Clinical Value of Pepsinogen in the Screening, Prevention, and Diagnosis of Gastric Cancer.

OBJECTIVES: To compare the levels of serum pepsinogen (PG) in patients with gastric cancer (GC), patients with atrophic gastritis (AG), and healthy donors. Also, we explored the clinical value of PG detection for the diagnosis and treatment of GC. METHODS: The PG level in peripheral blood from patients and heathy donors was determined using an Abbott automatic chemiluminescence instrument. The study included 117 patients with GC confirmed by gastroscopy and histopathology, of whom 13 patients had cancer at stage I, 47 at stage II, 41 at stage III, and 16 at stage IV. The AG group included 122 patients, and the control group had 120 healthy donors. The relationship between serum PG levels and the occurrence and development of GC, as well as the evaluation of the clinical value of diagnostic tests based on serum PG detection, were investigated by receiver operating characteristic (ROC) curve analyses. RESULTS: Pepsinogen I (PGI) levels gradually decreased from the control group, the AG group, and the GC group. PGI exhibited high diagnostic value for GC (area under the curve [AUC], 0.834; cutoff, 51.2 ng/mL, sensitivity, 81.7%; specificity, 68.4%), PGII (AUC, 0.587; cutoff value, 13.05 ng/mL; sensitivity, 65.8%; specificity, 53.8%), and PGR (AUC, 0.752; cutoff, 5.65; sensitivity, 54.2%; specificity, 87.2%). The occurrence of GC was negatively correlated with serum levels of PGI (B = -0.054; OR = 0.947; 95% confidence interval [CI], 0.925-0.970; P <.001) and PGR (B = -0.420; OR = 0.657; 95% CI, 0.499-0.864; P = .003). CONCLUSIONS: The combined detection of PGI, PGII, and PGR has important clinical value for the screening, prevention, and diagnosis of GC and could allow for earlier detection, diagnosis, and treatment of GC.

Effect of preservation time of formalin-fixed paraffin-embedded tissues on extractable DNA and RNA quantity.

OBJECTIVES: This study aimed to investigate the factors affecting the quantity of DNA and RNA extractable from human formalin-fixed paraffin-embedded (FFPE) tissues stored for different lengths of time. METHODS: We randomly selected 20 FFPE specimens harvested from hysteromyoma patients with uterine fibroids during 2010, 2015, and 2017 at the Department of Pathology, Jiading District Central Hospital Affiliated Shanghai University of Medicine and Health Sciences. DNA and RNA extractions were performed using a DNA/RNA FFPE kit. DNA and RNA concentrations and their OD260/OD280 ratios were determined by a NanoDrop 2000 spectrophotometer. The human ß-globin gene and aldehyde dehydrogenase-2 (ALDH2) gene were amplified from nucleic acids using a LightCycler 480 Real-Time PCR System, and PCR amplification products were electrophoresed on 1% agarose gels. RESULTS: Specimens that were stored for longer showed more degradation and a reduced concentration of DNA and RNA after nucleic acid extraction. However, there was no significant difference in DNA or RNA purity. ß-globin and ALDH2 genes could be amplified from more than 99% of specimens. CONCLUSION: We found that FFPE tissues stored for longer had a reduced quantity of extractable DNA and RNA. However, these tissues could be used for the analysis of some small target genes.

Hepatitis B X protein upregulates decoy receptor 3 expression via the PI3K/NF-κB pathway.

Chronic hepatitis B (CHB) is associated with the development of hepatocellular carcinoma (HCC). Decoy receptor 3 (DcR3) is a tumor necrosis factor receptor that promotes tumor cell survival by inhibiting apoptosis and interfering with immune surveillance. Previous studies showed that DcR3 was overexpressed in HCC cells and that short hairpin RNA (shDcR3) sensitizes TRAIL-resistant HCC cells. However, the expression of DcR3 during hepatitis B virus (HBV) infection has not been investigated. Here, we demonstrated that DcR3 was overexpressed in CHB patients and that DcR3 upregulation was positively correlated with the HBV DNA load and liver injury (determined by histological activity index, serum alanine aminotransferase level, and aspartate aminotransferase level). We found that hepatitis B virus X protein (HBx) upregulated DcR3 expression in a dose-dependent manner, but this increase was blocked by NF-κB inhibitors. HBx also induced the activation of NF-κB, and the NF-κB subunits p65 and p50 upregulated DcR3 by directly binding to the DcR3 promoters. Inhibition of PI3K significantly downregulated DcR3 and inhibited the binding of NF-κB to the DcR3 promoters. Our results demonstrate that the HBx induced DcR3 expression via the PI3K/NF-κB pathway; this process may contribute to the development of HBV-mediated HCC.

ShDcR3 sensitizes TRAIL-resistant HCC cells by inducing caspase-dependent apoptosis while suppressing NF-κB dependent cFLIPL expression.

Evidence has shown that most hepatocellular carcinoma (HCC) cells are resistant to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. However, the molecular mechanisms underlying TRAIL-mediated apoptosis resistance are not well understood. In this study, we reported that downregulation of Decoy receptor 3 (DcR3) expression by lentiviral vectors carrying shRNA against DcR3 (LV-ShDcR3, shDcR3) in Huh7 both greatly enhanced TRAIL-mediated apoptosis and reduced cell proliferation capability. In addition, silencing DcR3 resulted in upregulation of the cell apoptotic regulators including Bid, caspase-3, and caspase-8. Caspase inhibitors inhibited shDcR3-mediated cell death, which indicated that downregulation of DcR3 expression in Huh7 cells increased TRAIL-induced caspase-dependent apoptotic cell death. Furthermore, although the knockdown of DcR3 altered the expression of some Bcl-2- and IAP-family proteins, this change was inhibited by pretreatment with a pancaspase inhibitor, which indicated the cytotoxic effect of shDcR3 was not due to the expression of these proteins. In contrast, shDcR3 significantly inhibited TRAIL-induced transcription factor nuclear κB (NF-κB) activation through the IκB kinase (IKK) pathway, as well as inhibited TRAIL-induced increases in FLICE-inhibitory protein long form (cFLIPL) expression at the transcriptional level. Silencing cFLIPL expression mimicked the cytotoxic effect of shDcR3 on TRAIL-mediated cell apoptosis. Moreover, overexpression of cFLIPL effectively prevented the increase in cell apoptosis in Huh7 cells co-treated with TRAIL and shDcR3. Taken together, our findings indicated that silencing DcR3 sensitizes TRAIL-mediated apoptosis in HCC cells by inhibiting NF-κB.

IRF3 is an important molecule in the UII/UT system and mediates immune inflammatory injury in acute liver failure.

The urotensin II/urotensin receptor (UII/UT) system can mediate inflammatory liver injury in acute liver failure (ALF); however; the related mechanism is not clear. In this study, we confirmed that lipopolysaccharide/D-galactosamine (LPS/D-GalN) induced up-regulation of liver interferon regulatory factor 3 (IRF3) in ALF mice, whereas the UT antagonist urantide inhibited the up-regulated liver IRF3. LPS stimulation induced IRF3 transcription and nuclear translocation and promoted the secretion of interleukin-6 (IL-6), interferon (IFN)-ß, and IFN-γ in Kupffer cells (KCs); these effects in LPS-stimulated KCs were inhibited by urantide. Knockdown of IRF3 using an adenovirus expressing an IRF3 shRNA inhibited IFN-ß transcription and secretion as well as tumor necrosis factor (TNF)-α and IL-1ß secretion from LPS-stimulated KCs; additionally, IL-10 transcription and secretion were promoted in response to LPS. However, LPS-stimulated TNF-α and IL-1ß mRNA was not affected in the KCs. The IRF3 shRNA also did not have a significant effect on the NF-κB p65 subunit and p38MAPK protein phosphorylation levels in the nuclei of LPS-stimulated KCs. Therefore, IRF3 expression and activation depended on the signal transduction of the UII/UT system, and played important roles in UII/UT-mediated immune inflammatory injury in the liver but did not affect NF-κB and p38 MAPK activity.

Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome.

Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA-based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P < .05). Branched DNA-based Alu assays are feasible and useful to quantify serum cf-DNA levels. Increased cf-DNA levels in septic patients might complement C-reactive protein and procalcitonin in a multiple marker format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS.

The UII/UT system mediates upregulation of proinflammatory cytokines through p38 MAPK and NF-κB pathways in LPS-stimulated Kupffer cells.

The urotensin II (UII)/UII receptor (UT) system is closely related to immune inflammation. In acute liver failure (ALF), the UII/UT system can promote the production and release of proinflammatory cytokines, inducing an inflammatory injury response in liver tissue. However, the mechanism by which the hepatic UII/UT system promotes proinflammatory cytokine production and release is not clear. To solve this problem, we used primary Kupffer cells (KCs) as the model system in the current study. The results showed that after lipopolysaccharide (LPS) stimulation, KCs showed significantly increased expression and release of UII/UT and proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß). Pretreatment with urantide, which is a UT receptor antagonist, significantly inhibited the LPS-stimulated expression and release of UII/UT, TNF-α, and IL-1ß by KCs. In addition, LPS stimulation induced nuclear p38 mitogen-activated protein kinase (MAPK) protein phosphorylation and expression of the nuclear nuclear factor κB (NF-κB) p65 subunit in KCs and enhanced the binding activity of NF-κB to DNA molecules, whereas urantide pretreatment significantly inhibited the LPS-stimulated nuclear expression and activity of these molecules in KCs. Therefore, our conclusion is that the UII/UT system mediates LPS-stimulated production and release of proinflammatory cytokine by KCs, and this mediating effect at least partially relies on the inflammatory signaling pathway molecules p38 MAPK and NF-κB.

Effects of urotensin-II on cytokines in early acute liver failure in mice.

AIM: To investigate urotensin-II (UII) and its effects on tumor necrosis factor (TNF)-α and interleukin (IL)-1ß in early acute liver failure (ALF). METHODS: We investigated the time-dependent alteration in UII levels and its effects on TNF-α and IL-1ß in liver and blood in the early stage of lipopolysaccharide/D-galactosamine-induced ALF. RESULTS: After lipopolysaccharide/D-galactosamine challenge, UII rose very rapidly and reached a maximal level 0.5 h, and the level remained significantly elevated after 2 h (P < 0.05). Six hours after challenge, UII began to degrade, but remained higher than at 0 h (P < 0.05). Pretreatment with urantide, an inhibitor of the UII receptor, suppressed the degree of UII increase in liver and blood at 6 h after challenge (P < 0.05 vs paired controls). In addition, liver and blood TNF-α increased from 1 to 6 h, and reached a peak at 1 and 2 h, respectively; however, IL-1ß did not rise until 6 h after challenge. Urantide pretreatment inhibited the degree of TNF-α and IL-1ß increase following downregulation of UII post-challenge (all P < 0.05). CONCLUSION: UII plays a role in the pathogenesis and priming of ALF by triggering an inflammatory cascade and driving the early release of cytokines in mice.

Inhibition of UII/UTR system relieves acute inflammation of liver through preventing activation of NF-κB pathway in ALF mice.

Urotensin II (UII) is implicated in immune inflammatory diseases through its specific high-affinity UT receptor (UTR). Enhanced expression of UII/UTR was recently demonstrated in the liver with acute liver failure (ALF). Here, we analysed the relationship between UII/UTR expression and ALF in lipopolysaccharide (LPS)/D-galactosamine (GalN)-challenged mice. Thereafter, we investigated the effects produced by the inhibition of UII/UTR system using urantide, a special antagonist of UTR, and the potential molecular mechanisms involved in ALF. Urantide was administered to mice treated with LPS/GalN. Expression of UII/UTR, releases of proinflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and interferon-γ (IFN-γ), and activation of nuclear factor κB (NF-κB) signaling pathway were assessed in the lethal ALF with or without urantide pretreatment. We found that LPS/GalN-challenged mice showed high mortality and marked hepatic inflammatory infiltration and cell apoptosis as well as a significant increase of UII/UTR expression. Urantide pretreatment protected against the injury in liver following downregulation of UII/UTR expression. A close relationship between the acutely flamed hepatic injury and UII/UTR expression was observed. In addition, urantide prevented the increases of proinflammatory cytokines such as TNF-α, IL-1ß and IFN-γ, and activation of NF-κB signaling pathway induced by LPS/GalN in mice. Thus, we conclude that UII/UTR system plays a role in LPS/GalN-induced ALF. Urantide has a protective effect on the acutely inflamed injury of liver in part through preventing releases of proinflammatory cytokines and activation of NF-κB pathway.

Serum decoy receptor 3, a potential new biomarker for sepsis.

BACKGROUND: Sepsis, a common deadly systemic infection caused by a variety of pathogens, has some clinical symptoms similar to the systemic inflammatory response syndrome (SIRS), a whole-body non-infectious inflammatory reaction to severe insults, such as burn, trauma, hypotensive shock and so on. Treatment of sepsis depends mainly on anti-microbial, while remedy for SIRS might require steroids that could possibly enhance the spread of microbes. Unfortunately, it is very difficult to distinguish these two completely different serious conditions without blood culture, which takes days to grow and identify causative pathogens. We examined a biomarker, serum decoy receptor 3 (DcR3), was evaluated for its utility in the differential diagnosis between sepsis and SIRS. METHODS: Serum DcR3 level in 118 healthy controls, 24 sepsis patients and 43 SIRS patients, was quantitatively measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The serum DcR3 was significantly increased in sepsis patients compared with SIRS patients and healthy controls (6.11±2.58 ng/ml vs 2.62±1.46 ng/ml, and 0.91±0.56 ng/ml, respectively, p<0.001). The areas under the receiver operating characteristic curve of DcR3 for the normal vs. SIRS, normal vs. sepsis and SIRS vs. sepsis were 0.910 (0.870-0.950), 0.992 (0.984-1.000) and 0.896 (0.820-0.973), respectively. In addition, the DcR3 exhibited a positive correlation coefficient with APACHE II score, a most commonly used index for the severity of sepsis (r=0.556, p=0.005). CONCLUSION: The serum DcR3 has a potential to serve as a new biomarker for sepsis with its high specificity and sensitivity.

[Effects of urantide, a urotensin receptor inhibitor, on acute hepatocyte apoptosis in mice].

OBJECTIVE: To explore the effects of urantide, a urotensin II receptor (UT) inhibitor, on lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute hepatocyte apoptosis in mice. METHODS: Male BALB/c mice were randomly divided into 4 groups (n = 6 each): normal control, pre-treatment control, model and pre-treatment model. The pre-treatment control and pre-treatment model groups received urantide (0.6 mg/kg body weight) by a caudal vein injection. At 30 minutes post-injection, the model and pre-treatment model groups were treated with LPS/D-GalN to induce acute hepatocyte apoptosis via an intraperitoneal injection. Hepatocyte apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase-3 colorimetric assay. The expressions of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interferon-γ (IFN-γ) and interleukin-1 beta (IL-1ß), were detected by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. RESULTS: Massive hepatocyte apoptosis were detected in the model group. The apoptotic index was clearly reduced in the pre-treatment model group [(26 ± 11)% vs (77 ± 20)%, P < 0.01]. And the activity of caspase-3 was also lower in the pre-treatment model group than that in the model group [(2.50 ± 0.83) pmol · min(-1) · mg(-1) vs (3.76 ± 0.42) pmol · min(-1) · mg(-1), P < 0.01]. In addition, the serum and liver tissue levels of TNF-α, IL-1ß and IFN-γ in the pre-treatment model group were significantly lower than those in the model group[1.69 ± 0.47 vs 3.57 ± 0.79, 0.31 ± 0.02 vs 0.46 ± 0.06, 2.81 ± 0.72 vs 3.35 ± 0.84, (233 ± 36) pg/ml vs (441 ± 157) pg/ml, (228 ± 21) pg/ml vs (364 ± 20) pg/ml, (93.8 ± 5.2) pg/ml vs (180.3 ± 4.3) pg/ml, all P < 0.01]. CONCLUSION: LPS/D-GalN-induced acute hepatocyte apoptosis can be inhibited by a pretreatment of urantide through an inhibition of expression and secretion of proinflammatory cytokines. The UII/UT receptor system plays a pivotal role in the liver immuno-inflammatory injury of acute liver failure (ALF). And it may become a new drug target of ALF therapy.