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Introduction: Hepatitis delta virus (HDV) far exceeds our expected level. There remains a lack of reliable quantitative assays for HDV RNA detection. We sought to develop a new method based on digital droplet polymerase chain reaction (ddPCR) for HDV quantitative detection., Methods: With plasmid (pMD19T) containing HDV full genome, we determined the method for ddPCR-based HDV RNA quantification. To compare various assays for HDV detection, 30 cases diagnosed with hepatitis D and 14 controls were examined using enzyme-linked immunosorbent assay, reverse-transcriptase PCR (RT-PCR), and ddPCR. A total of 728 hepatitis B virus-related patients, including 182 patients with chronic hepatitis B, 182 with liver cirrhosis, 182 with hepatocellular carcinoma, and 182 with liver failure, were screened for HDV infection., Results: The detection limit of ddPCR for HDV is significantly low, with lower limit of detection and lower limit of quantitation of 0.29 IU/mL (95% confidence interval: 1.93 × 10-3-1.22 IU/mL) and 8.76 IU/mL (95% confidence interval: 1.83-1.03 × 106 IU/mL), respectively. Among the 44 samples, the enzyme-linked immunosorbent assay detected 30 cases positive, ddPCR reported 24 samples, and RT-PCR reported 10 samples positive for HDV RNA. Moreover, the positive rates of anti-HDV were 1.1%, 3.3%, 2.7%, and 7.1% in patients with chronic hepatitis B, liver cirrhosis, hepatocellular carcinoma, and liver failure, respectively; the detection rates of RT-PCR in HDV RNA were 0%, 16.67%, 15.4%, and 20%, respectively. However, the detection rates of ddPCR were 0%, 33.33%, 30.77%, and 60%, respectively., Discussion: We establish a high sensitivity and specificity quantitative HDV RNA detection method based on ddPCR. Hepatitis B virus-related end-stage liver diseases, especially liver failure, are associated with a remarkably high rate of HDV infection., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Objective: Peroxisome proliferator-activated receptor α (PPARα) activation has been reported to ameliorate liver injury in cases of acute liver failure (ALF). However, its intrinsic protective molecular mechanisms remain largely undetermined. C/EBP homologous protein (CHOP) is an important mediator of lipopolysaccharide (LPS)-induced inflammation. The aim of the present study was to test the hypothesis that PPARα activation alleviates liver inflammation to protect mice from acute liver failure (ALF) mediated by CHOP. Methods: In a murine model induced by d-galactosamine ( d-GalN, 700 mg/kg) and LPS (10 μg/kg), Wy-14643 (6 mg/kg) was administered to activate PPARα. The mice of different groups were killed 6 h after d-GalN/LPS injection, and the liver and blood were collected for analysis. To find out whether PPARα activation protects the liver from injury due to inflammation by regulating CHOP, we used expression plasmid to increase CHOP expression and demonstrated how PPARα mediated CHOP to regulate inflammation in vivo and in vitro. Results: The expression of PPARα was downregulated and the expression of CHOP was upregulated with the development of d-GalN/LPS-induced liver injury. The protective molecular mechanisms of PPARα activation were dependent on the expression of CHOP. Indeed, (1) PPARα activation decreased the expression of CHOP; on the other hand, PPARα knockdown increased the expression of CHOP in vivo; (2) the depressed liver inflammation by PPARα activation was due to the downregulation of CHOP expression, because overexpression of CHOP by transfect plasmid reversed liver protection and increased liver inflammation again; (3) in vitro, PPARα inhibition by siRNA treatment increased the expression of proinflammatory cytokines, and CHOP siRNA co-transfection reversed the expression of proinflammatory cytokines. Conclusions: Here, we demonstrated that PPARα activation contributes to liver protection and decreases liver inflammation in ALF, particularly through regulating CHOP. Our findings may provide a rationale for targeting PPARα as a potential therapeutic strategy to ameliorate ALF. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum stress (ER stress) has been increasingly recognized as an important mechanism in various liver diseases. However, its intrinsic physiological role in acute liver failure (ALF) remains largely undetermined. This study aimed to examine how ER stress orchestrates glycogen synthase kinase 3β (GSK3β) and inflammation to affect ALF. In a murine ALF model induced by d-galactosamine ( d-GalN) and lipopolysaccharide (LPS), 4-phenylbutyric acid (4-PBA) is to be administered to relieve ER stress. The lethality rate, liver damage, cytokine expression, and the activity of GSK3β were evaluated. How to regulate LPS-induced inflammation and TNF-α-induced hepatocyte apoptosis by ER stress was investigated in vitro. In vivo, ER stress was triggered in the liver with the progression of mice ALF model. ER stress was essential for the development of ALF because ER stress inhibition by 4-PBA ameliorated the liver damage through decreasing liver inflammation and hepatocyte apoptosis. 4-PBA also decreased GSK3β activity in the livers of ALF mice. In vitro, ER stress induced by tunicamycin synergistically increased LPS-triggered pro-inflammatory cytokine induction and promoted the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathway in bone marrow-derived macrophages; moreover, tunicamycin also cooperated with TNF-α to increase hepatocyte apoptosis. ER stress promoted LPS-triggered inflammation depending on GSK3β activation because inhibition of GSK3β by SB216763, the specific inhibitor of GSK3β, resulted in downregulation of pro-inflammatory genes. ER stress contributes to liver inflammation and hepatotoxicity in ALF, particularly by regulating GSK3β, and is therefore a potential therapeutic target for ALF. [ABSTRACT FROM AUTHOR]

Objective: Our previous studies have demonstrated that glycogen synthase kinase 3β (GSK3β) activity is increased in the progression of acute liver failure (ALF), which aggravates liver injury, while its regulatory mechanism remains elusive. This study is designated to address whether oxidative stress activates GSK3β to promote ALF. Methods: In a murine model induced by d-galactosamine ( d-GalN) (700 mg/kg) and LPS (10 μg/kg), N-acetylcysteine (300 mg/kg) or SB216763 (25 mg/kg) was used to inhibit oxidative stress or GSK3β activity, respectively. Serum alanine aminotransferase and aspartate aminotransferase levels were assessed. The parameters of oxidative stress were evaluated in liver tissue. Whether GSK3β inhibition protects hepatocytes from oxidative stress-induced cell apoptosis was investigated in vitro. Moreover, the activity of GSK3β was measured in the liver of chronic hepatitis B (CHB) patients and ALF patients. Results: In vivo, N-acetylcysteine ameliorated the d-GalN/LPS-induced hepatotoxicity and reduced GSK3β activity; GSK3β inhibition increased hepatic superoxide dismutase activity and the glutathione content, decreased malondialdehyde production in the liver tissues; while GSK3β inhibition suppressed the JNK activation in the liver and decreased cytochrome c release from mitochondria. In vitro, GSK3β inhibition lessened hepatocytes apoptosis induced by HO or Antimycin A, as demonstrated by decreased LDH activity, and reduced cleavage of caspase-3 expression. Furthermore, GSK3β activity in the CHB patients was increased in the phase of ALF. Conclusions: Results indicate that GSK3β activation contributes to liver injury by participating in oxidative stress response in ALF and is, therefore, a potential therapeutic target for ALF. [ABSTRACT FROM AUTHOR]

Kaempferol, a flavonoid compound, has various biological functions, such as anti-inflammatory and antitumor activities. Acute liver failure (ALF) is a lethal clinical syndrome that occurs due to severe damage of the liver function. In the present study, the mechanisms underlying the therapeutic effects of kaempferol in ALF were evaluated. An ALF mouse model was established using D-galactosamine (D-GalN; 700 mg/kg)/lipopolysaccharide (LPS; 10 µg/kg). A total of 2 h before the administration of D-GalN/LPS, mice were pretreated with different doses of kaempferol (2.5, 5, 10, 20 and 40 mg/kg), and 6 h after injection of D-GalN/LPS, mice were euthanized. The survival rate, liver function and levels of inflammatory cytokines were assessed. The results demonstrated that kaempferol pretreatment protected hepatocytes from ALF induced by D-GalN/LPS via regulation of the autophagy pathway, both in vivo and in vitro. Pretreatment with a high dose of kaempferol significantly decreased the survival rates and increased severe liver damage; however, pretreatment with a low dose of kaempferol had the opposite effect. Furthermore, pretreatment with a high dose of kaempferol enhanced the levels of proinflammatory cytokines [TNF-α, IL-6, IL-12p40, IL-1β, C-X-C motif chemokine ligand (CXCL)-2, CXCL-10] and markers of the MAPK signaling pathway [phosphorylated (p)-JNK, p-ERK, p-p38], whereas pretreatment with a low dose of kaempferol had the opposite effect. Pretreatment with a high dose of kaempferol decreased autophagy, whereas pretreatment with a low dose of kaempferol increased autophagy in vivo and in vitro. It was also shown that pretreatment with 3-methyadenine or autophagy related 7 small interfering RNA, to inhibit autophagy, partially abrogated the hepatoprotective effects of pretreatment with 5 mg/kg kaempferol in the ALF mouse model. These results demonstrate that the effects of different doses of kaempferol on D-GalN/LPS-induced ALF varies based on the dose, and that kaempferol exerted its effects via regulation of the autophagy pathway. [ABSTRACT FROM AUTHOR]

Background: Caspase-1 is an evolutionarily conserved enzyme that proteolytically cleaves the precursors of the inflammatory cytokines interleukin 1β and interleukin 18. However, the role of caspase-1 in determining the severity of acute-on-chronic liver failure (ACLF) has yet to be elucidated. We evaluated the expression levels of caspase-1 in HBV-related liver disease and assessed its utility as a biomarker predicting the severity of ACLF.Methods: The gene, protein and activity levels of caspase-1 were measured in the liver and/or serum of subjects with HBV-related disease. We also analysed the correlation between the expression levels of caspase-1 and liver injury of ACLF.Results: Compared with the values observed in normal subjects, the relative caspase-1 mRNA and protein levels in livers were decreased in patients with CHB, LC, and HCC but increased in those with ACLF; moreover, ACLF patients had the lowest serum level and hepatic activity of caspase-1 among the five groups. The serum caspase-1 levels in ACLF patients showed a negative correlation with total serum bilirubin and a positive correlation with serum total protein and albumin. Importantly, the serum caspase-1 levels in the surviving group with ACLF were higher than those in the non-surviving group and showed different dynamic trends. Analyses of the area under the receiver operating characteristic curve indicated that caspase-1 (AUC = 0.84, AUC of MELD score = 0.72) may be a useful marker for independently predicting ACLF.Conclusion: Caspase-1 is a potential non-invasive biomarker of disease progression and prognosis in ACLF. [ABSTRACT FROM AUTHOR]

Abstract Kaempferol is a flavonoid compound that has many functions, such as anti-inflammation and antioxidation. Acute liver failure (ALF) is a life-threatening illness accompanied by serious inflammation and extensive hepatocyte apoptosis. The aim of this study was to examine the therapeutic potential of kaempferol and its mechanism in ALF. In a murine ALF model induced by d -galactosamine (d -GalN, 700 mg/kg) / lipopolysaccharide (LPS, 10 μg/kg), mice were pretreated with kaempferol at 2 h before d -GalN/LPS administration and then sacrificed 6 h after d -GalN/LPS injection. Lethality, liver damage, endoplasmic reticulum(ER) stress, hepatocyte viability and apoptosis were evaluated. Whether pretreatment of kaempferol protected hepatocytes from ER stress-induced apoptosis was detected in vitro. Pretreatment of kaempferol decreased lethality, prolonged the survival time and significantly protected against liver injury, which was indicated by decreased transaminase levels and the well-preserved liver structure. The protective effect of kaempferol on the ALF mouse model was achieved by inhibiting hepatocyte apoptosis. Moreover, pretreatment of kaempferol increased the expression of glucose-regulated/binding immunoglobulin protein 78 (Grp78), decreased the expression of C/EBP-homologous protein (CHOP), and protected hepatocytes from ER stress-induced apoptosis in vitro. Our results showed that pretreatment of Grp78 siRNA partially negated the hepatic protection from kaempferol and reversed the inhibition of CHOP protein expression in d -GalN/LPS-induced ALF mice. In conclusion, kaempferol inhibits hepatocyte apoptosis to protect mice from liver failure by regulating the ER stress-Grp78-CHOP signaling pathway. Therefore, kaempferol may be used to treat ALF. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance The Qingchangligan formula, a traditional Chinese medicine comprising five herbs, is useful for treatment of patients with liver failure; however, its protective and regulatory mechanisms remain elusive. Aim of the study To test the hypothesis that the Qingchangligan formula protects mice against acute liver failure by inhibiting liver inflammation. Materials and methods Acute liver failure (ALF) was induced by intraperitoneal injection of D -GalN (700 mg/kg) plus LPS (10 μg/kg). The Qingchangligan formula was administered to mice in three doses of 50 mg/kg (on day 1, day 2, and day 3) prior to D -GalN/LPS injection by intragastric administration. The mice in different groups were sacrificed at 6 h after D -GalN/LPS injection, and liver samples and blood were collected for analysis. Results Administration of the Qingchangligan formula not only ameliorated liver injury, as evidenced by reduced transaminase levels and well-preserved liver architecture, but also decreased the lethality in ALF mice. Moreover, in the ALF model, pretreatment with the Qingchangligan formula alleviated liver inflammation and decreased hepatocyte apoptosis. Further demonstrating the protective effects of the Qingchangligan formula, we found that pretreatment with the Qingchangligan formula reduced the expression of inflammatory cytokines by decreasing the expression of components of the mitogen-activated protein kinase (MAPK) pathway and promoting autophagy in vitro and in vivo . Conclusions Our findings demonstrated that the Qingchangligan formula exerts a protective effect against the pathophysiology of ALF, especially in regulating liver inflammation, and provide a rationale for using the Qingchangligan formula as a potential therapeutic strategy to ameliorate ALF. [ABSTRACT FROM AUTHOR]

Abstract: Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, has been shown to be induced during oxidative injury, and its induction acts as an important cellular defense mechanism against such injuries. In this study, we examined the functional roles of HO-1 induction in a rat model of d-galactosamine (GalN) and lipopolysaccharide (LPS)-induced liver injury. We found that GalN/LPS treatment of rats produced severe hepatic injury, whereas upregulation of HO-1 by hemin pretreatment prevented rats from liver damage, as evidenced by decreased serum ALT, AST levels and ameliorated histological signs in the liver. Induction of HO-1 resulted in a significant decrease in hepatic malondialdehyde (MDA) contents, tumor necrosis factor-α (TNF-α) levels, iNOS/NO production, as well as the levels of caspase-3. In contrast, inhibition of HO activity by zinc protoporphyrin-9 (ZnPP, a specific inhibitor of HO) completely reversed HO-1-induced hepatoprotective effect. These data therefore suggested that HO-1 induction provided critical protection against GalN/LPS-induced liver injury, and the protection seemed to be mediated through the anti-oxidant, anti-inflammatory and anti-apoptotic functions. [Copyright &y& Elsevier]