Role In Pathogenesis Of Nonalcoholic Steatohepatitis Research Articles

TIR8 protects against nonalcoholic steatohepatitis by antagonizing lipotoxicity-induced PPARα downregulation and reducing the sensitivity of hepatocytes to LPS

In up to one-third of nonalcoholic fatty liver disease (NAFLD) patients, simple steatosis progresses to its more severe form, nonalcoholic steatohepatitis (NASH), but the precise mechanisms underlying this transition are not fully understood. Toll/interleukin-1 receptor 8 (TIR8), a conventional innate immune regulator highly expressed in hepatic tissue, has shown potential for ameliorating various inflammation-related disorders. However, its role in NASH pathogenesis, especially its regulatory effects on lipid metabolism and inflammatory responses, is still unclear. Here, using a TIR8 knockout (TIR8KO) mouse model and mass spectrometry analyses, we found that TIR8KO mice displayed aggravated hepatic steatosis and inflammation, whereas TIR8 overexpression attenuated these adverse effects. Ectopic TIR8 expression counteracts free fatty acid (FFA)-induced PPARα inhibition and downstream signaling. A decrease in TIR8 levels in hepatocytes heightened lipopolysaccharide (LPS) sensitivity. Notably, FFA stimulation led to a direct interaction between TIR8 and proteasome subunit alpha type 4 (PSMA4), facilitating TIR8 degradation. These results revealed that TIR8 safeguards PPARα-regulated lipid metabolism and mitigates inflammation induced by external factors during NASH progression. Our study highlights TIR8 as a promising target for NASH therapy, indicating the potential of TIR8 agonists in treatment strategies.

BRP39 Regulates Neutrophil Recruitment in NLRP3 Inflammasome-Induced Liver Inflammation

Background and AimsBRP-39 (Chi3L1) and its human homolog YKL-40, is an established biomarker of liver fibrosis in nonalcoholic steatohepatitis (NASH) patients, but its role in NASH pathogenesis remains unclear. We have recently identified Chi3L1 as one of the top upregulated genes in mice with inducible gain-of-function NOD-like receptor protein 3 (NLRP3) activation that mimics several liver features of NASH. This study aimed to investigate the effects of BRP39 deficiency on NLRP3-induced liver inflammation using Tamoxifen-inducible Nlrp3 knock-in mice sufficient (Nlrp3A350V CRT) and deficient for BRP39 (Nlrp3A350V /BRP-/- CRT). MethodsUsing Tamoxifen-inducible Nlrp3A350V knock-in mice (Nlrp3A350V CRT) mice and Tamoxifen-inducible Nlrp3A350V knock-in BRP39 deficient mice (Nlrp3A350V BRP-/- CRT), we investigated the consequence of BRP39 deficiency influencing NLRP3 induced liver inflammation. ResultsOur results showed that BRP39 deficiency in NLRP3 induced inflammation improved body weight and liver weight. Moreover, liver inflammation, fibrosis and HSC activation were significantly reduced, corresponding to significantly decreased Ly6C+ infiltrating macrophages, CD68+ OPN+ hepatic LAMs (lipid associated macrophages), and activated Ly6G+ and H3Cit+ neutrophils accumulation in the liver. Further investigation revealed that circulatory neutrophils from NLRP3 induced BRP39 deficient mice have impaired chemotaxis and migration ability, and this was confirmed by RNA-bulk sequencing showing reduced immune activation, migration and signaling responses in neutrophils. ConclusionThese data showcase the importance of BRP39 in regulating the NLRP3 inflammasome during liver inflammation and fibrotic NASH by altering cellular activation, recruitment and infiltration during disease progression, and revealing BRP39 to be a potential therapeutic target for future treatment of inflammatory NASH and its associated diseases.

Comparison of the Effect of 8- and 12-Week Aerobic Exercise with Calorie Restriction on the Bax and Bcl-2 Expression in Rats with Non-alcoholic Fatty Liver Disease

Background: In addition to oxidative stress, the apoptosis of liver cells plays a main role in non-alcoholic steatohepatitis pathogenesis. Nevertheless, the main mechanisms of the response of liver cell apoptosis in non-alcoholic steatohepatitis (NASH) models caused by a high-fat diet, as well as the effects of exercise with and without calorie restriction on these mechanisms, have not been assessed to date. Objectives: The present study aimed to investigate the effects of two exercise protocols with and without calorie restriction on apoptosis and liver damage in rats with non-alcoholic fatty liver disease (NAFLD). Methods: Sixty-four male Wistar rats were subjected to a high-fat diet for eight weeks and were divided randomly into eight groups: Control, calorie restriction (CR), aerobic exercise (AE), and aerobic exercise + calorie restriction (AC) for eight and twelve weeks. Also, two groups of rats that had normal and free access to food were named sham groups. The training groups exercised on the treadmill for five sessions a week for eight and twelve weeks. Two-way ANOVA was utilized for data analysis at a significance level of 0.05. Results: According to the findings, in both 8- and 12-week protocols, the expression of Bax proteins in the exercise and exercise + calorie restriction groups showed a significant decrease compared to the control group (P < 0.05). Also, Bcl-2 protein expression significantly increased in the exercise and exercise + calorie restriction groups than the control group (P < 0.05). The training groups showed no significant difference between the 8- and 12-week protocols. Conclusions: It was revealed that exercise with and without caloric restriction and independent of the protocol duration can improve the apoptosis of hepatocytes in NAFLD.

LILRB2/PirB mediates macrophage recruitment in fibrogenesis of nonalcoholic steatohepatitis

Inhibition of immunocyte infiltration and activation has been suggested to effectively ameliorate nonalcoholic steatohepatitis (NASH). Paired immunoglobulin-like receptor B (PirB) and its human ortholog receptor, leukocyte immunoglobulin-like receptor B (LILRB2), are immune-inhibitory receptors. However, their role in NASH pathogenesis is still unclear. Here, we demonstrate that PirB/LILRB2 regulates the migration of macrophages during NASH by binding with its ligand angiopoietin-like protein 8 (ANGPTL8). Hepatocyte-specific ANGPTL8 knockout reduces MDM infiltration and resolves lipid accumulation and fibrosis progression in the livers of NASH mice. In addition, PirB−/− bone marrow (BM) chimeras abrogate ANGPTL8-induced MDM migration to the liver. And yet, PirB ectodomain protein could ameliorate NASH by sequestering ANGPTL8. Furthermore, LILRB2-ANGPTL8 binding-promoted MDM migration and inflammatory activation are also observed in human peripheral blood monocytes. Taken together, our findings reveal the role of PirB/LILRB2 in NASH pathogenesis and identify PirB/LILRB2-ANGPTL8 signaling as a potential target for the management or treatment of NASH.

1570-P: Myeloid-Specific Inactivation of Basp1 Protects Mice from Diet-Induced NASH through Attenuating NLRP3 Inflammasome Activation

Nonalcoholic steatohepatitis (NASH) is characterized by persistent liver injury, inflammation, and fibrosis. Myeloid cells, including macrophages and dendritic cells, play an integral role in host defense, tissue homeostasis, and disease progression. Despite this, how myeloid cells contribute to NASH pathogenesis remains obscure. We previously identified NASH-associated macrophages (NAMs) as a unique population of monocyte-derived macrophages in NASH liver. We observed that hepatic NAMs exhibited abundant expression of Brain acid-soluble protein 1 (Basp1), which was markedly induced in the liver upon diet-induced NASH in mice. We generated myeloid-specific Basp1 knockout (Mye-Basp1 KO) mice to determine its role in NASH pathogenesis. Compared to control, Mye-Basp1 KO mice displayed significantly improved liver injury and fibrosis following NASH diet feeding, as shown by lower plasma ALT/AST levels and reduced Sirius Red staining on liver sections, respectively. Hepatocytes from Mye-Basp1 KO mice contained smaller lipid droplets without altering the total liver triglyceride content. Mechanistically, bone marrow-derived macrophages (BMDMs) lacking Basp1 exhibited diminished response to pro-inflammatory stimuli. Importantly, Basp1-deficient BMDMs displayed impaired activation of the NLRP3 inflammasome and secretion of interleukin-1 beta. Together, we propose that Basp1 is a novel regulator of inflammatory signaling in myeloid cells that plays a critical role in NASH pathogenesis. Disclosure Z.Meng: None. L.Zhou: None. S.Hong: None. T.Liu: None. X.Qiu: None. Z.Chen: None. S.Li: None. K.Inoki: None. J.Lin: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK102456)

Intestinal B cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling

The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and μMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis.

Ferroptosis plays a novel role in nonalcoholic steatohepatitis pathogenesis.

Ferroptosis relies on iron, and ferroptotic cell death is triggered when the balance of the oxidation-reduction system is disrupted by excessive lipid peroxide accumulation. A close relationship between ferroptosis and nonalcoholic steatohepatitis (NASH) is formed by phospholipid peroxidation substrates, bioactive iron, and reactive oxygen species (ROS) neutralization systems. Recent studies into ferroptosis during NASH development might reveal NASH pathogenesis and drug targets. Our review summarizes NASH pathogenesis from the perspective of ferroptosis mechanisms. Further, we discuss the relationship between mitochondrial dysfunction, ferroptosis, and NASH. Finally, potential pharmacological therapies directed to ferroptosis in NASH are hypothesized.

Deficiency of CD147 Attenuated Non-alcoholic Steatohepatitis Progression in an NLRP3-Dependent Manner.

Cluster of differentiation 147 (CD147) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD147 overexpression has been reported to facilitate the development of hepatocellular carcinoma (HCC) and influence immunologic disorders. Although increased expression of CD147 was reported in non-alcoholic steatohepatitis (NASH), functions of CD147 in NASH have not been evaluated. Firstly, we confirmed that CD147 expression was increased in the liver tissues from methionine-choline-deficient (MCD) diet-induced NASH model mice and NASH patients. Mice with hepatocyte-specific CD147 deletion exhibited attenuated NASH phenotypes, including reduced steatosis, liver injury, hepatocyte apoptosis and inflammatory cytokines IL-1β/IL-18 secretion. Following the administration of the MCD diet, NLRP3 expression was increased gradually along with CD147 expression. Furthermore, CD147 deletion inhibited the NF-κB/NLRP3 signaling pathway in both MCD diet-induced mice and primary hepatocytes. Finally, CypA inhibitor TMN355 attenuated liver steatosis and injury and inhibited NF-κB/NLRP3 signaling pathway. Therefore, our results suggest that CD147 played a vital role in NASH pathogenesis by regulating the inflammatory response, and CypA/CD147 could be attractive therapeutic targets for NASH treatment.

Multicenter Validation Study of a Diagnostic Algorithm to Detect NASH and Fibrosis in NAFLD Patients With Low NAFLD Fibrosis Score or Liver Stiffness

OBJECTIVES:Nonalcoholic steatohepatitis (NASH) and fibrosis play critical roles for the prognosis of patients with nonalcoholic fatty liver disease (NAFLD). Identification of patients at risk of NASH and fibrosis is therefore critical for disease management. NAFLD Fibrosis Score (NFS) and transient elastography (TE) have been suggested to exclude advanced fibrosis. However, there is increasing evidence that also patients with NASH and early fibrosis are at risk of disease progression and complications, emphasizing the need for improved noninvasive risk stratification in NAFLD.METHODS:Because hepatocyte apoptosis plays an early role in NASH pathogenesis, we evaluated whether the apoptosis biomarker M30 might identify NAFLD patients who are at risk of NASH and fibrosis despite low NFS or TE values. Serum M30 levels were assessed by enzyme-linked immunosorbent assay in combination with NFS and/or TE in an exploration (n = 103) and validation (n = 100) cohort of patients with biopsy-proven NAFLD.RESULTS:Most patients with low NFS (cutoff value < −1.455) revealed increased M30 levels (>200 U/L) in the exploration (62%) and validation (67%) cohort, and more than 70% of them had NASH, mostly with histological fibrosis. Vice versa, most patients with NFS < −1.455 but nonelevated M30 levels showed no NASH. NASH was also detected in most patients with indeterminate NFS (−1.455 to 0.676) but elevated M30 levels, from which ∼90% showed fibrosis. Similar results were obtained when using TE instead of NFS.DISCUSSION:The combination of the M30 biomarker with NFS or TE enables a more reliable identification of patients with an increased risk of progressed NAFLD and improves patient stratification.

HSPA12A Is a Novel Player in Nonalcoholic Steatohepatitis via Promoting Nuclear PKM2-Mediated M1 Macrophage Polarization.

Nonalcoholic steatohepatitis (NASH) is the most prevalent cause of chronic liver disease worldwide. Macrophage-mediated inflammation plays a critical role in NASH pathogenesis; however, optimum therapies for macrophage activation and NASH remain elusive. HSPA12A encodes a novel member of the HSP70 family. Here, we report that NASH patients showed increased hepatic HSPA12A expression and serum HSPA12A contents. Intriguingly, knockout of HSPA12A (Hspa12a-/- ) in mice attenuated high-fat diet (HFD)-induced hepatic steatosis and injury. HFD-induced macrophage polarization toward an M1 phenotype and inflammatory responses in the liver of Hspa12a-/- mice were also attenuated. Loss- and gain-of-function studies revealed that the de novo lipogenesis in hepatocytes was regulated by the paracrine effects of macrophage HSPA12A rather than by hepatocyte HSPA12A. In-depth molecular analysis revealed that HSPA12A interacted with the M2 isoform of pyruvate kinase (PKM2) in macrophages and increased its nuclear translocation, thereby promoting M1 polarization and secretion of proinflammatory M1 cytokines; this led, ultimately, to hepatocyte steatosis via paracrine effects. Taken together, these findings show that HSPA12A acts as a novel regulator of M1 macrophage polarization and NASH pathogenesis by increasing nuclear PKM2. Strategies that inhibit macrophage HSPA12A might be a potential therapeutic intervention for NASH.

Are N-acetylcysteine and adalimumab effective for non-alcoholic steatohepatitis?

Due to the lack of effective medical treatment for non-alcoholic steatohepatitis (NASH), we aimed to evaluate new treatment options. In particular, our goal was to investigate and compare the effects of N-acetylcysteine (NAC) and Adalimumab treatment on tumor necrosis factor alpha (TNF-α) and oxidative stress during the development of NASH in a rat model of the disease. Our study included a total of 35 female Wistar albino rats that were divided into 5 groups of 7 each, and evaluated over a 6 week period. One group received a normal diet, while the other four groups received a methionine and choline deficient (MCD) diet. One of the groups receiving the MCD diet did not take any medicine, while the other three were administered NAC, adalimumab, or a NAC/adalimumab combination therapy. NASH was successfully established in the MCD diet group. Levels of TNF-α were effectively suppressed in the three groups that received therapy. Even though adalimumab significantly enhanced suppression of TNF-α, the NASH score was suppressed to a more statistically significant extent in the groups receiving NAC. Our study showed that TNF-α and oxidative stress play an important role in NASH pathogenesis. The antioxidant agent, NAC, was found to be superior to the anti-TNF agent, Adalimumab, in the improvement of total NASH score. Although these drugs did not prevent the development of NASH, it was shown that they mildly reverse the NASH histopathology score, suggesting improvement of and overall liver function.

Hepatocyte X-box binding protein 1 deficiency increases liver injury in mice fed a high-fat/sugar diet.

Fatty liver is associated with endoplasmic reticulum stress and activation of the hepatic unfolded protein response (UPR). Reduced hepatic expression of the UPR regulator X-box binding protein 1 spliced (XBP1s) is associated with human nonalcoholic steatohepatitis (NASH), and feeding mice a high-fat diet with fructose/sucrose causes progressive, fibrosing steatohepatitis. This study examines the role of XBP1 in nonalcoholic fatty liver injury and fatty acid-induced cell injury. Hepatocyte-specific Xbp1-deficient (Xbp1(-/-)) mice were fed a high-fat/sugar (HFS) diet for up to 16 wk. HFS-fed Xbp1(-/-) mice exhibited higher serum alanine aminotransferase levels compared with Xbp1(fl/fl) controls. RNA sequencing and Gene Ontogeny pathway analysis of hepatic mRNA revealed that apoptotic process, inflammatory response, and extracellular matrix structural constituent pathways had enhanced activation in HFS-fed Xbp1(-/-) mice. Liver histology demonstrated enhanced injury and fibrosis but less steatosis in the HFS-fed Xbp1(-/-) mice. Hepatic Col1a1 and Tgfβ1 gene expression, as well as Chop and phosphorylated JNK (p-JNK), were increased in Xbp1(-/-) compared with Xbp1(fl/fl) mice after HFS feeding. In vitro, stable XBP1-knockdown Huh7 cells (Huh7-KD) and scramble control cells (Huh7-SCR) were generated and treated with palmitic acid (PA) for 24 h. PA-treated Huh7-KD cells had increased cytotoxicity measured by lactate dehydrogenase release, apoptotic nuclei, and caspase3/7 activity assays compared with Huh7-SCR cells. CHOP and p-JNK expression was also increased in Huh7-KD cells following PA treatment. In conclusion, loss of XBP1 enhances injury in both in vivo and in vitro models of fatty liver injury. We speculate that hepatic XBP1 plays an important protective role in pathogenesis of NASH.

The role of interleukin-6 and interleukin-8 gene polymorphisms in non-alcoholic steatohepatitis.

Background:Genetic polymorphisms may play role in the pathophysiology of nonalcoholic steatohepatitis (NASH).Objectives:We purposed to assess the role of interleukin 6 (IL 6) and interleukin 8 (IL 8) gene polymorphisms in the pathogenesis of NASH.Patients and Methods:Consecutive patients with biopsy proven NASH and age- and gender-matched healthy individuals with normal liver function tests and normal ultrasonography were enrolled in the study. Histopathological findings were recorded according to nonalcoholic fatty liver disease activity score (NAS). Patients were classified according to fibrosis scores as fibrosis score < 2 (mild fibrosis group) and fibrosis score ≥ 2 (significant fibrosis group). Blood samples were collected and genomic DNA isolation kit was used to evaluate genetic polymorphisms.Results:Of thirty-eight patients, 27 (71%) were in mild fibrosis group and 11 (29%) in significant fibrosis group. Thirty-eight age- and gender-matched healthy controls were enrolled in the study. The frequencies of genotypes G/C and G/G of IL 6 among the NASH group and healthy controls were 39.5% and 60.5% vs. 53.6% and 46.4%, respectively (P = 0.32). The frequencies of the genotypes of IL 8 among the NASH group were 47.2%, 44.6%, and 8.2% for T/T, A/T, and A/A, and in healthy controls were 50%, 28.6% and 21.4%, respectively, (P = 0.568). The differences between IL 8 gene T/A and T/T genotypes were not significant statistically (P > 0.05). However, the frequency of A/A genotype in significant fibrosis group was higher than the mild fibrosis group (P = 0.0016). The differences of -251 A/T polymorphism in the IL 8 and -174 C/G polymorphism in the IL 6 were not statistically significant between fibrosis groups (P > 0.05).Conclusions:IL6 and IL8 gene polymorphisms have no role in NASH pathogenesis and liver fibrosis process, but presence of the A/A genotype in the IL8 gene is associated with disease progression.

Differential Intrahepatic Phospholipid Zonation in Simple Steatosis and Nonalcoholic Steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) occurs frequently in a setting of obesity, dyslipidemia and insulin resistance, but the etiology of the disease, particularly the events favoring progression to nonalcoholic steatohepatitis (NASH) as opposed to simple steatosis (SS), are not fully understood. Based on known zonation patterns in protein, glucose and lipid metabolism, coupled with evidence that phosphatidylcholine may play a role in NASH pathogenesis, we hypothesized that phospholipid zonation exists in liver and that specific phospholipid abundance and distribution may be associated with histologic disease. A survey of normal hepatic protein expression profiles in the Human Protein Atlas revealed pronounced zonation of enzymes involved in lipid utilization and storage, particularly those facilitating phosphatidylcholine (PC) metabolism. Immunohistochemistry of obese normal, SS and NASH liver specimens with anti-phosphatidylethanomine N-methyltransferase (PEMT) antibodies showed a progressive decrease in the zonal distribution of this PC biosynthetic enzyme. Phospholipid quantitation by liquid chromatography mass spectrometry (LC-MS) in hepatic extracts of Class III obese patients with increasing NAFLD severity revealed that most PC species with 32, 34 and 36 carbons as well as total PC abundance was decreased with SS and NASH. Matrix assisted laser desorption ionization - imaging mass spectrometry (MALDI-IMS) imaging revealed strong zonal distributions for 32, 34 and 36 carbon PCs in controls (minimal histologic findings) and SS that was lost in NASH specimens. Specific lipid species such as PC 34∶1 and PC 36∶2 best illustrated this phenomenon. These findings suggest that phospholipid zonation may be associated with the presence of an intrahepatic proinflammatory phenotype and thus have broad implications in the etiopathogenesis of NASH.

Profiling microRNA expression: A snapshot of nonalcoholic steatohepatitis and a recording of its pathogenesis

We read with great interest the article by Cheung et al.1 In this report, the authors analyzed the expression profile of microRNAs in liver from patients affected by nonalcoholic steatohepatitis (NASH), and sought to find differentially expressed microRNAs and their potential role in NASH pathogenesis. Using microarray technology, microRNA profiles of 15 subjects with biopsy-proven NASH were normalized against 15 control subjects with a normal liver histology. Out of a total of 474 microRNAs analyzed, 46 were found differentially expressed in NASH patients. In particular, 23 were up-regulated (i.e., miR-34a and miR-146b), while 23 were down-regulated (i.e., miR-122) and a selection of differentially expressed microRNAs were further validated using quantitative real-time polymerase chain reaction (qRT-PCR). As expected, the predicted targets of these microRNAs include proteins belonging to complex intracellular pathways, which have been previously reported altered in NASH.2 In fact, the most recognized model of NASH pathogenesis, which is an advanced form of nonalcoholic fatty liver disease (NAFLD), considers the elevated plasma levels of free fatty acids as a first “hit” able to induce several secondary hits, including insulin resistance, increased oxidative stress on hepatocytes, and induction of proinflammatory cytokines.3 Interestingly, the authors demonstrated that miR-122 was significantly decreased (63%) in patients with NASH. The in vitro silencing of miR-122 affected the messenger RNA expression levels of predicted targets like FAS, sterol regulatory element binding protein-1c (SREBP-1c), SREBP-2, and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). These proteins, which play roles in the lipid metabolism, have been already associated to NASH both in in vitro and in vivo models.4-6 The authors conclude that human NASH is associated with unbalanced hepatic microRNA expression and that the alteration of lipid metabolism, linked to miR-122 down-regulation, could contribute to development of NASH. Cheung's work is a clear example of the basic application of microRNA profiling to study a pathology such as NASH. However, although the reported results connected to miR-122 expression are sound, their significance is difficult to interpret because the modulation of this microRNA is not a specific sign of NASH. In fact, down-regulation of miR-122 has been previously described in hepatocarcinoma, suggesting that its function is associated with liver cancer development.7, 8 As previously underlined, we strongly believe in the power of bioinformatics in determining the interconnections among genes, proteins, and pathways potentially involved in NAFLD/NASH pathogenesis.9 However, microRNA profiling and validation is only the first step toward the comprehension of NAFLD/NASH pathogenesis. The experimental validation of target gene (putative targets) predictions is not an easy issue. Several software and algorithms based on different rationales have been proposed in the last several years (i.e., MiRanda, TargetScan, Diana-microT, and PicTar) to help find candidate targets regulated by microRNAs.10 Also the performance of individual programs and their combined use to achieve a common list of targets has been evaluated and reported.11 The development of such algorithms indeed represents a hot topic in biomedical informatics. Moreover, the scarcity of biological data linking specific microRNA action to the fate of their respective messenger RNA targets so far represents the most serious limitation. In fact, as correctly indicated by Cheung and colleagues, their findings “do not provide direct proof of the involvement of microRNAs, but they serve as a broad blueprint and resource for future hypothesis generation and hypothesis-driven studies of the role of microRNAs in the development of NASH.” Finally, we believe that microRNA technology could provide not only a snapshot of the current condition of NASH, but also a record of early pathogenetic events. Thus, a complete profiling of microRNAs in patients with simple hepatic steatosis normalized against NASH, or animal models that provide the possibility to study the evolution to NASH, might help solve these intricate molecular mechanisms. Anna Alisi Ph.D.*, Andrea Masotti Ph.D. , Valerio Nobili M.D.*, * Liver Unit, Bambino Gesù Children's Hospital and Research Institute, Rome, Italy, Gene Expression–Microarrays Laboratory, Bambino Gesù Children's Hospital and Research Institute, Rome, Italy.

Effects of vitamin E treatment on peroxisome proliferator‐activated receptor‐α expression and insulin resistance in patients with non‐alcoholic steatohepatitis: results of a pilot study

Insulin resistance (IR) is commonly associated with non-alcoholic steatohepatitis (NASH). Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) may also play a role in the pathogenesis of NASH. A pivotal role in NASH pathogenesis depends on the hypothesis of increased oxidative stress. The aim of our study was to evaluate the effects of supplemental oral vitamin E, a potent antioxidant, on liver functions, PPAR-alpha expression and IR in patients with NASH. Nine patients with biopsy-proven NASH were given oral vitamin E 800 mg daily for 24 weeks. Liver functions, lipid parameters, IR index with homeostatic metabolic assessment and liver histology and PPAR-alpha staining index in biopsy specimens were detected before and after the treatment. Seven patients (78%) had IR initially. After 6 months of therapy in nine patients, fasting insulin improved (P = 0.01), but serum cholesterol, triglyceride, fasting blood glucose levels and body mass index remained unchanged. Aspartate aminotransferase and alanine aminotransferase levels decreased (P = 0.01 and P = 0.01, respectively). IR index with homeostatic metabolic assessment resistance improved (P = 0.01), but PPAR-alpha staining index did not change (P = 0.37). Although the histological grade of steatosis decreased (P = 0.01), necroinflammation and fibrosis remained unchanged. In seven patients with IR, however, necroinflammation and PPAR-alpha staining index were improved (P = 0.04 and P = 0.02). Vitamin E treatment, in addition to its previously shown beneficial effect by suppressing oxidative stress, may also achieve improvement by reducing IR and PPAR-alpha expression in NASH.