BMSC cells alleviate liver injury induced by ulcerative colitis via repairing the intestinal-liver barrier and activating hepatocyte growth factor.

Ulcerative colitis (UC), which belongs to inflammatory bowel diseases (IBD), frequently induces liver inflammation and injury. Previous studies have proved that bone marrow-derived mesenchymal stem cells (BMSCs) can suppress inflammation and improve intestinal mucosal injury in colitis, however, the effects of BMSCs on colitis-induced liver injury and the underlying molecular mechanisms remain unclear. Here, we investigated the effects and mechanisms of BMSCs in acute ulcerative colitis BALB/c mice, which were induced by 4 % dextran sodium sulphate (DSS). In this study, BMSCs derived from BALB/c mice were administrated by single intravenous injection with a dose of 5*10^7 cells/kg. And then, the effects and underlying molecular mechanisms were investigated. Firstly, the degree of liver injury in colitis mice was evaluated by hepatic ALT, AST, ALP and TBIL levels, which were measured by specific determination kits, the levels of TNF-α, IL-6, IFNγ and LPS were examined by ELISA. Secondly, as the indicator of intestinal-liver barrier disorder, tight junction proteins were analyzed by western blot. Thirdly, the pathological changes in the colon and liver were detected by H&E staining. At last, homing of BMSCs to lesion tissues was investigated by Immunofluorescence. The results indicated that histopathological changes in model mice had been greatly alleviated, BMSCs infusion remarkably decreased the serum ALT, AST, ALP and TBIL levels, and meanwhile reduced pro-inflammatory cytokines in liver tissues. Furthermore, homing of BMSCs was observed in the colon and liver, and the disorder of the intestinal-liver barrier declined significantly. In conclusion, BMSCs alleviate liver injury induced by ulcerative colitis via repairing the intestinal-liver barrier and activating hepatocyte growth factor, it has potential application prospects in the treatment of liver injury induced by ulcerative colitis.

[Mechanism of bone marrow-derived mesenchymal stem cell-promoted apoptosis of hepatic stellate cells].

OBJECTIVE: To explore the role of the Rho pathway in the hepatocyte growth factor (HGF) paracrine signal-mediated bone marrow-derived mesenchymal stem cell (BMSC) promotion of apoptosis of hepatic stellate cells (HSCs). METHODS: A BMSC-HSC co-culture system was established using plates with transwell inserts. Dynamic changes in response to pretreatment with the c-met blocker PHA665752 and the Rho pathway inhibitor Y-27632 were observed under an inverted phase contrast microscope at 24, 48 and 72 h of culture. Optimal intervention concentrations of Y-27632 and PHA665752 were determined by MTT assay. Expression of alpha-smooth muscle actin in HSCs was evaluated by immunohistochemistry, and the apoptosis rate of HSCs was measured by Annexin-V-FITC/propidium iodide. RhoA protein and mRNA levels were measured by western blot and quantitative real-time PCR respectively. Concentrations of HGF and hepatocyte growth factor activator (HGFA) were quantified by enzyme-linked immunosorbent assay. Between-group differences were evaluated by one-way ANOVA with P less than 0.05 indicating significance. RESULTS: The apoptosis rates of HSCs gradually and steadily increased in a time-dependent manner. The apoptosis rate of the PHA665752 pretreated group was lowest and that of the Y-27632 pretreated group was highest, with the most robust difference occurring at the 72 h time point (P less than 0.05). The mRNA and protein expression levels of RhoA decreased in a time-dependent manner in the Y-27632 pretreated group (all time points, P less than 0.05) but the expression levels increased in a time-dependent manner in the PHA665752 pretreated group (all time points, P less than 0.05). For both the PHA665752 and the Y-27632 pretreated groups, the concentration of HGF decreased in a time-dependent manner, but the concentrations in both remained significantly higher than that in the control group at all time points examined (P less than 0.05). The concentration of HGFA increased in a time-dependent manner, and the PHA665752 pretreated group showed significantly higher levels than any of the other groups at all time points examined (P less than 0.01). CONCLUSION: BMSC promotes HSC apoptosis in a co-culture system by activating HGF and down-regulating the RhoA signaling pathway.

[Activation of hepatocyte growth factor promotes apoptosis of hepatic stellate cells via the Rho pathway].

OBJECTIVE: To investigate the role of activated hepatocyte growth factor (HGF) in apoptosis of hepatic stellate cells (HSCs) and in modulating the Rho signaling pathway. METHODS: HSCs were divided into the following groups: blank control, consisting of HSCs without treatment; two treatment controls, consisting of HSCs exposed to exogenous HGF at 50 ng/ml and HSCs exposed to exogenous HGF activator (HGFA) at 70 ng/ml; three experimental groups, consisting of HSCs exposed to both exogenous HGF and HGFA, HSCs pre-incubated with the HGF inhibitor c-met at 500 ng/ml for 6 hours and then exposed to exogenous HGF and HGFA, and HSCs pre-incubated with the Rho pathway inhibitor Y-27632 at 10 ng/ml and then exposed to exogenous HGF and HGFA. Activation status of the cultured HSCs was determined by change in expression of alpha-smooth muscle actin (SMA). The optimal intervention concentration of Y-27632 was determined by MTT assay. The apoptotic status of HSCs was determined by flow cytometry. Expression of the HGF-alpha chain was detected by immunofluorescence. The expression of RhoA was evaluated by PCR (for mRNA) and by immunohistochemical staining and Western blot analysis (for protein). RESULTS: Exposure to 10 mumol/L Y-27632 led to obvious growth inhibition of HGF + HGFA-induced HSCs, compared with the other concentrations tested (P less than 0.05). HGF + HGFA induced the expression of the HGF-alpha chain in a time-dependent manner (P less than 0.01); however, the increases in expression of HGF-alpha chain induced by HGF alone and HGFA alone were not significantly different from the level in the blank controls (P more than 0.05). Exposure to HGF alone and HGFA alone led to a time-dependent increase in apoptosis (24 h, 48 h, 72 h) but exposure to HGF + HGFA led to the highest levels of apoptosis (P less than 0.05). Exposure to HGF + HGFA led to a time-dependent decrease in RhoA mRNA and protein expression (P less than 0.01). CONCLUSION: Activation of hepatocyte growth factor promotes apoptosis of hepatic stellate cells by suppressing RhoA expression and down-regulating the Rho signaling pathway.

[Activation of hepatocyte growth factor-induced apoptosis in hepatic stellate cells].

OBJECTIVE: To determine whether apoptosis is induced in rat hepatic stellate cells (HSCs) in response to activation of the hepatocyte growth factor (HGF) by hepatocyte growth factor activator (HGFA) by using a co-culture system of bone marrow mesenchymal stem cells (BMSCs) and HSCs. METHODS: In this study, cells were divided into the following five groups: HSC control group: HSCs co-cultured with fibroblast cells; HSCs blank group: HSCs cultured alone; BMSCs blank group: BMSCs cultured alone; Experimental group: BMSCs + HSCs; HGFA intervention group: HSCs treated with 70 ng/mL of HGFA. The culture systems were established in culture plates with transwell inserts, and cells were assessed at 24, 48, and 72 h of growth. Dynamic changes in cell morphology were observed under an inverted phase contrast microscope. The surface markers of BMSCs and the apoptosis rate of HSCs were detected by Annexin-V-FITC/propidium iodide (PI). Expression of a-smooth muscle actin (SMA) in HSCs was evaluated by immunohistochemistry. The presence of activated HGF (HGF-a chain) was determined by immunofluorescent staining. HSC proliferation was measured by MTT assay, and the concentrations of HGF and HGFA were quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: MTT results indicated that treatment with HGF alone had no effect on HSC proliferation rate (vs. HSC blank group, P more than 0.05), but that 24 h treatment with HGFA significantly inhibited the proliferation rate (0.26 ± 0.00 vs. blank group: 0.13 ± 0.04, P = 0.02); moreover, this effect was concentration-dependent. Expression of HGF-a was lower in the experimental group than in the HGFA intervention group at 72 h (37.24 ± 1.03 vs. 40.44 ± 0.77, P = 0.04), and both of these groups had higher expression than the control group at all time points examined (P less than 0.05). The apoptosis rate was consistently higher in the experimental group than in the HGFA intervention group, but most robustly at 72 h (40.77 ± 1.16% vs. 33.35 ± 2.04%, P = 0.00); moreover, the apoptosis rate was significantly higher than that in the control group at all time points examined (P less than 0.01). The concentration of HGF in the experimental group and the HGFA intervention group showed a time-dependent reduction, and was consistently lower than that in the HSCs control group (P less than 0.05). Finally, the concentration of HGFA was higher in the experimental group than in the blank group at all time points examined (P less than 0.05). CONCLUSION: The BMSC-HSC co-culture system can promote secretion of HGFA from HSCs and HGF activation, thereby inducing apoptosis of HSCs.

Prognostic Model and Immune Infiltration of Ferroptosis Subcluster-Related Modular Genes in Gastric Cancer.

Background: Gastric cancer (GC) is one of the gastrointestinal tumors with the highest mortality rate. The number of GC patients is still high. As a way of iron-dependent programmed cell death, ferroptosis activates lipid peroxidation and accumulates large reactive oxygen species. The role of ferroptosis in GC prognosis was underrepresented. The objective was to investigate the role of ferroptosis-related genes (FRGs) in the prognosis and development of GC. Methods: Datasets of GC patients were obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) database that include clinical information and RNA seq data. Through nonnegative matrix factorization (NMF) clustering, we identified and unsupervised cluster analysis of the expression matrix of FRGs. And we constructed the co-expression network between genes and clinical characteristics by consensus weighted gene co-expression network analysis (WGCNA). The prognostic model was constructed by univariate and multivariate regression analysis. The potential mechanisms of development and prognosis in GC were explored by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, gene ontology (GO), tumor immune microenvironment (TIME), and tumor mutation burden (TMB). Results: Two molecular subclusters with different expression patterns of FRGs were identified, which have significantly different survival states. Ferroptosis subcluster-related modular genes were identified by WGCNA. Based on 8 ferroptosis subcluster-related modular genes (collagen triple helix repeat containing 1 (CTHRC1), podoplanin (PDPN), procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2), glutamine-fructose-6-phosphate transaminase 2 (GFPT2), ATP-binding cassette subfamily A member 1 (ABCA1), G protein-coupled receptor 176 (GPR176), serpin family E member 1 (SERPINE1), dual specificity phosphatase 1 (DUSP1)) and clinicopathological features, a nomogram was constructed and validated for their predictive efficiency on GC prognosis. Through receiver operating characteristic (ROC) analysis, the results showed that the area under the curve (AUC) of 1-, 3-, and 5-year survival were 0.721, 0.747, and 0.803, respectively, indicating that the risk-scoring model we constructed had good prognosis efficacy in GC. The degree of immune infiltration in high-risk group was largely higher than low-risk group. It indicated that the immune cells have a good response in high-risk group of GC. The TMB of high-risk group was higher, which could generate more mutations and was more conducive to the body's resistance to the development of cancer. Conclusion: The risk-scoring model based on 8 ferroptosis subcluster-related modular genes has shown outstanding advantages in predicting patient prognosis. The interaction of ferroptosis in GC development may provide new insights into exploring molecular mechanisms and targeted therapies for GC patients.