# Protocatechuic Acid Prevents oxLDL-Induced Apoptosis by Activating JNK/Nrf2 Survival Signals in Macrophages

Authors: Rosaria Varì; Beatrice Scazzocchio; Carmela Santangelo; Carmelina Filesi; Fabio Galvano; Massimo D’Archivio; Roberta Masella; Claudio Giovannini
Journal: Oxidative Medicine and Cellular Longevity
 (2015)

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## Abstract

Protocatechuic acid (PCA), one of the main metabolites of complex polyphenols, exerts numerous biological activities including antiapoptotic, anti-inflammatory, and antiatherosclerotic effects. Oxidised LDL have atherogenic properties by damaging arterial wall cells and inducing p53-dependent apoptosis in macrophages. This study was aimed at defining the molecular mechanism responsible for the protective effects of PCA against oxidative and proapoptotic damage exerted by oxLDL in J774 A.1 macrophages. We found that the presence of PCA in cells treated with oxLDL completely inhibited the p53-dependent apoptosis induced by oxLDL. PCA decreased oxLDL-induced ROS overproduction and in particular prevented the early increase of ROS. This decrease seemed to be the main signal responsible for maintaining the intracellular redox homeostasis hindering the activation of p53 induced by ROS, p38MAPK, and PKCδ. Consequently the overexpression of the proapoptotic p53-target genes such as p66Shc protein did not occur. Finally, we demonstrated that PCA induced the activation of JNK, which, in turn, determined the increase of nuclear Nrf2, leading to inhibition of the early ROS overproduction. We concluded that the antiapoptotic mechanism of PCA was most likely related to the activation of the JNK-mediated survival signals that strengthen the cellular antioxidant defences rather than to the PCA antioxidant power.


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## Body

## 1. Introduction

The adherence to a Mediterranean-type diet characterized by high intake of fruit, vegetables, fish, and extra virgin olive oil has been demonstrated to exert beneficial effects on human health most likely because of the high content in antioxidant compounds, for example, antioxidant vitamins and polyphenols [1]. Current evidence strongly supports a preventive role of polyphenols against several chronic degenerative processes, diseases, and syndromes, mostly thanks to their strong antioxidant power [2–4]. Oxidation-linked diseases or disorders, including cell transformation and cancer, atherosclerosis and cardiovascular diseases (CVD), central nervous system disorders, and a variety of age-related disorders [5–7], can be exacerbated and perhaps even initiated by numerous prooxidant agents to be found in the environment, drugs, and food.Many of the biological actions of polyphenols have been attributed to their capacity to protect lipids, proteins, and DNA from oxidative damage. However, a variety of potential mechanisms of action of polyphenols may be independent of their conventional antioxidant activities [8, 9].The 3,4-dihydroxybenzoic acid, protocatechuic acid (PCA), is a phenolic acid found in fruit, vegetables, and extra virgin olive oil but also in plant-derived beverages such as tea and red and white grape wine and in herbal medicine [10–12]. The PCA content varies considerably depending on the type of food; for example, raspberries contain up to 100 mg/kg fresh weight of PCA, while in extra virgin olive oil its concentration is about 0.22 mg/kg [13, 14].Despite its low concentration in food, PCA is of great nutritional interest since it is one of the main metabolites of complex polyphenols [15, 16]. This is the case with anthocyanins normally present at high concentrations in vegetables and fruit. The daily intake of anthocyanins has been estimated to be much higher than of other polyphenols such as quercetin, kaempferol, or myricetin (180–250 mg/d versus 20–30 mg/d, resp.) [17]. Anthocyanins are absorbed by animals and humans, but their bioavailability has been demonstrated to be very poor. In fact, only a small portion of dietary anthocyanins are absorbed in the gastrointestinal tract, and in addition at physiological pH (e.g., in the bloodstream) anthocyanins easily convert to PCA [18, 19]. Most part of the ingested compounds enter the colon, where they undergo structural modifications by colonic microflora that hydrolyze glycosides and break down anthocyanins into simple phenolic acids, such as PCA and aldehydes [15, 16, 20], which are then absorbed by colonocytes. PCA has been demonstrated to have antiatherosclerosis properties inin vitro andin vivo studies mainly via its antioxidant and anti-inflammatory activities, by preventing macrophage-mediated oxidation of LDL, monocyte/macrophage endothelial infiltration, and expression of cytokines and adhesion/chemoattractant molecules, thus inhibiting the formation of the early vessel lesion and the severity of atherosclerosis [21–24].Atherosclerosis, one of the prevalent causes of morbidity and mortality in Western countries, is an inflammatory process triggered by the presence of lipids in the vascular wall. Subendothelial retention of lipoproteins such as LDL is one of the key events that set off the atherosclerosis process. Oxidized LDL (oxLDL) contain various toxic oxidized lipids (e.g., lipid peroxides, oxysterols, and aldehydes) [25] and exhibit a wide range of biological effects, such as proinflammatory, anti-inflammatory, and proliferative or apoptotic effects on the endothelial cells and leukocytes in the atherosclerotic plaque microenvironment [25, 26].The pathophysiology of atherosclerosis involves both apoptosis and proliferation at different stages of the vessel lesion. Apoptosis is likely involved in the progression or regression of lesions, vascular remodelling, and plaque instability. Indeed, apoptosis is frequently observed in endothelial cells, macrophages, and vascular smooth muscle cells (VSMC) in atherosclerotic plaques [27]. In advanced atherosclerotic plaques, up to 50% of the apoptotic cells are macrophages, which may promote core expansion and plaque instability. In advanced human atheromata the defective phagocytic clearance of dead macrophages leads to plaque necrosis, which triggers acute atherothrombotic vascular events [28, 29].We have previously demonstrated that oxLDL induce p53-dependent apoptosis by activating p38MAPK and PKCδ signalling pathways in J774.A1 macrophage-like cell line [30].The present study was aimed at evaluating the potential protective effect of PCA against oxLDL-induced cytotoxicity in macrophages. We demonstrated that, by activating JNK/Nrf2-mediated survival signals, PCA completely counteracted both ROS- and kinase-induced activation of p53 and the consequent p66Shc-mediated oxidative stress that eventually led to the apoptotic process.


## 2. Materials and Methods

### 2.1. Plasma LDL Isolation and Oxidation

LDL (1.019–1.063 g/mL), kindly provided by the Centro Trasfusionale, University of Rome La Sapienza, were isolated by density gradient ultracentrifugation in vertical rotor from fresh pooled plasma of healthy volunteers as described elsewhere [21]. Protein content was measured by Lowry’s method using BSA as standard [31]. Native LDL (nLDL) were oxidized with 20 μM CuSO4 for 18 h at 37°C. Oxidation was stopped by 1 mM EDTA. The degree of LDL oxidation was assessed by determining TBARS content according to Yagi [32] and the increase in electrophoretic mobility on agarose gel. The TBARS content of oxLDL was 45±7 nmol malondialdehyde equivalent/mg LDL protein (versus 4±0.3 nmol in nLDL); the relative electrophoretic mobility of oxLDL versus nLDL was 1.9±0.2.


### 2.2. Cell Cultures

J774A.1 cells, purchased from the American Tissue Culture Collection, were seeded (500,000) in 25 cm2 flasks and grown in RPMI 1640 medium containing 0.2 mM glutamine, 10 U/mL antibiotics, and 10% FCS, at 37°C, 5% CO2. J774A.1 is a useful tool to investigate in-depth the molecular mechanisms potentially involved in the atherosclerosis pathological processes, and it is widely used as anin vitro model [33, 34].


### 2.3. Experimental Procedure

Twenty-four hours before the experiments, cell cultures were washed twice with serum-free medium. The medium was then replaced with RPMI 1640 containing 20 mL/L Ultroser G (Flow), a lipoprotein-free serum substitute, and exposed to nLDL or oxLDL.Preliminary experiments were performed to evaluate optimal cell treatment conditions regarding oxLDL exposure time, phenol incubation time, and the most effective antioxidant concentrations (different exposure times of treatments and different concentrations of PCA were assessed).On the basis of these preliminary experiments (data not shown) we determined the following: (i) the lowest concentration of oxLDL still effective in causing a significant increase in cell apoptosis, but less than 10% cell necrosis (cells positive for both annexin-V and propidium iodide); (ii) the most effective PCA concentration, and (iii) the length of preincubation time.All the experiments herein presented were time-course experiments (0–48 h) carried out with (i) untreated cells, (ii) cells treated with 0.1 mg/mL nLDL, (iii) cells treated with 0.1 mg/mL oxLDL, and (iv) cells treated with nLDL or oxLDL in the presence of 25μM PCA. The phenolic acid was added 1 h before treatments with the lipoproteins and was present in the culture medium for the entire experimental period in all the experiments. Since the values obtained in untreated cells overlapped those obtained in cells treated with nLDL, the figures show only the results obtained in nLDL-treated cells, which were considered as controls and shown as CTR in all the figures.


### 2.4. Cytotoxicity

Cytotoxicity was measured by incorporation of the radiolabelled precursor of DNA synthesis14C Thymidine and by trypan blue exclusion method.Cells were exposed to LDL or oxLDL 5 days after seeding in 24 multiwell plates in the presence or absence of PCA. For measuring proliferating activity, 1.85 kBq of [14C] thymidine (Amersham, Buckinghamshire, UK; special activity: 2.09 GBq/mmol) was added to each well. After 4 h, [14C] thymidine incorporation was stopped and cells were prepared as previously reported [35]. Radioactivity was evaluated in 0.2 mL aliquots of NaOH extracts by a liquid scintillation spectrometer. [14C] thymidine incorporation was expressed as percentage of values observed in control cells. For assessing cell viability, an aliquot of the cell suspension was mixed with an equal volume of trypan blue. Nonviable cells stained with the dye were counted under light microscope. Data were reported as percent of the total number of cells displayed.


### 2.5. Evaluation of Apoptosis

Apoptosis was evaluated with the ApoAlert Annexin V apoptosis Kit (Clontech Laboratories, Palo Alto, CA, USA) following the manufacturer’s instructions. The two-colour cytometric analysis (fluorescence-activated cell sorting (FACS)) was performed on a Coulter Epics Elite ESP cell Sorter with an argon-ion laser tuned to 488 nm.


### 2.6. ROS Assay

Intracellular ROS levels were determined using a fluorescence probe, 5-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), which is converted to highly fluorescent dichlorofluorescein (DCF) in the presence of intracellular ROS. The probe, as other DCFDA-based fluorophores, is widely used to indirectly detect ROS that lead to cell peroxide formation and is a suitable indicator of oxidative cell status. Cells were washed with PBS and incubated with freshly diluted CM-H2DCFDA (25μM) in PBS for 1 h and then washed twice with PBS. Cell fluorescence intensity was measured on a spectrofluorometer with excitation wavelength of 485 nm and emission wavelength of 530 nm.


### 2.7. GSH and GSSG Determination

The levels of reduced glutathione (GSH) and oxidized GSH (GSSG) and the GSH/GSSG ratio were determined by the Bioxytech GSH/GSSG-412 assay kit (OXIS International, Inc., Portland, OR, USA) according to the manufacturer’s instructions.


### 2.8. Protein Determination by Immunoblotting Analysis

Whole cell extracts were prepared from cells collected and washed twice in ice-cold PBS, suspended in 50μL 1% TRITON X (Sigma) containing 2 mM Na3VO4 and 5 μL of a mixture of protease inhibitors (Sigma), and incubated on ice for 20 min. Cells were centrifuged at 18,000 g for 10 min at 4°C. Supernatants, assessed for protein concentration, were used for western blot analysis as described elsewhere [36]. Nuclear protein extracts were prepared by the Nuclear/Cytosol fractionation Kit (Medical & Biological Laboratories, Watertown, LA, USA) according to the manufacturer’s instructions. For immunoprecipitation, whole-cell lysates containing 500 μg of protein were incubated for 2 h at 4°C with specific antibodies, namely, anti-p53, anti-p38, anti-PKCδ, or anti-JNK. Then the samples were incubated with protein G-Sepharose for 30 min and the beads washed thrice with the same lysis buffer. Immunoblotting analyses were carried out using specific antibodies for p53, PKCδ, caspase-3 active form (caspase-3 p11), Bax, p66Shc, Nrf2 (Santa Cruz), phospho-PKCδ, p38, phospho-p38, JNK, phospho-JNK (Cell Signaling), and serine-phosphorylated proteins (Sigma). Blots were treated with appropriate secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology) followed by ECL detection (Amersham Bio-sciences, UK). Densitometric analysis was performed with a molecular imager FX (Bio-Rad, Hercules CA). Equal loading of proteins was verified by immunoblotting with anti-cyclophilin and anti-lamin B antibodies for whole cell and nuclear extracts, respectively.


### 2.9. Evaluation of Inhibitor Effects

In a set of experiments aimed at defining whether JNK plays the kinase responsible for the protective effects of PCA, the cells were treated with SP600125, a specific JNK1/2 inhibitor, one hour before PCA and oxLDL addition, at a concentration (50μM) that effectively inhibited targeted pathways without any signs of cytotoxicity.


### 2.10. Gene Silencing and Small Interfering RNA

Nrf2 expression was inhibited with Nrf2-directed siRNA reagents (Nrf2 siRNA mouse; Santa Cruz). Briefly, J774A.1 cells were transfected with 100 nM Nrf2-siRNA mixed with Lipofectamine 2000 transfection reagent (Invitrogen) in the absence of antibiotics, according to the manufacturer’s instructions. Scrambled nontargeting siRNA was introduced in the cells following the same protocol and used as negative control. At selected time points after transfection, proteins were extracted to assess phospho-p53, Bax, and the active form of caspase-3 expressions.


### 2.11. Statistical Analysis

Student’st-test and one-way analysis of variance on the Statview programme for Macintosh were used to analyse data. Values of P lower than 0.05 were considered statistically significant.


## 2.1. Plasma LDL Isolation and Oxidation

LDL (1.019–1.063 g/mL), kindly provided by the Centro Trasfusionale, University of Rome La Sapienza, were isolated by density gradient ultracentrifugation in vertical rotor from fresh pooled plasma of healthy volunteers as described elsewhere [21]. Protein content was measured by Lowry’s method using BSA as standard [31]. Native LDL (nLDL) were oxidized with 20 μM CuSO4 for 18 h at 37°C. Oxidation was stopped by 1 mM EDTA. The degree of LDL oxidation was assessed by determining TBARS content according to Yagi [32] and the increase in electrophoretic mobility on agarose gel. The TBARS content of oxLDL was 45±7 nmol malondialdehyde equivalent/mg LDL protein (versus 4±0.3 nmol in nLDL); the relative electrophoretic mobility of oxLDL versus nLDL was 1.9±0.2.


## 2.2. Cell Cultures

J774A.1 cells, purchased from the American Tissue Culture Collection, were seeded (500,000) in 25 cm2 flasks and grown in RPMI 1640 medium containing 0.2 mM glutamine, 10 U/mL antibiotics, and 10% FCS, at 37°C, 5% CO2. J774A.1 is a useful tool to investigate in-depth the molecular mechanisms potentially involved in the atherosclerosis pathological processes, and it is widely used as anin vitro model [33, 34].


## 2.3. Experimental Procedure

Twenty-four hours before the experiments, cell cultures were washed twice with serum-free medium. The medium was then replaced with RPMI 1640 containing 20 mL/L Ultroser G (Flow), a lipoprotein-free serum substitute, and exposed to nLDL or oxLDL.Preliminary experiments were performed to evaluate optimal cell treatment conditions regarding oxLDL exposure time, phenol incubation time, and the most effective antioxidant concentrations (different exposure times of treatments and different concentrations of PCA were assessed).On the basis of these preliminary experiments (data not shown) we determined the following: (i) the lowest concentration of oxLDL still effective in causing a significant increase in cell apoptosis, but less than 10% cell necrosis (cells positive for both annexin-V and propidium iodide); (ii) the most effective PCA concentration, and (iii) the length of preincubation time.All the experiments herein presented were time-course experiments (0–48 h) carried out with (i) untreated cells, (ii) cells treated with 0.1 mg/mL nLDL, (iii) cells treated with 0.1 mg/mL oxLDL, and (iv) cells treated with nLDL or oxLDL in the presence of 25μM PCA. The phenolic acid was added 1 h before treatments with the lipoproteins and was present in the culture medium for the entire experimental period in all the experiments. Since the values obtained in untreated cells overlapped those obtained in cells treated with nLDL, the figures show only the results obtained in nLDL-treated cells, which were considered as controls and shown as CTR in all the figures.


## 2.4. Cytotoxicity

Cytotoxicity was measured by incorporation of the radiolabelled precursor of DNA synthesis14C Thymidine and by trypan blue exclusion method.Cells were exposed to LDL or oxLDL 5 days after seeding in 24 multiwell plates in the presence or absence of PCA. For measuring proliferating activity, 1.85 kBq of [14C] thymidine (Amersham, Buckinghamshire, UK; special activity: 2.09 GBq/mmol) was added to each well. After 4 h, [14C] thymidine incorporation was stopped and cells were prepared as previously reported [35]. Radioactivity was evaluated in 0.2 mL aliquots of NaOH extracts by a liquid scintillation spectrometer. [14C] thymidine incorporation was expressed as percentage of values observed in control cells. For assessing cell viability, an aliquot of the cell suspension was mixed with an equal volume of trypan blue. Nonviable cells stained with the dye were counted under light microscope. Data were reported as percent of the total number of cells displayed.


## 2.5. Evaluation of Apoptosis

Apoptosis was evaluated with the ApoAlert Annexin V apoptosis Kit (Clontech Laboratories, Palo Alto, CA, USA) following the manufacturer’s instructions. The two-colour cytometric analysis (fluorescence-activated cell sorting (FACS)) was performed on a Coulter Epics Elite ESP cell Sorter with an argon-ion laser tuned to 488 nm.


## 2.6. ROS Assay

Intracellular ROS levels were determined using a fluorescence probe, 5-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), which is converted to highly fluorescent dichlorofluorescein (DCF) in the presence of intracellular ROS. The probe, as other DCFDA-based fluorophores, is widely used to indirectly detect ROS that lead to cell peroxide formation and is a suitable indicator of oxidative cell status. Cells were washed with PBS and incubated with freshly diluted CM-H2DCFDA (25μM) in PBS for 1 h and then washed twice with PBS. Cell fluorescence intensity was measured on a spectrofluorometer with excitation wavelength of 485 nm and emission wavelength of 530 nm.


## 2.7. GSH and GSSG Determination

The levels of reduced glutathione (GSH) and oxidized GSH (GSSG) and the GSH/GSSG ratio were determined by the Bioxytech GSH/GSSG-412 assay kit (OXIS International, Inc., Portland, OR, USA) according to the manufacturer’s instructions.


## 2.8. Protein Determination by Immunoblotting Analysis

Whole cell extracts were prepared from cells collected and washed twice in ice-cold PBS, suspended in 50μL 1% TRITON X (Sigma) containing 2 mM Na3VO4 and 5 μL of a mixture of protease inhibitors (Sigma), and incubated on ice for 20 min. Cells were centrifuged at 18,000 g for 10 min at 4°C. Supernatants, assessed for protein concentration, were used for western blot analysis as described elsewhere [36]. Nuclear protein extracts were prepared by the Nuclear/Cytosol fractionation Kit (Medical & Biological Laboratories, Watertown, LA, USA) according to the manufacturer’s instructions. For immunoprecipitation, whole-cell lysates containing 500 μg of protein were incubated for 2 h at 4°C with specific antibodies, namely, anti-p53, anti-p38, anti-PKCδ, or anti-JNK. Then the samples were incubated with protein G-Sepharose for 30 min and the beads washed thrice with the same lysis buffer. Immunoblotting analyses were carried out using specific antibodies for p53, PKCδ, caspase-3 active form (caspase-3 p11), Bax, p66Shc, Nrf2 (Santa Cruz), phospho-PKCδ, p38, phospho-p38, JNK, phospho-JNK (Cell Signaling), and serine-phosphorylated proteins (Sigma). Blots were treated with appropriate secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology) followed by ECL detection (Amersham Bio-sciences, UK). Densitometric analysis was performed with a molecular imager FX (Bio-Rad, Hercules CA). Equal loading of proteins was verified by immunoblotting with anti-cyclophilin and anti-lamin B antibodies for whole cell and nuclear extracts, respectively.


## 2.9. Evaluation of Inhibitor Effects

In a set of experiments aimed at defining whether JNK plays the kinase responsible for the protective effects of PCA, the cells were treated with SP600125, a specific JNK1/2 inhibitor, one hour before PCA and oxLDL addition, at a concentration (50μM) that effectively inhibited targeted pathways without any signs of cytotoxicity.


## 2.10. Gene Silencing and Small Interfering RNA

Nrf2 expression was inhibited with Nrf2-directed siRNA reagents (Nrf2 siRNA mouse; Santa Cruz). Briefly, J774A.1 cells were transfected with 100 nM Nrf2-siRNA mixed with Lipofectamine 2000 transfection reagent (Invitrogen) in the absence of antibiotics, according to the manufacturer’s instructions. Scrambled nontargeting siRNA was introduced in the cells following the same protocol and used as negative control. At selected time points after transfection, proteins were extracted to assess phospho-p53, Bax, and the active form of caspase-3 expressions.


## 2.11. Statistical Analysis

Student’st-test and one-way analysis of variance on the Statview programme for Macintosh were used to analyse data. Values of P lower than 0.05 were considered statistically significant.


## 3. Results

### 3.1. Cytotoxicity

PCA completely counteracted the cytostatic/cytotoxic effects induced by oxLDL in A.1 cells. OxLDL reduced cell growth showing a dramatic cytostatic effect: about 30% and 50% decrease of relative incorporation of [14C] thymidine after 18 and 24 h exposure, respectively, with respect to control cells. OxLDL exposure augmented the percentage of dead cells measured by counting trypan blue positive cells. PCA reversed oxLDL-induced cytotoxicity (Figures 1(a) and 1(b)).Figure 1
Protocatechuic acid completely protects macrophage cells from oxLDL-cytotoxic activity. J774A.1 cells were exposed to native LDL or oxLDL (0.2 mg/mL) in the presence or absence of 25μM protocatechuic acid. (a) Relative incorporation of [14C] thymidine. Each column represents the relative incorporation after 18 and 24 h treatment as compared with control values (100%). (b) Trypan blue staining. Percentages of dead cells (trypan blue positive cells) are reported at 6, 18, and 24 h. The data are expressed as mean ± S.E.M. of three independent experiments. P∗<0.001 versus controls.
(a)
(b)


### 3.2. Apoptosis and Oxidative Stress

To assess whether PCA was able to counteract oxLDL-induced apoptosis in macrophages, experiments were carried out to identify annexin V positive cells in (i) cells exposed to oxLDL, (ii) control cells, and (iii) cells exposed to oxLDL in the presence of PCA. Cells treated with oxLDL underwent apoptosis after 18 h exposure, while treatment with PCA prevented the death of the cells (Figure2(a)).Figure 2
Protocatechuic acid inhibited OxLDL-induced apoptosis and the onset of oxidative stress in macrophage cells. Time-course experiments. (a) Percentage of apoptotic annexin-V positive cells, (b) intracellular ROS production, and (c) intracellular reduced glutathione (GSH) content in J774A.1 cells. Values are the mean ± S.E.M. of four independent experiments. Values obtained in control cells (CTR), oxLDL-treated cells (oxLDL), and oxLDL-treated cells preincubated with 25μM PCA (PCA + oxLDL) were compared. P∗<0.05 versus controls; P∗∗<0.001 versus controls. P∗#<0.05 versus oxLDL-treated cells and versus controls, for each time point.
(a)
(b)
(c)Since the first toxic effect of oxLDL was an early and progressive increase in ROS production followed by a dramatic decrease in GSH content in macrophage-like cells [30], the effect of PCA on cellular oxidative stress was evaluated. The early increase in ROS production was completely abrogated in cells treated with PCA until hour 6 (Figure 2(b)). ROS production increased in PCA-treated cultures only after 6 h of oxLDL-exposure, and, although significantly higher with respect to control cultures, it was significantly lower than in oxLDL-treated cells. Notably, PCA treatment completely counteracted GSH depletion induced by oxLDL (Figure 2(c)), clearly indicating that it was able to prevent the onset of oxidative stress induced by oxLDL.


### 3.3. PCA Inhibited p53 Apoptotic Signal

Western blotting analyses were performed to elucidate if the antiapoptotic activity of PCA depended on the ability to modulate p53 expression and activation induced by oxLDL. For this purpose phosphorylated and nuclear p53 and protein expression of two of the main p53 target genes, p66Shc and BAX, both responsible for the execution of p53-mediated apoptosis, were evaluated in oxLDL-treated cells in the presence of PCA. The obtained results demonstrated that PCA was able to counteract all the changes induced by oxLDL in p53 pathway. In particular, PCA prevented (i) the significant overexpression of p53 protein observed throughout the time period considered (data not shown), (ii) the early (1 h) significant increase of the phosphorylated form, and (iii) the increase in p53 nuclear translocation that occurred after 3 h of oxLDL exposure (Figures3(a) and 3(b)). Moreover, PCA completely counteracted the overexpression of both p66Shc, which was significantly higher at hour 6 in oxLDL exposed cells and then dropped to control level, and Bax, which significantly increased after 18 h in oxLDL-treated cells (Figures 3(c) and 3(d)).Figure 3
Protocatechuic acid counteracted oxLDL-induced activation of p53 and expression of its target genes p66Shc and Bax. Blotting analyses of (a) serine-phosphorylated p53 (1 h after oxLDL treatment) detected in anti-p53 immunoprecipitate and normalized to the nonphosphorylated protein. (b) p53 content determined in nuclear extract and normalized to lamin B protein, (c) p66Shc protein expression, and (d) Bax protein expression, normalized to cyclophilin protein. Values obtained in control cells (CTR), oxLDL-treated cells (oxLDL), and oxLDL-treated cells preincubated with 25μM PCA (PCA + oxLDL) were compared. Representative blots are shown. Results are expressed as mean ± S.E.M. (P∗<0.05 versus controls) of at least three independent experiments.
(a)
(b)
(c)
(d)


### 3.4. PCA Reverted p38 MAPK and PKCδ Activation Induced by oxLDL

Since the p53 activation depends on the activation of both p38MAPK and PKCδ [30], we investigated PCA effect on the kinase activation/phosphorylation. While macrophage treated with oxLDL exhibited an early (30 minutes after cell exposure) activation/phosphorylation of both the kinases, the presence of PCA completely reverted the kinase activation to control levels (Figures 4(a) and 4(b)).Figure 4
Protocatechuic acid prevented oxLDL-induced p38 and PKCδ activation. (a) phospho-p38 expression or (b) phospho-PKCδ expression (30′ after oxLDL treatment) in control cells (CTR), oxLDL-treated cells (oxLDL), and oxLDL-treated cells preincubated with 25 μM PCA (PCA + oxLDL). Results were normalized to the respective nonphosphorylated proteins. Each panel shows representative blots. Values are the mean ± SEM. P∗<0.05 versus controls of at least three independent experiments.
(a)
(b)


### 3.5. PCA Activated JNK/Nrf2 Pathway

PCA,per se, is able to potentiate the antioxidant cell system by activating JNK/Nrf2 pathway, which upregulates phase-2 detoxifying enzymes [37].Thus, we investigated whether this was the mechanism responsible for PCA-mediated inhibition of oxLDL-induced apoptosis. Blotting experiments demonstrated an early (15 min after treatment), transient increase in phosphorylated-JNK and a significant and prolonged increase in nuclear Nrf2 expression (within 30′ after treatment) in oxLDL-treated cells incubated with PCA with respect to oxLDL-treated cells (Figures5(a) and 5(b)). The increase of nuclear Nrf2 by PCA was inhibited when the cells were pretreated with SP600125, a specific inhibitor of JNK, strongly suggesting that the observed upregulation of Nrf2 was due to the activation of JNK (Figure 5(b)).Figure 5
Protocatechuic acid activated JNK/Nrf2 pathway in J774A.1 cells treated with oxLDL. (a) Phospho-JNK expression normalized to the nonphosphorylated form in oxLDL-treated cells or in oxLDL-treated cells incubated with PCA, with respect to controls. (b) Nrf2 content determined in nuclear extract and normalized to lamin B protein in oxLDL-treated cells incubated with PCA in the presence or absence of the JNK1/2 inhibitor with respect to oxLDL-treated cells. The inhibitor tested on control cells and oxLDL-treated cells did not show any effect (data not shown). Representative blots from at least three independent experiments are shown. Results are expressed as mean ± S.E.M.P∗<0.05 versus controls.
(a)
(b)To definitively assess the crucial role of JNK and Nrf2 in counteracting oxLDL-induced apoptosis by PCA, we inhibited the activation of Nrf2 also by silencing Nrf2 (siNrf2). Both SP600125 and siNrf2 treatments determined expression levels of phosphorylated-p53, Bax, and the active form of caspase-3 completely comparable to those obtained in cells treated with oxLDL only. These findings supported the hypothesis that the inhibitory effects triggered by PCA were due to the activation of JNK and the consequent increase in nuclear Nrf2 (Figures6(a), 6(b), and 6(c)).Figure 6
Protocatechuic acid activation of JNK and JNK-dependent activation of Nrf2 lead p53-dependent apoptosis inhibition. Blotting analyses of (a) serine-phosphorylated p53 (1 h after oxLDL treatment) detected in anti-p53 immunoprecipitate and normalized to the nonphosphorylated protein. (b) Bax protein expression (18 h after oxLDL treatment), normalized to cyclophilin protein. (c) caspase-3 active form expression (18 h after oxLDL treatment) normalized to cyclophilin protein. Values obtained in oxLDL-treated cells were compared with values obtained in (i) cells treated with oxLDL and PCA or (ii) cells treated with oxLDL and PCA in the presence of the JNK1/2 inhibitor SP600125 or cells treated with (iii) oxLDL and PCA transfected with anti-Nrf2 siRNA or (iv) the corresponding scrambled RNAs. The inhibitor tested on control cells and oxLDL-treated cells did not show any effect (data not shown). Representative blots from at least three independent experiments are shown. Results are expressed as mean ± S.E.M.;P∗<0.05 versus controls; P#<0.05 versus oxLDL-treated cells.
(a)
(b)
(c)


### 3.6. Effect of the JNK Inhibitor on ROS Production

The JNK/Nrf2 signalling pathway was responsible for the antiapoptotic effect of PCA. These results, together with those regarding the modulation of ROS production, suggested that PCA exerted its protective action mainly by maintaining cell redox balance rather than to directly inhibit cell macromolecule oxidation. To define the actual mechanism by which PCA impacted on the cellular redox status, we measured ROS in cells treated with oxLDL and PCA in the presence of the JNK inhibitor. We found that SP600125 completely hindered PCA protection against ROS production so that their levels were comparable to those found in cells treated with oxLDL only. Notably, JNK inhibitor mostly affected early ROS production, which was thus allowed to trigger the apoptotic signal (Figure7).Figure 7
Protocatechuic acid activation of JNK is responsible for the inhibition of ROS hyperproduction. Time course of intracellular ROS production in control cells (CTR), oxLDL exposed cells (oxLDL), oxLDL and PCA-treated cells (PCA + oxLDL), and cells treated with oxLDL and PCA in the presence of the JNK-inhibitor (JNK-inhibitor + PCA + oxLDL). The inhibitor tested on control cells and oxLDL-treated cells did not show any effect (data not shown). Values are the mean ± S.E.M. of three independent experiments.P∗<0.05 versus controls; P∗∗<0.001 versus controls; P∗#<0.05 versus oxLDL-treated cells and versus controls.


## 3.1. Cytotoxicity

PCA completely counteracted the cytostatic/cytotoxic effects induced by oxLDL in A.1 cells. OxLDL reduced cell growth showing a dramatic cytostatic effect: about 30% and 50% decrease of relative incorporation of [14C] thymidine after 18 and 24 h exposure, respectively, with respect to control cells. OxLDL exposure augmented the percentage of dead cells measured by counting trypan blue positive cells. PCA reversed oxLDL-induced cytotoxicity (Figures 1(a) and 1(b)).Figure 1
Protocatechuic acid completely protects macrophage cells from oxLDL-cytotoxic activity. J774A.1 cells were exposed to native LDL or oxLDL (0.2 mg/mL) in the presence or absence of 25μM protocatechuic acid. (a) Relative incorporation of [14C] thymidine. Each column represents the relative incorporation after 18 and 24 h treatment as compared with control values (100%). (b) Trypan blue staining. Percentages of dead cells (trypan blue positive cells) are reported at 6, 18, and 24 h. The data are expressed as mean ± S.E.M. of three independent experiments. P∗<0.001 versus controls.
(a)
(b)


## 3.2. Apoptosis and Oxidative Stress

To assess whether PCA was able to counteract oxLDL-induced apoptosis in macrophages, experiments were carried out to identify annexin V positive cells in (i) cells exposed to oxLDL, (ii) control cells, and (iii) cells exposed to oxLDL in the presence of PCA. Cells treated with oxLDL underwent apoptosis after 18 h exposure, while treatment with PCA prevented the death of the cells (Figure2(a)).Figure 2
Protocatechuic acid inhibited OxLDL-induced apoptosis and the onset of oxidative stress in macrophage cells. Time-course experiments. (a) Percentage of apoptotic annexin-V positive cells, (b) intracellular ROS production, and (c) intracellular reduced glutathione (GSH) content in J774A.1 cells. Values are the mean ± S.E.M. of four independent experiments. Values obtained in control cells (CTR), oxLDL-treated cells (oxLDL), and oxLDL-treated cells preincubated with 25μM PCA (PCA + oxLDL) were compared. P∗<0.05 versus controls; P∗∗<0.001 versus controls. P∗#<0.05 versus oxLDL-treated cells and versus controls, for each time point.
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(c)Since the first toxic effect of oxLDL was an early and progressive increase in ROS production followed by a dramatic decrease in GSH content in macrophage-like cells [30], the effect of PCA on cellular oxidative stress was evaluated. The early increase in ROS production was completely abrogated in cells treated with PCA until hour 6 (Figure 2(b)). ROS production increased in PCA-treated cultures only after 6 h of oxLDL-exposure, and, although significantly higher with respect to control cultures, it was significantly lower than in oxLDL-treated cells. Notably, PCA treatment completely counteracted GSH depletion induced by oxLDL (Figure 2(c)), clearly indicating that it was able to prevent the onset of oxidative stress induced by oxLDL.


## 3.3. PCA Inhibited p53 Apoptotic Signal

Western blotting analyses were performed to elucidate if the antiapoptotic activity of PCA depended on the ability to modulate p53 expression and activation induced by oxLDL. For this purpose phosphorylated and nuclear p53 and protein expression of two of the main p53 target genes, p66Shc and BAX, both responsible for the execution of p53-mediated apoptosis, were evaluated in oxLDL-treated cells in the presence of PCA. The obtained results demonstrated that PCA was able to counteract all the changes induced by oxLDL in p53 pathway. In particular, PCA prevented (i) the significant overexpression of p53 protein observed throughout the time period considered (data not shown), (ii) the early (1 h) significant increase of the phosphorylated form, and (iii) the increase in p53 nuclear translocation that occurred after 3 h of oxLDL exposure (Figures3(a) and 3(b)). Moreover, PCA completely counteracted the overexpression of both p66Shc, which was significantly higher at hour 6 in oxLDL exposed cells and then dropped to control level, and Bax, which significantly increased after 18 h in oxLDL-treated cells (Figures 3(c) and 3(d)).Figure 3
Protocatechuic acid counteracted oxLDL-induced activation of p53 and expression of its target genes p66Shc and Bax. Blotting analyses of (a) serine-phosphorylated p53 (1 h after oxLDL treatment) detected in anti-p53 immunoprecipitate and normalized to the nonphosphorylated protein. (b) p53 content determined in nuclear extract and normalized to lamin B protein, (c) p66Shc protein expression, and (d) Bax protein expression, normalized to cyclophilin protein. Values obtained in control cells (CTR), oxLDL-treated cells (oxLDL), and oxLDL-treated cells preincubated with 25μM PCA (PCA + oxLDL) were compared. Representative blots are shown. Results are expressed as mean ± S.E.M. (P∗<0.05 versus controls) of at least three independent experiments.
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## 3.4. PCA Reverted p38 MAPK and PKCδ Activation Induced by oxLDL

Since the p53 activation depends on the activation of both p38MAPK and PKCδ [30], we investigated PCA effect on the kinase activation/phosphorylation. While macrophage treated with oxLDL exhibited an early (30 minutes after cell exposure) activation/phosphorylation of both the kinases, the presence of PCA completely reverted the kinase activation to control levels (Figures 4(a) and 4(b)).Figure 4
Protocatechuic acid prevented oxLDL-induced p38 and PKCδ activation. (a) phospho-p38 expression or (b) phospho-PKCδ expression (30′ after oxLDL treatment) in control cells (CTR), oxLDL-treated cells (oxLDL), and oxLDL-treated cells preincubated with 25 μM PCA (PCA + oxLDL). Results were normalized to the respective nonphosphorylated proteins. Each panel shows representative blots. Values are the mean ± SEM. P∗<0.05 versus controls of at least three independent experiments.
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## 3.5. PCA Activated JNK/Nrf2 Pathway

PCA,per se, is able to potentiate the antioxidant cell system by activating JNK/Nrf2 pathway, which upregulates phase-2 detoxifying enzymes [37].Thus, we investigated whether this was the mechanism responsible for PCA-mediated inhibition of oxLDL-induced apoptosis. Blotting experiments demonstrated an early (15 min after treatment), transient increase in phosphorylated-JNK and a significant and prolonged increase in nuclear Nrf2 expression (within 30′ after treatment) in oxLDL-treated cells incubated with PCA with respect to oxLDL-treated cells (Figures5(a) and 5(b)). The increase of nuclear Nrf2 by PCA was inhibited when the cells were pretreated with SP600125, a specific inhibitor of JNK, strongly suggesting that the observed upregulation of Nrf2 was due to the activation of JNK (Figure 5(b)).Figure 5
Protocatechuic acid activated JNK/Nrf2 pathway in J774A.1 cells treated with oxLDL. (a) Phospho-JNK expression normalized to the nonphosphorylated form in oxLDL-treated cells or in oxLDL-treated cells incubated with PCA, with respect to controls. (b) Nrf2 content determined in nuclear extract and normalized to lamin B protein in oxLDL-treated cells incubated with PCA in the presence or absence of the JNK1/2 inhibitor with respect to oxLDL-treated cells. The inhibitor tested on control cells and oxLDL-treated cells did not show any effect (data not shown). Representative blots from at least three independent experiments are shown. Results are expressed as mean ± S.E.M.P∗<0.05 versus controls.
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(b)To definitively assess the crucial role of JNK and Nrf2 in counteracting oxLDL-induced apoptosis by PCA, we inhibited the activation of Nrf2 also by silencing Nrf2 (siNrf2). Both SP600125 and siNrf2 treatments determined expression levels of phosphorylated-p53, Bax, and the active form of caspase-3 completely comparable to those obtained in cells treated with oxLDL only. These findings supported the hypothesis that the inhibitory effects triggered by PCA were due to the activation of JNK and the consequent increase in nuclear Nrf2 (Figures6(a), 6(b), and 6(c)).Figure 6
Protocatechuic acid activation of JNK and JNK-dependent activation of Nrf2 lead p53-dependent apoptosis inhibition. Blotting analyses of (a) serine-phosphorylated p53 (1 h after oxLDL treatment) detected in anti-p53 immunoprecipitate and normalized to the nonphosphorylated protein. (b) Bax protein expression (18 h after oxLDL treatment), normalized to cyclophilin protein. (c) caspase-3 active form expression (18 h after oxLDL treatment) normalized to cyclophilin protein. Values obtained in oxLDL-treated cells were compared with values obtained in (i) cells treated with oxLDL and PCA or (ii) cells treated with oxLDL and PCA in the presence of the JNK1/2 inhibitor SP600125 or cells treated with (iii) oxLDL and PCA transfected with anti-Nrf2 siRNA or (iv) the corresponding scrambled RNAs. The inhibitor tested on control cells and oxLDL-treated cells did not show any effect (data not shown). Representative blots from at least three independent experiments are shown. Results are expressed as mean ± S.E.M.;P∗<0.05 versus controls; P#<0.05 versus oxLDL-treated cells.
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## 3.6. Effect of the JNK Inhibitor on ROS Production

The JNK/Nrf2 signalling pathway was responsible for the antiapoptotic effect of PCA. These results, together with those regarding the modulation of ROS production, suggested that PCA exerted its protective action mainly by maintaining cell redox balance rather than to directly inhibit cell macromolecule oxidation. To define the actual mechanism by which PCA impacted on the cellular redox status, we measured ROS in cells treated with oxLDL and PCA in the presence of the JNK inhibitor. We found that SP600125 completely hindered PCA protection against ROS production so that their levels were comparable to those found in cells treated with oxLDL only. Notably, JNK inhibitor mostly affected early ROS production, which was thus allowed to trigger the apoptotic signal (Figure7).Figure 7
Protocatechuic acid activation of JNK is responsible for the inhibition of ROS hyperproduction. Time course of intracellular ROS production in control cells (CTR), oxLDL exposed cells (oxLDL), oxLDL and PCA-treated cells (PCA + oxLDL), and cells treated with oxLDL and PCA in the presence of the JNK-inhibitor (JNK-inhibitor + PCA + oxLDL). The inhibitor tested on control cells and oxLDL-treated cells did not show any effect (data not shown). Values are the mean ± S.E.M. of three independent experiments.P∗<0.05 versus controls; P∗∗<0.001 versus controls; P∗#<0.05 versus oxLDL-treated cells and versus controls.


## 4. Discussion

In this study we demonstrated the ability of PCA to counteract the cytotoxic activity of oxLDL in murine macrophages. In particular, the treatment of the cells with PCA inhibited apoptosis not only by counteracting oxidative stress occurrence but also through the modulation of intracellular signalling pathways responsible for caspase activation. This can have great relevance to atherosclerosis development since the occurrence of apoptosis, which mainly concerns macrophages in advanced vessel lesions, is a crucial event in the progression of the disease.Modulation of apoptosis is one of the main mechanisms responsible for the protective action of polyphenols against degenerative diseases [8, 38, 39]. PCA exerts antiapoptotic activity by acting as antioxidant and by modulating a number of pro- and antiapoptotic factors [40, 41]. However, PCA is also able to induce apoptosis and its proapoptotic activity appears specifically directed towards transformed cells [42, 43].In the first place, this study demonstrated that the treatment with PCA of cells exposed to oxLDL prevented the decrease of cellular GSH, even if in the presence of ROS overproduction. In addition, it is worth noting that the biphasic increase of ROS found in oxLDL-exposed cells did not occur when the cells were treated with PCA. The inhibition of early ROS production and GSH saving by PCA treatment seem to be the key events responsible for the protective effect of PCA against oxLDL-induced apoptosis. Thus, PCA might act directly on the main factors that control the different stages of activation, induction, and execution of programmed cell death induced by oxLDL. Apoptosis induced by oxLDL in J774A.1 macrophages occurs mainly by activating p53 that regulates, positively or negatively, the transcription of tens of effector genes coding for proteins such as Bax, a proapoptotic member of the Bcl-2 protein family, and p66Shc. The latter is the oxidative stress-sensor that acts as a cytochrome c oxidoreductase and has been considered the main signal leading to irreversible cell apoptosis, because it triggers a further irreparable mitochondrial ROS hyperproduction [44, 45]. The presence of PCA inhibited p53 phosphorylation and, consequently, the expression of two of its target genes BAX and p66SHC.The activation of p53 in cells treated with oxLDL appears to be controlled by ROS hyperproduction and by the activation/phosphorylation of p38MAPK and PKCδ both necessary for oxLDL-induced proapoptotic signal [30].In addition to reduce ROS production, we showed that PCA completely decreased the cellular content of phosphorylated p38MAPK and PKCδ to the levels observed in control cells, thus blocking the entire signalling pathway responsible for apoptosis induced by oxLDL.Furthermore, since GSH concentration in PCA-treated cells exposed to oxLDL remained as high as in the controls, notwithstanding the presence of significantly increased ROS, we hypothesized that PCA might strengthen the endogenous antioxidant defences of the cells.In this regard, we have demonstrated that PCA,per se, like other polyphenols [37], can induce the ARE-dependent antioxidant/detoxifying phase II enzymes such as GR and GPx by activating the transcription factor Nrf2 through JNK-mediated phosphorylation [45]. Herein, in the presence of oxLDL-induced oxidative stress, PCA determined a significant increase of nuclear Nrf2. This most likely depended on JNK activation since it was abolished when the specific JNK1/2 inhibitor was added to the cell culture. This finding is also consistent with our previous data obtained by silencing JNK in. A.1 incubated with PCA alone [37].It is worth noting that the early (15 min) and transient activation of JNK by PCA seems to play a prominent role in PCA-promoted cell survival. In fact, when JNK was inhibited by SP600125 in oxLDL-treated cells, the ROS overproduction, the activation of p53, the expression of BAX, and the active form of caspase-3 took place even in the presence of PCA. The antiapoptotic effects of PCA were also hindered by transfecting the cells with anti-Nrf2 siRNA. Taken together, these results strongly suggest that JNK/Nrf2 pathway played a major role in counteracting the apoptotic cell death induced by oxLDL in macrophages and add new evidence of the dual role, proapoptotic or antiapoptotic, of JNK signalling pathway.To the best of our knowledge, this is the first demonstration of PCA-dependent activation of antiapoptotic cell survival signals via the JNK/Nrf2 pathway in macrophages. Furthermore, only few studies have shown that JNK may be responsible for cell survival, proliferation, and differentiation [46]. In fact, the bulk of the studies have provided evidence of a functional role of JNK signalling pathway in promoting proapoptotic responses to stress. The different cellular context as well as the time at which activation takes place, the type of stimulation (transient and prolonged in time), and the ability of JNK to interact with other procell survival signalling pathways has been indicated as responsible for the different activity of JNK [47–49]. In our study, JNK activation might act as a temporary, limited, intracellular signal capable of regulating the cellular antistress and antiapoptotic responses induced by PCA.In conclusion, our study gives convincing evidence of the essential role of JNK/Nrf2 signalling pathway in the antiapoptotic activity exerted by PCA in oxidatively stressed macrophages by improving the endogenous cellular antioxidant system.PCA deserves great nutritional interest as the main human metabolite of cyanidin 3-glucoside (C3G), which is in turn the most representative dietary anthocyanins (ACN). Actually, few studies have specifically investigated ACN and phenolic acid bioavailability in humans [16, 50, 51]. PCA has been demonstrated to account for almost 73% of ingested C3G [16]. After ingestion of 1 L of blood orange juice containing 71 mg C3G, the serum maximal concentrations of C3G and PCA were 1.9 nmol/L and 492 nmol/L, respectively [16]. However, it should be considered that the bioavailability can be affected by “chronic” exposure to polyphenols, as the daily consumption of ACN-rich food can provide. Furthermore, the polyphenols might concentrate in the tissue microenvironment [37]. From this point of view, the concentration of PCA tested in our experiments, although higher than that reported after ingestion of food rich in ACN, can be reached plausibly at cellular level. Thus, although the physiologicalin vivo context in which dietary polyphenols exert their influence is undoubtedly much more complex than that available from anin vitro system, it could be argued that micromolar concentrations of PCA, which are comparable to those achievedin vivo following a Mediterranean type diet, can help fight cell damage, associated with the onset and progression of atherosclerosis.The new knowledge achieved on the molecular mechanism that allows PCA to exert protective effects against oxidative injury might represent a useful basis for furtherin vivo studies necessary to design novel diet-based preventive and therapeutic strategies aimed at counteracting oxidative stress-induced pathologies.



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*Source: 351827-2015-06-09.xml*