Collagen Peptides as a Hypoxia-Inducible Factor-2α-Stabilizing Prolyl Hydroxylase Inhibitor to Stimulate Intestinal Iron Absorption by Upregulating Iron Transport Proteins.

Iron intervention is not always safe and effective to correct iron deficiency. Host iron absorption stimulation is emerging as a promising adjunctive/alternative treatment. Here, porcine collagen hydrolysate (CH) and collagen-derived dipeptide prolyl-hydroxyproline, rather than collagen amino acids, namely, glycine, proline, and hydroxyproline, were found to increase cellular iron reduction, absorption, and transportation, to upregulate duodenal cytochrome b (Dcytb), divalent metal transporter 1 (DMT1), ferroportin (FPN), and hephaestin, and to nongenomically activate hypoxia-inducible factor-2α signaling in polarized Caco-2 cells. Prolyl-hydroxyproline showed both competitive and uncompetitive inhibition of recombinant human prolyl hydroxylase-3 activity with EC50 and Ki values of 10.62 and 6.73 µM, respectively. Docking simulations revealed collagen peptides as iron chelators and/or steric hindrances for prolyl hydroxylase-3. CH and prolyl-hydroxyproline acutely increased duodenal hypoxia-inducible factor-2α stability and Dcytb, DMT1, FPN, and hephaestin transcription in rats. Overall, collagen peptides act as a hypoxia-inducible factor-2α-stabilizing prolyl hydroxylase inhibitor to stimulate intestinal iron absorption.

Rare Case of Paraneoplastic Pemphigus Associated With Prostatic Cancer.

ABSTRACT: The combination of paraneoplastic pemphigus and prostate cancer is extremely unusual and has not been reported yet. Paraneoplastic pemphigus is caused by tumor-induced autoantibodies, which cause damage to the skin and mucosa. The essential treatment is active tumor control. Our patient received a robot-assisted radical prostatectomy and glucocorticoid therapy to improve his condition and relieve his skin lesions.

Crocetin Protected Human Hepatocyte LO2 Cell From TGF-ß-Induced Oxygen Stress and Apoptosis but Promoted Proliferation and Autophagy via AMPK/m-TOR Pathway.

Objective: To investigate the protective effects of crocetin against transforming growth factor-ß (TGF-ß)-induced injury in LO2 cells. Methods: Human hepatocyte LO2 cells were pre-treated with crocetin (10 µM) for 6, 12, and 24 h, and then induced by TGF-ß. Proliferation, oxidative stress, apoptosis, autophagy, and related proteins were assessed. Results: Crocetin pre-treating promoted proliferation but suppressed apoptosis in TGF-ß-induced LO2 cells. Crocetin protected LO2 cells from TGF-ß-induced inflammation and oxygen stress by reducing reactive oxygen species (ROS) and malondialdehyde (MDA) but enhancing superoxide dismutase (SOD) and glutathione (GSH). Autophagy was suppressed in TGF-ß but crocetin promoted autophagy in LO2 cells by mediating Adenosine 5'-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (m-TOR) signaling pathway via upregulating p-AMPK and p-Beclin-1 but downregulating p-mTOR. Conclusions: Crocetin protected LO2 cells from TGF-ß-induced damage by promoting proliferation and autophagy, and suppressing apoptosis and anti-inflammation via regulation of AMPK/m-TOR signaling pathway.

An extracellular matrix-based signature associated with immune microenvironment predicts the prognosis of patients with hepatocellular carcinoma.

OBJECTIVE: Increased data showed that genes related to extracellular matrix (ECM) are important to hepatocellular carcinoma (HCC) development. In contrast, no research was carried out that proposed that ECM-related genes should be reliable prognostic signature. METHODS: This study used data from The Cancer Genome Atlas along with The International Cancer Genome Consortium to gather ECM-related gene expression as well as clinical information related to the extracellular matrix. The least absolute shrinkage, Cox analysis, along with selection operator Cox regression and random forest have been utilized for establishing an ECM-related prognostic models. RESULTS: A series of investigations led us to identify 13 ECMs which we utilized to construct a prognostic signature with a larger area under the curve of 0.808. HCC patients have been categorized into 2 main groups based on the risk score formula: low risk along with high risk. The findings of the Kaplan-Meier curve revealed that there had been a statistically significant difference between these two groups. Our ECM-related signature can be utilized as independent predictor of survival in HCC. Low-risk patients stratified by the final model presented higher sensitivity to 8 targeted drugs (especially sorafenib) and 2 common chemo-drugs. Our gene set enrichment analysis outcomes recommended that high-risk group have been enriched in ECM, tumorigenesis, as well as immune-related pathways. Immune cell analysis showed that high-risk group had lower cell fraction of CD8+ T cells, Macrophages M1, B naïve cells, memory resting CD4+ T cells, Monocytes, resting Dendritic cells and activated Mast cells, along with higher PD-1 and CTLA4 expression levels as compared to low-risk group. CONCLUSION: Our identified ECM-related signature can also give new insight into underlying mechanisms along with therapeutic strategies in order to treat HCC.

Crocetin protects against fulminant hepatic failure induced by lipopolysaccharide/D-galactosamine by decreasing apoptosis, inflammation and oxidative stress in a rat model.

Fulminant hepatic failure (FHF) is a clinical syndrome characterized by sudden and severe liver dysfunction. Apoptosis and inflammation are essential for the pathogenesis of FHF. Crocetin, the major component present in saffron, has been reported to possess anti-inflammatory and antioxidant functions; however, its role in FHF is poorly understood. The aim of this study was to explore the protective effects of crocetin against lipopolysac§§charide (LPS)/D-galactosamine (D-GalN)-induced FHF and the underlying mechanisms in a rat model. For the in vivo study, rats were assigned to the LPS/D-GalN group or to the crocetin pre-treatment+LPS/D- GalN group. Each group was then further divided according to the different LPS/D-GalN treatment times of 0, 6, 12 or 48 h. The results demonstrated that crocetin pre-treatment efficiently protected against LPS/D-GalN-induced FHF by improving liver tissue morphology, reducing total bilirubin generation and decreasing the activities of alanine transaminase and aspartate aminotransferase. Moreover, crocetin pre-treatment significantly decreased hepatocyte apoptosis, p53 mRNA expression and the expression of proteins in the caspase family and the Bcl-2 pro-apoptotic family following LPS/D-GalN treatment. Furthermore, crocetin also decreased the secretion of pro-inflammatory cytokines in the serum and in the liver via suppression of NF-κB activation, and also suppressed hepatic oxidative stress. In conclusion, crocetin protected against LPS/D-GalN-induced FHF and inhibited apoptosis, inflammation and oxidative stress. The underlying mechanisms may be related to the regulation of apoptotic proteins in the caspase family and the Bcl-2 family, as well as the modulation of NF-κB expression. Therefore, crocetin may be used as a novel therapy for preventing FHF.

Antioxidant Activity Evaluation of Dietary Flavonoid Hyperoside Using Saccharomyces Cerevisiae as a Model.

Oxidative stress leads to various diseases, including diabetes, cardiovascular diseases, neurodegenerative diseases, and even cancer. The dietary flavonol glycoside, hyperoside (quercetin-3-O-galactoside), exerts health benefits by preventing oxidative damage. To further understand its antioxidative defence mechanisms, we systemically investigated the regulation of hyperoside on oxidative damage induced by hydrogen peroxide, carbon tetrachloride, and cadmium in Saccharomyces cerevisiae. Hyperoside significantly increased cell viability, decreased lipid peroxidation, and lowered intracellular reactive oxygen species (ROS) levels in the wild-type strain (WT) and mutants gtt1∆ and gtt2∆. However, the strain with ctt1∆ showed variable cell viability and intracellular ROS-scavenging ability in response to the hyperoside treatment upon the stimulation of H2O2 and CCl4. In addition, hyperoside did not confer viability tolerance or intercellular ROS in CdSO4-induced stress to strains of sod1∆ and gsh1∆. The results suggest that the antioxidative reactions of hyperoside in S. cerevisiae depend on the intercellular ROS detoxification system.

[Crocetin promotes autophagy in injured rat hepatocytes induced by lipopolysaccharide and D-galactosamine in vitro].

OBJECTIVE: To observe the effect of crocetin on autophagy in rat hepatocytes exposed to lipopolysaccharide (LPS) and D-galactosamine (D-gal) and explore the mechanism. METHODS: Cultured rat hepatocytes were exposed to LPS (1 mg/L) and Dgal (60 mg/L) to induce cell injury and treated with crocetin, 3MA, or crocetin+3MA. Twelve hours after the treatments, the cells were examined for levels of ALT, AST and LDH in the supernatant using ELISA. LC3 fluorescence in the cells following immunofluorescence staining was observed using fluorescence microscopy. Autophagosomes in the cells were observed by transmission electron microscopy, and the cellular expressions of LC3, p62 and SIRT1 were detected using Western blotting. RESULTS: The levels of ALT, AST and LDH in the hepatocytes were elevated after LPS- and D-gal-induced injury, reached the highest levels after 3MA treatment, but were decreased significantly by crocetin treatment. LC3 fluorescence increased obviously in the injured hepatoctyes, and the increment was the most obvious in crocetin-treated cells; LC3 fluorescence was decreased significantly after 3MA treatment. Cell injury induced obvious increase in autophagy in the hepatocytes, and the number of autophagosomes increased significantly after crocetin treatment but was reduced significantly after 3MA treatment. The cell injury caused an obvious up-regulation of LC3 and SIRT1 expression and down-regulated p62 expression. LC3 and SIRT1 expression levels were the highest and the expression of p62 was the lowest in cells with crocetin treatment. 3MA treatment significantly reduced the expression of LC3 and SIRT1 and increased the expression of p62 in the injured cells. CONCLUSIONS: Autophagy is increased in injured rat hepatocytes, and crocetin can promote autophagy in the injured cells to reduce further cell injury.

miR-224 suppresses HBV replication posttranscriptionally through inhibiting SIRT1-mediated autophagy.

Hepatitis B virus (HBV) enters the host and successfully completes replication by using several mechanisms, including autophagy. However, previous studies revealed that microRNAs (miRNAs) widely participate in regulation of various cellular processes, such as autophagy and viral replication. Hence, the purpose of this study was to investigate the role of miR-224 in HBV infection and to determine whether its role depended on the miR-224/SIRT1/autophagy axis. Our results show that secretions of HBeAg and HBsAg, and HBV replication significantly declined in Huh7-1.3 cells, established by transfecting recombinant pcDNA 3.0-1.3 mer containing the 1.3 mer fragment of HBV genomic DNA,with miR-224 mimic transfection as compared to the Huh7-1.3 group. Moreover, it was discovered that HBV could induce autophagy, while miR-224 inhibited autophagy caused by HBV. Additionally, miR-224 could suppress SIRT1, LC3 expression, and facilitate p62 expression. SIRT1 was identified as the target gene of miR-224 and down-regulation of SIRT1 via miR-224 or si-SIRT1 transfected treatment in Huh7-1.3 cells repressed LC3 expression and enhanced p62 expression. In conclusion, these results suggest that miR-224 might hinder HBV replication through attenuating SIRT1-mediated autophagy, thereby these findings open a new avenue for the treatment of HBV infection.

Surface expression of anti-CD3scfv stimulates locoregional immunotherapy against hepatocellular carcinoma depending on the E1A-engineered human umbilical cord mesenchymal stem cells.

Tumor antigens is at the core of cancer immunotherapy, however, the ideal antigen selection is difficult especially in poorly immunogenic tumors. In this study, we designed a strategy to modify hepatocellular carcinoma (HCC) cells by surface expressing anti-CD3scfv within the tumor site strictly, which depended on the E1A-engineered human umbilical cord mesenchymal stem cells (HUMSC.E1A) delivery system. Subsequently, membrane-bound anti-CD3scfv actived the lymphocytes which lysed HCC cells bypassing the expression of antigens or MHC restriction. First, we constructed the anti-CD3scfv gene driven by human α-fetoprotein (AFP) promoter into an adenoviral vector and the E1A gene into the lentiviral vector. Our results showed that anti-CD3scfv could specifically express on the surface of HCC cells and activate the lymphocytes to kill target cells effectively in vitro. HUMSC infected by AdCD3scfv followed by LentiR.E1A could support the adenoviral replication and packaging in vitro 36 h after LentiR.E1A infection. Using a subcutaneous HepG2 xenograft model, we confirmed that AdCD3scfv and LentiR.E1A co-transfected HUMSC could migrate selectively to the tumor site and produce considerable adenoviruses. The new generated AdCD3scfv infected and modified tumor cells successfully. Mice injected with the MSC.E1A.AdCD3scfv and lymphocytes significantly inhibited the tumor growth compared with control groups. Furthermore, 5-fluorouracil (5-FU) could sensitize adenovirus infection at low MOI resulting in improved lymphocytes cytotoxicity in vitro and in vivo. In summary, this study provides a promising strategy for solid tumor immunotherapy.

Dendritic cell-derived exosomes elicit tumor regression in autochthonous hepatocellular carcinoma mouse models.

BACKGROUND & AIMS: Dendritic cell (DC)-derived exosomes (DEXs) form a new class of vaccines for cancer immunotherapy. However, their potency in hepatocellular carcinoma (HCC), a life-threatening malignancy with limited treatment options in the clinic that responds poorly to immunotherapy, remains to be investigated. METHODS: Exosomes derived from α-fetoprotein (AFP)-expressing DCs (DEXAFP) were investigated in three different HCC mouse models systemically. Tumor growth and microenvironment were monitored. RESULTS: DEXAFP elicited strong antigen-specific immune responses and resulted in significant tumor growth retardation and prolonged survival rates in mice with ectopic, orthotopic and carcinogen-induced HCC tumors that displayed antigenic and pathological heterogeneity. The tumor microenvironment was improved in DEXAFP-treated HCC mice, demonstrated by significantly more γ-interferon (IFN-γ)-expressing CD8+ T lymphocytes, elevated levels of IFN-γ and interleukin-2, and fewer CD25+Foxp3+ regulatory T (Treg) cells and decreased levels of interleukin-10 and transforming growth factor-ß in tumor sites. Lack of efficacy in athymic nude mice and CD8+ T cell-depleted mice showed that T cells contribute to DEXAFP-mediated antitumor function. Dynamic examination of the antitumor efficacy and the immune microenvironment in DEXAFP-treated orthotopic HCC mice at different time-points revealed a positive correlation between tumor suppression and immune microenvironment. CONCLUSIONS: Our findings provide evidence that AFP-enriched DEXs can trigger potent antigen-specific antitumor immune responses and reshape the tumor microenvironment in HCC mice and thus provide a cell-free vaccine option for HCC immunotherapy. Lay summary: Dendritic cell (DC)-derived exosomes (DEXs) form a new class of vaccines for cancer immunotherapy. However, their potency in hepatocellular carcinoma (HCC) remains unknown. Here, we investigated exosomes from HCC antigen-expressing DCs in three different HCC mouse models and proved their feasibility and capability of treating HCC, and thus provide a cell-free vaccine for HCC immunotherapy.

[Early acute liver injury in paraquat poisoning rats].

OBJECTIVE: To observe hepatocellular apoptosis and inflammatory cytokines expression and their mechanisms after paraquat poisoning in rat. METHODS: Forty Wistar rats were divided into control group (n=8) and model group (n=32) by random number table. Rats in model group were intraperitoneally injected with 30 mg/kg 20% paraquat concentrate, while those in control group were injected with normal saline. 0.5, 1, 3, 7 days after reproduction of the model, 8 rats were sacrificed, and blood was collected from inferior vena cava and hepatic tissue was harvested. The serum levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of IL-1ß, TNF-α, inducible nitric oxide synthase (iNOS) and p53 were determined by reverse transcription-polymerase chain reaction (RT-PCR). Cysteine-containing aspartate-specific proteases (caspase-3, -8, -9, -12) activity in hepatic tissue was determined on the 3rd day with chromogenic substrate method. The liver histopathological changes were observed after hematoxylin-eosin (HE) staining. RESULTS: In model group, hepatic tissue showed extensive necrosis with inflammatory cell infiltration in time dependant manner. Serum IL-1ß and TNF-α levels were significantly higher in model group half a day after reproduction than those in control group (IL-1ß: 220.13 ± 69.74 ng/L vs. 0.14 ± 0.03 ng/L, TNF-α: 102.66 ± 26.43 ng/L vs. 0.16 ± 0.02 ng/L, P<0.01 and P<0.05), and peaked on the 3rd day and 1st day (IL-1ß: 423.72 ± 153.11 ng/L, TNF-α: 690.35 ± 229.64 ng/L). They then decreased gradually, but were still significantly higher than those in control group on the 7th day (IL-1ß: 357.47 ± 87.28 ng/L, TNF-α: 12.39 ± 5.06 ng/L, both P<0.05). The contents of IL-1ß, TNF-α and iNOS mRNA expressions in hepatic tissue were significantly higher than those in control group, and the highest values were seen on the 1st day, the 1st day, and the 3rd day [IL-1ß mRNA (gray value): 1.569 ± 0.057 vs. 0.123 ± 0.016, TNF-α mRNA (gray value): 0.683 ± 0.077 vs. 0.261 ± 0.025, iNOS mRNA (gray value): 3.259 ± 0.135 vs. 0.002±0.001, P<0.05 or P<0.01]. There was no difference in p53 mRNA expression between model group and control group at early stage, and both of them showed low expression, and p53 mRNA expression was significantly higher in model group on the 7th day (gray value: 2.959±0.086 vs. 0.263±0.032, P<0.01). In model group, caspase activity in liver tissue were significantly higher on the 3rd day than those in control group (caspase-3: 857.25±309.26 pmol/mg vs. 169.73±48.21 pmol/mg, caspase-8: 199.18±61.41 pmol/mg vs. 32.26±11.09 pmol/mg, caspase-9: 321.62±80.73 pmol/mg vs. 90.38±29.76 pmol/mg, caspase-12: 413.13±89.77 pmol/mg vs. 26.73±9.86 pmol/mg, all P<0.01). CONCLUSIONS: Paraquat can cause acute liver injury in rats, with caspase-3, -8, -9, -12 activities markedly enhanced, and liver injury may be associated with an early high expression of TNF-α, iNOS and p53 gene.

Extracellular domain of human 4-1BBL enhanced the function of cytotoxic T-lymphocyte induced by dendritic cell.

Interaction of costimulatory molecules and their receptors is crucial for tumor lysate-pulsed dendritic cells (sensitized DC, sDC) to promote T cell activation, clonal expansion and its antitumor immunity. To augment the costimulatory signal may regulate the interaction between DC and cytotoxic T lymphocyte (CTL) and consequently enhance the antitumor response. The costimulatory ligand and receptor pair of 4-1BB/4-1BBL is one of the main factors in the costimulation of CTL. We explored the adjuvant role of a recombinant human 4-1BBL extracellular domain (ex4-1BBL) in modulating CTL activation induced by HepG2 antigen-loaded DC (sDC). The augment effects of sDC in combination with ex4-1BBL on the proliferation, activation, cell survival and cytotoxicity against HepG2 cells of CTL were examined. In the presence of ex4-1BBL, sDC exhibited markedly augmented effects on the above four functions of CTL. These results demonstrate that ex4-1BBL plays an important role in the costimulation pathway for DC-mediated CTL's activation, which might be a useful adjuvant factor for DC-based cancer biotherapy.