M.B. Afonso

miR-34a/SIRT1/p53 is suppressed by ursodeoxycholic acid in the rat liver and activated by disease severity in human non-alcoholic fatty liver disease

BACKGROUND & AIMS Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of stages from simple steatosis to non-alcoholic steatohepatitis (NASH). However, disease pathogenesis remains largely unknown. microRNA (miRNA or miR) expression has recently been reported to be altered in human NASH, and modulated by ursodeoxycholic acid (UDCA) in the rat liver. Here, we aimed at evaluating the miR-34a/Sirtuin 1(SIRT1)/p53 pro-apoptotic pathway in human NAFLD, and to elucidate its function and modulation by UDCA in the rat liver and primary rat hepatocytes. METHODS Liver biopsies were obtained from NAFLD morbid obese patients undergoing bariatric surgery. Rat livers were collected from animals fed a 0.4% UDCA diets. Primary rat hepatocytes were incubated with bile acids or free fatty acids (FFAs) and transfected with a specific miRNA-34a precursor and/or with a p53 overexpression plasmid. p53 transcriptional activity was assessed by ELISA and target reporter constructs. RESULTS miR-34a, apoptosis and acetylated p53 increased with disease severity, while SIRT1 diminished in the NAFLD liver. UDCA inhibited the miR-34a/SIRT1/p53 pathway in the rat liver in vivo and in primary rat hepatocytes. miR-34a overexpression confirmed its targeting by UDCA, which prevented miR-34a-dependent repression of SIRT1, p53 acetylation, and apoptosis. Augmented apoptosis by FFAs in miR-34a overexpressing cells was also inhibited by UDCA. Finally, p53 overexpression activated miR-34a/SIRT1/p53, which in turn was inhibited by UDCA, via decreased p53 transcriptional activity. CONCLUSIONS Our results support a link between liver cell apoptosis and miR-34a/SIRT1/p53 signaling, specifically modulated by UDCA, and NAFLD severity. Potential endogenous modulators of NAFLD pathogenesis may ultimately provide new tools for therapeutic intervention.

Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis

Hepatocyte cell death, inflammation and oxidative stress constitute key pathogenic mechanisms underlying non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the role of necroptosis in human and experimental NAFLD and its association with tumour necrosis factor α (TNF-α) and oxidative stress. Serum markers of necrosis, liver receptor-interacting protein 3 (RIP3) and phosphorylated mixed lineage kinase domain-like (MLKL) were evaluated in control individuals and patients with NAFLD. C57BL/6 wild-type (WT) or RIP3-deficient (RIP3(-/-)) mice were fed a high-fat choline-deficient (HFCD) or methionine and choline-deficient (MCD) diet, with subsequent histological and biochemical analysis of hepatic damage. In primary murine hepatocytes, necroptosis and oxidative stress were also assessed after necrostatin-1 (Nec-1) treatment or RIP3 silencing. We show that circulating markers of necrosis and TNF-α, as well as liver RIP3 and MLKL phosphorylation were increased in NAFLD. Likewise, RIP3 and MLKL protein levels and TNF-α expression were increased in the liver of HFCD and MCD diet-fed mice. Moreover, RIP3 and MLKL sequestration in the insoluble protein fraction of NASH (non-alcoholic steatohepatitis) mice liver lysates represented an early event during stetatohepatitis progression. Functional studies in primary murine hepatocytes established the association between TNF-α-induced RIP3 expression, activation of necroptosis and oxidative stress. Strikingly, RIP3 deficiency attenuated MCD diet-induced liver injury, steatosis, inflammation, fibrosis and oxidative stress. In conclusion, necroptosis is increased in the liver of NAFLD patients and in experimental models of NASH. Further, TNF-α triggers RIP3-dependent oxidative stress during hepatocyte necroptosis. As such, targeting necroptosis appears to arrest or at least impair NAFLD progression.

c-Jun N-Terminal Kinase 1/c-Jun Activation of the p53/MicroRNA 34a/Sirtuin 1 Pathway Contributes to Apoptosis Induced by Deoxycholic Acid in Rat Liver

ABSTRACT MicroRNAs (miRs) are increasingly associated with metabolic liver diseases. We have shown that ursodeoxycholic acid, a hydrophilic bile acid, counteracts the miR-34a/sirtuin 1 (SIRT1)/p53 pathway, activated in the liver of nonalcoholic steatohepatitis (NASH) patients. In contrast, hydrophobic bile acids, particularly deoxycholic acid (DCA), activate apoptosis and are increased in NASH. We evaluated whether DCA-induced apoptosis of rat hepatocytes occurs via miR-34a-dependent pathways and whether they connect with c-Jun N-terminal kinase (JNK) induction. DCA enhanced miR-34a/SIRT1/p53 proapoptotic signaling in a dose- and time-dependent manner. In turn, miR-34a inhibition and SIRT1 overexpression significantly rescued targeting of the miR-34a pathway and apoptosis by DCA. In addition, p53 overexpression activated the miR-34a/SIRT1/p53 pathway, further induced by DCA. DCA increased p53 expression as well as p53 transcriptional activation of PUMA and miR-34a itself, providing a functional mechanism for miR-34a activation. JNK1 and c-Jun were shown to be major targets of DCA, upstream of p53, in engaging the miR-34a pathway and apoptosis. Finally, activation of this JNK1/miR-34a proapoptotic circuit was also shown to occur in vivo in the rat liver. These results suggest that the JNK1/p53/miR-34a/SIRT1 pathway may represent an attractive pharmacological target for the development of new drugs to arrest metabolism- and apoptosis-related liver pathologies.

Circulating microRNAs as Potential Biomarkers in Non-Alcoholic Fatty Liver Disease and Hepatocellular Carcinoma.

Obesity and metabolic syndrome are growing epidemics worldwide and greatly responsible for many liver diseases, including nonalcoholic fatty liver disease (NAFLD). NAFLD often progresses to cirrhosis, end-stage liver failure and hepatocellular carcinoma (HCC), the most common primary liver cancer and one of the leading causes for cancer-related deaths globally. Currently available tools for the diagnosis of NAFLD staging and progression towards HCC are largely invasive and of limited accuracy. In light of the need for more specific and sensitive noninvasive molecular markers, several studies have assessed the potential of circulating microRNAs (miRNAs) as biomarkers of liver injury and hepatocarcinogenesis. Indeed, extracellular miRNAs are very stable in the blood, can be easily quantitated and are differentially expressed in response to different pathophysiological conditions. Although standardization procedures and larger, independent studies are still necessary, miRNAs constitute promising, clinically-useful biomarkers for the NAFLD-HCC spectrum.

miR-21 ablation and obeticholic acid ameliorate nonalcoholic steatohepatitis in mice

microRNAs were recently suggested to contribute to the pathogenesis of nonalcoholic fatty liver disease (NAFLD), a disease lacking specific pharmacological treatments. In that regard, nuclear receptors are arising as key molecular targets for the treatment of nonalcoholic steatohepatitis (NASH). Here we show that, in a typical model of NASH-associated liver damage, microRNA-21 (miR-21) ablation results in a progressive decrease in steatosis, inflammation and lipoapoptosis, with impairment of fibrosis. In a complementary fast food (FF) diet NASH model, mimicking features of the metabolic syndrome, miR-21 levels increase in both liver and muscle, concomitantly with decreased expression of peroxisome proliferator-activated receptor α (PPARα), a key miR-21 target. Strikingly, miR-21 knockout mice fed the FF diet supplemented with farnesoid X receptor (FXR) agonist obeticholic acid (OCA) display minimal steatosis, inflammation, oxidative stress and cholesterol accumulation. In addition, lipoprotein metabolism was restored, including decreased fatty acid uptake and polyunsaturation, and liver and muscle insulin sensitivity fully reinstated. Finally, the miR-21/PPARα axis was found amplified in liver and muscle biopsies, and in serum, of NAFLD patients, co-substantiating its role in the development of the metabolic syndrome. By unveiling that miR-21 abrogation, together with FXR activation by OCA, significantly improves whole body metabolic parameters in NASH, our results highlight the therapeutic potential of nuclear receptor multi-targeting therapies for NAFLD.

Activation of necroptosis in human and experimental cholestasis

Cholestasis encompasses liver injury and inflammation. Necroptosis, a necrotic cell death pathway regulated by receptor-interacting protein (RIP) 3, may mediate cell death and inflammation in the liver. We aimed to investigate the role of necroptosis in mediating deleterious processes associated with cholestatic liver disease. Hallmarks of necroptosis were evaluated in liver biopsies of primary biliary cholangitis (PBC) patients and in wild-type and RIP3-deficient (RIP3−/−) mice subjected to common bile duct ligation (BDL). The functional link between RIP3, heme oxygenase-1 (HO-1) and antioxidant response was investigated in vivo after BDL and in vitro. We demonstrate increased RIP3 expression and mixed lineage kinase domain-like protein (MLKL) phosphorylation in liver samples of human PBC patients, coincident with thioflavin T labeling, suggesting activation of necroptosis. BDL resulted in evident hallmarks of necroptosis, concomitant with progressive bile duct hyperplasia, multifocal necrosis, fibrosis and inflammation. MLKL phosphorylation was increased and insoluble aggregates of RIP3, MLKL and RIP1 formed in BLD liver tissue samples. Furthermore, RIP3 deficiency blocked BDL-induced necroinflammation at 3 and 14 days post-BDL. Serum hepatic enzymes, fibrogenic liver gene expression and oxidative stress decreased in RIP3−/− mice at 3 days after BDL. However, at 14 days, cholestasis aggravated and fibrosis was not halted. RIP3 deficiency further associated with increased hepatic expression of HO-1 and accumulation of iron in BDL mice. The functional link between HO-1 activity and bile acid toxicity was established in RIP3-deficient primary hepatocytes. Necroptosis is triggered in PBC patients and mediates hepatic necroinflammation in BDL-induced acute cholestasis. Targeting necroptosis may represent a therapeutic strategy for acute cholestasis, although complementary approaches may be required to control progression of chronic cholestatic liver disease.

Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis.

MicroRNAs (miRNAs/miRs) are key regulators of liver metabolism, while toxic bile acids participate in the development of several liver diseases. We previously demonstrated that deoxycholic acid (DCA), a cytotoxic bile acid implicated in the pathogenesis of non-alcoholic fatty liver disease, inhibits miR-21 expression in hepatocytes. Here, we investigated the mechanisms by which DCA modulates miR-21 and whether miR-21 contributes for DCA-induced cytotoxicity. DCA inhibited miR-21 expression in primary rat hepatocytes in a dose-dependent manner, and increased miR-21 pro-apoptotic target programmed cell death 4 (PDCD4) and apoptosis. Both miR-21 overexpression and PDCD4 silencing hampered DCA-induced cell death. Further, DCA decreased NF-κB activity, shown to represent an upstream mechanism leading to modulation of the miR-21/PDCD4 pathway. In fact, NF-κB overexpression or constitutive activation halted miR-21-dependent apoptosis by DCA while opposite results were observed upon NF-κB inhibition. In turn, DCA-induced oxidative stress resulted in caspase-2 activation and NF-κB/miR-21 inhibition, in a PIDD-dependent manner. Finally, modulation of the NF-κB/miR-21/PDCD4 pro-apoptotic pathway by DCA was also shown to occur in the rat liver in vivo. These signalling circuits may constitute appealing targets for bile acid-associated liver pathologies.

miRNA-21 ablation protects against liver injury and necroptosis in cholestasis

Inhibition of microRNA-21 (miR-21) prevents necroptosis in the mouse pancreas. Necroptosis contributes to hepatic necro-inflammation in the common bile duct ligation (BDL) murine model. We aimed to evaluate the role of miR-21 in mediating deleterious processes associated with cholestasis. Mechanistic studies established a functional link between miR-21 and necroptosis through cyclin-dependent kinase 2-associated protein 1 (CDK2AP1). miR-21 expression increased in the liver of primary biliary cholangitis (PBC) patients and BDL wild-type (WT) mice at both 3 and 14 days. Notably, under BDL, miR-21−/− mice displayed decreased liver injury markers in serum compared with WT mice, accompanied by reduced hepatocellular degeneration, oxidative stress and fibrosis. Hallmarks of necroptosis were decreased in the liver of BDL miR-21−/− mice, via relieved repression of CDK2AP1. Further, miR-21−/− mice displayed improved adaptive response of bile acid homeostasis. In conclusion, miR-21 ablation ameliorates liver damage and necroptosis in BDL mice. Inhibition of miR-21 should arise as a promising approach to treat cholestasis.

On the way to understand antioxidants: chromanol and dimethoxyphenols gas-phase acidities

Some antioxidant mechanisms displayed by several phenolic compounds relate with OH bond dissociation energy. One way for its determination, in the gas-phase, relies on acidity measurements. Gas-phase acidities were determined experimentally, applying the kinetic method, for chromanol and four dimethoxyphenols, and theoretically through quantum chemical DFT calculations for chromanol, six dimethoxyphenols and 3,4,5-trimethoxyphenol. The experimental acidity order, 2,3-dimethoxyphenol > 3,5-dimethoxyphenol > 2,6-dimethoxyphenol > 3,4-dimethoxyphenol ≈ phenol > chromanol shows good agreement with the theoretical acidity order, 2,5-dimethoxyphenol > 2,3-dimethoxyphenol > 3,4,5-trimethoxyphenol > 3,5-dimethoxyphenol ≈ 2,4-dimethoxyphenol > 2,6-dimethoxyphenol > 3,4-dimethoxyphenol > phenol > chromanol. These acidity trends were rationalized in terms of the substituent effects on the thermodynamic stabilities both of the parent phenols and the corresponding phenoxide ions. DFT calculations also evidenced the occurrence of intramolecular C-H···O hydrogen bonds whenever there are vicinal substituents (either OH, O(-) or OCH(3)) which induce further stabilization of the geometries.

Corrigendum: Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis

Corrigendum: Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis