Micro-RNA 21 inhibition of SMAD7 enhances fibrogenesis via leptin-mediated NADPH oxidase in experimental and human nonalcoholic steatohepatitis

Abstract

Hepatic fibrosis in nonalcoholic steatohepatitis (NASH) is the common pathophysiological process resulting from chronic liver inflammation and oxidative stress. Although significant research has been carried out on the role of leptin-induced NADPH oxidase in fibrogenesis, the molecular mechanisms that connect the leptin-NADPH oxidase axis in upregulation of transforming growth factor (TGF)-β signaling have been unclear. We aimed to investigate the role of leptin-mediated upregulation of NADPH oxidase and its subsequent induction of micro-RNA 21 (miR21) in fibrogenesis. Human NASH livers and a high-fat (60% kcal) diet-fed chronic mouse model, where hepatotoxin bromodichloromethane was used to induce NASH, were used for this study. To prove the role of the leptin-NADPH oxidase-miR21 axis, mice deficient in genes for leptin, p47phox, and miR21 were used. Results showed that wild-type mice and human livers with NASH had increased oxidative stress, increased p47phox expression, augmented NF-κB activation, and increased miR21 levels. These mice and human livers showed increased TGF-β, SMAD2/3-SMAD4 colocalizations in the nucleus, increased immunoreactivity against Col1α, and α-SMA with a concomitant decrease in protein levels of SMAD7. Mice that were deficient in leptin or p47phox had decreased activated NF-κB and miR21 levels, suggesting the role of leptin and NADPH oxidase in inducing NF-κB-mediated miR21 expression. Further miR21 knockout mice had decreased colocalization events of SMAD2/3-SMAD4 in the nucleus, increased SMAD7 levels, and decreased fibrogenesis. Taken together, the studies show the novel role of leptin-NADPH oxidase induction of miR21 as a key regulator of TGF-β signaling and fibrogenesis in experimental and human NASH.

Fatty Liver and Chronic Kidney Disease: Novel Mechanistic Insights and Therapeutic Opportunities

“…Consistently, in experimental models of NASH and CKD, anti-miRNA21 antisense oligonucleotides induced weight loss, normalized metabolic dysregulation, and improved hepatic and renal inflammation and fibrosis, effects at least partly mediated by PPAR-a upregulation (59,60). Despite these premises, several issues remain, including the stability and selective delivery of the pharmacological modulators to the target organs and long-term safety of this approach because miRNAs also regulate cell proliferation and cell cycle progression in diverse tissues and longterm consequences of its modulation on tumor onset and progression are unclear (15,59).…”

“…On this basis, approaches inhibiting overexpressed miRNAs by antisense oligonucleotides or restoring the expression of downregulated miRNAs by synthetic miRNA mimics have been attempted. miRNA21 in particular is an attractive target because it regulates key metabolic, proinflammatory, and profibrogenic pathways and its hepatic and renal overexpression in NASH and CKD leads to PPAR-a downregulation, SREBP-2 upregulation, mitochondrial dysfunction, and profibrogenic hepatic stellate cell activation and proximal tubular cell epithelialto-mesenchymal transition (EMT) (59,60). Consistently, in experimental models of NASH and CKD, anti-miRNA21 antisense oligonucleotides induced weight loss, normalized metabolic dysregulation, and improved hepatic and renal inflammation and fibrosis, effects at least partly mediated by PPAR-a upregulation (59,60).…”

“…miRNA21 in particular is an attractive target because it regulates key metabolic, proinflammatory, and profibrogenic pathways and its hepatic and renal overexpression in NASH and CKD leads to PPAR-a downregulation, SREBP-2 upregulation, mitochondrial dysfunction, and profibrogenic hepatic stellate cell activation and proximal tubular cell epithelialto-mesenchymal transition (EMT) (59,60). Consistently, in experimental models of NASH and CKD, anti-miRNA21 antisense oligonucleotides induced weight loss, normalized metabolic dysregulation, and improved hepatic and renal inflammation and fibrosis, effects at least partly mediated by PPAR-a upregulation (59,60). Despite these premises, several issues remain, including the stability and selective delivery of the pharmacological modulators to the target organs and long-term safety of this approach because miRNAs also regulate cell proliferation and cell cycle progression in diverse tissues and longterm consequences of its modulation on tumor onset and progression are unclear (15,59).…”

Global Deletion of Glutathione S-Transferase A4 Exacerbates Developmental Nonalcoholic Steatohepatitis

“…In this regard, a recent study has linked increased leptin, the essential Nox2 cofactor p47phox, and miR-21 expression with increased tumor growth factor beSmad signaling and fibrosis in adult NASH patients and in adult mice developing NASH after high-fat feeding and treatment with the hepatotoxin bromochloromethane. 23 Moreover, the presence of p47phox in liver parenchymal cells has also been shown to be a key mediator of liver pathology in alcoholic liver disease. 24 In addition, recent studies from Bettaieb et al 25 suggest that hepatocyte Nox4 may also play an important role in development of oxidative stress and progression of NASH pathology to inflammation and fibrosis.…”

Non-alcoholic fatty liver disease: an emerging driving force in chronic kidney disease

“…Anti-miRNA21 antisense oligonucleotides induced weight loss, normalized metabolic dysregulation, and improved hepatic and renal inflammation and fibrosis, effects at least partly mediated by PPAR-alpha upregulation 116,117 .…”