**8.1 Effect of alcohol on miR-212 expression in intestinal epithelial cells and its potential role in alcoholic liver disease (Tang et al., 2008)**

Alcoholic liver disease (ALD) is commonly associated with intestinal barrier dysfunction. Alcohol-induced dysregulation of tight junction proteins such as zonula occluden 1 (ZO-1) in intestinal epithelial cells, plays an important role in regulation of intestinal permeability. MicroRNAs (miRNAs) are recently discovered small noncoding RNAs that can regulate gene expression by targeting mRNAs and triggering either translational repression or RNA degradation. ZO-1 is predicted to be a target gene of one such miRNA, miR-212. We previously showed that miR-212 levels in colon biopsy samples in ALD patients were higher than in healthy controls; while ZO-1 protein levels were lower. Here, we studied the mechanisms for the involvement of miR-212 in alcohol-induced gut leakiness. We showed that miR-212 is highly expressed in intestinal tissues using a TaqMan microRNA real time PCR assay. Alcohol-induced miR-212 over-expression is accompanied by reductions in ZO-1 protein expression, disruption of tight junction protein (ZO-1), and increased permeability of Caco-2 cell monolayers. Alcohol-induced

Using miRNA as Biomarkers to Evaluate the Alcohol-Induced Oxidative Stress 329

We previously reported that oats supplementation prevents gut leakiness and alcoholic steatohepatitis in our rat model of ALD. Since oxidative stress is implicated in the pathogenesis of both alcohol-induced gut leakiness and ASH, and since oats have antioxidant properties, we tested the hypothesis that oats protect by preventing alcohol-induced oxidative damage to the intestine. Oxidative stress and injury were assessed by measuring colonic mucosal iNOS (by immunohistochemistry), nitric oxide, (colorimetric assay) and protein carbonylation and nitrotyrosination (immunoblotting). Colonic barrier integrity was determined by assessing the integrity of the actin cytoskeleton (immunohistochemistry) and the integrity of tight junctions (electron microscopy). Oats supplementation prevented alcohol-induced upregulation of iNOS, nitric oxide overproduction in the colonic mucosa, and increases in protein carbonyl and nitrotyrosine levels. This protection was associated with prevention of EtOH-induced disorganization of the actin cytoskeleton and disruption of tight junctions. We conclude that oats supplementation attenuates EtOH-induced disruption of intestinal barrier integrity, at least in part, by inhibiting EtOH-induced increases in oxidative stress and oxidative tissue damage. This inhibition prevents alcoholinduced disruption of the cytoskeleton and tight junctions. This study suggests that oats may be a useful therapeutic agent – a nutriceutical – for the prevention of alcohol-induced

Our research results demonstrated that the differentially expressed miRNAs are the sensitive and specific biomarkers for alcohol-induced oxidative stress. MiRNAs are potential biomarkers to accurately measure the degree of oxidative stress, early detect the indication of disease, and evaluate the effectiveness of antioxidant therapy (fig.5). The validation of miRNAs as biomarkers for alcohol-induced oxidative stress requires further studying. The key steps for validation of miRNA as suitable biomarkers for alcohol-induced oxidative stress are summarized in Fig.6. The application of miRNA as new biomarkers will lead to: a) identification susceptible individuals who are at risk for oxidative stress and would thus benefit from interventions that provide antioxidants; b) novel strategies to prevent and treat

> 1. Accurate assessment of the degree of oxidative stress 2. Early indication of disease 3. Indication of disease progress 4. Evaluation of the effectiveness

> > of antioxidant therapy

**8.4 Oats supplementation prevents alcohol-induced oxidative tissue damage in rats** 

**(Tang et al., 2009b)** 

oxidative stress and organ dysfunction.

**miRNAs**

Fig. 5. Potential uses of miRNA as biomarkers for oxidative stress

**9. Conclusion** 

oxidative injury.

miR-212 over-expression correlated with alcohol-induced disruption of monolayer integrity. To demonstrate that miR-212 acts directly at the ZO-1 3'UTR, we inserted the miR-212 target site of ZO-1 3'UTR into luciferase reporter construct and transfected it into Caco-2 cells. The expression of luciferase was significantly decreased when cotransfected with miR-212. This suppression was relieved by a single base mutantion in the UTR binding site. To see if miR-212 regulates ZO-1 levels, we did both overexpression studies using miR-212 precursors and inhibition studies using miR-212-specific antisense oligonucleotide inhibitors (anti-miR-212). miR-212 over-expression significantly inhibited ZO-1 protein expression. Knocking down of miR-212 expression in Caco-2 cells using antimiR-212 inhibited alcohol-induced hyperpermeability by 50% (p<0.05). Our studies suggest a novel mechanism for alcohol-induced gut leakiness. Alcohol induces miR-212 over-expression which disrupts intestinal barrier integrity by inhibiting ZO-1 translation. This cascade could lead to dysfunction of tight junction and increase intestinal permeability. This mechanism provides a potential therapeutic target for preventing the leaky gut in patients with ALD.

#### **8.2 Oxidative stress is required for alcohol-induced gut leakiness and liver damage (ALD model)(Keshavarzian et al., 2009)**

Thus ALD model is excellent model for studying the biomarkers of oxdative stress. Time courses for development of gut hyperpermeability, nitric oxide production, oxidative injury to the gut, endotoxemia, and liver injury were assessed in these ALD medol. Liver fat and serum transaminase increased after 2 weeks, but evidence of liver cell injury and inflammation occurred after 8 weeks. Gut leakiness, intestinal oxidative injury, and endotoxemia occurred in weeks 2-4 and progressed thereafter. Our data support the hypothesis that oxidative stress is a key co-factor (trigger) for ALD.

#### **8.3 Nitric oxide mediated oxidative injury is required for ALD (Tang et al., 2009c)**

We hypothesized that iNOS inhibitors (L-NAME, L-NIL) in vivo will inhibit the above cascade and liver injury in an animal model of alcoholic steatohepatitis (ASH). Male Sprague-Dawley rats were gavaged daily with alcohol (6 g/kg/day) or dextrose for 10 weeks ± L-NAME, L-NIL or vehicle. Systemic and intestinal NO levels were measured by nitrites and nitrates in urine and tissue samples, oxidative damage to the intestinal mucosa by protein carbonyl and nitrotyrosine, intestinal permeability by urinary sugar tests, and liver injury by histological inflammation scores, liver fat, and myeloperoxidase activity. The results showed that alcohol caused tissue oxidation, gut leakiness, endotoxemia and ASH. L-NIL and L-NAME, but not the D-enantiomers, attenuated all steps in the alcohol-induced cascade including NO overproduction, oxidative tissue damage, gut leakiness, endotoxemia, hepatic inflammation and liver injury. *Conclusions:* The mechanism we reported for alcoholinduced intestinal barrier disruption in vitro – NO overproduction, oxidative tissue damage, leaky gut, endotoxemia and liver injury – appears to be relevant in vivo in an animal model of alcohol-induced liver injury. That iNOS inhibitors attenuated all steps of this cascade suggests that prevention of this cascade in alcoholics will protect the liver against the injurious effects of chronic alcohol and that iNOS may be a useful target for prevention of ALD.

#### **8.4 Oats supplementation prevents alcohol-induced oxidative tissue damage in rats (Tang et al., 2009b)**

We previously reported that oats supplementation prevents gut leakiness and alcoholic steatohepatitis in our rat model of ALD. Since oxidative stress is implicated in the pathogenesis of both alcohol-induced gut leakiness and ASH, and since oats have antioxidant properties, we tested the hypothesis that oats protect by preventing alcohol-induced oxidative damage to the intestine. Oxidative stress and injury were assessed by measuring colonic mucosal iNOS (by immunohistochemistry), nitric oxide, (colorimetric assay) and protein carbonylation and nitrotyrosination (immunoblotting). Colonic barrier integrity was determined by assessing the integrity of the actin cytoskeleton (immunohistochemistry) and the integrity of tight junctions (electron microscopy). Oats supplementation prevented alcohol-induced upregulation of iNOS, nitric oxide overproduction in the colonic mucosa, and increases in protein carbonyl and nitrotyrosine levels. This protection was associated with prevention of EtOH-induced disorganization of the actin cytoskeleton and disruption of tight junctions. We conclude that oats supplementation attenuates EtOH-induced disruption of intestinal barrier integrity, at least in part, by inhibiting EtOH-induced increases in oxidative stress and oxidative tissue damage. This inhibition prevents alcoholinduced disruption of the cytoskeleton and tight junctions. This study suggests that oats may be a useful therapeutic agent – a nutriceutical – for the prevention of alcohol-induced oxidative stress and organ dysfunction.
