Introduction

Inflammatory pain, resulting from inflammation and tissue injury, constitutes a prominent facet of nociceptive experiences (Devries et al. 2017). Notably, women exhibit a higher prevalence and heightened severity of pain as compared to men (Mogil 2020). Injuries, infections, autoimmune illnesses, and chronic inflammatory diseases have been linked to pain (Pahwa et al. 2023). The precise prevalence of inflammatory pain across the human populace remains intricate to ascertain, given its contingent nature, dictated by underlying causative factors and demographics under examination. Notably, the Global Burden of Disease Study found that musculoskeletal illnesses, frequently entailing manifestations of inflammatory pain, accounted for 7.8% of years lived with disability on a global scale (Cieza et al. 2021). This suggests a substantial problem imposed by inflammatory pain worldwide. Chronic inflammatory pain is characterized by diverse manifestations such as spontaneous pain, allodynia, and hyperalgesia (Pahwa et al. 2023). While non-steroidal anti-inflammatory drugs (NSAIDs) serve as a cornerstone therapy for chronic inflammatory pain, their repetitive usage has been linked to unpleasant cardiovascular and gastrointestinal effects (Orlando et al. 2010). As a result, the quest for novel therapeutic avenues that exhibit a favorable side effect profile within the context of inflammatory pain management is necessary.

Nowadays, we rely on models that emulate inflammatory processes and possess translational significance to better understand inflammatory pain. In particular, the utilization of carrageenan or complete Freund’s adjuvant (CFA; a suspension of desiccated mycobacterium in paraffin oil and mannide monooleate that induces inflammation, tissue necrosis, and ulceration) stands as pertinent strategies, given their propensity to evoke mechanical allodynia and edema, mirroring aspects of inflammatory pain (Morales-Medina et al. 2019; McCarson and Fehrenbacher 2021). Both models have been extensively employed to scrutinize the efficacy of prospective analgesic and anti-inflammatory agents (Stein et al. 1988; Bautista-Carro et al. 2021).

Numerous secondary plant metabolites may have a plethora of physiological effects in humans (Flores et al. 2016; Wang et al. 2017). Chinese traditional medicine is replete with a diverse array of substances bearing analgesic and anti-inflammatory attributes, including alkaloids, saponins, flavonoids, terpenoids, coumarin, among others (Wang et al. 2017). Notably, the phytoalexin trans-resveratrol (1; 3,5,4′-trihydroxystilbene) is present in sources such as grapes, peanuts, berries, and turmeric, constituting a promising candidate (Bastianetto et al. 2015; Flores et al. 2016). Resveratrol has been recognized to elicit anti-inflammatory and antioxidant effects in vivo and in vitro (Bastianetto et al. 2015). Therefore, we conducted the current investigation to assess the plausible anti-inflammatory and analgesic attributes of resveratrol in two rat models of inflammatory pain, encompassing male and female rats. These well-recognized models serve as valuable tools for translational inquiries aimed at unraveling the complex landscape of inflammatory pain and its modulation.

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Materials and Methods

Animals

Male (n = 42) and female (n = 43) Wistar rats (Rattus norvegicus), 2–3 months of age, were sourced from the Centro de Investigación en Reproducción Animal as part of CINVESTAV, Mexico, animal facility and were housed three to four per cage in a room maintained on a 12-h light/dark cycle with ad libitum access to food and water. Two groups of rats were used for our experiments to test biological properties of resveratrol. The first group was treated with carrageenan and the second group with CFA. All procedures complied with ARRIVE guidelines (Kilkenny et al. 2010). Every effort was made to minimize animal suffering. If animals showed signs of suffering before or during experimentation, they were immediately evaluated by a veterinarian and, if necessary, euthanized.

Mechanical Threshold Testing

Rats were acclimated to a stainless-steel grid inside individual Plexiglas boxes (20 × 13 × 13 × 10 cm; three opaque white walls and one transparent wall) for 60 min, and then basal mechanical thresholds were assessed with von Frey monofilaments (Stoelting Inc., IL, USA) (Morales-Medina et al. 2019; Bautista-Carro et al. 2021; Morales-Medina et al. 2023). For each animal, the medial plantar region of the left hind paw was stimulated with an incremental series of eight monofilaments of logarithmic stiffness. The 50% withdrawal threshold was determined using Dixon’s up-down method, modified by Chaplan et al. (1994). First, von Frey monofilament number 4.31 was applied exerting 2 g of force perpendicular to the plantar skin, causing slight flexion. If a positive response consisting of a rapid withdrawal of the paw within 6 s was produced, a smaller filament was applied. If a negative response was produced within 6 s, a larger filament was used. Once the basal mechanical thresholds were assessed, the paw thickness was determined with a caliper according to McCarson and Fehrenbacher (2021).

Administration of Inflammogens

After assessment of basal mechanical threshold and paw thickness, complete Freund’s adjuvant (CFA; Sigma, St. Louis, MO; 50 µl) was administered intradermally into the ventral mid-plantar left hind paw of rats (Stein et al. 1988). Mechanical responses, which were assessed as described above for the basal mechanical threshold tests and left hind paw edema, were measured 3 days after injection. A 3% carrageenan solution was prepared by dissolving carrageenan (Sigma, C1867-5G) in saline, heating to 37 °C, and vortexing (Morales-Medina et al. 2019; Bautista-Carro et al. 2021). After basal measurements, rats received an intradermal injection of the carrageenan solution (50 µl) into their ventral mid-plantar left hind paw. Mechanical responses and edema of the left hind paw were measured 4 h after injection.

Oral Administration

In rats treated with CFA on day 3 and carrageenan at 4 h post-administration, trans-resveratrol (purity 72%; batch number 2022838.3; Resvitále, Boca Raton, FL, USA) dosed at 50 mg/kg or vehicle (0.5% gum tragacanth in 0.1 M phosphate buffer saline pH 7.4) were administered orally by gavage after performing mechanical threshold tests, as previously described in Navarro-Cruz et al. (2017). Oral administration was performed by an intragastric tube (16 gauge with ball-shaped tip). von Frey filament assessment was performed at 15, 30, 60, 90, 120, 150, and 180 min after resveratrol administration. Finally, paw edema was measured.

Statistical Methods

The data was analyzed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). Mechanical threshold, paw edema, and area under the curve (AUC) data were analyzed using a two-way ANOVA followed by Sidak’s post hoc test. In all cases, p < 0.05 was considered statistically significant. The maximum achievable response (Emax %) was calculated for each animal as the maximum percentage change in mechanical threshold response from baseline after treatment (Morales-Medina et al. 2019). Subsequently, the Emax (%) data was analyzed using an unpaired t-test to compare the effects of resveratrol and control vehicle for male and female rats, separately.

Results and Discussion

Carrageenan and CFA Effects

Carrageenan administration reduced the mechanical thresholds as assessed by two-way ANOVA (Fig. 1A) [Treatment F (1, 76) = 586.0, p < 0.0001; Sex F (1, 76) = 0.8730, p = 0.3531; Interaction F (1, 76) = 0.01106, p = 0.9165] in both sexes. Increased the paw size as assessed by two-way ANOVA [Treatment F (1, 76) = 1239, p < 0.0001; Sex F (1, 76) = 15.54, p = 0.0002; Interaction F (1, 76) = 0.5622, p = 0.4557] in male and female rats and Sidak’s post hoc test shows differences between sexes 4 h post carrageenan **p = 0.0028 (Fig. 1B). Complete Freund’s adjuvant administration resulted in mechanical allodynia as assessed by two-way ANOVA [Treatment F (1, 86) = 346.0, p < 0.0001; Sex F (1, 86) = 0.8369, p = 0.3628; Interaction F (1, 86) = 0.01142, p = 0.9151] (Fig. 1C). Complete Freund’s adjuvant increased the paw size as assessed by two-way ANOVA [Treatment F (1, 86) = 1093, p < 0.0001; Sex F (1, 86) = 58.51, p < 0.0001; Interaction F (1, 86) = 17.22, p < 0.0001] in male and female rats and Sidak’s post hoc test shows differences between sexes (Fig. 1D).

Fig. 1
figure 1

Administration of inflammogens into the hind paw induced mechanical allodynia and paw edema. A significant effect of carrageenan on mechanical thresholds and paw size was observed at 4 h post-treatment compared to basal mechanical threshold (A and B). A reduction in mechanical thresholds and an increase in paw size were also observed at 3 days post-CFA (C and D) compared to BL. Sex differences in paw size were observed. The results are presented as mean ± SEM. ***p < 0.001 compared to the BL test. ##p < 0.01, ###p < 0.001 compared between sexes. Abbreviations: baseline, BL; 4 h, 4 h; and 3 days, 3d

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Effects of Resveratrol on the Carrageenan Model

Resveratrol increased mechanical thresholds at 15, 30, 60, 90, 120, 150, and 180 min post resveratrol administration. A two-way ANOVA showed a significant effect by increasing the mechanical thresholds at 15 and 120 min post-carrageenan between resveratrol and vehicle in females [Treatment F (1, 144) = 18.12, p < 0.0001; Time F (7, 144) = 2.465, p = 0.0203; Interaction F (7, 144) = 2.161, p = 0.04101], and males [Treatment F (1, 144) = 27.93, p < 0.0001; Time F (7, 144) = 2.039, p = 0.0540; Interaction F (7, 144) = 3.150, p = 0.0040] (Fig. 2A). The area under the curve of the effect of resveratrol post-carrageenan administration on mechanical allodynia, between 15- and 120-min post resveratrol or vehicle administration as shown by two-way ANOVA [Treatment F (1, 36) = 18.38, p = 0.0001; Sex F (1, 36) = 3.491, p = 0.0699; Interaction F (1, 36) = 0.006462, p = 0.9364] (Fig. 2B). When analyzing the Emax (%) data, a comparable pattern was observed with a significant effect in male rats after resveratrol administration but not in females (p = 0.0231 and 0.2925 respectively; Fig. 4A). Figure 2C shows the mean diameter of the left hind paw 4 h post-carrageenan administration and 180 min post resveratrol administration [Treatment F (1, 36) = 3.476, p = 0.0704; Sex F (1, 36) = 2.780, p = 0.1041; Interaction F (1, 36) = 2.934, p = 0.0954]. Resveratrol reduced paw diameter in males; Sidak’s post hoc test shows differences between males 180 min after resveratrol (Fig. 2C).

Fig. 2
figure 2

Temporal effect of resveratrol (1) administration on mechanical allodynia and paw edema in the rat carrageenan model. Mechanical allodynia caused by carrageenan is reduced 15 and 120 min after resveratrol administration in male and female rats (A). Resveratrol increased mechanical thresholds compared to vehicle in carrageenan-treated rats (B). Modifications in paw diameter after resveratrol treatment were only observed in male rats (C). The results are presented as mean ± SEM, n = 10 rats per group, *p = 0.05, **p < 0.01, and ***p < 0.001 compared to the BL test; #p = 0.05, ##p < 0.01, and ###p < 0.001 compared between sexes treated. Abbreviations: baseline, BL; 4 h, 4 h; and area under the curve, AUC

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Effects of Resveratrol on the CFA Model

Resveratrol increased mechanical thresholds (Fig. 3A) and showed mechanical response threshold at baseline at 3 days (3D) post-CFA administration and at 15, 30, 60, 90, 120, 150, and 180 min post resveratrol administration. A two-way ANOVA showed a significant effect increasing the mechanical thresholds at 15 and 120 min post resveratrol between resveratrol and vehicle in females [Treatment F (1, 168) = 42.92, p < 0.0001; Time F (7, 168) = 1.431, p = 0.1959; Interaction F (7, 168) = 2.418, p = 0.0220] and males [Treatment F (1, 160) = 60.03, p < 0.0001; Time F (7, 160) = 2.269, p = 0.0315; Interaction F (7, 160) = 3.319, p = 0.0025] (Fig. 3A). The area under the curve of the effect of resveratrol post CFA administration on mechanical allodynia, between 15- and 120-min post resveratrol or vehicle administration as shown by two-way ANOVA [Treatment F (1, 41) = 64.51, p < 0.0001; Sex F (1, 41) = 0.003254, p = 0.9548; Interaction F (1, 41) = 0.09397, p = 0.7607] (Fig. 3B). When analyzing the Emax (%) data, a similar pattern was observed, with a significant effect in female rats but not in males (p = 0.0161 and 0.1130, respectively; Fig. 4B). The mean diameter of the left hind paw 3 days post-CFA administration and 180 min post resveratrol or saline administration in male and female rats is illustrated in Fig. 3C [Treatment F (1, 41) = 0.01080, p = 0.9177; Sex F (1, 41) = 44.03, p < 0.0001; Interaction F (1, 41) = 0.1765, p = 0.6766] and Sidak’s post hoc test shows differences between sexes treated vehicle and resveratrol ###p =  < 0.0001.

Fig. 3
figure 3

The effect of oral resveratrol (1) on CFA-induced mechanical allodynia and paw edema. Mechanical allodynia caused by CFA is reduced 15 and 120 min after resveratrol administration in male and female rats (A). Resveratrol increased mechanical thresholds compared to vehicle in CFA-treated rats. The AUC analysis revealed an effect of resveratrol treatment (B). No between-group changes in paw diameter were observed after resveratrol treatment (C). The results are presented as mean ± SEM, n = 10 rats per group, *p = 0.05, **p < 0.01, and ***p < 0.001 compared to the BL test; #p = 0.05, ##p < 0.01, and ###p < 0.001 compared between sexes treated. Abbreviations: complete Freund’s adjuvant, CFA; baseline, BL; 3 days, 3d; and area under the curve, AUC

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Fig. 4
figure 4

The maximum response achievable (Emax%) resulting from the administration of resveratrol (1) in the carrageenan and CFA models. This assessment represents the highest percentage of alteration in mechanical threshold observed in the ipsilateral hind paw after resveratrol treatment calculated at the time points of 4 h and 3d for the carrageenan and CFA models (A and B, respectively). * indicates p < 0.05 for resveratrol versus vehicle in female and male rats. All data is presented as mean ± standard error of the mean (SEM). Abbreviations: complete Freund’s adjuvant, CFA; 4 h, 4 h; and 3 days, 3d

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Plant metabolites have been postulated as prospective reservoirs of medicinal solutions in pain research. As previously documented, carrageen and CFA administration has been observed to induce swelling and mechanical allodynia. In the present study, we evaluated the acute effects of resveratrol in nociception and inflammatory processes. In carrageen-treated rats, resveratrol increased mechanical thresholds within both sexes while notably reducing paw diameter exclusively among males. Furthermore, in rats subjected to CFA, resveratrol demonstrated an analogous elevation in mechanical thresholds in both male and female. Thereby, these results suggest that resveratrol possesses analgesic and anti-inflammatory attributes.

Increment of Mechanical Thresholds

In the present study, we observed that resveratrol elicited an elevation in mechanical thresholds among rats subjected to CFA treatment. A similar trend was observed using the Emax (%) analysis although this yielded statistical significance only in female rats. It is acknowledged that the administration of CFA is associated with sustained inflammation and a heightened state of mechanical hypersensitivity in rodent models. The peak of mechanical allodynia is observed on the third day following CFA administration in rats (Morales-Medina et al. 2019; Bautista-Carro et al. 2021). Within the context of the present investigation, we ascertained that resveratrol engendered a period of increased mechanical thresholds lasting 120 min within both male and female rats treated with CFA. Previous studies have examined the iterative administration of resveratrol in rats treated with CFA across distinct anatomical regions. Notably, intraplantar administration of CFA resulted in thermal hyperalgesia spanning from 2 to 48 h post-administration, concurrently inducing increased levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in the dermal tissue of male rats (Singh and Vinayak 2016). This treatment also correspondingly accentuated the concentration of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 within the spinal cord (Singh and Vinayak 2016). Acute administration of resveratrol mitigated CFA-induced thermal hyperalgesia, concurrently dampening iNOS and COX-2 levels in the dermal tissue and TNF-α, IL-1β, and IL-6 within the spinal cord (Singh and Vinayak 2016). Furthermore, CFA administration into the temporomandibular joint of male mice induced facial mechanical allodynia, a decline in short-chain fatty acids (acetic acid, propionic acid, and butyric acid) within the gut milieu, and induced microglia activation within the spinal cord (Ma et al. 2020). Sequential resveratrol administration was shown to alleviate mechanical allodynia and increased the concentration of short-chain fatty acids within the gut (Ma et al. 2020).

The increased levels of short-chain fatty acids being associated with the presence of Bacteroidetes and Lachnospiraceae within the gut (Ma et al. 2020). Likewise, CFA administered in the lumbar intervertebral disk, a model simulating lower back pain, elicited the activation of multiple pro-inflammatory agents, including iNOS, COX-2, PGE2, and engendering mechanical allodynia. Importantly, resveratrol intervention was observed to attenuate the expression of these inflammatory molecules at the site of injury, concurrently alleviating the mechanical allodynia (Genovese et al. 2022). Consequently, the present investigation stands as a pioneering endeavor, representing the first instance wherein the analgesic effects of resveratrol have been evaluated in both male and female rats following CFA administration.

Nonetheless, our study did not show that resveratrol exhibited anti-inflammatory properties in CFA-treated rats. In contrast, resveratrol demonstrated a reduction in paw edema, alongside a mitigation of elevated IL-6 and IL-10 concentration within the paw subsequent to intraplantar CFA administration in male rats (Silva et al. 2020). Furthermore, the induction of arthritis via subcutaneous CFA injection in female rats was linked to an increase in serum levels of TNF-α, malondialdehyde (MDA), and metalloprotinease-3 (MMP3) and reductions in IL-10 and glutathione (GSH) (Wahba et al. 2016). Remarkably, resveratrol administration exhibited a capacity to reduce the serum levels of TNF-α, MDA, and MMP3, while increasing the levels of IL-10 and GSH (Wahba et al. 2016). This study also revealed the protective effect of resveratrol against joint cartilage degradation (Wahba et al. 2016), suggesting a potential anti-inflammatory effect. Notably, CFA administered within the lumbar intervertebral disk increased oxidative stress markers, including MDA, GSH, catalase (CAT), and superoxide dismutase (SOD), with resveratrol administration being observed to counteract the elevation of these molecular species (Genovese et al. 2022). Moreover, the attenuation of several inflammatory agents linked to the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) pathway, including IL-18, TNF-α, and IL-1β, was noted as a result of resveratrol intervention (Genovese et al. 2022). The difference between our study and the others stems from the temporal aspect of resveratrol administration; while our study engaged resveratrol in an acute manner, most investigations encompassed a repetitive administration regimen. It is plausible that repetitive administration may be a requisite to elicit an anti-inflammatory response, whereas an acute application might solely elicit an anti-nociceptive effect.

In the quest for novel therapeutic avenues within pain research, the present findings centered on the utility of resveratrol as an anti-nociceptive agent hold notable significance. Importantly, the temporal effectiveness of resveratrol in this study appears noteworthy, particularly when compared with the outcomes stemming from the administration of Curcuma longa L., Zingiberaceae, or cerebrolysin (a multi-modal neuropeptide drug), wherein mechanical allodynia amelioration was observed at earlier time points (Morales-Medina et al. 2019; Bautista-Carro et al. 2021). These results suggest that resveratrol displays better analgesic-related effects in contrast to other agents tested with our laboratory setting.

Resveratrol’s influence on mechanical thresholds was demonstrated in rats subjected to carrageen treatment. Resveratrol evoked an elevation in mechanical thresholds in male and female rats, concomitantly leading to a reduction in male paw diameter. A similar trend was observed using the Emax (%) analysis although this yielded statistical significance only in male rats. Notably, a discernible sex-associated effect was observed in the anti-inflammatory effect. These findings align with prior research indicating that resveratrol, whether administered prophylactically or therapeutically, elicited a reduction in paw edema in male rats treated with carrageenan (Silva et al. 2020). Additionally, resveratrol exhibited a capacity to attenuate thermal hyperalgesia, paw edema, and the levels of 4-hydroxynoneal (4-HNE) and SIRT3 within the spinal cord of male rats exposed to carrageenan (Ilari et al. 2020). Moreover, the edema-inducing agents (votalin or carrageenan) in the ear or paw of female mice were evaluated (Yu et al. 2018). In both models, resveratrol inhibited edema while concurrently reducing prostaglandin E2 (PGE2) and COX-2 levels in the paw of carrageenan-treated mice.

Putative Mechanisms of Resveratrol

Various conceivable mechanisms may underlie resveratrol’s induction of analgesic effects. Resveratrol acts on diverse targets such as angiotensin-converting enzyme 2 (ACE2), sirtuins, COX-2, NFkB, reactive oxygen species (ROS), and NOS at different levels (Russo et al. 2023). It is important to consider that inflammation has been hypothesized as a mechanism linked to pain (Pahwa et al. 2023). Notably, resveratrol has exhibited the capacity to mitigate microglial cell activation within the spinal cord (Ma et al. 2020). Additionally, microglia’s propensity to induce oxidative stress–related molecules (MMP3, MDA, GSH, 4-HNE, CAT, and SOD) and their involvement in the NFkB pathway, facilitating the release of pro-inflammatory cytokines, is pivotal in the context of inflammation. Recently, sirtuins have been implicated in modulating ROS and neuroinflammation in microglial cells (Kincaid and Bossy-Wetzel 2013). Resveratrol’s consistent reduction of the mentioned molecules offers a proof to its potential in tempering microglial activation. Moreover, resveratrol can activate sirtuin 1 and SOD, promoting ACE2 upregulation and decreasing inflammatory markers (Russo et al. 2023). The rapid upregulation of COX-2 serves as a primary harbinger of inflammation, leading to the production of inflammatory mediators, including PGE2, and resveratrol reduces COX-2 levels, thereby inhibiting the NFkB pathway and the induction of various pro-inflammatory cytokines. Furthermore, emerging evidence underscores the capacity of repeated resveratrol administration to reshape the gut microbiota, revealing an innovative therapeutic modality (Ma et al. 2020). Significantly, perturbations in gut microbial balance are frequent among individuals experiencing chronic pain (Minerbi et al. 2019). These mechanisms may elucidate how resveratrol reduces paw edema in carrageen-treated but not in CFA-treated male rats. While inflammation is established in the CFA-treated rats, the inflammation is very active in carrageen-treated animals.

In the clinic, there is a preponderance of pain prevalence and heightened sensitivity in women; however, the preclinical milieu largely relies on male rodent models (Mogil 2020). Remarkably, recent research by Mogil (2020) reported that male rodents presented heightened sensitivity to novel pain-relieving pharmaceuticals. In this regard, resveratrol’s anti-nociceptive potential has been evidenced across diverse animal models of inflammatory pain (Singh and Vinayak 2016; Wahba et al. 2016; Genovese et al. 2022). However, most studies have been only carried out in male rodents, omitting a direct comparison between sexes (Singh and Vinayak 2016; Wahba et al. 2016; Genovese et al. 2022), thus accentuating a partial and oversimplification of pain mechanisms that inadequately accounts for sex as a relevant biological variable.

Conclusion

Resveratrol’s manifestation of analgesic effects within diverse models of inflammatory pain is well-established. Our present findings show that resveratrol induces these effects devoid of sexual differences or model disparities, accentuating its viability as a reliable therapeutic candidate for the multifaceted landscape of inflammatory pain.