Protocatechuic Acid-Mediated miR-219a-5p Activation Inhibits the p66shc Oxidant Pathway to Alleviate Alcoholic Liver Injury

Alcohol abuse has become common worldwide and has been recognized as a major cause of chronic alcoholic liver disease (ALD). ALD encompasses a complex process that includes a broad scope of hepatic lesions, ranging from steatosis to cirrhosis. In particular, reactive oxygen species (ROS) are mainly involved. Numerous studies have shown that p66shc plays a significant role in ALD. Protocatechuic acid (PCA), a dihydroxybenzoic acid that is naturally found in green tea, vegetables, and fruits, has efficient free radical scavenging effects. In this study, we aimed to assess the protective effect of PCA on ALD and to evaluate the microRNA- (miRNA-) p66shc-mediated reduction of ROS formation in ALD. Our results demonstrated that PCA treatment significantly decreased p66shc expression and downstream ROS formation in ALD. miR-219a-5p, which was identified by bioinformatics and experimental analysis, was enhanced by PCA and subsequently suppressed p66shc expression. Importantly, p66shc played an essential role in the protection of PCA-stimulated miR-219a-5p overexpression. Overall, these findings show that PCA-stimulated miR-219a-5p expression mitigates ALD by reducing p66shc-mediated ROS formation. This study may contribute to the development of therapeutic interventions for ALD.


Introduction
Alcoholic liver disease (ALD), which is caused by alcohol consumption, has been recognized as a significant risk factor for morbidity and mortality worldwide [1,2]. ALD encompasses a complex process involving a broad disease spectrum, which includes steatosis, hepatitis, fibrosis, and cirrhosis [3][4][5]. Also, ROS formation reportedly plays a vital role in regulating ALD [6,7]. Accordingly, decreasing ROS formation may be effective in preventing and treating alcoholic liver injury.
p66shc, as an isoform of the ShcA adaptor protein family [8], has been suggested to play a pivotal role in inducing oxidative stress, oxidant-induced inflammation, and cell death [9][10][11]. It has been reported that p66shc regulates ROS for-mation and causes oxidative cell damage in the liver due to chronic ethanol exposure [12,13].
MicroRNAs (miRNAs) are short, endogenous, noncoding RNAs that modulate pathophysiological processes [14] and modulate gene expression by binding to complementary sites on target mRNAs [15,16]. In recent years, studies on p66shc regulation have been in progress, and several miR-NAs have been shown to modulate p66shc expression in tissues [17]. Also, miRNAs play predominant roles in many liver diseases, including ALD [18][19][20]. Despite these observations, whether a miRNA-modulating p66shc is involved in ROS formation in ALD remains unknown.
The expression of p66shc has been remarkably reduced in the lung and intestine by pretreatment with protocatechuic acid, a polyphenolic compound that is commonly present in tea, fruits, and vegetables [21,22]). In recent years, PCA has been recognized as a chemopreventive compound that possesses biological activities, such as antiatherosclerotic, antioxidant, antiapoptotic, and neuroprotective activities [23,24]. However, the efficiency of PCA treatment in ALD is currently unclear.
The aims of this study were as follows: (1) to investigate whether PCA prevents ALD by reducing p66shc-mediated ROS formation, (2) to examine the role of miR-219a-5p in regulating p66shc in ALD, and (3) to clarify whether PCA prevents ALD by reducing ROS formation via a miR-219a-5p-p66shc signaling pathway.

Methods and Materials
2.1. Animals and Treatment. Protocatechuic acid (PCA, 98% purity) was purchased from Shanghai Winherb Medical Science Technology Co. Ltd. (Shanghai, China) and dissolved in water. Male Sprague Dawley rats (n = 8, 180-220 g) were housed in a special cage with free access to feed and water under controlled conditions at a temperature of 22 ± 2°C, humidity of 60 ± 10%, and a 12 h light/dark cycle. Rats were fed either a normal or a Lieber-DeCarli liquid diet (Dytes, USA) with 5% ethanol (v/v) for eight weeks. All the liquid diets were freshly prepared before distribution. The control group was pair-fed with an isocaloric liquid diet, where ethanol calories were replaced with maltose-dextrin. During this time, the experimental rats were either not administered PCA or orally administered PCA (10 or 20 mg/kg/d). The dosages of PCA were determined according to previous studies [25,26], combined with our preliminary animal experiments. Animal models demonstrating the features of chronic ethanol consumption were divided into the following groups: (1) control, (2) control+PCA (20 mg/kg/d), (3) Liber-DeCarli liquid diet, (4) Liber-DeCarli liquid diet+PCA (10 mg/kg/d), and (5) Liber-DeCarli liquid diet+PCA (20 mg/kg/d). Subsequently, liver tissue and blood were harvested for subsequent analysis. The blood samples were centrifuged at 500 g for 15 min, and then serum was collected. All animals received care by the protocols authorized by the Experimental Animal Center of Dalian Medical University (Dalian, China).

Liver Histological Analysis.
Paraffin-embedded liver tissue samples were cut into 5 μm thick sections for hematoxylin and eosin staining (H&E staining), and the sections were then examined by light microscopy.
2.4. Immunohistochemistry Analysis. Liver tissues were fixed and embedded in paraffin, and antigen retrieval was constructed by heat mediation in a Tris/EDTA buffer (pH 9). Then, the samples were incubated with a p66shc primary antibody (Abcam, EP332Y, UK). An undiluted HRP-conjugated mouse anti-rabbit IgG antibody was used as the secondary antibody. The liver tissues were counterstained with hematoxylin.
2.5. Immunofluorescence Staining. After AML-12 cells were grown in 24-well plates, the cells were fixed with methanolfree 4% formaldehyde for 30 min at room temperature. Then, the cells were washed three times in PBS, blocked with 1% bovine serum albumin in 0.1% Triton X-100, and incubated with an anti-p66shc polyclonal antibody (Proteintech, 10054-1-AP, Wuhan, China) at 4°C overnight. Afterward, the cells were incubated with FITC-conjugated AffiniPure goat anti-rabbit IgG secondary antibodies (Proteintech, FITC-10835, Wuhan, China) for 2 h at room temperature and then counterstained with DAPI for nuclei (Beyotime Institute of Biotechnology, Hangzhou, China). The colors of the images were checked by Vischeck software.
2.12. Statistical Analysis. Statistical analysis was performed using GraphPad Prism software (version 5.0; GraphPad Prism Software, La Jolla, CA, USA). All experimental data are reported as the mean ± SD. Statistical analyses were performed using multifactorial one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. The data were analyzed to determine the statistical significance between the groups. Differences with P < 0 05 were considered statistically significant.

PCA Protected Rats against Lieber-DeCarli Diet-Induced
Alcoholic Liver Injury. To ascertain the effects of PCA on rat liver injury triggered by ALD, we determined the levels of serum ALT, AST, TC, and TG and the levels of liver TC and TG and we conducted H&E staining to confirm the degree of liver injury. Serum ALT, AST, TC, and TG ( Figure 1(a)-1(d)) and liver TC and TG (Figures 1(e) and 1(f)) levels were increased in response to alcoholic liver injury, compared with the pair-fed group. These effects were abolished by PCA treatment. Furthermore, PCA treatment attenuated chronic ethanol-exposed structural alterations, according to H&E staining ( Figure 1(g)). These data confirmed that PCA protected rats against ALD.

PCA Reduced p66shc-Mediated ROS Formation in Rat
Livers and Ethanol-Exposed AML-12 Cells. Our previous studies and other reports have verified that p66shc expression can be stimulated in ALD [12,13,28] and PCA can modulate p66shc expression in different organs [21,22]. To determine whether PCA treatment could attenuate alcoholic liver injury through p66shc, hepatic p66shc expression was assayed by western blotting and immunohistochemistry ( Figure 2(a)-2(c)). The results showed that p66shc expression was increased in ALD and decreased by PCA treatment.
Because p66shc-mediated ROS formation has been confirmed [8,10,28], even in chronic alcohol-intake rats [12,13,28], we further evaluated the function of PCA on decreasing p66shc-mediated ROS formation in ALD. Rats that consumed alcohol showed decreased levels of MnSOD protein expression (Figures 2(a) and 2(b)) and hepatic GSH and CAT (Figures 2(d) and 2(f)) but displayed high levels of hepatic MDA (Figure 2(e)). In contrast, PCA treatment significantly reversed the decrease in MnSOD expression and GSH and CAT levels and caused the decrease in MDA levels ( Figure 2(a)-2(f)).
Based on the in vivo findings, AML-12 cells were transfected with si-p66shc, and as anticipated, MnSOD protein expression (Figures 3(a) and 3(b)) and GSH and CAT levels were increased and MDA and H 2 O 2 levels were decreased ( Figure 3(d)-3(g)). DCFH-DA staining, which was used to determine intracellular ROS formation, also showed that the levels of ROS were attenuated when p66shc was silenced ( Figure 3(h)). In vitro, 10 μM was selected as the primary concentration of PCA (Supplementary Figure 1). PCApretreated cells decreased p66shc expression levels and the ROS formation induced by ethanol exposure (Figure 3).

miR-219a-5p Decreased p66shc Expression in ALD.
It has been reported that miRNA plays an important role in ALD [19,20,29], and some studies have shown that p66shc could be regulated by miRNA [17]. Accordingly, we hypothesized that miRNAs might be implicated in the modulation of p66shc in ALD. To investigate the miRNA expression profiles in ALD, we referred to the literature associated with the ALD model [18,30,31] (Table 2). The miRNA target prediction program TargetScan (http://www.targetscan.org/) was used to identify miRNAs that target the p66shc 3 ′  The data are presented as the mean ± SD (n = 10). * * P < 0 01 and * P < 0 05 compared with ND; ## P < 0 01 and # P < 0 05 compared with ALD. untranslated region (3 ′ -UTR). Among the 10 downregulated miRNAs, miR-183-5p.2 and miR-219a-5p, with high conservation, were predicted to bind to the 3′-UTR of p66shc mRNA. The expression of the two miRNAs was further assayed by qPCR in vivo and in vitro (Figures 4(a) and 4(b)). The in vivo qPCR results indicated that both miRNA expression levels were significantly suppressed in the ALD rats, but miR-183-5p.2 showed no response to PCA pretreatment in ALD (groups 4 and 5, Figure 4(a) and Table 3). Fur-thermore, miR-183-5p.2 expression was undetectable by qPCR in AML-12 cells exposed to ethanol (Table 4). Compared with the relative miRNAs, ago-219a-5p decreased p66shc expression, while ago-219a-2-3p and ago-219b-5p failed to regulate p66shc expression (Supplementary Figure 3). Then, we examined whether p66shc could be regulated by miR-219a-5p modulation. AML-12 cells were transfected with ago-219a or ant-219a. Along with marked changes in the miR-219a-5p expression (Figures 4(d) and 4(e)), p66shc  , and (f) hepatic CAT levels (n = 10). * * P < 0 01 and * P < 0 05 compared with ND; ## P < 0 01 and # P < 0 05 compared with ALD.  3.4. miR-219a-5p Exerted Protective Effects on ALD in a p66shc-Dependent Manner. Based on the findings mentioned above, we examined whether miR-219a-5p exerted protection against ALD via p66shc. As shown in Figures 5(a) and 5(d), p66shc silencing had no effect on miR-219a-5p expression in the ethanol-exposed AML-12 cells. Then, we transfected AML-12 cells with si-p66shc and ago-219a either together or apart. As anticipated, ago-219a decreased the p66shc expression and increased the MnSOD expression, but si-p66shc transfected together with ago-219a inhibited the ago-219a-mediated protection (Figure 5(a)-5(c)). Mean-while, when AML-12 cells were transfected with si-p66shc and ant-219a either together or apart, ant-219a increased the p66shc expression and decreased the MnSOD expression and with p66shc silencing partly alleviated the increase in p66shc and loss in MnSOD ( Figure 5(d)-5(f)). Overall, on the basis of p66shc silencing, further modulation of the miR-219a-5p expression on p66shc and MnSOD protein levels, ago-219a, or ant-219a was mitigated compared to the respective transfected group. These results indicate that miR-219a-5p at least partly affects ALD in a p66shcdependent manner.
To further examine the PCA-regulated protective functions of miR-219a-5p on p66shc, we transfected AML-12 cells with ant-219a in either the presence or absence of PCA and measured the expression of miR-219a-5p and p66shc. Consistent with the luciferase assays, the miR-219a-5p expression was substantially decreased after ant-219a transfection compared with the control group, while PCA enhanced miR-219a-5p expression (Figure 6(c)). Further-more, silencing miR-219a-5p increased the p66shc expression in AML-12 cells (Figure 6(d)). Notably, PCA reversed the increase of p66shc induced by miR-219a-5p silencing. Thus, we concluded that PCA might target the miR-219a-5p/p66shc pathway in AML-12 cells.
3.6. PCA Exerted an Antioxidant Effect through the miR-219a-5p/p66shc Pathway with Ethanol Exposure. To further investigate the PCA-mediated miR-219a-5p/p66shc antioxidant signaling on ALD, AML-12 cells were transfected with the miR-219a-5p agomir in either the presence or absence of PCA with ethanol exposure, and the expression levels of p66shc and MnSOD were determined.
PCA and ago-219a significantly inhibited ROS formation in ALD (Figure 7(b)-7(g)). Consistent with the in vivo results, miR-219a-5p was decreased in ethanol-exposed cells, while miR-219a-5p was upregulated with PCA treatment or ago-219a transfection (Figure 7(a)). Correspondingly, the p66shc protein expression and MDA and H 2 O 2 levels were increased, and MnSOD protein expression levels and GSH and CAT levels were downregulated with ethanol exposure. DCFH-DA staining also indicated that intracellular ROS formation was increased. However, these effects were abrogated by PCA or ago-219a (Figure 7(b)-7(g)). Consequently, it was inferred that PCA might increase the miR-219a-5p expression to inhibit p66shc protein levels and downstream ROS formation in ALD. Meanwhile, ethanol metabolism might be partly linked to the miR-219a-5p/p66shc pathway in ALD (Supplementary Figure 4).

Discussion
Chronic alcoholic liver disease is widely identified as an important contributor to clinical morbidity; therefore, efficient and appropriate therapeutic agents and insight into the molecular mechanisms are necessary for the development of potential treatments. This the first study to demonstrate the following: (1) PCA contributes to the protection against ALD by reducing   6). (e, f) Protein levels of p66shc and MnSOD (n = 3). * * P < 0 01 and * P < 0 05 compared with the control group; ## P < 0 01 and # P < 0 05 compared with the ethanol group; && P < 0 01 and ROS formation and hepatocellular injury, (2) the miR-219a-5p/p66shc pathway is involved in ALD, and (3) the protective and antioxidant effect of PCA is mainly related to the modulation of the miR-219a-5p/p66shc pathway.
Protocatechuic acid (PCA), a compound that is commonly present in teas, vegetables, fruits, and many Chinese herbal medicines, is generally accepted as an antioxidant and anticarcinogenic agent [32]. In recent years, PCA has reportedly ameliorated chronic liver diseases and has been shown to protect against intestine and lung injuries [21,22]. Also, PCA has been recognized as an efficient regulator of lipid profiles and oxidative stress [23,24]. However, whether PCA treatment attenuates ALD remains unclear. In this study, we generated a Lieber-DeCarli diet-intake ALD model, and for the first time, we found that ALD was suppressed by PCA treatment, as indicated by the decrease in the serum index levels and the improvement of liver pathology. Moreover, we revealed the functional mechanisms through which PCA protects against ALD.
The p66shc molecule, an intracellular mediator, is related to ROS formation [22][23][24]28]. p66Shc functions as an inducible redox enzyme, which is activated by stress and generates hydrogen peroxide (H 2 O 2 ) [10,33]. The deletion of p66shc in cells and tissues results in reduced levels of ROS and oxidative stress [34]. An increasing number of studies have shown that p66shc and downstream ROS formation are involved in ALD [1,8,10,11]. Also, the generation and elimination of ROS is a dynamic equilibrium process [35,36]. Ethanol exposure induces oxidative stress and stimulates ROS generation, which breaks the dynamic equilibrium balance and results in liver injury [37,38]. In this study, p66shc expression assayed by western blotting, immunohistochemistry, and immunofluorescence analysis was increased in ALD and greatly reduced by the antioxidant PCA treatment. Moreover, p66shc-mediated ROS formation, indicated by DCFH-DA staining and H 2 O 2 levels (Figures 3(g) and 3(h) and Figures 7(g) and 7(h)), was attenuated in ALD after PCA treatment. These data indicate that the PCA-mediated protection against ALD includes the downregulation of the p66shc expression and a reduction in downstream ROS formation.
miRNAs are potent gene regulators that have been associated with multiple pathophysiological processes [12,14,15]. Recently, it has been reported that miRNAs regulate the key pathogenic events and effect on ALD [17][18][19]. However, few reports have examined whether miRNAs mitigate ALD by reducing ROS formation. In this study, p66shc was predicted to be a potential target gene of miR-219a-5p with bioinformatics analysis, which was further determined in vivo and in vitro. And the results of the luciferase reporter assay showed that p66shc is a downstream target gene of   miR-219a-5p. The miR-219a-5p binding sequences in the p66shc 3 ′ -UTR are highly conserved across various species (Figure 4(c)). Studies regarding the silencing and overexpression of miRNA demonstrated that miR-219a-5p directly repressed the p66shc expression. These data revealed that miR-219a-5p might play an indispensable role in the control of the p66shc expression.
Consistently, the in vivo experiments revealed that PCA treatment could significantly alleviate Lieber-DeCarli dietintake ALD and reverse the suppression of miR-219a-5p. Therefore, we hypothesized that the PCA-induced protection against ALD was related to the induction of miR-219a-5p. We then investigated this assumption in vitro and found that the miR-219a-5p expression was increased in PCA- treated AML-12 cells. Additionally, a decreased p66shc expression was observed in the ago-219a group compared with the control group. miR-219a-5p silencing reversed the PCA-mediated suppression of the p66shc expression and downstream ROS formation. Therefore, PCA stimulates the miR-219a-5p expression to inhibit the p66shc expression and downstream ROS formation, thereby attenuating alcoholic liver injury.
In summary, our results demonstrated for the first time that PCA could be a promising therapeutic agent for ALD via the miR-219a-5p/p66shc signaling pathway, which reduces ROS formation. The antioxidant functions of PCA treatment require further investigation, and this study may provide a new miRNA-based strategy for the treatment of alcoholic liver disease.  Figure S1: AML-12 cells were grown in 96-well plates, and after being exposed with 0, 25, 50, 100, and 200 mM ethanol for 24 h, the cells were incubated with a CCK-8 assay.