Oral administration of boldine reduces neuropathic pain After spared nerve injury in female mice

Supplemental Digital Content is Available in the Text.

Boldine inhibits glia activation and reduces neuropathic pain after peripheral nerve injury.

Abstract

Introduction:

Chronic pain is present in approximately 20% of the population and is a major burden to the healthcare system. Approximately 30% to 40% of these patients report neuropathic pain. Neuropathic pain is defined as pain caused by injury or disease of the somatosensory nervous system. Current available treatments for neuropathic pain have limited efficacy and substantial side effects.

Objective:

To address the need for more effective and safer treatments for neuropathic pain, this study aimed to test whether boldine, a naturally occurring alkaloid, could attenuate neuropathic pain in a murine model of spared nerve injury (SNI).

Methods:

Von Frey filament test, hot/cold plate test, and dynamic weight-bearing test were used to assess pain phenotypes and functional outcomes after SNI. Multiplex enzyme-linked immunosorbent assay, quantitative PCR, and immunostainings were performed to evaluate cellular and molecular changes after boldine treatment.

Results:

Oral administration of boldine at 50 mg/kg body weight/day resulted in significant reduction of SNI-induced mechanical and thermal hypersensitivities. Functional outcome measurements showed that boldine corrected SNI-induced weight-bearing deficits. Boldine significantly inhibited SNI-induced elevation of some pain-related inflammatory cytokines/chemokines in the serum. Immunofluorescence studies revealed that boldine reduced the number of reactive astrocytes and inhibited microglia activation in the lumbar spinal cord. In vitro studies showed that boldine inhibited the lipopolysaccharide-induced elevation of inflammatory markers in BV-2 microglial cells.

Conclusion:

Our findings suggest that boldine may be a promising therapeutic candidate for the treatment of neuropathic pain, possibly through inhibition of glia activation and neuroinflammation.

1. Introduction

As described by the International Association for the Study of Pain, neuropathic pain is “pain caused by a lesion or a disease of the somatosensory nervous system.” It affects 7% to 10% of the general population globally.¹⁷ Neuropathic pain can be central or peripheral, and tissue damage is not always apparent. Studies have shown that neuropathic pain has multiple adverse effects in both humans and rodents that include impaired sleep, depression, anxiety, and lower quality of life.^5,20,41,48 Common causes of neuropathic pain include trigeminal neuralgia, postamputation pain, persistent postoperative or post-traumatic pain, pain after peripheral nerve injury, painful diabetic neuropathy, cancer pain, and chemotherapy-induced peripheral neuropathy. Owing to its subjective nature, diagnosing neuropathic pain is challenging. Typical characteristics of neuropathic pain include allodynia, where normally nonpainful stimuli cause pain; hyperalgesia, an exaggerated response to painful stimuli; and spontaneous pain.^3,33,39,47

Currently, the first line of choice for neuropathic pain treatment includes: anticonvulsants, tricyclic antidepressants, serotonin and norepinephrine reuptake inhibitors, and topical agents.⁶ Opioids, such as morphine and oxycodone, can be administered under careful medical supervision for very severe cases, although their long-term efficacy in treating neuropathic pain is uncertain, and they can be associated with severe side effects and risks, including sedation, respiratory depression, potential dependence, and addiction.²³ Thus, there is an urgent need to develop novel, efficacious, and safe interventions for treating neuropathic pain. Neuroinflammation, characterized by the activation of immune cells in the nervous system, plays a significant role in the development and persistence of neuropathic pain.²¹ This inflammatory response, involving microglia and astrocytes in the central nervous system (CNS) and macrophages in the peripheral nervous system (PNS), releases inflammatory mediators such as cytokines and chemokines, contributing to pain hypersensitivity and central sensitization. Targeting neuroinflammation may offer potential therapeutic strategies for neuropathic pain management.

Recently, the beneficial effect of some nutraceuticals and herbal remedies have gained attention in combating neuropathic pain.¹ Curcumin for instance, reduced neuropathic pain in streptozotocin-induced diabetic neuropathy⁵³ and peripheral nerve injury rodent models.³⁰ Boldine is the most abundant alkaloid isolated from the leaves and bark from boldo (Peumus boldus), a widely distributed evergreen tree native to Chile.⁵² Boldo extracts containing boldine has been used since the past century in Europe and North/South America for the treatment of digestive problems, headache, rheumatism, and urinary tract inflammation.⁵⁴ Boldine exhibits hepatoprotective, anti-inflammatory, and antipyretic activities in animal models.^4,10 Recent studies conducted by us and others have shown the beneficial effect of boldine in neurological conditions. Boldine reduces neuroinflammation and improves locomotor function in rodent models of spinal cord injury.^22,56 Oral administration of boldine decreases intramuscular connexin expression and promotes neuromuscular recovery in a rat model of delayed nerve repair.⁹ Boldine provides neuroprotection in Alzheimer's disease models by both direct interactions with Aβ and by preventing oxidative stress and mitochondrial dysfunction.⁵⁵ Boldine accumulates in brain tissues after oral administration.³⁵ Considering the anti-inflammatory activity of boldine and the key role of neuroinflammation in the development and maintenance of neuropathic pain, we administered boldine in a mouse model of spared nerve injury to test the hypothesis that boldine could alleviate neuropathic pain through reduction of neuroinflammation.

2. Material and methods

2.1. Reagents

Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), Dulbecco's Phosphate-Buffered Saline, penicillin, and streptomycin were purchased from GIBCO (Waltham, MA). Lipopolysaccharides (LPS), boldine, dimethyl sulfoxide (DMSO), triton X-100, and paraformaldehyde (PFA) were purchased from Sigma-Aldrich (St. Louis, MO). Lipopolysaccharides was dissolved in PBS and boldine was first dissolved in DMSO then diluted in culture medium.

2.2. Cell culture, treatment, cytotoxicity, and enzyme-linked immunosorbent assay

BV-2 cells were obtained from AcceGen (Fairfield, NJ) and maintained in DMEM supplemented with 10% FBS and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL) at 37°C with 5% CO₂. Cells (in triplicates) were treated with 100 ng/mL LPS with or without boldine (25 or 50 µg/mL) for 6 hours, and 100 μL of the supernatant was sampled for cytotoxicity essay before cells were harvested for RNA extraction or enzyme-linked immunosorbent assay (ELISA). Cell viability was assessed using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Fitchburg, WI) following manufacturer's instructions. For ELISA, BV-2 cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer (Pierce Biotechnology, Waltham, MA) and protein concentration was measured using the Bicinchoninic acid (BCA) Protein Assay Kits (Pierce Biotechnology). Interleukin-1β (IL-1β) protein levels were assessed using the Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) following the manufacturer's directions. Optical densities were measured using a SpectraMax iD5 plate reader (Molecular Devices, San Jose, CA).

2.3. Animal, surgery, and boldine administration

Animals were maintained on a 12:12-h light/dark cycle in a temperature-controlled (20 ± 2°C) vivarium, with food and water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee at James J. Peters Veterans Affairs Medical Center (#1585874) and conformed to all guidelines and regulations for the protection of the welfare of animals. C57BL6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and pair-housed. Spared nerve injury (SNI) surgery was conducted on the left hind limb under anesthesia (3%–5% isoflurane) by cutting the peroneal and tibial nerves and leaving an intact sural nerve. Sham animals underwent a similar procedure where the branches of the sciatic nerve were exposed but left intact. Boldine was administered to the animal right after surgery through a customized chow (Research Diets, New Brunswick, NJ) to reach a dose of 50 mg/kg BW/d. A control diet (OpenStandard Diet With 15 Kcal% Fat, Research Diets) was provided to sham-operated or SNI group. Animals were killed at 15 days postsurgery. Lumbar spinal cords (L3-5) were dissected and stored at −80°C for further analysis. Blood samples were collected and left at room temperature for 1 hour to allow it to clot. Blood was then centrifuged at 1,000g for 10 minutes and supernatant (serum) was stored at −80°C for further analysis.

2.4. RNA isolation, cDNA synthesis, and quantitative Polymerase Chain Reaction (PCR)

BV-2 cells were harvested and RNAs were extracted using the RNeasy Kit (Qiagen, Hilden, Germany) following the manufacturer's directions. Frozen tissues were homogenized in TRI-Reagent (Sigma-Aldrich) using a FastPrep-24 5 G Bead Beating Grinder and Lysis System (MP Biomedicals, Solon, OH). Total RNA was isolated following the manufacturer's instructions. RNA concentrations were measured using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). One microgram of total RNA was reverse transcribed with High-Capacity cDNA Reverse Transcription Kit using random hexamers in a 20 μL reaction according to the manufacturer's protocol. Quantitative PCR was performed using the Taqman Universal PCR Master Mix on a ViiA 7 Real-Time PCR System. Relative expression levels were calculated using the 2^–ΔΔCt method with hypoxanthine phosphoribosyltransferase 1 as an internal control. A list of the Taqman probes used in this study is summarized in Supplemental Table 1, http://links.lww.com/PR9/A368 (all from Applied Biosystems, Waltham, MA).

2.5. Behavioral tests

Mechanical pain responses were assessed by von Frey filament test performed on the ipsilateral and contralateral hind paws.¹⁵ In brief, The von Frey hair (Bioseb, Vitrolles, France) is inserted perpendicularly from below an elevated wire platform into the lateral plantar surface of the hind paw, starting with the smallest gauge filament. The pressure was increased slowly, until the hair just began to bend (approximately 2–3 seconds). The test was then repeated with the next largest gauge filament until withdrawal responses including paw flicking, paw lifting, paw licking, or possibly vocalizing was observed. The withdrawal threshold, defined as the smallest gauge wire that produced a withdrawal response, was recorded.

Thermal pain responses were measured using a hot/cold pate apparatus (Maze Engineers, Skokie, IL). Mice were allowed to move freely on a hot/cold plate enclosure for a maximum of 30 seconds. For assessing pain responses to heat, the plate temperature was set at 50°C. For testing pain responses to cold, the plate temperature was set at 15°C. Once paw retraction, paw licking, or escaping behaviors were observed, mice were immediately taken out of the testing apparatus and the time latency was recorded.⁶³

Weight-bearing behaviors were measured using the Dynamic Weight Bearing 2.0 system (DWB2.0) (Bioseb). In brief, the animal was placed into the DWB enclosure for a test duration of 5 minutes. The floor sensors automatically recorded the weight and surface area distributed between the paws and other body parts (eg, tail) during the 5-minute data acquisition period. Weight distribution was analyzed automatically using the accompanied tracking software. A blinded observer manually validated each automatically detected paw position to avoid subsequent error identification afterward.

2.6. Multiplex enzyme-linked immunosorbent assay

MILLIPLEX Mouse Cytokine/Chemokine Magnetic Bead Panel (Millipore, Billerica, MA) was used to measure the levels of 32 cytokines/chemokines in mouse serum as previously described.^59,60,67 In brief, 25 µL of serum sample was diluted with assay buffer and incubated with magnetic beads overnight at 4°C. The samples were then washed, incubated with detection antibodies for 1 hour at room temperature followed by streptavidin–phycoerythrin incubation for 30 minutes. The plate was run on a Luminex 200 instrument (Millipore), and the median fluorescent intensity was used to calculate the concentration of each cytokine/chemokine.

2.7. Immunostaining and microscopy

At 15 dpi, mice were transcardially perfused with saline followed by 4% PFA. Lumbar spinal cords (L3-5) were harvested and transversely sectioned using a LEICA 1000S vibratome (Leica, Wetzlar, Germany). For immunostaining, slides were first incubated with blocking buffer (1 × PBS + 5% goat serum + 0.3% Triton X-100) for 2 hour at room temperature, followed by incubation with primary antibodies of astrocyte marker glial fibrillary acidic protein (GFAP, Abcam, Cambridge, United Kingdom) or microglia marker Ionized calcium-binding adaptor molecule 1 (IBA1, Abcam) overnight at 4°C. After incubation, the slides were washed 3 times in PBS and incubated with goat antimouse Alexa Fluor 488 secondary antibody (Abcam) for 2 hours at room temperature. Slides were then washed with PBS 3 times and mounted with VECTASHIELD Antifade Mounting Medium with DAPI (4′,6-diamidino-2-phenylindole) (Vector Laboratories, Newark, CA). Images were acquired using the ZEISS LSM 700 confocal imaging system (Carl Zeiss, Oberkochen, Germany) with ×20 objective. Mean fluorescent intensity was quantified using ImageJ (3 sections per mice, 4 mice per group).

2.8. Statistical analysis

Data were analyzed using GraphPad Prism 10 (San Diego, CA) and were presented as the mean ± standard error of the mean. Two-way repeated-measures analysis of variance (ANOVA) followed by the multiple comparison test was performed to compare the mean differences in mechanical thresholds, thermal thresholds, and weight bearing among the experimental groups. One-way ANOVA followed by multiple comparison test was performed for the comparison of mean difference in cytotoxicity and RNA or protein expression levels among experimental groups. P-value of <0.05 was considered statistically significant.

3. Results

3.1. Boldine alleviates spared nerve injury–induced mechanical allodynia

To assess the role of boldine in peripheral nerve injury–induced neuropathic pain, we conducted studies in a mouse model of SNI, which produces persistent peripheral hypersensitivity to mechanical stimuli.¹⁸ In brief, 10-week-old female C57BL6 mice were subjected to SNI or sham surgery. Customized diet containing boldine or control diet was administered immediately after surgery. Pain phenotyping tests including von Frey filament test, hot/cold plate test, and dynamic weight-bearing test were performed before injury for baseline and starting from 3 days after injury (Fig. 1A, experiment design). The dosage of boldine was monitored by weekly measurement of food consumption (Fig. 1B). The average food intake is 2.5∼2.7 gram per animal per day, which is equivalent to 50∼54 mg/kg BW/d of boldine. No significant difference in food intake was observed among experimental groups (Fig. 1B).

Figure 1.

Boldine prevents spared nerve injury-induced mechanical allodynia. Female C57BL6 mice were subjected to spared nerve injury (SNI). Boldine was administered right after injury through a customized diet (Research Diet) to reach a dose of 50 mg/kg/d. A control diet was provided to the sham or SNI group. Von Frey filament tests were performed to evaluate mechanical pain responses. (A) Diagram of experiment design. (B) Food intake was monitored weekly and average daily intake was calculated and plotted in GraphPad Prism 10. (C) Withdraw threshold to mechanical stimuli on the ipsilateral hind paw before and postinjury; (D) Paw withdraw threshold to mechanical stimuli on the contralateral hind paw. Data represent mean ± SEM, N = 8 per group, ***P < 0.001 (SNI vs sham), ###P < 0.001 (SNI + boldine vs SNI) by 2-way ANOVA with Tukey post-tests. ANOVA, analysis of variance; SEM, standard error of the mean.

As expected, SNI significantly induced mechanical pain hypersensitivity on the ipsilateral hind limb at 3-, 7-, and 14-day post-injury (dpi) when compared with sham-operated animals. Oral administration of boldine significantly attenuated this injury-induced mechanical allodynia at 7 and 14 dpi (Fig. 1C). There was no change of responses to mechanical stimuli after nerve injury or boldine treatment on the contralateral hind limb (Fig. 1D), suggesting that the boldine-mediated prevention of mechanical-induced allodynia is specific to the region innervated by the injured nerve.

3.2. Boldine reduces spared nerve injury–induced thermal pain

Rodents with SNI often exhibit increased sensitivity to thermal stimuli, manifesting as decreased withdrawal latencies in response to heat or cold compared with control animals.¹⁸ To test the effect of boldine in altering thermal pain phenotypes, we performed the hot plate test and the cold plate test after SNI. At 7- and 14-dpi, SNI animals developed increased sensitivities to both warm (Fig. 2A) and cold stimuli (Fig. 2B). Administration of boldine significantly attenuated the SNI-induced hypersensitivity to warm temperature at both time points (Fig. 2A) and to cold temperatures at 7 dpi (Fig. 2B).

Figure 2.

Boldine reduces SNI-induced thermal pain hypersensitivity. Hot/cold plate tests were performed to evaluate thermal pain responses before (Pre) and after (7 dpi and 14 dpi) injury in sham animals (Sham) and animals with (SNI + boldine) or without (SNI) boldine treatment. (A) Latency to nociceptive responses by the hot plate test (50°C). (B) Latency to nociceptive responses by the cold plate test (15°C). Data represent mean ± SEM, N = 8 to 10 per group, *P < 0.05, **P < 0.01, ***P < 0.001 by 2-way ANOVA with Tukey post-test. ANOVA, analysis of variance; SEM, standard error of the mean; SNI, spared nerve injury.

3.3. Boldine prevents spared nerve injury–induced weight-bearing deficits

Alterations in weight-bearing behavior can be functional outcome measurements of peripheral nerve injury. In the context of SNI, changes in weight-bearing distribution are often observed as part of the behavioral phenotypes.⁴⁹ To evaluate the effect of boldine on potential SNI-induced weight-bearing deficits, we performed weight-bearing tests using the dynamic weight bearing 2.0 system, an observer-independent system to measure weight-bearing behavior phenotypes in animal models.^19,44 As compared with sham-operated mice, injured mice tended to use the ipsilateral hind limb less for weight support at 3 and 7 dpi, and boldine administration significantly alleviated the SNI-induced weight-bearing deficit at 7 dpi (Fig. 3A). We also measured some additional parameters including the hind limb weight support area ratio and rearing duration. Rearing behavior, where a mouse stands on its hind legs with its forelimbs raising and head extending, has been used as a spontaneous pain-like behavior indicator in mice.⁶² Spared nerve injury led to significantly decreased ipsilateral/contralateral weight support area ratio (Fig. 3B) and markedly lower rearing duration (Fig. 3C). Boldine did not prevent SNI-induced deficits in weight support area or rearing duration (Figs. 3B and C).

Figure 3.

Effect of boldine on SNI-induced weight-bearing deficits. Assessment of weight-bearing behavior measuring the changes in postural equilibrium was achieved using the Dynamic Weight Bearing System 2.0 (Bioseb) in sham animals (Sham) and animals with (SNI + boldine) or without (SNI) boldine treatment. (A) Weight support ratio of the ipsilateral hind limb vs the contralateral hind limb; (B) Support area ratio of the ipsilateral hind limb vs the contralateral hind limb; (C) Rearing duration. Data represent mean ± SEM, N = 8 to 10 per group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-way ANOVA with Tukey post-tests. ANOVA, analysis of variance; SEM, standard error of the mean; SNI, spared nerve injury.

3.4. Boldine reduces the expression of some cytokines/chemokines in the serum

To evaluate the effect of boldine on peripheral inflammation markers, serum samples were subjected to a multiplex ELISA using the MILLIPLEX Mouse Cytokine/Chemokine Magnetic Bead Panel (Millipore). Spared nerve injury induced the elevation of interleukin-12 (IL-12p40), interleukin-15 (IL-15), macrophage colony-stimulating factor (M-CSF), and C-C motif chemokine ligand 5 (CCL5) in the serum, which was prevented by boldine treatment (Fig. 4).

Figure 4.

Levels of serum inflammation markers after spared nerve injury (SNI) and boldine treatment. Serum samples from sham (Sham) animals and mice subjected to SNI or SNI plus 50 mg/kg boldine (SNI + Boldine) collected at 15 dpi were subjected to a multiplex ELISA using the MILLIPLEX Mouse Cytokine/Chemokine Magnetic Bead Panel (Millipore) and the median fluorescent intensity was used to calculate the concentration of each cytokine/chemokine. (A) IL-12p40; (B) IL-15; (C) CCL5; (D) M-CSF. Data represent mean ± SEM, N = 8 to 10 per group, *P < 0.05, **P < 0.01 by 1-way ANOVA with Tukey post-test. ANOVA, analysis of variance; CCL5, C-C Motif chemokine ligand 5; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; M-CSF, macrophage colony-stimulating factor; SEM, standard error of the mean.

3.5. Boldine inhibits glia activation in the lumbar spinal cord

In pain conditions, nociceptive signals are transmitted from peripheral nerves to the spinal cord. Glial cells in the spinal cord dorsal horn play complex and dynamic roles in pain processing. Both activated microglia and reactive astrocytes have been implicated in pathogenesis of pain after nerve injuries.^{12,14,29,36,45} To test the effect of boldine on glia activation, transverse lumbar spinal cord sections were immuno-stained with the astrocyte marker GFAP or the microglia marker IBA1. As compared with sham-operated mice, significantly more reactive astrocytes were detected at the ipsilateral dorsal horn of the lumbar spinal cord after SNI (Fig. 5A). Consistent with previous reports,^26,50 microglia in the spinal cord dorsal horn were activated after SNI (Fig. 5B). Boldine treatment significantly reduced the SNI-induced astrocyte activation (Fig. 5C) and microglia activation (Fig. 5D) in the ipsilateral dorsal horn.

Figure 5.

Boldine decreases levels of reactive astrocytes in the lumbar spinal cord after SNI. Fifty-micrometer thick transverse lumbar spinal cord sections were immuno-stained with astrocyte marker GFAP or microglia marker IBA1 to evaluate the effect of SNI and boldine treatment on glia activation. Images were acquired using the ZEISS LSM 700 confocal imaging system with ×20 objective. (A) Representative images of GFAP staining of the lumbar spinal cord. (B) Representative images of IBA1 staining of the lumbar spinal cord. Red box: ipsilateral dorsal horn. Scale bar: 10 µm. (C) Quantification of GFAP mean fluorescent intensity; (D) Quantification of IBA1 mean fluorescent intensity. Data represent mean ± SEM, N = 4 mice per group. **P < 0.01 by 1-way ANOVA with Tukey post-test. ANOVA, analysis of variance; GFAP, glial fibrillary acidic protein; SEM, standard error of the mean; SNI, spared nerve injury.

3.6. Boldine inhibits the elevation of neuroinflammation markers in the spinal cord in female mice

Glia mediators, such as IL-1β and tumor necrosis factor-α (TNF-α), are key factors in the glial control of pain. It has been reported that overexpression of IL-1β in the injured sciatic nerve after nerve injury may be a common mechanism underlying the generation of neuropathic pain.²⁷ C-C motif chemokine ligand 2 (CCL2), also known as monocyte chemotactic protein 1, and its receptor C-C motif chemokine receptor 2 (CCR2) may play a role in neuroinflammation by participating in neuron–glial cell interactions.^7,46,57 To evaluate the effect of boldine on these inflammatory markers in the spinal cord after SNI, we extracted RNA from lumbar spinal cord collected at 15 dpi and performed quantitative PCR using the standard Taqman method. Significantly elevated levels of TNF-α, IL-1β, and CCL2 (Figs. 6A–C) mRNA were detected in the lumbar spinal cord after SNI. Oral administration of boldine decreased the levels of TNF-α (Fig. 6A) and IL-1β (Fig. 6B). We also examined the mRNA levels of the receptors of TNF-α, IL-1β, and CCL2, and found no difference (Figs. 6D–F).

Figure 6.

Boldine decreases mRNA levels of inflammation markers in the spinal cord. Total RNA was extracted from lumbar spinal cords collected at 15 dpi and subjected to gene expression analysis by quantitative PCR using standard Taqman methods. Relative levels were calculated using the 2^–ΔΔCt method with HPRT as an internal control. (A) TNF-α; (B) IL-1β; (C) CCL2; (D) TNFR; (E) IL1R; (F) CCR2 mRNA levels. Data represent mean ± SEM, N = 6 per group, *P < 0.05, **P < 0.01, ***P < 0.001 by the unpaired t test. CCL, C-C motif chemokine ligand; CCR, C-C motif chemokine receptor; HPRT, hypoxanthine phosphoribosyltransferase 1; IL, interleukin; SEM, standard error of the mean; TNF-α, tumor necrosis factor-α; TNFR, tumor necrosis factor receptor.

3.7. Boldine inhibits lipopolysaccharides-induced elevation of inflammation mediators in microglial cells

Neuroinflammation acts as both a driver and an amplifier of pain states, particularly in chronic conditions. To test whether boldine could reduce neuroinflammation, BV-2 microglia cells were treated with LPS with or without boldine. After treatment, medium was collected and subjected to cell viability assays. Neither the 25 µg/mL or the 50 µg/mL dose of boldine showed any cytotoxicity (Supplemental Figure 1A, http://links.lww.com/PR9/A368). Cells were then harvested and subjected to gene expression analysis by quantitative PCR. Lipopolysaccharides treatment led to significantly elevated mRNA levels of chemokines (CCL2, CCL3, and CCL5), cytokines (IL-1β, IL-6, and IL-18), macrophage and microglia marker CD11b, toll-like receptors (TLR2 and TLR3), and NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) (Fig. 7). Coincubation with boldine inhibited the LPS-induced upregulation of the abovementioned molecules, suggesting a beneficial role of boldine in LPS-induced inflammation. We also found that IL-1β protein levels were drastically increased after LPS treatment, which was prevented by boldine cotreatment (Supplemental Figure 1B, http://links.lww.com/PR9/A368).

Figure 7.

Boldine prevents lipopolysaccharides (LPS)-induced inflammation in vitro. BV-2 microglia cells were treated with 100 ng/mL LPS with or without boldine (25 µg/mL or 50 µg/mL) for 6 hours. Cells were harvested and subjected to RNA extraction followed by cDNA synthesis and quantitative PCR using standard Taqman methods. Taqman probes include CCL2, CCL3, CCL5, CD11b, IL-1β, IL-6, IL-18, TLR2, TLR3, and NLRP3. Relative levels were calculated using the 2^–ΔΔCt method with HPRT as an internal control. Data represent mean ± SEM, N = 3 per condition, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 1-way ANOVA followed by Tukey post-tests. ANOVA, analysis of variance; CCL, C-C Motif chemokine ligand; HPRT, hypoxanthine phosphoribosyltransferase 1; IL, interleukin; NLRP3, nod-like receptor pyrin containing 3; SEM, standard error of the mean; TLR, toll-like receptor.

4. Discussion

The SNI model is a commonly used model in rodents to study neuropathic pain. Spared nerve injury leads to mechanical allodynia, which develops 3 days after injury and lasts between 4 weeks and 12 months in mice.²⁸ Spared nerve injury also leads to thermal hypersensitivity and weight-bearing deficits. In this study, we administered boldine through a customized diet to avoid potential stress associated with gavage or injections. Boldine is a bitter alkaloid and there is no added flavor in the diet, and one potential risk is that the bitter taste might negatively affect food intake in the boldine treatment group. We monitored food consumption and did not observe any difference between experimental groups, suggesting that the bitter taste does not have any influence on food intake. We found that oral administration of boldine could prevent SNI-induced mechanical allodynia at 3, 7, and 14 dpi. A potential confound is whether boldine could impair locomotor function which could slow or weaken paw withdrawal responses to mechanical stimulus. Several lines of evidence argue against this possibility. First, boldine did not alter the withdraw threshold on the contralateral hind paw; second, a recent study from our laboratory showed no effect of boldine (50 mg/kg/d) on muscle weights in a mouse model of spinal cord injury⁵¹; third, the pharmacological effects of boldine have been studied in naive and multiple rodent models of disease without any reported defects in motor function.⁵² Boldine prevented SNI-induced thermal hypersensitivity and weight-bearing deficit at 7 dpi. We did not observe boldine-mediated prevention of mechanical allodynia or weight-bearing deficits at 3 dpi, which might be partially attributed to possible neophobia to the diet (ie, receiving a lower dose of boldine for the first several days). Unlike mechanical allodynia, which is likely to have central mechanisms,¹⁸ thermal pain responses or weight-bearing deficits started to recover at 14 dpi, suggesting potential different underlying mechanisms, possibly peripheral in origin.

Inflammatory responses are of great significance in the development of neuropathic pain. A large body of evidence indicates that inflammatory mediators in local inflamed tissues, peripheral nerves, and spinal cord make important contributions to the initiation and maintenance of chronic pain.^32,34 In this study, we have observed significantly elevated expression of cytokine/chemokines including IL-12p40, IL-15, CCL5, and M-CSF in the serum after SNI. IL12p40 is a subunit of IL12. Application of IL-12p40 has been shown to induce analgesia to mechanical stimulation in rats exposed to neuropathic pain.¹³ IL-15 has been linked with nerve injury-induced neuropathic pain in a rodent CCI models²⁴ as well as rheumatoid arthritis- and osteoarthritis-induced joint pain in humans.⁶¹ Intradermal injection of CCL5 has been shown to induce pain and administration of Met-CCL5, an antagonist of CCL5 receptor CCR5, attenuates inflammatory and nociceptive responses after peripheral nerve injury.⁴² In humans, upregulated CCL5 has been detected in patients with various pain conditions including small fiber neuropathy,³⁷ atypical facial pain and trigeminal neuralgia,⁴⁰ and discogenic back pain.^25,38 Macrophage colony-stimulating factor is also an important contributing factor to the development of neuropathic pain⁶⁵ and arthritic pain.¹⁶ Our data suggested that boldine administration prevented the SNI-mediated induction of these pain-related inflammatory mediators in the periphery.

Neuroglial cells, including microglia, astrocytes, oligodendrocytes, and ependymal cells, play complex and dynamic roles in pain processing. Their activation and interactions with neurons contribute to the amplification and maintenance of pain signals, as well as the development of chronic pain conditions.³¹ Our immunofluorescence data confirmed an increased number of reactive astrocytes and activated microglia in the ipsilateral spinal cord at 15 dpi. Boldine treatment significantly prevented the injury-induced glia activation. Notably, the activation of astrocytes and microglia seems weaker than what is generally reported, and no detectable signal of TNFα and IL-1β was identified by immunostaining (data not shown), possibly due to the peak of glia activation occurring at a more acute phase. In the future, additional time-course studies could be performed to evaluate the effect of boldine at various time points starting from 1 day post-SNI (acute to chronic) to address this possibility. The prolonged glia activation in the ipsilateral spinal cord and some levels of reactive astrocytes in the contralateral spinal cord detected at 15 dpi could possibly be attributed to the central sensitization process in pain chronification and maintenance. Further experiments could also be conducted to evaluate the effect of boldine in brain regions that are involved in pain perception. In addition to CNS, immune cells in the PNS also play an important role in pain modulation.⁴³ It would be of interest to evaluate the effect of boldine treatment on the cellular and molecular levels in the peripheral nerve and dorsal root ganglia after SNI.

Recent studies have shown that after nerve injury, TLRs are involved in the generation of neuropathic pain.³⁹ In this study, we found decreased expression of toll-like receptors (eg, TLR2, TLR3), and NLRP3, as well as multiple inflammatory cytokines and chemokines including CCL2, IL-1β, IL-6, and IL-18 after boldine treatment in BV-2 microglial cells, suggesting that inhibiting TLR/NLRP3 inflammasome signaling could be a potential mechanism of action of boldine. However, the direct target of boldine still remains to be characterized. Boldine has been shown to interact with several cell surface proteins, including connexin hemichannels,^11,56,64 serotonergic receptor,⁵⁸ and dopaminergic receptor.² Receptor binding and target engagement essays could provide better understanding of the underlying mechanism.

In summary, we report for the first time that oral administration of boldine, a naturally occurring alkaloid, reduced neuroinflammation and attenuated neuropathic pain in a mouse model of spared nerve injury, suggesting that boldine might be considered as a novel therapeutic agent for the treatment of neuropathic pain. Sex differences in neuropathic pain are observed at many levels, including prevalence, intensity, and underlying biological mechanisms. We focused on female mice in this study because females are more likely to report chronic pain men,⁶⁶ but males may be more susceptible to certain pain-related cognitive impairments.⁸ Future studies will be conducted across sexes to explore the underlying mechanisms how the boldine and its derivatives differentially influence pain perception in the central nervous system and peripheral nervous system, and possibly extend our research to other pain conditions, including arthritic pain, back pain, and diabetic pain.

Disclosures

C.A.T., J.C.S., C.P.C., and W.Z. are coinventors for US Patent No. 11,707,459 for the use of boldine to treat neurological injuries. This study falls within the claims of the patent. The remaining authors declare no conflict of interest.

Supplemental digital content

Supplemental digital content associated with this article can be found online at http://links.lww.com/PR9/A368.