Introduction: Inflammatory pain is a significant global clinical challenge that involves both unpleasant sensory and emotional experiences. The treatment of pain is imminent, and we are committed to seeking new analgesics for pain relief. Transcrocetin meglumine salt (TCMS), a saffron metabolite derived from the crocin apocarotenoids, has exhibited the ability to cross the blood-brain barrier and exert neuroprotective effects. In this study, we aimed to investigate whether TCMS could ameliorate complete Freund’s adjuvant (CFA)-induced inflammatory pain in mice and elucidate its underlying mechanisms. Methods: Here, we established an inflammatory pain model in mice by injecting CFA into the left hind paw. Three days later, we administered intraperitoneal injections of TCMS (10 mg/kg) or saline to the animals. We examined mechanical allodynia, thermal hypersensitivity, and anxiety behavior. Furthermore, the activation of glial cells and proinflammatory cytokines in the spinal cord were detected. Results: Our results showed that TCMS significantly reversed the mechanical allodynia and thermal hypersensitivity in the CFA-injected mice. Furthermore, TCMS administration effectively inhibited the activation of microglia and astrocytes in the spinal cord induced by CFA. Additionally, TCMS suppressed the production and release of spinal proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, in CFA-injected mice. Conclusion: Taken together, our findings demonstrate that TCMS holds promise as an innovative analgesic due to its ability to ameliorate inflammatory reactions.

Inflammatory pain poses a substantial clinical challenge, severely impacting patients’ quality of life and imposing a significant economic burden. It is characterized by a notable decrease in the threshold for pain induction, resulting in persistent nociceptive hypersensitivity, including allodynia and hyperalgesia [1]. Individuals experiencing inflammatory pain often endure unpleasant subjective sensory and emotional sensations, which frequently restrict their daily activities and work capabilities, leading to productivity loss [2, 3]. Unfortunately, the current range of clinical analgesic medications for chronic pain is limited, with some exhibiting serious side effects [4, 5]. Consequently, there is an urgent need to explore novel analgesic compounds for the treatment of chronic pain.

Accumulating evidence underscores the crucial involvement of glial cells, particularly astrocytes and microglia, in the pathogenesis of chronic pain. In the central nervous system (CNS), microglia serve as the first immune cells that respond to changes, whereas astrocytes represent the most abundant glial cell population [6]. Moreover, activated microglia and astrocytes release inflammatory cytokines and mediators that enhance the neuroexcitability of nociceptive pathways, thereby contributing to both peripheral and central sensitization [7, 8].

In recent years, there has been significant interest in the use of traditional Chinese herbal medicine for pain management. Previous studies have demonstrated that saffron has anti-inflammatory [9], anti-nociceptive [9], and anxiolytic effects [10, 11]. Moreover, saffron has been found to downregulate the expression of proinflammatory cytokines, inducible enzymes, adhesion molecules, and chemokines, all of which play crucial roles in regulating various inflammatory processes within the immune system [12]. Additionally, saffron exhibits neuroprotective properties, including the improvement of cognitive dysfunction, enhancement of memory and learning abilities, inhibition of neuronal apoptosis, promotion of angiogenesis, and prevention of cerebral ischemia-reperfusion injury [13‒17]. In our study, we have specifically chosen transcrocetin meglumine salt (TCMS) (Fig. 1a) extracted from saffron (Crocus sativus L.) to investigate its potential impact on pain management [18]. Lautenschlager et al. [19] have reported that transcrocetin can penetrate the blood-brain barrier and reach the CNS, which accounts for saffron’s activity within the CNS. Collectively, these findings suggest that TCMS may play a significant role in the CNS regulation. However, it remains unclear whether TCMS is involved in the regulation of inflammatory pain, and this aspect requires further investigation.

Fig. 1.

TCMS attenuates mechanical allodynia and thermal hypersensitivity under CFA-induced inflammatory pain conditions. a TCMS chemical structure. b Schematic representation of the experimental protocols. c Significant swelling of the left hind paw of mice was observed 3 days after CFA injection. Mechanical allodynia (d) and thermal hyperalgesia (e) in mice were detected at baseline, before the test, and 1 d and 2 d after administration of TCMS/saline (10 mg/kg). Results are expressed as mean ± SEM. n = 8 mice per group. *p < 0.05, ****p < 0.0001, CON + Veh versus CFA + TCMS.

Fig. 1.

TCMS attenuates mechanical allodynia and thermal hypersensitivity under CFA-induced inflammatory pain conditions. a TCMS chemical structure. b Schematic representation of the experimental protocols. c Significant swelling of the left hind paw of mice was observed 3 days after CFA injection. Mechanical allodynia (d) and thermal hyperalgesia (e) in mice were detected at baseline, before the test, and 1 d and 2 d after administration of TCMS/saline (10 mg/kg). Results are expressed as mean ± SEM. n = 8 mice per group. *p < 0.05, ****p < 0.0001, CON + Veh versus CFA + TCMS.

Close modal

The purpose of our study was to evaluate the analgesic effect of TCMS on an inflammatory pain model induced by intraplantar injection of complete Freund’s adjuvant (CFA). We observed that TCMS had some analgesic-compatible effect and showed a certain anxiolytic-like tendency in CFA-injected mice. Moreover, we conducted investigations to elucidate the underlying mechanism and discovered that TCMS could effectively reduce the activation of spinal glial cells following CFA treatment, as well as suppress the expression of spinal inflammatory cytokines, namely, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). Thus, these results indicate that TCMS has the potential to serve as an efficacious analgesic for pain management in the future.

Animals

The experiments were performed using male C57BL/6 mice, aged 6–8 weeks and weighing 20–30 g. The mice were housed under standard conditions, which included a 12-h light/dark cycle with lights on at 07:00, a controlled temperature of 25 ± 1°C, and a relative humidity of 60 ± 5%. Throughout the study, the animals had unrestricted access to food and water. We made every effort to minimize the number of animals used and to ensure their well-being, thus minimizing any potential suffering.

Experimental Procedure

Mice were injected with 10 μL CFA (Sigma) into the left hind paw to induce inflammatory pain model, while the control groups received 10 μL of sterile saline in the same paw. TCMS, purchased from MCE (Cat. No.: HY-42937, USA), was dissolved in sterile DMSO (final concentration 0.1%) and then diluted with sterile saline to achieve a concentration of 1 mg/mL and injected intraperitoneally into the experimental group. Two days after establishing the model, mice in the experimental group were administered the same dose (10 mg/kg) of either TCMS or sterile saline (vehicle) via intraperitoneal injection. Prior to performing any experiments, animals were allowed to acclimate to the laboratory conditions for approximately 1 week. All behavioral experiments were conducted at the same time of day, and mice were adapted to the test room for 30 min before each behavioral test. At the end of the behavioral experiment, following 3 days of continuous TCMS administration (10 mg/kg), the animals were euthanized under deep anesthesia, and the L4–5 spinal cord was collected for Western blot and immunofluorescence detection. A schematic representation of the experimental protocols is shown in Figure 1b.

Mechanical Allodynia

The mechanical allodynia, indicated by the paw withdrawal threshold (PWT), was assessed using the von Frey filament following the up-down algorithm as described by Chaplan [20]. Prior to the test, the animals were individually placed in a clear plastic box with a raised metal mesh grid and allowed to acclimate to the test room for 30 min. Through the mesh floor, the von Frey filament was applied vertically to the center of the left hind paw surface and held there for three to 4 s. A minimum interval of 5 min was observed between two consecutive stimuli, and each animal underwent five tests. The 50% withdrawal threshold values were calculated and considered as the PWT.

Thermal Hyperalgesia (Hargreaves Test)

The measurement of thermal hypersensitivity, referred to as the paw withdrawal latency (PWL), was conducted following a previously described method [21]. A plantar hot plate instrument (Ugo Basile, Italy) was utilized to assess thermal hypersensitivity. Prior to applying the thermal stimulus, animals were placed in a transparent plastic case for a 30-min adaptation period. The radiant heat source was stimulating the middle of the plantar surface of the left hind paw. The heat was automatically turned off when the animal licked or lifted its heated paw, and the PWL was a plantar hot plate instrument (Ugo Basile, Italy) was utilized to assess thermal hypersensitivity. Prior to applying the thermal stimulus, animals were placed in a transparent plastic case for a 30-min adaptation period.

Open Field Test

As reported previously [22], the open field test (OFT) was done in a white opaque plastic box with dimensions of 45 × 45 × 45 cm3. Prior to the experiments, mice were allowed to acclimate to the testing condition and light level for 30 min. Subsequently, the mice were individually placed in the box and given a 15-min exploration period. After each test, the box was cleaned using a 75% ethanol solution. The ANY-maze software (Stoelting, Wood Dale, USA) was utilized to record and quantify the movement of the mice. Assessment of the total distance traveled for locomotor activity.

Elevated plus Maze Test

The elevated plus maze test (EPM) was conducted following a previously described procedure [23]. Briefly, the animals were positioned at the junction of the open and closed arms, facing away from the experimenter. A camera positioned above the maze was used to record the entries into and duration spent in each arm for a duration of 5 min. Clean the maze with 75% ethanol before the next mouse was trained. The movements were traced and analyzed by the ANY-maze software.

Immunofluorescence Staining

The animals were administered intraperitoneal injection of sodium pentobarbital (50 mg/kg) for anesthesia. They were then transcardially perfused with 20 mL of phosphate-buffered saline (PBS), followed by 20 mL of 4% paraformaldehyde. The L4–5 spinal cord was carefully removed and fixed in paraformaldehyde at 4°C for additional 48 h. Afterward, the spinal cord was sequentially transferred to 10%, 15%, and 30% sucrose solutions until saturation. The samples were then freeze-mounted in optimal cutting temperature compound and sliced into 30 μm at −20°C using a freezing microtome (NX50, Thermo, USA). The slices were blocked at room temperature for 1 h with 5% normal donkey serum in 0.3% Triton-X in PBS (TPBS). Next, the slices were incubated overnight at 4°C with primary antibodies at the following dilution ratios: anti-rabbit ionized calcium binding adapter molecule 1 (Iba-1) (1:100; 17,198, Cell Signaling Technology, USA) and anti-mouse glial fibrillary acidic protein (GFAP) (1:200; 3,670, Cell Signaling Technology, USA). After being washed three times with PBS, the slices were incubated at room temperature for 2 h with the appropriate secondary antibodies at 1:200 (donkey anti-rabbit, ab150064 and donkey anti-mouse, ab150105, Abcam, USA). The slices were then washed 3 times with PBS. Before visualization, DAPI (ab104139, Abcam, USA) was applied to stain the nuclei. Images were acquired by a fluorescence microscope (VS120, Olympus, Japan) and analyzed by ImageJ (NIH, Bethesda, MD, USA).

Western Blot Analysis

Following anesthesia exposure, animals were sacrificed for harvesting of the spinal cord L4–5. The tissues were homogenized with RIPA buffer (Cat. No.: HY-K1001, MCE) with PMSF (CAS No.: 329-98-6, MCE). After homogenization, the protein extracts were centrifuged at 12,000 g for 15 min at 4°C, and the supernatants were collected. The protein concentrations in the supernatants were determined using the bicinchoninic acid assay (Beyotime, China). Protein sample was separated by 10% SDS-PAGE gel and then transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in PBS containing Tween-20 for 2 h at room temperature and subsequently incubated at 4°C overnight with appropriate primary antibodies: anti-rabbit TNF-α (1:1,000; A11534, ABclonal, China), anti-rabbit IL-1β (1:1,000; A16288, ABclonal, China; 27,989, CST, USA), anti-rabbit IL-6 (1:1,000; ab259341, Abcam, USA; A2447, ABclonal, China), and anti-rabbit β-actin (1:3,000; AC026, ABclonal, China). After three washes with TBST, the membranes were incubated with HRP-conjugated secondary goat anti-rabbit antibody (1:5,000; HA1001, HuaBio, China) for 2 h at RT, and then washing 3 times in TBST. Signal detection was performed using the ECL luminescence reagent (Absin, China) and acquired by ChemiDoc Touch Imaging System (Bio-Rad, USA). The results were analyzed and quantified by ImageJ (NIH, Bethesda, MD, USA).

Data Analyses

All results were expressed as mean ± SEM and analyzed with GraphPad Prism 9.0. Two-way repeat measure ANOVA followed by Sidak’s post hoc test was used to compare the differences in mechanical allodynia and thermal hypersensitivity. And other data were analyzed by one-way ANOVA with Tukey’s post hoc test. p < 0.05 was set statistical significance.

TCMS Alleviates CFA-Induced Mechanical Allodynia and Thermal Hypersensitivity

The inflammatory pain model was established by injecting CFA into the left hind paw of mice, resulting in significant swelling observed in the left hind paw (Fig. 1c) compared to the saline group. The behavioral tests revealed a noticeable reduction in PWT (Fig. 1d) and PWL (Fig. 1e) following CFA injection, as compared to the control group. Moreover, when compared to saline injection, TCMS alleviated mechanical allodynia (Fig. 1d) and thermal hypersensitivity pain (Fig. 1e) on the day of administration. These results indicate that TCMS treatment temporarily alleviates CFA-induced inflammatory pain.

Anxiolytic Effects of TCMS on Mice Injected with CFA

Meanwhile, we employed the EPM and OFT to estimate the effects of TCMS on anxiety-like behavior after CFA treatment. In the EPM, CFA-treated mice exhibited no significant differences in the entries for the open arms compared to the control groups; however, there was a notable decrease in the percentage of time spent in the open arms (Fig. 2a–c). Similarly, the OFT results showed that CFA-injected mice reduced the distance moved in the center area (Fig. 2e, f) compared to the control group. These results indicate that CFA-treated mice exhibited anxiety-like behavior compared with the control group. After TCMS treatment, CFA-treated mice spent a longer time in the open arms of the EPM (Fig. 2c). Regarding the OFT, TCMS exhibited a tendency to increase the distance traveled in the central area compared with the control group (Fig. 2f). Notably, no significant differences were observed among all groups in terms of the total distance moved in both the EPM (Fig. 2d) and OFT (Fig. 2g), suggesting that the locomotor activity of mice was unaffected by CFA and TCMS. All these findings suggest that TCMS shows promising potential in ameliorating CFA-induced anxiety-like behavior.

Fig. 2.

Effects of TCMS on emotional functions measured by the EPM (a-d) and OFT (e-g). TCMS tends to alleviate the anxiety-like behaviors on CFA-injected mice. a Representative tracks in the EPM among the four groups. b Number of open arms entries in the EPM. c Percentage of time spent in the open arm in the EPM. d Total distance tracked in the EPM. e Representative trajectory diagram in the OFT for the four groups. f Distance moved in the central area in the OFT. g Total distance traveled in the OFT. Data are presented as mean ± SEM. n = 8 mice per group. *p < 0.05, **p < 0.01.

Fig. 2.

Effects of TCMS on emotional functions measured by the EPM (a-d) and OFT (e-g). TCMS tends to alleviate the anxiety-like behaviors on CFA-injected mice. a Representative tracks in the EPM among the four groups. b Number of open arms entries in the EPM. c Percentage of time spent in the open arm in the EPM. d Total distance tracked in the EPM. e Representative trajectory diagram in the OFT for the four groups. f Distance moved in the central area in the OFT. g Total distance traveled in the OFT. Data are presented as mean ± SEM. n = 8 mice per group. *p < 0.05, **p < 0.01.

Close modal

TCMS Application Antagonizes Microglia and Astrocyte in the Spinal Cord Activation following CFA Injection

Previous studies have demonstrated the significant involvement of spinal glial cell activation, particularly astrocytes and microglia, in the initiation and progression of CFA-induced inflammatory pain [24, 25]. To further clarify the mechanism of TCMS-mediated anti-inflammatory effects, immunostaining was conducted in the spinal cord to detect the presence of GFAP as a marker for astrocytes and Iba-1 for microglia. Our results demonstrated that mice injected with CFA displayed a notable increase in the fluorescence intensity of Iba-1 (Fig. 3a, c) and GFAP (Fig. 3b, d) compared to the control group. However, treatment with TCMS significantly reduced the fluorescence intensity of Iba-1 (Fig. 3a, c) and GFAP (Fig. 3b, d) compared to the CFA-injected mice. These results strongly suggest that TCMS has the ability to inhibit glial activation triggered by inflammatory pain, thereby exerting analgesic effects.

Fig. 3.

Effect of TCMS treatment on the activation of microglia and astrocytes in the spinal cord of CFA-induced inflammatory pain mice. Representative images for Iba-1 (a) and GFAP (b) in the spinal cord after CFA injection and TCMS (10 mg/kg) administration suppressed the enhanced activation of Iba-1 and GFAP. Mean immunofluorescence intensity of Iba-1 (c) and GFAP (d). Scale bars = 200 μm and 20 μm (magnification). Data are expressed as mean ± SEM. n = 3 per group. *p < 0.05.

Fig. 3.

Effect of TCMS treatment on the activation of microglia and astrocytes in the spinal cord of CFA-induced inflammatory pain mice. Representative images for Iba-1 (a) and GFAP (b) in the spinal cord after CFA injection and TCMS (10 mg/kg) administration suppressed the enhanced activation of Iba-1 and GFAP. Mean immunofluorescence intensity of Iba-1 (c) and GFAP (d). Scale bars = 200 μm and 20 μm (magnification). Data are expressed as mean ± SEM. n = 3 per group. *p < 0.05.

Close modal

TCMS Administration Mitigates CFA-Induced Expression of Proinflammatory Cytokines TNF-α, IL-6, and IL-1β in the Spinal Cord

In response to activation of spinal astrocytes and microglia, the production and release of proinflammatory cytokines occur, which can directly sensitize nociceptive sensory neurons and contribute to the development of pain [26]. Following our demonstration that TCMS suppressed spinal microglia and astrocyte activation, we wondered whether it could also downregulate the expressions of proinflammatory cytokines released by activated glial cells. Thus, we detected spinal protein levels of TNF-α, IL-6, and IL-1β. As shown in Fig. 4a–c, the protein level of proinflammatory cytokines TNF-α (Fig. 4a), IL-6 (Fig. 4b), and IL-1β (Fig. 4c) exhibited a significant increase in mice treated with CFA compared to the control group. Conversely, in the TCMS-treated group, the levels of proinflammatory cytokine proteins were lower than those observed in CFA-treated mice (Fig. 4a–c). The original WB images are shown in the Figure S1 (for all online suppl. material, see https://doi.org/10.1159/000534607), and the expected protein molecular weights are as described previously [27]. Our results indicate that TCMS exerts anti-inflammation effects and subsequently attenuates CFA-triggered inflammatory pain.

Fig. 4.

TCMS inhibited the upregulation of TNF-α (a), IL-6 (b), and IL-1β (c) in the spinal cord of CFA-injected mice. Data are expressed as mean ± SEM. n = 6 mice per group. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 4.

TCMS inhibited the upregulation of TNF-α (a), IL-6 (b), and IL-1β (c) in the spinal cord of CFA-injected mice. Data are expressed as mean ± SEM. n = 6 mice per group. *p < 0.05, **p < 0.01, and ***p < 0.001.

Close modal

In the current study, we evaluated the analgesic potential and mechanism of TCMS. Our results suggest that TCMS effectively alleviated mechanical allodynia and thermal hypersensitivity in CFA-injected mice and exhibited a tendency to improve anxiety-like behavior. Next, we investigated the inflammatory pathway and found that TCMS suppressed the activation of spinal microglia and astrocytes and reduced the release of proinflammatory cytokines (such as TNF-α, IL-1β, and IL-6) induced by CFA administration.

Spinal glial cells, especially astrocytes and microglia, play a crucial role in chronic pain by being activated in response to noxious stimuli and inflammation, as manifested by their morphological changes, increased the expression of their specific markers GFAP or Iba-1, and activation of glia-specific proinflammatory pathways [28]. Consistent with previous studies, our finding showed that CFA injection upregulated the level of GFAP and Iba-1 in the spinal cord. Activated spinal glial cell lose their homeostatic functions and contribute to inflammation, which subsequently result in microglia, astrocyte, and neuron damages [29, 30]. Likewise, inflammatory conditions also facilitate the development of mechanical allodynia and thermal hypersensitivity and accelerate disease progression. Therefore, maintaining the resting state of microglia and astrocytes is very important for CNS homeostasis. In this study, we first showed that intraperitoneally administration of TCMS (10 mg/kg) could suppress the activation of spinal astrocytes and microglia, thus playing an analgesic role in CFA-injected mice.

Growing evidence suggests that inflammatory cytokines are produced in response to various noxious stimuli and play a crucial role in the initiation and maintenance of pain by promoting neutrophil recruitment and inflammatory propagation [31, 32]. Activated spinal astrocytes and microglia release numerous inflammatory cytokines such as TNF-α, IL-6, and IL-1β, which subsequently act on glial cells or neurons to enhance neuronal excitability or synaptic transmission, playing a significant role in pain states [28, 33, 34]. Furthermore, it has also been reported that spinal injection of TNF-α, IL-6, and IL-1β produces marked pain behavior by activating astrocytes [35‒37]. The interaction between glial cells and proinflammatory cytokines contributes to pain development. Consistently, our results suggested that CFA injection induced an increase in proinflammatory cytokines in the spinal cord, and TCMS attenuated the production of proinflammation cytokines, which may be a potential mechanism for its analgesic effect.

It has been reported that TCMS acts as a high-affinity antagonist of the NMDA receptor [38]. The NMDA receptor in the spinal cord has been implicated in the transmission of inflammatory pain, and inhibiting its activation has shown to reduce inflammatory pain [39]. Consistent with this, our study has demonstrated that TCMS could alleviate inflammatory pain and inhibit the inflammatory reaction. Furthermore, Wang et al. [40] have demonstrated that crocetin effectively reduced neuropathic pain induced by spared nerve injury while also significantly suppressing oxidative stress and neuroinflammation. In clinical trials, saffron has shown efficacy not only in relieving symptoms of chemotherapy-induced peripheral neuropathy [41], but also in exerting anxiolytic and antidepressant effects [42, 43]. In conclusion, saffron and its related compounds have the potential to be involved in the development of chronic pain.

In conclusion, our study showed that application with TCMS reduced mechanical allodynia and thermal hyperalgesia induced by CFA and then restrained the spinal glial cell activation and proinflammatory cytokine production. These findings elucidate the molecular mechanisms underlying the analgesic effect of TCMS. Thus, our results suggest that TCMS could be a promising candidate for pain management in the future.

All animal experiments were performed in accordance with the guidelines approved by the Animal Care and Use Committee of the Zhejiang University (protocol number ZJU20230108).

The authors have no conflicts of interest to declare.

This study was supported by the National Natural Science Foundation of China (No. 82171176 and No. 82001424) and the Key Program of the Natural Science Foundation of Zhejiang, China (No. LZ19H090003).

Qing Qiao and Gang Chen designed the study, wrote the manuscript, analyzed the data, and reviewed and edited the draft. Qing Qiao, Dandan Yao, Yongjie Wang, and Shuxia Zhang performed the experiments. All the authors read and approved the final manuscript.

The data used to support the findings of this study are included within the article. Further inquiries can be directed to the corresponding author.

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