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Introduction

Chronic pain often lasts months or years, affecting >20% of adults worldwide and developing into a widespread public health issue. Chronic pain significantly affects the patients' ability to sleep, work and study, which lowers their quality of life and ability to function (1). Spontaneous pain and evoked pain in reaction to non-noxious or noxious stimuli are characteristics of chronic pain (2). Preferred pharmacologic treatments consist of opioids, acetaminophen and nonsteroidal anti-inflammatory drugs. Due to gastrointestinal discomfort along with other side effects of drugs, nearly one-third of patients were unsatisfied with their treatment (3). Thus, developing new analgesic drugs with fewer side effects is essential.

Neuroinflammation in the spinal cord participates in and modulates chronic pain, which is marked by the release of inflammatory mediators and glial cell activation (4,5). During pain processing, tissue injury generates a number of nociceptive stimuli that cause primary afferent fibers. After transmitting to the spinal cord, these nociceptive signals activate astrocytes and nociceptors (6). Activated astrocytes undergo hyperplasia and hypertrophy and release several pro-inflammatory mediators, for instance IL-1β, which is known to mediate pain and sensitize nociceptors directly (7). The Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is well-characterized for IL-1β maturation (8). NLRP3 inflammasome assembly allows the cleavage of caspase-1 and in turn leads IL-1β secretion and NF-κB activation (9,10). The pathophysiology of chronic pain is influenced by the dysregulation of NLRP3 inflammasome (11). IL-1β, apoptosis-associated speck-like protein (ASC) and caspase-1 are among the NLRP3 inflammasome components whose spinal expression is greatly elevated in the complete Freund's adjuvant (CFA)-induced inflammatory pain model (12). MCC950 (a specific inhibitor of NLRP3) or Nlrp3 gene knockout therapy reduces inflammatory pain in mice (13). Hence, targeting NLRP3 inflammation activation is a potential therapy for chronic pain.

Traditional Chinese medicine uses emodin (1,3,8-trihydroxy-6-methylanthraquinone), which is a natural anthraquinone derivative and found in Rheum palmatum and a number of other plants. Emodin possesses a number of benefits, including those against cancer, inflammation, viruses and bacteria (14). In several pain models, such as capsaicin, acetic acid, carrageenan and formalin-induced pain, emodin decreases nociceptive and inflammatory responses (15). Emodin treatment alleviates hyperalgesia by decreasing the P2X (2/3) expression in chronic constriction injury neuropathic pain model rats (16). Consequently, targeting HDAC6 is favorable for managing chronic pain as well as neuroinflammation.

In order to establish a chronic inflammatory pain model an intraplantar injection of CFA in the left foot of C57BL/6J mice was performed. For three successive days intraperitoneal injections of emodin were performed. The related protein expression levels, changes in behaviors and spinal inflammation were detected. It was intended to explain how emodin works to reduce chronic pain and provide theoretical and data support for emodin application in chronic pain treatment.

Materials and methods

Animal model and drug administration

Male C57BL/6J mice (6-8 weeks old; 18-20 g; n=30) were bought from the Hubei Province Experimental Animal Centre. Animals were kept under 12 h light/dark environment and unlimited access to food and water. The experiment was approved by the Laboratory Animal Ethics Committee of Hubei University of Science and Technology (approval no 2019-03-021).

Prior to the test, mice were randomly divided into Control, CFA and CFA + emodin groups, 10 mice for each group. There was a 7-day period of acclimation to the surroundings. Then on days 0 and 7, left hind paws of mice received intraplantar subcutaneous injections of 10 µl CFA and the equal amount of saline was administered in mice from control group (17). On days 0, 7 and 14, behavioral assessments were conducted. On days 15-17 following the CFA injection, vehicle and emodin (10 mg/kg) were intraperitoneally injected in CFA and CFA + emodin groups for 3 days. Prior to use, emodin was dissolved in DMSO which diluted with 0.9% NaCl. After 4 h of emodin treatment, the behavioral tests were conducted.

Antibodies and reagents

Anti-NLRP3 (cat. no. DF7438), anti-HDAC6 (cat. no. AF6485), anti-glial fibrillary acidic protein (GFAP, cat. no. BF0345), anti-IL-1β (cat. no. AF5103), anti-β-actin (cat. no. AF7018) and anti-cleaved caspase-1 (cat. no. AF4022) antibodies were purchased from Affinity Biosciences. Anti-caspase-1 (cat. no. A0964), HRP Goat anti-rabbit IgG (H+L; cat. no. AS014) and HRP Goat anti-mouse IgG (H+L; cat. no. AS003) antibodies were acquired from ABclonal Biotech Co., Ltd. H&E staining kit (cat. no. BL735B) was bought from Biosharp Life Sciences. Emodin was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Goat Anti-Rabbit IgG H&L (FITC; cat. no. ab6717) and Goat Anti-Mouse IgG H&L (TRITC; cat. no. ab6786) were from Abcam.

Mechanical threshold test

Mice were housed in a plexiglass container to acclimatize for almost 30 min. Then, von Frey filaments (Stoelting Co.) were applied to stimulate the left hind paw. The filaments were briefly bent by being pushed firmly vertically on the plantar surfaces for 3-5 sec. Paw flinching and brisk withdrawal were regarded as positive responses in this circumstance. The patterns of withdrawal responses were then translated into mechanical threshold values (18).

Spontaneous flinch test

Mice were housed for nearly 30 mins in a plexiglass chamber. Within 5 min, numbers of flinches were counted 3 times individually (19).

Rotarod test

Mice were trained three days at a fixed pace for 10 min before the examinations. In the experiments, the test was initially set at a constant speed at 10 revolutions per min for 10 sec, then at an increasing speed to 20 revolutions per min for 30 sec. The latency to fall was measured to evaluate the balance and motor coordination of the animals (20).

H&E staining

Following the behavior test, mice were given a deep anesthetic dose of 60 mg/kg sodium pentobarbital and transcardially perfused using 4% PFA. After collection and post-fixation using 4% PFA for 12 h at 4˚C, the spinal cords were embedded with paraffin and cutting into 4-µm sections. The sections were treated with xylene, 100, 90 and 70% ethanol and dyed with H&E staining kit. Briefly, paraffin sections were treated with xylene (5 min, twice), 100% ethanol (10 min, twice), 90% ethanol (10 min) and 70% ethanol (10 min) for dewaxing, then stained with hematoxylin solution for 3 min at 25˚C and eosin for 3 min at 25˚C. The slices were treated with 70% ethanol (10 sec), 80% ethanol (10 sec), 90% ethanol (30 sec), 100% ethanol (1 min, twice) and xylene (1 min, twice) to dehydrate the sample and render it transparent, sealed with neutral balsam, images captured under a fluorescence microscope (IX73; Olympus Corporation) and analyzed using ImageJ 1.48v (National Institutes of Health). Inflammatory cell infiltration was categorized as 0 (normal); 1 (meningeal and perivascular lymphocytic infiltration); 2 (1-10 lymphocytes present); 3 (11-100 lymphocytes); and 4 (>100 lymphocytes).

Immunofluorescence

The sections of spinal cord tissue were hydrated and subjected to antigen retrieval using antigen retrieval solution (cat. no. P0083; Beyotime Institute of Biotechnology) for 10 min at 95˚C. The sections were blocked with immunofluorescence blocking solution (cat. no. P0102; Beyotime Institute of Biotechnology) for 1h before incubating with primary antibody at 4˚C overnight and fluorescent secondary antibodies for 1 h at room temperature. The images were captured by a fluorescence microscope and analyzed using ImageJ 1.48v (National Institutes of Health). The primary antibodies were anti-HDAC6, anti-IL-1β, anti-caspsae-1, anti-GFAP and anti-NLRP3, the dilution ratio was 1:100.

Western blotting

Mice were intraperitoneally injected with 150 mg/kg pentobarbital sodium after the behavioral tests. The lumbar spinal cords were homogenized in RIPA lysis buffer containing 1% protease inhibitors (MilliporeSigma) and centrifuged for 20 min (12,000 x g, 4˚C). The supernatant was quantified using a BCA analysis kit (cat. no. P0012; Beyotime Institute of Biotechnology). Protein mixture samples were loaded at 20 µg/lane and separated using 10-15% SDS-PAGE. The blots were transferred to PVDF membranes. Following a blocking using QuickBlock Blocking Buffer (cat. no. P0220; Beyotime Institute of Biotechnology) at 4˚C for 15 min, the membranes were incubated with primary antibodies overnight at 4˚C and secondary antibodies (1:5,000) for 1 h (room temperature). The bands were visualized using ECL solution (cat. no. P0018M; Beyotime Institute of Biotechnology) and analyzed using ImageJ 1.48v (National Institutes of Health). β-actin was used as a loading control. The following primary antibodies were used: anti-GFAP (1:1,000), anti-IL-1β (1:1,000), anti-cleaved-caspsae-1 (1:1,000), anti-HDAC6 (1:1,000), anti-NLRP3 (1:1,000) and anti-β-actin (1:50,000).

Molecular docking

HDAC6 X-ray crystal structure was acquired through Protein Data Bank (PDB ID, 5B8D; https://www.rcsb.org/structure/5B8D). The structure of emodin was optimized and retrieved from the PubChem compound database (PubChem CID, 3220; https://pubchem.ncbi.nlm.nih.gov/compound/3220). Docking conformation among emodin and HDAC6 was employed by Auto Dock Vina 1.2.0 software (Center for Computational Structural Biology, https://vina.scripps.edu/downloads/). The conformation was seen using PyMOL 2.2.3(21).

Statistical analysis

All statistical analyses were performed using SPSS 26.0 (IBM Corp.). Data of H&E staining, behaviors, western blotting and immunofluorescence were examined by a one-way ANOVA followed by Tukey's post-hoc test. Data for paw withdrawal threshold (PWT), flinches and latency to fall are expressed as mean ± SEM. Data for histology, morphology and western blotting are expressed as the mean ± SD.

Results

Emodin decreases pain sensitivity in CFA-induced mice

Fig. 1A indicates the experiment process including the behavioral tests. As shown in Fig. 1B, mechanical threshold values in the CFA mice were lowered with the PWT values of day 0, 7 and 14 at 1.15 ± 0.12, 0.55 ± 0.06 and 0.39 ± 0.03, respectively. The number of flinches (Fig. 1C) raised in CFA mice, from 3.20 ± 0.36 (day 0) to 11.60 ± 1.01 (day 7), 12.80 ± 0.89 (day 14), compared with control mice. In contrast to control mice, the latency to fall (Fig. 1D) in CFA mice decreased from 480.03 ± 33.27 (day 0) to 238.53 ± 20.75 (day 7) and 217.90 ± 23.67 (day 14). As suggested by the data, CFA results in motor disability as well as nociceptive hyperalgesia in mice, which indicated that the mouse model of chronic pain had been constructed effectively. The impact of emodin on motor function and pain sensitivity was then evaluated. The values of mechanical threshold in CFA + emodin mice were higher after emodin treatment on days 15-17, which are 0.56 ± 0.07, 0.64 ± 0.06 and 0.70 ± 0.09, respectively (Fig. 1B). There were significantly fewer flinches in the CFA + emodin mice, 11.10 ± 0.62 (day 15), 9.80 ± 0.70 (day 16) and 9.90 ± 0.57 (day 17), respectively (Fig. 1C). The emodin treatment also increased the latency to fall in CFA-induced mice, 354.27 ± 35.95, 367.16 ± 33.36 and 382.72 ± 32.39, on days 15 to 17, respectively (Fig. 1D). As a result, emodin alleviated pain behavior and improved motor ability in the CFA mice.

Figure 1 Effect of emodin on pain behaviors. (A) Schematic diagram of the experimental process. Changes of (B) PWT values, (C) numbers of spontaneous flinches and (D) latency to fall in different groups. Data are presented as the mean ± SEM (n=10). *P<0.05 vs. control group; #P<0.05 vs. CFA group. PWT, paw withdrawal threshold; CFA, complete Freund's adjuvant.

Emodin reduces spinal cord inflammatory response by inhibiting spinal cord astrocyte activation

For CFA mice, a significant inflammatory infiltration was found in the spinal dorsal horn with the inflammatory scores at 2.65 ± 0.06, whereas emodin treatment reduced the inflammatory infiltration to 2.45 ± 0.05 (Fig. 2A and B). Activated astrocytes are a major source of a number of pro-inflammatory cytokines and GFAP is used as a marker of abnormal proliferation and activation of astrocytes (22). The fluorescence intensities of IL-1β and GFAP in spinal dorsal horn of CFA group were enhanced, with the relative intensity at 2.26 ± 0.17 and 1.90 ± 0.28, respectively. The emodin treatment decreased the IL-1β and GFAP intensity to 1.62 ± 0.29 and 1.14 ± 0.09, respectively (Fig. 2C and E). As revealed by western blotting, the CFA group had higher spinal expression levels of IL-1β and GFAP than the control group, with the relative grey values of 1.45 ± 0.06 and 1.28 ± 0.06, respectively. The levels of IL-1β and GFAP were reduced to 1.04 ± 0.10 and 1.10 ± 0.05, respectively (Fig. 2D and F) by emodin treatment.

Figure 2 Emodin reduces spinal cord inflammatory response by inhibiting spinal cord astrocyte activation. Representative (A) hematoxylin and eosin-stained images and (B) inflammation score analysis of the spinal dorsal horn in each group. Scale bar, 20 µm. Representative (C) immunofluorescence staining images of IL-1β and GFAP and (E) quantitative fluorescence intensity analysis (overall magnification, x400; scale bar, 20 µm). (D) Western blot analysis and (F) quantification of the relative grey value of IL-1β and GFAP. Data are presented as the mean ± SD (n=5). *P<0.05 vs. control group; #P<0.05 vs. CFA group. GFAP, glial fibrillary acidic protein; CFA, complete Freund's adjuvant.

Emodin decreases the activity of spinal NLPR3 inflammasome

IL-1β maturation is promoted by NLRP3 inflammasome activation (23). The fluorescence intensities of NLRP3 and caspase-1 in spinal dorsal horn of CFA group were enhanced, with the relative intensity at 1.60 ± 0.11 and 1.33 ± 0.08, respectively. The emodin treatment decreased the NLRP3 and caspase-1 intensity to 1.28 ± 0.13 and 1.13 ± 0.07, respectively (Fig. 3A-D). As revealed by western blotting, the CFA group had higher spinal expression levels of NLRP3 and cleaved caspase-1 than control group, with the relative grey values of 1.40 ± 0.11 and 2.35 ± 0.16, respectively. The levels of NLRP3 and cleaved caspase-1 were reduced to 0.97 ± 0.10 and 2.03 ± 0.15, respectively (Fig. 3E and F) by emodin administration.

Figure 3 Changes in NLRP3 inflammasome components upon emodin treatment. (A-D) Representative immunofluorescence staining images and quantitative analysis of spinal NLRP3 and caspase-1 in the control, CFA and CFA + emodin groups. Scale bar, 20 µm. (E) Representative western blots and (F) quantitation examination of NLRP3 and cleaved caspase-1 expression levels the in spinal cords of the control, CFA and CFA + emodin groups. Data are presented as the mean ± SD (n=5). *P<0.05 vs. control group, #P<0.05 vs. CFA group. NLRP3, Nod-like receptor family pyrin domain-containing 3; CFA, complete Freund's adjuvant.

Emodin prevents spinal HDAC6 activity

The ligand emodin and the X-ray crystal structures of HDAC6 were used in a molecular docking test (Fig. 4A-C). According to Auto Dock data, four electrovalent bonds are produced by emodin and HDAC6 at residues ARG-1155, TYR-1189, GLU-1141 and TRP-1143. The electrovalent bond distances were 1.9, 3.2, 2.4 and 2.1 Å between HDAC6 and emodin, while the binding affinity was -8.7 kcal/mol. The increased HDAC6 fluorescence intensity in the spinal dorsal horn was observed in the CFA group, with a relative intensity of 2.04 ± 0.13. HDAC6 intensity was decreased to 1.52 ± 0.13 upon emodin treatment (Fig. 4D and E). The spinal HDAC6 protein level of CFA group was higher than the control group; the relative grey value was 1.52 ± 0.16. HDAC6 protein level was downregulated by emodin treatment to 0.88 ± 0.10 (Fig. 4F and G).

Figure 4 Effect of emodin treatment on HDAC6 activity. (A) The modeled 3D structure of HDAC6 docked with emodin. (B) An enlarged view of the binding site is displayed in the inset box. (C) The interaction bonds of HDAC6 with emodin. The HDAC6 protein is shown in cyan, emodin is colored green, the interacting residues as red, bonds are shown as yellow dotted lines and bond lengths are depicted as numbers. (D) Representative immunofluorescence staining images and (E) quantitative intensity analysis of HDAC6 of each group. Scale bar, 20 µm. Representative (F) western blots and (G) quantitation of HDAC6 expression levels. Data are presented as the mean ± SD (n=5). *P<0.05 vs. control group, #P<0.05 vs. CFA group. HDAC6, histone deacetylase 6; CFA, complete Freund's adjuvant.

Discussion

The present study discovered that emodin reduced CFA-induced pain by controlling HDAC6. HDAC6 works as a therapeutic target for chronic pain (24). HDAC6 inhibitor SW-100 treatment restores deacetylation of α-tubulin in sciatic nerve and alleviates mechanical allodynia and hyperalgesia in the peripheral neuropathy mouse model (25). Chemotherapy-induced peripheral neuropathy model mice treated with HDAC6 inhibitor recover from mechanical allodynia and spontaneous pain caused by cisplatin (26). HDAC6 genetic deletion prevents the mechanical allodynia that cisplatin causes (27). The present study found increased spinal HDAC6 expression in CFA-induced inflammatory pain model mice. Emodin binds with HDAC6 at a high binding affinity, producing a comparatively stable docking outcome (28). In the meantime, in the cardiac hypertrophy model, emodin inhibits HDAC activity and increases histone acetylation, while blocking pathological cardiac hypertrophy (29). Hence, it was hypothesized that emodin binds with HDAC6 and inhibits its activity.

Emodin suppresses spinal inflammation by regulating HDAC6. It is reported that HDAC6 serves as a dynein adaptor for the purpose of facilitating transport and assembling NLRP3 inflammasome (30). HDAC6 inhibition decreases the NLRP3 and IL-1β levels and suppresses nicotine-induced pyroptosis in atherosclerosis model mice (31). In LPS-induced mice, HDAC6 degrader application lessened activation of NLRP3 inflammasome (32). In a Parkinson's disease mouse model, the pharmacological suppression of HDAC6 by tubastatin A reduces NLRP3 expression and the maturation of IL-1β (33). The present study suggested that emodin treatment disrupted the binding of HDAC6 and NLRP3 and suppressed NLRP3 inflammasome activation. Furthermore, HDAC6 participates in pro-inflammatory interleukin expression through NF-κB signaling, which induces the pro-IL-1β and NLRP3 transcription expression (34). In arthritis model animals, HDAC6 depletion post-transcriptionally upregulates NF-κB inhibitor and downregulates NF-κB reporter activation and prevents the nuclear translocation of NF-κB subunits (35). HDAC6 mediates deacetylation of NF-κB and plays an inhibitory effect on invasion (36). The present study indicated that emodin administration reduced NF-κB-mediated neuroinflammation in chronic pain.

During chronic pain processing, NLRP3 inflammasome is activated and spinal inflammation is triggered. Emodin treatment binds HDAC6, weakens the HDAC6-NLRP3 interaction, reduces NLRP3 inflammasome reaction and suppresses spinal inflammation while alleviating chronic inflammatory pain.

Acknowledgements

Not applicable.

Funding

Funding: The present study was funded by the Hubei University of Science and Technology Program (grant nos. BK202213, 2021WG06, 2022YKY02 and 2022YKY09), as well as the Research Project of the Hubei Provincial Department of Education (grant nos. ZY2023F109, B2022186 and B2022185).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

Each author significantly contributed to the present study. MX and FZ conceived and designed the study. The experiments were conducted by DC, YX and TC. SZ, WM and HZ gathered and analyzed the data. The manuscript was drafted and edited by MX and FZ. The final manuscript was reviewed and approved by all authors. DC, YX and TC confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The Hubei University of Science and Technology Ethics Committee approved the present study (approval number 2020-01-900). All experiments were performed in accordance with international and local guidelines on the ethical use of animals.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References