Construction of A375·S2 Melanoma Cell Line with High Sensibility to IL-1 by Overexpressing IL-1 Receptor.

We described an operation that co-overexpress interleukin receptor 1 (IL-1R1) and its co-receptor (IL-1R1AcP) genes in wild-type A375·S2 cells in order to increase their sensibility to IL-1. Firstly, laser scanning confocal microscope observed that IL-1R1 could be expressed on the surface of A375·S2 cells. qPCR was performed to estimate the ratio of two genes and result showed the ratio was almost 4.57:1. Then two genes were linked to vectors and co-transfected into A375·S2 cells. qPCR and Western blotting showed the protein content improved markedly. Finally, MTS assay was executed and the sensitivity of A375·S2 cells that co-transfected receptors to IL-1ß increased significantly. Another MTS assay showed the cell activity variation changed significantly (P < 0.05) and the reliability of the experiment was high, indicating that cell line established in this study could be further used for the activity test of IL-1Ra. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01027-8.

FAM83D promotes the proliferation and migration of hepatocellular carcinoma cells by inhibiting the FBXW7/MCL1 pathway.

Background: Hepatocellular carcinoma (HCC) is one of the most malignant tumors and has a poor 5-year survival rate. Family with sequence similarity 83, member D (FAM83D) is characterized as an oncogenic gene related to cell proliferation in many tumors, but the role and underlying mechanism of FAM83D in the development of HCC are still unclear. Methods: FAM83D expression profiles and clinicopathological data were obtained from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC). Additionally, 2 data sets from the Gene Expression Omnibus (GEO) database were used to further validate the FAM83D profile in HCC. We then downregulated the expression of FAM83D in HCC cells transfected with FAM83D small-interfering ribonucleic acid (siRNA) and upregulating its expression by FAM83D-overexpression transfection for further in vitro studies. Results: TCGA and the GEO databases showed that FAM83D was significantly more upregulated in tumor tissues than non-tumor tissues. The high expression of FAM83D in HCC is associated with poor prognostic clinical factors. The knockdown of FAM83D in SNU449 and HUH7 cells in vitro impaired cell proliferation and migration, and promoted apoptosis, while the overexpression of FAM83D in BEL7402 cells had the opposite effect. Further, combined transfection with FBXW7 siRNA or MCL1-overexpression reversed the role of FAM83D knockdown on cell proliferation, migration, and apoptosis in vitro, while FBXW7 expression was negatively correlated with both the FAM83D and MCL1 levels in TCGA-LIHC patients. Conclusions: FAM83D played a significant role in HCC progression by enhancing cell proliferation and migration and inhibiting apoptosis, which may have been caused by the inhibition of the FBXW7/MCL1 signaling pathway. Thus, FAM83D may be a promising therapeutic target for HCC.

Expression and Immunogenicity of SARS-CoV-2 Virus-Like Particles based on Recombinant Truncated HEV-3 ORF2 Capsid Protein.

COVID-19 is an emerging disease that poses a severe threat to global public health. As such, there is an urgent demand for vaccines against SARS-CoV-2, the virus that causes COVID-19. Here, we describe a virus-like nanoparticle candidate vaccine against SARS-CoV-2 produced by an E. coli expression system. The fusion protein of a truncated ORF2-encoded protein of aa 439~608 (p170) from hepatitis E virus CCJD-517 and the receptor-binding domain of the spike protein from SARS-CoV-2 were expressed, purified and characterized. The antigenicity and immunogenicity of p170-RBD were evaluated in vitro and in Kunming mice. Our investigation revealed that p170-RBD self-assembled into approximately 24 nm virus-like particles, which could bind to serum from vaccinated people (p < 0.001) and receptors on cells. Immunization with p170-RBD induced the titer of IgG antibody vaccine increased from 14 days post-immunization and was significantly enhanced after a booster immunization at 28 dpi, ultimately reaching a peak level on 42 dpi with a titer of 4.97 log10. Pseudovirus neutralization tests showed that the candidate vaccine induced a strong neutralizing antibody response in mice. In this research, we demonstrated that p170-RBD possesses strong antigenicity and immunogenicity and could be a potential candidate for use in future SARS-CoV-2 vaccine development.

Effect of Curing Temperature on the Properties of a MgO-SiO2-H2O System Prepared Using Dead-Burned MgO.

The hydration of M-S-H prepared using silica fume (SF) and dead-burned MgO cured at 20 °C, 50 °C, and 80 °C was investigated, and the properties and performance of this M-S-H were measured. The formation of M-S-H was characterized using XRD, FTIR, TGA, and 29Si MAS-NMR. Results show that the compressive strength of paste prepared using MgO calcined at 1450 °C for 2 h reached 25 MPa after 28 d. The shrinkage of mortar made with low reactivity MgO was lower than that made with high reactivity MgO. The pH value of MgO/SF paste mixed with dead-burned MgO did not exceed 10.4 at room temperature. The shrinkage of M-S-H prepared using dead-burned MgO was less than that prepared using more active MgO, and its strength did not decrease over time. No (or only a small amount of) Mg(OH)2 was formed, which is why the strength of M-S-H prepared with dead-burned MgO continually increased, without decreasing. The promotion of curing temperature favor process of MgO hydration and is beneficial for degree of silica polymerization. The sample cured in 50 °C water showed the highest relative degree of reaction.

Prevalence and Associated Factors of Sexual Dysfunction in Patients With Inflammatory Bowel Disease.

Background: Sexual dysfunction (SD) in patients who suffer from inflammatory bowel disease (IBD) has not attracted widespread attention, and thus research studies are scarce. Objective: This study aims to evaluate the rates of SD in IBD compared with healthy individuals and elucidate the associated factors. Methods: In this cross-sectional study, the Female Sexual Function Index (FSFI) and the simplified version of the International Index of Erectile Function (IIEF-5) were filled by IBD patients, as well as healthy control individuals. Results: A total of 208 IBD patients, including 133 with Crohn's disease (CD) and 75 with ulcerative colitis (UC), and 190 healthy individuals filled out the questionnaires. In women, SD rates were 61.9% in the patients with IBD vs. 24.4% in the healthy controls (p < 0.01). In men, the rates of erectile dysfunction (ED) were 43.5% in the patients with IBD vs. 12.5% in the healthy controls (p < 0.01). Anxiety (OR, 3.092; 95%CI: 1.033-9.252, p = 0.044) and active perianal disease (OR, 4.481; 95%CI: 1.055-19.029, p = 0.042) were independent risk factors for SD in female IBD patients. age (OR, 1.050; 95%CI: 1.007-1.095, p = 0.022), depression (OR, 5.763; 95%CI: 1.864-17.821, p = 0.002) and active perianal disease (OR, 7.117; 95%CI: 1.747-28.983, p = 0.006) were independent risk factors for ED in male patients. Conclusions: In the IBD patients, 62% of women reported having SD, and 44% of men reported having ED. These higher rates, as compared to the healthy controls, are mostly driven by active perianal disease and psychological factors.

Efficacy and Safety of Endoscopic Submucosal Dissection for Rectal Tumors Extending Versus Not to the Dentate Line: A Systematic Review and Meta-Analysis.

GOALS: To evaluate the outcomes of endoscopic submucosal dissection (ESD) for rectal tumors extending to the dentate line (RTDLs) compared with rectal tumors not extending to the dentate line (non-RTDLs). BACKGROUND: There is limited composite data on the outcomes of ESD for RTDLs versus non-RTDLs. STUDY: We performed a systematic review and meta-analysis of studies that reported the clinical outcomes of ESD for RTDLs and non-RTDLs. Main outcomes were pooled estimated rates of en bloc/complete/curative resection, local recurrence, and incidence of bleeding, perforation, stricture, anal pain, and fever. RESULTS: Six studies were enrolled, including 265 cases of RTDLs and 788 cases of non-RTDLs. The en bloc resection rate was comparable for RTDLs and non-RTDLs [odds ratio (OR), 1.04; 95% confidence interval (CI), 0.55-1.95; P=0.90]. The complete resection rate was significantly lower for RTDLs (OR, 0.59; 95% CI, 0.41-0.83; P=0.003), as well as the curative resection rate (OR, 0.57; 95% CI, 0.38-0.87; P=0.010). The rates of stricture, postoperative anal pain and local recurrence were significantly higher for RTDLs than non-RTDLs (OR, 3.07; 95% CI, 1.01-9.31; P=0.05) (OR, 42.10; 95% CI, 4.73-374.97; P=0.0008) (OR, 3.00; 95% CI, 1.13-7.96; P=0.03), but the higher rates of postoperative bleeding and fever for RTDLs were not significantly (OR, 1.33; 95% CI, 0.53-3.30; P=0.54) (OR, 2.23; 95% CI, 0.55-9.07; P=0.26), as well as its lower perforation rate (OR, 0.85; 95% CI, 0.27-2.63; P=0.78). CONCLUSIONS: Despite its inferior outcomes than non-RTDLs, ESD is still a feasible and safe treatment for RTDLs if appropriate lesions are treated by experienced operators.

Efficacy and Safety of Endoscopic Submucosal Dissection for Dysplasia in Ulcerative Colitis Patients: A Systematic Review and Meta-Analysis.

BACKGROUND AND AIMS: Ulcerative colitis (UC) is associated with an increased risk of colorectal cancer. Current guidelines recommend endoscopic resection if the lesion is visible with distinct margins and a complete resection can be achieved. However, submucosal fibrosis due to chronic inflammation may increase the procedural risk and reduce the complete resection rate. The aim of this study is to assess the efficacy and safety of endoscopic submucosal dissection (ESD) for dysplasia in UC patients. MATERIALS AND METHODS: A systematic search of databases was performed until May 30, 2021. Studies that reported the resection rates and complication rates of ESD for dysplasia in UC patients were included. A random-effects model was used to generate conservative estimates of the prevalence of the outcome variables. All data analyses were performed using software Stata (version 15). RESULTS: 8 studies were enrolled in the meta-analysis, with a total of 203 dysplastic lesions in 192 UC patients. The mean lesion size was 26.7 mm. About 83% of the lesions were located in the left-side colon, and 90% of the lesions were nonpolypoid, and about 71% of the lesions had submucosal fibrosis. The mean procedural time of ESD was 83 minutes. The en bloc resection rate, complete resection rate, and curative resection rate were 94%, 84%, and 81%, respectively, with a local recurrence rate of 5%. The pooled prevalence of bleeding and perforation were 8% and 6%, respectively. The rates of metachronous tumors and additional surgery after ESD were 6% and 10%, respectively. CONCLUSION: Despite some limitations, our study suggests that ESD is an effective and safe treatment for dysplasia in UC patients. However, randomized controlled multicenter studies with less heterogeneity and longer follow-up are needed to better assess the clinical outcomes of ESD in UC patients.

Value of peripheral perfusion index to predict acute limb ischaemia in newborns after peripheral artery cannulation.

AIMS AND OBJECTIVES: To investigate the feasibility of using peripheral perfusion index (PPI) to monitor acute limb ischaemia (ALI) in newborns after catheterisations. BACKGROUND: ALI is common complication of neonatal peripheral artery cannulation. It is important to address as soon as the early signs of ALI. PPI could aid in noninvasive evaluation of distal extremity perfusion in an effort to notify risk of potential ischaemic injury from catheterisations. DESIGN: A nested case-control study. METHODS: Clinical information of newborns who had been admitted to the Neonatal Intensive Care Unit of Jiangxi Provincial Children's Hospital and had received peripheral artery cannulation from January 2018 to January 2020 was prospectively collected. Transcutaneous blood oxygen saturation (TcSO2 ), PPI and delta-PPI (ΔPPI1; the difference in PPI values of the two arms. ΔPPI2; difference in the PPI values before and after cannulation) were recorded. We used STROBE checklist as an EQUATOR in this study. RESULTS: A total of 25 newborns with ALI were included in the study. These were then paired with 100 newborns without ALI. The PPI and TcSO2 of the cannulated limb were significantly lower in the ALI group than in the non-ALI (NALI) group (p < .05). The area under the receiver-operating characteristic curve was significant for ΔPPI1. The ΔPPI1 had a sensitivity and specificity of 92% and 87%, respectively, for diagnosing ALI. ΔPPI1 greater than 0.315 suggested that the infant was at risk of ALI. CONCLUSIONS: Monitoring the change in the PPI in newborns after catheterisations helped in the early assessment of ALI. RELEVANCE TO CLINICAL PRACTICE: Drops in the PPI and TcSO2 of the cannulated limbs might, to some extent, reflect the possibility of ALI in newborns. ΔPPI1 (the difference in PPI values of the two arms) proved to be a simple, objective parameter to predict the presence of ALI.

The Role of E3 Ubiquitin Ligases and Deubiquitinases in Inflammatory Bowel Disease: Friend or Foe?

Inflammatory bowel disease (IBD), which include Crohn's disease (CD) and ulcerative colitis (UC), exhibits a complex multifactorial pathogenesis involving genetic susceptibility, imbalance of gut microbiota, mucosal immune disorder and environmental factors. Recent studies reported associations between ubiquitination and deubiquitination and the occurrence and development of inflammatory bowel disease. Ubiquitination modification, one of the most important types of post-translational modifications, is a multi-step enzymatic process involved in the regulation of various physiological processes of cells, including cell cycle progression, cell differentiation, apoptosis, and innate and adaptive immune responses. Alterations in ubiquitination and deubiquitination can lead to various diseases, including IBD. Here, we review the role of E3 ubiquitin ligases and deubiquitinases (DUBs) and their mediated ubiquitination and deubiquitination modifications in the pathogenesis of IBD. We highlight the importance of this type of posttranslational modification in the development of inflammation, and provide guidance for the future development of targeted therapeutics in IBD.

Targeting Gremlin 1 Prevents Intestinal Fibrosis Progression by Inhibiting the Fatty Acid Oxidation of Fibroblast Cells.

Intestinal fibrosis is a consequence of continuous inflammatory responses that negatively affect the quality of life of patients. By screening altered proteomic profiles of mouse fibrotic colon tissues, we identified that GREM1 was dramatically upregulated in comparison to that in normal tissues. Functional experiments revealed that GREM1 promoted the proliferation and activation of intestinal fibroblast cells by enhancing fatty acid oxidation. Blocking GREM1 prevented the progression of intestinal fibrosis in vivo. Mechanistic research revealed that GREM1 acted as a ligand for VEGFR2 and triggered downstream MAPK signaling. This facilitated the expression of FAO-related genes, consequently enhancing fatty acid oxidation. Taken together, our data indicated that targeting GREM1 could represent a promising therapeutic approach for the treatment of intestinal fibrosis.

YAP-TEAD up-regulates IRS2 expression to induce and deteriorate oesophageal cancer.

Oesophageal cancer (EC) represents a significant cause of cancer worldwide. Yes-associated protein (YAP) is reported to correlate with the initiation of multiple cancers including EC, but the underlying mechanism remains elusive. The current study aimed to investigate the molecular mechanism of YAP-TEAD in the occurrence and progression of EC. EC tissues and cells were obtained, followed by determination of the expression of YAP, c-Jun, pc-Jun and IRS2. The effect of YAP-TEAD on the biological EC cell processes was explored through gain- and loss-of-function approaches. The interaction between YAP and TEAD was detected by co-immunoprecipitation. The binding of TEAD to the c-Jun promoter was determined using chromatin immunoprecipitation. Tumour formation in the nude mice was detected in order to ascertain the effect of YAP and IRS2 in vivo. We found elevated YAP in the EC tissues and cells. YAP silencing led to a decrease in EC cell proliferation, invasion and sphere formation. YAP-TEAD complex bound to the promotor of c-Jun, and c-Jun led to an increase in the expression of IRS2 through the JNK/c-Jun pathway. Additionally, pc-Jun and phosphorylated JNK were localized in the nuclear in addition to displaying enhanced expression in the EC tissues. IRS2 overexpression negated the inhibition of cell proliferation, invasion and sphere formation triggering YAP silencing. YAP up-regulated IRS2 and aggravated EC in vivo. Taken together, YAP-TEAD activates the JNK/c-Jun pathway to up-regulate IRS2, ultimately promoting EC progression. Therefore, YAP-TEAD inhibition could be a promising therapeutic approach for EC treatment.

LINC00337 promotes tumor angiogenesis in colorectal cancer by recruiting DNMT1, which suppresses the expression of CNN1.

Colorectal cancer (CRC) is one of the most common human malignancies. An increasing body of evidence has revealed the important roles long noncoding RNA (lncRNA) plays in the growth dynamics of CRC cells. In this study, we aimed to define the role of LINC00337 in the malignant phenotypes, especially angiogenesis, of CRC and clarify the underlying molecular basis. Bioinformatic analyses identified promoter region methylation of CNN1 in CRC, which was further validated by BSP and MSP assays. Loss- and gain- of function approaches were used to determine the roles of CNN1 and LINC00337 in vitro and in vivo. MTT-based method, Transwell migration/invasion assays, and tube formation assay were adopted to evaluate the cancer cell proliferation, migration/invasion, and proangiogenetic potency respectively in vitro. The tumor growth, microvascular density (MVD) and markers of proliferation (Ki67) and angiogenesis (VEGF) were quantified in nude mice xenografted with CRC cells. It was found that CNN1 downregulation and LINC00337 overexpression occurred in CRC tissues and cells. Besides, the CNN1 promoter region was hypermethylated in CRC. CNN1 overexpression or LINC00337 knockdown restricted CRC cell proliferation, migration/invasion, and proangiogenetic potency in vitro, which was substantiated by the in vivo experiments evidenced by facilitated tumor growth and MVD as well as elevated Ki67 and VEGF. Furthermore, our mechanistic evidence revealed that LINC00337 recruited DNMT1 to the promoter region of CNN1 and restricted the transcription of CNN1. Taken together, this study indicates that LINC00337 facilitates the tumorigenesis and angiogenesis in CRC via recruiting DNMT1 to inhibit the expression of CNN1.

Analysis of non­alcoholic fatty liver disease microRNA expression spectra in rat liver tissues.

The prevalence of non-alcoholic fatty liver disease (NAFLD) has been increasing in recent years. Previous studies have suggested that micro (mi)RNAs may be involved in the pathogenesis of NAFLD. To investigate the role of miRNAs in rat NAFLD, a total of 16 male Sprague Dawley rats were randomly divided into a control group and a model group. Rats in the control group were fed a normal diet for 12 weeks, whereas the rats in the model group were fed a high­fat and high­sugar diet for 12 weeks. Following this, the animals were sacrificed and liver tissues were rapidly removed to investigate the severity of NAFLD. Blood samples were collected to investigate liver function, in addition to total cholesterol, total triglyceride and fasting plasma glucose levels. Total RNA from three fresh liver samples per experimental group was extracted for subsequent miRNA gene chip analysis using GeneChip miRNA 4.0 to investigate differentially expressed miRNAs, and miRNA expression was further verified via reverse transcription­quantitative polymerase chain reaction (RT­qPCR). Compared with the control group, the results revealed that there were 10 differentially expressed miRNAs in the model group, five of which were overexpressed and five of which were underexpressed compared with the control group. The results of the RT­qPCR analysis revealed that miR­182, miR­29b­3p and miR­741­3p were significantly overexpressed in the model group compared with the control group, which was largely consistent with the results of the microarray analysis. The results suggested that the differentially expressed microRNAs demonstrated in the present study may be involved in the pathogenesis of NAFLD; however, the mechanism underlying the differential expression of miRNAs in NAFLD requires further investigation.

Mutation of miR-21 targets endogenous lipoprotein receptor-related protein 6 and nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a chronic disorder characterized by hepatic fat accumulation and abnormal lipid metabolism. Although miR-21 has been implicated in nonalcoholic fatty liver disease, it is unknown whether miR-21 could function as a therapeutic target. Here, we perform transfection analysis of miR-21 mimic or control mimic to evaluate the effects of miR-21 expression levels on human HepG2 nonalcoholic fatty liver cells. We used siRNA techniques to knock down miR-21 in HepG2 and control 293T cell lines, and then monitored lipid production and the expression levels of genes involved in lipid metabolism. The effects of miR-21 expression levels on LDL receptor-related protein 6 (LRP6) expression were evaluated using qRT-PCR and western blot analyses. Luciferase reporter assays were conducted to confirm the effects of miR-21 expression levels on LRP6. The results indicated that transfection of miR-21 mimic induced changes in the expression levels of lipogenic enzymes, including acetyl-CoA carboxylase 1 (ACC1), stearoyl CoA desaturase (1SCD1), sterol regulatory element-binding protein 1 (SREBP1), and liver X receptor alpha (LXRα). Transfection of miR-21 mimic suppressed the transcription and translation of LRP6 at the mRNA and protein levels, whereas miR-21 knockdown increased the expression levels of LRP6. Transfection of miR-21 mimic in HepG2 cells also induced lipid production and triggered the expression of critical lipid metabolic enzymes. These data suggest that mutation of miR-21 may be a new therapeutic strategy to treat nonalcoholic fatty liver diseases by targeting endogenous LRP6.

Forsythoside A Controls Influenza A Virus Infection and Improves the Prognosis by Inhibiting Virus Replication in Mice.

OBJECTIVE: The objective of this study was to observe the effects of forsythoside A on controlling influenza A virus (IAV) infection and improving the prognosis of IAV infection. METHODS: Forty-eight SPF C57BL/6j mice were randomly divided into the following four groups: Group A: normal control group (normal con); Group B: IAV control group (V con); Group C: IAV+ oseltamivir treatment group (V oseltamivir; 0.78 mg/mL, 0.2 mL/mouse/day); Group D: IAV+ forsythoside A treatment group (V FTA; 2 µg/mL, 0.2 mL/mouse/day). Real-time fluorescence quantitative PCR (RT-qPCR) was used to measure mRNA expression of the TLR7, MyD88, TRAF6, IRAK4 and NF-κB p65 mRNA in TLR7 signaling pathway and the virus replication level in lung. Western blot was used to measure TLR7, MyD88 and NF-κB p65 protein. Flow cytometry was used to detect the proportion of the T cell subsets Th1/Th2 and Th17/Treg. RESULTS: The body weight began to decrease after IAV infection, while FTA and oseltamivir could reduce the rate of body weight loss. The pathological damages in the FTA and oseltamivir group were less serious. TLR7, MyD88, TRAF6, IRAK4 and NF-κB p65 mRNA were up-regulated after virus infection (p < 0.01) while down-regulated after oseltamivir and FTA treatment (p < 0.01). The results of TLR7, MyD88 and NF-κB p65 protein consisted with correlative mRNA. Flow cytometry showed the Th1/Th2 differentiated towards Th2, and the Th17/Treg cells differentiated towards Treg after FTA treatment. CONCLUSIONS: Our study suggests forsythoside A can control influenza A virus infection and improve the prognosis of IAV infection by inhibiting influenza A virus replication.

Efficacy of traditional Chinese medicine in treating cancer.

The morbidity associated with cancer has rapidly increased in recent years, and in the previous 5 years has had a tendency to be the leading cause of fatality compared with cardiovascular disease. Therefore, effective measures are required with an aim to reduce the incidence. Based on the results of clinical investigation, a multidisciplinary treatment strategy for cancer, which includes radiotherapy, chemotherapy, surgery, targeted therapy and immunotherapy, are prominently used in clinical practice. However, the therapies are insufficient due to multidrug resistance, adverse effects and the presence of the root of the cancer. Therefore, there is a necessity to develop more effective or adjunctive therapies for cancer prevention and treatment. Cancer is now widely recognized as a systemic humoral disease. Similarly, the function of herbal drugs is to modulate the whole body system in a more holistic way. Recently, herbal drugs have been applied to one of the efficient approaches for cancer therapy. Furthermore, there is evidence that various herbal medicines have been proven to be useful and effective in sensitizing the conventional agents against the various factors at the cellular and molecular levels that are associated with the occurrence of cancer and in prolonging survival time, alleviating side effects of chemotherapy and radiotherapy and improving the quality of life in cancer patients.

[TLR4 contributes to intestinal hyperpermeability in alcoholic liver disease].

OBJECTIVE: To determine whether Toll-like receptor 4 (TLR4) is involved in development of gut leakiness in alcoholic steatohepatitis using an in vivo animal model and an in vitro cell culture system. METHODS: Mice were fed an alcohol (ethanol group, EtOH) or isocaloric liquid diet (control group, Ctrl). Successful establishment of the alcoholic steatohepatitis model was assessed at week 6 by measuring serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and evaluating the liver pathology using hematoxylin and eosin (HandE) staining of liver tissues. Gut permeability was assessed by measuring serum endotoxin and urine lactulose/mannitol (L/M) levels and evaluating HandE-stained colon tissues. Intestinal and colon tissue expression levels of TLR4 were assessed by immunohistochemistry. Cultured Caco-2 cells were exposed to 25 - 400 mmol/L EtOH and changes in TLR4 were assessed by enzyme-linked immunoassay and in permeability were assessed by intracellular uptake of FD4. RESULTS: The mice in the EtOH group had significantly higher levels of serum ALT (46.5 +/- 6.9 U/L vs. Ctrl: 30.9 +/- 4.4 U/L, P less than 0.01), serum AST (53.3 +/- 7.9 U/L vs. Ctrl: 29.3 +/- 3.8 U/L, P less than 0.01), serum endotoxin (0.33 +/- 0.05 Eu/L vs. Ctrl: 0.27 +/- 0.04 Eu/L, P less than 0.01), and urine L/M (2.59 +/- 0.44% vs. Ctrl: 2.17 +/- 0.31%, P less than 0.05). The mice in the EtOH group also had significantly higher expression levels of TLR4 in intestinal tissues (13.1 +/- 2.0 ng/ml vs. Ctrl: 7.4 +/- 1.2 ng/L, P less than 0.01) and in colonic tissues (18.5 +/- 2.7 ng/ml vs. Ctrl: 9.1 +/- 1.6 ng/ml, P less than 0.01). The intestinal histopathology of the two groups was not different. Immunohistochemical staining of colonic tissues showed brown particles distributed in the endochylema and membrane of the EtOH group, which was almost completely absent in the Ctrl group. EtOH treatment of Caco-2 cells led to a dose-dependent increase in TLR4 expression and in cellular permeability. CONCLUSION: Chronic alcohol exposure induced TLR4 expression and cellular permeability in gut tissues. Activation of TLR4 may be involved in development of gut leakiness in alcoholic liver disease.

Toll-like receptor 4 increases intestinal permeability through up-regulation of membrane PKC activity in alcoholic steatohepatitis.

Intestinal hyperpermeability is a causal factor for the development of alcoholic endotoxemia and steatohepatitis. However, the mechanisms governing this link remain unknown. The purpose of this study was to determine whether toll-like receptor 4 (TLR4) is involved in ethanol's deleterious effects on the intestinal barrier. Caco-2 cells were incubated in vitro with 1-10% ethanol. The results indicated that ethanol had a dose-dependent effect in increasing TLR4 expression and intercellular permeability. Then the effects of TLR4 on protein kinase C (PKC) and the intercellular junction protein occludin were assessed with and without pretreatment with a TLR4 inhibitor. The results indicated that TLR4 increased nonspecific PKC activity and reduced the expression of phosphorylated occludin in the membrane, which increased intercellular permeability. These effects were prevented by pretreatment with TLR4 mAb. Wild-type C57BL/6 mice were fed an ethanol or isocaloric liquid diet for 6 weeks. Hepatitis was diagnosed by the presence of an associated elevated blood endotoxin level. Chronic ethanol treatment significantly elevated blood endotoxin levels, intestinal permeability, and the expression of TLR4 in the ileum and colon. Moreover, ethanol exposure reduced the distribution of phosphorylated occludin in the intestinal epithelium because of PKC activation. In conclusion, chronic ethanol exposure induces a high response of TLR4 to lipopolysaccharide (LPS), and TLR4 increases intestinal permeability through down-regulation of phosphorylated occludin expression in the intestinal epithelial barrier, accompanied by membrane PKC hyperactivity.