Phosphorylation of eIF2α mitigates endoplasmic reticulum stress and hepatocyte necroptosis in acute liver injury.

INTRODUCTION AND OBJECTIVES: Necroptosis and endoplasmic reticulum (ER) stress has been implicated in acute and chronic liver injury. Activated eukaryotic initiation factor 2 alpha (eIF2α) attenuates protein synthesis and relieves the load of protein folding in the ER. In this study, we aimed to analyze the impact of eIF2α phosphorylation on hepatocyte necroptosis in acute liver injury. MATERIALS AND METHODS: Male BALB/c mice were injected with tunicamycin or d-galactosamine, and LO2 cells were incubated with tunicamycin to induce acute liver injury. 4-Phenylbutyric acid (PBA) and salubrinal were used to inhibit ER stress and eIF2α dephosphorylation, respectively. We analyzed the eIF2α phosphorylation, ER stress, and hepatocyte necroptosis in mice and cells model. RESULTS: Tunicamycin or d-galactosamine significantly induced ER stress and necroptosis, as well as eIF2α phosphorylation, in mice and LO2 cells (p<0.05). ER stress aggravated tunicamycin-induced hepatocyte necroptosis in mice and LO2 cells (p<0.05). Elevated eIF2α phosphorylation significantly mitigated hepatocyte ER stress (p<0.05) and hepatocyte necroptosis in mice (34.37±3.39% vs 22.53±2.18%; p<0.05) and LO2 cells (1±0.11 vs 0.33±0.05; p<0.05). Interestingly, tumor necrosis factor receptor (TNFR) 1 protein levels were not completely synchronized with necroptosis. TNFR1 expression was reduced in d-galactosamine-treated mice (p<0.05) and cells incubated with tunicamycin for 12 and 24h (p<0.05). ER stress partially restored TNFR1 expression and increased necroptosis in tunicamycin-incubated cells (p<0.05). CONCLUSIONS: These results imply that ER stress can mediate hepatocyte necroptosis independent of TNFR1 signaling and elevated eIF2α phosphorylation can mitigate ER stress during acute liver injury.

Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression.

AIM: To investigate the protective effect of prostaglandin E1 (PGE1) against endoplasmic reticulum (ER) stress-induced hepatocyte apoptosis, and to explore its underlying mechanisms. METHODS: Thapsigargin (TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinoma-derived cell line HepG2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RT-PCR. Apoptotic index and cell viability of L02 cells and HepG2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. RESULTS: Pretreatment with 1 µmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and HepG2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein (CHOP), glucose-regulated protein (GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and mRNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phospho-eukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A (PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78. CONCLUSION: Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathway-dependent induction of GRP78 expression.

[The impact of electrophysiology of central dopaminergic neurons and the depression-state induced by chronic neuropathic pain].

OBJECTIVE: To explore the underlying electrophysiological mechanism of depression induced by chronic pain in dopaminergic neurons in midbrain ventral tegmental area (VTA) of rats. METHODS: Twenty-four healthy adult rats were divided into two groups randomly(n=12):12 rats formed the sham group by exposing the spared nerve, and another 12 rats were served as the spared nerve injury (SNI)group by branchedness sciatic nerve injury surgery. The mechanical allodynia test were detected on the day of 3, 7, 14, 28, 42 and 56 after surgery, and the depressive-like behaviors such as open-field test, sucrose preference and forcedswim test were detected at the same time. Then we used the Multichannel Acquisition Processor (MAP) system to record the firing activity of neurons in VTA in both Sham rats and SNI rats. RESULTS: ①Comparing to sham rats, the paw withdrawalmechanical threshold of SNI rats was decreased significantly (P< 0.01);② According to depression-related behavioral test, SNI rats showed significant difference in open field text, sucrose preference, focus swim text comparing with Sham rats (P< 0.01);③ The firing rate and burst activity of dopaminergic neuronsin midbrain VTA are increased in depression rats compare to sham rats(P<0.05). CONCLUSIONS: Chronic pain could induce depression, and the increase of spontaneous firing rate of dopaminergic neurons in midbrain VTA might be contribute to the depression induced by the chronic neuropathic pain.

An experimental study of preventing and treating acute radioactive enteritis with human umbilical cord mesenchymal stem cells.

OBJECTIVE: To test the curative effect of human umbilical cord-derived mesenchymal stem cells on rat acute radioactive enteritis and thus to provide clinical therapeutic basis for radiation sickness. METHODS: Human umbilical cord-derived mesenchymal stem cells were cultivated in vitro and the model of acute radioactive enteritis of rats was established. Then, the umbilical cord mesenchymal stem cells were injected into the rats via tail vein. Visual and histopathological changes of the experimental rats were observed. RESULTS: After the injection, the rats in the prevention group and treatment group had remarkably better survival status than those in the control group. The histological observations revealed that the former also had better intestinal mucosa structure, more regenerative cells and stronger proliferation activity than the latter. CONCLUSIONS: Human umbilical cord-derived mesenchymal stem cells have a definite therapeutic effect on acute radioactive enteritis in rats.

Ratio of circulating follistatin and activin A reflects the severity of acute liver injury and prognosis in patients with acute liver failure.

BACKGROUND AND AIM: The activin A-follistatin system is known to play a critical role in hepatocyte regeneration during the repair of liver tissue. However, the relationship between blood levels of these compounds and the severity and prognosis of acute liver injury remains unclear. The aim of this study was to evaluate the clinical significance of circulating activin A and follistatin in patients with acute liver disease. METHODS: Serum activin A and plasma follistatin levels were determined on admission by enzyme-linked immunosorbent assay in 32 patients with acute hepatitis (AH), 23 patients with acute severe hepatitis (ASH) and 16 patients with acute liver failure (ALF). RESULTS: Both serum activin A and plasma follistatin levels were significantly elevated in patients with ASH and ALF when compared with those in patients with AH and normal controls (NC). Although plasma follistatin levels were significantly and positively correlated with serum activin A levels (r = 0.413, P < 0.001), the follistatin and activin A (F/A) ratio showed distinct deviation from NC between AH and ALF patients. The F/A ratio in AH patients was significantly elevated when compared with NC, but was significantly reduced in ALF patients. Furthermore, the F/A ratio in non-surviving ALF patients was significantly lower than that in survivors. Levels of serum activin A and plasma follistatin were significantly and negatively correlated with prothrombin time (PT) and normotest (NT) levels, while the F/A ratio showed significant and positive correlations with PT and NT. CONCLUSIONS: Decreased blood F/A ratio in ALF patients may be a reliable indicator of the severity of acute liver injury and prognosis in ALF.

Prostaglandin E2 receptor EP4 agonist induces Bcl-xL and independently activates proliferation signals in mouse primary hepatocytes.

BACKGROUND: To improve the survival rate of fulminant hepatic failure (FHF), we examined the mechanism of the antiapoptotic effect, and the possible proliferative effect, of a specific agonist of prostaglandin E2 receptor EP4 (PGEP4-A) on mouse primary hepatocytes, as a candidate for a new therapeutic agent. METHODS: The expression of four PGE2 receptor subtypes was detected by a reverse transcriptase polymerase chain reaction (PCR) method. Hepatocytes were stimulated with PGEP4-A, ONO-AE1-437, and changes in the expression levels of Bcl-xL and cyclin D1 and in the phosphorylation of epidermal growth factor receptor (EGF-R) and extracellular-signal related kinase (ERK) were examined by Western blot analysis. RESULTS: Mouse primary hepatocytes constitutively expressed the mRNAs of all four PGE2 receptor subtypes, including that of PGEP4. PGEP4-A induced not only Bcl-xL protein expression (as we had previously demonstrated in HepG2 cells) but also induced cyclin D1 protein expression in mouse primary hepatocytes as well as the phosphorylation of EGF-R and ERK. The inhibition of ERK phosphorylation by a specific inhibitor, PD98059, did not affect the increase in Bcl-xL expression level. CONCLUSIONS: PGEP4-A may be a therapeutic agent for FHF because of its antiapoptotic and regenerative effects on hepatocytes.

Proinflammatory cytokines up-regulate synthesis and secretion of urinary trypsin inhibitor in human hepatoma HepG2 cells.

Background and Aims: The urinary trypsin inhibitor (UTI), a wide range protein inhibitor synthesized by hepatocytes, is considered to play an important role not only in the protection of organ injury during severe inflammation but also in the inhibition of tumor invasion and metastasis. However, the precise mechanisms underlying control of its synthesis, secretion, and processing remain unclarified. The aim of this study is to determine whether human hepatoma HepG2 cells secrete UTI in free form and whether its synthesis and secretion are regulated by proinflammatory cytokines. Methods: Cultured HepG2 cells were stimulated using different concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). The concentration of free UTI in the medium was measured by ELISA and the intracellular UTI precursor was identified by western blotting. UTI mRNA expression was studied by reverse transcription polymerase chain reaction (RT-PCR). Results: HepG2 cells constantly secreted free UTI and this secretion was significantly up-regulated by IL-6, IL-1beta, and TNF-alpha. IL-6, IL-1beta, and TNF-alpha enhanced the synthesis of the intracellular UTI precursor protein, and IL-1beta up-regulated UTI mRNA expression. Conclusions: HepG2 cells constantly secrete free UTI. The proinflammatory cytokines, IL-1beta, IL-6, and TNF-alpha, up-regulate UTI synthesis and secretion by up-regulating UTI mRNA expression.

Induction of Bcl-xL is a possible mechanism of anti-apoptotic effect by prostaglandin E2 EP4-receptor agonist in human hepatocellular carcinoma HepG2 cells.

Because fulminant hepatic failure has a poor prognosis without liver transplantation, it is required to develop new therapies directed toward hepatocyte protection and regeneration. Previously, we showed the anti-apoptotic effects of a prostaglandin E2 EP4-receptor agonist (PGE2R-A) in a rat model of acute liver failure. The aim of this study is to determine the anti-apoptotic mechanism underlying the hepatocyte protective effect of PGE2R-A in vitro. Method: (1) Apoptosis was induced in HepG2 cells using CH11, an agonistic anti-Fas antibody. The apoptosis index (percentage of apoptotic cells with respect to the total number of cells) was sequentially estimated after the administration of CH11 alone or CH11 together with indomethacin or PGE2R-A (ONO-AE1-437). (2) The expression levels of Bcl-xL and Mcl-1, members of the anti-apoptotic Bcl-2 family, were sequentially determined by western blot analysis after treatment with PGE2R-A. Results: (1) Apoptosis indexes 6h after treatment with CH11 alone, CH11 plus indomethacin, and CH11 plus PGE2R-A were 24, 42, and 16%, respectively. (2) The expression level of the Bcl-xL protein and mRNA significantly increased 30-180min after treatment with PGE2R-A, while indomethacin decreased the expression levels of Mcl-1 proteins. Conclusion: Direct induction of Bcl-xL plays an important role in the hepatocyte protective effects induced by PGE2R-A.

Plasma and urine levels of urinary trypsin inhibitor in patients with chronic liver diseases and hepatocellular carcinoma.

BACKGROUND AND AIM: Because urinary trypsin inhibitor (UTI) is synthesized by hepatocytes and excreted into the urine, plasma and urine levels of UTI may alter in liver diseases. However, there are few reports on the changes in these levels in chronic liver diseases and hepatocellular carcinoma (HCC). The aim of the present study was to evaluate the relationships between plasma and urine UTI levels and the severity of liver damage or progression of HCC in patients with chronic liver diseases and HCC. METHODS: Plasma and urine UTI levels were measured by a newly developed enzyme-linked immunosorbent assay in 16 patients with chronic hepatitis (CH), 19 patients with liver cirrhosis (LC) and 39 patients with HCC. RESULTS: Plasma UTI level exhibited a significant positive correlation with the urine UTI level. Plasma and urine UTI levels significantly decreased in LC patients compared with those of normal controls. In contrast, the plasma UTI level in HCC patients was higher than that in LC patients, but there was no difference between the groups in the urine UTI level. Plasma and urine UTI levels in LC and HCC patients were significantly correlated with the degree of liver damage according to the Child-Pugh classification. Although neither the plasma nor urine level of UTI in HCC patients were related to the clinical stage of HCC, both levels were closely associated with the level of protein induced by vitamin K absence or antagonist-II. CONCLUSIONS: The present findings indicate that the levels of plasma and urine UTI in patients with LC reflect the severity of liver damage. In HCC patients, these levels may also reflect progression of HCC, although further study is required.