VX-765 inhibits pyroptosis and reduces inflammation to prevent acute liver failure by upregulating PPARα expression.

INTRODUCTION AND OBJECTIVES: As a fatal clinical syndrome, acute liver failure (ALF) is characterized by overwhelming liver inflammation and hepatic cell death. Finding new therapeutic methods has been a challenge in ALF research. VX-765 is a known pyroptosis inhibitor and has been reported to prevent damage in a variety of diseases by reducing inflammation. However, the role of VX-765 in ALF is still unclear. MATERIALS AND METHODS: ALF model mice were treated with D-galactosamine (D-GalN) and lipopolysaccharide (LPS). LO2 cells were stimulated with LPS. Thirty subjects were enrolled in clinical experiments. The levels of inflammatory cytokines, pyroptosis-associated proteins and peroxisome proliferator-activated receptor α (PPARα) were detected using quantitative reverse transcription-polymerase chain reaction (qRT‒PCR), western blotting and immunohistochemistry. An automatic biochemical analyzer was used to determine the serum aminotransferase enzyme levels. Hematoxylin and eosin (HE) staining was used to observe the pathological features of the liver. RESULTS: With the progression of ALF, the expression levels of interleukin (IL) -1ß, IL-18, caspase-1, and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were increased. VX-765 could reduce the mortality rate of ALF mice, relieve liver pathological damage, and reduce inflammatory responses to protect against ALF. Further experiments showed that VX-765 could protect against ALF through PPARα, and this protective effect against ALF was reduced in the context of PPARα inhibition. CONCLUSIONS: As ALF progresses, inflammatory responses and pyroptosis deteriorate gradually. VX-765 can inhibit pyroptosis and reduce inflammatory responses to protect against ALF by upregulating PPARα expression, thus providing a possible therapeutic strategy for ALF.

[The protective role of peroxisome proliferator activated receptors-α and pathomechanism in D-galactosamine/lipopolysaccharide- induced acute liver failure in rats].

OBJECTIVE: To determine the role and mechanism of peroxisome proliferator activated receptors (PPAR) α in a mouse model of D-galactosamine/lipopolysaccharide (D-GalN/LPS)-induced acute liver failure (ALF). METHODS: Firstly, C57BL/6 mice were randomly divided into control group (n = 8), ALF 2h group (n = 8), ALF 4h group (n = 8), ALF 6h group (n = 8). Secondly C57BL/6 mice were randomly divided into control group (n = 8), ALF group (n = 8), WY14643 group (n = 8). To induce ALF, the mice were injected intraperitoneally with D-GalN (700 mg/kg) and LPS (10 µg/kg). WY14643 (6 mg/kg), the selective agonist of PPAR α, was administered via tail vein two hours prior to D-GalN/LPS exposure. Two, four, and six hours after D-GalN/LPS treatment in the first study, mice were anesthetized and blood was collected, 6h after D-GalN/LPS treatment in the second study, blood was collected. The liver tissue was harvested for histology and mRNA extraction. Serum levels of ALT and AST were measured to evaluate the hepatic damage. Inflammatory cytokines (TNFα, IL-1ß, IL-6) and chemokines (CXCL-1, CXCL-10) were detected by real-time quantitative PCR. Differential protein expression of p-NF-κBp65, p-JNK, p-ERK, p-p38 in inflammatory pathways was detected by Western blotting. Significance of inter-group differences was assessed by one-way ANOVA, and pairwise comparison was performed by the least significant difference test. RESULTS: The gene and protein expression of PPAR α were gradually reduced during the development of ALF. Compared with the model group, the liver architecture was better preserved almost with normal morphology in WY14643-treated mice. Serum ALT and AST levels in WY14643-treated group were significantly lower [ALT: (555 ± 62)U/L vs (2 898 ± 822) U/L, P < 0.05; AST: (791 ± 58) U/L vs (3 013 ± 997) U/L, P < 0.05]. The expression of proinflammatory cytokines and chemokines was significantly suppressed during the activation of PPAR α. In the second study, the levels of gene expression of proinflammatory cytokines and chemokines were detected in control group, ALF group and WY14643 group respectively as followings: TNFα (0.161 ± 0.085, 7.996 ± 1.068, 3.346 ± 0.94, P < 0.05), IL-1ß (0.041 ± 0.002, 3.657 ± 0.904, 0.176 ± 0.089, P < 0.01), IL-6 (0.018 ± 0.008, 1.762 ± 0.589, 0.163 ± 0.0487, P < 0.05), CXCL-1 (0.063 ± 0.008, 7.881 ± 0.966, 2.737 ± 0.864, P < 0.01), CXCL-10 (0.054 ± 0.005, 5.671 ± 0.948, 2.578 ± 0.804, P < 0.05). CONCLUSION: Our findings first demonstrate that PPARα protects liver from injury in an ALF mouse model by suppressing inflammatory response, indicating PPARα as a potential new therapeutic target for ALF.

[Roles of peroxisome proliferator-activated receptor-α in acute liver failure and its pathogenetic mechanism in mice].

OBJECTIVE: To explore the protective effects of peroxisome proliferator-activated receptor-α (PPAR α) in D-GalN/LPS-induced acute liver failure (ALF) and its pathogenetic mechanism. METHODS: C57BL/6 mice were randomly divided into control, ALF, WY14643, 3-MA+WY14643, siAtg7+WY14643 and control siRNA+WY14643 groups (n = 8 each).For inducing ALF, the mice were injected intraperitoneally with D-GalN (700 mg/kg) and LPS (10 µg/kg). The selective activator of PPAR α-WY14643 (6 mg/kg) and autophagy inhibitor 3-MA (10 mg/kg) were administered via tail vein at 2 hours prior to D-GalN/LPS exposure. The autophagy inhibitor siAtg7 (50 µmol × L⁻¹ × kg⁻¹) and control siRNA (50 µmol × L⁻¹ × kg⁻¹) were dosed via tail vein at 48 hours prior to D-GalN/LPS exposure. At 6 hours after D-GalN/LPS dosing, the mice were anesthetized and blood sample collected.Liver samples were freshly harvested for preparing mRNA.Liver histology and serum levels of aminotransferase (ALT), aspartate aminotransferase (AST) were measured as markers of hepatic damage. Autophagy related genes (ATG5, ATG7, LAMP1), inflammatory cytokines (TNF-α, IL-1ß, IL-6) and chemokines (CXCL-1, CXCL-10) were detected by real-time quantitative PCR. Differential protein expressions of LC3, ATG5, ATG7, LAMP1 in autophagy pathways were detected by Western blotting. RESULTS: Compared with the model group, the hepatic architecture of WY14643-treated mice was better preserved. And the serum levels of ALT and AST were significantly lower ((486 ± 56) vs (2 705 ± 423) U/L, (795 ± 115) vs (3 709 ± 820) U/L, both P < 0.05) while the expression of autophagy related gene LAMP1 and protein levels were significantly higher (mRNA:4.28 ± 0.57 vs 2.67 ± 0.43, P < 0.05) . As compared with WY14643-treated ALF, the mice receiving 3-MA or ATG7-specific siRNA suffered severe acute liver failure again as evidenced by worse preserved hepatic architecture, significantly higher levels of ALT and AST ((2 563 ± 576), (2 148 ± 221) U/L and (3 474 ± 858), (3 305 ± 632) U/L, all P < 0.05) and mRNA levels of proinflammatory cytokines and chemokines (all P < 0.05). CONCLUSIONS: PPARα-mediated induction of autophagy ameliorates liver injury in ALF by attenuating inflammatory responses. Thus it may become a potential target for ALF treatment.

NKG2D modulates aggravation of liver inflammation by activating NK cells in HBV infection.

Hepatitis B virus (HBV) infection is thought to be an immune-mediated liver disease. The mechanisms underlying natural killer (NK) cell group 2D receptor (NKG2D) that activates NK cells and participates in anti-HBV immunity and immunopathology has not been thoroughly elucidated. Peripheral NKG2D+ and IFN-γ+ NK cells frequencies and intrahepatic NKG2D and IFN-γ mRNA and protein expressions were determined in HBV-infected patients. Levels of NKG2D and IFN-γ mRNA and protein in NK cells, co-cultured with HBV-replicating HepG2 cells with or without NKG2D blockade, were analyzed. Serum and supernatant IFN-γ, TNF-α, perforin and granzyme B were measured. In results, peripheral NKG2D+ and IFN-γ+ NK cells frequencies, intrahepatic NKG2D and IFN-γ mRNA and protein levels, and serum IFN-γ, TNF-α, perforin and granzyme B levels were all highest in HBV-related acute-on-chronic liver failure group, followed by chronic hepatitis B and chronic HBV carrier groups. In vitro, NKG2D and IFN-γ mRNA and protein levels were higher in NK cells with IFN-α stimulation than without stimulation. Supernatant IFN-γ, TNF-α, perforin and granzyme B levels were increased under co-culture or IFN-α stimulating conditions, but were partially blocked by NKG2DmAb. In conclusion, NKG2D regulates immune inflammation and anti-viral response partly through activation of NK cells during HBV infection.