for selecting high-fertility rams. The hypothalamus-pituitary-gonadal (HPG) axis plays an important role in regulating reproductive capacity in vertebrates, while key genes and regulatory pathways within the HPG axis associated with testicular size remain largely unknown in sheep. This study comprehensively compared the transcriptomic profiles in the hypothalamus, pituitary and testis of rams after sexual maturity between the large-testis group (LTG, testicular weight = 454.29 ± 54.24 g) and the small-testis group (STG, testicular weight = 77.29 ± 10.76 g). In total, 914, 795 and 10518 differentially expressed genes (DEGs) were identified in the hypothalamus, pituitary and testis between LTG and STG, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that these DEGs were mainly involved in the biological processes of reproduction, biological regulation, and development process. Notably, the neuroactive ligand-receptor interaction and cAMP signaling pathways, commonly enriched by the DEGs in the hypothalamus and pituitary between two groups, were considered as two key signal pathways regulating testicular development through the HPGs axis. Weighted gene co-expression network analysis (WGCNA) identified two modules that were significantly associated with testicular size, and 97 key genes were selected with high module membership (MM) and gene significance (GS) in these two modules. Finally, a protein-protein interaction (PPI) network was constructed, and ten genes with the highest degree were represented as hub genes, including FOS, NPY, SST, F2, AGT, NTS, OXT, EDN1, VIP and TAC1. Taken together, these results provide new insights into the molecular mechanism underlying the HPG axis regulating testicular size of Hu sheep.

High-throughput screening and quantification of pesticides in Lilii Bulbus using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Lilii Bulbus is a notable flower in Chinese cuisine, and has also been used as a Chinese herbal medicine for over 2000 years. This work presents an analytical method for rapidly screening multiple pesticide residues in Lilii Bulbus using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). For sample pre-treatment, the QuEChERS method is employed, and targeted MS/MS is adopted for data acquisition. Moreover, a database containing 515 pesticides with accurate mass database and a high-resolution fragment ion spectrum library is established in this work. In addition, the qualitative and quantitative results of the screening method are validated. The results show that within the linear concentration range of 2 to 200 µg L-1, for each pesticide, 89.3% of the pesticides exhibit linear correlation coefficients R2 equal to or exceeding 0.990. The limit of quantification for all pesticides is below 50 µg kg-1. With a recovery of 70% to 120% and RSD ≤ 20% as the satisfactory standards, 387 (75.0%), 411 (79.7%) and 420 (81.4%) pesticides meet the standards at the three addition levels of 10 µg kg-1, 20 µg kg-1, and 100 µg kg-1, respectively. By utilizing the proposed method, pesticide residues in 100 samples are investigated, providing scientific data to ensure the safety of pesticide residues and demonstrating the general applicability of the method for routine monitoring of pesticide residues in Lilii Bulbus.

Synthesis and Biological Evaluation of Gentiopicroside Derivatives as Novel Cyclooxygenase-2 Inhibitors with Anti-Inflammatory Activity.

Purpose: Nonsteroidal anti-inflammatory drugs cause a series of adverse reactions. Thus, the search for new cyclooxygenase-2 selective inhibitors have become the main direction of research on anti-inflammatory drugs. Gentiopicroside is a novel selective inhibitor of cyclooxygenase-2 from Chinese herbal medicine. However, it is highly hydrophilic owing to the presence of the sugar fragment in its structure that reduces its oral bioavailability and limits efficacy. This study aimed to design and synthesize novel cyclooxygenase-2 inhibitors by modifying gentiopicroside structure and reducing its polarity. Materials and Methods: We introduced hydrophobic acyl chloride into the gentiopicroside structure to reduce its hydrophilicity and obtained some new derivatives. Their in vitro anti-inflammatory activities were evaluated against NO, TNF-α, PGE2, and IL-6 production in the mouse macrophage cell line RAW264.7 stimulated by lipopolysaccharide. The in vivo inhibitory activities were further tested against xylene-induced mouse ear swelling. Molecular docking predicted that whether new compounds could effectively bind to target protein cyclooxygenase-2. The inhibitory activity of new compounds to cyclooxygenase-2 enzyme were verified by the in vitro experiment. Results: A total of 21 novel derivatives were synthesized, and exhibit lower polarities than the gentiopicroside. Most compounds have good in vitro anti-inflammatory activity. The in vivo activity results demonstrated that 8 compounds were more active than gentiopicroside. The inhibition rate of some compounds was higher than celecoxib. Molecular docking predicted that 6 compounds could bind to cyclooxygenase-2 and had high docking scores in accordance with their potency of the anti-inflammatory activity. The confirmatory experiment proved that these 6 compounds had significant inhibitory effect against cyclooxygenase-2 enzyme. Structure-activity relationship analysis presumed that the para-substitution with the electron-withdrawing groups may benefit the anti-inflammatory activity. Conclusion: These gentiopicroside derivatives especially PL-2, PL-7 and PL-8 may represent a novel class of cyclooxygenase-2 inhibitors and could thus be developed as new anti-inflammatory agents.

Quantitative Analysis of the Multicomponent and Spectrum-Effect Correlation of the Antispasmodic Activity of Shaoyao-Gancao Decoction.

Shaoyao-Gancao Decoction (SGD) is a well-known classic traditional Chinese medicine (TCM) with antispasmodic, anti-inflammatory, and analgesic effects. This preparation has been widely used to treat spasticity diseases in the clinic. To date, the material basis of SGD remains unclear, and the spectrum-effect correlation of its antispasmodic activity has not been reported yet. In this study, high-performance liquid chromatography (HPLC) was used to establish the fingerprint and determine the multiple components of SGD. The common peaks of fingerprints were evaluated by the similarity with the chromatographic fingerprints of the TCM. Meanwhile, the multiple components were quantified and analysed using the heatmap and box size analysis. Furthermore, data on the antispasmodic effect were extracted through in vitro smooth muscle contraction assay. Grey relational analysis combined with partial least square regression was used to study the spectrum-effect correlation of SGD. Finally, the potential antispasmolytic components were validated using an isolated tissue experiment. The HPLC fingerprint was established, and 20 common peaks were identified. The similarities of 15 batches of SGD were all above 0.965. The HPLC method for simultaneous determination of the multiple components was accurate and reliable. The contents of albiflorin, paeoniflorin, liquiritin, and glycyrrhizic acid were higher than the other components in SGD. The heatmap and box size also showed that X3 (albiflorin), X4 (paeoniflorin), X5 (liquiritin), X11 (liquirtigenin), and X16 (glycyrrhizic acid) could be used as quality indicators in the further establishment of quality standards. The spectrum-effect correlation results indicated that X4, X11, and X16 were highly correlated with antispasmolytic activity. Verification tests showed that paeoniflorin (11.7-29.25 µg/mL) and liquirtigenin (17.19-28.65 µg/mL) could significantly reduce the maximum contractile (P < 0.01). These compounds exerted concentration-dependent spasmolytic effects with the inhibitory response for acetylcholine (Ach)-evoked contraction. Thus, SGD had a significant antispasmodic effect, which resulted from the synergistic activity of its multiple components. These findings can be used for the pharmacodynamics study of SGD and are of great significance for the determination of quality markers and quality control.

Epigallocatechin Gallate Protects against MNNG-Induced Precancerous Lesions of Gastric Carcinoma in Rats via PI3K/Akt/mTOR Pathway.

OBJECTIVE: To evaluate the therapeutic effect of epigallocatechin gallate (EGCG) on precancerous lesions of gastric carcinoma (PLGC) and to determine whether EGCG protects against PLGC by regulating PI3K/Akt/mTOR pathway. METHODS: Twenty-four male Wistar rats were randomly divided into 3 groups: normal control group (NC), PLGC model group (MC), and group of PLGC rats treated with EGCG (MC + EGCG). 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG) and sodium salicylate were combined and used to establish the PLGC rat animal model. The therapeutic effect of EGCG on PLGC was evaluated by body weight and pathological lesions of gastric mucosa in PLGC rats. Quantitative polymerase chain reaction (qPCR) was applied to measure the mRNA expressions of PI3K, Akt, and mTOR. The protein expressions of cleaved caspase-3, PTEN, PI3K, p-PI3K, Akt, p-Akt, p-mTOR, and mTOR were determined by automated western immunoblotting. RESULTS: The body weight decreased in PLGC rats while EGCG significantly increased body weight. The gastric mucosa of PLGC rats exhibited the pathological lesions of atrophy, intestinal metaplasia, and atypical hyperplasia while EGCG could ameliorate the pathological lesions. EGCG could upregulate the expressions of cleaved caspase-3 and PTEN and reduce the expressions of PI3K, Akt, and mTOR. CONCLUSIONS: EGCG ameliorated pathological lesions of PLGC and exerted the effect of apoptosis promotion in PLGC rats. The apoptotic pathway triggered by EGCG may be related to inhibition of PI3K/Akt/mTOR pathway. It provided a theoretical basis for the PLGC treatment and gastric cancer prevention.

Anti-tumor effect of the extract from qingyihuaji formula on pancreatic cancer by down-regulating Notch-4 and Jagged-1.

OBJECTIVE: To investigate, in terms of Notch signaling pathway, the effect on pancreatic cancer of the extract of an anti-tumor prescription--Qingyi-huaji formula (QYHJ)--from Traditional Chinese Medicine (TCM). METHODS: Nude mice were implanted subcutaneously with human pancreatic cancer cell line SW1990 and then randomly divided into four groups: Control, QYHJ extract, Gemcitabine, and Combination of QYHJ extract and gemcitabine. Treatments were given for 21 days and tumor growth was evaluated simultaneously. Then, expression of Notch receptors (Notch-1, Notch-2, Notch-3, and Notch-4) and their Jagged ligands (Jagged-1 and Jagged-2) in dissected tumor tissue were detected by real-time quantitative reverse transcription-polymerase chain reaction and Western blot. Finally, immunohistochemistry was performed to detect CD133, a marker of pancreatic cancer stem cells (CSCs), to evaluate the impact of QYHJ extract on pancreatic CSCs. RESULTS: QYHJ extract treatment effectively inhibited the tumor growth in nude mice. The expression of both Notch-4 and Jagged-1 were decreased significantly in QYHJ treatment groups (P < 0.05), while gemcitabine alone had no significant effect in down-regulating Jagged-1 (P > 0.05). No significant difference was observed in the ex- pression of Notch-1, Notch-2, Notch-3, and Jagged-2 between three treatment groups and control group (P > 0.05). Moreover, immunohistochemical analysis showed that the number of CD133 positive cells was significantly reduced by QYHJ treatment (P < 0.05), and the combined treatment was more effective than gemcitabine alone (P < 0.05). CONCLUSION: The role of the extract in pancreatic cancer treatment was associated with down-regulation of Notch-4 and Jagged-1 in Notch signaling pathway. The extract could enhance the antitumor activity of gemcitabine and was more effective than gemcitabine in regulating Notch signaling pathway to some extent.

Qingyihuaji Formula Inhibits Pancreatic Cancer and Prolongs Survival by Downregulating Hes-1 and Hey-1.

The dire prognosis of pancreatic cancer has not markedly improved during past decades. The present study was carried out to explore the effect of Qingyihuaji formula (QYHJ) on inhibiting pancreatic cancer and prolonging survival in related Notch signaling pathway. Proliferation of pancreatic cancer cells (SW1990 and PANC-1) was detected by MTT assay at 24, 48, and 72 h with exposure to various concentrations (0.08-50 mg/mL) of QYHJ water extract. Pancreatic tumor models of nude mice were divided into three groups randomly (control, QYHJ, and gemcitabine). mRNA and protein expression of Notch target genes (Hes-1, Hey-1, Hey-2, and Hey-L) in dissected tumor tissue were detected. Results showed that proliferation of SW1990 cells and PANC-1 cells was inhibited by QYHJ water extract in a dose-dependent and time-dependent manner. QYHJ effectively inhibited tumor growth and prolonged survival time in nude mice. Expression of both Hes-1 and Hey-1 was decreased significantly in QYHJ group, suggesting that Hes-1 and Hey-1 in Notch signaling pathway might be potential targets for QYHJ treatment. This research could help explain the clinical effectiveness of QYHJ and may provide advanced pancreatic cancer patients with a new therapeutic option.

Discovery of potent and selective sirtuin 2 (SIRT2) inhibitors using a fragment-based approach.

Sirtuin 2 (SIRT2) is one of the sirtuins, a family of NAD(+)-dependent deacetylases that act on a variety of histone and non-histone substrates. Accumulating biological functions and potential therapeutic applications have drawn interest in the discovery and development of SIRT2 inhibitors. Herein we report our discovery of novel SIRT2 inhibitors using a fragment-based approach. Inspired by the purported close binding proximity of suramin and nicotinamide, we prepared two sets of fragments, namely, the naphthylamide sulfonic acids and the naphthalene-benzamides and -nicotinamides. Biochemical evaluation of these two series provided structure-activity relationship (SAR) information, which led to the design of (5-benzamidonaphthalen-1/2-yloxy)nicotinamide derivatives. Among these inhibitors, one compound exhibited high anti-SIRT2 activity (48 nM) and excellent selectivity for SIRT2 over SIRT1 and SIRT3. In vitro, it also increased the acetylation level of α-tubulin, a well-established SIRT2 substrate, in both concentration- and time-dependent manners. Further kinetic studies revealed that this compound behaves as a competitive inhibitor against the peptide substrate and most likely as a noncompetitive inhibitor against NAD(+). Taken together, these results indicate that we have discovered a potent and selective SIRT2 inhibitor whose novel structure merits further exploration.

Determination of aflatoxins B1, B2, G1, and G2 in olive oil, peanut oil, and sesame oil using immunoaffinity column cleanup, postcolumn derivatization, and liquid chromatography with fluorescence detection: first action 2013.05.

A collaborative study of a method for determination of aflatoxins (AFs) B1, B2, G1, and G2 in olive oil, peanut oil, and sesame oil using immunoaffinity column cleanup, postcolumn derivatization, and LC with fluorescence detection, previously published in J. AOAC Int. 95, 1689-1700 (2012), was approved as First Action 2013.05 on March 29, 2013 by the Method-Centric Committee for Aflatoxins in Edible Oils. The method uses methanol for extraction followed by filtration. The extract is applied to an immunoaffinity column with antibodies specific for AFs, which are then eluted from the column with a methanol solution. Determination and quantification occur using RP-LC with fluorescence detection after postcolumn derivatization. The average recovery of AFs in olive, peanut, and sesame oils in spiked samples (levels between 2.0 and 20.0 microg/kg) ranged from 84 to 92%. The recoveries for AFs B1, B2, G1, and G2 were 86-93, 89-95, 85-97, and 76-85%, respectively. Within-laboratory RSD (RSDr) values for AFs ranged from 3.4 to 10.2%. RSDr values forAF B1, B2, G1, and G2 were 3.5-10.9, 3.2-9.5, 6.5-14.9, and 4.8-14.2%, respectively. Between-laboratory RSD (RSDR) values for AFs were 6.1-14.5%. RSD, values for AFs B1, B2, G1, and G2 were 7.5-15.4, 7.1-14.6, 10.8-18.1, and 7.6-23.7%, respectively. Horwitz ratio values were < or =2 for the analytes in the three matrixes.

Influence of epidermal growth factor receptor inhibitor AG1478 on epithelial-mesenchymal transition in endometrial carcinoma cells.

OBJECTIVE: To analyze the influence of epidermal growth factor receptor inhibitor AG1478 on the proliferation and epithelial-mesenchymal transition in endometrial carcinoma cells. MATERIALS AND METHODS: The inhibitory effect of different concentrations of AG1478 on the proliferation of endometrial carcinoma cells was detected by tetrazolium-based assay. Western blot was applied to investigate the influence of AG1478 on expressions of epithelium-cadherin, α-catenin, neural cadherin, vimentin, matrix metalloproteinase 9 (MMP9), and MMP2 protein in endometrial carcinoma cells. The influence of AG1478 on migration and invasion of endometrial carcinoma cells was examined by Transwell migration and invasion assay, respectively. RESULTS: AG1478 could suppress the proliferation of different endometrial carcinoma cells, and cells transfected with epidermal growth factor receptor were more sensitive (P < 0.05). The expression of an increase in epithelial marker proteins and a decrease in mesenchymal marker proteins, MMP9, MMP2 were observed in endometrial carcinoma cells after AG1478 treated. This effect was more obvious in cells transfected with epidermal growth factor receptor (P < 0.05). The migration and invasion ability of endometrial carcinoma cells were suppressed by AG1478, and Ishikawa cells transfected with epidermal growth factor receptor were demonstrated to be more sensitive to AG1478 (P < 0.05). CONCLUSIONS: Epidermal growth factor receptor inhibitor AG1478 could effectively inhibit the proliferation and epithelial-mesenchymal transition of endometrial carcinoma cells.

Determination of aflatoxins B1, B2, G1, and G2 in olive oil, peanut oil, and sesame oil using immunoaffinity column cleanup, postcolumn derivatization, and liquid chromatography/fluorescence detection: collaborative study.

The accuracy, repeatability, and reproducibility characteristics of a method using immunoaffinity column (IAC) cleanup with postcolumn derivatization and LC with a fluorescence detector (FLD) for determination of aflatoxins (AFs; sum of AFs B1, B2, G1, and G2) in olive oil, peanut oil, and sesame oil have been established in a collaborative study involving 15 laboratories from six countries. Blind duplicate samples of blank, spiked at levels ranging from 0.25 to 20.0 microg/kg for AF, were analyzed. A naturally contaminated peanut oil sample was also included. Test samples were extracted with methanol-water (55 + 45, v/v). After shaking and centrifuging, the lower layer was filtered, diluted with water, and filtered through glass microfiber filter paper. The filtrate was then passed through an IAC, and the toxins were eluted with methanol. The toxins were then subjected to RPLC-FLD analysis after postcolumn derivatization. Average recoveries of AFs from olive oil, peanut oil, and sesame oil ranged from 84 to 92% (at spiking levels ranging from 2.0 to 20.0 microg/kg); of AFB1 from 86 to 93% (at spiking levels ranging from 1.0 to 10.0 microg/kg); of AFB2 from 89 to 95% (at spiking levels ranging from 0.25 to 2.5 microg/kg); of AFG1 from 85 to 97% (at spiking levels ranging from 0.5 to 5.0 microg/kg); and of AFG2 from 76 to 85% (at spiking levels ranging from 0.25 to 2.5 microg/kg). RSDs for within-laboratory repeatability (RSD(r)) ranged from 3.4 to 10.2% for AF, from 3.5 to 10.9% for AFB1, from 3.2 to 9.5% for AFB2, from 6.5 to 14.9% for AFG1, and from 4.8 to 14.2% for AFG2. RSDs for between-laboratory reproducibility (RSDR) ranged from 6.1 to 14.5% for AF, from 7.5 to 15.4% for AFB1, from 7.1 to 14.6% for AFB2, from 10.8 to 18.1% for AFG1, and from 7.6 to 23.7% for AFG2. Horwitz ratio values were < or = 2 for the analytes in the three matrixes.

Synthesis and evaluation of novel neamine derivatives effectively targeting to RNA.

Three new derivatives of neamine, 3 (NE), 6 (NEA) and 9 (NEL), were synthesized by connecting arginine or lysine to 5-hydroxyl group of neamine using ethylenediamine as a linker. The binding affinities of these derivatives to A site of 16S RNA and TAR RNA indicate that the modification on 5-hydroxyl of neamine by amino acid can enhance the binding affinity of neamine. Compound 9 (NEL) shows some antibacterial activities. These results demonstrate that modification on 5-hydroxyl group of neamine may provide a promising way for the development of potential candidates effectively targeting to RNAs.

[Effects of radix Astragali injection on apoptosis of lymphocytes and immune function in patients with systemic lupus erythematosus].

OBJECTIVE: To investigate the effect of Radix Astragali Injection on apoptosis of lymphocytes and immune function in treating patients with systemic lupus erythematosus (SLE). METHODS: Eighty SLE patients were randomly assigned into the routine treatment group (RT) treated with conventional therapy and the Radix Astragali treated group (RA) treated with Radix Astragali Injection besides routine treatment. The expressions of Fas and Bcl-2 antigen on lymphocytes and the changes of T lymphocyte subsets in peripheral blood before and after treatment were observed. RESULTS: After treatment, the expression of Fas antigen on lymphocytes significantly lowered (P < 0.01), and that of Bcl-2 antigen, CD4+ lymphocyte subset and CD4+ / CD8+ ratio significantly increased in both groups (all P < 0.01). However, the changes of Fas antigen expression, CD4+ and CD4+ / CD8+ ratio were more significant in the RA group than those in the RT group (P < 0.05). CONCLUSION: Radix Astragli Injection can enhance the inhibitory function of corticosteroid/immunosuppressant on apoptosis, and regulate the ratio and function of T lymphocyte subsets to normal range, which may be a useful approach for enhancing the efficacy of treatment to SLE.

Clinical observation on treatment of chronic aplastic anemia by Shengxuening and cyclosporin A.

OBJECTIVE: To explore the therapy to further elevate the efficacy of the treatment of chronic aplastic anemia (CAA). METHODS: Forty-five patients with CCA were assigned into two groups, the 26 patients in the treated group were treated by Shengxuening (a Chinese herbal preparation) and cyclosporin A (CsA), and the 19 patients in the control group were treated with androgen alone, with the therapeutic course lasting for over 3 months. Changes of peripheral blood picture, and the colony productivity of burst forming unit-erythroid (BFU-E), colony forming unit-erythroid (CFU-E) and colony forming unit-granulocyte macrophage (CFU-GM) in bone marrow were observed before and after 3 months treatment. The amount of erythrocyte and platelet infusion, frequency of infection, condition of hemorrhage and relevant death were also observed. The follow-up study was conducted for over half a year. RESULTS: The total effective rate in the treated group was 84.6%, which was significantly higher than that in the control group (52.6%, P < 0.05). Levels of hemoglobin, reticulocyte, neutrophil and platelet increased after treatment in the treated group, as compared with those before treatment, with significant difference (P < 0.05), and the colony productivity of BFU-E, CFU-E and CFU-GM in bone marrow also got significantly increased (P < 0.01), and showed significant difference from those in the control group (P < 0.05). CONCLUSION: Shengxuening-assisting CsA therapy is an effective measure for treatment of CAA.

[Effects of Lycium barbarum polysaccharide on tumor microenvironment T-lymphocyte subsets and dendritic cells in H22-bearing mice].

OBJECTIVE: To study the effects of Lycium barbarum polysaccharide (LBP) on tumor microenvironment T-lymphocyte subsets and dendritic cells in H22-bearing mice and the mechanisms for intervention of tumor immune escape by LBP. METHODS: H22-bearing mice were given LBP orally for two weeks. T-lymphocyte subsets and the phenotypes of dendritic cells in tumor-infiltrating lymphocytes (TIL) were detected by flow cytometry (FCM). RESULTS: LBP could significantly increase the numbers of CD4(+) and CD8(+) T cells in TIL as compared with those in model control group (P<0.05). In model control group, the number of dendritic cells in tumor microenvironment decreased markedly, while in LBP-treated group, the increased number of dendritic cells and B7-1 expression were observed, but there were no significant differences between these two groups. CONCLUSION: LBP has anti-tumor effect probably by increasing the numbers of CD4(+) and CD8(+) T cells in TIL to relieve the immunosuppression and enhance the anti-tumor function of the immune system. But whether LBP can recover the phenocyte and function of dendritic cells in H22-bearing mice should be further studied.