Neohesperidin Alleviates the Neuropathic Pain Behavior of Rats by Downregulating the P2X4 Receptor

Neuropathic pain (NP) is a type of chronic pain affecting 6–8% of human health as no effective drug exists. The purinergic 2X4 receptor (P2X4R) is involved in NP. Neohesperidin (NH) is a dihydroflavonoside compound, which has anti-inflammatory and antioxidative properties. This study aimed to investigate whether NH has an effect on P2X4R-mediated NP induced by chronic constriction injury (CCI) of the sciatic nerve in rats. In this study, the CCI rat model was established to observe the changes of pain behaviors, P2X4R, and satellite glial cells (SGCs) activation in dorsal root ganglion (DRG) after NH treatment by using RT-PCR, immunofluorescence double labeling and Western blotting. Our results showed CCI rats had mechanical and thermal hyperalgesia with an increased level of P2X4R. Furthermore, SGCs were activated as indicated by increased expression of glial fibrillary acidic protein and increased tumor necrosis factor-alpha receptor 1and interleukin-1β. In addition, phosphorylated extracellular regulated protein kinases and interferon regulatory factor 5 in CCI rats increased. After NH treatment in CCI rats, the levels of above protein decreased, and the pain reduced. Overall, NH can markedly alleviate NP by reducing P2X4R expression and SGCs activation in DRG.


Introduction
Neuropathic pain (NP) is a type of chronic pain caused by nervous system injury [1,2]. Because of its complicated mechanism and involvement of multiple factors, effective drugs are still lacking. Chronic pain considerably affects patients' quality of life and can lead to clinical depression [3,4]. Therefore, it is necessary to explore the pathogenesis of chronic NP and identify effective drugs for treatment. Previous studies have focused on abnormal changes in neuron function. However, the common clinical analgesic drugs that target neurons are not ideal for NP, suggesting that certain non-neurological mechanisms may cause NP. An increasing number of studies have shown that activation of glial cells is the key to pain development [5]. The interaction between neurons and satellite glial cells (SGCs) in primary sensory ganglia was enhanced after nerve injury [6][7][8], and the mechanisms involved were closely associated with purinergic receptors, activation of glial cells, and release of inflammatory factors [9][10][11].
The purinergic 2X4 receptor (P2X4R) is one of the P2X receptors belonging to ligand-gated ion channels [12].
Stressed and damaged cells and nerve endings release a higher number of pain-induced molecules such as adenosine triphosphate (ATP). ATP binding P2 receptors resulted in NP transmission [13][14][15]. P2X4R expressed in the microglia of the spinal cord is associated with the transmission and occurrence of NP [16][17][18]. Furthermore, dorsal root ganglion (DRG), as a primary sensory ganglion, can transmit peripheral information to the spinal dorsal horn [19]. In DRG, SGCs are closely arranged around the cell body of the neuron and express P2X4Rs [11,20,21]. Studies have shown that P2X4Rs in DRG were upregulated, and SGCs were activated after chronic constrictive injury (CCI) of the sciatic nerve in rats. SGC activation is marked by increased glial fibrillary acidic protein (GFAP) and the release of inflammatory elements from activated SGC, which is associated with NP [6,22]. Moreover, activated SGCs release tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), etc. TNF-α can act on TNF-α receptor 1 (TNF-R1) in DRG neurons and increase neuronal sensitivity to injurious stimuli [17,23], eventually contributing to NP. Transcription factor, interferon regulatory factor 5 (IRF5), belonging to the IRF family (IRF1-9), exists in microglia [24]. After peripheral nerve injury, IRF5 in microglia directly binds to the promoter region of the P2X4R gene to control P2X4R expression [25].
Neohesperidin (NH) is a dihydroxyflavone rich in the peels of oranges, lemons, and grapefruit and is widely used in traditional Chinese medicine. NH has reactive oxygen species scavenging activity [26] and neuroprotective effects [27]. However, its effect on CCI-induced NP is still unclear. On the basis of the above-mentioned pharmacological effects and functions of NH, this study aimed to explore whether NH can reduce the pain behavior of CCI rats by affecting P2X4R in the DRG. We believe that the results of this study will provide a creative, experimental systemic basis for the effective treatment of NP.

CCI Model Establishment
Male Sprague-Dawley rats weighing 180-220 g were obtained from the Center of Laboratory Animal Science of Nanchang University. The rats were acclimatized for 1 week before trial procedures and randomly assigned to each group. The rats were fed freely with water and food. Temperature, humidity, and light/dark cycles remained constant (23 ± 2 °C, 60 ± 10%, and 12/12 h, respectively). The steps of CCI model establishment were as follows [28]: first, the rats were anesthetized with intraperitoneal injection using 10% chloral hydrate (3 mL/kg), and then the skin on the back of the thigh was disinfected with iodophor. Second, the right sciatic nerve of the rat was exposed and ligated four times using 4-0 catguts at 1 mm intervals, starting at 7 mm proximal to the bifurcation of the sciatic nerve. Third, the surgical incision was sutured and disinfected. At last, the sham rats underwent the sham operation without ligation. All the animal handling procedures were approved by the Animal Protection and Use Committees of the Medical School of Nanchang University. Animal pain research abides by the ethical guidelines of the International Association for the Study of Pain. The animal trials followed the Animal Research Reporting of In Vivo Experiments guidelines.

Experimental Design
To assess the analgesic effect of NH on CCI, three concentrations (10, 30, and 100 mg/kg) of NH were used. The doses were selected according to our preliminary experiments and published reference [29]. Moreover, 42 rats were divided into seven groups (n = 6 each group), including the sham group, CCI group, CCI + NH10 group, CCI+NH30 group, CCI+NH100 group, CCI+dimethyl sulfoxide (DMSO) group, and CCI+5-BDBD. The rats in the CCI+NH10, CCI+NH30, and CCI+NH100 groups received 10, 30, 100 mg/kg of NH, respectively, by intragastric (i.g.) administration after CCI. The CCI + DMSO group received an equal volume of 0.5% DMSO. To evaluate the involvement of P2X4Rs in CCI-induced NP, 5-BDBD as the P2X4 antagonist was added to the experiment. The CCI+5-BDBD group was intrathecally injected with 5-BDBD (10 µL, 100 µM dissolved in 0.5% DMSO). The above drugs were given daily for 2 weeks from the day after CCI. To further verify the effect and mechanism of NH on P2X4R and SGC activation in DRG, another 60 rats were divided into six groups (n = 10): control group (Ctrl), Ctrl+NH10, sham group, CCI group, CCI+NH10 group, and CCI+DMSO group. The rats in the control group did not undergo the operation. The control rats in the Ctrl+NH10 group received NH (10 mg/kg) i.g. daily for 2 consecutive weeks. Treatments for the other four groups were the same as above. The pain behaviors, including mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL), were tested half an hour after injecting the drug on CCI 0, 1, 3, 5, 7, 9, 11, and 13 days. After 14 days, L4-L6 DRGs were obtained from each rat. In each group, six DRGs from six rats were used for immunofluorescence assay of GFAP, six DRGs from six rats were used for real-time quantitative polymerase chain reaction (qPCR), and the DRGs from the remaining rats were used for western blotting analysis.

Mechanical and Thermal Hyperalgesia Tests
The MWT was tested using an electronic, mechanical pain detector, BME-404 (Tianjin Bern Technology Co., LTD) [11]. The rat was kept in a cage with a hollow bottom for 15 min so that it could acclimatize to the test environment. Mechanical stimulus at 1 min intervals was given five times from the bottom of the cage to the rat's right rear foot. The minimum intensity of the mechanical stimulus that caused the foot retraction reflex was the MWT. The mean of the five stimulating strengths was considered the MWT of each rat.
The TWL was measured using the BME-410C thermal Paw Stimulation System (Tianjin Bern Technology Co., LTD) [11]. It emits a light source to stimulate the plantar of rats. The rat was placed in a transparent glass cage for 30 min for acclimatization, and the plantar was exposed to the light. When the rats withdrew their feet because of pain caused by heat stimulation, the light source was automatically cut off, and the time from the beginning to cutoff was recorded as TWL. To avoid damage caused by prolonged exposure, the cutoff time was set at 30 s. Each rat was tested five times at an interval of 5 min, and the average value was considered the TWL of the rat.

Immunofluorescence
The separated L4-6 DRGs were fixed in 4% paraformaldehyde for 2 h and then dehydrated with 20% sucrose for 12 h after rinsing with 0.1 M phosphate-buffered saline (PBS). DRG was mixed with an optimal cutting temperature compound and placed in a frozen cryostat (Leyca Biosystems, Wetzlar, Germany) for cutting into 5-µm thick slices. DRG slice was washed three times in PBS for 10 min. Nonspecific staining was blocked using 3% bovine serum albumin containing 0.3% Triton X-100 for 1 h. Both the primary antibodies of GFAP (chicken anti-GFAP, Abcam, Cambridge, UK) and P2X4 (rabbit anti-P2X4, Abcam, Cambridge, UK) were diluted to 1:100 and incubated overnight at 4 °C. After rinsing three times in PBS, the slice was incubated in the corresponding secondary antibodies, including goat anti-rabbit tetraethyl rhodamine isothiocyanate (Jackson ImmunoResearch Inc., West Grove PA, USA) and goat anti-chicken fluorescein isothiocyanate (Beijing Zhongshan Biotech Co.) at the dilution of 1:200 for 45 min at 37 °C. Subsequently, wash it in PBS three times.
An immunofluorescence image was obtained using the fluorescence microscope (Olympus). Moreover, three to five areas were selected for quantitative analysis in each section, and the average intensity of the fluorescence signal was calculated. The negative control slice at the background level followed the same steps as the other groups, except for no primary antibody. The image background was then corrected or subtracted by handling the scaling of the image histograms. All slices were obtained under the same parameters in the experiment. In this study, the microscope showed full stability, and the acquisition parameters held the same samples.

Western Blotting
After the isolated DRG was washed in precooled PBS, it was transferred to a glass grinder containing lysate to grind on ice. The lysate contains 250 mM Tris-HCl, 200 mM dithiothreitol, 10% sodium dodecyl sulfate, 0.5% bromophenol blue, and 50% glycerol. After DRG tissue was thoroughly grounded and centrifuged, the supernatant was retained, and the total protein was obtained. Extracted protein was separated using electrophoresis with 10% sodium dodecyl sulfate-polyacrylamide gel. The protein in the gel was then transferred to a polyvinylidene difluoride membrane for further immunoassay. It was then incubated in the primary antibody. The dilution ratio of primary antibodies was as follows: 1:10000 for β-actin (Long Beach, CA), 1:200 for P2X4 (Abcam, Cambridge, UK), 1:1000 for extracellular-regulated protein kinases1/2 (ERK1/2) (Cell Signaling Technology, Danvers, Massachusetts, USA), 1:2000 for phosphorylated ERK1/2 (p-ERK1/2) (Cell Signaling Technology,Danvers, Massachusetts, USA), 1:800 for TNF-R1 (Abcam, Cambridge, UK), 1:800 for IL-1β (Boster, Wuhan, China), 1:1000 for GFAP (Abcam, Cambridge, UK), and 1:800 for IRF5 (Proteintech, Chicago, USA). They were then incubated with corresponding secondary antibodies (1:2000, Cell Signaling Technology), and chemiluminescence substrate (Thermo Scientific, USA) was added to view the protein bands. The band intensity was obtained using Pro-Plus image software. The relative expression of target protein was expressed as the intensity ratio of target proteins/β-actin.

Statistical Analysis
Data were presented as means ± standard error of the mean. The SPSS software (version 11.5) was used to analyze all the data. The behavioral data were analyzed using a two-way repeated method analysis of variance (ANOVA). One-way ANOVA was used for western blotting, qPCR, and immunofluorescence analysis. A p-value of < 0.05 was considered statistically significant.

Effects of Different Concentrations of NH on Pain Behaviors and P2X4 Level
Before the operation, there was no difference in pain behaviors, including MWT (Fig. 1A) and TWL (Fig. 1B), among all the groups. However, after CCI, lower MWT and TWL were noted in CCI rats than in sham rats. On administration of different concentrations of NH, the pain of rats was significantly improved. In addition, the pain behavior in CCI rats could be significantly alleviated by 5-BDBD, a P2X4R-specific antagonist. The results revealed that the lowest concentration of NH (10 mg/ kg) inhibited mechanical and thermal hyperalgesia in Fig. 1 Effects of different concentrations of NH on pain behaviors and P2X4 level. The mechanical withdrawal threshold (MWT) (A) and thermal withdrawal latency (TWL) (B) of chronic constriction injury (CCI) rats were markedly lower than those of sham rats. Different concentrations of neohesperidin (NH) increased the above pain threshold of CCI rats. The MWT and TWL increased after using 5-BDBD in CCI rats. The arrow indicates the start time of drug treat-ment from the first day after CCI. The level of P2X4R mRNA (C) and protein (D) increased in CCI rats compared with sham rats and reduced after administration of different NH concentrations. n = 6 for MWT and TWL test, three independent experiments for mRNA and protein measurement (n = 6). ***p < 0.001 vs. Sham group, ### p < 0.001 vs. CCI group 1 3 CCI rats. Meanwhile, P2X4 mRNA (Fig. 1C) and protein (Fig. 1D) levels increased after CCI but decreased dramatically after the administration of different NH concentrations. Furthermore, the expression of P2X4R reduced with increasing NH concentrations. NH dose of 10 mg/ kg was considered an effective concentration to be used in the subsequent experiments.

NH Relieved Pain Behaviors in CCI Rats
The MWT ( Fig. 2A) and TWL (Fig. 2B) of rats did not differ among all the groups before the operation (p > 0.05). After CCI, the MWT and TWL decreased significantly compared with control rats (Ctrl) (p < 0.001), whereas they considerably increased after NH10 treatment from day 3 to 13 after the first gavage. Control, sham, and NH-treated control groups showed no difference (p > 0.05). DMSO had no effect on the pain behaviors of CCI rats (p > 0.05).

NH Reduced the Expression of P2X4R in CCI Rats
Real-time quantitative PCR was used to evaluate P2X4R mRNA levels in the DRG of all groups. The P2X4R mRNA level in the CCI group was approximately 1.6 times that of the control group, whereas it significantly decreased in the NH-treated CCI group compared with the untreated CCI group (Fig. 3A).
The P2X4R protein level was detected by western blotting. The relative expression of P2X4R in each group was expressed by normalizing to β-actin. The P2X4R protein level was higher in the CCI group than in the control group, whereas it considerably decreased in the NHtreated CCI group (Fig. 3B).

NH Reduced SGC Activation in CCI Rats
GFAP, the marker of SGC activation, is commonly used to detect SGC activation. The co-location of P2X4R and GFAP in the DRG of each group was detected using immunofluorescence. Fluorescence results showed that P2X4R was co-located with GFAP (Fig. 4A). Furthermore, the coexpression level in the CCI group was significantly higher than the control group but was significantly decreased after NH administration (Fig. 4B). In addition, the protein level of GFAP was evaluated by western blotting. The results showed that the GFAP protein level in the CCI group was significantly higher than the control group but could be reduced by NH treatment (Fig. 4C).

NH Decreased the Levels of TNF-R1 and IL-1β in CCI Rats
The levels of TNF-R1 and IL-1β in DRG were investigated by western blotting. Results showed increased levels of TNF-R1 (Fig. 5A) and IL-1β (Fig. 5B) in CCI rats but no change in sham rats. Treatment with NH significantly reduced TNF-R1 and IL-1β levels in CCI rats, whereas DMSO treatment had no effect.

NH Decreased p-ERK1/2 Level in CCI Rats
To verify whether the ERK pathway is associated with the P2X4 signaling pathway, the ERK1/2 and p-ERK1/2 levels were detected by western blotting. The p-ERK level indicates ERK pathway activation. No difference was found in the total ERK1/2 level, expressed as the ratio of ERK to β-actin among all the groups (Fig. 6A, B). Nevertheless, the ratio of p-ERK1/2 to ERK1/2 was significantly higher in CCI rats than in control rats (Fig. 6C). In addition, treatment Fig. 2 NH relieved pain behaviors in CCI rats. The MWT (A) and TWL (B) of rats in each group were measured before CCI operation, and no difference was found between the groups (p > 0.05). In the CCI group, both pain thresholds decreased significantly compared with control (Ctrl) rats, whereas they considerably increased from day 3 to 13 after the first gavage of NH. Control, sham, and NH-treated control groups showed no difference (p > 0.05). Dimethyl sulfoxide (DMSO) had no effect on pain behavior (p > 0.05). The arrow indicates the start time of drug treatment from the first day after CCI. n = 6, ***p < 0.001 vs. Ctrl group, ###p < 0.001 vs. CCI group Fig. 3 NH reduced the expression of P2X4R in CCI rats. The expression of P2X4R mRNA and protein levels in DRG were evaluated using real-time quantitative PCR and western blotting, respectively. A The P2X4R mRNA level in the CCI group was approximately 1.6 times that of the control group, whereas it significantly decreased in the NH-treated CCI group compared with the untreated CCI group.
B The P2X4R protein level was higher in the CCI group than in the control group, whereas it considerably decreased in the NH-treated CCI group. DMSO showed no effect on the P2X4R level of CCI rats. Three independent experiments for each bar (n = 6 for mRNA, n = 10 for protein). ***p < 0.001 vs. Ctrl group, ###p < 0.001 vs. CCI group Fig. 4 NH reduced SGC activation in CCI rats. A The co-expression of P2X4R and glial fibrillary acidic protein (GFAP) was evaluated using immunofluorescence. The green light indicates GFAP, red indicates P2X4R, and yellow in the Merge image indicates colocation. The fluorescence results showed that P2X4R is co-located with GFAP in DRG. Scale bar: 20 μm. B The histogram shows the fluorescence intensity of each group. The co-expression level of the CCI group was significantly higher than that of the control group; however, NH could significantly reduce the co-expression level of CCI rats. Sham operation and solvent DMSO did not change the coexpression level. C Protein quantification of GFAP was also detected by western blotting. The results showed that the GFAP level in the CCI group was significantly higher than the control group but could be decreased by NH drug treatment. No change was observed in sham and DMSO groups. Three independent experiments for western blotting (n = 6 rats for immunofluorescence and n = 10 rats for protein). ***p < 0.001 vs. Ctrl group, ###p < 0.001 vs. CCI group with NH significantly decreased p-ERK1/2 levels in CCI rats, whereas DMSO treatment had no effect (Fig. 6A, C).

Effect of NH on IRF5 Level in CCI Rats
Western blotting was used to detect IRF5 protein levels in DRG. The IRF5 level of the CCI group was approximately five times higher than that of the control group and decreased by approximately 1.4 times after NH treatment (Fig. 7). No statistical difference was observed between the control and sham operation groups, and between the DMSO and CCI groups.

Discussion
Chronic NP often causes tremendous pain to the patient. Chronic pain has various forms, including spontaneous pain, hyperalgesia, allodynia, etc. [31]. The CCI is a widely used NP model in the development and research of analgesic drugs [6]. In this study, the pain behavior, including MWT and TWL, of CCI rats improved, indicating that CCI rats showed clinical symptoms of NP. In the CCI model, different concentrations of NH reduced the pain of rats, indicating that NH has a certain analgesic A Tumor necrosis factor-alpha receptor 1 (TNF-R1) increased in CCI rats compared with the control group and decreased after treatment with NH. B Interleukin-1β (IL-1β) increased in CCI rats com-pared with the control group and decreased after treatment with NH. Whereas DMSO treatment had no effect. Three independent experiments for each bar (n = 10 rats). ***p < 0.001 vs. Ctrl group, ###p < 0.001 vs. CCI group Fig. 6 NH decreased p-ERK1/2 level in CCI rats. A Phosphorylated extracellular-regulated protein kinases (p-ERK), ERK, and β-actin protein bands are shown in the figure. B ERK levels did not differ between groups. C The p-ERK1/2 level increased in CCI rats compared with that in control rats, whereas decreased in NH-treated CCI rats. Data were obtained from three separate experiments for each bar (n = 10 rats). ***p < 0.001 vs. Ctrl group, ###p < 0.001 vs. CCI group effect; however, the potential mechanism is unknown. Therefore, we need to further explore the mechanism of NH analgesia.
Damaged cells and nerve endings release a large number of ATP that can activate P2X receptors expressed in the primary sensory ganglion. Purinergic signaling refers to the nociceptive signaling transmission of NP [13][14][15]. Studies have shown that CCI upregulates the P2X4R levels. In addition, P2X4R selective antagonist 5-BDBD can relieve the pain, indicating that P2X4R in DRG was involved in NP induced by CCI. Therefore, blocking P2X4R was considered an effective treatment method for NP. In addition, we observed an increased level of P2X4R in the DRG of CCI rats, which was consistent with the above results. However, the upregulated expression of P2X4R in CCI rats was downregulated after NH administration. The results suggest that NH alleviates pain behaviors in CCI rats by decreasing P2X4R levels.
Plenty of evidence has shown that microglia cells played a key role in NP by directly interacting with neurons. Therefore, inhibiting glial cell activation and reducing the release of inflammatory factors may be a potential treatment for NP. Numerous studies have demonstrated that P2X4R is extensively expressed in SGCs rather than neurons in DRG [17,20]. Nerve injury upregulates P2X4R, followed by SGC activation, which releases cytokines to increase the sensitivity of DRG neurons to noxious stimuli resulting in hyperalgesia [11,17]. In this study, the coexpression of P2X4R and GFAP was enhanced in CCI rats along with the expression level of GFAP protein, suggesting that P2X4R was expressed on the SGC and SGCs were activated by CCI. Moreover, when CCI rats were treated with NH, both the upregulated levels of P2X4R and GFAP induced by CCI decreased. This result revealed that NH controls the nociceptive signals transmission of NP by suppressing P2X4R expression and SGC activation in DRG, thus alleviating pain behavior in rats.
During NP, activated glial cells released TNF-α and IL-1β that sensitize neurons [16,17] and facilitate pain development. TNF-α could act on TNF-R1 of neurons and promote pain signal transmission [17,23]. In this study, a significant increase in the levels of TNF-R1 and IL-1β in CCI rats supports the correlation between inflammatory cytokines and NP. NH has anti-inflammatory and antioxidant effects [32]. The levels of TNF-R1 and IL-1β declined after NH treatment in CCI rats. NH reduces the level of inflammatory factors probably by reducing the expression of P2X4R and inhibiting SGC activation, thereby reducing NP in rats.
Several researchers have revealed that the activation of P2X4R in microglia involves ERK signaling [9,23,33]. Activation of ERK signaling promotes cytokine production associated with NP [34,35]. Our previous study found the antagonist of P2X4R 5-BDBD inhibited the upregulated p-ERK in CCI rats [28]. Moreover, this study found that p-ERK1/2 protein remarkably increased in CCI rats, indicating that the activation of the ERK1/2 pathway participated in P2X4-mediated NP induced by CCI. In addition, the activation of the ERK pathway increased P2X4R expression in glial cells [17,23,33]. Therefore, activation of ERK signaling can strengthen the pathological changes mediated by P2X4R. NH inhibits the upregulation of p-ERK1/2 level in CCI rats, thereby inhibiting the positive feedback between P2X4R and ERK signaling. These results showed that NH alleviates pain by inhibiting P2X4Rs and reducing ERK phosphorylation.
Transcription factors IRF8 and IRF5 promoted P2X4R transcription [24]. In addition, IRF5 is regulated by IRF8 in an IRF8-dependent pattern in spinal microglia cells [25]. In peripheral nerve injury, IRF5 enters the nucleus from the cytoplasm and directly regulates P2X4R expression in microglia [24]. In addition, IRF5-deficient mice do not upregulate P2X4R expression and show hyperalgesia after peripheral nerve injury [24]. On the basis of the collective studies, the IRF5 transcriptional axis regulates microglia activation and P2X4R levels and induces pain after peripheral nerve injury [36]. In this study, the upregulation of P2X4R expression was accompanied by an increase in IRF5 expression level after CCI, suggesting that the increased expression of P2X4R may be influenced by the upregulation of transcription factor IRF5. However, NH treatment decreased the IRF5 level in CCI rats, indicating that NH may reduce P2X4R expression by decreasing the IRF5 level. Fig. 7 NH decreased the IRF5 level in CCI rats. The level of interferon regulatory factors (IRF5) in the CCI group was approximately five times higher than that of the control group and decreased by approximately 1.4 times after NH treatment. No statistical difference was observed between the control and sham operation groups and between the DMSO and CCI groups. Data were obtained from three separate experiments for each bar (n = 10 rats). ***p < 0.001 vs. Ctrl group, ###p < 0.001 vs. CCI group

Conclusion
In conclusion, the upregulation of P2X4R expression and overactivation of SGCs in DRG play a key role in the occurrence and persistence of CCI-induced NP. NH can alleviate CCI-induced mechanical and thermal hyperalgesia, and its possible analgesic mechanism includes the inhibition of P2X4R expression and SGC activation. This study broadens the medicinal value of NH and provides the experimental basis for the potential therapeutic effect of NH in NP. Informed Consent Not applicable.