ROS are metabolites from molecular oxygen as a result of normal cellular metabolism, and are essential signaling molecules that regulate various physiological processes, such as activation of transcription factors, kinase phosphorylation, host defense genes, and mobilization of ion transport systems (Touyz and Schiffrin, 2004; Schramm et al., 2012). However, the overproduction of ROS causes adverse modifications in macromolecules, and the development of several diseases (Checa and Aran, 2020). The imbalance between oxidants and antioxidants in favor of oxidants is called oxidative stress (Birben et al., 2012). Oxidative stress contributes for many pathological conditions, such as hypertension (Rodrigo et al., 2011). Hypertension is an important risk factor for the development of cardiovascular diseases (Fuchs and Whelton, 2020), which are the leading cause of deaths worldwide (GBD 2013 Mortality and Causes of Death Collaborators, 2015).

In the vascular system, ROS regulates endothelial function and vascular tone under physiological conditions, but in pathological situations, ROS play an important role in angiogenesis and rarefaction, inflammation, hypertrophy, proliferation, migration, fibrosis, vascular remodeling, and endothelial dysfunction associated with hypertension (Touyz and Briones, 2011). During hypertension, in addition to the multifactorial processes that lead to blood pressure elevation (interaction among multiple organ systems, many genes, environmental stimuli, activation of the renin-angiotensin system, inflammation, and others) (Touyz and Briones, 2011), oxidative stress contributes to excessive ROS production, decreased NO^• bioavailability and decreased antioxidant capacity (Checa and Aran, 2020).

There are two ways for the elimination of ROS. The first is their scavenging by the stimulation of endogenous antioxidant systems or through the administration of antioxidants. The second is more complex and involves the inhibition of enzymes that catalyze the production of ROS (Schramm et al., 2012). Nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidases (NOX) are the major source of ROS in the vascular tissues (Sedeek et al., 2009; Dikalov et al., 2014). NOX family comprises seven isoforms, each based on a distinct core catalytic subunit, and all of them use NADPH as an electron donor and catalyze electrons transfer to molecular oxygen to generate superoxide anion (O₂ ^•-) (Sirker et al., 2011).

Several approaches to overcome oxidative stress in the vascular wall have been studied as a possible treatment for hypertension (Schramm et al., 2012), and the compounds apocynin and protocatechuic acid are potential candidates.

Apocynin is an acetophenone initially extracted from the root extracts of the medicinal herb Picrorhiza kurroa in the Himalayan Mountains (Petrônio et al., 2013). Our group has studied the effects of apocynin in normotensive and hypertensive rats. In aortas from Wistar rats, apocynin promoted a long-lasting hypotensive effect, and it was more potent inducing endothelium-dependent and independent relaxation than its active dimer, diapocynin (Potje et al., 2017). Furthermore, in SHR, chronic treatment with apocynin reduced blood pressure and oxidative stress biomarkers, reversed endothelial dysfunction (Perassa et al., 2016), increased NO^• production and reduced ROS generation in blood vessels (Graton et al., 2019). Apocynin has a controversial mechanism of action. Some studies suggest that its effects depend only on its antioxidant properties (Heumüller et al., 2008), whereas other studies suggest its ability to inhibit NOX in phagocytic (Simons et al., 1990) and cardiovascular cells (El-Sawalhi and Ahmed, 2014). Protocatechuic acid is a naturally occurring phenolic acid, widely distributed and present in most edible plants used in folk medicine, being one of the biologically active components of some medicinal plants, fruits and honey (Kakkar and Bais, 2014). There is evidence that it reduces the risk of cardiovascular diseases due to its anti-inflammatory, antioxidant and free radical scavenger properties (Semaming et al., 2015).

The aim of this study was to compare the antioxidant and/or NOX inhibitory properties of apocynin with those of protocatechuic acid, and their potential effect in vascular dysfunction in SHR.

The Animal Research Ethics Committee of the School of Dentistry of Araçatuba, UNESP, approved all the procedures used in this study (Process CEUA FOA 2016-00130).

The main findings of this study are that apocynin had a weak scavenger effect and reduced antioxidant capacity in vitro when compared to protocatechuic acid, but in vascular cells, the effects of apocynin are greater than or equal to the effects of protocatechuic acid. Apocynin and protocatechuic acid showed free radical scavenging capacity and an effective inhibitory effect on ROS production. Furthermore, NO^• and ROS concentration were modulated by both compounds in isolated endothelial cells, and phenylephrine and acetylcholine vasomotor effects were altered by these compounds.

The reducing ability of a compound may be a significant indicator of its potential antioxidant activity (Kilic et al., 2014). Only higher concentrations (100 μmol/L and 1 mmol/L) of the apocynin and protocatechuic acid compounds exhibited antioxidant capacity. In addition, at the concentration of 1 mmol/L protocatechuic acid had higher antioxidant capacity than apocynin (Figure 2). Antioxidants are defined as molecules with the ability to donate electrons to eliminate the unpaired condition of the radical and neutralize the free radicals. These molecules can directly react with ROS creating less harmful compounds or can regulate the (dys)function of ROS generating enzymes, by inhibiting their activity or expression (Lü et al., 2010). NOX enzymes are the major source of vascular superoxide anion in the organism (Dikalov et al., 2014) and recently we showed that in vivo treatment with apocynin decreased the expression of NOX2 in aortas and increased plasma antioxidant capacity in SHR (Perassa et al., 2016; Graton et al., 2019). Protocatechuic acid showed a concentration-dependent antioxidant capacity (Figure 2), as observed previously (Li et al., 2011), and its pharmacological actions were closely correlated with its antioxidant activities (Kakkar and Bais, 2014). Based on these results, we chose the concentration (100 μmol/L) of the both compounds for the subsequent experiments of TBARS, lucigenin chemiluminescence, cell culture and vascular reactivity.

Considering the importance of discriminating between the direct free radical scavenging capacity and the inhibition of NOX activity, the antioxidant potency of the studied compounds was evaluated by three different additional assays. The first was the oxygen radical absorbance capacity (ORAC) assay, which is based on the efficacy of the studied compounds to scavenge peroxyl free radicals (ROO^•) generated by thermolysis of AAPH at 37°C (Ou et al., 2001). The second method for evaluation of antiradical efficacy was the CAT degradation assay. This method is based on the measurement of the capacity of the studied compounds act as inhibitor of the degradation of the unsaturated fatty acid, eleostearic acid, provoked by ROO^• (Laguerre et al., 2008). The third method for evaluation of the antioxidant efficacy was the DPPH method, which is based on the reduction of this stable free radical compound. Figure 3 shows the kinetic profile of fluorescein degradation, triene degradation and antioxidant capacity of the compounds. Each of the compounds were a better scavenger of ROO^• compared to Trolox, a water-soluble derivative of vitamin E and protocatechuic acid was more efficient scavenger than apocynin (Table 1). In addition, the inhibitory efficacy of protocatechuic acid was also superior that of apocynin (Table 1). As we can see, when comparing the effects of both compounds by the different methods used, apocynin proved to be a weaker antioxidant than protocatechuic acid.

TBARS is a well-established method for measuring lipid peroxidation, an indicator of oxidative stress (Yagi, 1998) and results are expressed by MDA levels. We did not observe alterations of MDA levels in the Wistar rats aortas incubated with apocynin and protocatechuic acid (Figure 4A). Other studies have also reported no differences following treatment with antioxidants in normotensive rats, presumably due to the absence of oxidative stress in these animals (Tain et al., 2012; Perassa et al., 2016). On the other hand, in aortas from SHR, a model with vascular oxidative stress (Suzuki et al., 1995), apocynin and protocatechuic acid were able to reduce MDA levels (Figure 4B), confirming that these compounds present antioxidant capacity, which can be defined as the ability of a substance to inhibit oxidative degradation, such as lipid peroxidation in vascular cells. The reduction of lipid peroxidation appears to be associated with the antioxidant capacity of these compounds, since other antioxidants, such as ascorbic acid and α-tocopherol, were also able to decrease this marker of oxidative stress (Frei, 1991; Motoyama et al., 1998). It is important to note that although apocynin has a lower antioxidant effect than protocatechuic acid, its effect in reducing MDA levels was similar to that of protocatechuic acid.

Lucigenin is a valid probe for superoxide production detection by enzymatic and cellular sources (Li et al., 1998). Apocynin and protocatechuic acid were able to decrease the lucigenin signal in vascular cells in the aortas from normotensive Wistar rats and SHR (Figure 5), suggesting that both compounds reduces. superoxide production in cells with absence or presence of oxidative stress. From these results, along with the previous ones (Figure 3), we can predict that the compounds here studied showed antioxidants characteristics, due to the ability to scavenging ROS, but also to inhibit their formation.

Among ROS, O₂ ^•- has a critical importance in the blood vessel due to its rapid reaction with NO^• leading to peroxynitrite formation and marked alteration of vascular tone promoting vasoconstriction and altering vasodilatation (Münzel et al., 2002). DHE is an additional probe for the detection of intracellular O₂ ^•- levels (Chen et al., 2013). To minimize the detection of the nonspecific DHE oxidation product ethidium (E+), we used settings of the excitation (405 nm) and emission (570 nm) to optimize the measurement of the O₂ ^•- specific product 2-hydroxyethidium (2-OH-E+) (Nazarewicz et al., 2013). Previously it was shown that another phenolic compound known as triazolo pyrimidine VAS3947, the specific NOX inhibitor, was able to inhibit DHE-detectable ROS in SHR aortas (Wind et al., 2010). In addition, apocynin treatment reduced DHE-detectable ROS in fresh aortic endothelial cells of SHR (Perassa et al., 2016). Furthermore, apocynin was able to decrease ROS production in HUVEC due reduced expression and translocation of p47phox (Oliveira et al., 2018). Corroborating these previous studies, we showed that apocynin and protocatechuic acid were able to reduce the fluorescence intensity of DHE in HUVEC.

We next used the membrane-permeable DAF-2 DA probe for detection of intracellular NO^• levels. DAF-2 DA is hydrolyzed to DAF-2 and trapped inside the cell after reacting with NO^• (Kojima et al., 1998; Zhou and He, 2011). Apocynin and protocatechuic acid increased the fluorescence intensity of DAF-2DA in endothelial cells when compared to control group (Figure 7). Han and others (2019) observed similar results which protocatechuic acid was also able to increase NO^• generation in HUVEC. NO^• is an important factor that regulates endothelial function, including prevention of platelet adhesion and aggregation, limitation of cholesterol oxidation, vascular smooth muscle cell proliferation and promotes vasodilation (Tousoulis et al., 2012).

In the next step, we analyzed the effects of apocynin and protocatechuic acid on vascular reactivity. Our results demonstrated that apocynin decreased the Emax and potency of phenylephrine in Wistar and SHR aortic rings. In the pathology of hypertension, one of the characteristics is sympathetic hyperactivity (Julius and Nesbitt, 1996). In that sense, an increase in the concentration of circulating noradrenaline and adrenaline was demonstrated in hypertensive animals and patients (Julius and Nesbitt, 1996; Mancia et al., 1999). Phenylephrine is a sympathomimetic drug, therefore, evaluating whether apocynin and protocatechuic acid have the ability to modulate the contraction induced by phenylephrine is an indication that these compounds can be used in pathologies such as hypertension. As observed in our results, apocynin reduced the Emax and potency induced by phenylephrine in aortic rings of normotensive and hypertensive rats. On the other hand, protocatechuic acid reduced the potency of phenylephrine only in the aortic rings of normotensive rats and had no effect on the vessels of hypertensive rats.

It has been reported that phenylephrine-induced contraction activates superoxide anion (Tsai and Jiang, 2010; Silva et al., 2013) and hydrogen peroxide (Silva et al., 2013) production in arteries of rats. It was demonstrated previously by our group that apocynin and its active dimer, diapocynin, promoted hyporeactivity to phenylephrine (Potje et al., 2017), associated with decreased ROS generation in aortic rings of Wistar rats. In addition, the aortic rings from normotensive rats incubated with superoxide scavenger (Tiron) or aortic rings from renal hypertensive rats incubated with peg-catalase, which degrades hydrogen peroxide (H₂O₂), showed reduced contractile effect stimulated by phenylephrine (Silva et al., 2013). Moreover, apocynin or VAS2870 (NOX inhibitor) attenuated the contractile effect induced by phenylephrine in rat-tail artery strips. The authors showed that ROS from NADPH oxidase and mitochondria, changed myosin phosphatase-mediated MLC20 phosphorylation through both RhoA/Rho kinase- and CPI-17-dependent pathways, thus regulating α1-adrenoceptor-activated smooth muscle contraction (Tsai and Jiang, 2010). Based on these results, we can conclude that apocynin reduced phenylephrine-induced concentration-dependent contraction in aortic rings of normotensive and hypertensive rats associated with ROS scavenger. However, protocatechuic acid had no anti-contractile effect on the phenylephrine-stimulated concentration-dependent curves even being a more potent ROS scavenger than apocynin as shown by the ORAC assay. More experiments are needed to elucidate the underlying mechanisms of protocatechuic acid on vascular smooth muscle cells.

Endothelial dysfunction plays a central role in the pathogenesis of hypertension (Konukoglu and Uzun, 2017), being characterized by impaired bioavailability/production of endothelium-derived relaxing factors and associated with oxidative stress (Dharmashankar and Widlansky, 2010; Flammer et al., 2012). In addition, endothelial dysfunction favors the thrombotic and inflammatory process in blood vessels. SHR aorta have impaired acetylcholine-induced relaxation due to endothelial dysfunction (Potje et al., 2019). As observed in this present study, acetylcholine promoted relaxation in aortic rings from Wistar rats and SHR, but we can clearly see the impairment of potency induced by acetylcholine in aortic rings of SHR, confirming endothelial dysfunction. Subsequently, we observed that apocynin and protocatechuic acid increased the potency of acetylcholine only in aortic rings of hypertensive rats, in other words, these compounds were efficient in improving endothelial function. In aortic rings from normotensive rats, which do not show endothelial dysfunction, both compounds did not alter acetylcholine-induced relaxation.

It was reported that the chronic treatment with apocynin prevented hypertension in fructose fed rat models (Unger and Patil, 2009). Later, our group also reported that the chronic treatment with apocynin prevented development of hypertension in SHR (Perassa et al., 2016). Moreover, in the aortic rings of fructose-fed rats, apocynin increased the Emax of the acetylcholine curves, but the Emax values were not similar to the control curve, indicating that apocynin improved endothelial function, but did not recover the basal endothelial function (Unger and Patil, 2009). However, in our previously study, the chronic treatment with apocynin shifted the acetylcholine curve to the left in aortas of SHR and these curves were similar to the acetylcholine-curves constructed in the aorta of normotensive rats, confirming that apocynin recovered endothelial function in vessels of SHR (Perassa et al., 2016). Therefore, it is clearly shown that apocynin has a physiological effect on the smooth muscle vascular cell.

Different groups have shown different effects of protocatechuic acid. The chronic treatment (20 weeks) with protocatechuic acid in the apolipoprotein E (ApoE)-deficient mouse model was able to decrease the aortic vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression and plasma-soluble VCAM-1 and ICAM-1 levels, inhibiting atherosclerosis development (Wang et al., 2010). In addition, the HUVECs treated with protocatechuic acid (100 μmol/L) showed a reduction of two biomarkers of oxidative stress, 3-nitrotyrosine (3-NT) and 8-hydroxydeoxyguanosine (8-OHdG), and the nitric oxide levels were increased (Han et al., 2019). In our present study, we show that protocatechuic acid has a physiological effect, increasing the relaxation induced by acetylcholine in aortic rings of SHR, but the pathway mechanism of action needs to be investigated.

Therefore, O₂ ^•- scavenging appears to be useful in the restoring of endothelium-dependent dilatation as demonstrated in SHR blood vessels (Figure 9B). In addition, even though SHR showed increased ROS production, both compounds were able to oxidize the ROS and decrease the lucigenin chemiluminescence. Our findings are in accordance with previous results showing the beneficial effects of antioxidants on the biological activity of NO^• and endothelium-dependent relaxation (Carr and Frei, 2000). As endothelial dysfunction is an independent predictor of cardiovascular risk (Schiffrin, 2008), these data provide new information on the benefits of using drugs that act as antioxidants or inhibit the production of ROS, particularly in disorders with marked endothelial dysfunction. Taken together, the compounds tested in this study should be considered in the treatment of these vascular diseases, mainly apocynin, which seems to present a higher efficacious therapeutic in cases of endothelial dysfunction as observed in this study and reported by other groups.

In vitro assays, protocatechuic acid showed greater ability to act as a ROS scavenger and to have a greater antioxidant capacity. However, when we tested the compounds in isolated blood vessels, apocynin showed a more effective pharmacological function, since it reduced the contraction induced by phenylephrine with greater magnitude in aortas of normotensive and hypertensive vessels and it also improved the endothelium-dependent relaxation in SHR, a model of endothelial dysfunction.

Our study reinforces the importance of using substances that combine the ability to scavenge and/or prevent the ROS generation, and the reestablishment of NO^• bioactivity. Based on the data presented here apocynin and protocatechuic acid are candidates for future therapeutic approaches of cardiovascular pathological conditions where oxidative stress is a key feature. Future studies involving the in vivo treatment should be performed to evaluate other potential effects of these drugs, as well, for the better description and elucidation of mechanism of action of these drugs, in order to check potential improvements on the cardiovascular function, mainly in hypertension.