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. 2024 Feb 15;7(3):797–808. doi: 10.1021/acsptsci.3c00321

Linderane Attenuates Complete Freund’s Adjuvant-Induced Pain and Anxiety in Mice by Restoring Anterior Cingulate Cortex Microglia M2 Polarization through Activating Cannabinoid 2 Receptor

Yue Chen †,‡, Yan Qin ‡, Ying Gao ‡, Saiying Wang ‡, Qinhui Wang ‡, Xiuling Tang ‡, Zheng Rong ‡, Caiyan Cheng ‡, Longfei Li ‡, Yuan Xu ‡, Qi Yang ‡, Yuping Tang †,*, Minggao Zhao ‡,*, Le Yang ‡,*
PMCID: PMC10928880  PMID: 38481693

Abstract

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Chronic pain is a common condition that causes negative emotions as the disease progresses. The anterior cingulate cortex (ACC) is a key region in the integration of nociceptive perception and emotional response in chronic pain. Linderane (LDR) is an active ingredient from Linderae radix, a traditional Chinese medicine with anti-inflammatory, analgesic, and antibacterial properties. In this study, the analgesic and antianxiety effects of LDR were evaluated using a complete Freund’s adjuvant (CFA)-induced inflammatory pain model in C57BL/6 male mice. Mechanical and thermal pain sensitivity were measured through plantar mechanical analgesia and hot plate apparatus, and anxiety-like behavior was evaluated by open field and elevated plus maze tests. The results showed that LDR-alleviated CFA-induced pain and anxiety, reduced the release of inflammatory cytokines, and inhibited ACC microglial activation. Target prediction, molecular docking, and cellular thermal shift assay demonstrated that LDR could bind to the cannabinoid 2 receptor (CB2R), a key component of the endocannabinoid system with an important role in regulating pain and related emotions. Moreover, both the analgesic effect of LDR and its regulation of microglia polarization were reversed by a CB2R antagonist (SR144528) treatment. Therefore, our results suggested that LDR exerted analgesic effects by regulating microglial polarization in ACC via CB2R activation.

Keywords: linderane, chronic pain, anterior cingulate cortex, microglia, cannabinoid 2 receptor

Introduction

Chronic pain is a painful experience associated with tissue damage or potential tissue damage that manifests as hyperalgesia and allodynia.1 Epidemiological surveys indicate that the prevalence of chronic pain is approximately 35% in China.2 Chronic pain can also cause emotional disorders, such as anxiety and depression, affecting the quality of life and, in severe cases, increasing the risk of suicide, imposing a huge burden on families and society.3 Drug therapy is the primary treatment for chronic pain. Common analgesics include nonsteroidal anti-inflammatory drugs and opioids, but long-term use causes drug addiction and other adverse reactions.4 Therefore, identifying safe and effective drugs for the treatment of chronic pain is crucial.

The anterior cingulate cortex (ACC) is a key region in the integration of nociceptive perception and emotional response in chronic pain.5,6 The ACC primarily receives the projections from the mediodorsal thalamic nucleus, and is significantly activated during chronic pain, evidenced by functional imaging data, electrophysiological and behavior studies.7 Furthermore, ACC is also involved in the emotional response of pain. For example, oxytocin in the anterior cingulate cortex attenuates neuropathic pain and emotional anxiety by inhibiting presynaptic long-term potentiation.8 Taken together, regulating the function of ACC can produce analgesic and antianxiety effects.

In the central nervous system (CNS), microglia respond to neuroinflammation and regulate pain and affective disorders by phagocytosis or releasing pro-inflammatory cytokines and their metabolites.9 For instance, microglia polarize toward the M1 or M2 phenotypes at different stages of CNS injury. M1 microglia release pro-inflammatory factors and free radicals that impair brain repair and regeneration, such as tumor necrosis factor-a (TNF-α), inducible nitric oxide synthase (iNOS), interleukin (IL)-6, and IL-1β. In contrast, M2 microglia help decrease inflammation by enhancing phagocytosis and releasing anti-inflammatory factors such as arginase-1 (Arg-1), mannose receptor (i.e., CD206), and IL-10.10 Therefore, promoting the polarization of microglia from the M1 to the M2 phenotype could promote CNS repair while limiting secondary inflammation-mediated damage.11

The endocannabinoid system (ECS) is a neuromodulatory system that plays an important role in neural transmission and neuroinflammation. Cannabinoid receptors include cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R).12 Among them, CB2R belongs to G protein-coupled receptor and is mainly involved in immune regulation in the brain.13 Selective activation of CB2R can reduce neuroinflammation after traumatic brain injury through macrophage polarization14 and alleviate neuropathic pain by preventing the transition of microglia to proinflammatory stage.15 These findings suggest the important role of CB2R in regulating neuro-inflammation and pain.

Linderae radix, the dried root of Lindera aggregata (Sims) Kosterm, is a traditional Chinese folk medicine possessing anti-inflammatory, analgesic, hepatoprotective and neuroprotective effects.16−19 Linderane (LDR), a furan sesquiterpenoid (Figure 1A), is the main bioactive component isolated from Linderae radix.20,21 Previous studies have shown that LDR could suppress hepatic gluconeogenesis and protect pancreatic β cells from streptozotocin (STZ)-induced oxidative damage.22,23 However, the effects of LDR on regulating sensation and emotion in chronic pain are still unclear. Therefore, the effects of LDR on pain and anxiety were evaluated using the complete Freund’s adjuvant (CFA)-induced inflammatory pain mouse model,24 and the role of LDR in microglia polarization in the ACC region was also explored in this research.

Figure 1.

Figure 1

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LDR could cross the blood–brain barrier. (A) Chemical structure of LDR. (B) UPLC analyses showed that LDR could cross the blood–brain barrier.

Results

LDR Alleviates CFA-Induced Hyperalgesia

First, ultra-performance liquid chromatography (UPLC) indicated that LDR could cross the blood–brain barrier (Figure 1B). CFA-induced chronic inflammatory pain is a commonly used model for studying chronic pain;25 thus, we selected this model to investigate the effects of LDR on chronic pain (Figure 2A). As expected, mice in the CFA-treated group had lower mechanical and thermal pain thresholds than those in the control group [F (4, 35) = 50.05, P < 0.001, F (4, 35) = 39.31, P < 0.001, F (4, 35) = 64.52, P < 0.001, F (4, 35) = 70.55, P < 0.001, Figure 2B, F (4, 35) = 147.4, P < 0.001, F (4, 35) = 195.6, P < 0.001, F (4, 35) = 124.2, P < 0.001, F (4, 35) = 92.32, P < 0.001, Figure 2C], and CFA treatment caused significant swelling of the hind paws [F (4, 35) = 55.99, P < 0.001, F (4, 35) = 63.47, P < 0.001, F (4, 35) = 53.55, P < 0.001, F (4, 35) = 48.79, P < 0.001, Figure 2D]. However, LDR administration (50 mg/kg) significantly reversed these effects (Figure 2B,C). In addition, the hind paw was significantly thinner in the LDR group than in the CFA group. Therefore, LDR significantly alleviated inflammatory pain in the CFA-induced mice.

Figure 2.

Figure 2

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LDR alleviated hyperalgesia in CFA-induced inflammatory pain mice model. (A) The schedule of the experiments. (B) Mechanical pain threshold and (C) thermal pain threshold in mice after CFA injection; (D) swelling thickness of mouse PAWS after CFA injection. (n = 8, ***p < 0.001 versus control group; #p < 0.05, ###p < 0.001 versus CFA group).

LDR Alleviates CFA-Induced Anxiety-like Behavior

Previous studies demonstrated that pain and mood disorders coexist.26 Therefore, we explored whether CFA treatment induced anxiety-like behaviors in mice and if LDR elicited therapeutic effects. Open field test (OFT) and elevated plus maze (EPM) were performed 1 day after the end of the nociceptive tests. It was found that in the OFT (Figure 3A), CFA administration reduced the length of stay in the central region compared to the control group [F (4, 35) = 11.98, P < 0.001, Figure 3B, F (4, 35) = 4.488, P = 0.005, Figure 3C, F (4, 35) = 2.203, P = 0.0888 Figure 3D], but the duration and distance of stay in the central region were greater in the 50 mg/kg LDR group than in the CFA group. In the EPM experiment (Figure 3E), the CFA group made fewer trips to the open arms [F (4, 35) = 4.243, P = 0.007, Figure 3F] and spent significantly less time in the open arms and at shorter distances from the open arms [F (4, 35) = 11.68, P < 0.001, Figure 3G, F (4, 35) = 12.00, P < 0.001, Figure 3H] than the control group. However, 50 mg/kg LDR prolonged the time and frequency of the mice entering the open arm compared to those of the CFA group (Figure 3F,G). Therefore, LDR alleviated CFA-induced anxiety-like behavior in mice.

Figure 3.

Figure 3

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LDR could improve CFA-induced anxiety-like behavior. (A) OFT representative activity track. (B) Total distance of the mouse within the OFT central region. (C) Amount of time the mice spent in the OFT central region. (D) Total distance the mice traveled in the OFT. (E) EPM representative activity track. (F) Number of times mice entered the open arm of EPM. (G) Time spent in the EPM open arm. (H) Total distance of mice in the open arm of EPM. (n = 8, *p < 0.05, **p < 0.01, ***p < 0.001).

LDR Reduces the Levels of CFA-Induced Inflammatory Factors

During inflammatory pain, the expressions of many pro-inflammatory cytokines increase.27 Thus, we assessed the serum levels of pro-inflammatory cytokines to determine the anti-inflammatory effects of LDR. Serum IL-1β, IL-6, and TNF-α levels were significantly higher in the CFA group than in the control group [F(4, 20) = 59.91, P < 0.001, Figure 4A, P < 0.001, Figure 4B, F(4, 20) = 43.34, , P < 0.001, Figure 4C] and significantly lower in the LDR group than in the CFA group. Therefore, LDR inhibited the release of inflammatory factors.

Figure 4.

Figure 4

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LDR inhibited levels of serum inflammatory cytokines in CFA-induced inflammatory pain mice. (A) Serum IL-6 content was detected by ELISA. (B,C) Levels of serum IL-1β and TNF-a were detected by ELISA. (n = 5, *p < 0.05, ***p < 0.001).

LDR Attenuates CFA-Triggered Microglia Activation in ACC

Microglia are the resident immune cells of the CNS. Normally, microglia are in a resting state but they are activated and release pro-inflammatory cytokines and chemokines in a state of chronic pain.28 We evaluated microglial activation by immunofluorescence (IF) staining of Iba1 in the ACC of mice, finding that the fluorescence intensity of Iba1 was significantly higher in the CFA group than in the control group and significantly lower in the LDR group than in the CFA group [Figure 5A, F (2, 6) = 30.26, P < 0.01, Figure 5B]. These results suggested that LDR reverses ACC microglial activation after inflammatory pain in CFA-induced mice.

Figure 5.

Figure 5

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LDR inhibited the activation of microglia in the CFA-induced inflammatory pain mice model. (A) Change of Iba1+ microglia in the ACC area in each group was determined by IF staining. (B) Quantification of fluorescence intensity of Iba1. (n = 3 slices per group, *p < 0.05, **p < 0.01).

LDR Directly Targets the Cannabinoid 2 Receptor

The Super-PRED database was used to analyze the possible downstream targets of the LDR (Table 1). The cannabinoid 2 receptor (CB2R), which modulates acute, chronic inflammatory, postoperative, cancer, and nerve damage-associated pain,29 might be a target of LDR. Therefore, we verified the binding ability of LDR to CB2R by molecular docking, finding that LDR formed key hydrogen bonds with CYS288 of CB2R and π–π bonds with PHE883. Their binding energy was −8.367 kcal/mol, indicating that LDR and CB2R had good binding activity (Figure 6A,B). Cell thermal migration experiments were also performed to verify the thermal stability of LDR and CB2R. We found enhanced LDR and CB2R thermal stability after LDR administration to BV2 cells compared to that in the DMSO group (Figure 6C). Thus, these results suggested that LDR was combined with CB2R.

Table 1. Target of LDR Predicted by the Database.
target name probability (%) model accuracy (%)
transcription intermediary factor 1-alpha 92.8 96
DNA-(apurinic or apyrimidinic site) lyase 89 91
cannabinoid CB2 receptor 88 97
LSD1/CoREST complex 87 97
casein kinase II alpha/beta 87 99
DNA topoisomerase II alpha 86 89
cyclooxygenase-2 83 90
pregnane X receptor 82 95
IgG receptor FcRn large subunit p51 82 91
monoamine oxidase B 80 93
cyclin-dependent kinase 5/CDK5 activator 1 80 93
endoplasmic reticulum-associated amyloid betapeptide-binding protein 80 70
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Figure 6.

Figure 6

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LDR might interact with CB2R in the agonist-binding pocket. (A) Schematic diagram of molecular docking between LDR and CB2R. (B) Schematic diagram of LDR interaction at CB2R binding sites. The purple lines represent hydrogen bonds. (C) CETSA experiment to evaluate the binding ability of LDR and CB2R interaction.

Activation of CB2R by LDR Treatment Promotes the Polarization of Microglia from the Pro-inflammatory to Anti-inflammatory State

First, we used BV2 cells to verify the role of LDR in inflammation. After 24 h of LPS and LDR treatment, the IL-6, TNF-a and IL-1β levels in the supernatant of BV2 cells were significantly higher in the LPS group than in the control group [F (4, 10) = 53.87 , P < 0.001, Figure 7A, F (4, 10) = 76.78,P < 0.001, Figure 7B, F(4, 10) = 38.89,P < 0.001, Figure 7C], but their levels were lower in the LDR group than in the LPS group. Thus, LDR treatment inhibited the release of LPS-induced inflammatory factors.

Figure 7.

Figure 7

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Activation of CB2R by LDR treatment could promote the polarization of microglia from a pro-inflammatory to an anti-inflammatory state. (A) The levels of IL-6, TNF-a (B), and IL-1β (C) in the supernatant of BV2 cells were detected by ELISA. (D) Confocal images of Arg-1 and iNOS expression in BV2 cells after treatment with LPS and LDR (10 μM) for 24 h. (E) and (G) Western blot detection of iNOS and Arg-1 protein expression in BV2 cells. (F) and (H) Quantified of the expression of iNOS and Arg-1 related proteins. (n = 3, **p < 0.01, ***p < 0.001).

CB2R activation could shift microglia from an inflammatory M1 state to an anti-inflammatory M2 state.30 Therefore, BV2 cells were used to verify the expression of iNOS (M1 marker) and Arg-1 (M2 marker) in microglia. The IF results showed significantly increased iNOS protein levels in the LPS group compared to those in the control group and lower levels in the LPS + LDR than in the LPS group. The trend was also observed for Arg-1 (Figure 7D). The Weston Blot results showed (Figure 7E) that the iNOS protein level was significantly higher in the LPS group compared to the control group and the Arg-1 level was not statistically significant. Administration of LDR alone did not affect the levels of INOS and Arg-1, while the LDR + LPS group significantly increased the level of Arg-1 and decreased INOS level, compared with LPS simulation [F (3, 8) = 643.6, P < 0.001, F (3, 8) = 282.5, P < 0.001, Figure 7F]. These results suggested that LDR could promote microglia polarization from pro-inflammatory M1 to anti-inflammatory M2 state.

Then, LPS-induced BV2 cells were cotreated with LDR and a CB2R antagonist SR144528. Based on the results of Weston Blot, the upregulation of iNOS and downregulation of Arg-1 expression by LDR were reversed via SR144528 treatment [Figure 7G, F (4, 10) = 9.309, P < 0.001, F (4, 10) = 10.18, P < 0.001 in Figure 7H], illustrating that LDR might activate CB2R to promote microglia polarization from M1 to M2 state.

Analgesic and Antianxiety Effects of LDR were Partially Reduced through Antagonizing CB2R

To better explore the direct effect of LDR on CB2R, a CB2R antagonist SR144528 (3 mg/kg) was injected intraperitoneally 30 min before LDR administration in a CFA-induced inflammatory pain mouse model. The experimental paradigm is shown in Figure 8A. Compared with the control group, 50 mg/kg LDR significantly reduced CFA-induced hind paws swelling [F (3, 20) = 82.40, F (3, 20) = 107.4, F (3, 20) = 58.71, F (3, 20) = 49.50, P < 0.001, Figure 8D], and increased the mechanical abnormal pain threshold and thermal hyperalgesia threshold [F (3, 20) = 81.24, F (3, 20) = 71.10, F (3, 20) = 52.20, F (3, 20) = 66.11, P < 0.001, Figure 8B, F (3, 20) = 95.71, F (3, 20) = 109.4, F (3, 20) = 101.6, F (3, 20) = 109.6, P < 0.001, Figure 8C]. However, the preadministration of SR144528 weakened the analgesic effect of LDR.

Figure 8.

Figure 8

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Analgesic and antianxiety effects of LDR were partially reduced through antagonizing CB2R. (A) Schedule of the experiments. (B) Mechanical pain threshold and (C) thermal pain threshold in mice after CFA injection; (D) swelling thickness of mouse PAWS after CFA injection. (***p < 0.001 versus control group; #p < 0.05, ###p < 0.001 versus CFA group) (E) OFT representative activity track. (F) Total distance the mice traveled in the OFT. (G) Amount of time the mice spent in the OFT central region. (H) Total distance of the mouse within the OFT central region. (I) EPM representative activity track. (J) Number of times mice entered the open arm of EPM. (K) Time spent in the EPM open arm. (L) Total distance of mice in the open arm of EPM. (n = 6, *p < 0.05, **p < 0.01, ***p < 0.001).

The results of OFT (Figure 8E) showed that the LDR + LPS group had significantly increased time at the center [F (3, 20) = 6.820, P < 0.001, Figure 8G] and distance [F (3, 20) = 11.15, P < 0.001, Figure 8H], compared with the LDR + SR + CFA group. Similarly, the results of EPM (Figure 8I) showed that the LDR + LPS group had more trips to open arm [F (3, 20) = 5.475, P < 0.001, Figure 8J], and significantly increased time spent on open arm [F (3, 20) = 30.27, P < 0.001, Figure 8K] and distance [F (3, 20) = 20.62, P < 0.001, Figure 8L]. These results suggest that LDR reduced pain-associated anxiety-like behavior, while the CB2R antagonist SR144528 weakened this effect.

Discussion

This study found that LDR alleviated CFA-induced mechanical and thermal nociceptive hypersensitivity and anxiety-like behaviors in mice. In addition, LDR treatment decreased microglia activation in the ACC brain region and the release of pro-inflammatory factors, such as IL-1β, IL-6, and TNF-α. Molecular docking and cellular thermal displacement experiments showed that LDR has a high affinity for CB2R. Finally, LDR administration to BV2 cells decreased LPS-induced iNOS levels (the microglial M1 marker), but treatment with SR144528, a CB2R antagonist, and LDR increased the iNOS level. The results of animal experiments showed that the analgesic and antianxiety effects of LDR were canceled after SR144528 antagonized CB2R. In summary, LDR exerts its analgesic effects by activating CB2R.

The ACC plays a vital role in perceiving noxious signals,31 and altered ACC function exacerbates anxiety-like behaviors induced by chronic or visceral pain in animal models.32 Intraperitoneal injections of Cannabidiol reduce chronic neuropathic pain aversion behaviors by attenuation of neuroinflammation markers and neuronal activity in ACC.33 Therefore, we selected ACC as the target brain region. Microglia are key cells that cause acute and chronic pain after peripheral and central nerve injury.34 Under inflammatory conditions, microglia exist in an activated state, presenting a pro-inflammatory (M1) phenotype, releasing inflammatory cytokines and maintaining the ability to change their functional phenotype during an inflammatory response.35 We found that CFA injections into the hind paw of mice activated the microglia, increasing the levels of pro-inflammatory cytokine mRNAs (i.e., IL-1β, IL-6, and TNF-α) released by the activated microglia, enhancing pain.36 Consistent with previous studies, our study showed that LDR treatment reduced microglial activation, which significantly decreased the levels of inflammatory cytokines released by activated microglia, perhaps explaining the analgesic and anxiolytic effects.

CB2R is expressed in neurons and microglia, and its activation regulates neuronal excitability and neuroinflammation.30,37 CB2R agonists reduce pain sensitivity and the number of microglia that increases under inflammatory conditions.38 For example, HU-210 reverses carrageenin-induced inflammatory hypersensitivity via CB2R,39 and in a postoperative painful foot incision model, the CB2R selective agonist HU-308 reduced abnormal pain caused by the incision, which was blocked by the CB2R selective antagonist SR144528.40 In CB2 receptor knockout mice, CFA significantly reduced thermal and mechanical withdrawal thresholds.41 Therefore, we investigated whether LDR exerts its analgesic effects through CB2R. Molecular docking and CESTA verified that LDR could bind to the CB2R protein.

Reports indicate that the microglia of CB2R-deficient mice (CB2–/−) had reduced Arg-1 expression after IL-4/IL-3 stimulation, and they could not polarize to the M2 state.42 However, pharmacological activation of CB2R increases the expression of M2 microglial markers but decreases the expression of M1 inflammatory markers,43 suggesting that CB2R is essential for microglial activation and that its deficiency impairs microglial activation to pro-inflammatory or anti-inflammatory phenotypes. Our in vitro experiments showed that microglial cells treated with the CB2R antagonist, SR144528, released less of the M1 marker, Arg-1, and more of the M2 marker, iNOS. Thus, LDR activates CB2R to induce the transition of microglia from the M1 pro-inflammatory to the M2 anti-inflammatory state. In vivo experiments showed that when CB2R antagonist SR144528 was used to verify the analgesic and antianxiety effects of LDR in CFA-induced inflammatory pain model mice, SR144528 could reverse the analgesic and antianxiety effects of LDR, indicating that LDR may activate CB2R to exert analgesic and antianxiety effects.

In summary, our results suggest that LDR improved mechanical and thermal hyperalgesia and alleviated pain-induced anxiety-like behaviors in a CFA-induced inflammatory pain mouse model. LDR also decreased IL-1β, IL-6, and TNF-α levels in LPS-induced BV2 cells and polarized microglia to an anti-inflammatory M2 state by activating CB2R. The potent analgesic effect of LDR provides a new strategy for the clinical development of analgesic drugs.

Materials and Methods

Animals

Adult male C57BL/6 mice (aged 6–8 weeks and weighed 18.0 ± 2.0 g) were obtained from the Experimental Animal Center of Air Force Military Medical University. All animals were housed in a room with 24 ± 2 °C, a relative humidity of 50 ± 5%, and a 12 h day/night cycle and were provided with adequate food and water. Animals were acclimated to the laboratory environment for at least 1 week before the experiment began. All experimental procedures were approved by the Air Force Military Medical University Animal Care and Use Committee (ethical approval reference number no. KY20193145).

Experimental Procedure

Mice were randomly divided into control, CFA and CFA + LDR groups (2, 10, 50 mg/kg, n = 8 for each group). CFA (50% in saline, 10 μL, Sigma) was injected into the left hind paw of mice to induce inflammatory pain. Consistently, the control animals were given the same volume of saline in their left hind paw. One day after the CFA injection, mice in the CFA + LDR group were given LDR orally (2, 10, 50 mg/kg, dissolved in 5% DMSO and 0.5% CMC-Na solution) once a day for 14 days. Mechanical thresholds and paw swelling were measured at 0, 1, 3, 7, and 14 days after CFA injection.

Cell Culture and Treatment

The mouse BV2 microglia immortalized cell line was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The cells were cultured in high-glucose DMEM medium (Cytiva, Utah, USA) containing 10% fetal bovine serum (Gibco, NY, USA) and 1% penicillin/streptomycin (Sangon, Shanghai, China) in a CO2 incubator at 37 °C. LDR was purchased from Shanghai Yuanye Biotechnology (B21311). Lipopolysaccharide was purchased from Sigma-Aldrich (L2630, USA), and the CB2R antagonist SR144528 was purchased from MedChemExpress (HY-13439). The cells were treated with lipopolysaccharides (LPS) (1 μg/mL, Sigma-Aldrich, cat. no.: L2630) and SR144528 (10 μM) for 24 h and LDR at different concentrations for 24 h.

Mechanical Pain Threshold Determination

Before the experiment, the mice were habituated alone on a metal grid rack for half an hour. After the mice quieted, the automatic plantar pain meter (Jiangsu Cylon Biological Technology Co., Ltd. SA502) was used to stimulate the hind paws of the mice. When the mice had a rapid foot retraction response, the corresponding gram numbers were recorded as the mechanical threshold of the mice. Three measurements were taken for each animal with a 10 min interval allowed between trials, and the mean value was used for the analysis.

Thermal Threshold Determination

The mice were acclimated in a plexiglass box for 30 min. The hot plate pain meter (Nanjing Calvin Biotechnology Co., Ltd.KW-LB) was adjusted to 50 °C, and transparent perforated glass was covered around the hot plate. The mice were placed on a hot plate, and the timing began. When paw-licking, jumping, or significantly leg-shrinking of mice were observed, the timing was immediately terminated and the reaction latency was recorded. A cutoff time of 40 s was used to avoid a local burn injury. Three measurements were taken for each animal, with a 10 min interval allowed between trials, and the mean value was used for the analysis.

Open Field Test

The open field (40 × 40 × 40 cm, JLBehv-LAM-4, Shanghai Jiliang software, China) was placed in a dimly lit isolation room. Before each experiment, the mice were acclimated in the laboratory for an hour. During the formal tests, the mice were placed individually in the center of the box. The tracks of mice traveling in 15 min were recorded by a camera fixed above the box and analyzed with a video tracking system (MedAssociates, St. Albans, VT, USA).

EPM Test

The elevated plus maze system (DigBehv-EPMG, Shanghai Jiliang software) was used with two closed arms with high walls of 15 cm (30 × 5 cm) and two open arms at a height of 60 cm from the floor. The mice were placed in the testing room to acclimate for an hour. At the start of each session, the mice were placed in the EPM center, and each one was allowed to explore in the maze for 5 min. The timing and number of times the mice entered the open and closed arms were recorded and analyzed using a video tracking system (MedAssociates). Between sessions, the maze was thoroughly cleaned with 75% ethanol.

Enzyme-Linked Immunosorbent Assay

At the end of the animal experiment, the mouse blood was collected. After standing at room temperature for 1 h, blood samples were centrifuged at 3000 rpm for 10 min, and the serum was obtained for enzyme-linked immunosorbent assay (ELISA) tests. According to the manufacturer’s instructions (Thermo Fisher), the concentrations of IL-1β (BMS6002TEN), IL-6 (BMS603–2), and TNF-α (BMS607–3) were detected by ELISA kits. BV2 cells were treated with LPS and LDR for 24 h, and the supernatant was collected. The total protein concentration of inflammatory factors in the supernatant was also detected by the above ELISA kits.

Immunofluorescence

The mice were anesthetized with pentobarbital sodium and then underwent cardiac perfusion with normal saline and 4% paraformaldehyde (PFA). Brain tissues were taken and put in 4% PFA overnight and then transferred to 10 and 20% sucrose solutions successively for dehydration. Coronal sections containing ACC were cut on a low-temperature thermostat (Leica Microsystems, 25 μm). Sections were sealed with 10% goat serum containing 0.1% Triton X-100 at room temperature for 1 h. The primary antibody Iba1 (cat. no. ab2Ib89874, 1:200, Abcam) was incubated overnight at 4 °C. After rinsing sections with phosphate buffer saline (PBS) for 3 times (once for 5 min), the sections were paired with FITC (Zhuangzhi bio, China, 1:200) and incubated at room temperature for 1 h. The nucleus was stained with DAPI (Beyotime, China; C1006) for 10 min. Finally, the film was sealed with IF sealing solution and images were taken with a Fluo View FV1000 microscope (Olympus, Tokyo, Japan).

BV2 cells were spread on round plates (Biosharp, Hefei, China) and treated with LPS and LDR for 24 h. The samples were washed with PBS and fixed with 4% PFA for 30 min. The cells were incubated with an IF permeability solution (Beyotime, Shanghai, China) for 1 h. After washing with PBS three times, the primary antibodies iNOS (Abcam, UK, ab178945, 1:200) and Arg-1 (Abcam, UK, ab203490, 1:200) were incubated at 4 °C overnight. Finally, the fluorescently labeled secondary antibodies FITC (Zhuangzhi bio, China, 1:200) and CY3 (Zhuangzhi bio, China, 1:200) were incubated at room temperature for 1 h, and then, DAPI was incubated for 5 min. Finally, the film was sealed with IF sealing solution, and images were taken with a Fluo View FV1000 microscope (Olympus, Tokyo, Japan).

Western Blot Analysis

Cells were homogenized in a RIPA-lysis buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Sangon Biotech, Shanghai, China). BCA Protein Assay (Thermo Scientific, 23227) was used to check protein concentrations. After denaturation, the protein was isolated by SDS-PAGE and transferred to a PVDF membrane. The PVDF membrane was sealed in 5% skim milk in Tris-buffered saline with Tween (TBST) at room temperature for 1 h and incubated overnight at 4 °C with the following primary antibodies: beta-actin (Novus, NB600–501, 1:10,000), Arg-1 (Abcam, UK, ab203490, 1:5000), iNOS (Abcam, UK, ab178945, 1:1000), and CB2R (Affinity, DF8646, 1:1000). After TBST irrigation for 3 times (once for 10 min), the corresponding secondary antibodies (Goat Anti-Rabbit IgG, HRP Conjugated, Zhuangzhi bio, China, 1:5000, Goat Anti-Mouse IgG, HRP Conjugated, Zhuangzhi bio, China, 1:5000) were incubated for 1 h at room temperature. The signal was detected with an ECL plus kit (Shanghai Life iLab Biotech Co., Ltd. LFH23082) and captured by a ChemiScope 6600 PLUS imaging system (Clinx, Shanghai)

Molecular Docking

The X-ray diffraction structure of target protein CB2R (PDB: 6KPC) was retrieved from the Protein Data Bank with a 3.20 Å resolution. The protein was prepared using the Protein Preparation Wizard in Schrödinger Maestro. After the missing side chains were added, water molecules were removed from the protein structure. The 3D structure of Linderane (LDR) was downloaded from the PubChem database (6915739) and was prepared using the Ligprep tool in Schrödinger Maestro. The docking simulation was performed using the Schrödinger Glide module, and the binding site of the receptor was centered on the agonist bound in the active site.

Cellular Thermal Shift Assay

BV2 microglial cells were treated with LDR (50 μM) for 2 h, while another group of cells were incubated for the same time duration with an equal amount of DMSO (0.1%) as control. After cells were collected, the cell suspensions containing protease inhibitors were divided into 9 equal parts. Each one was repeatedly frozen in liquid nitrogen and then thawed at room temperature 3 times. Subsequently, these samples were heated at the gradient temperature for 3 min and then cooled to room temperature. The temperature was set from 37 to 61 °C: 37, 40, 43, 46, 49, 52, 55, 58, 61 °C. At last, proteins were denaturized for Western blotting detection.

Ultra-Performance Liquid Chromatography

UPLC was performed using a liquid chromatography–mass spectrometer (Shimazu UFLC30A + AB SCIEX API400). Chromatographic conditions: Waters-XSelect R HSS-T3 column (2.1 × 150 mm, 2.5 μm), flow rate: 0.3 mL/min, mobile phases: acetonitrile (A phase) and 0.1% formic acid solution (B phase), injection volume: 1 μL. Analysis time: 6.5 min. Gradient elution mode: 0–1 min, 70% B; 1–2.5 min, 2% B; 2.5–4.5 min, 2% B; 4.5–6.5 min, 95% B.

Statistical Analysis

The data were expressed as the SEM ± mean, and multiple groups were statistically analyzed using one-way ANOVA in GraphPad Prism 8.0 software. In all cases, P < 0.05 was considered statistically significant.

Acknowledgments

The study was funded by the National Natural Science Foundation of China (nos 31800887, 32241007, and 31972902), and partially supported by the Shaanxi Province Key Industry Innovation Chain Project (grant no. 2023-ZDLSF-59).

Glossary

Abbreviations

Arg-1

arginase-1

ACC

anterior cingulate cortex

CFA

complete Freund’s adjuvant

CB1R

cannabinoid 2 receptor

CB1R

cannabinoid 2 receptor

CNS

central nervous system

CETSA

cellular thermal shift assay

ECS

endocannabinoid system

ELISA

enzyme-linked immunosorbent assay

EPM

elevated plus maze

iNOS

inducible nitric oxide synthase

IL-6

interleukin-6

IL-1β

interleukin-1β

IL-10

interleukin-10

IF

immunofluorescence

IL-4

interleukin-4

IL-3

interleukin-3

LDR

Linderane

LPS

lipopolysaccharides

OFT

open field test

PFA

paraformaldehyde

PBS

phosphate buffer saline

SR

SR144528 (CB2R antagonist)

TNF-α

tumor necrosis factor-α

TBST

Tris-buffered saline with Tween

UPLC

ultra-performance liquid chromatography

Data Availability Statement

Data will be made available on request.

Author Contributions

§ Y.C., Y.Q., and Y.G. contribute equally to this work. Yue Chen: conceptualization, methodology, software, and writing–original draft. Yan Qin, Ying Gao: visualization, investigation, validation, and methodology. Saiying Wang, Qinhui Wang, Xiuling Tang: visualization, investigation, supervision. Zheng Rong, Caiyan Cheng, Longfei Li: investigation, validation. Yuan Xu, Qi Yang: writing–review and editing, software, and formal analysis. Le Yang, Minggao Zhao, Yuping Tang: conceptualization, methodology, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

The authors declare no competing financial interest.

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Data Availability Statement

Data will be made available on request.

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