Lycium barbarum polysaccharide alleviates DSS-induced chronic ulcerative colitis by restoring intestinal barrier function and modulating gut microbiota.

PURPOSE: This study examined the protective effects and mechanism of Lycium barbarum polysaccharides (LBP) in the context of intestinal barrier function and intestinal microbiota in mice with dextran sulfate sodium (DSS)-induced chronic ulcerative colitis (UC). METHODS: C57BL/6J male mice were assigned to a standard normal diet without DSS (control group), a normal diet with DSS (DSS group, 2% DSS given discontinuously for 3 weeks) or a normal diet supplemented with LBP (1% dry feed weight, LBP group, 2% DSS given discontinuously for 3 weeks) for a total of 8 weeks, at which point colonic tissues and caecal contents were collected. RESULTS: LBP exerted a significant effect against colitis by increasing body weight, colon length, DAI and histopathological scores. LBP inhibited proinflammatory cytokines (IL-1ß, IL-6, iNOS and TNF-α) expression, improved anti-inflammatory cytokine (IL-10) expression, promoted the expression of tight junction proteins (Occludin and ZO-1) via nuclear factor erythroid 2-related factor 2 (Nrf2) activation and decreased Claudin-2 expression to maintain the intestinal mucosal barrier. In addition, the abundances of some probiotics (Ruminococcaceae, Lactobacillus, Butyricicoccus, and Akkermansia) were decreased with DSS treatment but increased obviously with LBP treatment. And LBP reduced the abundance of conditional pathogens associated with UC (Mucispirillum and Sutterella). Furthermore, LBP improved the production of short-chain fatty acids (SCFAs), including acetic acid, propionic acid, butyric acid and isobutyric acid. CONCLUSION: LBP can alleviate DSS-induced UC by regulating inflammatory cytokines and tight junction proteins. Moreover, LBP promotes probiotics, suppresses conditional pathogens and increases SCFAs production, showing a strong prebiotic effect.

PIWI-interacting RNA-17458 is oncogenic and a potential therapeutic target in cervical cancer.

Cervical cancer (CC) is one of the leading cancers among the female reproductive system. The piwi-interacting RNA (piRNA) function and biogenesis has been studied in various cancers, including CC. But the precise mechanism of piRNA in CC is still unknown. In our study, we found that piRNA-17458 was overexpressed in CC tissues and cells. piRNA-17458 mimic and inhibitor promoted and suppressed proliferation, migration and invasion ability of CC cells, respectively. We also demonstrated that piRNA-17458 mimic could contribute to tumor growth in mice xenograft models. Besides, we also found that the piRNA-17458 mimic could enhance mRNA N6-methyladenosine(m6A) levels and increase WTAP stability in CC cells, while the effects of the mimic was reversed by the WTAP knockdown. The results of dual luciferase reporter assay showed that WTAP was a direct target of piRNA-17458. Knockdown of WTAP attenuated proliferation, migration and invasion of CC cells in piRNA-17458 mimic group. Our finding not only demonstrates for the first time that piRNA-17458 is overexpressed in CC tissues and cells, but also shows that piRNA-17458 promotes tumorigenesis of CC in a WTAP-mediated m6A methylation manner.

Artesunate delays the dysfunction of age-related intestinal epithelial barrier by mitigating endoplasmic reticulum stress/unfolded protein response.

The impairment of the intestinal epithelial barrier and subsequent bacterial translocation are common in aging individuals, contributory to several local and systematic disorders. However, the underlying mechanism of the age-related degeneration has not been fully understood. In this study, we demonstrated that the intestinal KIT signaling declined and de-activated with aging, parallel with epithelial barrier dysfunction. Endoplasmic reticulum stress (ERS)/unfolded protein response (UPR) was obviously increased during aging. The ERS and its downstream IRE1α were highly activated in the aging colonic epithelium. Furthermore, by the use of Tunicamycin (Tm)-induced ERS mouse and cell models, we uncovered that the activity of the ERS/IRE1α accelerated the protein degradation of KIT via ubiquitin-proteasome pathway. The deficiency of KIT signaling further reduced the transcription of the tight junction protein Claudin-3. Of significance, Artesunate (ART) could be capable of ameliorating the detrimental effect of ERS/IRE1α, indicated by the re-gained KIT and Claudin-3 expressions and the restoration of the intestinal epithelial barrier. In conclusion, our present study provided novel evidence elucidating the ERS/IRE1α-induced loss of KIT and Claudin-3 in the aging colonic epithelium and also shed light on the protective effect of Artesunate on the intestinal epithelial barrier by blocking ERS/IRE1α activity during aging.

Intense endoplasmic reticulum stress (ERS) / IRE1α enhanced Oxaliplatin efficacy by decreased ABCC10 in colorectal cancer cells.

BACKGROUND: Attenuated Oxaliplatin efficacy is a challenge in treating colorectal cancer (CRC) patients, contributory to the failure in chemotherapy and the risks in relapse and metastasis. However, the mechanism of Oxaliplatin de-efficacy during CRC treatment has not been completely elucidated. METHODS: Microarray screening, western blot and qPCR on clinic CRC samples were conducted to select the target gene ABCC10 transporter. The Cancer Genome Atlas data was analyzed to figure out the correlation between the clinical manifestation and ABCC10 expression. ABCC10 knock-down in CRC cells was conducted to identify its role in the Oxaliplatin resistance. Cell counting kit-8 assay was conducted to identify the CRC cell viability and Oxaliplatin IC50. Flow cytometry was conducted to detect the cell apoptosis exposed to Oxaliplatin. The intracellular Oxaliplatin accumulation was measured by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. RESULTS: CRC patients with higher ABCC10 were prone to relapse and metastasis. Differential ABCC10 expression in multiple CRC cell lines revealed a strong positive correlation between ABCC10 expression level and decreased Oxaliplatin response. In ABCC10 knock-down CRC cells the Oxaliplatin sensitivity was evidently elevated due to an increase of intracellular Oxaliplatin accumulation resulted from the diminished drug efflux. To explore a strategy to block ABCC10 in CRC cells, we paid a special interest in the endoplasmic reticulum stress (ERS) / unfolded protein response (UPR) that plays a dual role in tumor development. We found that neither the inhibition of ERS nor the induction of mild ERS had anti-CRC effect. However, the CRC cell viability was profoundly decreased and the pro-apoptotic factor CHOP and apoptosis were increased by the induction of intense ERS. Significantly, the Oxaliplatin sensitivity of CRC cells was enhanced in response to the intense ERS, which was blocked by inhibiting IRE1α branch of UPR. Finally, we figured out that the intense ERS down-regulated ABCC10 expression via regulated IRE1-dependent decay activity. CONCLUSION: Oxaliplatin was a substrate of ABCC10 efflux transporter. The intense ERS/IRE1α enhanced Oxaliplatin efficacy through down-regulating ABCC10 in addition to inducing CHOP. We suggested that introduction of intense ERS/UPR could be a promising strategy to restore chemo-sensitivity when used in combination with Oxaliplatin or other chemotherapeutic drugs pumped out by ABCC10.

Propofol-based total intravenous anesthesia is associated with better survival than desflurane anesthesia in limb-salvage surgery for osteosarcoma: A retrospective analysis.

Previous studies have demonstrated that anesthetic techniques can affect the outcomes of cancer surgery. We investigated the association between anesthetic techniques and patient outcomes after elective limb-salvage surgery for osteosarcoma (OS). This was a retrospective cohort study of patients who underwent elective limb-salvage surgery for OS between January 2007 and December 2018. Patients were grouped according to the administration of propofol-based total intravenous anesthesia (TIVA) or desflurane (DES) anesthesia. Kaplan-Meier analysis was performed, and survival curves were constructed from the date of surgery to death. Univariate and multivariate Cox regression models were applied to compare the hazard ratios (HRs) for death after propensity matching. Subgroup analyses were done for postoperative recurrence, metastasis, and tumor-node-metastasis (TNM) staging. A total of 30 patients (17 deaths, 56.7%) who received DES anesthesia and 26 (4 deaths, 15.4%) who received TIVA were eligible for analysis. After propensity matching, 22 patients were included in each group. In the matched analysis, patients who received TIVA had better survival with a HR of 0.30 (95% confidence interval [CI], 0.11-0.81; P = .018). Subgroup analyses also showed significantly better survival in the presence of postoperative metastasis (HR, 0.24; 95% CI, 0.06-0.87; P = .030) and with TNM stage II to III (HR, 0.26; 95% CI, 0.09-0.73; P = .011) in the matched TIVA group. In addition, patients administered with TIVA had lower risks of postoperative recurrence and metastasis than those administered with DES anesthesia in the matched analyses. Propofol-based TIVA was associated with better survival in patients who underwent elective limb-salvage surgery for OS than DES anesthesia. Prospective studies are needed to assess the effects of TIVA on oncological outcomes in patients with OS.

Risk Factors Associated with Cartilage Defects after Anterior Cruciate Ligament Rupture in Military Draftees.

This study aimed to evaluate the different clinical results and factors associated with cartilage defects in military draftees who underwent different treatments after anterior cruciate ligament (ACL) rupture. Overall, 105 patients who had sustained ACL rupture were military draftees who underwent a conscription examination for physical status assessment from January 2012 to December 2020. Patients were divided into three groups: conservative treatment after ACL rupture, status post-anterior cruciate ligament reconstruction (ACLR), but graft rupture, and status post-ACLR with graft intact. Inter-group comparisons and statistical analyses were performed for age, body mass index (BMI), thigh circumference difference, side-to-side difference in anterior knee translation by KT-2000, meniscus tear, and cartilage defect. Multivariate logistic regression analysis was used to determine the factors associated with cartilage defects. The multivariable regression model showed that BMI (odds ratio OR: 1.303; 95% CI: 1.016-1.672; p = 0.037), thigh circumference difference (OR: 1.403; 95% CI: 1.003-1.084; p = 0.034), tear of lateral meniscus (LM) and medial meniscus (MM) (OR: 13.773; 95% CI: 1.354-140.09; p = 0.027), and graft rupture group (OR: 5.191; 95% CI: 1.388-19.419; p = 0.014) increased the risk of cartilage defects. There was no correlation between cartilage defects and age, KT-2000 difference, tear of LM or MM, or graft intact group. Progression of osteoarthritis was concerned after ACL rupture, and this study identified several factors of post-ACLR graft rupture, greater thigh circumference difference, BMI, and meniscus tear of both LM and MM affecting cartilage defects, which represent early degenerative osteoarthritis changes of the knee. The results of this study should be customized for rehabilitation and military training, especially in military draftees with ACL injuries.

piRNA-14633 promotes cervical cancer cell malignancy in a METTL14-dependent m6A RNA methylation manner.

BACKGROUND: Cervical cancer (CC) is one of the most common gynecological tumors that threatens women's health and lives. Aberrant expression of PIWI-interacting RNA (piRNA) is closely related with a range of cancers and can serve as a tumor promoter or suppressor in proliferation, migration and invasion. In this study, the aim was not only to discover differential expression of piRNA in CC tissue (CC cells) and normal cervical tissue (normal cervical epithelium cells), but also to investigate the biological function and action mechanism of piRNA in CC. METHODS: The DESeq2 approach was used to estimate fold change in piRNA between CC tissue and normal cervical tissue. The relative expressions of piRNAs (piRNA-20657, piRNA-20497, piRNA-14633 and piRNA-13350) and RNA m6A methyltransferases/demethylases were detected using RT-qPCR. After intervention with piRNA-14633 and METTL14 expression, the viability of CaSki cells and SiHa cells was detected by CCK8. CC cell proliferation was detected by colony formation assay. Apoptosis rate and cell cycle were detected by flow cytometry. Transwell assay was performed to detect cell migration and invasion. EpiQuik m6A RNA Methylation Quantification Kit was used to evaluate m6A RNA methylation levels. Expression of methyltransferase-like protein 14 (METTL14), PIWIL-proteins and CYP1B1 were detected by RT-qPCR and western blot. The effect of piRNA-14633 on METTL14 was evaluated by a dual-luciferase reporter assay. The in vivo effects of piRNA-14633 on CC was assessed by nude mice experiments. RESULTS: piRNA-14633 showed high expression in CC tissues and cells, piRNA-14633 mimic (piRNA-14633 overexpression) promoted viability, proliferation, migration and invasion of CaSki cells and SiHa cells. Besides, piRNA-14633 mimic increased m6A RNA methylation levels and METTL14 mRNA stability. Results of dual luciferase reporter assays indicated that METTL14 was a directed target gene of piRNA-14633. Knockdown of METTL14 with siRNA attenuated proliferation, migration and invasion of CC cells. piRNA-14633 increased CYP1B1 expression, while silencing of METTL14 impaired its expression. The effect of piRNA overexpression on METTL14 expression has concentration-dependent characteristics. Results from in vivo experiment indicated that piRNA-14633 promoted cervical tumor growth. CONCLUSION: piRNA-14633 promotes proliferation, migration and invasion of CC cells by METTL14/CYP1B1 signaling axis, highlighting the important role of piRNA-14633 in CC.

MiR-1-3p and MiR-124-3p Synergistically Damage the Intestinal Barrier in the Ageing Colon.

BACKGROUND AND AIMS: Disruption of the intestinal barrier of the digestive tract is a common pathophysiological change in the elderly, which may partly contribute to gut dysfunction and inflammatory bowel disease [IBD]. This study aimed to discover new interactive epigenetic regulation patterns involved in intestinal barrier dysfunction and colitis in elderly populations. METHODS: Intestinal barrier function and structure were evaluated in naturally ageing mice and elderly people. High-throughput analysis was performed on colonic tissues from humans and mice. The synergistic roles of miR-1-3p and miR-124-3p were identified using microRNA mimic/agomirs. Related genes were examined in biopsies of old IBD patients. RESULTS: A defective mucus barrier was observed before mucosal microstructural damage during ageing. Elevated miR-1-3p expression in the colons of older individuals impaired the mucus barrier by directly targeting T-synthase, similarly to the mechanism of miR-124-3p, which we reported previously. Importantly, the synergistic effect of a half dose of each microRNA supplement on T-synthase and CDK4/6 was stronger than that of a full dose of miR-1-3p or miR-124-3p alone, and mice co-treated with two microRNAs showed greater susceptibility to chemical-induced colitis than mice treated with either microRNA alone. These two microRNAs were up-expressed in old IBD patients. CONCLUSIONS: The slight increases in miR-1-3p and miR-124-3p expression with ageing may be important contributors to the breakdown of intestinal homeostasis by targeting divergent genes in different cells. These data reveal the potential ability of multiple microRNAs to exert synergistic effects to damage the intestinal barrier and promote inflammatory bowel disease development in elderly populations.

TP53 Co-Mutational Features and NGS-Calibrated Immunohistochemistry Threshold in Gastric Cancer.

PURPOSE: TP53 is the most frequently mutated gene in gastric cancer and it can be potentially used for gastric cancer diagnosis and screening. However, standardized clinical approaches that could accurately and cost-effectively detect TP53 mutations in gastric cancer are largely lagged behind. PATIENTS AND METHODS: We conducted next-generation sequencing (NGS) analysis of 425 cancer-related genes in 42 gastric cancer patients in our cohort. A 1313-patient cohort derived from the cBioPortal database was used for validation. We performed immunohistochemistry (IHC) staining with four commonly used p53 antibodies, and the NGS results were used as the gold standard to optimize the IHC threshold for each antibody. RESULTS: By NGS analysis, we found that around 80% of gastric cancer patients in our cohort harbored TP53 alterations. Genetic alterations of BRCA1/2 or KMT2B were mostly exclusive with TP53 mutations, so were the MSI status or low grade of tumors. These results were further validated using the data from cBioPortal. We then used the NGS-derived TP53 status to optimize four commonly used IHC antibodies for detecting TP53 mutations. We showed that all antibodies could achieve more than 93% accuracy when proper IHC positivity thresholds were used, especially for the SP5 antibody that could reach 100% sensitivity and specificity with the 20% threshold. CONCLUSION: Our results indicated that exclusivity between TP53 and BRCA mutations could be potentially used as a cost-effective way to predict BRCA status. Also, setting proper IHC thresholds for each specific antibody is critical to accurately detect TP53 mutations and facilitate disease diagnosis.

Scavenger receptor MARCO contributes to macrophage phagocytosis and clearance of tumor cells.

Macrophage receptor with collagenous structure (MARCO) is a member of the class A scavenger receptor family which is expressed on the cell surface of macrophages. It is well known that MARCO avidly binds to unopsonized pathogens, leading to its ingestion by macrophages. However, the role of MARCO in the recognition and phagocytosis of tumor cells by macrophages remains poorly understood. In this study, we used lentiviral technology to knockdown and overexpress MARCO and investigated the ability of macrophages to phagocytose tumor cells. Our results showed that MARCO expression was correlated with the ability of macrophages to carry on phagocytosis. MARCO knockdown led to significant decreases in the number of engulfment pseudopodia of macrophages and inhibition of the phagocytosis of tumor cells. On the other hand, MARCO overexpression elevated activity of SYK, PI3K and Rac1 in macrophages, which led to changes in macrophage morphology and enhanced phagocytosis by promoting formation of stress fibers and pseudopodia. By Co-IP analysis we showed that MARCO directly binds to the ß5 integrin of SL4 tumor cells. In summary, these results demonstrated the important role for MARCO in demonstrated tumor cells uptake and clearance by macrophages.

Proteome profiling of formalin-fixed, paraffin-embedded lung adenocarcinoma tissues using a tandem mass tag-based quantitative proteomics approach.

Over the past few decades, increasing efforts have been made to improve the understanding of, and treatment options for, lung adenocarcinoma (LUAD). However, considering the heterogeneity of LUAD, precise proteomics-based characterization at the molecular level is an urgent clinical requirement for effective treatment. Formalin-fixed, paraffin-embedded (FFPE) tissue is a good option as the working tool for proteomics studies. The present study aimed to obtain a global protein profile using LUAD FFPE tissue samples. Using a quantitative proteomics approach, the study revealed that 360 proteins were significantly more highly expressed in LUAD than in adjacent nontumor lung tissues. Also, 19 differentially expressed membrane proteins were found to be primarily responsible for immune processes. Epidermal growth factor (EGF)-like domain and laminin EGF domain showed markedly different expression levels between cancer tissues and tumor-adjacent normal tissues. Furthermore, Gene Ontology functional enrichment analysis showed that significantly upregulated proteins were associated with the endoplasmic reticulum lumen, protein disulfide isomerase activity, vitamin binding, cell cycle G1/S phase transition, to name but a few. Also, numerous kinases and post-translational modification enzymes were significantly upregulated across all eight LUAD samples compared with paracarcinoma tissues. Proteomics analysis revealed that AAA domain containing 3A (ATAD3a), a member of the ATPase family, was highly expressed in LUAD tissues, which was supported by immunohistochemical analysis. Furthermore, the study confirmed that ATAD3a enhanced the cisplatin sensitivity of LUAD cells. Collectively, the findings of the present study provide new potential candidate targets in patients with LUAD, and may aid auxiliary LUAD diagnosis and surveillance in a noninvasive manner.

c-KIT-ERK1/2 signaling activated ELK1 and upregulated carcinoembryonic antigen expression to promote colorectal cancer progression.

Carcinoembryonic antigen (CEA) is highly expressed in embryo and colorectal cancer (CRC) and has been widely used as a marker for CRC. Emerging evidence has demonstrated that elevated CEA levels promote CRC progression. However, the mechanism of the increased CEA expression in patients with primary and recurrent CRC is still an open question. In this study, we showed that c-KIT, ELK1, and CEA were hyperexpressed in patients with CRC, especially patients with recurrent disease. From bioinformatics analysis, we picked ELK1 as a candidate transcription factor (TF) for CEA; the binding site of ELK1 within the CEA promoter was confirmed by chromatin immunoprecipitation and dual luciferase reporter assays. Overexpression of ELK1 increased CEA expression in vitro, while knockdown of ELK1 decreased CEA. Upregulated ELK1 promoted the adhesion, migration, and invasion of CRC cells, however knockdown of CEA blocked the activities of ELK1-overexpressed CRC cells. Furthermore, we explored the role of c-KIT-ERK1/2 signaling in activation of ELK1. Blocking c-KIT signaling using Imatinib or ISCK03 reduced p-ELK1 expression and consequently decreased CEA levels in CRC cells, as did blocking the ERK1/2 pathway by U0126. Compared with wild type littermates, the c-kit loss-of-functional Wadsm/m mice showed lowered c-KIT, ELK1, and CEA expression. In conclusion, our study revealed that ELK1, which was activated by c-KIT-ERK1/2 signaling, was a key TF for CEA expression. Blocking ELK1 or its upstream signaling could be an alternative way to decelerate CRC progression. Besides being a biomarker for CRC, CEA could be used for guiding targeted therapy.

Elevated miR-124-3p in the aging colon disrupts mucus barrier and increases susceptibility to colitis by targeting T-synthase.

The risk of colitis and colorectal cancer increases markedly throughout adult life, endangering the health and lives of elderly individuals. Previous studies have proposed that bacterial translocation and infection are the main risk factors for these diseases. Therefore, in the present study, we aimed to identify the underlying mechanism by focusing on the mucus barrier function and mucin-type O-glycosylation. We evaluated alterations in the colon mucus layer in 2-, 16-, and 24-month-old mice and aged humans. Aged colons showed defective intestinal mucosal barrier and changed mucus properties. The miR-124-3p expression level was significantly increased in the aged distal colonic mucosa, which was accompanied by an increase in pathogens and bacterial translocation. Meanwhile, T-synthase, the rate-limiting enzyme in O-glycosylation, displayed an age-related decline in protein expression. Further experiments indicated that miR-124-3p modulated O-glycosylation by directly targeting T-synthase. Moreover, young mice overexpressing miR-124-3p exhibited abnormal glycosylation, early-onset, and more severe colitis. These data suggest that miR-124-3p predisposes to senile colitis by reducing T-synthase, and the miR-124-3p/T-synthase/O-glycans axis plays an essential role in maintaining the physiochemical properties of colonic mucus and intestinal homeostasis.

Esophageal carcinoma: Intravoxel incoherent motion diffusion-weighted MRI parameters and histopathological correlations.

BACKGROUND: The pathological grade of esophageal carcinoma is highly determinant of patient prognosis, but it still cannot be adequately evaluated preoperatively. Compared with conventional diffusion-weighted imaging (DWI), intravoxel incoherent motion (IVIM) diffusion-weighted MRI can separate true molecular diffusion and perfusion in tissues and has been shown to be useful in characterizing malignant tumors. There is no report that compared IVIM and conventional DWI in grading esophageal carcinoma. PURPOSE: To prospectively determine the diagnostic performance of conventional DWI and IVIM models in differentiating the pathological differentiated grade of esophageal carcinoma. STUDY TYPE: Prospective. POPULATION: A cohort comprising 81 patients with newly diagnosed esophageal squamous cell carcinoma (ESCC) between December 2015 and August 2017 were evaluated. FIELD STRENGTH/SEQUENCE: 3.0T, axial echo-planer imaging, fast spin echo (FSE) sequence, IVIM sequence (b = 0, 20, 50, 80, 100, 150, 200, 400, 600, 800, 1000, 1200). ASSESSMENT: Apparent diffusion coefficient (ADC), true ADC (ADCslow ), pseudo ADC (ADCfast ), and perfusion fraction (f) of each tumor were calculated by two independent radiologists. Histopathologic grade was used as the reference standard. STATISTICAL TESTS: Games-Howell test; diagnostic accuracy; Spearman correlation; intraclass correlation coefficient; and Bland-Altman analysis. Receiver operating characteristics (ROC) curves. RESULTS: ADCslow demonstrated the highest area under curve (AUC) with a value of 0.830 (95% confidence interval [CI]: 0.730-0.904) and 0.816 (95% CI: 0.714-0.893) by two radiologists, followed by ADC with a value of 0.754 (95% CI: 0.646-0.843) and 0.761 (95% CI: 0.653-0.848). Good correlation was obtained between the histologic grade and ADCslow (r(R1) = 0.748, r(R2) = 0.720) and ADC (r(R1) = 0.576, r(R2) = 0.571). DATA CONCLUSION: ADCslow and ADC had a significantly higher performance than the ADCfast and f, and ADCslow had a significantly higher performance than the ADC. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;49:253-261.

Lipopolysaccharide induces the early enhancement of mice colonic mucosal paracellular permeability mainly mediated by mast cells.

The alteration of intestinal mucosal barrier is considered to be the central pathophysiological process in response to gastrointestinal infections, and mucosal microstructural damage is a major factor for enhancing epithelial permeability in persistent bacterial infections. However, the mechanism involved in hyperpermeability in the early stage of acute bacterial infections is not fully understood. In the present study, fluorescein isothiocyanate-dextran across and transepithelial resistance measured in Ussing chambers were used to assess the intestinal paracellular permeability. Mast cell activation was evaluated by western blotting for the presence of tryptase released from mast cells. Serum levels of interleukin-6 were evaluated using enzyme-linked immunosorbent assay. Our results indicated that mast cells played a pivotal role in colonic mucosal hyperpermeability in wild type mice treated with lipopolysaccharide (LPS) for 2 h. And the effect of LPS was mainly dependent on mast cell degranulation, while no change in permeability was observed in the mast cell-deficient mice (Wads⁻/⁻) after LPS administration. No obvious changes of the mucosal structure including histomorphological architecture and expression of intercellular junction proteins were obtained in either wild type or Wads⁻/⁻ mice after LPS stimulation by hematoxylin and eosin staining, immunofluorescence staining and western blot analysis. Furthermore, the self-renewal of intestinal epithelia, detected by using proliferation marker 5'-bromo-2'-deoxyuridine, was not involved in increased permeability. Collectively, activation of mast cells induced by LPS mediated intestinal hyperpermeability in the initial stage, and played a crucial role in barrier dysfunction rather than mucosal microstructural damage in acute enterogenous bacterial infection.

Aging-dependent decrease in the numbers of enteric neurons, interstitial cells of Cajal and expression of connexin43 in various regions of gastrointestinal tract.

Aging is a significant risk factor for gastrointestinal dysmotility, but aging-associated differences between different organs and the exact time to start degenerating have remained obscure. Here we evaluated alterations of interstitial cells of Cajal, enteric neurons and connexin43 expression in the stomach, jejunum and colon in 2-, 12-, 16-, 20- and 24-month-old mice, as well as in aged human colon. Interstitial cells of Cajal, cholinergic and nitrergic neurons within the whole digestive tract were reduced over time, but their loss first appeared in stomach, then in intestine, helping to understand that gastric function was first impaired during aging. The decrease of connexin43 expression occurred before interstitial cells of Cajal and neurons loss, suggesting that connexin43 might be the major target influenced during senescence. Furthermore, changes in expressions of pro-inflammatory cytokines (tumour necrosis factor-α, interleukin-1ß, interleukin-6) and apoptosis-related proteins (B-cell lymphoma-2, caspase-3) which indicated "inflammaging", might contribute to the loss of enteric neurons and interstitial cells of Cajal in aged gastrointestinal tract. Our results provide possible therapeutic time window for beneficial intervention for geriatric patients with gastrointestinal motility disorders.

SCF/c-KIT signaling promotes mucus secretion of colonic goblet cells and development of mucinous colorectal adenocarcinoma.

Mucinous colorectal adenocarcinoma (MCA) is characterized by a great mount of extracellular mucus fundamentally composed of Mucin2 (MUC2) which is significantly correlated with the high malignancy and strong invasive ability of MCA. However, rare is known about the underlying mechanism of the mucus accumulation in MCA. Our latest study demonstrated that SCF/c-KIT signaling was highly activated in MCA patients and mouse model, which up-regulated MUC2 transcription. In the present study, we paid a special interest in whether and how SCF/c-KIT signaling promoted mucus secretion by using wild-type (WT) C57BL mice and their littermates who harbor mutational c-kit gene (Wadsm/m), clinical colorectal cancer (CRC) samples, as well as human CRC cell lines. Our results clearly showed that the inner mucus layer of colon was thinner and the intracellular mucin residual was more in Wadsm/m mice than those in WT mice by Alcian blue and PAS staining, suggesting that the mucus secretion process was crippled when SCF/c-KIT signaling was hypo-activated. Inhibiting SCF/c-KIT signaling by Imatinib also resulted in weakened mucus secretion in WT mice. Intraperitoneal administration of MANS which competitively inhibits the activity of the vesicular transport protein MARCKS efficiently reduced mucus secretion in colonic goblet cells of WT mice. Significantly, phosphorylated MARCKS (p-MARCKS) was overtly decreased in colonic mucosa of Wadsm/m mice compared with WT mice, indicating that SCF/c-KIT signaling-regulated mucus secretion was probably mediated by MARCKS activation. Similar results were obtained in MCA patients and mouse model. Moreover, SCF/c-KIT signaling was activated or inhibited in HT-29 and LS174T CRC cells, which potently increased or decreased MARCKS activity, respectively. Finally, we found that PKCδ, a known kinase for MARCKS, was activated in WT and MCA mice along with MARCKS. Inhibition or activation of SCF/c-KIT signaling resulted in decreased or increased PKCδ activity respectively in vitro. In conclusion, we demonstrated that SCF/c-KIT signaling can promote the mucus secretion by activating PKCδ-MARCKS, which provided a new insight into understanding the mechanism of mucus secretion of goblet cells and MCA development.

SCF/c-KIT Signaling Increased Mucin2 Production by Maintaining Atoh1 Expression in Mucinous Colorectal Adenocarcinoma.

Mucinous colorectal adenocarcinoma (MCA) patients often a show high risk of malignant potential and a poorer survival rate. Given that the pathological feature and oncobiological characteristics of MCA are correlated with its abundant extracellular mucin2 (MUC2), we paid interest toward investigating the key factor that promotes MUC2 production exposure to highly-activated stem cell factor (SCF)/c-KIT signaling, which we believed to contribute to MCA formation. Long-term azoxymethane and dextran sodium sulfate treatment successfully induced MCA only in wild-type (WT) mice at week 37 and 43, while all c-kit loss-of-function mutant mice (Wadsm/m) developed non-MCA. Significantly, MUC2 and its key transcriptional factor Atonal homologue 1 (Atoh1) were remarkably expressed in MCA mice compared with non-MCA mice. Atoh1 was significantly elevated in colorectal cancer (CRC) cells stimulated by exogenous SCF or overexpressing c-KIT in vitro, while decreased by the blockage of SCF/c-KIT signaling with Imatinib. Furthermore, the maintained Atoh1 protein level was due to the inactive glycogen synthase kinase 3ß (p-GSK3ß) by virtue of the activated SCF/c-KIT-Protein Kinase B (AKT) signaling. Similar results were obtained from the ONCOMINE database and CRC patients. In conclusion, we suggested that SCF/c-KIT signaling promoted MUC2 production and MCA tumorigenesis by maintaining Atoh1 expression. Therefore, targeting the related key molecules might be beneficial for treating MCA patients.

Intravoxel incoherent motion diffusion-weighted magnetic resonance imaging for predicting histological grade of hepatocellular carcinoma: Comparison with conventional diffusion-weighted imaging.

AIM: To compare intravoxel incoherent motion (IVIM)-derived parameters with conventional diffusion-weighted imaging (DWI) parameters in predicting the histological grade of hepatocellular carcinoma (HCC) and to evaluate the correlation between the parameters and the histological grades. METHODS: A retrospective study was performed. Sixty-two patients with surgically confirmed HCCs underwent diffusion-weighted magnetic resonance imaging with twelve b values (10-1200 s/mm2). The apparent diffusion coefficient (ADC), pure diffusion coefficient (D), pseudo-diffusion coefficient (D*), and perfusion fraction (f) were calculated by two radiologists. The IVIM and conventional DWI parameters were compared among the different grades by using analysis of variance (ANOVA) and the Kruskal-Wallis test. Receiver operating characteristic (ROC) analysis was performed to evaluate the diagnostic efficiency of distinguishing between low-grade (grade 1, G1) and high-grade (grades 2 and 3, G2 and G3) HCC. The correlation between the parameters and the histological grades was assessed by using the Spearman correlation test. Bland-Altman analysis was used to evaluate the reproducibility of the two radiologists' measurements. RESULTS: The differences in the ADC and D values among the groups with G1, G2, and G3 histological grades of HCCs were statistically significant (P < 0.001). The D* and f values had no significant differences among the different histological grades of HCC (P > 0.05). The ROC analyses demonstrated that the D and ADC values had better diagnostic performance in differentiating the low-grade HCC from the high-grade HCC, with areas under the curve (AUCs) of 0.909 and 0.843, respectively, measured by radiologist 1 and of 0.911 and 0.852, respectively, measured by radiologist 2. The following significant correlations were obtained between the ADC, D, and D* values and the histological grades: r = -0.619 (P < 0.001), r = -0.628 (P < 0.001), and r = -0.299 (P = 0.018), respectively, as measured by radiologist 1; r = -0.622 (P < 0.001), r = -0.633 (P < 0.001), and r = -0.303 (P = 0.017), respectively, as measured by radiologist 2. The intra-class correlation coefficient (ICC) values between the two observers were 0.996 for ADC, 0.997 for D, 0.996 for D*, and 0.992 for f values, which indicated excellent inter-observer agreement in the measurements between the two observers. CONCLUSION: The IVIM-derived D and ADC values show better diagnostic performance in differentiating high-grade HCC from low-grade HCC, and there is a moderate to good correlation between the ADC and D values and the histological grades.