Asperuloside suppressing oxidative stress and inflammation in DSS-induced chronic colitis and RAW 264.7 macrophages via Nrf2/HO-1 and NF-κB pathways.

BACKGROUND: Inflammatory bowel diseases (IBDs), which mainly include Crohn's disease (CD) and ulcerative colitis (UC), are chronic idiopathic inflammatory disease of the gastrointestinal tract for which effective pharmacological treatments are lacking or options are very limited. PURPOSE: Here, we aim to investigate the therapeutic effects of an iridoid glycoside, asperuloside (ASP) on mice experimental chronic colitis induced by dextran sulfate sodium (DSS) and further explore underlying mechanisms in vitro and in vivo. METHODS: LPS-treated RAW 264.7 cells showed inflammation and were assessed for various physiological, morphological and biochemical parameters in the absence or presence of ASP. Chronic colitis was induced by 2% DSS in mice, which were used as an animal model to explore the pharmacodynamics of ASP. We detected p65 and Nrf2 pathway proteins via Western blot and RT-PCR analysis, assessed the cytokines TNF-α and IL-6 via ELISA, tested p65 and Nrf2 nuclear translocation via fluorescence. In addition, the docking affinity of ASP and p65 or Nrf2 proteins in the MOE 2015 software. RESULTS: We found that ASP attenuated weight loss, disease activity index (DAI) and colonic pathological damage in colitis mice and restored the expressions of inflammatory cytokines in the colon. In addition, ASP restored antioxidant capacity in DSS-induced chronic colitis mice and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Furthermore, ASP suppressed oxidative stress through increasing Nrf2, HO-1 and NQO-1 proteins expressions, and down-regulated nuclear levels of p65 to inhibit DSS-induced colonic oxidative stress and inflammation. Validation of the molecular docking results also indicated that ASP interacts with Nrf2 or p65 proteins. In summary, ASP improved DSS-induced chronic colitis by alleviating inflammation and oxidative stress, activating Nrf2/HO-1 signaling and limiting NF-κB signaling pathway, which may be an effective candidate for the treatment of IBD.

International consensus on natural orifice specimen extraction surgery (NOSES) for gastric cancer (2019).

At present, natural orifice specimen extraction surgery (NOSES) has attracted more and more attention worldwide, because of its great advantages including minimal cutaneous trauma and post-operative pain, fast post-operative recovery, short hospital stay, and positive psychological impact. However, NOSES for the treatment of gastric cancer (GC) is still in its infancy, and there is great potential to improve its theoretical system and clinical practice. Especially, several key points including oncological outcomes, bacteriological concerns, indication selection, and standardized surgical procedures are raised with this innovative technique. Therefore, it is necessary to achieve an international consensus to regulate the implementation of GC-NOSES, which is of great significance for healthy and orderly development of NOSES worldwide.

Mechanistic toxicity of DEHP at environmentally relevant concentrations (ERCs) and ecological risk assessment in the Three Gorges Reservoir Area, China.

Di-(2-ethylhexyl) phthalate (DEHP) associated in vitro/vivo toxicity at current environmentally relevant concentration (ERC) with attendant ecological risks in the Three Gorges Reservoir Area (TGRA) is still elusive. Responding to this challenge, a novel integrated study based on analytical and biological assays was designed to elucidate the underlying mechanisms for toxicity of DEHP and its ecological risks at ERC. In this study, GC-MS analysis showed that the highest environmental concentration of DEHP in the TGRA surface water was nearly double that of WHO and USEPA standards. Both distribution and ecological risk decreased from the upper to middle and lower reaches of the TGRA. In vitro toxicity was assessed by cell viability and DNA damage assays: DEHP exposure at ERCs (100-800 µg/L) caused significant reduction in cell viability and elevated DNA damage. Further, DEHP exposure above 400 µg/L resulted in enhanced migration behavior of cancer cells. For in vivo toxicity assessment, short term acute exposure (7 d, 400 µg/L) apparently activated the PI3K-AKT-mTOR pathway, and chronic low-level exposure (3 months, 10-33 µg/L) suppressed the hypothalamus pituitary thyroid (HPT) axis pathway in zebrafish. In addition, acute low-level exposure (5 d, 33-400 µg/L) to DEHP increased aryl hydrocarbon receptor (AhR) activity in Tg(cyp1a:gfp) zebrafish in a concentration-dependent manner. In short, DEHP at ERC has extended potential to induce diverse in vitro and in vivo toxicity at concentrations that also cause impairment of biochemical function in aquatic species of the TGRA.

New toxicogenetic insights and ranking of the selected pharmaceuticals belong to the three different classes: A toxicity estimation to confirmation approach.

Tetracycline hydrochloride (TH), indomethacin (IM), and bezafibrate (BF) belong to the three different important classes of pharmaceuticals, which are well known for their toxicity and environmental concerns. However, studies are still elusive to highlight the mechanistic toxicity of these pharmaceuticals and rank them using both, the toxicity prediction and confirmation approaches. Therefore, we employed the next generation toxicity testing in 21st century (TOX21) tools and estimated the in vitro/vivo toxic endpoints of mentioned pharmaceuticals, and then confirmed them using in vitro/vivo assays. We found significant resemblance in the results obtained via both approaches, especially in terms of in vivo LC50 s and developmental toxicity that ranked IM as most toxic among the studied pharmaceuticals. However, TH appeared most toxic with the lowest estimated AC50s, the highest experimental IC50s, and DNA damages in vitro. Contrarily, IM was found as congener with priority concern to activate the Pi3k-Akt-mTOR pathway in vitro at concentrations substantially lower than that of TH and BF. Further, IM exposure at lower doses (2.79-13.97 µM) depressed the pharmaceuticals detoxification phase I (CYP450 s), phase II (UGTs, SULTs), and phase III (TPs) pathways in zebrafish, whereas, at relatively higher doses, TH (2.08-33.27 µM) and BF (55.28-884.41 µM) partially activated these pathways, which ultimately caused the developmental toxicity in the following order: IM > TH > BF. In addition, we also ranked these pharmaceuticals in terms of their particular toxicity to myogenesis, hematopoiesis, and hepatogenesis in zebrafish embryos. Our results revealed that IM significantly affected myogenesis, hematopoiesis, and hepatogenesis, while TH and BF induced prominent effects on hematopoiesis via significant downregulation of associated genetic markers, such as drl, mpx, and gata2a. Overall, our findings confirmed that IM has higher toxicity than that of TH and BF, therefore, the consumption of these pharmaceuticals should be regulated in the same manner to ensure human and environmental safety.

CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.

Numerous feasible methods for inserting large fragments of exogenous DNA sequences into the zebrafish genome have been developed, as has genome editing technology using programmable nucleases. However, the coding sequences of targeted endogenous genes are disrupted, and the expression patterns of inserted exogenous genes cannot completely recapitulate those of endogenous genes. Here we describe the establishment of a novel strategy for endogenous promoter-driven and microhomology-mediated end-joining-dependent integration of a donor vector using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9. We successfully integrated mCherry into the final coding sequence of targeted genes to generate seamless transgenic zebrafish lines with high efficiency. This novel seamless transgenesis technique not only maintained the integrity of the endogenous gene but also did not disrupt the function of targeted gene. Therefore, our microhomology-mediated end-joining-mediated transgenesis strategy may have broader applications in gene therapy. Moreover, this novel seamless gene-editing strategy in zebrafish provides a valuable new transgenesis technique, which was driven by endogenous promoters and in vivo animal reporter modes for translational medicine. It is expected to be a standard gene-editing technique in the field of zebrafish, leading to some important breakthroughs for studies in early embryogenesis.-Luo, J.-J., Bian, W.-P., Liu, Y., Huang, H.-Y., Yin, Q., Yang, X.-J., Pei, D.-S. CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.