Algerian bee Venom attenuates formaldehyde-induced acute inflammation and oxidative stress in male rats

Abstract

Formaldehyde exposure is associated with inflammation and oxidative stress, leading to systemic toxicity. Natural products such as bee venom have been proposed as alternative therapeutic agents due to their anti-inflammatory and antioxidant properties. This study investigated the potential of Algerian Apis mellifera intermissa bee venom to attenuate formaldehyde-induced acute inflammation and oxidative stress in male rats. Inflammation was induced by subplantar injection of 0.1 mL of a 2.5% formaldehyde solution into the hind paw. One hour later, rats received either subcutaneous bee venom (0.76 mg/kg) or oral diclofenac sodium (10 mg/kg) as a reference drug. Paw edema was quantified using ImageJ software. Blood samples were analyzed for erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), white blood cell count (WBC), platelet count (PLT), and albumin levels. Oxidative stress biomarkers including malondialdehyde (MDA), catalase (CAT), glutathione (GSH), and glutathione S-transferase (GST) were assessed in erythrocytes. Formaldehyde exposure produced sustained paw edema, impaired body weight gain, reduced food and water intake, systemic inflammatory alterations, and pronounced oxidative stress characterized by increased lipid peroxidation and depletion of erythrocyte antioxidant defenses. Bee venom treatment significantly attenuated paw swelling, improved systemic inflammatory alterations, and partially restored redox balance, with effects comparable to those of diclofenac. The overall protective effects of bee venom were comparable to those of diclofenac. These findings demonstrate that A. mellifera intermissa venom exerts significant anti-inflammatory and antioxidant actions and may represent a promising natural therapeutic candidate for inflammation associated with oxidative stress.

Introduction

The western honeybee (Apis mellifera L.) is an omnipresent species, mainly as a pollinator and a producer of diverse hive products[1]. Apis mellifera intermissa is commonly referred to as the North African bee, and is indigenous to Algeria, Tunisia, and Morocco[2]. In Algeria, A. mellifera intermissa is the dominant native subspecies and is well adapted to the country’s varied climatic zones, ranging from humid Mediterranean areas to arid inland regions[1–3]. These adaptations are thought to enhance their survival in the challenging Algerian environment and may also influence the chemical composition and bioactivity of their secretions, including venom [4].

In addition to honey production and pollination, honeybees provide several biologically active products such as honey, propolis, pollen, royal jelly, and venom[1]. These products have been used in traditional medicine for centuries and are increasingly studied for their therapeutic potential [5]. Among them, bee venom (BV) stands out for its wide range of pharmacological properties, including anti-inflammatory, antioxidant, antimicrobial, and analgesic effects [6,7].

Bee venom is a complex mixture of biologically active components. It consists predominantly of peptides such as melittin (40–60% of the dry weight), apamin, adolapin, and mast cell-degranulating peptide, as well as enzymes including phospholipase A2 (PLA2) and hyaluronidase [8,9]. Melittin exerts potent anti-inflammatory and lytic activities, primarily by disrupting cellular membranes and modulating key inflammatory signaling pathways such as NF-κB and MAPKs [10,11]. PLA2 contributes to the hydrolysis of membrane phospholipids, influencing prostaglandin synthesis and leukocyte recruitment [10]. Apamin, a neurotoxin, modulates calcium-activated potassium channels and exhibits anti-inflammatory effects in neuroinflammation models [12,13].

In traditional apitherapy, bee venom has been employed to treat ailments such as arthritis, neuralgia, multiple sclerosis, and other inflammatory conditions[14]. Modern research supports its role in reducing oxidative damage by enhancing endogenous antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [15]. These effects suggest its potential as a natural alternative or complementary agent for inflammation-related pathologies.

Inflammation is a complex physiological response triggered by various stimuli, including pathogens, toxins, and tissue injury [16]. Chronic or dysregulated inflammation contributes to the progression of diseases such as cancer, cardiovascular disorders, and neurodegeneration [17]. One well-established experimental model for inducing localized inflammation in rodents involves the subcutaneous injection of formaldehyde (FA) [18]. FA, a ubiquitous environmental and industrial pollutant, induces inflammation and oxidative stress through the excessive generation of reactive oxygen species (ROS), lipid peroxidation, and cytokine release [19,20].

Given the therapeutic promise of bee venom and the need to explore its activity in inflammation models, especially using venom from region-specific subspecies such as A. mellifera intermissa, experimental validation is necessary. Therefore, this study, to our knowledge, is the first to evaluate the anti-inflammatory and antioxidant effects of Algerian Apis mellifera intermissa bee venom in a formaldehyde-induced inflammation model in male rats. This research seeks to elucidate the therapeutic potential of locally sourced bee venom and support its application in natural medicine.

Materials and Methods

Twenty-four adult male Wistar rats weighing 150–155 g were obtained from the Pasteur Institute of Algiers. The animals were housed under standard laboratory conditions (temperature 22 ± 2°C, 12-h light/dark cycle, relative humidity 50–60%) with free access to standard laboratory chow and water ad libitum. All experimental procedures were conducted in accordance with the guidelines of the Ethical Guidelines of our Institution for the experimental protocol and animal handling (Approval number PRFU/2022; D01N01EN210120220001).

The rats were randomly divided into four groups of six animals each (n = 6 per group):

Control Group (C): Received a saline injection only.

Formaldehyde Group (FA): Received subplantar injections of 0.1 mL of 2.5% formaldehyde solution (v/v in normal saline, Sigma-Aldrich, USA) into the right hind paw on day 1 and day 3 to induce subacute inflammation. The solution was prepared fresh daily by diluting a 37% formaldehyde stock solution in 0.9% sterile saline to ensure concentration accuracy. This repeated-injection protocol has been previously described as a model of subacute/chronic inflammation characterized by sustained edema, leukocyte infiltration, and oxidative stress, distinct from acute carrageenan-induced edema models [18,19].

Formaldehyde + Bee Venom Group (FA+BV): Received formaldehyde injections as above, followed by bee venom treatment. Crude bee venom was collected from Apis mellifera intermissa colonies located in Northeast Algeria (Annaba region) during the spring season (April-May 2023) using electrical stimulation. The venom used in this study was from the same characterized batch described in our previous publication [21], where HPLC analysis revealed the presence of the primary bioactive component: melittin (approx. 38%), and the SDS PAGE revealed the presence of melittin, phospholipase A2, and hyaluronidase with molecular weights of 3-5kDa, 15-20kDa and 44kDa respectively. The venom was stored as a lyophilized powder at −20 °C and freshly dissolved in sterile saline prior to administration. To ensure reproducibility, all experiments were performed using venom from the same batch. A dose of 0.76 mg/kg body weight was administered subcutaneously, starting one hour after the first formaldehyde injection, and continued once daily for three consecutive days.

Formaldehyde + Diclofenac Group (FA+DC): Received formaldehyde injections followed by diclofenac sodium treatment. As a standard anti-inflammatory reference, diclofenac sodium (Novartis Pharma, Switzerland) was administered at a dose of 10 mg/kg body weight via oral gavage, once daily for three days, starting one hour after the first formaldehyde injection. The drug was freshly prepared in distilled water immediately before administration.

Throughout the treatment period, animals were monitored daily for changes in body weight and food intake using a digital balance (±0.01 g accuracy). Paw edema was assessed by measuring paw area from standardized digital photographs. To minimize variability, all photographs were taken by the same investigator using a fixed camera setup (Canon EOS 2000D, 24.1 MP) positioned perpendicular to the plantar surface at a constant distance of 20 cm.

A ruler was included in each frame for scale calibration. Animals were gently positioned with minimal restraint to ensure consistent paw placement without applied pressure. Images were captured at baseline, 1 h, 3 h, 6 h, 24 h, 48 h, and 72 h post-formaldehyde injection. Paw area was quantified using ImageJ software (version 1.53k, NIH, USA) by a blinded investigator who manually outlined the plantar surface boundary, and the software calculated the area in mm² after scale calibration . While paw volume measurement via plethysmometry is considered the gold standard for edema quantification, our photographic method has been validated in previous studies[22,23] and provides a reasonable alternative when plethysmometry is unavailable. Percentage of swelling was calculated as:

[(Area at time t-Baseline area) / Baseline area] × 100.

At the end of the treatment period, animals were sacrificed, and blood samples were collected and transferred into plain and EDTA-coated tubes for the determination of inflammatory and oxidative stress biomarkers.

Determination of inflammatory parameters

Erythrocyte sedimentation rate (ESR)

The determination of erythrocyte sedimentation rate (ESR) follows the standardized Westergren method, where blood samples were collected in EDTA-coated tubes and transferred to Westergren pipettes (internal diameter 2.5 mm), which were placed vertically and allowed to stand undisturbed for exactly one hour at room temperature (20-25°C). The distance (in millimeters) that erythrocytes descended during this one-hour period was recorded as the ESR value (mm/h).

C-reactive protein (CRP)

C-reactive protein (CRP), White Blood Cell (WBC) Count, Platelet Count (PLT), and albumin levels were determined in rat plasma using a colorimetric assay on a fully automated biochemical analyzer (Mindray BS-120, Roche Cobas C111) with a CRP reagent kit designed for use with such analyzers (LiquiColor® CRP reagent kit, Stanbio Laboratory).

Determination of erythrocyte oxidative stress markers

Erythrocyte levels of malondialdehyde (MDA), an indicator of lipid peroxidation, were determined using the thiobarbituric acid (TBA) method. This assay is based on the reaction of MDA with TBA to form a colored adduct that is measured spectrophotometrically at 532 nm [24]. The activity of the antioxidant enzyme catalase (CAT) in red blood cells was assessed spectrophotometrically by monitoring the decomposition of hydrogen peroxide (H2O2)[25]. Cellular levels of reduced glutathione (GSH), a critical non-enzymatic antioxidant, were quantified following deproteinization of erythrocyte lysates[26]. The activity of glutathione S-transferase (GST), a key detoxification enzyme, was determined by measuring the conjugation of L-glutathione to the substrate 1-chloro-2,4-dinitrobenzene (CDNB) [27].

Statistical Analysis

Data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism software version 9.0 (GraphPad Software, San Diego, CA, USA). For data meeting parametric assumptions, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons was used. For paw edema measurements over time, two-way repeated measures ANOVA followed by Bonferroni post hoc test was employed. Statistical significance was set at p < 0.05. All tests were two-tailed.

Determination of inflammatory parameters

Erythrocyte sedimentation rate (ESR)

The determination of erythrocyte sedimentation rate (ESR) follows the standardized Westergren method, where blood samples were collected in EDTA-coated tubes and transferred to Westergren pipettes (internal diameter 2.5 mm), which were placed vertically and allowed to stand undisturbed for exactly one hour at room temperature (20-25°C). The distance (in millimeters) that erythrocytes descended during this one-hour period was recorded as the ESR value (mm/h).

C-reactive protein (CRP)

C-reactive protein (CRP), White Blood Cell (WBC) Count, Platelet Count (PLT), and albumin levels were determined in rat plasma using a colorimetric assay on a fully automated biochemical analyzer (Mindray BS-120, Roche Cobas C111) with a CRP reagent kit designed for use with such analyzers (LiquiColor® CRP reagent kit, Stanbio Laboratory).

Determination of erythrocyte oxidative stress markers

Erythrocyte levels of malondialdehyde (MDA), an indicator of lipid peroxidation, were determined using the thiobarbituric acid (TBA) method. This assay is based on the reaction of MDA with TBA to form a colored adduct that is measured spectrophotometrically at 532 nm [24]. The activity of the antioxidant enzyme catalase (CAT) in red blood cells was assessed spectrophotometrically by monitoring the decomposition of hydrogen peroxide (H2O2)[25]. Cellular levels of reduced glutathione (GSH), a critical non-enzymatic antioxidant, were quantified following deproteinization of erythrocyte lysates[26]. The activity of glutathione S-transferase (GST), a key detoxification enzyme, was determined by measuring the conjugation of L-glutathione to the substrate 1-chloro-2,4-dinitrobenzene (CDNB) [27].

Statistical Analysis

Data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism software version 9.0 (GraphPad Software, San Diego, CA, USA). For data meeting parametric assumptions, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons was used. For paw edema measurements over time, two-way repeated measures ANOVA followed by Bonferroni post hoc test was employed. Statistical significance was set at p < 0.05. All tests were two-tailed.

Results and Discussion

Body weight, food intake, and water consumption

In Table 1, FA treatment resulted in a highly significant decrease in final body weight, food intake, and water intake when compared with the control group (p < 0.001). Co-administration of bee venom (FA + BV) significantly ameliorated these FA-induced impairments relative to the FA group (p < 0.001 for body weight, to p < 0.01 for food and water intake), although some parameters remained significantly different from control values (p < 0.01 for body weight and p < 0.05 for food and water intake). Similarly, diclofenac co-treatment (FA + DC) produced a significant improvement versus the FA group (p<0.01 for body weight and p < 0.05 for food and water intake), but the recovery was less pronounced, with parameters still showing significant deviation from control levels (p < 0.01). Direct comparison between the two treatments demonstrated no statistical significance between FA + BV and FA + DC groups. The decline in these physiological parameters in FA treated rats as previously also found [28] can be attributed to the well-recognized phenomenon of inflammation-induced sickness behavior, in which pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 act on hypothalamic centers to suppress appetite and thirst while simultaneously promoting catabolic processes that drive weight loss [18–20]. In addition, formaldehyde-induced oxidative stress, further disrupts mitochondrial energy metabolism, increasing energetic costs of the immune response and shifting metabolic balance toward tissue breakdown [29]. Further, this protective effect may be attributed to the venom's reported anti-inflammatory and antioxidant properties [21]. Previous studies have shown that melittin can suppress NF-κB–mediated cytokine production and restore redox balance[30]. However, these specific mechanisms were not directly examined in the present study and remain to be confirmed.

Table 1.

Changes in body weight, food and water intake in control and rats with subplantar injection of 0.1 mL of 2.5% formaldehyde (FA), bee venom (subcutaneous injection of 0.76 mg/kg body weight) + FA, and Diclofenac sodium (DC, oral administration at a dose of 10 mg/kg body weight) + FA.

	Group	Control	FA	FA+ BV	FA+ DC
Parameter
Initial body weight (g)		154.95±1.74	152.58 ± 1.51	153.67 ± 1.63	155.04 ± 1.12
Final body weight (g)		201.32±10.06	136.32± 6.88***	181.79± 5.72**c	161.44± 5.35**b ns
Food intake (g)		47.48±3.73	23.74± 5.87***	40.75± 2.88*b	31.19± 2.79**a ns
Water intake (ml)		66.34±1.41	29.20± 3.70***	57.23± 2.29*b	33.51± 2.08**a ns

Each value is displayed as mean ± SEM (n = 6). Values with superscripts are statistically different, p-value,

^*p< 0.05,

^**p<0.01, and

^***p< 0.001

Treatments versus the control group,

^ap< 0.05,

^bp<0.01, and

^cp< 0.001

FA+ BV and FA+ DC versus FA group, and ns = not significant (p > 0.05) FA+ BV versus FA+ DC group.

Paw edema measurements

As shown in Figure 1, Panel (a) shows the hind paws of a control rat with no signs of inflammation or swelling. Panel (b) displays a swollen and erythematous paw following FA injection, indicating significant edema. In contrast, panels (c) and (d) show paws treated with bee venom (BV) + FA and diclofenac (DC) + FA, respectively; both demonstrate visibly reduced swelling compared to the FA group. Panel (e) presents the time-course of paw swelling (%) across four groups: Control (●), FA (■), FA+BV (▲), and DC+FA (▼), measured up to 72 hours. The time-course curves show a stable baseline with no paw swelling in the control group throughout the 72-h observation period (ns, p > 0.05). In contrast, FA injection produced a rapid and sustained increase in paw swelling, which was significant from the earliest time point and progressively increased over time compared with control (p < 0.01 and p < 0.001). Treatment with BV or DC significantly attenuated FA-induced paw edema at multiple time points, showing a gradual decline in swelling over time compared with the FA group (p < 0.05, p < 0.01, and p < 0.001 vs. FA). The BV-treated group exhibited a slight greater reduction but not significant in paw swelling compared with FA+DC. These findings are in line with the well-established ability of FA to elicit robust inflammatory edema through protein and nucleic acid adduct formation, activation of nociceptive fibers, and induction of cytokine-mediated vascular permeability [31,32]. Treatment with bee venom (BV) significantly reduced paw edema at multiple time points (p < 0.05), with effects comparable to diclofenac (DC). At 48 h and 72 h, BV showed numerically lower edema values compared to DC; however, direct pairwise statistical comparison between the two treatment groups did not reach significance (p > 0.05). These findings indicate that BV and DC exert comparable anti-edematous effects in this model.

Figure 1.

Comparative effects of bee venom (BV) and diclofenac (DC) on formaldehyde (FA)-induced paw edema in rats: (a)–(d) Representative photographs of hind paws from control, FA, FA+BV, and FA+DC groups, respectively, taken 24 h post-injection. Scale bar = 10 mm. The hind paw region is indicated by a red circle in all groups; edema is evident in the FA and combined treatment groups (FA+BV and FA+DC), whereas the control group shows a normal hind paw without edema. Images are representative of six animals per group showing median edema response. (e) Time-course of paw swelling expressed as percentage change from baseline (% change = [(Area_t - Area_baseline) / Area_baseline] × 100), measured over 72 h following FA injection (blue arrow at 0 h). Treatments with BV (0.76 mg/kg, s.c.) and DC (10 mg/kg, p.o.) were administered 1 h post-FA injection (green arrow) and continued once daily. Control (●), FA (■), FA+BV (▲), and FA+DC (▼). Values are expressed as mean ± SEM (n = 6); ^***p < 0.001 vs. control group; Values are expressed as mean ± SEM; p-value, Each value is displayed as mean ± SEM (n = 6). Values with superscripts are statistically different, p-value, ^*p < 0.05, ^**p <0.01, and ^***p < 0.001 Treatments versus the control group, ap < 0.05, bp < 0.01, and cp < 0.001 FA + BV and FA + DC versus FA group, and ns = not significant (p > 0.05) FA + BV versus FA + DC group.

Notably, numerical trends suggested potential differences at later stages (48 and 72 h), which may indicate that BV not only suppresses the initial inflammatory response but also exerts longer-lasting regulatory effects on edema resolution. The anti-inflammatory effects of BV observed in this study aligns with previous studies reporting that bee venom and its principal component, melittin, exert potent anti-inflammatory effects in diverse models of inflammation. For instance, The anti-edematous effects of BV were comparable to those of diclofenac, despite their distinct pharmacological mechanisms of action, though these were not directly investigated in our study. Previous literature indicates that diclofenac primarily inhibits cyclooxygenase enzymes (COX-1 and COX-2), reducing prostaglandin synthesis [30]. In contrast, bee venom contains multiple bioactive components including melittin, apamin, and phospholipase A2, which have been reported in prior studies to modulate multiple inflammatory pathways including NF-κB, MAPK signaling, and cytokine production [31–34]. However, to confirm whether these specific pathways mediate the effects observed in our model, future studies should directly measure inflammatory mediators such as TNF-α, IL-1β, IL-6, NF-κB activation, and COX-2 expression in paw tissues. Additionally, Western blotting and immunohistochemical analyses would help elucidate the precise molecular mechanisms underlying the therapeutic effects of bee venom in formaldehyde-induced inflammation [33,34].

Apamin, another peptide component, modulates calcium-activated potassium channels and influences immune cell activation, while phospholipase A₂ (PLA₂) regulates eicosanoid production and inflammasome activity [35]. Collectively, these components not only dampen vascular permeability and leukocyte infiltration, thereby reducing edema, but also interfere with oxidative stress–inflammation crosstalk by restoring redox homeostasis. Thus, the present findings confirm that FA-induced paw edema is a robust model of inflammation and demonstrate that bee venom exerts anti-inflammatory efficacy comparable to that of diclofenac. Although BV showed a numerical trend toward greater edema reduction at later time points, these differences were not statistically significant and should be interpreted cautiously. The dual capacity of BV to suppress both inflammatory mediators and oxidative stress likely underlies its sustained and more pronounced reduction in paw swelling. These results support previous reports and further position bee venom as a promising natural therapeutic candidate with broader mechanistic activity than conventional NSAIDs.

Hematological and inflammatory biomarkers

In Table 2, subplantar injection of formaldehyde (FA) produced a marked inflammatory response compared with the control group. FA significantly increased ESR (p < 0.001), CRP (p < 0.001), total WBC count (p < 0.001), neutrophil percentage (p < 0.001), and platelet count (p < 0.001), while significantly reducing lymphocyte percentage (p < 0.001), eosinophil percentage (p < 0.001), and serum albumin levels (p<0.001) relative to control rats. These alterations are consistent with previous studies showing that formaldehyde induces oxidative stress, cytokine release, and activation of inflammatory pathways leading to hematological disturbances and acute-phase protein changes [36,37]. Further, Combined treatment with bee venom (FA + BV) significantly attenuated FA-induced inflammatory changes. ESR, CRP, total WBC count, neutrophils, and platelet count were significantly reduced compared with the FA group (p < 0.01– 0.001), while lymphocyte percentage, eosinophil percentage, and albumin levels were significantly restored toward control values (p < 0.05 – 0.001 vs FA). Despite this improvement, several parameters in the FA + BV group remained significantly different from control values, including ESR (p < 0.05), CRP (p < 0.05), and total WBC count (p < 0.05). Similarly, diclofenac treatment (FA + DC) significantly improved FA-induced alterations compared with the FA group, as evidenced by reduced ESR, CRP, WBC count, neutrophil percentage, and platelet count, along with increased lymphocyte percentage, eosinophils, and albumin levels (p<0.01–0.001 vs FA). However, most parameters in the FA + DC group also remained significantly different from controls (p < 0.01–0.001). Direct comparison between the two combined treatments showed that FA + BV exerted a not significant improvement in these parameters as compared with FA + DC. Overall, bee venom demonstrated a comparable efficacy to diclofenac in mitigating formaldehyde-induced systemic inflammation and restoring hematological and biochemical parameters toward normal levels.

Table 2.

Changes in Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), White Blood Cell (WBC) Count, Platelet Count (PLT), and albumin levels in control and rats with subplantar injection of 0.1 mL of 2.5% formaldehyde (FA), bee Venom (subcutaneous injection of 0.76 mg/kg body weight) + FA, and Diclofenac sodium (DC, oral administration at at a dose of 10 mg/kg body weight) + FA.

	Group	Control	FA	FA+ BV	FA+ DC
Parameter
ESR (mm/h)		1.06±0.2	2.66 ± 0.57***	1.67 ± 0.46*b	2.03 ± 0.72**b ns
CRP (mg/L)		0.91 ± 0.03	3.60 ± 0.08***	1.86 ± 0.04* c	2.11 ± 0.05** b ns
Total WBC count (×10³ cells/μL)		7.2 ± 0.11	11.8 ± 0.16***	8.1 ± 0.08*c	8.7 ± 0.14** c ns
Neutrophils Count (%)		22.5 ± 0.43	46.5 ± 0.43***	29.0 ± 0.37*c	31.5 ± 0.43** b ns
Lymphocytes Count (%)		71.5 ± 0.37	48.5 ± 0.34***	64.0 ± 0.31*c	61.5 ± 0.34** b ns
Eosinophils percentage		2.0 ± 0.06	1.52 ± 0.03***	2.17 ± 0.05*b	2.33 ± 0.04**c ns
Platelet Count (PLT)(×10³cells/μL)		642.5 ± 3.64	736.7 ± 4.24***	666.7 ± 2.36*b	680.0 ± 2.89**c ns
Albumin (g/dL)		4.08 ± 0.03	3.12 ± 0.03***	3.88 ± 0.02**a	3.68 ± 0.03**a ns

Each value is displayed as mean ± SEM (n = 6). Each value is displayed as mean ± SEM (n = 6). Values with superscripts are statistically different, p-value,

^*p< 0.05,

^**p<0.01, and

^***p< 0.001

Treatments versus the control group,

^ap< 0.05,

^bp<0.01, and

^cp< 0.001

FA+ BV and FA+ DC versus FA group, and ns = not significant (p > 0.05) FA+ BV versus FA+ DC group.

The ability of bee venom to markedly attenuate these changes aligns with earlier reports describing its potent anti-inflammatory and immunomodulatory properties. As reported [14,38], bee venom has been reported to suppress NF-κB activation, reduces pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, and enhances anti-inflammatory mediators like IL-10. The normalization of leukocyte subsets and albumin levels observed here suggests that bee venom not only suppresses inflammatory signaling but also restores immune homeostasis and mitigates acute-phase protein imbalance. Diclofenac, a conventional NSAID, also conferred protective effects, likely through inhibition of cyclooxygenase-mediated prostaglandin synthesis [39], however, its effects were comparatively moderate. The comparable anti-inflammatory efficacy of bee venom relative to diclofenac, despite its numerical trend toward higher values in certain parameters, may be attributed to its multi-target mechanisms, including modulation of arachidonic acid metabolism, suppression of reactive oxygen species, and regulation of immune cell recruitment [40]. Taken together, the results corroborate earlier findings that natural products with pleiotropic bioactive components can exert broader immunoregulatory effects than single-target synthetic anti-inflammatory drugs. Importantly, FA -induced thrombocytosis and hypoalbuminemia were significantly reversed by both BV and DC, with BV showed numerically greater normalization of platelet counts and albumin levels than DC (p < 0.01 vs. p < 0.05 for DC). This suggests broader regulation of cytokine-driven acute-phase responses. The improved efficacy of BV may reflect its multi-target actions, including modulation of arachidonic acid metabolism, oxidative pathways, and immune cell recruitment [40,41]. Collectively, these findings highlight BV as a comparable or slightly greater numerical anti-inflammatory and immunomodulatory agent than DC in formaldehyde-induced inflammation.

Erythrocyte oxidative stress markers

Figure 2 showed that exposure to formaldehyde (FA) induced marked oxidative stress, as evidenced by a significant elevation in malondialdehyde (MDA) levels alongside a depletion of key antioxidant defenses, including glutathione (GSH), glutathione S-transferase (GST), and catalase (CAT), compared with controls (p < 0.001 for all). Co-treatment with bee venom (FA + BV) or a drug compound (FA + DC) significantly ameliorated these biochemical disturbances, though with varying efficacy. MDA, which was markedly elevated by FA, was significantly reduced by BV and DC (p < 0.001), but MDA levels were still elevated versus control (FA + BV p < 0.05 ; FA + DC p < 0.01). Importantly, BV treated FA mice decreased significantly (p < 0.05) MDA levels compared with DC supplemented FA treatment. Regarding antioxidant parameters, FA-induced depletion of GSH was significantly reversed by BV and DC co-treatment (p< 0.01 - p< 0.001 vs FA); however, GSH levels in both FA + BV and FA + DC groups remained significantly lower than control (p<0.001). Similarly, GST activity, which was markedly suppressed by FA (p < 0.001 vs control), was significantly elevated following BV or DC supplementation (p<0.01 and p< 0.001 vs FA), yet values remained significantly reduced compared with controls (p < 0.05 p< 0.01). In the same manner, CAT activity was significantly diminished by FA exposure (p < 0.001 vs control) and was significantly restored by both BV and DC (p < 0.001 vs FA), although CAT levels remained significantly different from control in FA + DC treatment groups (p < 0.01). Notably, CAT activity was slightly higher, but not significant in the FA + BV group compared with FA + DC, further supporting the enhanced antioxidant efficacy of bee venom in mitigating FA-induced oxidative stress. Collectively, these findings emphasize BV’s enhanced protective efficacy against FA-induced oxidative injury, particularly in lipid peroxidation suppression and antioxidant enzyme recovery. Comparable results have been reported in earlier studies showing that FA exposure disrupts the oxidant–antioxidant equilibrium through excessive reactive oxygen species (ROS) generation and depletion of GSH, GST, superoxide dismutase (SOD), and CAT, ultimately leading to oxidative damage in hepatic, renal, and neural tissues [42,43]. In a related FA-induced arthritis model, BV treatment ameliorated oxidative and hematological alterations more effectively than prednisolone, supporting its strong antioxidant potential [44]. Recent reviews further confirm that BV components such as melittin, apamin, and phospholipase A2 exert antioxidant and anti-inflammatory activities by directly scavenging ROS, boosting endogenous antioxidant defenses, and suppressing pro-oxidant inflammatory cascades [40,45]. Mechanistically, the oxidative burden induced by FA is largely attributed to its metabolism into reactive intermediates such as formate and free radicals, which interact with lipids, proteins, and nucleic acids, leading to lipid peroxidation and depletion of thiol-based antioxidants. FA also activates redox-sensitive signaling pathways, including NF-κB and MAPKs, thereby amplifying oxidative and inflammatory responses. BV appears to counteract these effects through synergistic actions of its bioactive peptides, which not only neutralize ROS and upregulate GSH and phase II detoxifying enzymes (e.g., GST, CAT, SOD) but also downregulate NF-κB-mediated inflammatory signaling. These complementary mechanisms explain why BV demonstrated stronger protection than DC in the current model and underscore its potential as a therapeutic antioxidant against FA-induced toxicity. Several limitations of this study should be acknowledged. First, we used a photographic method for paw edema measurement rather than the gold-standard plethysmometric volume assessment, which may have introduced measurement variability despite our standardization efforts. Second, bee venom and diclofenac were administered via different routes (subcutaneous vs. oral), precluding direct pharmacokinetic comparisons and potentially confounding efficacy assessments. Third, while we observed significant anti-inflammatory and antioxidant effects, we did not directly measure key mechanistic mediators such as pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), NF-κB activation, or COX-2 expression in tissue samples. Therefore, our mechanistic interpretations remain inferential and based on existing literature. Fourth, this study used only male rats; sex-related differences in inflammatory responses and bee venom effects warrant investigation. Fifth, we examined only a single dose of bee venom; dose-response studies would help optimize therapeutic dosing. Finally, longer-term studies are needed to assess sustained efficacy, potential toxicity, and effects in chronic inflammation models. Future research should address these limitations through direct mechanistic measurements, standardized dosing comparisons, inclusion of both sexes, and extended observation periods.

Figure 2.

Comparative effects of bee venom (BV) and diclofenac (DC) on formaldehyde (FA)-induced oxidative stress in rat erythrocytes: (A) Malondialdehyde (MDA, nmol/mg protein), (B) Reduced glutathione (GSH, nmol/mg protein), (C) Glutathione S-transferase (GST, nmol/min/mg protein), and (D) Catalase (CAT, μmol H2O2/min/mg protein) levels measured 72 h post-FA injection in control, FA, FA+BV, and FA+DC groups. BV (0.76 mg/kg, s.c.) and DC (10 mg/kg, p.o.) were administered 1 h post-FA exposure and continued once daily for 3 days. Values are expressed as mean ± SEM; Each value is displayed as mean ± SEM (n = 6). Values with superscripts are statistically different, p-value, ^*p < 0.05, ^**p <0.01, and ^***p < 0.001 Treatments versus the control group, ap < 0.05, bp < 0.01, and cp < 0.001 FA + BV and FA + DC versus FA group, and ns = not significant (p > 0.05) FA + BV versus FA + DC group.

Conclusions

This study provides the first experimental evidence that Algerian Apis mellifera intermissa bee venom exerts significant anti-inflammatory and antioxidant effects against formaldehyde-induced toxicity in rats. Formaldehyde exposure produced pronounced systemic inflammation, oxidative stress, and physiological disturbances, while bee venom treatment effectively reduced paw edema, normalized inflammatory biomarkers (CRP, ESR, leukocyte profile, platelets, and albumin), and restored redox balance by lowering lipid peroxidation and enhancing endogenous antioxidant defenses (GSH, GST, CAT). Compared with diclofenac, bee venom demonstrated comparable therapeutic efficacy despite different routes of administration, which can be attributed to its complex mixture of bioactive components such as melittin, apamin, and phospholipase A2 that act synergistically to inhibit pro-inflammatory signaling pathways and reinforce antioxidant defenses. These findings highlight the therapeutic promise of Algerian bee venom as a natural, multifunctional agent for mitigating oxidative stress and inflammation-related disorders. Nonetheless, further studies are warranted to explore dose-response relationships, sex-related variations, molecular mechanisms, and potential applications in chronic disease models, as well as to ensure translational relevance for clinical use. An important limitation of this study is that bee venom and diclofenac were administered via different routes (subcutaneous vs. oral, respectively), which precludes direct pharmacokinetic comparison. The subcutaneous route may result in more sustained local tissue exposure, while oral administration involves hepatic first-pass metabolism and systemic distribution. Therefore, the observed differences in efficacy may partly reflect these pharmacokinetic distinctions rather than inherent differences in anti-inflammatory potency. Future comparative studies should employ identical routes of administration and conduct dose-response analyses to enable valid efficacy comparisons.