Sinomenine hydrochloride improves DSS-induced colitis in mice through inhibition of the TLR2/NF-κB signaling pathway.

BACKGROUND: Sinomenine hydrochloride (SH) has anti-inflammatory and immunosuppressive effects, and its effectiveness in inflammatory diseases, such as rheumatoid arthritis, has been demonstrated. However, whether SH has a therapeutic effect on dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) in mice and its mechanism of action have not been clarified. This study aimed to investigate the therapeutic effects and mechanism of action of SH on UC. METHODS: Twenty-four mice were randomly divided into control, model, SH low-dose (SH-L, 20 mg/kg), and SH high-dose (SH-H, 60 mg/kg) groups with six mice in each group. Disease activity index (DAI), colonic mucosal damage index, and colonic histopathology scores were calculated. The expression levels of related proteins, genes, and downstream inflammatory factors in the Toll-like receptor 2/NF-κB (TLR2/NF-κB) signaling pathway were quantified. RESULTS: SH inhibited weight loss, decreased DAI and histopathological scores, decreased the expression levels of TLR2, MyD88, P-P65, P65 proteins, and TLR2 genes, and also suppressed the expression of inflammatory factors TNF-α, IL-1 ß, and IL-6 in the peripheral blood of mice. CONCLUSION: The therapeutic effect of SH on DSS-induced UC in mice may be related to the inhibition of the TLR2/NF-κB signaling pathway.

Risk prediction of cholangitis after stent implantation based on machine learning.

The risk of cholangitis after ERCP implantation in malignant obstructive jaundice patients remains unknown. To develop models based on artificial intelligence methods to predict cholangitis risk more accurately, according to patients after stent implantation in patients' MOJ clinical data. This retrospective study included 218 patients with MOJ undergoing ERCP surgery. A total of 27 clinical variables were collected as input variables. Seven models (including univariate analysis and six machine learning models) were trained and tested for classified prediction. The model' performance was measured by AUROC. The RFT model demonstrated excellent performances with accuracies up to 0.86 and AUROC up to 0.87. Feature selection in RF and SHAP was similar, and the choice of the best variable subset produced a high performance with an AUROC up to 0.89. We have developed a hybrid machine learning model with better predictive performance than traditional LR prediction models, as well as other machine learning models for cholangitis based on simple clinical data. The model can assist doctors in clinical diagnosis, adopt reasonable treatment plans, and improve the survival rate of patients.

A Retrospective Analysis of the Efficacy of Endoscopic Variceal Ligation versus Endoscopic Tissue Adhesive Injection in the Treatment of Esophagogastric Variceal Bleeding.

BACKGROUND: The aim of the study was to investigate the effectiveness and safety of endoscopic variceal ligation (EVL) and endoscopic tissue adhesive injection (TAI) in the treatment of esophagogastric variceal bleeding (EVB). METHODS: A total of 245 patients with EVB who attended the First Affiliated Hospital of Bengbu Medical College from December 2017 to June 2021 were retrospectively collected. The participants were divided into the esophageal EVL (E-EVL) + gastric EVL (G-EVL) group (n = 103) and E-EVL + gastric TAI (G-TAI) group (n = 142), according to the procedure, comparing and assessing the clinical characteristics, laboratory results, operation time, rebleeding rate, efficacy, and complications. RESULTS: The E-EVL + G-EVL group had significantly less varicose vein diameter and operative time than the E-EVL + G-TAI group (p < 0.05). No statistical difference in the length of hospital stay between the two groups was noted (p > 0.05). The total rebleeding rate in the E-EVL + G-EVL group was 9.7%, whereas that of the E-EVL + G-TAI group was 11.9%; no statistical difference between the two groups was noted (p > 0.05). The overall effective rate of the E-EVL + G-EVL group was 90.21%, whereas that of the E-EVL + G-TAI group was 92.81%; no statistical difference between the two groups was observed (p > 0.05). The postoperative ulcer in the E-EVL + G-EVL group was smaller and more superficial than that in the E-EVL + G-TAI group, and the wound surface was smoother. CONCLUSION: Both EVL and TAI have good therapeutic effects on EVB. Furthermore, owing to its effectiveness in preventing rebleeding, no reduction in efficacy and no increase in complications, shortened operative time, smaller and superficial ulcer, and smoother wounds, gastric EVL is worthy of further clinical promotion.

miR-30c inhibits angiogenesis by targeting delta-like ligand 4 in liver sinusoidal endothelial cell to attenuate liver fibrosis.

Liver fibrosis is a common feature of liver dysfunction during chronic liver diseases and is frequently associated with angiogenesis, a dynamic process that forms new blood vessels from preexisting vasculature. MicroRNAs (miRNAs), which act as posttranscriptional regulators of gene expression, have been shown to regulate liver fibrosis; however, how miRNAs regulate angiogenesis and its mechanism in fibrosis are not well understood. We aimed to elucidate the role and mechanism of miR-30c in attenuating liver fibrosis. Using miRNA profiling of fibrotic murine livers, we identified differentially regulated miRNAs and discovered that miR-30c is aberrantly expressed and targets endothelial delta-like ligand 4 (DLL4) in either carbon tetrachloride-treated or bile duct ligated fibrotic mice, as well as in patients with liver fibrosis. Using CCK-8, wound healing and Matrigel tube formation assays, we found that miR-30c inhibited liver sinusoidal endothelial cell (LSEC) proliferation, migration, and angiogenesis capacity by targeting DLL4 in vitro. Importantly, nanoparticle-based delivery of miR-30c to LSECs inhibited the DLL4/Notch pathway and angiogenesis, thereby ameliorating liver fibrosis in vivo. Collectively, our findings demonstrate a protective role of miR-30c in liver fibrosis by regulating DLL4/Notch signaling and angiogenesis. Thus, miR-30c may serve as a potential treatment for chronic liver diseases.

Y-box binding protein 1 regulates liver lipid metabolism by regulating the Wnt/ß-catenin signaling pathway.

BACKGROUND: We mainly investigated how y-box binding protein 1 (YB-1) regulates liver lipid metabolism through the Wnt/ß-catenin signaling pathway using multiple models. METHODS: The LO2 cells were treated with palmitic acid (PA) to create an NAFLD model in vitro. Immunohistochemistry and Western blotting assays were used to detect the expression of YB-1, ß-catenin, SREBP-1c, LXRa, FXR1 and PPARα protein, and RNAs of them was detected by qRT-PCR. Oil Red O assay was applied to observe lipid droplets in LO2 cells and liver tissues. H&E staining was performed to observe the degree of liver inflammation. Proteomics in LO2 cells were conducted by Tandem mass tag proteomics assay. Co-immunoprecipitation and Western blotting assays were used to verify YB-1 complexed pGSK3ß. ELISA and Western blotting assays were used to detect the concentrations of TNFα and IL-6 in LO2 cells and liver tissues, respectively. RESULTS: We found that YB-1 and ß-catenin were highly expressed in the LO2 cell NAFLD model, and that the expression of TNFα and IL-6 also increased. Lipid synthases (SREBP-1c and LXRa) expression were decreased, while ß-oxidation-related factors (FXR1 and PPARα) expression were increased. The expression of SREBP-1c and LXRa were increased while FXR1 and PPARα were decreased, though such responses were rescued through inhibiting ß-catenin expression. Finally, tandem mass tag proteomics, co-immunoprecipitation, and Western blotting demonstrated that YB-1 could form a protein complex with phosphorylated glycogen synthase kinase 3 beta (pGSK3ß) to regulate Wnt/ß-catenin. In mouse NAFLD livers, immunohistochemistry and Western blotting validated the finding of YB-1 gene downregulation leading to the inhibition of Wnt/ß-catenin pathway activation, ultimately inhibiting lipid synthesis and reducing the inflammatory response. Similar to the in vitro investigation, ß-catenin overexpression reversed such YB-1 downregulation-induced downstream effects. Upregulation of the YB-1 gene promoted the activation of the Wnt/ß-catenin pathway, thus increasing lipid synthesis and the inflammatory response. However, downregulation of ß-catenin reversed this phenomenon caused by upregulating YB-1. CONCLUSIONS: In summary, these results demonstrate that YB-1 regulates liver lipid metabolism by regulating the Wnt/ß-catenin signaling pathway.

RNF8 induces autophagy and reduces inflammation by promoting AKT degradation via ubiquitination in ulcerative colitis mice.

RING finger protein 8 (RNF8) is an E3 ligase that is pivotal for DNA repair. However, the role of RNF8 in ulcerative colitis (UC) remains unclear. The aim of this study is to investigate the effect and the mechanism of RNF8 on UC model induced by trinitrobenzene sulfonic acid (TNBS) in mice. Lentiviruses overexpressing RNF8 were injected into mice after the induction of UC. The histopathological changes in colon tissues were assessed by haematoxylin and eosin staining. The mRNA level of RNF8 was detected by real-time quantitative polymerase chain reaction. The protein levels of RNF8, autophagy-related proteins (LC3 and P62) and AKT/mammalian target of rapamycin (mTOR) signalling-related proteins were measured by Western blot. The pro-inflammatory cytokines (tumour necrosis factor-α and interleukin-1ß) were examined by immunohistochemical analysis. Immunoprecipitation was performed to analyse the interaction between RNF8 and AKT1. The TNBS-induced UC mice exhibited colonic damage and inflammation, accompanied by decreased RNF8 expression, impaired autophagy and increased phosphorylation levels of AKT and mTOR in the colon. However, these alterations were reversed by RNF8 overexpression. Furthermore, RNF8 bound to AKT1 and mediated its ubiquitination. Collectively, RNF8 overexpression protects against TNBS-induced UC, which might be due to its enhancement of autophagy by suppressing the AKT/mTOR signalling via AKT1 ubiquitination.

Delta-like ligand 4/DLL4 regulates the capillarization of liver sinusoidal endothelial cell and liver fibrogenesis.

Liver sinusoidal endothelial cells (LSECs) undergo capillarization, or loss of fenestrae, and produce basement membrane during liver fibrotic progression. DLL4, a ligand of the Notch signaling pathway, is predominantly expressed in endothelial cells and maintains liver sinusoidal homeostasis. The aim of this study was to explore the role of DLL4 in LSEC capillarization. The expression levels of DLL4 and the related genes, capillarization markers and basement membrane proteins were assessed by immunohistochemistry, immunofluorescence, RT-PCR and immunoblotting as appropriate. Fenestrae and basement membrane formation were examined by electron microscopy. We found DLL4 was up-regulated in the LSECs of human and CCl4-induced murine fibrotic liver, consistent with LSEC capillarization and liver fibrosis. Primary murine LSECs also underwent capillarization in vitro, with concomitant DLL4 overexpression. Bioinformatics analysis confirmed that DLL4 induced the production of basement membrane proteins in LSECs, which were also increased in the LSECs from 4 and 6-week CCl4-treated mice. DLL4 overexpression also increased the coverage of liver sinusoids by hepatic stellate cells (HSCs) through endothelin-1 (ET-1) synthesis. The hypoxic conditions that was instrumental in driving DLL4 overexpression in the LSECs. Consistent with the above findings, DLL4 silencing in vivo alleviated LSEC capillarization and CCl4-induced liver fibrosis. In conclusion, DLL4 mediates LSEC capillarization and the vicious circle between fibrosis and pathological sinusoidal remodeling.

Autophagy promotes hepatic differentiation of hepatic progenitor cells by regulating the Wnt/ß-catenin signaling pathway.

Hepatic progenitor cells (HPCs) can be activated when the liver suffers persistent and severe damage and can differentiate into hepatocytes to maintain liver regeneration and homeostasis. However, the molecular mechanism underlying the hepatic differentiation of HPCs is unclear. Therefore, in this study, we aimed to investigate the roles of autophagy and the Wnt/ß-catenin signaling pathway during hepatic differentiation of HPCs in vivo and in vitro. First, immunohistochemistry, immunofluorescence and electron microscopy showed that Atg5 and ß-catenin were highly expressed in human fibrotic liver and mouse liver injury induced by feeding a 50% choline-deficient diet plus 0.15% ethionine solution in drinking water (CDE diet) for 21 days; in addition, these factors were expressed in CK19-positive HPCs. Second, Western blotting and immunofluorescence confirmed that CK19-positive HPCs incubated in differentiation medium for 7 days can differentiate into hepatocytes and that differentiated HPCs were able to take up ICG and secrete albumin and urea. Further investigation via Western blotting, immunofluorescence and electron microscopy revealed autophagy and the Wnt/ß-catenin pathway to be activated during hepatic differentiation of HPCs. Next, we found that inhibiting autophagy by downregulating Atg5 gene expression impaired hepatic differentiation of HPCs and inhibited activation of the Wnt/ß-catenin pathway, which was rescued by overexpression of the ß-catenin gene. Moreover, downregulating ß-catenin gene expression without inhibiting autophagy still impeded the differentiation of HPCs. Finally, coimmunoprecipitation demonstrated that P62 forms a complex with phosphorylated glycogen synthase kinase 3 beta (pGSK3ß). Third, in mouse CDE-induced liver injury, immunohistochemistry and immunofluorescence confirmed that downregulating Atg5 gene expression inhibited autophagy, thus impeding hepatic differentiation of HPCs and inhibiting activation of the Wnt/ß-catenin pathway. As observed in vitro, overexpression of ß-catenin rescued this phenomenon caused by autophagy inhibition, though decreasing ß-catenin levels without autophagy inhibition still impeded HPC differentiation. We also found that HPCs differentiated into hepatocytes in human fibrotic liver tissue. Collectively, these results demonstrate that autophagy promotes HPC differentiation by regulating Wnt/ß-catenin signaling. Our results are the first to identify a role for autophagy in promoting the hepatic differentiation of HPCs.

[Expression of miR-454-3p and its effect on proliferation, invasion and metastasis of colon cancer].

OBJECTIVE: To study the expression of miR-454-3p in colon cancer and its effect on colon cancer proliferation, invasion and hepatic metastasis. METHODS: Specimens of tumor tissues and paired adjacent tissues were collected from 20 patients with colorectal cancer for detecting the expression levels of miR-454-3p using in situ hybridization. Colon cancer cell line SW480 was transfected with a lentiviral vector to induce miR-454-3p over-expression, and the changes in cell proliferation and invasion were observed using cell counting kit-8 (CCK-8), clone formation assay and Transwell experiment. The effect of miR- 454-3p over-expression on hepatic metastasis of colon cancer was assessed in BALB/c mouse models. RESULTS: The results of in situ hybridization showed a significantly increased expression level of miR-454-3p in colon cancer tissues as compared with normal colon tissues (P < 0.05). In SW480 cells, over-expression of miR-454-3p significantly promoted the cell proliferation and invasion (P < 0.05). In BALB/c mice, SW480 cells over-expressing miR-454-3p showed a significantly higher potential for liver metastases than the control cells (P < 0.05). CONCLUSIONS: miR-454-3p is overexpressed in the tumor tissues in patients with colorectal cancer and participates in the progression of colorectal cancer by promoting cancer cell proliferation, invasion, and liver metastasis. miR-454-3p may serve as a novel biomarker for colorectal cancer diagnosis and prognostic evaluation.

Y-box Protein-1 Regulates the Expression of Collagen I in Hepatic Progenitor Cells via PDGFR-ß/ERK/p90RSK Signalling.

Y-box protein-1 (YB-1) is a highly conserved transcription factor that is involved in multiple biological processes via transcriptional regulation of several genes, including p53, cyclin D1, and EGFR. YB-1 has been reported to be overexpressed in injured livers. This study aims to explore the functions of YB-1 in hepatic progenitor cells (HPCs). Herein, chromatin immunoprecipitation sequencing (ChIP-sequencing) and RNA-sequencing assays identified that YB-1 participated in the biological adhesion process and ECM-receptor interactions in HPCs. Further study demonstrated that YB-1 modulated the expression of extracellular matrix components in HPCs. ChIP-sequencing assays established that PDGFR-ß was a target gene of YB-1, and luciferase reporter assays confirmed that YB-1 negatively regulated PDGFR-ß promoter activity in HPCs. In addition, PDGFR-ß can regulate the expression of collagen I through ERK/p90RSK signalling, and disruption of the signalling pathway with a PDGFR-ß inhibitor or ERK1/2 inhibitor abolished the regulatory effect of PDGFR-ß on collagen I expression in HPCs. Conclusively, YB-1 can modulate the expression of collagen I in HPCs via direct binding to the PDGFR-ß promoter, negatively regulating its expression. In addition, the ERK/p90RSK axis serves as the downstream signalling pathway of PDGFR-ß.

Exosomes derived from miR-181-5p-modified adipose-derived mesenchymal stem cells prevent liver fibrosis via autophagy activation.

Proliferating hepatic stellate cells (HSCs) respond to liver damage by secreting collagens that form fibrous scar tissue, which can lead to cirrhosis if in appropriately regulated. Advancement of microRNA (miRNA) hepatic therapies has been hampered by difficulties in delivering miRNA to damaged tissue. However, exosomes secreted by adipose-derived mesenchymal stem cells (ADSCs) can be exploited to deliver miRNAs to HSCs. ADSCs were engineered to overexpress miRNA-181-5p (miR-181-5p-ADSCs) to selectively home exosomes to mouse hepatic stellate (HST-T6) cells or a CCl4-induced liver fibrosis murine model and compared with non-targeting control Caenorhabditis elegans miR-67 (cel-miR-67)-ADSCs. In vitro analysis confirmed that the transfer of miR-181-5p from miR-181-5p-ADSCs occurred via secreted exosomal uptake. Exosomes were visualized in HST-T6 cells using cyc3-labelled pre-miRNA-transfected ADSCs with/without the exosomal inhibitor, GW4869. The effects of miRNA-181-5p overexpression on the fibrosis associated STAT3/Bcl-2/Beclin 1 pathway and components of the extracellular matrix were assessed. Exosomes from miR181-5p-ADSCs down-regulated Stat3 and Bcl-2 and activated autophagy in the HST-T6 cells. Furthermore, the up-regulated expression of fibrotic genes in HST-T6 cells induced by TGF-ß1 was repressed following the addition of isolated miR181-5p-ADSC exosomes compared with miR-67-ADSCexosomes. Exosome therapy attenuated liver injury and significantly down-regulated collagen I, vimentin, α-SMA and fibronectin in liver, compared with controls. Taken together, the effective anti-fibrotic function of engineered ADSCs is able to selectively transfer miR-181-5p to damaged liver cells and will pave the way for the use of exosome-ADSCs for therapeutic delivery of miRNA targeting liver disease.