The protective effect of intranasal immunization with influenza virus recombinant adenovirus vaccine on mucosal and systemic immune response.

Influenza virus is a kind of virus that poses several hazards of animal and human health. Therefore, it is important to develop an effective vaccine to prevent influenza. To this end we successfully packaged recombinant adenovirus rAd-NP-M2e-GFP expressing multiple copies of influenza virus conserved antigens NP and M2e and packaged empty vector adenovirus rAd-GFP. The effect of rAd-NP-M2e-GFP on the activation of dendritic cell (DC) in vitro and in vivo was detected by intranasal immunization. The results showed that rAd-NP-M2e-GFP promoted the activation of DC in vitro and in vivo. After the primary immunization and booster immunization of mice through the nasal immune way, the results showed that rAd-NP-M2e-GFP induced enhanced local mucosal-specific T cell responses, increased the content of SIgA in broncho alveolar lavage fluids (BALF) and triggered the differentiation of B cells in the germinal center. It is proved that rAd-NP-M2e-GFP can significantly elicit mucosal immunity and systemic immune response. In addition, rAd-NP-M2e-GFP could effectively protect mice after H1N1 influenza virus challenge. To lay the foundation and provide reference for further development of influenza virus mucosal vaccine in the future.

Caffeic acid alleviates cerebral ischemic injury in rats by resisting ferroptosis via Nrf2 signaling pathway.

There are few effective and safe neuroprotective agents for the treatment of ischemic stroke currently. Caffeic acid is a phenolic acid that widely exists in a number of plant species. Previous studies show that caffeic acid ameliorates brain injury in rats after cerebral ischemia/reperfusion. In this study we explored the protective mechanisms of caffeic acid against oxidative stress and ferroptosis in permanent cerebral ischemia. Ischemia stroke was induced on rats by permanent middle cerebral artery occlusion (pMCAO). Caffeic acid (0.4, 2, 10 mg·kg-1·d-1, i.g.) was administered to the rats for 3 consecutive days before or after the surgery. We showed that either pre-pMCAO or post-pMCAO administration of caffeic acid (2 mg·kg-1·d-1) effectively reduced the infarct volume and improved neurological outcome. The therapeutic time window could last to 2 h after pMCAO. We found that caffeic acid administration significantly reduced oxidative damage as well as neuroinflammation, and enhanced antioxidant capacity in pMCAO rat brain. We further demonstrated that caffeic acid down-regulated TFR1 and ACSL4, and up-regulated glutathione production through Nrf2 signaling pathway to resist ferroptosis in pMCAO rat brain and in oxygen glucose deprivation/reoxygenation (OGD/R)-treated SK-N-SH cells in vitro. Application of ML385, an Nrf2 inhibitor, blocked the neuroprotective effects of caffeic acid in both in vivo and in vitro models, evidenced by excessive accumulation of iron ions and inactivation of the ferroptosis defense system. In conclusion, caffeic acid inhibits oxidative stress-mediated neuronal death in pMCAO rat brain by regulating ferroptosis via Nrf2 signaling pathway. Caffeic acid might serve as a potential treatment to relieve brain injury after cerebral ischemia. Caffeic acid significantly attenuated cerebral ischemic injury and resisted ferroptosis both in vivo and in vitro. The regulation of Nrf2 by caffeic acid initiated the transcription of downstream target genes, which were shown to be anti-inflammatory, antioxidative and antiferroptotic. The effects of caffeic acid on neuroinflammation and ferroptosis in cerebral ischemia were explored in a primary microglia-neuron coculture system. Caffeic acid played a role in reducing neuroinflammation and resisting ferroptosis through the Nrf2 signaling pathway, which further suggested that caffeic acid might be a potential therapeutic method for alleviating brain injury after cerebral ischemia.

Triosephosphate isomerase 1 may be a risk predictor in laryngeal squamous cell carcinoma: a multi-centered study integrating bulk RNA, single-cell RNA, and protein immunohistochemistry.

BACKGROUND: Although great progress has been made in anti-cancer therapy, the prognosis of laryngeal squamous cell carcinoma (LSCC) patients remains unsatisfied. Quantities of studies demonstrate that glycolytic reprograming is essential for the progression of cancers, where triosephosphate isomerase 1 (TPI1) serves as a catalytic enzyme. However, the clinicopathological significance and potential biological functions of TPI1 underlying LSCC remains obscure. METHODS: We collected in-house 82 LSCC tissue specimens and 56 non-tumor tissue specimens. Tissue microarrays (TMA) and immunohistochemical (IHC) experiments were performed. External LSCC microarrays and bulk RNA sequencing data were integrated to evaluate the expression of TPI1. We used a log-rank test and the CIBERSORT algorithm to assess the prognostic value of TPI1 and its association with the LSCC microenvironment. Malignant laryngeal epithelial cells and immune-stromal cells were identified using inferCNV and CellTypist. We conducted a comprehensive analysis to elucidate the molecular functions of TPI1 in LSCC tissue and single cells using Pearson correlation analysis, high dimensional weighted gene co-expression analysis, gene set enrichment analysis, and clustered regularly interspaced short palindromic repeats (CRISPR) screen. We explored intercellular communication patterns between LSCC single cells and immune-stromal cells and predicted several therapeutic agents targeting TPI1. RESULTS: Based on the in-house TMA and IHC analysis, TPI1 protein was found to have a strong positive expression in the nucleus of LSCC cells but only weakly positive activity in the cytoplasm of normal laryngeal cells (p < 0.0001). Further confirmation of elevated TPI1 mRNA expression was obtained from external datasets, comparing 251 LSCC tissue samples to 136 non-LSCC tissue samples (standardized mean difference = 1.06). The upregulated TPI1 mRNA demonstrated a high discriminative ability between LSCC and non-LSCC tissue (area under the curve = 0.91; sensitivity = 0.87; specificity = 0.79), suggesting its potential as a predictive marker for poor prognosis (p = 0.037). Lower infiltration abundance was found for plasma cells, naïve B cells, monocytes, and neutrophils in TPI-high expression LSCC tissue. Glycolysis and cell cycle were significantly enriched pathways for both LSCC tissue and single cells, where heat shock protein family B member 1, TPI1, and enolase 1 occupied a central position. Four outgoing communication patterns and two incoming communication patterns were identified from the intercellular communication networks. TPI1 was predicted as an oncogene in LSCC, with CRISPR scores less than -1 across 71.43% of the LSCC cell lines. TPI1 was positively correlated with the half maximal inhibitory concentration of gemcitabine and cladribine. CONCLUSIONS: TPI1 is dramatically overexpressed in LSCC than in normal tissue, and the high expression of TPI1 may promote LSCC deterioration through its metabolic and non-metabolic functions. This study contributes to advancing our knowledge of LSCC pathogenesis and may have implications for the development of targeted therapies in the future.

Longitudinal surveillance of three biomarkers to predict recurrence of hepatocellular carcinoma after radical resection.

BACKGROUND: Radical resection is a curative treatment for patients with hepatocellular carcinoma (HCC), but the incidence of recurrence remains high. We aimed to explore the performance of predicting HCC recurrence by longitudinal surveillance of the protein induced by vitamin K absence (PIVKA-II), alpha- fetoprotein (AFP), and lectin-reactive AFP (AFP-L3) during postoperative follow-up. METHODS: Patients who underwent radical resection for HCC at the Ningbo Medical Centre Lihuili Hospital between January 2015 and December 2020 were included. All enrolled patients regularly monitor PIVKA-II, AFP, AFP-L3 every 3 months during postoperative follow-up. The surveillance performance of PIVKA-II, AFP, AFP-L3 during follow-up for the prediction of HCC recurrence was compared in patients. The generalized estimation equation (GEE) was used to analyze the trends of the tumor biomarkers and interactions with time. Area under the receiver operator characteristic (AUROC) curves, the optimal cut-off value, the sensitivity and specificity were calculated to evaluate the performance of the three biomarkers. The recurrence-free survival (RFS) and overall survival (OS) of patients with any of the elevated biomarkers was analyzed by Kaplan-Meier curves and the log-rank test. Multivariate logistic regression models were used to analyze potential risk factors for recurrence. RESULTS: The GEE analysis indicated that PIVKA-II, AFP, AFP-L3 in the recurrence patients were higher than the no recurrence patients during follow-up, PIVKA-II and AFP showed increasing trends from 6 months before recurrence. In predicting recurrence, the AUROCs for PIVKA-II, AFP, AFP-L3 and their combination were 0.885, 0.754, 0.781 and 0.885 respectively, the optimal cut-off value for PIVKA-II, AFP, AFP-L3 was 29.5 mAU/ml, 10.7 ng/L, 1.5% respectively. The sensitivity in predicting recurrence for PIVKA-II, AFP, AFP-L3 and combination were 75.0, 54.7, 57.8 and 79.7% respectively. The RFS and the OS of patients with any of the biomarkers elevated during the follow-up was significantly shorter than that without elevated biomarkers ( P  < 0.001). Multivariate analysis showed that any of the biomarkers elevated was the independent risk factor of recurrence. CONCLUSION: Longitudinal surveillance of PIVKA-II, AFP and AFP-L3 can effectively predict recurrence of HCC after operation.

Clinicopathological value of hematopoietic cell kinase overexpression in laryngeal squamous cell carcinoma tissues.

Laryngeal squamous cell carcinoma (LSCC) is the most lethal cancer in head and neck tumors. Although hematopoietic cell kinase (HCK) has been proven to be an oncogene in several solid tumors, its roles in LSCC remain obscure. This is the first study to evaluate the clinical value of HCK in LSCC, with the aim of exploring its expression status and potential molecular mechanisms underlying LSCC. LSCC tissue-derived gene chips and RNA-seq data were collected for a quantitive integration of HCK mRNA expression level. To confirm the protein expression level of HCK, a total of 82 LSCC tissue specimens and 56 non-tumor laryngeal epithelial controls were collected for in-house tissue microarrays and immunohistochemical staining. Kaplan-Meier curves were generated to determine the ability of HCK in predicting overall survival, progress-free survival, and disease-free survival of LSCC patients. LSCC overexpressed genes and HCK co-expressed genes were intersected to preliminarily explore the enriched signaling pathways of HCK. It was noticed that HCK mRNA was markedly overexpressed in 323 LSCC tissues compared with 196 non-LSCC controls (standardized mean difference = 0.81, p < 0.0001). Upregulated HCK mRNA displayed a moderate discriminatory ability between LSCC tissues and non-tumor laryngeal epithelial controls (area under the curve = 0.78, sensitivity = 0.76, specificity = 0.68). The higher expression level of HCK mRNA could predict worse overall survival and disease-free survival for LSCC patients (p = 0.041 and p = 0.013). Lastly, upregulated co-expression genes of HCK were significantly enriched in leukocyte cell-cell adhesion, secretory granule membrane, and extracellular matrix structural constituent. Immune-related pathways were the predominantly activated signals, such as cytokine-cytokine receptor interaction, Th17 cell differentiation, and Toll-like receptor signaling pathway. In conclusion, HCK was upregulated in LSCC tissues and could be utilized as a risk predictor. HCK may promote the development of LSCC by disturbing immune signaling pathways.

Is Helicobacter pylori infection associated with pancreatic cancer? A systematic review and meta-analysis of observational studies.

Background and Objectives: Recent observational studies have investigated the association between Helicobacter pylori (H. pylori) infection and pancreatic cancer with conflicting data. Therefore, we conducted a systematic review and meta-analysis to assess the potential association. Design: This is a systematic review and meta-analysis. Methods: We searched three databases (PubMed, Embase, and Web of Science) from inception to 30 August 2022. The summary results as odds ratio (OR) or hazard ratio (HR) with 95% confidence interval (CI) were pooled by generic inverse variance method based on random-effects model. Results: A total of 20 observational studies involving 67,718 participants were included in the meta-analysis. Meta-analysis of data from 12 case-control studies and 5 nested case-control studies showed that there was no significant association between H. pylori infection and the risk of pancreatic cancer (OR = 1.20, 95% CI = 0.95-1.51, p = 0.13). Similarly, we also did not find significant association between cytotoxin-associated gene A (CagA) positive strains, CagA negative strains, vacuolating cytotoxin gene A (VacA) positive strains H. pylori infection, and the risk of pancreatic cancer. Meta-analysis of data from three cohort studies showed that H. pylori infection was not significantly associated with an increased risk of incident pancreatic cancer (HR = 1.26, 95% CI = 0.65-2.42, p = 0.50). Conclusion: We found insufficient evidence to support the proposed association between H. pylori infection and increased risk of pancreatic cancer. To better understand any association, future evidence from large, well-designed, high-quality prospective cohort studies that accounts for diverse ethnic populations, certain H. pylori strains, and confounding factors would be useful to settle this controversy.

Gene mutations in the PI3K/Akt signaling pathway were related to immune thrombocytopenia pathogenesis.

BACKGROUND: Immune thrombocytopenic (ITP) is an autoimmune bleeding disease with genetic susceptibility. Twenty newly diagnosed active primary ITP patients who had not been treated with glucocorticosteroids, immune globulin or immunosuppressants prior to sampling were enrolled in this study. Bone marrow blood mononuclear cells were used for whole exome sequencing to further elucidation the variant genes of ITP. METHODS: High-molecular-weight genomic DNA was extracted from freshly frozen bone marrow blood mononuclear cells from 20 active ITP patients. Next, the samples were subjected to molecular genetic analysis by whole-exome sequencing, and the results were confirmed by Sanger sequencing. The signaling pathways and cellular processes associated with the mutated genes were identified with gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. RESULTS: The results showed that there were 3998 missense mutations involving 2269 genes in more than 10 individuals. Unique genetic variants including phosphatase and tensin homolog, insulin receptor, and coagulation factor C homology were the most associated with the pathogenesis of ITP. Functional analysis revealed these mutation genes mainly affect Phosphatidylinositol 3 kinase/serine/threonine kinase B signaling pathways (signal transduction) and platelet activation (immune system). CONCLUSION: Our finding further demonstrates the functional connections between these variant genes and ITP. Although the substantial mechanism and the impact of genetic variation are required further investigation, the application of next generation sequencing in ITP in this paper is a valuable method to reveal the genetic susceptibility.

Mesenchymal Stem Cells Promote Intestinal Mucosal Repair by Positively Regulating the Nrf2/Keap1/ARE Signaling Pathway in Acute Experimental Colitis.

BACKGROUND/AIMS: Mesenchymal stem cells (MSCs) are a type of adult pluripotent stem cell that has anti-inflammatory and immunomodulatory effects, and whose conditioned medium (CM) has also been found to be effective. We used MSC and CM enemas to investigate their ameliorative effects in a mouse model of colitis. METHODS: We employed MSCs, CM, and MSCs + ML385 (an inhibitor of Nrf2) in dextran sodium sulfate (DSS)-induced colitis. Mice were sacrificed on day 8, and the effects of MSC or CM treatment on the levels of inflammation and oxidative stress in colonic epithelial cells were evaluated by histological analyses. RESULTS: MSCs inhibited inflammatory cell infiltration and proinflammatory cytokine expression in the colon. In addition, MSCs reduced extracellular matrix deposition and maintained the mechanical barrier and permeability of colonic epithelial cells. Mechanistically, MSCs activated Nrf2, which then increased HO-1 and NQO-1 levels and downregulated the expression of Keap1 to suppress reactive oxygen species production and MDA generation, accompanied by increases in components of the enzymatic antioxidant system, including SOD, CAT, GSH-Px, and T-AOC. However, after administering an Nrf2 inhibitor (ML385) to block the Nrf2/Keap1/ARE pathway, we failed to observe protective effects of MSCs in mice with colitis. CM alone also produced some of the therapeutic benefits of MSCs but was not as effective as MSCs. CONCLUSIONS: Our data confirmed that MSCs and CM can effectively improve intestinal mucosal repair in experimental colitis and that MSCs can improve this condition by activating the Nrf2/Keap1/ARE pathway.

Specific antagonist of receptor for advanced glycation end­products attenuates delirium­like behaviours induced by sevoflurane anaesthesia with surgery in aged mice partially by improving damage to the blood­brain barrier.

Postoperative delirium (POD), which occurs in hospital up to 1-week post-procedure or until discharge, is a common complication, especially in older adult patients. However, the pathogenesis of POD remains unclear. Although damage to blood-brain barrier (BBB) integrity is involved in the neuropathogenesis of POD, the specific role of the BBB in POD requires further elucidation. Anaesthesia using 2% isoflurane for 4 h results in the upregulation of hippocampal receptor for advanced glycation end-products (RAGE) expression and ß-amyloid accumulation in aged rats. The present study investigated the role of RAGE in BBB integrity and its mechanisms in POD-like behaviours. The buried food, open field and Y maze tests were used to evaluate neurobehavioural changes in aged mice following 2.5% sevoflurane anaesthesia administration with exploratory laparotomy. Levels of tight junction proteins were assessed by western blotting. Multiphoton in vivo microscopy was used to observe the ultrastructural changes in the BBB in the hippocampal CA1 region. Anaesthesia with surgery decreased the levels of tight junction proteins occludin and claudin 5, increased matrix metalloproteinases (MMPs) 2 and 9, damaged the ultrastructure of the BBB and induced POD-like behaviour. FPS-ZM1, a specific RAGE antagonist, ameliorated POD-like behaviour induced by anaesthesia and surgery in aged mice. Furthermore, FPS-ZM1 also restored decreased levels of occludin and claudin 5 as well as increased levels of MMP2 and MMP9. The present findings suggested that RAGE signalling was involved in BBB damage following anaesthesia with surgery. Thus, RAGE has potential as a novel therapeutic intervention for the prevention of POD.

Conditioned medium of mesenchymal stem cells pretreated with H2O2 promotes intestinal mucosal repair in acute experimental colitis.

Mesenchymal stem cells (MSCs) are a new therapeutic strategy for inflammatory bowel disease (IBD), and their efficacy has been widely recognized. However, there are still some challenges in cell therapy, including stable cell passage, laboratory conditions for cell culture, high-cost burden, and poor transplantation. The conditioned medium (CM) of MSCs is considered be an excellent alternative to cell transplantation, but the paracrine group in MSC-CM is limited in variety and low in concentration, which cannot meet the therapeutic needs of injured tissues and needs to be optimized. Pretreatment with low concentration of hydrogen peroxide (H2O2) can not only protect cells from oxidative damage, but also play a role similar to growth factors and regulate the physiological function of stem cells, to obtain an improved conditioned medium. To determine the optimal protocol for pretreatment of MSCs with H2O2, and to study the efficacy and potential mechanism of MSC-CM pretreated with H2O2 on Dextran Sulfate Sodium (DSS)-induced acute experimental colitis. MSCs were exposed to different concentrations of H2O2, and the optimal H2O2 pretreatment conditions were determined by evaluating their critical cell functional properties. H2O2-pretreated MSC-CM was transplanted into experimental mouse colitis by enema at 2, 4, and 6 days in modeling, and the changes of colonic tissue structure, the levels of inflammation and oxidative stress, the molecular changes of Nrf2/Keap1/ARE axis, and the related indicators of apoptosis in colonic epithelial cells were observed in each group. In vitro, Pretreated MSCs with 25 µM H2O2 significantly enhanced cell proliferation, migration, and survival, but had no effect on apoptosis. In vivo, MSC-CM treatment decreased apoptosis and extracellular matrix deposition, and maintained the mechanical barrier and permeability of colonic epithelial cells in experimental mouse colitis. Mechanistically, H2O2-pretreated MSC-CM against reactive oxygen species (ROS) production and MDA generation, accompanied by increases in components of the enzymatic antioxidant system includes SOD, CAT, GSH-PX, and T-AOC, which is through the up-regulation of the Nrf2, HO-1, and NQO-1 antioxidant genes. Our data confirmed that 25 µM H2O2 pretreated MSC-CM treatment could effectively improve intestinal mucosal repair in experimental colitis, which may be achieved by activating Nrf2/Keap1/ARE pathway.

N6-methyladenosine reader YTHDF3 regulates melanoma metastasis via its 'executor'LOXL3.

BACKGROUND: A number of studies have demonstrated that N6-methyladenosine (m6A) plays a vital role in the pathological process of various tumours. Recently, it was found that m6A writers or erasers affect the tumourigenesis of melanoma. However, the relationship between m6A readers such as YTH domain family (YTHDF) proteins and melanoma was still elusive. METHODS: RT-qPCR, Western blot and immunohistochemistry were conducted to measure the expression level of YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) and lysyl oxidase-like 3 (LOXL3) in melanoma tissues and cells. The effects of YTHDF3 and LOXL3 on melanoma were verified in vitro and in vivo. Multi-omics analysis including RNA-seq, MeRIP-seq, RIP-seq and mass spectrometry analyses was performed to identify the target. The interaction between YTHDF3 and LOXL3 was verified by RT-PCR, Western blot, MeRIP-qPCR, RIP-qPCR and CRISPR-Cas13b-based epitranscriptome engineering. RESULTS: In this study, we found that m6A reader YTHDF3 could affect the metastasis of melanoma both in vitro and in vivo. The downstream targets of YTHDF3, such as LOXL3, phosphodiesterase 3A (PDE3A) and chromodomain helicase DNA-binding protein 7 (CHD7) were identified by means of RNA-seq, MeRIP-seq, RIP-seq and mass spectrometry analyses. Besides, RT-qPCR, Western blot, RIP-qPCR and MeRIP-qPCR were performed for subsequent validation. Among various targets of YTHDF3, LOXL3 was found to be the optimal target of YTHDF3. With the application of CRISPR-Cas13b-based epitranscriptome engineering, we further confirmed that the transcript of LOXL3 was captured and regulated by YTHDF3 via m6A binding sites. YTHDF3 augmented the protein expression of LOXL3 without affecting its mRNA level via the enrichment of eukaryotic translation initiation factor 3 subunit A (eIF3A) on the transcript of LOXL3. LOXL3 downregulation inhibited the metastatic ability of melanoma cells, and overexpression of LOXL3 ameliorated the inhibition of melanoma metastasis caused by YTHDF3 downregulation. CONCLUSIONS: The YTHDF3-LOXL3 axis could serve as a promising target to be interfered with to inhibit the metastasis of melanoma.

miR-99a-5p: A Potential New Therapy for Atherosclerosis by Targeting mTOR and Then Inhibiting NLRP3 Inflammasome Activation and Promoting Macrophage Autophagy.

Objective: MicroRNAs have been revealed to be involved in the development of atherosclerosis. The present study is aimed at exploring the potential of miR-99a-5p as a therapy for atherosclerosis. We suspected that miR-99a-5p might inhibit NLRP3 inflammasome activation and promote macrophage autophagy via constraining mTOR, therefore, alleviating atherosclerosis. Methods: The cell viability in ox-LDL-induced THP-1 macrophages was assessed by CCK-8 assay. Bioinformatic analysis was used to predict the target genes of miR-99a-5p. The binding between miR-99a-5p and mTOR was confirmed by luciferase reporter assay. In vivo, a high-fat-diet-induced atherosclerosis model was established in apolipoprotein E knockout mice. Hematoxylin-eosin, oil red O, and Sirius red staining were performed for the determination of atherosclerotic lesions. MTOR and associated protein levels were detected by Western blot analysis. Results: miR-99a-5p inhibited NLRP3 inflammasome activation and promoted macrophage autophagy by targeting mTOR. Enforced miR-99a-5p significantly reduced the levels of inflammasome complex and inflammatory cytokines. Furthermore, miR-99a-5p overexpression inhibited the expression of mTOR, whereas mTOR overexpression reversed the trend of the above behaviors. In vivo, the specific overexpression of miR-99a-5p significantly reduced atherosclerotic lesions, accompanied by a significant downregulation of autophagy marker CD68 protein expression. Conclusion: We demonstrated for the first time that miR-99a-5p may be considered a therapy for atherosclerosis. The present study has revealed that miR-99a-5p might inhibit NLRP3 inflammasome activation and promote macrophage autophagy by targeting mTOR, therefore, alleviating atherosclerosis.

Protective Effect of Anwulignan on Gastric Injury Induced by Indomethacin in Mice.

Anwulignan (AN) is a monomer lignan from Schisandra sphenanthera Rehd. et Wits (Schisandra sphenanthera fructus, Schisandra sphenanthera). The protective effect of AN against the indomethacin (IND)-induced gastric injury to mice and the related mechanism of action was investigated in this study. The effect of AN was mainly assessed by observing the gastric tissue morphology, gastric ulcer index (GUI), ulcer inhibition rate (UIR), gastric juice volume (GJV) and pH value. Chemical colorimetry, immunofluorescence, ELISA, and Western blot were used to detect related factors in the gastric tissue. The results showed that AN reduced the GUI, increased the UIR, inhibited the GJV, and increased the gastric pH value. AN significantly increased cyclooxygenase-1, cyclooxygenase-2, and prostaglandin E2 expression levels in the gastric tissue, activated nuclear factor (erythroid-derived 2)-like 2 (Nrf2), increased heme oxygenase-1 expression, enhanced the activity of superoxide dismutase and glutathione peroxidase, and decreased the malondialdehyde content. AN reduced the phosphorylation of nuclear factor-κ gene binding (NF-κB) p65 and its nuclear translocation, the key protein of NF-κB signaling pathway in the gastric tissue, and the content of the pathway downstream signaling molecules, including interleukin-6, interleukin-1ß, and tumor necrosis factor-α, to play an anti-inflammatory role. AN inhibited the downstream signals B-cell lymphoma 2-associated x protein and cleaved caspase-3 in gastric tissue, and activated B-cell lymphoma 2, to play an antiapoptotic role, which were further verified by Hoechst staining. Therefore, AN has a significant protection against the gastric injury induced by IND in mice, and the mechanism may be concerned in its activation of Nrf2, inhibition of NF-κB signaling pathway, and anti-apoptotic effect. SIGNIFICANCE STATEMENT: Anwulignan (AN) significantly reduced the indomethacin-induced gastric injury in mice, and its antioxidation, anti-inflammation, and antiapoptosis were considered to be involve in the effect, suggesting that AN should be a potential drug or food supplement for gastric injury induced by indomethacin.

[Chemical constituents of Scrophulariae Radix and their antitumor activities in vitro].

The present study investigated the chemical constituents of Scrophulariae Radix and their antitumor activities in vitro. The compounds in the ethyl acetate extract were separated and purified by conventional column chromatographies(such as silica gel, Sephadex LH-20, and ODS column) and semi-preparative high-performance liquid chromatography(HPLC), and their structures were identified by various spectral techniques such as nuclear magnetic resonance(NMR) and mass spectrometry(MS). Twenty-three compounds were isolated and identified as benzyl-ß-D-(3',6'-di-O-acetyl) glucoside(1), 5-O-p-methoxybenzoyl kojic acid(2), 5-O-methoxybenzoyl kojic acid(3), 7-O-methylbenzoyl kojic acid(4), 5-O-benzoyl kojic acid(5), methyl ferulate ethyl ether(6), trans-ferulic acid(7), trans-isoferulic acid(8), trans-caffeic acid(9), trans-caffeic acid methyl ester(10), caffeic acid ethyl ester(11), trans-cinnamic acid(12), trans-p-methoxycinnamic acid(13), trans-p-hydroxycinnamic acid(14), trans-p-hydroxycinnamic acid methyl ester(15), 2-(3,4-dihydroxyphenethyl) alcohol(16),(p-hydroxyphenyl) propanoic acid(17), coniferaldehyde(18), sinapaldehyde(19), benzyl ß-primeveroside(20), 5-(hydroxymethyl) furfural(21), furan-2-carboxylic acid(22), and decanedioic acid(23). Among them, compound 1 is a new benzyl glucoside, compounds 2-4 are new pyranone compounds, compound 5 is a new natural product of pyranone. The NMR data of compounds 5 and 6 are reported for the first time. Compounds 6 and 20 were isolated from the Scrophularia plant for the first time. Compounds 8, 11, 14, 16, 18, 19, 22, and 23 were isolated from this plant for the first time. The in vitro cytotoxic activities of these compounds against three tumor cell lines(HepG2, A549, and 4 T1) were evaluated. The results showed that compounds 10 and 15 showed cytotoxic activities against HepG2 cells with IC_(50) values of(19.46±0.48) µmol·L~(-1) and(46.10±1.21) µmol·L~(-1).

Isolation of Novel Mycobacterium Species from Skin Infection in an Immunocompromised Person.

We investigated a case of cutaneous infection in an immunocompromised patient in China that was caused by a novel species within the Mycobacterium gordonae complex. Results of whole-genome sequencing indicated that some strains considered to be M. gordonae complex are actually polyphyletic and should be designated as closely related species.

Long non-coding RNA HOXA-AS3 facilitates the malignancy in colorectal cancer by miR-4319/SPNS2 axis.

Growing evidence has shown the oncogenic role of long non-coding RNA HOXA-AS3 in the progression of several types of cancers, while the effect of HOXA-AS3 on colorectal cancer (CRC) remains unclear. In this study, HOXA-AS3 was significantly over-expressed in CRC clinical samples and human CRC cell lines (SW480, SW620, HCT116, COLO205, and LOVO). HOXA-AS3 knockdown was further achieved by specific siRNAs in COLO205 and LOVO cell lines. The depletion of HOXA-AS3 remarkably inhibited cell proliferation, induced cell cycle arrest, and promoted cell apoptosis in CRC cell lines. Additionally, HOXA-AS3 knockdown was determined to facilitate miR-4319 expression and reduce expression level of sphingolipid transporter 2 (SPNS2) in CRC cell lines. The dual luciferase reporter assay suggested that HOXA-AS3 acted as a sponge of miR-4319, and miR-4319 further directly targeted SPNS2 for expression regulation. Besides, HOXA-AS3 was determined to mediate CRC cell proliferation and apoptosis via miR-4319/SPNS2 axis. Moreover, tumorigenesis experiment validated that HOXA-AS3 promoted CRC progression in vivo by regulating miR-4319, SPNS2, and protein kinase B (AKT) signaling. In summary, this study reveals the novel role of HOXA-AS3 in pathogenesis of CRC and provides a candidate for CRC therapeutic target.

Protective Effects of Anwulignan against HCl/Ethanol-Induced Acute Gastric Ulcer in Mice.

Gastric ulcer is one of the most common gastrointestinal diseases. Anwulignan (AN) is a major active component of Schisandra sphenanthera Rehd. This study was designed to evaluate the protective effect of AN against the acute gastric ulcer induced by HCl/ethanol in mice. The mice were given HCl/ethanol by gavage to establish an acute gastric ulcer model. Then, the serum and gastric tissue samples were taken for biochemical analyses. The results showed that the pretreatment with AN could significantly reduce the gastric ulcer index (GUI) and increase the ulcer inhibition rate, indicating that AN can protect against gastric ulcers. AN showed its antioxidant roles by decreasing the content of reactive oxygen species (ROS), malondialdehyde (MDA), and 8-hydroxydeoxyguanosine (8-OHdG) and increasing the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) and anti-inflammatory roles by decreasing the content of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and myeloperoxidase (MPO) and increasing the content of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), prostaglandin E2 (PGE2), and nitric oxide (NO) in both serum and gastric tissue. Furthermore, AN also activated the NRF2/ARE signaling pathway and inhibited the MAPK/NF-κB signaling pathway. AN improves the acute gastric ulcer induced by HCl/ethanol in mice, which may be mainly through its antioxidant capacity and anti-inflammatory effect.

Associations of Serum Magnesium With Insulin Resistance and Testosterone in Women With Polycystic Ovary Syndrome.

Objective: This article aimed to investigate whether serum magnesium is associated with insulin resistance index and testosterone level in women with polycystic ovary syndrome (PCOS). Materials and Methods: Overall 1000 women with PCOS were enrolled in a randomized controlled trial and a cross-sectional analysis of the association of serum magnesium with glucose metabolism markers and testosterone was performed. Serum magnesium, glucose metabolism markers and testosterone were measured. Insulin resistance was evaluated by homeostatic model assessment of insulin resistance (HOMA-IR) and quantitative insulin-sensitivity check index (QUICKI). Multivariable linear regression and logistic regression models were used to estimate the association between serum magnesium, insulin resistance and testosterone. Results: In comparative analyses, women with higher quartile of serum magnesium had significantly lower fasting glucose, HOMA-IR and testosterone. Multiple linear regression showed serum magnesium was independently negatively associated with insulin, glucose, HOMA-IR, testosterone and positively associated with QUICKI (P for trend <0.05) after adjusting confounding covariates. Logistic regression showed serum magnesium in quartile 1 and 2 were independently associated with insulin resistance status (Quartile 1: OR: 2.15, 95%CI: 1.35-3.40, P = 0.001; Quartile 2: OR: 1.90, 95%CI: 1.20-3.02, P = 0.006), while quartile 1 was marginally associated with hyperandrogenemia status (Quartile 1: OR: 1.45, 95%CI: 0.99-2.11, P = 0.055) after adjusting confounding covariates. Conclusion: The current findings suggest that lower serum magnesium was associated with aggravated insulin resistance and higher testosterone levels among women with PCOS.

A novel twin-column continuous chromatography approach for separation and enrichment of monoclonal antibody charge variants.

Downstream processing of mAb charge variants is difficult owing to their similar molecular structures and surface charge properties. This study aimed to apply a novel twin-column continuous chromatography (called N-rich mode) to separate and enrich acidic variants of an IgG1 mAb. Besides, a comparison study with traditional scaled-up batch-mode cation exchange (CEX) chromatography was conducted. For the N-rich process, two 3.93 mL columns were used, and the buffer system, flow rate and elution gradient slope were optimized. The results showed that 1.33 mg acidic variants with nearly 100% purity could be attained after a 22-cycle accumulation. The yield was 86.21% with the productivity of 7.82 mg/L/h. On the other hand, for the batch CEX process, 4.15 mL column was first used to optimize the separation conditions, and then a scaled-up column of 88.20 mL was used to separate 1.19 mg acidic variants with the purity of nearly 100%. The yield was 59.18% with the productivity of 7.78 mg/L/h. By comparing between the N-rich and scaled-up CEX processes, the results indicated that the N-rich method displays a remarkable advantage on the product yield, i.e. 1.46-fold increment without the loss of productivity and purity. Generally, twin-column N-rich continuous chromatography displays a high potential to enrich minor compounds with a higher yield, more flexibility and lower resin cost.

Anwulignan Ameliorates the Intestinal Ischemia/Reperfusion.

Anwulignan is one of the monomer compounds in the lignans from Schisandra sphenanthera In this study, we observed the effect of anwulignan on intestinal ischemia/reperfusion (II/R) injury in male Sprague-Dawley rats and explored the underlying mechanisms. The results showed that pretreatment with oral anwulignan could significantly increase the mesenteric blood microcirculatory flow velocity; relieve the congestion and pathologic injury of jejunum; enhance the autonomic tension of jejunum smooth muscle and its reactivity to acetylcholine; increase the activities of superoxide dismutase, catalase, glutathione S-transferase, and choline acetyltransferase; increase the contents of acetylcholine and glutathione in the serum or jejunal tissue; decrease the activities of myeloperoxidase, protein kinase C, and nicotinamide adenine dinucleotide phosphate oxidase; reduce the contents of malondialdehyde, 8-hydroxy-2-deoxyguanosine, nicotinamide adenine, reactive oxygen species, tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß; increase the expression levels of muscarinic receptor 3, PI3K, phosphorylation protein kinase B, p-GSK3ß Ser9, Nrf2, p-Nrf2, heme oxygenase (decycling) 1, and b-cell lymphoma 2 in the jejunal tissue; and decrease the expression levels of p-GSK3ß Tyr216, kelch-like ECH-associated protein 1, Bax, and cleaved caspase-3, suggesting that anwulignan can ameliorate II/R-induced jejunal tissue injury in rats and that the mechanism may be related to its activating the PI3K/protein kinase B pathway and then regulating the Nrf2/Anti-oxidative Response Element signaling pathway and the expression of apoptosis-related proteins to play antioxidant and antiapoptotic roles. SIGNIFICANCE STATEMENT: Anwulignan can significantly reduce jejunal tissue injury and the production of inflammatory factors in rats with intestinal ischemia-reperfusion injury, improve the antioxidant capacity, and reduce the apoptosis of jejunal tissue, and it has the effect of significantly improving intestinal ischemia-reperfusion injury in rats, suggesting that anwulignan may be used as a potential drug for the prevention and treatment of intestinal ischemia-reperfusion injury or a resource for the development of health food.