Viburnum opulus fruit extract-capped gold nanoparticles attenuated oxidative stress and acute inflammation in carrageenan-induced paw edema model

ABSTRACT This study designed, characterized, and investigated the antioxidant and anti-inflammatory effects of gold nanoparticles coated with Viburnum opulus extract (AuNP-VO) compared to those of Viburnum opulus fruit extract (VO) and indomethacin, on experimental inflammation in Wistar rats. The structure and properties of the synthesized AuNPs-VO were evaluated by UV-Vis spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurements. Their biological effects on paw inflammation were assessed at 2, 24, and 48 h after the administration of plantar carageenan by quantifying oxidative stress and inflammation and by histopathological analysis. TEM micrographs confirmed the spherical shape, homogeneous distribution, and nano-size of 65 nm. AuNP-VO and VO showed antioxidant and anti-inflammatory effects on plantar inflammation with different dynamics of action. AuNP-VO exerted antioxidant effects, especially, at 48 h and diminished the plantar inflammation in the early stages (IL-1α, IL-1β, IL-6, TNF-α and IL-10) and late stages (IL-1β, p < 0.01) whereas VO reduced the inflammatory response, predominantly in the early stages (p < 0.01; p < 0.001), and maintained reduced IL-1β values (p < 0.001) and a lower histopathological inflammatory score at 48 h. In conclusion, AuNP-VO and VO extract can be used as valuable agents for the treatment of diseases associated with inflammation. GRAPHICAL ABSTRACT


Introduction
Inflammation is a complex biological phenomenon that plays a protective role in maintaining the integrity of the body against physical, chemical, or infectious agents. Numerous studies have shown that inflammation has been involved in the pathogenesis of different diseases (1) such as: diabetes (2), obstructive pulmonary disease (3), asthma (4), arthritis (5), infectious diseases (6) and malignant tumors (7).
In inflammation, polymorphonuclear neutrophils (PMNs) play an important role because of their migration in inflamed tissues, attraction to chemokines, and due to their ability to eliminate pathogens by degranulation, phagocytosis, neutrophil extracellular traps, and cytokine release (8). Endogenous reactive oxygen species (ROS), released by NADPH-oxidase activity regulate pathways dependent on tyrosine phosphorylation, control phagocytosis, and modulate the expression of cytokines and chemokines involved in the inflammatory response (9).
The key mediator of inflammation is the nuclear transcription factor (NF)-κB. This factor, after activation by oxidizing factors, lipid mediators, bacterial products, cytokines, and viral proteins, amplifies the inflammatory response by overregulating inflammatory mediators including cyclooxygenase-2 (COX-2), inflammatory cytokines, and inducible nitric oxide synthase (iNOS).
Proinflammatory cytokines are immunoregulatory mediators secreted by immune cells (macrophages and monocytes) and non-immune cells (fibroblasts and endothelial cells) in response to various stimuli (10). TNF-α and IL-1β stimulate PGE 2 formation after COX-2 activation. In addition, TNF-α triggers iNOS expression, increases neutrophil migration (11) and initiates nitric oxide (NO) synthesis. The constitutive isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS), produce small amounts of NO, which acts as a neurotransmitter and vasodilator. Inducible isoform (iNOS) generates large amounts of NO, especially during inflammatory reactions (12)(13)(14). At high concentrations, NO reacts with superoxide anions to form peroxynitrite, which is responsible for oxidative stress and inflammatory cellular changes (15). To counteract the inflammation, the anti-inflammatory cytokines such as IL-10, IL-11, IL-13, IL-4 and IL-5 are secreted (16,17).
Conventional therapies used in inflammation are mainly non-steroidal anti-inflammatory drugs, which are very effective agents but have many side effects. Because oxidative mechanisms are at the origin of inflammation, it has been suggested that antioxidants have anti-inflammatory effects and no side effects. In this context, natural compounds from plants and fruits have been increasingly used in medical practice as adjuvants for inflammatory conditions.
The rapid development of nanotechnology in recent years has made nanoparticles ubiquitous. There is a wide range of metal nanoparticles, made of various materials, having different shapes and sizes (23,24,25,26). Gold nanoparticles are frequently used in medicine for diagnostic or therapeutic purposes, for the administration of drugs or as new therapeutic agents, in the treatment of some forms of cancer: lung cancer (27,28), colorectal cancer (27,29), breast cancer (30,31), cervical cancer (32,33), leukaemia (34).
Nanoparticles reach the body in different ways: oral, injectable, inhaler or transdermal. Their absorption and solubility depend on their size, surface charge, concentration, and pH (35). They enter the circulatory system and interact with blood plasma proteins, which form a protein crown around nanoparticles. Immunoglobulins G, M, and fibrinogen have been detected in almost all nanoparticles (36).
AuNPs have been shown to selectively inhibit the production of IL-1β, IL-17, and TNF-α, decrease IL-12, and alter the cellular immune response from TH1 (proinflammatory) to TH2 (anti-inflammatory). Several studies have demonstrated that IL-1β aggregates around AuNPs, thus decreasing the number of available IL-1β molecules that interact with the interleukin cellular receptor and altering its biological activity (38,39).
Based on these data, the present study evaluated the antioxidant and anti-inflammatory effects of Viburnum opulus extract-capped gold nanoparticles (AuNP-VO) in a model of experimental carrageenan-induced plantar inflammation in Wistar rats. The effects were assessed by oxidative stress and inflammation markers, and by histopathological analysis of inflamed plantar tissue.

Preparation and characterization of fruit extract
Viburnum opulus (VO) fruits were harvested in August 2019, packed in polyethylene bags, and frozen until use. The concentrated fruit extract was obtained as follows: 100 g of fruit puree was stirred for one hour at room temperature with 300 mL of acetone, the mixture was filtered, and the acetone was completely removed under vacuum. The resulting concentrated fruit extract was further used for the in vivo antioxidant and anti-inflammatory evaluation of VO fruits. The concentrated fruit extract was characterized using the total phenolic content, determined by the Folin-Ciocalteu method (40), slightly modified by Moldovan et al. (41). Thus, 250 μL of diluted fruit extract (256 fold) was mixed with 1.5 mL of Folin-Ciocalteu reagent. After 5 minutes, 1.2 mL of 0.7M sodium carbonate solution was added to the mixture. The resulting solution was kept at room temperature in the dark for 2 h and then spectrophotometrically evaluated by measuring absorbance at 765 nm. A calibration curve of the gallic acid standard was used to determine the total phenolic content of the investigated fruit extract.

Preparation and characterization of gold nanoparticles
AuNPs were obtained by a green procedure that involves the reduction of Au 3+ ions using the antioxidant phytocompounds of VO fruits. To this end, at 25 mL diluted fruit extract 100 mL of boiling 1 mM HAuCl 4 solution were added and the resulting mixture was stirred for 1 h. The progress of the reduction reaction was visually and spectrophotometrically evaluated using a Perkin-Elmer Lambda 25 UV-Vis double-beam spectrophotometer. The color change from faint red to intense purple indicates the formation of a colloidal gold solution. The AuNPs were purified by repeated centrifugation at 10.000 rpm and washed. Several consecrated methods have been used to evaluate the properties of the synthesized gold nanoparticles, including UV-Vis spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential. An H-7650 120 kV automatic TEM Hitachi microscope on a carbon-coated copper greed was used to investigate the size and morphology of the AuNPs, and the hydrodynamic diameter and zeta potential were determined using a Zetasizer Nano ZS-90 instrument (Malvern Instruments Ltd., Malvern, UK) equipped with a He-Ne laser.

Experimental design
28 male Wistar rats, weighing 110-130 g, from the Biobase of the University of Medicine and Pharmacy 'Iuliu Haţieganu' Cluj-Napoca were used in the study. For acclimatization, the animals were kept for one week before the start of the experiments in the Biobase of the Department of Physiology under the following conditions: light/dark cycle of 12 h/12 h, controlled temperature of 21 ± 2°C, and water and food ad libitum. The experiments were carried out in compliance with the protocols approved by the Ethics Commission of UMF 'Iuliu Haţieganu' in accordance with internationally accepted principles for the use and handling of laboratory animals (European Community Guidelines, EEC Directive of 1986; 86/609/EEC).
The experimental model used was carrageenaninduced paw inflammation, as described by Winter et al. (42). Animals were randomized into 4 groups (n = 7) as follows: control group, treated with vehicle (phosphate buffered saline -PBS); group treated with 5 mg/kg b.w. Indomethacin; group treated with 15 mg/kg b.w. Viburnum opulus (VO) extract; group treated with 0.3 mg/kg b.w. gold nanoparticles (AuNP-VO). All compounds were orally administered daily for 4 days. On day 5, inflammation of the paw with sterile 1% carrageenan suspension was induced in all groups. Under intraperitoneal anesthesia with ketamine/ xylazine (90 mg/kg b.w./10 mg/kg b.w. xylazine), each animal was injected into the right paw under the aponeurosis with 100 μL carrageenan suspension. The left paw was injected with an equal volume (100 µL) of PBS.
The doses used were selected based on previous pilot studies on Wistar rats (43). Indomethacin (no. I7378), a potent nonsteroidal anti-inflammatory agent, was suspended in 1.5% carboxymethylcellulose. The dose was chosen based on previous studies and served as the positive control (44). Saline phosphate buffer (PBS) was used as a negative control.
At 2, 24, and 48 h after carrageenan administration, blood and plantar tissues were collected under anesthesia. Part of the plantar tissues was immersed in formalin and processed for histopathological examination, and part was homogenized with a Polytron homogenizer (Brinkman Kinematica, Switzerland) for 3 min, on ice, in phosphate buffered saline (pH 7.4) in a 1:4 ratio (w/v) for biochemical evaluation. COX-2 and iNOS expression was evaluated by western blotting, and the profile of proinflammatory and anti-inflammatory cytokines in plantar tissue was quantified by ELISA. Oxidative stress markers were determined from blood and plantar tissue homogenates. Protein concentration in the homogenate was measured using the Bradford method (45).

Measurement of paw edema
Paw edema was assessed using a plethysmometer (Ugo Basile, 37140, Italy). For this, paw volume was measured initially and then at 2, 24, and 48 h after carrageenan injection. The anti-inflammatory activity was assessed as a percentage of the reduction in edema in the treated groups, compared to the control group (46), according to the formula: Oxidative stress assessment Malondialdehyde (MDA) (47), reduced glutathione (GSH) (48), catalase (CAT) (49), and glutathione peroxidase (GPx) activities (50) were measured in the blood and skin tissue homogenates. The reduced glutathione/oxidized glutathione ratio (GSH/GSSG) was calculated.

Histopathology exam and lesion grading
Paw biopsies were fixed in 10% neutral-buffered formalin for at least 24 h before trimming and routine processing. Serial tissue sections (5 µm thick) were stained with hematoxylin and eosin (HE). Each case had five different sections of plantar tissue examined using an Olympus system (microscope, digital camera, and image software). Histology scores were assigned on a scale from 0 (normal) to 5 (most severe), based on the presence, intensity, and type of cell infiltrate, edema, necrosis, and hemorrhage (51). The paw inflammation scores for each group were averaged and analyzed.

The assessment of COX-2 and iNOS in the paw tissue
For quantification of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS), western blotting was performed. For western blotting, lysates (20 μg protein/ band) were separated by electrophoresis (SDS-PAGE) and transferred by adsorption onto a polyvinylidene difluoride membrane using the Bio-Rad Miniprotean system (Bio-Rad, Hercules, CA, USA) as previously described (52). The blots were blocked at room temperature for one hour and incubated overnight with primary anti-COX-2, anti-iNOS, and anti-GAPDH antibodies. After washing, the blots were incubated with specific secondary antibodies for 1.5 h. Protein visualization was accomplished using the West Femto Chemiluminescent Supersignal substrate (Thermo Fisher Scientific, Rockford, IL, USA), and the measurement was performed using Image Lab analysis software (Bio-Rad, Hercules, CA, USA). GAPDH was used as a control for protein loading.

Cytokine profile analysis
IL-1α, IL-1β, IL-6, IL-10, and TNFα levels in skin tissue were determined using a multiplex cytokine kit (Luminex 200; Luminex Corporation, Austin, TX, USA). The assay was performed according to the manufacturer's instructions.

Data analysis
The obtained data were statistically processed using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA), performing one-way ANOVA followed by Tukey's post-hoc test. The threshold significance level was set at p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001).

Synthesis and characterization of gold nanoparticles
In the present study, the VO fruit extract was used as a source of reducing agents for gold ions and stabilizing agents for the synthesized nanoparticles. Viburnum opulus L. fruits are well known for their high antioxidant properties because of their high content of polyphenolic compounds (53,54). The determined value of The TPC of the fruit extract was 22.54 GAE/L. This high content enabled us to select this extract for the synthesis of AuNPs. By adding the VO fruit extract to the gold ion solution, a rapid color change from faint red to intense purple was observed, indicating the reduction of metallic ions and the synthesis of gold nanoparticles. The UV-Vis absorption spectrum of AuNP-VO ( Figure 1A) displays an intense peak at λ max = 536 nm, confirming the formation of a gold colloidal solution. This maximum absorption peak is due to the SPR band characteristic of the nanogold, which is consistent with other studies on green synthesis of AuNP-VO (55,56).
The size and morphology of the synthesized AuNP-VO were evaluated by TEM. The results are shown in Figure 1. As shown in Figure 1B, the obtained AuNPs were spherical in shape, homogeneous, and not aggregated, with an average size of 65 nm ( Figure 1C).
The hydrodynamic size and stability of the AuNP-VO were assessed using DLS/zeta potential analysis. The obtained value of the zeta potential (−20.7 mV, Figure 2A) clearly indicates the negative charge of the surface of the nanoparticles due to the presence of biomolecules from the fruit extract proving their good stability. The average hydrodynamic size determined by DLS was 83 nm ( Figure 2B).

Measurement of paw edema
Plantar injection of carrageenan induced a significant increase in paw edema, which peaked after 2 h. Paw edema persisted for up to 48 h after treatment. Pre-administration of the VO extract for 4 days significantly decreased plantar edema only at 2 h (p < 0.01), and the values were close to those obtained after administration of indomethacin at 2 h (p < 0.05). After 24 and 48 h,  none of the treatments administered did not changed the clinical plantar edema (p > 0.05) ( Figure 3).

Biochemical assays
In the serum, MDA decreased significantly in all treated groups compared to the control group, both at 2, 24, and 48 h after carrageenan administration (p < 0.001) ( Figure 4A).
In the plantar tissue, MDA levels increased significantly at 2 h in the groups treated with indomethacin and AuNP-VO (p < 0.001), while the VO extract reduced lipid peroxidation close to the control level. MDA values decreased after AuNP-VO treatment only at 48 h, similar to indomethacin (p < 0.001) ( Figure 4B). The VO extract maintained low levels of MDA up to 24 h after carrageenan injection, while at 48 h, its protective effect was minimal.
In serum, the GSH/GSSG ratio was enhanced 2 h after carrageenan administration in all treated groups compared to the control group, with an increase maintained in the VO and AuNP-VO groups up to 24 h (p < 0.001) and 48 h (p < 0.01) ( Figure 5A).
In paw tissue, at 2 and 24 h, the GSH/GSSG ratio was improved after AuNP-VO treatment compared to the control group (p < 0.001), while at 48 h, the values were close to those of the control group. At 24 h, after the administration of carrageenan, the GSH/GSSG ratio increased in the indomethacin-treated group, and this increase was maintained at 48 h (p < 0.001) (Figure 5B), suggesting a beneficial effect on endogenous antioxidant defense.
CAT activity was enhanced at 2 h in VO-treated animals compared with that in animals receiving indomethacin or AuNP-VO (p < 0.01). Enzymatic activity was amplified at 24 h in the paw tissue of AuNP-VO-treated animals (p < 0.05) and maintained for up to 48 h (p < 0.001) ( Figure 6A).
GPx activity decreased at 2 and 24 h in the indomethacin-treated group (p < 0.01) and in the VO group (p < 0.05) compared to that in the control. At 48 h, AuNP-VO administration significantly increased enzymatic activity compared to that in the control group (p < 0.001) and the other groups (p < 0.01) ( Figure 6B).

Cytokine profile analysis
At 2 h after the induction of inflammation, IL-1α levels decreased after treatment with VO (p < 0.01) and AuNP-VO (p < 0.001) compared to controls, while indomethacin administration significantly increased IL-1α secretion (p < 0.01). At 48 h, AuNP-VO induced a significant increase in IL-1α levels compared with all other groups (p < 0.001) ( Figure 7A).
IL-1β decreased in all treated groups compared to the control group 2 h after induction of inflammation; the decrease was more significant in the group that received VO (p < 0.001). At 48 h, this decrease was maintained in the VO group compared to that in all other groups (p < 0.001) ( Figure 7B). IL-6 levels diminished in all groups only at 2 h (indomethacin p < 0.05; VO p < 0.001; AuNP p < 0.01, vs. control), whereas at 24 h and 48 h, there were no significant differences between these groups ( Figure 7C). IL-10, an anti-inflammatory cytokine, and TNFα secretion decreased in the VO and AuNP-VO groups (p < 0.01) only 2 h after inflammation. At 24 and 48 h, there were no significant differences between the groups ( Figure 8A, 8B).
The assessment of COX-2 and iNOS in the paw tissue iNOS expression decreased at 2 h in the indomethacintreated group (p < 0.001) and the AuNP-VO group (p < Figure 3. Paw volume at 2 h, 24 h and 48 h after carrageenan administration. At 2 h, paw edema was lower in the Indomethacin (p < 0.05) and VO (p < 0.01) groups compared to the control group. The statistical significance between the compared groups was evaluated with one-way ANOVA followed by the Tukey-test, *p < 0.05, **p < 0.01 vs control group. 0.05) compared with the control group. At 24 h, iNOS expression increased in the VO-and AuNP-VO-treated groups compared to the control group or the indomethacin group (p < 0.001) (Figure 9). COX-2 expression decreased significantly at 2 h in all treated groups (p < 0.001) compared with that in the control group. At 24 h, COX-2 levels remained low in the indomethacin and AuNP-VO groups (p < 0.001), while at 48 h, COX-2 levels increased in the indomethacin-treated group (p < 0.001) and AuNP-VO-treated group (p < 0.05) compared to those in the control group or the VO-treated group ( Figure 10).

Histopathology analysis
Minimal histological changes in paw biopsies were observed in all animals from the treated groups, but not in the untreated control. They varied among individuals and were composed of a combination of the following: focal to multifocal to diffuse inflammatory infiltrates (mainly macrophages, lymphocytes, and plasma cells) affecting all layers, increased numbers of mast cells, edema, necrosis, and focal hemorrhage (Figure 11). At 2 and 24 h, subcutaneous edema was more severe in the control, VO, and AuNP-VO groups, while it was less prominent in the indomethacin group. Overall edema was reduced at 48 h in all groups. Inflammatory infiltrates, mainly lymphocytes and plasma cells, with rare neutrophils and macrophages, were observed in all groups at all experimental times. They were minimal in the indomethacin-treated group across all experimental times (p < 0.01, 2 h; p < 0.001 at 24 h and p < 0.001 at 48 h vs. control group) and most severe in the control group, whereas VO and AuNP-VP were of intermediate intensity. Thus, in the VO and AuNP-VO groups, the average inflammatory score was lower than that in the control group at 24 h (p < 0.05, p < 0.01) and was maintained at 48 h after induction of inflammation (p < 0.001 and p < 0.01).
The histopathological quantification of the paw changes is illustrated in Table 1.

Discussion
Carrageenan-induced paw edema is an experimental model of acute inflammation used to quantify the potential anti-inflammatory effects of drugs or natural compounds. In the present study, we evaluated the In the plantar homogenates, MDA level increased at 2 h and then decreased at 48 h in the Indomethacin and AuNP-VO groups, compared to the control group and VO group (p < 0.001). The statistical significance between the compared groups was evaluated with one-way ANOVA followed by the Tukey-test, ***p < 0.001 vs control group; ### p < 0.001 vs Indomethacin group;^^^p < 0.001 between VO and AuNP-VO.
anti-inflammatory and antioxidant effects of gold nanoparticles phytoreduced with Viburnum opulus fruit extract compared to indomethacin and Viburnum opulus fruit extract.
A green synthesis method of gold nanoparticles was applied, by exploiting the reducing potential of the bioactive compounds from the above-mentioned fruits. Numerous plant extracts have been used for the green phytomediated synthesis of AuNPs. The involvement of biomolecules from plants throughout the synthesis process is an efficient and eco-friendly method for obtaining metal nanoparticles (44,57). Apart plants (58,59,27,33), different biological sources such as algae, fungi, yeasts, bacteria or viruses can be used for the intra-or extracellular biosynthesis of metallic nanoparticles (24,25,60,61,62,63). This eco-friendly method for fabrication of metallic nanoparticles presents numerous benefits over traditional chemical and physical methods, offering a clean, benign, nontoxic, cost-effective and relatively easy to scale up method of obtaining well-defined nanoparticles with a wide range of shapes, sizes, compositions and properties (26,61).
The results showed that the effect of AuNP-VO on plantar oxidative stress appeared 48 h after the induction of inflammation. Thus, AuNP-VO reduced skin MDA formation and increased CAT and GPx activities as well as the GSH/GSSG ratio, particularly in late-stage inflammation. Additionally, AuNP-VO diminished the local secretion of IL-1α, IL-1β, IL-6, and TNF-α compared to the control group at 2 h after induction of inflammation with moderate reduction in iNOS and COX-2 expression. The effect was persistent for COX-2 for up to 24 h. VO treatment diminished MDA levels in the paw, especially at 2 and 24 h after carrageenan injection, in parallel with the increase in CAT activity and reduced IL-1α, IL-1β, IL-6, and TNF-α levels, mainly at 2 h. In addition, the VO extract diminished the expression of iNOS and COX-2 in plantar tissue at 2 and 48 h after carrageenan injection and reduced IL-10 secretion. Moreover, protection against oxidative stress was important in serum for Figure 5. GSH/GSSG ratio in serum (A) and plantar tissue (B) at 2 h, 24 h and 48 h after carrageenan administration. A. In serum, the GSH/GSSG ratio increased at 2 h in all treated groups compared to the control group; the VO and AuNP-VO groups showed increases in the GSH/GSSG ratio at 24 h (p < 0.001) and at 48 h (p < 0.01). B. In the paw tissue, the GSH/GSSG ratio enhanced at 2 h in the AuNP-VO group compared to the control group (p < 0.001) and at 24 h compared to all groups (p < 0.001). At 48 h, the GSH/GSSG ratio decreased in AuNP-VO-treated group while in the Indomethacin group, the values were maintained increased compared to the other groups (p < 0.001). The statistical significance between the compared groups was evaluated with one-way ANOVA followed by the Tukey-test, *p < 0.05, **p < 0.01, ***p < 0.001 vs control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs Indomethacin group;^^p < 0.001 between VO and AuNP-VO.
both VO and AuNP-VO, suggesting a beneficial effect on systemic changes induced by local inflammation. Histologically, paw edema was reduced in the indomethacin, VO, and AuNP-VO groups at all experimental times, with a progressive reduction in intensity from 2 h to 24 h to 48 h in each group. Inflammatory infiltrates showed an opposite trend to edema in all groups, with mixed inflammatory cells increasing progressively with time. However, when edema and inflammation, together with other histological changes, were combined to obtain the inflammatory score, there was a steady increase in the score from 2 h to 24 h to 48 h in all groups except VO. All treated groups showed a reduction in the inflammatory score, and indomethacin showed the best protective effect histologically. At 48 h, VO and indomethacin had similar and low scores, and exerted the same morphological protection.
Carrageenan administration induces a biphasic inflammatory response; in the early phase, histamine, serotonin, and bradykinin are released, while the late phase includes the production of free radicals from neutrophils and macrophages, the secretion of cytokines, NO synthesis, neutrophil infiltration, COX-2 activation, and the subsequent production of prostaglandins (11).
Previous studies have shown that carrageenan administration in mice triggers intense inflammatory events characterized by excessive excretion of various inflammatory mediators, temporary vasoconstriction, and vasodilation with edema, which peaks after 8 h (64,65). In our study, edema was maximum at 2 h after carrageenan administration and then decreased at 24 h and 48 h. VO extract significantly reduced clinical edema at 24 h, while at 48 h, the effect was not significant.
Redox imbalance was quantified by MDA level and GSH/GSSG ratio in serum and plantar tissue and by evaluation of antioxidant enzyme activities. MDA is a specific marker of lipid peroxidation and its production is a valuable marker for monitoring oxidative stress. The GSH system represents the first line of endogenous antioxidant defense against free radicals and is mostly found in the reduced form (GSH) and only a small part in the oxidized form (GSSG). The GSH/GSSG ratio reflects the redox equilibrium. Catalase (CAT) and glutathione peroxidase (GPx) are among the primary antioxidant enzymes that detoxify excessive hydrogen peroxide and lipid peroxides in cells.
The results of our study showed an improvement in systemic antioxidant defense in the groups treated CAT activity increased at 2 h in the VO-treated group (p < 0.01) and at 48 h in the group receiving AuNP-VO (p < 0.001), compared to controls. GPx activity was improved 48 h after AuNP-VO treatment (p < 0.001 vs. control group; p < 0.01 vs. Indomethacin group). The statistical significance between the compared groups was evaluated with one-way ANOVA followed by the Tukey-test, *p < 0.05, **p < 0.01, ***p < 0.001 vs control group; ## p < 0.01, ### p < 0.001 vs Indomethacin group;^^^p < 0.001 between VO and AuNP-VO.
with VO and AuNP-VO compared to the control group. In the plantar tissue, the effect was dependent on the compound administered and the time of evaluation. VO extract reduced lipid peroxidation and enhanced antioxidant defense early after induction of inflammation, whereas AuNP-VO exerted a late effect 48 h after the induction of inflammation.
The literature on the protective effect of VO extract in pathological conditions is contradictory. Thus, some studies showed favorable effects of VO fruit extract while the others exhibited the lack of its effect. In an in vivo rat experiment, Eken et al. (66) demonstrated protective properties against ischemia/reperfusioninduced oxidative stress during lung transplantation.
Intraperitoneal administration of 200 mg/kg b.w. VO dried fruit extract resulted in significant increases in superoxide dismutase (SOD), CAT, GPx, and GSH levels, and decreased MDA and carbonylated protein levels.
In vitro studies have also shown favorable effects of VO fruit extract, with decreased intracellular reactive oxygen species production in human Caco-2 adenocarcinoma cells (19), MIN6 mouse cells, and 3T3-L1 cells (21,22).
In contrast, Altun et al. (67), in rats and mice with plantar inflammation induced by carrageenan, VO leaf extract did not have a protective effect on inflammation compared to Indomethacin, independent of dose used (50 mg, 100 mg or 200 mg/kg b.w.). The divergent Figure 7. IL-1α (A), IL-1β (B), and IL-6 (C) levels in plantar tissue at 2, 24, and 48 h after carrageenan administration. The levels of IL-1α, IL-1β, and IL-6 were significantly reduced at 2 h in the groups treated with VO extract (p < 0.01) and AuNP-VO (p < 0.001) compared to the control group. Low IL-1β levels were maintained at 24 h (p < 0.01) in the AuNP-VO-treated group compared with indomethacin, while at 48 h, cytokine secretion decreased in the VO-treated group (p < 0.001) compared to the other groups. IL-1α levels increased at 24 h in the VO-and AuNP-VO-treated groups (p < 0.001), and increased at 48 h in the AuNP-VO-treated group (p < 0.001). The statistical significance between the compared groups was evaluated with one-way ANOVA followed by Tukey's test, **p < 0.01, ***p < 0.001 vs. control group; ### p < 0.001 vs. indomethacin group;^^^p < 0.001 between VO and AuNP-VO. TNFα (p < 0.01 and p < 0.05) levels decreased at 2 h after carrageenan administration in animals treated with VO and AuNP-VO compared to Indomethacin group. The statistical significance between the compared groups was evaluated with one-way ANOVA followed by the Tukey-test, *p < 0.05, ***p < 0.001 vs control group; # p < 0.05, ## p < 0.01 vs Indomethacin group. Figure 9. iNOS expressions in animal paw tissues at 2 h, 24 h and 48 h after carrageenan administration. Image analysis of WB bands was performed through densitometry (lower panel). Results normalized to GAPDH were shown in upper panel. At 2 h, iNOS expression decreased in the Indomethacin (p < 0.001) and AuNP-VO (p < 0.05) groups compared to the control group. At 24 h, iNOS increased in VO and AuNP-VO groups compared to control group or Indomethacin group (p < 0.001). At 48 h, Indomethacin, VO and AuNP-VO did not induce the iNOS expression The statistical significance between the compared groups was evaluated with one-way ANOVA followed by the Tukey-test, *p < 0.05, ***p < 0.001 vs control group; ## p < 0.01, ### p < 0.001 vs Indomethacin group;^^^p < 0.001 between VO and AuNP-VO.
literature data can be explained by the different contents of polyphenols in the VO extract used and the time of quantification of the effects, especially as its effect is short-lived.
IL-1α, IL-1β, IL-6, and TNF-α are the main pro-inflammatory cytokines responsible for the early inflammatory responses. Early stage mediators are generally produced by platelets and mast cells and play a role in inducing At 48 h, COX-2 increased in the Indomethacin group (p < 0.001) and in the AuNP-VO group (p < 0.05) compared to the control group or the VO group. The statistical significance between the compared groups was evaluated with one-way ANOVA followed by the Tukey-test, *p < 0.05, ***p < 0.001 vs control group; # p < 0.05, ### p < 0.001 vs Indomethacin group;^p < 0.05,^^p < 0.01,^^^p < 0.001 between VO and AuNP-VO. Figure 11. Paw changes histologically at 2 h, 24 h and 48 h after carrageenan administration. Prominent subcutaneous edema and mixed inflammatory infiltrates in the dermis and subcutis in the control, VO and AuNP-VO groups at 2 and 24 h, and minimal in the Indomethacin treated group. Edema and inflammation were reduced at 48 h across all groups but were significant compared to control group. HE stain x ob 40. and activating the inflammatory process. Late-stage mediators are involved in controlling vascular phenomena. They appear later, 6-12 h after the onset of inflammation and are represented by various arachidonic acid derivatives (16,17,68).
The results of our study confirmed important antiinflammatory effects compared to indomethacin, both for VO extract and AuNP-VO 2 h after induction of inflammation. This effect was not persistent at 24 or 48 h, less in terms of COX-2, which was reduced after carrageenan administration.
Jeon et al. (69) showed that AuNPs exert immunomodulatory effects through their ability to block NF-κB activation by suppressing the production of proinflammatory cytokines IL-1β and TNF-α. In another study, Ma et al. (70) showed that AuNPs decreased iNOS-induced gene expression and NO production by blocking NF-κB and STAT1 activation in lipopolysaccharide-stimulated RAW264.7 cells. Sumbayev et al. (38) demonstrated the size-dependent anti-inflammatory activity of AuNPs by downregulating IL-1β-induced cellular responses in both in vitro and in vivo studies. AuNPs with a diameter of 5 nm completely inhibited the inflammatory process, those with a diameter of 15 nm showed moderate efficacy, and those with a diameter of 35 nm did not show any statistically significant effect. The large size of our AuNPs could explain the moderate anti-inflammatory effect obtained in the experiment.
Several in vitro studies have shown antioxidant and anti-inflammatory effects of 3-5 nm AuNP (HUVECs, VECs) (71), or for 15 nm, 20 nm, and 50 nm AuNPs (RAW264.7 cells) (39,70,72). Khan and Khan (73) also observed the antioxidant and anti-inflammatory effects of AuNPs with sizes of 20 nm in rats with collageninduced arthritis. They exhibited a decrease in MDA levels and a significant decrease in the mRNA expression of NF-κB, TNF-α, COX-2, iNOS, and IL-1β. Dohnert et al. (74) showed that 10 nm AuNP have anti-inflammatory effects by significantly decreasing IL-1β and TNF-α in an experimental model of tendinopathy in rats. Our results confirmed the moderate anti-inflammatory action of AuNP-VO with a diameter of 65 nm, especially in the early stages of inflammation, suggesting a protective effect on the secretion of histamine, bradykinin, and serotonin from inflammatory cells. AuNP-VO inhibited cyclooxygenase-2 activity and decreased its expression both at 2 and 48 h after induction of inflammation, suggesting protection of arachidonic acid derivative generation in the late stages of paw inflammation. These results were also confirmed by histopathology, which revealed a lower inflammation score at 24 and 48 h after induction of inflammation in animals treated with AuNP-VO.
IL-10 is a cytokine with anti-inflammatory properties that limits excessive immune responses. It suppresses the release of immune mediators, antigen expression and phagocytosis (75). In vitro inflammation models showed that IL-10 prevents the activation of PMNs and TNF-α, as well as the release of IL-8 (76). In our study, IL-10 decreased in the groups treated with VO and AuNP-VO 2 h after induction of inflammation and was not influenced by treatment at other monitoring intervals. These data showed no effect on the secretion of anti-inflammatory cytokines.
Although several studies have reported antioxidant and anti-inflammatory effects of biologically synthesized metal nanoparticles, there are also studies that have shown genotoxic effects, which are mainly dependent on the type of metal, synthesis parameters, dose, time of administration or biological source used (61,77). Therefore, supplementary investigations are required to decipher all mechanisms and signaling pathways involved, especially in long-term exposure, the appropriate dose used and time of administration in order to minimize side effects and increase administration safety and real benefit.

Conclusion
The results of our study demonstrate the antioxidant and anti-inflammatory effects of AuNP-VO and VO extracts on plantar inflammation, with different dynamics of action. AuNP-VO exerted antioxidant effects, especially at 48 h, and diminsihed the plantar inflammation in the early stages (IL-1α, IL-1β, IL-6, TNFα and IL-10) but also in the late stages of inflammation (IL-1β). The protective effects were more persistent in the plantar tissue after AuNP-VO treatment, mainly in terms of oxidative stress and arachidonic acid generation and increased local antioxidant defense. The biochemical changes were correlated with the results of the histopathological examination, where the inflammatory changes in the plantar tissue were more discrete at 24 h and 48 h after carrageenan injection. The VO extract reduced the biochemical inflammatory response, predominantly in the early stages (IL-1α, IL-1β, IL-6, TNF-α and IL-10), and maintained reduced IL-1β secretion and a lower histopathological inflammatory score at 48 h, similar to indomethacin. Because of these properties, gold nanoparticles capped with Viburnum opulus and Viburnum opulus fruit extract can be valuable agents for the treatment of diseases associated with inflammation. Further investigations are required to accurately decipher all aspects and mechanisms involved, especially in long-term exposure to reduce side effects and increase therapeutic benefit.