Dexmedetomidine alleviates inflammation-induced neuropathic pain by suppressing NLRP3 via activation of Nrf2


 Objective: To investigate the mechanism of dexmedetomidine (DEX) involving Nrf2-dependent inhibition of NLRP3 in relieving neuropathic pain in chronic constriction injury (CCI) rat models.Methods: The CCI rat models were constructed through sciatic nerve ligation. The CCI rats were treated with DEX, Nrf2 inhibitor (ML385), NLRP3 antagonist (MCC950) and NLRP3 activator (Nigericin). Mechanical withdrawal threshold (MWT) was measured to test the pain sensitivity of CCI rats. H&E staining detected spinal injury of the rats and TUNEL staining was applied to test apoptosis in the spinal cords. ELISA measured the expressions of inflammatory factors. The expressions of Nrf2 and NLRP3 were also detected.Results: Decreased MWT, enhanced spinal cord injury, promoted apoptosis and increased inflammatory factors were detected in CCI rats. The expressions of the above indicators were retraced in DEX-treated CCI rats. Increased MWT, reduced spinal cord injury, inhibited apoptosis and decreased inflammatory factors were detected in rats treated with MCC950 or ML385 while opposite expression patterns were found in rats treated with Nigericin. The expressions of these indicators were retraced in both DEX+ML385 group and MCC950+ML385 group compared to ML385 group and MCC950 group respectively.Conclusion: DEX reduces neuropathic pain of CCI rats by suppressing NLRP3 through activation of Nrf2.


Introduction
In ammatory responses play a pivotal role in various neurodegenerative diseases such as Alzheimer's disease, multiple sclerosis and Parkinson's disease [1]. Neuroin ammation is characterized by activation of pro-in ammatory cytokines and chemokines which promote chronic widespread pain in central nervous system [2]. Chronic neuropathic pain may complicate neuropathic symptoms and treatment decisions, leading to poor outcomes and impaired life quality of patients [3]. Diagnosis of neuropathic pain is complex for its diverse clinical features and pharmacological treatment is the most widely used approach in neuropathic pain [4]. Further research and clinical trials should be implemented to improve the diagnosis and treatment of neuropathic pain. The present study intends to validate the antiin ammatory effect of dexmedetomidine (DEX) on in ammation-induced neuropathic pain in chronic constriction injury (CCI) rat models.
DEX is a potent and highly selective α2-adrenoceptor agonist with sedative, anxiolytic, sympatholytic, and analgesic abilities [5]. This drug is used to induce short-and longer-term sedation among patients in intensive care unit and has effective suppression on delirium [6]. DEX was reported to have the potential for prevention of acute pain in adults performed with abdominal surgery [7]. Moreover, a steadily growing number of studies have investigated the inhibitory effect of DEX on neuropathic pain. For instance, DEX suppressed neuropathic pain by inhibiting P2 × 7R through regulation of ERK in a rat model of CCI [8].
NLRP3, belonging to the protein family of nucleotide-binding oligomerization domain-like receptor (NLR), is one of the pattern recognition receptors which contribute to the formation of in ammasomes [9].
Among those in ammasomes, NLRP3 in ammasome has been intensively investigated and veri ed to play a critical role in innate immunity and pathology of human diseases [10]. NLRP3 functions as an in ammation promoter through cleavage of pro-in ammatory cytokines IL-1β and IL-18 by stimulating caspase-1 [11]. Peter M. Grace et al. found that morphine promoted the intensity and duration of neuropathic pain by activating NLRP3 in ammasome [12]. However, little attention has been attached to whether NLRP3 was implicated in DEX-induced reduction of in ammatory responses and neuropathic pain.
Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a multifunctional protein that modulates antioxidant as well as other cytoprotective genes and is involved in in ammatory processes [13,14]. Up-regulation of Nrf2 by Plumbagin was found in the alleviation of CCI-induced neuropathic pain [15]. In addition, a new biphenyl diester derivative AB-38b attenuated diabetic nephropathy by suppressing NLRP3 via Nrf2 activation [16]. However, the regulation of NLRP3 through Nrf2 signaling is rarely discussed in neuropathic pain.
A recent study claimed that DEX exerted neuroprotective effect via Nrf2 signaling pathway [17]. The present research utilizes NLRP3 antagonist MCC950, NLRP3 activator Nigericin and Nrf2 inhibitor ML385 to determine the effects of NLRP3 and Nrf2 in neuropathic pain, and veri es that DEX exerted suppression on neuropathic pain by inhibiting NLRP3-mediated in ammation via activation of Nrf2.

Animal experiments
Male Sprague-Dawley rats (n = 108, 200 ~ 220 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. The rats were subjected to a 12 h/12 h light-dark cycle (light: 8 am to 8 pm; dark: 8 pm to 8 am) at room temperature (23 ± 1 °C) and fed with standard food and water for at least one week. All the animal experiments were approved by the Animal Protection and Use Committee of Hunan Provincial People's Hospital and strictly followed the "Guidelines for the Use and Management of Laboratory Animals" issued by the National Institutes of Health (NIH).
The rats were subjected to sciatic nerve ligation operation after being anesthetized using pentobarbital sodium (40 mg/kg). An incision was made at the biceps femoris in the left thigh to expose the sciatic nerve. Four sterile 4 − 0 chromic gut sutures (ethicon, Somerville, NJ), 1 mm apart, were loosely tied to the proximal end of the trifurcation of the sciatic nerve so long as each suture could detect small twitches of the hind limb. After ligation, the muscle and skin were immediately stitched. Rats that received sciatic nerve ligation were established as chronic constriction injury (CCI) models (Model group). Rats in sham group were sham-operated (no nerve ligation was performed). Rats in Control group were left totally untreated (no surgery or drug treatment).
After model establishment, rats were subjected to corresponding treatments and accordingly grouped into DEX group (intraperitoneally injected with 5 µg/kg DEX for post-operational 7 days, Nhwa Pharmaceutical, Jiangxi, China), MCC950 group (intraperitoneally injected with 50 µg/kg NLRP3 antagonist MCC950 for post-operational 7 days, MedChemExpress, Shanghai, China), Nigericin group (intraperitoneally injected with 1 mg/kg NLRP3 activator Nigericin for post-operational 7 days, MedChemExpress, Shanghai, China), DEX + ML385 group (intraperitoneally injected with 5 µg/kg DEX and 30 mg/kg Nrf2 inhibitor ML385 for post-operational 7 days, Selleck, Shanghai, China), DEX + Nigericin group (intraperitoneally injected with 5 µg/kg DEX and 1 mg/kg Nigericin for post-operational 7 days) and MCC950 + ML385 group (intraperitoneally injected with 50 µg/kg MCC950 and 30 mg/kg ML385 for post-operational 7 days). The animal groups and treatment are listed in Table 2. Rats subjected to behavioral tests received drug treatment for post-operational 14 days. Experiment timetable is shown in Fig. 1. Rats were subjected to behavioral tests 0, 1, 3, 7 and 14 after operation. These rats were accordingly assigned into Control, Sham, Model, DEX, MCC950, Nigericin, DEX + ML385, MCC950 + ML385 and DEX + Nigericin groups (n = 6 in each group). The behavioral tests were conducted from 9 am to 12 am in a noiseless environment. The rats were placed in a wire cage for 30 minutes to adjust to the environment before experiment. Mechanical withdrawal threshold (MWT) of the rats was measured every 5 min for three times (each time for 2 s) using automated dynamic plantar aesthesiometer (Ugo Basile, Varese, Italy). Von Frey laments were vertically placed on the inner plantar surface of rat's right hind paw and increasing pressure was put on to bend the laments (from 0.6, 1, 2, 4, 6, 8, 10, 15, 25 to 60 g; break force = 60 g). The minimum retraction force (G) of rat's right hind paw was recorded and the average value of the power that induced a reliable retreat was recorded as threshold. Quick withdrawal or licking of paws in response to stimuli was considered a positive reaction.
The spinal cords were xed in 4% paraformaldehyde and then dehydrated in 30% sucrose at 4 °C overnight. The spinal cords were embedded in para n and cut into 5 µm sections (10 slices of each spinal cord) for hematoxylin and eosin staining. Finally, the spinal cord sections were observed using a microscope (Olympus ABX50, Tokyo, Japan). The scoring criteria of spinal cord injury are as follows: 0 = no lesion; 1 = gray matter contained 1 ~ 5 eosinophilic neurons; 2 = gray matter contained 5 ~ 10 eosinophilic neurons; 3 = gray matter contained more than 10 eosinophilic neurons; 4 = infarction of less than 1/3 of the gray matter area; 5 = infarction of 1/3 to 1/2 of the gray matter area; 6 = infarction of more than 1/2 of the gray matter area.  Table 1. and allowed for color development with ECL. The brands were then detected using chemiluminescence imaging system (GE Healthcare, Beijing, China).

ELISA
The whole blood of rats from the Control, Sham, Model, DEX, MCC950, Nigericin, DEX + ML385, MCC950 + ML385 and DEX + Nigericin groups (n = 6 in each group) was mixed with EDTA and then centrifuged at 1000 g for 10 min at 4℃ to remove the yellow supernatant. The expressions of TNF-α, IL-1β, IL-6 and IL-10 were detected using a commercial ELISA kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer's instructions.

Statistical analysis
The statistics were analyzed by GraphPad Prism 7.0. T was used to compare differences between two groups. One-way analysis of variance was applied for multi-group comparison. P < 0.05 was considered statistically signi cant.

DEX reduces neuropathic pain of CCI rats
Mechanical withdrawal threshold (MWT) was measured to assess rats' sensitivity to pain. MWT of rats was decreased in Model group compared with Control group while increased in DEX group compared with Model group (Fig. 2A, P < 0.05). This result indicated that rats in Model group had the highest pain sensitivity whereas DEX reduced the pain of CCI rats.
H&E staining showed that the spinal injury area was expanded in Model group compared with Control group while lessened in DEX group compared with Model group (Fig. 2B, P < 0.05). TUNEL positive cells were decreased in Model group compared with Control group while increased in DEX group compared with Model group (Fig. 2C, P < 0.05), suggesting DEX could decrease cell apoptosis in spinal cord tissues of CCI rats. TNF-α, IL-1β and IL-6 were increased in Model group compared with Control group while decreased in DEX group compared with Model group (Fig. 2D, P < 0.05). The expression pattern of IL-10 was different from those pro-in ammatory cytokines (Fig. 2D, P < 0.05). According to the results of qRT-PCR and Western blot, Nrf2 was down-regulated in Model group compared with Control group while upregulated in DEX group compared with Model group (Fig. 2E-F, P < 0.05) Meanwhile, different expression pattern of NLRP3 was also detected (Fig. 2F, P < 0.05). There was no signi cant difference between Sham group and Control group. The above results demonstrate that DEX suppresses in ammation in the spinal cords to reduce neuropathic pain of CCI rats.

NLRP3 increases neuropathic pain of CCI rats
After CCI rats were injected with NLRP3 antagonist MCC950 or NLRP3 activator Nigericin, MWT was found to be increased in MCC950 group while decreased in Nigericin group (Fig. 3A, P < 0.05, vs Model group). Western blot detected down-regulated NLRP3 in MCC950 group and up-regulated NLRP3 in Nigericin group (Fig. 3B, P < 0.05, vs Model group). The result demonstrated the successful suppression or activation of NLRP3 in CCI rats. H&E staining showed that the spinal injury was reduced in MCC950 group while expanded in Nigericin group (Fig. 3C, P < 0.05, vs Model group). According to the results of TUNEL staining, apoptosis was suppressed in MCC950 group while promoted in Nigericin group (Fig. 3D, P < 0.05, vs Model group). Decreases of TNF-α, IL-1β and IL-6 and increase of IL-10 were found in MCC950 group; different expression patterns of these cytokines were detected in Nigericin group (Fig. 3E, P < 0.05, vs Model group). Given the above, NLRP3 can promote neuropathic pain of CCI rats.

DEX reduces neuropathic pain by activating Nrf2 in CCI rats
After CCI rats were injected with Nrf2 inhibitor ML385 followed by DEX treatment, rats in DEX + ML385 group had lower MWT compared to DEX group (Fig. 4A, P < 0.05). Western blot and qRT-PCR tested the expression of Nrf2 (Fig. 4B-C, P < 0.05). Nrf2 was down-regulated in DEX + ML385 group compared with DEX group, indicating the effectiveness of ML385 injection. According to H&E staining and TUNEL staining respectively, spinal injury and apoptosis were aggravated in DEX + ML385 group (Fig. 4D-E, P < 0.05, vs DEX group). TNF-α, IL-1β and IL-6 were over-expressed while IL-10 was under-expressed in DEX + ML385 group (Fig. 4F, P < 0.05, vs DEX group). Moreover, NLRP3 was also up-regulated in DEX + ML385 group (Fig. 4G, P < 0.05, vs DEX group). To conclude, DEX promotes the expression of Nrf2 to relieve neuropathic pain of CCI rats. DEX releases neuropathic pain of CCI rats by suppressing NLRP3 via activation of Nrf2 To investigate the pain relief mechanism of DEX, CCI rats were injected with MCC950 + ML385 or Nigericin + DEX. First, MWT was decreased in MCC950 + ML385 group and increased in Nigericin + DEX group (Fig. 5A, P < 0.05, vs MCC950 group and Nigericin group respectively). Western blot detected upregulated NLRP3 in MCC950 + ML385 group compared to MCC950 group and down-regulated NLRP3 in Nigericin + DEX group compared to Nigericin group (Fig. 5B, P < 0.05). Spinal cords injury and apoptosis were enhanced in MCC950 + ML385 group while suppressed in Nigericin + DEX group (Fig. 5C-D, P < 0.05, vs MCC950 group and Nigericin group respectively). Moreover, increases of TNF-α, IL-1β and IL-6 and decrease of IL-10 were found in MCC950 + ML385 group; different expression patterns of these cytokines were detected in Nigericin + DEX group (Fig. 5E, P < 0.05, vs MCC950 group and Nigericin group respectively). Taken together, DEX inhibits NLRP3 by activating Nrf2 to suppress neuropathic pain of CCI rats.

Discussion
Over the past decades, pharmacotherapy has been considerably recommended for management of neuropathic pain but still with clinical de ciency [18]. The present study is committed to discovery of the mechanism of DEX in relieving neuropathic pain. Collected evidence in this study supports that DEX inhibits NLRP3 by stimulating Nrf2 to suppress neuropathic pain in CCI rat models.
First of all, we demonstrated the neuroprotective role of DEX in a rat model of neuropathic pain. DEX reduced TNF-α, IL-1β and IL-6 while increased IL-10 in CCI rats; meanwhile, the down-regulation of NLRP3 and up-regulation of Nrf2 were also detected. DEX was found to attenuate neuropathic pain by progressing anti-in ammatory activity in chronic constriction injury [19]. Consistent with our results, H.S.M. Farghaly et al. demonstrated that DEX decreased TNF-α and IL-6 to relieve neuropathic pain [20].
Abnormal expression pattern of NLRP3 was also found in CCI rat model which encourage us to dig the possible relationship between NLRP3 and DEX or the mechanism for implication of NLRP3 in neuropathic pain. NLRP3 was found to promote neuropathic pain and in ammatory responses in CCI rats. Despite the absence of research on interaction between DEX and NLRP3, involvement of NLRP3 has been intensively investigated in research on neuropathic pain. For instance, microRNA-223 ameliorated morphine analgesic tolerance to neuropathic pain by down-regulating NLRP3 [21]. Paclitaxel activated neuropathic pain by stimulating NLRP3 in ammasome and pro-in ammatory factor IL-1β [22].
Afterwards, we con rmed that DEX relieved neuropathic pain via the activation of Nrf2. The DEX-treated CCI rats after receiving ML385 obtained an aggravation of neuropathic pain as well as up-regulation of NLRP3. In accordance with our study, the expression of Nrf2 was lowered in a rat model of chronic neuropathic pain [23]. Nrf2 may alleviate oxaliplatin-induced peripheral neuropathy by maintaining mitochondrial homeostasis and suppressing oxidative stress [24]. However, there is lack of research on the regulation of Nrf2 in DEX-induced neuroprotection.
The NLRP3/Nrf2 pathway has been considerably discussed in many pharmacological and pathological conditions such as liver injury [25] and colitis [26]. In the present study, NLRP3 was up-regulated in MCC950-treated CCI rats in response to ML385 injection, indicating the suppressive effect of Nrf2 on NLRP3 expression. Furthermore, DEX in this study was proved to down-regulate NLRP3 in Nigericintreated CCI rats with reduced MWT as well as attneuated spinal cord injury and in ammatory responses.
As the protective effect of Nrf2 is demonstrated in the above paragraph, we nally drew a conclusion that DEX released neuropathic pain of CCI rats by suppressing NLRP3 via activation of Nrf2. Additionally, Nrf2 was found to modulate HO-1 in the suppression of murine lupus nephritis by dietary oleuropein and peracetylated oleuropein [27]. Further research could be done to explore if there is a downstream target of Nrf2 in the management of neuropathic pain.

Conclusions
In summary, DEX ameliorates in ammation-induced neuropathic pain in CCI rat models. The NLRP3/Nrf2 pathway is veri ed to modulate neuropathic pain and in ammatory responses. We demonstrate that DEX inhibits neuropathic pain by activating Nrf2 via the suppression of NLRP3. The revealing of the mechanism of DEX may alleviate the burden of neuropathic pain in patients and improve the pharmacotherapeutic treatment of neuropathy. Availability of data and materials The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.
Ethical approval and consent to participate All the animal experiments were approved by the Animal Protection and Use Committee of Hunan Provincial People's Hospital and strictly followed the "Guidelines for the Use and Management of Laboratory Animals" issued by the National Institutes of Health (NIH).

Consent for publication
Not applicable