AIM: To investigate the role of Gadd45a in hepatic fibrosis and the transforming growth factor (TGF)-β/Smad signaling pathway. METHODS: Wild-type male BALB/c mice were treated with CCl4 to induce a model of chronic liver injury. Hepatic stellate cells (HSCs) were isolated from the liver of BALB/c mice and were treated with small interfering RNAs (siRNAs) targeting Gadd45a or the pcDNA3.1-Gadd45a recombinant plasmid. Cellular α-smooth muscle actin (α-SMA), β-actin, type I collagen, phospho-Smad2, phospho-Smad3, Smad2, Smad3, and Smad4 were detected by Western blots. The mRNA levels of α-SMA, β-actin, and type I collagen were determined by quantitative real-time (qRT)-PCR analyses. Reactive oxygen species production was monitored by flow cytometry using 2,7-dichlorodihydrofluorescein diacetate. Gadd45a, Gadd45b, anti-Gadd45g, type I collagen, and SMA local expression in liver tissue were measured by histologic and immunohistochemical analyses. RESULTS: Significant downregulation of Gadd45a, but not Gadd45b or Gadd45g, accompanied by activation of the TGF-β/Smad signaling pathways was detected in fibrotic liver tissues of mice and isolated HSCs with chronic liver injury induced by CCl4 treatment. Overexpression of Gadd45a reduced the expression of extracellular matrix proteins and α-SMA in HSCs, whereas transient knockdown of Gadd45a with siRNA reversed this process. Gadd45a inhibited the activity of a plasminogen activator inhibitor-1 promoter construct and (CAGA)9 MLP-Luc, an artificial Smad3/4-specific reporter, as well as reduced the phosphorylation and nuclear translocation of Smad3. Gadd45a showed protective effects by scavenging reactive oxygen species and upregulating antioxidant enzymes. CONCLUSION: Gadd45a may counteract hepatic fibrosis by regulating the activation of HSCs via the inhibition of TGF-β/Smad signaling.
AIM: To investigate the role of Gadd45a in hepatic fibrosis and the transforming growth factor (TGF)-β/Smad signaling pathway. METHODS: Wild-type male BALB/c mice were treated with CCl4 to induce a model of chronic liver injury. Hepatic stellate cells (HSCs) were isolated from the liver of BALB/c mice and were treated with small interfering RNAs (siRNAs) targeting Gadd45a or the pcDNA3.1-Gadd45a recombinant plasmid. Cellular α-smooth muscle actin (α-SMA), β-actin, type I collagen, phospho-Smad2, phospho-Smad3, Smad2, Smad3, and Smad4 were detected by Western blots. The mRNA levels of α-SMA, β-actin, and type I collagen were determined by quantitative real-time (qRT)-PCR analyses. Reactive oxygen species production was monitored by flow cytometry using 2,7-dichlorodihydrofluorescein diacetate. Gadd45a, Gadd45b, anti-Gadd45g, type I collagen, and SMA local expression in liver tissue were measured by histologic and immunohistochemical analyses. RESULTS: Significant downregulation of Gadd45a, but not Gadd45b or Gadd45g, accompanied by activation of the TGF-β/Smad signaling pathways was detected in fibrotic liver tissues of mice and isolated HSCs with chronic liver injury induced by CCl4 treatment. Overexpression of Gadd45a reduced the expression of extracellular matrix proteins and α-SMA in HSCs, whereas transient knockdown of Gadd45a with siRNA reversed this process. Gadd45a inhibited the activity of a plasminogen activator inhibitor-1 promoter construct and (CAGA)9 MLP-Luc, an artificial Smad3/4-specific reporter, as well as reduced the phosphorylation and nuclear translocation of Smad3. Gadd45a showed protective effects by scavenging reactive oxygen species and upregulating antioxidant enzymes. CONCLUSION:Gadd45a may counteract hepatic fibrosis by regulating the activation of HSCs via the inhibition of TGF-β/Smad signaling.
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