ABSTRACT
AIM:To evaluate the effect of exogenous hydrogen sulfide(H2S)on the expression of NLRP3 in-flammasome in hepatocytes.METHODS:The hepatocytes L 02 and SMMC-7721 were used to establish the model of inflam-mation by stimulating with lipopolysaccharide(LPS)at different concentrations in vitro.The expression of NLRP3 inflam-masome in the hepatocytes was detected by Western blot and the cell viability was measured by MTT assay for determining appropriate concentration of LPS.The hepatocytes were divided into 4 groups:the cells in control group were incubated with normal medium for 18.5 h;the cells in LPS group were incubated with normal medium for 0.5 h followed by 100 μg/L LPS for 18 h;the cells in LPS+H2 S group and H 2 S group were incubated with 200μmol/L sodium hydrosulfide hydrate(NaHS)for 0.5 h followed by 100 μg/L LPS or normal medium for 18 h,respectively.The protein expression of NLRP3 and caspase-1 in the cells of every group was determined by Western blot.RESULTS:Compared with control group ,the protein expression of NLRP3 and caspase-1 increased significantly in LPS group(P<0.05)and had no significant change in H2S group.Compared with LPS group,the protein expression of NLRP3 and caspase-1 in LPS+H2S group decreased significantly(P<0.05).CONCLUSION:In hepatocytes,exogenous H2S suppresses the expression of NLRP3 inflamma-some.
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AIM:To evaluate the effect of exogenous hydrogen sulfide ( H2 S) from GYY4137 on lipophagy in mouse primary hepatocytes .METHODS:The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion method were divided into 4 groups:the cells in control group were incubated with normal medium; the cells in model group were incubated with 1.2 mmol/L oleic acid (OA) for 48 h;the cells in H2S group or propargylglycine (PAG) group were incubated with 1.2 mmol/L OA for 48 h followed by serum-free phenol red-free RPMI-1640 medium which con-tained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h.The cells were collected to conduct immunofluorescence staining of LC3 and photography under fluorescence microscope , phase-contrast microscope or transmission electron microscope . The protein expression of LC 3-Ⅰ/Ⅱin the hepatocytes was determined by Western blot .RESULTS:In contrast with the model group, the fluorescent particles of LC3, the protein expression of LC3, the number of autophagic lysosome and vacuoles in hepatocytes in H 2 S group increased .CONCLUSION: In steatosis hepatocytes , exogenous H 2 S promotes the lipophagy .
ABSTRACT
AIM:To evaluate the effect of exogenous hydrogen sulfide ( H2 S) from GYY4137 on lipophagy in mouse primary hepatocytes .METHODS:The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion method were divided into 4 groups:the cells in control group were incubated with normal medium; the cells in model group were incubated with 1.2 mmol/L oleic acid (OA) for 48 h;the cells in H2S group or propargylglycine (PAG) group were incubated with 1.2 mmol/L OA for 48 h followed by serum-free phenol red-free RPMI-1640 medium which con-tained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h.The cells were collected to conduct immunofluorescence staining of LC3 and photography under fluorescence microscope , phase-contrast microscope or transmission electron microscope . The protein expression of LC 3-Ⅰ/Ⅱin the hepatocytes was determined by Western blot .RESULTS:In contrast with the model group, the fluorescent particles of LC3, the protein expression of LC3, the number of autophagic lysosome and vacuoles in hepatocytes in H 2 S group increased .CONCLUSION: In steatosis hepatocytes , exogenous H 2 S promotes the lipophagy .
ABSTRACT
<p><b>BACKGROUND</b>Pyroptosis is the term for caspase-1-dependent cell death associated with pro-inflammatory cytokines. The role of alveolar macrophage (AM) pyroptosis in the pathogenesis of the acute lung injury and acute respiratory distress syndrome (ALI/ARDS) remains unclear.</p><p><b>METHODS</b>C57BL/6 wild-type mice were assigned to sham, lipopolysaccharide (LPS) + vehicle, LPS + acetyl-tyrosyl-valyl- alanyl-aspartyl-chloromethylketone (Ac-YVAD-CMK) and LPS + Z-Asp-Glu-Val-Asp-fluoromethylketone groups. Mice were given intraperitoneal (IP) injections of LPS. Drugs were IP injected 1 h before LPS administration. Mice were sacrificed 16 h after LPS administration, and AMs were isolated. Western blot analysis for active caspase-1 and cleaved caspase-3, evaluation of lung injury and a cytokine release analysis were performed. AMs were treated with LPS and adenosine triphosphate (ATP); caspase-1-dependent cell death was evaluated using flow cytometry; the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) pyroptosomes were examined by immunofluorescence.</p><p><b>RESULTS</b>The expression of activated caspase-1 in AMs was enhanced following LPS challenge compared with the sham group. In the ex vivo study, the caspase-1/propidium iodide-positive cells, caspase-1 specks and ASC pyroptosomes were up-regulated in AMs following LPS/ATP stimulation. The specific caspase-1 inhibitor Ac-YVAD-CMK inhibited the activation of caspase-1 and pyroptotic cell death. Ac-YVAD-CMK also reduced the lung injury, pulmonary edema and total protein in bronchoalveolar lavage fluid (BALF). In addition, Ac-YVAD-CMK significantly inhibited interleukin-α2 (IL-1α2) release both in serum and BALF and reduced the levels of IL-18, tumor necrosis factor-α± (TNF-α±), High Mobility Group Box 1 (HMGB1) in BALF during LPS-induced ALI/ARDS.</p><p><b>CONCLUSIONS</b>This study reported AM pyroptosis during LPS-induced ALI/ARDS in mice and has demonstrated that Ac-YVAD-CMK can prevent AM-induced pyroptosis and lung injury. These preliminary findings may form the basis for further studies to evaluate this pathway as a target for prevention or reduction of ALI/ARDS.</p>