Fucoidan Ameliorates Ferroptosis in Ischemia-reperfusion-induced Liver Injury through Nrf2/HO-1/GPX4 Activation.

Background and Aims: Liver ischemia-reperfusion (IR) injury is a common pathological process in liver surgery. Ferroptosis, which is closely related to lipid peroxidation, has recently been confirmed to be involved in the pathogenesis of IR injury. However, the development of drugs that regulate ferroptosis has been slow, and a complete understanding of the mechanisms underlying ferroptosis has not yet been achieved. Fucoidan (Fu) is a sulfated polysaccharide that has attracted research interest due to its advantages of easy access and wide biological activity. Methods: In this study, we established models of IR injury using erastin as an activator of ferroptosis, with the ferroptosis inhibitor ferrostatin-1 (Fer-1) as the control. We clarified the molecular mechanism of fucoidan in IR-induced ferroptosis by determining lipid peroxidation levels, mitochondrial morphology, and key pathways in theta were involved. Results: Ferroptosis was closely related to IR-induced hepatocyte injury. The use of fucoidan or Fer-1 inhibited ferroptosis by eliminating reactive oxygen species and inhibiting lipid peroxidation and iron accumulation, while those effects were reversed after treatment with erastin. Iron accumulation, mitochondrial membrane rupture, and active oxygen generation related to ferroptosis also inhibited the entry of nuclear factor erythroid 2-related factor 2 (Nrf2) into the nucleus and reduced downstream heme oxygenase-1 (HO-1) and glutathione peroxidase 4 (GPX4) protein levels. However, fucoidan pretreatment produced adaptive changes that reduced irreversible cell damage induced by IR or erastin. Conclusions: Fucoidan inhibited ferroptosis in liver IR injury via the Nrf2/HO-1/GPX4 axis.

APCCDC20-mediated degradation of PHD3 stabilizes HIF-1a and promotes tumorigenesis in hepatocellular carcinoma.

CDC20 regulates cell cycle progression by targeting key substrates for destruction, but its role in hepatocellular carcinoma (HCC) tumorigenesis remains to be explored. Here, by using weighted gene co-expression network analysis (WGCNA), we identified CDC20 as a hub gene in HCC. We demonstrated that CDC20 expression is correlated with HIF-1 activity and overall survival (OS) of clinic HCC patients. The activity of HIF-1 is regulated by the stability of HIF-1a subunit, which is hydroxylated by oxygen-dependent prolyl hydroxylase enzymes, the PHDs. In addition, we show that genetic ablation or pharmacological inhibition of CDC20 can accelerate the degradation of HIF-1a and impair VEGF secretion in HCC cells. Mechanistically, we found that CDC20 binds to the destruction-box (D-box) motif present in the PHD3 protein to promote its polyubiquitination and degradation. The depletion of endogenous PHD3 in CDC20 knockdown HCC cells greatly attenuated the decline of HIF-1a protein and restored the secretion of VEGF. In contrast, overexpression of a non-degradable PHD3 mutant significantly inhibited the proliferation of HCC cells both in vitro and in vivo. Collectively, our findings indicate that CDC20 plays a crucial role in the development of HCC by governing PHD3 protein.

15-Deoxy-Δ12,14-prostaglandin J2 alleviates hepatic ischemia-reperfusion injury in mice via inducing antioxidant response and inhibiting apoptosis and autophagy.

Hepatic ischemia-reperfusion (I/R) injury is a common clinical impairment that occurs in many circumstances and leads to poor prognosis. Both apoptosis and autophagy have been shown to contribute to cell death in hepatic I/R injury. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is one of the best-studied anti-inflammatory prostaglandins, which has been verified to exert anti-inflammatory and cell-protective functions in various types of cells and animal models. In this study we explored the effects of 15d-PGJ2 on both apoptosis and autophagy in mouse hepatic I/R injury and its possible mechanisms. A model of segmental (70%) hepatic warm ischemia was established in Balb/c mice, and the pathological changes in serum and liver tissues were detected at 6, 12, and 24 h post-surgery, while 15d-PGJ2 (2.5, 7.5, 15 µg, iv) was administered 30 min prior the surgery. Pretreatment with 15d-PGJ2 (7.5, 15 µg) significantly ameliorated I/R-induced hepatic injury evidenced by dose-dependent reduction of serum ALT and AST levels as well as alleviated tissue damages. 15d-PGJ2 pretreatment significantly decreased the serum TNF-α and IL-1ß levels and the hepatic expression of F4/80, a major biomarker of macrophages. 15d-PGJ2 pretreatment upregulated the Bcl-2/Bax ratio, thus reducing the number of apoptotic cells in the livers. 15d-PGJ2 pretreatment considerably suppressed the expression of Beclin-1 and LC3, thus decreasing the number of autophagosomes in the livers. Furthermore, 15d-PGJ2 pretreatment activated Nrf2 and inhibited a ROS/HIF1α/BNIP3 pathway in the livers. Pretreatment with the PPARγ receptor blocker GW9662 (2 µg, ip) partly reversed the protective effects of 15d-PGJ2 on hepatic I/R injury. In conclusion, our results confirm the protective effect of 15d-PGJ2 on hepatic I/R injury, an effect that may rely on a reduction in the activation of Kupffer cells and on activation of the Nrf2 pathway, which lead to inhibition of ROS generation, apoptosis, and autophagy.

Meta-analysis of the efficacy of probiotics in Helicobacter pylori eradication therapy.

AIM: To evaluate the role of probiotics in the standard triple Helicobacter pylori therapy. METHODS: In this meta-analysis, we investigated the efficacy of probiotics in a standard triple H. pylori therapy in adults. Searches were mainly conducted in MEDLINE/PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials. Fourteen studies met our criteria, and the quality of these studies was assessed using the Jadad scale. We used STATA version 12.0 to extract data and to calculate the odds ratios (ORs), which are presented with the corresponding 95% confidence intervals (CIs). The data are presented as forest plots. RESULTS: The pooled ORs for the eradication rates calculated by intention-to-treat analysis and per-protocol analysis in the probiotic group vs the control group were 1.67 (95%CI: 1.38-2.02) and 1.68 (95%CI: 1.35-2.08), respectively, using the fixed-effects model. The sensitivity of the Asian studies was greater than that of the Caucasian studies (Asian: OR = 1.78, 95%CI: 1.40-2.26; Caucasian: OR = 1.48, 95%CI: 1.06-2.05). The pooled OR for the incidence of total adverse effects was significantly lower in the probiotic group (OR = 0.49, 95%CI: 0.26-0.94), using the random effects model, with significant heterogeneity (I (2) = 85.7%). The incidence of diarrhea was significantly reduced in the probiotic group (OR = 0.21, 95%CI: 0.06-0.74), whereas the incidence of taste disorders, metallic taste, vomiting, nausea, and epigastric pain did not differ significantly between the probiotic group and the control group. CONCLUSION: Supplementary probiotic preparations during standard triple H. pylori therapy may improve the eradication rate, particularly in Asian patients, and the incidence of total adverse effects.

Association of STAT4 gene rs7574865G > T polymorphism with ulcerative colitis risk: evidence from 1532 cases and 3786 controls.

INTRODUCTION: Several studies have reported the relationship between the STAT4 rs7574865G > T polymorphism as a susceptibility factor to ulcerative colitis (UC). However, the results have been controversial. Therefore, we conducted this meta-analysis to obtain the most reliable estimate of the association. MATERIAL AND METHODS: PubMed, Embase and Web of Science databases were searched. Crude odds ratios (OR) with 95% confidence intervals (CI) were extracted and pooled to assess the strength of the association between the STAT4 rs7574865G > T polymorphism and risk of UC. A total of five eligible studies including 1532 cases and 3786 controls based on the search criteria were involved in this meta-analysis. RESULTS: We observed that the STAT4 rs7574865G > T polymorphism was significantly correlated with UC risk when all studies were pooled into the meta-analysis (the allele contrast model: OR = 1.13, 95% CI = 1.02-1.25; the heterozygote codominant model: OR = 1.22, 95% CI = 1.04-1.43; the dominant model: OR = 1.25, 95% CI = 1.07-1.45). In the stratified analysis by ethnicity, significant associations were observed in Spanish for the allele contrast model (OR = 1.20; 95% CI = 1.04-1.39), for the homozygote codominant model (OR = 1.57; 95% CI = 1.07-2.31), for the dominant model (OR = 1.20; 95% CI = 1.01-1.43), and for the recessive model (OR = 1.50; 95% CI = 1.03-2.19). CONCLUSIONS: This meta-analysis suggests that the STAT4 rs7574865G > T polymorphism is a low-penetrant risk factor for UC, especially in Spanish.

The miR-146a rs2910164 G > C polymorphism and susceptibility to digestive cancer in Chinese.

BACKGROUND: Several studies have reported the role of the miR-146a rs2910164 G > C polymorphism as a susceptibility factor for several digestive cancers. However, the results have been controversial. Therefore, we conducted the present meta-analysis to obtain the most reliable estimate of the association. METHODS: PubMed, Embase and Web of Science databases were searched. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were extracted and pooled to assess the strength of the association between miR-146a rs2910164 G > C polymorphism and digestive cancer risk. A total of four eligible studies including 3,447 cases and 5,041 controls based on the search criteria were included. RESULTS: We observed that miR-146a rs2910164 G > C polymorphism was not significantly correlated with digestive cancer risks when all studies were pooled into the meta-analysis. While we found that miR-146a rs2910164 polymorphism was not associated with gastric cancer, it was significantly linked with hepatocellular cancer risk (the homozygote codominant model: OR = 1.40, 95% CI = 1.04-1.87). In the stratified analysis by ethnicity, significant associations were observed in Chinese population for the allele contrast model (OR = 1.25; 95% CI = 1.12-1.38), for the homozygote codominant model (OR = 1.62; 95% CI = 1.28-2.04), and for the recessive model (OR = 1.38; 95% CI = 1.16-1.64). However, studies with Asian groups presented no significant association for all genetic models. CONCLUSIONS: This meta-analysis suggests that the miR-146a rs2910164 G > C polymorphism is a low-penetrant risk factor for digestive cancers in Chinese.

Mechanism of action of salinomycin on growth and migration in pancreatic cancer cell lines.

OBJECTIVES: Pancreatic cancer is one of the most aggressive and lethal cancers worldwide and there are few effective treatments. Recently, salinomycin (Sal) was reported to alter proliferation and apoptosis in various tumors. This prompted us to investigate the effect of Sal on pancreatic cancer cells and to explore the possible molecular mechanism in vitro. METHODS: After treatment with Sal, pancreatic cancer cell viability and apoptosis were analyzed by Hoechst 33342 staining and flow cytometry, respectively. Invasion and metastasis of pancreatic cancer cells were assayed by a Transwell migration assay. Flow cytometry was also used to assessed the fraction of CD133(+) cell subpopulations. The expression of proliferating cell nuclear antigen (PCNA), Bcl-2, E-cadherin, and Wnt/ß-catenin signaling-related proteins were detected by RT-PCR and western blot. RESULTS: Sal inhibited the growth and migration of pancreatic cancer cells in vitro in a dose- and time-dependent manner. We found that the proportion of CD133(+) cell subpopulations decreased after treatment with Sal in pancreatic cancer cell lines at the same time. The percentage of apoptotic cells was increased after Sal treatment. Compared with control groups, Bax and E-cadherin were significantly upregulated, and Bcl-2 and PCNA were significantly downregulated in Sal-treated cells. The expression of Wnt/ß-catenin signaling-related proteins (ß-catenin and p-GSK-3ß) was inhibited. CONCLUSIONS: These results indicate that Sal could influence the cell growth and migration in pancreatic cancer cells in vitro, which may occur by inhibition of Wnt/ß-catenin signaling.

Effects of multiple-target anti-microRNA antisense oligodeoxyribonucleotides on proliferation and migration of gastric cancer cells.

BACKGROUNDS: To investigate the inhibiting effects of multi-target anti-microRNA antisense oligonucleotide (MTg-AMOs) on proliferation and migration of human gastric cancer cells. METHODS: Single anti-microRNA antisense oligonucleotides (AMOs) and MTg-AMOs for miR-221, 21, and 106a were designed and transfected into SGC7901, a gastric cancer cell line, to target the activity of these miRNAs. Their expression was analyzed using stem-loop RT-PCR and effects of MTg-AMOs on human gastric cancer cells were determined using the following two assay methods: CCK8 for cell proliferation and transwells for migration. RESULTS: In the CCK-8 cell proliferation assay, 0.6 µmol/L was selected as the preferred concentration of MTg-AMOs and incubation time was 72 hours. Under these experimental conditions, MTg-AMOs demonstrated better suppression of the expression of miR-221, miR-106a, miR-21 in gastric cancer cells than that of single AMOs (P = 0.014, 0.024; 0.038, respectively). Migration activity was also clearly decreased as compared to those in randomized and blank control groups (28 ± 4 Vs 54 ± 3, P <0.01; 28 ± 4 Vs 59 ± 4, P < 0.01). CONCLUSIONS: MTg-AMOs can specifically inhibit the expression of multiple miRNAs, and effectively antagonize proliferation and migration of gastric cancer cells promoted by oncomirs.

The eradicating Helicobacter pylori infection in duodenal ulcer patients by three short-term triple therapies in China: a multicenter clinical comparative study.

BACKGROUND/AIMS: This study aimed to compare the 7d triple therapy with 3d and 5d triple therapies, to observe the effect of eradicating Helicobacter pylori (Hp) on treating duodenal ulcers. METHODOLOGY: One hundred and sixteen patients who were confirmed duodenal ulcer active period and Hp positive were enrolled in the study. All the patients were divided into three groups: 3d group (n=39), 5d group (n=37) and 7d control group (n=40). All three groups were provided triple therapy first: rabeprazole, 10mg + furazolidone, 100mg + clarithromycin 250mg, twice a day for three days, five days and seven days, respectively. Then rabeprazole 10mg was provided once a day. Following the treatment, 13C urea breath test was performed to observe the Hp eradication rate. The symptoms of patients such as epigastralgia, burning pain and acidity were evaluated. RESULTS: The Hp eradication rate was: 3d group 76% (28/37), 5d 89% (31/35) and 7d 91% (32/35). There was no significant difference between 5d and 7d group (p>0.05). But the rate of groups 5d and 7d was significantly higher than group 3d (p<0.05). All the three groups showed an improvement in symptoms such as epigastralgia, burning pain and acidity. CONCLUSIONS: All three therapy schemes could alleviate symptoms of duodenal ulcer patients efficiently. But as far as eradicating Hp concerned, 5d and 7d therapies were better than 3d.

Salinomycin inhibits proliferation and induces apoptosis of human hepatocellular carcinoma cells in vitro and in vivo.

The anti-tumor antibiotic salinomycin (Sal) was recently identified as a selective inhibitor of breast cancer stem cells; however, the effect of Sal on hepatocellular carcinoma (HCC) is not clear. This study aimed to determine the anti-tumor efficacy and mechanism of Sal on HCC. HCC cell lines (HepG2, SMMC-7721, and BEL-7402) were treated with Sal. Cell doubling time was determinated by drawing growth curve, cell viability was evaluated using the Cell Counting Kit 8. The fraction of CD133(+) cell subpopulations was assessed by flow cytometry. We found that Sal inhibits proliferation and decreases PCNA levels as well as the proportion of HCC CD133(+)cell subpopulations in HCC cells. Cell cycle was analyzed using flow cytometry and showed that Sal caused cell cycle arrest of the various HCC cell lines in different phases. Cell apoptosis was evaluated using flow cytometry and Hoechst 33342 staining. Sal induced apoptosis as characterized by an increase in the Bax/Bcl-2 ratio. Several signaling pathways were selected for further mechanistic analyses using real time-PCR and Western blot assays. Compared to control, ß-catenin expression is significantly down-regulated upon Sal addition. The Ca(2+) concentration in HCC cells was examined by flow cytometry and higher Ca(2+) concentrations were observed in Sal treatment groups. The anti-tumor effect of Sal was further verified in vivo using the hepatoma orthotopic tumor model and the data obtained showed that the size of liver tumors in Sal-treated groups decreased compared to controls. Immunohistochemistry and TUNEL staining also demonstrated that Sal inhibits proliferation and induces apoptosis in vivo. Finally, the role of Sal on in vivo Wnt/ß-catenin signaling was evaluated by Western blot and immunohistochemistry. This study demonstrates Sal inhibits proliferation and induces apoptosis of HCC cells in vitro and in vivo and one potential mechanism is inhibition of Wnt/ß-catenin signaling via increased intracellular Ca(2+) levels.