DOI: https://doi.org/10.21203/rs.3.rs-1385762/v1
We aimed to examine the pain inhibitory effects of systemic administration of choline and parezoxib sodium in a rat model of neuropathic pain induced by a chronic constriction injury (CCI) of the sciatic nerve, and to determine the changes of high mobility protein-1 (HMGB1) and NF-κBp65 expression in the dorsal root ganglion (DRG) after drug treatment.
Adult rats were randomly divided into 5 groups (N = 8/group). Sham surgery group received an intraperitoneal (i.p.) injection of saline. CCI rats received saline, choline (cho, 6, 12 and 24 mg/kg), parecoxib sodium (pare, 3, 6, 12mg/kg), or a combination of choline and parecoxib sodium with each drug delivered at a sub-effective dose. Mechanical and heat pain thresholds were measured at 30 min after drug treatment at day 3, 5, 7, 10, and 14 after CCI. In a separate study, rats were also divided into 5 groups (N = 6/group) including sham, CCI + saline, CCI + cho-6 mg/kg, CCI + pare-3mg/kg, and CCI + cho-6mg/kg + pare-3mg/kg group. After repeated drug treatment for 7 days, five rats were randomly selected in each group, and the lumbar DRGs (L4-6) were harvested for Western blot study.
Choline significantly attenuated the mechanical and heat hypersensitivities in CCI rats at 12 and 24 mg/kg doses (P < 0.05), but was not effective at 6 mg/kg dose. Parecoxib sodium exerted significant pain inhibitory effects at 6 and 12 mg/kg doses (P < 0.05), but not at 3 mg/kg dose. Combining a low dose of choline (6 mg/kg) and parecoxib sodium (3 mg/kg) produced significant pain inhibition in CCI rats, and also reduced the expression of HMGB1 and NF-κBp65 in L4-6 DRGs.
A combination of choline and parecoxib sodium may effectively relieve neuropathic pain, and the underlying mechanisms may involve an inhibition of HMGB1/TLR4/NF-κB signaling pathway in DRG neurons.
Neuropathic pain is a chronic disease caused by the injury of the nervous system or functional disorders.[1] After the nervous system injury, the release of inflammatory factors and mediators will induce inflammatory responses. These changes may increase the excitability of nociceptive sensory neurons causing peripheral and central sensitization, which are involved in neural plasticity and contribute to the occurrence and maintenance of neuropathic pain.[2]
Because of its complex underlying mechanisms, the clinical treatment of neuropathic pain remains to be unsatisfactory. Previous studies showed that choline and paracetin sodium had synergistic analgesic effects in an acetic acid-induced pain model. Their interaction may play a role in modulating the NF-κB signaling pathway.[3] In this study, we examined the two drugs for the treatment of neuropathic pain, and determined whether they may exert additive or synergistic effect, which remain unknown. We further explored the mechanisms underlying the pain inhibitory drug action.
Adult male Sprague Dawley (SD) rats (6–8 weeks, 180-220g) were provided by the Academy of Military Medical Sciences Experimental Animal Center (SCXK-Jun 2012-0004). All experiment protocols were reviewed and approved by the PLA General Hospital Ethics Committee. Animals were acclimated to the testing environment for 2 days. Animals received sub-cage feeding, and were housed (room temperature 20–25 ℃, humidity 60% -70%) with free access to food and water.
Tartaric acid choline (CAS No. 87-67-2, Shanghai Aladdin Biotechnology Co., Ltd, China), parecoxib sodium (Pfizer Ltd, lot: J20080044, USA), pentobarbital sodium (Beijing Chemical Reagent Company, Beijing, China), 0.9% saline solution ( Shijiazhuang four drugs Co., Ltd., Hebei) were stored at 4 ℃.
The rat CCI model was prepared according to the methods described by Bennett et al.[4] Rats were deeply anesthetized with 2% pentobarbital sodium (45 mg/kg, i.p.), and secured on the operating table. Skin in the middle of the left limb was opened after disinfection. The left sciatic nerve was exposed at mid-thigh level by blunt dissection through the biceps femoris muscle. Four loosely constrictive ligatures (4 − 0 chrome line, bubble in sterile saline for 30 min 4) were tied around the nerve near the bifurcation site, and at spaces of ~ 1 mm. The nerve ligation was performed slowly which caused a mild fibrillation in calf muscle, but did not block blood circulation. The muscle layer was approximated and the skin was closed with sutures.
Rats were randomly divided into 5 groups (N = 8/group) including sham: rats received sham surgery and saline injection; CCI + saline: rats received CCI surgery and saline treatment; CCI + choline: CCI rats received choline (6, 12, 24 mg/ kg); CCI + parecoxib: CCI rats received parecoxib (3, 6, 12mg/kg); CCI + choline + parecoxib: CCI rats received both parecoxib and choline, each drug was delivered at a sub-effective dose. Drugs were given by intraperitoneal (i.p.) injection in the morning (9: 00–10: 00 AM) for 7 days (one injection/day).
Rats were tested on day 3, 5, 7, 10, and 14 after surgery. Behavior study was conducted between 8: 00 AM and 5: 00 PM. The room temperature was maintained at 20–24 ° C. The pain threshold was measured at 30 min after each drug injection.
To examine mechanical pain threshold, rats were placed in an empty cage and remained calm. Mechanical stimulation (von Frey filaments) was applied to the dorsal surface of the left hind paw, which was ipsilateral to side of nerve injury. Each von Frey filament was applied perpendicularly to the skin to slowly bend the cilia. There was a 30 sec interval between two tests. If a withdrawal response was evoked, the next stimulus was applied after the rat resettled.
To examine the thermal pain threshold, rats were placed in the plexiglass box which was put on a 6 mm thick glass plate. After 15 minutes of adaptation, rats were stimulated with a light beam applied to the plantar side of the left hind paw. The time (sec) between the starting of heat stimulation and an evoked paw withdrawal response was measured as the thermal withdrawal latency. The cut-off time was set to 20 sec to prevent tissue damage. The light intensity was set to 40% and kept consistent during the experiment. Each rat was measured 3 times at an interval of 5 minutes, and data were averaged for analysis.
Rats were divided into 5 groups (N = 6/group) including Sham (S), CCI + saline (M), CCI + cho-6mg/kg (P), CCI + pare-3mg/kg (C), and CCI + cho-6mg/kg + pare-3mg/kg (L). CCI rats received drug treatment for 7 days. After that, L4-6 DRGs were quickly removed and stored in liquid nitrogen. Tissues from five rats which were randomly selected in each group were used in Western blot study to examine the protein levels of HMGB1 and NF-κBp65.
All statistical analysis was carried out with SPSS22.0 statistical software. Comparisons between groups were made by using single factor analysis of variance (ANOVA), Data are expressed as mean ± standard error. Statistical significance was set at P < 0.05 in all tests.
Compared to the pre-injury condition, saline-treated CCI rats developed mechanical hypersensitivity on the ipsilateral hind paw, as indicated by a significant decrease of mechanical withdrawal threshold, which occurred at 3 day after injury and persisted till the end of the study. In contrast, saline-treated sham rats did not show mechanical hypersensitivity. Choline significantly inhibited mechanical hypersensitivity in CCI rats at 12 and 24 mg/kg doses (P༜0.05), but not at 6 mg/kg dose (Fig. 1A). Parecoxib induced significant anti-allodynic effects at 6 and 12 mg/kg doses (P༜0.05), but not at 3 mg/kg dose (Fig. 1B).
Saline-treated CCI rats also developed heat hypersensitivity at 3 day post-injury, and persisted till the end of the study. Saline-treated sham rats did not show heat hypersensitivity. In CCI rats, choline reduced heat hypersensitivity at 12 and 24 mg/kg doses (P༜0.05), but not at 6 mg/kg dose (Fig. 2A). Parecoxib reduced heat hypersensitivity at 6 and 12 mg/kg doses (P༜0.05, Fig. 2B).
Importantly, the combination of a low dose choline (6 mg/kg) and parecoxib (3 mg/kg) resulted in a significant inhibition of both mechanical and heat hypersensitivities (P < 0.05, Fig. 3A-B).
Compared to the sham group, the expression levels of HMGB1 and NF-κBp65 were significantly increased in CCI rats which received saline injection (Fig. 4). Compared to that in CCI rats after saline treatment, the levels of HMGB1 and NF-κBp65 were significantly decreased in CCI rats which received combined choline and parecoxib treatment for 7 days (P < 0.05). However, the levels of HMGB1 and NF-κBp65 were not significantly decreased in CCI rats after receiving single drug treatment.
Neuropathic pain is an intractable chronic pain condition. The efficacy of sodium channel inhibitors for treating neuropathic pain has been examined in the past decade.[5] Because current therapies for neuropathic pain are not suitable for some patients, the search for new pain medications and therapies must continue.[6]
In this study, we examined pain inhibitory effects of choline and parecoxib sodium in rats with neuropathic pain. We used the CCI model because it has been reported that this model may simulate some characteristics of neuropathic pain in humans, and CCI of the sciatic nerve induced rapid and significant mechanical and heat hypersensitivities in rats.[7] Similarly, our study showed that CCI rats began to develop pain hypersensitivity at 3 days after injury, and persisted through the study period. The affected hind limb (i.e., ipsilateral to the side of CCI) showed eversion, and were often lifted to avoid touching the ground. The paw withdrawal threshold to mechanical stimulation and paw withdrawal latency to heat stimulation were significantly decreased, as compared to that before the injury.
Parecoxib sodium is a highly selective COX-2 inhibitor. It inhibits the production of PGE2 and nitric oxide and regulates neurotransmission in the spinal cord dorsal horn.[8] Parecoxib sodium has been reported to reduce the accumulation of tumor necrosis factor -α (TNF-α) and neutrophils in inflammatory exudates (Add reference please). Compared to other commonly used NSAIDs (e.g., diclofenac and nimesulide), parecoxib sodium induces a quicker pain inhibition in humans.[9] Studies also showed that parecoxib sodium reduced pain and inflammatory responses in formalin and other inflammatory pain models.[10] Our study demonstrated that after 7 days of treatment with 6 mg/kg and 12 mg/kg parecoxib sodium, CCI rats showed significant reductions of mechanical and heat hypersensitivities, as compared to pre-drug condition. However, 3 mg/kg dose was not effective. Thus, moderate and high doses of parecoxib sodium reduced neuropathic mechanical and heat hypersensitivities, and these findings are consistent with a previous report.[11]
As a specific α7nAChR agonist, choline exerts anti-inflammatory and analgesic effects through activation of cholinergic anti-inflammatory pathway,[12] which was shown in several pain models and sepsis models.[13] In acetic acid torsion model, choline induced dose-dependent analgesic effects.[14] In current study, choline attenuated mechanical and heat hypersensitivity in CCI rats at 12 mg/kg and 24 mg/kg doses, but 6 mg/kg was not effective. Under chronic neuropathic pain condition, the decomposition and synthesis of ACh were reduced in the hypothalamus, causing a low level of cholinergic activity.[15] Our findings suggest that choline supplement may help to alleviate chronic pain after nerve injury.
In patients with diabetic neuropathy and postherpetic neuralgia, a previous study showed that morphine in combination with a low dose of gabapentin provided a better pain relief than morphine alone.[16] In current study, co-treatment of CCI rats with a low, sub-effective dose of choline (6 mg/kg) and parecoxib sodium (3 mg/kg) induced a significant pain inhibition, indicating that two drugs may exert an additive or synergistic pain inhibitory effect. Using low doses may limit the dose-limiting adverse effects for each drug .
After peripheral nerve injury, glial cells were activated and pro-inflammatory cytokines were released in the spinal cord.[17] HMGB1 is a late inflammatory factor. It can promote the release of pro-inflammatory factors and more HMGB1 from inflammatory cells, thus forming a positive feedback loop and aggravating inflammatory responses.[18] NF-κBp65 is an activated nuclear signal transduction protein, and mediates the production of inflammatory mediators.[19] Our study showed that the expressions of HMGB1 and NF-κBp65 were increased in DRGs after CCI, which may increase the inflammatory responses and pain.
Parecoxib sodium was shown to reduce the expression of HMGB1 in cerebral ischemia model, and hence it plays a neuroprotective role.[‘20] Choline-activated α7nAChR signaling pathway was suggested to be sensitive to control HMGB1 release.[21] In this study, although there was a trend that parecoxib sodium at 3 mg/kg dose reduced HMGB1 and NF-κBp65 levels, the difference was not statistically significant. Similarly, choline at 6 mg/kg dose did not significantly reduced the upregulated HMGB1 and NF-κBp65 expression in CCI rats. However, a combination of these drugs significantly reduced the expressions of HMGB1 and NF-κBp65, which was consistent with findings in our behavioral study. HMGB1 can bind and activate TLR4 receptors, and induces the production and release of inflammatory factors. NF-κB, as a major nuclear signal transduction protein that mediates the production of a variety of inflammatory mediators, is downstream of HMGB1/TLR4 pathway.[22–23]Therefore, these findings suggest that choline and parecoxib sodium may both inhibit HMGB1/TLR4/NF-κB signaling pathway, and may be used together for the treatment of neuropathic pain which remains a great clinical challenge.
Parecoxib sodium is frequently used in the clinic as an analgesic, but has dose-limiting side effects.[24]The cholinergic pathway may also be targeted for pain inhibition by inducing an anti-inflammatory effect.[25]Current findings suggest that combining choline and parecoxib sodium, which have different properties, may improve neuropathic pain inhibition and decrease the side effects of each drug by reducing the required dose. This notion needs to be further tested in future clinical studies.
1. In a rat model of chronic neuropathic pain(CCI),at a certain dose,choline or parecoxib sodium can alleviate mechanical pain and thermal hyperalgesia caused by CCI.
2. The combination of choline and parecoxib sudium in non-analgesic doses can effectively relieve neuropathic pain, and its mechanism may be related to the inhibition of HMGB1/TLR4/NF-κB pathway.
Acknowledgments
The author thanks all the participants, they made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data. We acknowledge the Academy of Military Medical Sciences for giving us the chance to conduct this research.
Funding
No funding is declared
Author information
Affiliations
Anesthesiology department, Civil Aviation General Hospital, No. 1 Gaojingjia, Chaoyang District, Beijing, 100123, China;
Na Zhang
First Medical Center, Chinese PLA General Hospital, No. 28, Fuxing Road, Haidian District, Beijing, 100853, China
Yang Li, Zeguo Feng
Contributions
Methodology and Original draft preparation: NZ; Statistical analysis and Review: YL; Supervision, Resource, and Review: ZF. All authors read and approved the final manuscript.
Corresponding author
Correspondence to Zeguo Feng
Ethics declarations
This study was approved by the Animal Care and Use Committee of Chinese PLA General Hospital on 16 February 2021 (ethic approval ID: 20210216–001) and performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study was carried out in compliance with the ARRIVE guidelines.
Consent for publication
Informed consent obtained from all the participants to publish the information/image(s) in an online open-access publication.
Competing interests
We declare that we have no conflicts of interest in this work.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.