Development and external validation of prognostic scoring models for portal vein thrombosis: a multicenter retrospective study.

BACKGROUND: Portal vein thrombosis is a common complication of liver cirrhosis and hepatocellular carcinoma; however, few studies have reported its long-term clinical prognosis. This study aimed to establish and validate easy-to-use nomograms for predicting gastrointestinal bleeding, portal vein thrombosis resolution, and mortality of patients with portal vein thrombosis. METHODS: This multicenter retrospective cohort study included 425 patients with portal vein thrombosis who were divided into training (n = 334) and validation (n = 91) sets. Prediction models were developed using multivariate Cox regression analysis and evaluated using the consistency index and calibration plots. RESULTS: Predictors of gastrointestinal bleeding included a history of gastrointestinal bleeding, superior mesenteric vein thrombosis, red color sign observed during endoscopy, and hepatic encephalopathy. Meanwhile, predictors of resolution of portal vein thrombosis included a history of abdominal infection, C-reactive protein and hemoglobin levels, and intake of thrombolytics. Predictors of death included abdominal infection, abdominal surgery, aspartate aminotransferase level, hepatic encephalopathy, and ascites. All models had good discriminatory power and consistency. Anticoagulation therapy significantly increased the probability of thrombotic resolution without increasing the risk of bleeding or death. CONCLUSIONS: We successfully developed and validated three prediction models that can aid in the early evaluation and treatment of portal vein thrombosis.

Circulating Bone morphogenetic protein 9 (BMP9) as a new biomarker for noninvasive stratification of nonalcoholic fatty liver disease and metabolic syndrome.

Nonalcoholic fatty liver disease (NAFLD) is closely related to metabolic syndrome (MetS). Bone morphogenetic protein 9 (BMP9) is an essential factor in glucose, lipid and energy metabolism. This study aims to investigate whether BMP9 can serve as a serological marker for the severity of NAFLD or MetS. Blood samples, clinical data and FibroTouch test were collected from consecutively recruited 263 individuals in Shanghai East hospital. All the participants were divided into three groups: the healthy controls, nonalcoholic fatty liver (NAFL) group and nonalcoholic steatohepatitis (NASH) at-risk group according to the results of FibroTouch test and liver function. Serum BMP9 levels were measured by enzyme-linked immunosorbent assay. Serum BMP9 levels were positively correlated with transaminase, triglyceride, fasting plasma glucose, glycated hemoglobin (HbA1c) and uric acid while it showed a downward trend as the increasing number of MetS components. Furthermore, it differentiated NASH at-risk (58.13 ± 2.82 ng/L) from the other groups: healthy control (70.32 ± 3.70 ng/L) and NAFL (64.34 ± 4.76 ng/L) (p < 0.0001). Controlled attenuation parameter of liver fat and liver stiffness measurement were negatively correlated with BMP9 levels, while high-density lipoprotein levels were positively correlated. The risk of developing NAFLD increased along with elevated serum BMP9 and BMI, and a significantly higher risk was observed in men compared to women. BMP9 should be considered a protective factor for the onset and development of NAFLD, as well as a promising biomarker for the severity of the NAFLD and MetS.

hUC-MSCs therapy for Crohn's disease: efficacy in TNBS-induced colitis in rats and pilot clinical study.

BACKGROUND: The use of mesenchymal stem cells (MSCs) has recently emerged as a promising new therapeutic strategy for many diseases including perianal fistulizing Crohn's disease (CD). Whether hUC-MSCs can promote the healing of luminal ulcer in CD has not been studied so far. METHODS: The model of TNBS-induced colitis in rats was used to confirm the efficacy of hUC-MSCs in the treatment of CD. Then, seventeen CD patients refractory to or unsuitable for currently available therapies were enrolled and received once submucosal local injection through colonoscopy combined with once intravenous drip on the next day. All patients received a 24-week follow-up. Clinical and laboratory assessments were monitored at baseline, week 4, 8, 12, and 24. Endoscopic evaluations were conducted at baseline and week 12. Mucosal specimens were obtained at the margin of lesions by endoscopy biopsies and used for RNA sequencing. Two hUC-MSCs co-culture systems were established in vitro, one with the mucosa specimens and the other with M1 macrophages induced from THP1. The expressions of genes representing inflammation (TNFα, IL-6, and IL-1ß) and intestinal barrier function (ZO1, CLAUDIN1, and CDH1) were tested by RT-PCR. FINDINGS: hUC-MSCs treatment increased body weight and decreased disease activity index (DAI), colon macroscopic damage index (CMDI), and histopathological score (HPS) of rats with TNBS-induced colitis. The results of the clinical study also showed that this mode of hUC-MSCs application was associated with regression of intestinal ulceration. Eight patients (47%) got endoscopic responses (SES-CD improvement of ≥50% from baseline) and three patients (17.65%) got mucosal healing (SES-CD is zero), with a parallel improvement of clinical and laboratory parameters without serious adverse events. RNA sequencing showed hUC-MSCs therapy was associated with an upregulation of transcripts linked to intestinal epithelial barrier integrity and a downregulation of inflammatory signaling pathways in the intestinal mucosa, especially the TNF signaling pathway, IL-17 signaling pathway, and TLR signaling pathway. RNA expression of intestinal epithelial tight junction protein (ZO1, CLAUDIN1, and CDH1), and the RNA expression of major intestinal inflammatory factors in CD (IL-1ß, IL-6, and TNFα, p < 0.001 for all) were improved significantly. Moreover, hUC-MSCs could attenuate the polarization of M1 macrophage induced from THP1, thereby decreasing the mRNA expression of IL-1ß, IL-6, and TNFα significantly (p < 0.05 for all). TSG-6 expression was evaluated in hUC-MSCs culture supernatant after treatment with TNFα, IFNγ, and LPS for 48 h. And hUC-MSCs could inhibit the phosphorylation of JAK/STAT1 in the intestinal mucosa of CD patients. INTERPRETATION: hUC-MSCs transplantation alleviated TNBS-induced colitis in rats. In this pilot clinical study, preliminary data suggested that this approach to administering hUC-MSCs might have potential for clinical efficacy and manageable safety in treating refractory CD, potentially providing hope for better outcomes. No serious adverse events were observed. FUNDING: This work was funded by General Program of National Natural Science Foundation of China (Grant No. 82270639), the Scientific research project of Shanghai Municipal Health Committee (Grant No. 202240001), Specialty Feature Construction Project of Shanghai Pudong New Area Health Commission (Grant No. PWZzb2022-05), Shanghai East Hospital Youth Research and Cultivation Foundation program (Grant No. DFPY2022015), Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai, Technology Development Project of Pudong Science, Technology and Economic Commission of Shanghai (Grant No. PKJ2021-Y08), Key Disciplines Group Construction Project of Shanghai Pudong New Area Health Commission (Grant No. PWZxq2022-06), Medical discipline Construction Project of Pudong Health Committee of Shanghai (Grant No. PWYgf2021-02) and National Natural Science Foundation of China (Grant No. 82300604).

A Novel Multiparameter Scoring Model for Noninvasive Early Prediction of Ischemic Colitis: A Multicenter, Retrospective, and Real-World Study.

INTRODUCTION: Ischemic colitis (IC) is a common gastrointestinal ischemic disease caused by hypoperfusion or reperfusion injury. However, there are few studies on risk factors associated with poor prognoses of the disease. This study aimed to determine the predictors of poor prognoses in patients with IC and establish a prognostic scoring method with good internal and external validity for identifying severe cases in an early stage. METHODS: We established a prognosis model by conducting a multicenter, retrospective study of patients hospitalized with IC between November 2008 and May 2020. Predictive power was tested using 5-fold internal cross-validation and external validation. RESULTS: The following 6 factors were included in the prognostic model: neutrophil count, D-dimer level, ischemia of the distal ileum, ischemia of the hepatic flexure, ulceration, and luminal stenosis. The area under the receiver-operating characteristic curve for internal cross-validation of the prediction model was 86%, and that for external validation was 95%. During internal validation, our model correctly identified 88.08% of the patients. It was further found that patients younger than 65 years with a higher neutrophil-to-lymphocyte ratio and higher heart rate had poor prognoses. Patients aged 65 years and older with ischemia of terminal ileum, hepatic flexure, splenic flexure, and intestinal stenosis had poor prognoses. DISCUSSION: Patients with ischemia in the hepatic flexure and the distal ileum, endoscopic evidence of ulcer or stenosis, higher neutrophil counts, and higher D-dimer levels have worse prognoses. This information could aid in the selection of timely and appropriate treatment.

The Role of Bone Morphogenetic Protein 9 in Nonalcoholic Fatty Liver Disease in Mice.

Background and Aims: It's reported that bone morphogenetic protein 9 (BMP9) played an important role in lipid and glucose metabolism, but the role of BMP9 in nonalcoholic fatty liver disease (NAFLD) is unclear. Here, we evaluated the therapeutic efficacy of recombined BMP9 in NAFLD mice and investigated the potential mechanism. Methods: The effects of recombinant BMP9 on NAFLD were assessed in HFD-induced NAFLD mice. C57BL/6 mice were administrated with high-fat diet (HFD) for 12 weeks. In the last 4 weeks, mice were treated with PBS or recombined BMP9 once daily. Insulin sensitivity was evaluated by glucose tolerance test (GTT) and insulin tolerance test (ITT) at the end of the 12th week. Then NAFLD related indicators were assessed by a variety of biological methods, including histology, western blotting, real-time PCR, RNA-seq and assay for transposase-accessible chromatin using sequencing (ATAC-seq) analyses. Results: BMP9 reduced obesity, improved glucose metabolism, alleviated hepatic steatosis and decreased liver macrophages infiltration in HFD mice. RNA-seq showed that Cers6, Cidea, Fabp4 involved in lipid and glucose metabolism and Fos, Ccl2, Tlr1 involved in inflammatory response downregulated significantly after BMP9 treatment in HFD mouse liver. ATAC-seq showed that chromatin accessibility on promoters of Cers6, Fabp4, Ccl2 and Fos decreased after BMP9 treatment in HFD mouse liver. KEGG pathway analysis of dysregulated genes in RNA-seq and integration of RNA-seq and ATAC-seq showed that TNF signaling pathway and Toll-like receptor signaling pathway decreased in BMP9 treated HFD mouse liver. Conclusion: Our data revealed that BMP9 might alleviate NAFLD via improving glucose and lipid metabolism, decreasing inflammatory response and reshaping chromatin accessibility in HFD mouse liver. BMP9 downregulate genes related to lipid metabolism, glucose metabolism and inflammation expression, at least partially via decreasing promoter chromatin accessibility of Cers6, Fabp4, Fos and Tlr1. BMP9 may also reduce the expression of liver Ccl2, thereby changing the number or composition of liver macrophages, and ultimately reducing liver inflammation. The effect of BMP9 on NAFLD might be all-round, and not limit to lipid and glucose metabolism. Therefore, the underlying mechanism needs to be studied in detail further.

[Inhibition of bFGF gene expression and tumor angiogenesis of orthotopic implantation of human gastric carcinoma by N-desulfated heparin].

OBJECTIVE: To investigate the effect of N-desulfated heparin on tumor metastasis, tumor angiogenesis and basic fibroblast growth factor(bFGF) gene expression of orthotopically implanted human gastric carcinoma in NOD-SCID mice. METHODS: Human gastric cancer SGC-7901 tissues were orthotopically implanted into the stomach of the NOD-SCID mice. Twenty mice were randomly divided into two groups which received either intravenous injection of 0.9% NaCl solution(0.9%NaCl solution group) or 10 mg/kg N-desulfated heparin (N-desulfated heparin group) twice a week for three weeks. Mice were sacrificed six weeks after tumor implantation. Tissues from stomach and other organs were obtained for histopathological evaluation. The intratumoral microvessel density (MVD) in tumor was evaluated immunohistochemically. Real time PCR was used to detect bFGF mRNA expression. RESULTS: The tumor metastasis rates were 9/10 in 0.9% NaCl solution group and 2/10 in N-desulfated heparin group(P<0.05).MVD was 9.1+/-3.4 in 0.9% NaCl solution group and 4.7+/-1.8 in N-desulfated heparin group (t=3.617,P<0.05). bFGF mRNA expression was lower in N-desulfated heparin group(2.60+/-0.56%)than that in 0.9% NaCl solution group(30.65+/-6.84%). CONCLUSION: N-desulfated heparin can inhibit the metastasis of gastric cancer through inhibiting tumor bFGF gene expression and tumor angiogenesis with no obvious anticoagulant activity.

MiR-629-5p promotes colorectal cancer progression through targetting CXXC finger protein 4.

MiR-629-5p has been shown to function as a tumor promoter in some types of cancer. However, the role of miR-629-5p in colorectal cancer remains unclear. Here, the significant up-regulation of miR-629-5p in colorectal cancer tissues and cell lines was observed. Overexpression of miR-629-5p showed a positive effect on cell proliferation and migration. The enhanced miR-629-5p level also suppressed cell apoptosis and resulted in a low Bax level and a high Bcl-2 level. Further down-regulating miR-629-5p demonstrated opposite effects. CXXC finger protein 4 (CXXC4) was predicted as a direct target of miR-629-5p Dual-luciferase reporter and Western blotting assays exhibited miR-629-5p directly bound to the 3'UTR of CXXC4 and then down-regulated its expression at post-transcriptional level. CXXC4 knockdown rescued the decreased cell proliferation and migration and the enhanced cell apoptosis induced by inhibiting miR-629-5p expression. Notably, overexpression of miR-629-5p also conferred 5-fluorouracil sensitivity, which was partly abrogated by coexpression of CXXC4. Overall, the results presented here suggest that miR-629-5p functions as a tumor promoter by improving proliferation and migration and repressing apoptosis and 5-FU sensitivity in colorectal cancer progression by directly down-regulating CXXC4.

Long non-coding RNA LINC01503 promotes colorectal cancer cell proliferation and invasion by regulating miR-4492/FOXK1 signaling.

Increasing evidence indicates that long non-coding RNAs (lncRNAs) are closely associated with the progression of human cancer, including colorectal cancer (CRC). A previous study suggested that lncRNA LINC01503 promotes squamous cell carcinoma progression. However, the function of LINC01503 in CRC has remained elusive. The present study indicated that LINC01503 was significantly upregulated in CRC tissues compared with that in adjacent normal tissues as detected by reverse transcription-quantitative polymerase chain reaction. It was demonstrated that knockdown of long intergenic non-protein coding RNA (LINC)01503 markedly inhibited the proliferation and invasion of CRC cells, whereas overexpression of LINC01503 had the opposite effects, as indicated by Cell Counting kit-8 and Transwell assays. Mechanistically, it was revealed that LINC01503 serves as a sponge for microRNA (miR)-4492, which targets forkhead box K1 (FOXK1) in CRC cells. In addition, luciferase reporter assays demonstrated the direct binding of miR-4492 mimics to LINC01503 and to a sequence in the 3'-untranslated region of FOXK1. Furthermore, it was demonstrated that overexpression of LINC01503 reduced the availability of miR-4492 in CRC cells. Furthermore, miR-4492 mimics inhibited FOXK1 expression, while simultaneous overexpression of LINC01503 abolished this effect. Finally, it was demonstrated that restoration of FOXK1 abolished the inhibitory effect of LINC01503 knockdown on CRC cell proliferation and invasion. Taken together, the present results suggested that LINC01503 promotes CRC progression via acting as a competing endogenous RNA for miR-4492/FOXK1.

Effect of 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid in combination with carboplatin on gastric carcinoma growth in vivo.

AIM: To investigate the effects of 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid (NM-3) alone and in combination with carboplatin on tumor growth and apoptosis in mouse models of human gastric cancer constructed by subcutaneous implantation of histologically intact tumor tissue. METHODS: Human gastric cancer SGC-7901 tissues were implanted into the dorsal subcutis of nude mice. One week after tumors reached to a volume of 50-100 mm(3) for around 1 wk, these mice were randomly divided into 8 groups (n = 10). NM-3 was injected peritoneally at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg every other day for 5 wk, combined with carboplatin (5 mg/kg) every third day for 4 wk. As controls of combined treatment, another 4 groups of mice were injected with either NM-3 at 10 mg/kg, 20 mg/kg or 40 mg/kg, or with carboplatin alone (5 mg/kg). The control mice received normal saline. Tumor weight, tumor growth inhibition (TGI), and intratumoral microvessel density (MVD) were evaluated. Apoptosis of human gastric cancer was detected by TUNEL method and flow cytometry analysis, respectively. RESULTS: The mean tumor volume (692.40 +/- 58.43 mm(3), 548.30 +/- 66.02 mm(3), 382.13 +/- 43.52 mm(3)) after treatment with carboplatin combined NM-3 at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg was lower than that after treatment with either NM-3 at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg or with carboplatin alone. Compared with the normal saline group, NM-3 administered at 10 mg/kg, 20 mg/kg or 40 mg/kg significantly reduced the tumor weight in these groups (P < 0.05). Carboplatin used alone at 5 mg/kg showed minimal effects. But NM-3 in combination with carboplatin had greater effects of tumor weight than either NM-3 or carboplatin alone. NM-3 alone at the dose 10 mg/kg or in combination with carboplatin had no obvious effects on body changes. Two mice died of diarrhea in each of the two groups treated with 40 mg/kg NM-3 or with 40 mg/kg NM-3 in combination with carboplatin. A significant increase in apoptosis was observed in the NM-3 treated groups, and the effect was more significant in the groups treated with carboplatin in combination with NM-3 at 10 mg/kg, 20 mg/kg and 40 mg/kg, than in the control group. The induction of apoptosis was positively associated with the dose of NM-3. NM-3 significantly reduced the neo-microvascular formation of gastric cancer. The MVD was lower in the groups treated with NM-3 or with NM-3 in combination with carboplatin than in the group treated with carboplatin or in the normal saline group (P < 0.05). CONCLUSION: The results suggest that the inhibitory effect of NM-3 on gastric cancer growth is mediated through decreased angiogenesis and the increased induction of apoptosis. Furthermore, NM-3 alone at the dose of 10 mg/kg or in combination with carboplatin has no obvious effects on body changes, indicating that NM-3 in combination with carboplatin may be effective in the treatment of gastric cancer. The toxicity of NM-3 needs further studies.

Effect of non-anticoagulant N-desulfated heparin on expression of vascular endothelial growth factor, angiogenesis and metastasis of orthotopic implantation of human gastric carcinoma.

AIM: To investigate the effect of N-desulfated heparin on tumor metastasis and angiogenesis, and expression of vascular endothelial growth factor (VEGF) of orthotopic implantation of human gastric carcinoma in male severe combined immune deficiency (SCID) mice. METHODS: Human gastric cancer SGC-7901 cells were orthotopically implanted into the stomach of SCID mice. The mice were randomly divided into normal saline group and N-desulfated heparin group. One week after operation, the mice in N-desulfated heparin group received i.v. injections of N-desulfated heparin (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, 10 mg/kg.d) twice weekly for 3 wk. The mice in normal saline group received i.v. injections of normal saline (100 microL) twice weekly for 3 wk. The mice were sacrificed six weeks after implantation. Tumor metastasis was evaluated histologically for metastasis under microscope. Intratumoral microvessel density (MVD) and VEGF expression were evaluated immuohistochemically. VEGF mRNA expression in gastric tissue of SCID mice was detected by real time PCR. RESULTS: The tumor metastasis rate was 80% in normal saline group and 20% in N-desulfated heparin group (P < 0.05). MVD was 8.0 +/- 3.1 in normal saline group and 4.3 +/- 1.8 in N-desulfated heparin group (P < 0.05). VEGF positive immunostaining was found in cytoplasm of cancer cells. The rate of VEGF positive expression was higher in normal saline group than in N-desulfated heparin treated group (90% vs 20%, P < 0.05). VEGF mRNA expression was significantly inhibited by N-desulfated heparin and was higher in normal saline group than in N-desulfated heparin group (Ct value 19.51 +/- 1.01 vs 22.55 +/- 1.36, P < 0.05). N-desulfated heparin significantly inhibited the expression of VEGF mRNA in cancer cells. No bleeding occurred in N-desulfated heparin group. CONCLUSION: N-desulfated heparin can inhibit metastasis of gastric cancer by suppressing tumor VEGF expression and tumor angiogenesis, but has no obvious anticoagulant activity.