Fasting-mimicking diet alleviates inflammatory pain by inhibiting neutrophil extracellular traps formation and neuroinflammation in the spinal cord

Background Neutrophil extracellular traps (NETs) promote neuroinflammation and, thus, central nervous system (CNS) disease progression. However, it remains unclear whether CNS-associated NETs affect pain outcomes. A fasting-mimicking diet (FMD) alleviates neurological disorders by attenuating neuroinflammation and promoting nerve regeneration. Hence, in this study, we explore the role of NETs in the CNS during acute pain and investigate the role of FMD in inhibiting NETs and relieving pain. Methods The inflammatory pain model was established by injecting complete Freund’s adjuvant (CFA) into the hind paw of mice. The FMD diet regimen was performed during the perioperative period. PAD4 siRNA or CI-amidine (PAD4 inhibitor) was used to inhibit the formation of NETs. Monoamine oxidase-B (MAO-B) knockdown occurred by AAV-GFAP-shRNA or AAV-hSyn-shRNA or was inhibited by selegiline (an MAO-B inhibitor). The changes in NETs, neuroinflammation, and related signaling pathways were examined by western blot, immunofluorescence, ELISA, and flow cytometry. Results In the acute phase of inflammatory pain, NETs accumulate in the spinal cords of mice. This is associated with exacerbated neuroinflammation. Meanwhile, inhibition of NETs formation alleviates allodynia and neuroinflammation in CFA mice. FMD inhibits NETs production and alleviates inflammatory pain, which is enhanced by treatment with the NETs inhibitor CI-amidine, and reversed by treatment with the NETs inducer phorbol 12-myristate 13-acetate (PMA). Mechanistically, the neutrophil-recruiting pathway MAO-B/5-hydroxyindoleacetic acid (5-HIAA) / G-protein-coupled receptor 35 (GPR35) and NETs-inducing pathway MAO-B/ Reactive oxygen species (ROS) are significantly upregulated during the development of inflammatory pain. MAO-B is largely expressed in astrocytes and neurons in the spinal cords of CFA mice. However, knockdown or inhibition of MAO-B effectively attenuates CFA-induced inflammatory pain, NETs formation, and neuroinflammation in the spinal cord. Moreover, within rescue experiments, MAO-B inhibitors synergistically enhance FMD-induced pain relief, NETs inhibition, and neuroinflammation attenuation, whereas supplementation with MAO-B downstream molecules (i.e., 5-HIAA and PMA) abolished this effect. Conclusions Neutrophil-released NETs in the spinal cord contribute to pain development. FMD inhibits NETs formation and NETs-induced neuroinflammation by inhibiting the MAO-B/5-HIAA/GPR35 and MAO-B/ROS pathways in astrocytes and neurons, thereby relieving pain progression. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12964-023-01258-2.


Background
Acute pain is normal but prone to transform into intractable persistent pain [1,2].In fact, a significant proportion of patients (ranging from 20 to 56%) undergo transition from acute postoperative pain to chronic pain, despite the active use of available medications [3].Persistent neuroinflammation triggered by trauma is the major contributing factor to the initiation and sensitization of pain [4].Timely and adequate treatment of neuroinflammation during acute pain reduces the risk of turning into chronic pain [2].
Emerging evidence has shown that the intricate bidirectional communication between immune cells infiltrating the central nervous system (CNS) and CNS resident cells, such as neurons and glial cells, plays a crucial role in pain progression [4,5].In neuropathic and inflammatory pain models, neutrophils infiltrating the CNS cause neuroinflammation and central sensitization by releasing proinflammatory mediators [4].In addition, activated neutrophils release neutrophil extracellular traps (NETs) which are web-like structures composed of double-stranded DNA, histones, and granule proteins, such as neutrophil elastase and myeloperoxidase (MPO) [6,7].These NETs are involved in traumatic brain injury, Alzheimer's disease, and stroke [8][9][10], with neuroinflammation and neurotoxicity identified as the primary mechanisms through which NETs exacerbate these neurological disorders [11,12].However, it is unclear whether NETs in the CNS affect pain outcomes by regulating neuroinflammation.
Fasting-mimicking diet (FMD) is a periodic feeding strategy based on a diet that provides limited calories, low sugar, low protein, and high unsaturated fat to minimize the burden of fasting and offer adequate micronutrients [13,14].Growing evidence indicates that different forms of dietary restrictions effectively limit the progression of various chronic diseases such as diabetes mellitus, cancer, and neurological disorders [14][15][16].In particular, FMD increases synaptic plasticity and neurogenesis and promotes axonal regeneration and recovery after sciatic nerve injury [17].Evidence from a clinical trial suggests that a modified fasting regimen with a cycle of 7-21 days is effective in treating chronic pain syndrome [18].Nevertheless, the molecular mechanisms underlying the pain-reducing effects of fasting remain largely elusive.
Therefore, in this study, we aim to explore the potential analgesic effects of FMD through the inhibition of neutrophil extracellular traps (NETs) and NETs-induced neuroinflammation, and further elucidate the role of spinal cord NETs in inducing pain, with the goal of providing novel insights for postoperative pain prevention and treatment.

Animals and surgery
Experiments were conducted with approval from the Experimental Animal Care and Use Committee of Zhongshan Hospital, Fudan University (Shanghai, China).Male C57 adult mice (6-8 weeks old) were sourced from Shanghai Jihui Laboratory Animal Care Center (Shanghai, China) and housed in a specific pathogen-free environment at the Department of Laboratory Animal Science, Fudan University.Mice in the complete Freund's adjuvant (CFA) model group were anesthetized with isoflurane before injecting CFA (20 μL, Sigma, St. Louis, MO) in the right hind paw to induce inflammatory pain.The mice in the sham-operated group received an injection of an equivalent volume of saline solution.

Mouse diet strategy
The mice in the normal diet group were fed AIN-93G purified rod chow containing 4 kcal/g of gross energy, with calories supplied by proteins, carbohydrates, and fat in a percentage ratio of 20:64:16.The mice in the  FMD group were fed a diet cycle comprising three periods (day 1, days 2-4, and days 5-7), as described previously [19,20].Day 1 diet was provided at 50% of the normal daily caloric intake (50% of AIN-93G intake, 0.11 kJ/g carbohydrates, 1.17 kJ/g protein, and 0.53 kJ/g fat).The diet provided on days 2-4 was 10% of the normal daily caloric intake (10% of AIN-93G intake, 0.34 kJ/g carbohydrates, and 0.01 kJ/g protein).On days 5-7, mice were fed a normal diet (AIN-93G), followed by a new round of FMD.Two FMD cycles were performed one week before and one week after CFA administration for a total of 14 days.The body weight of each mouse was monitored daily; weight losses did not exceed 20%.

Assessment of pain behaviors
The pain threshold was measured on the day before CFA injection as the baseline value.The mice were tested for pain behavior at 9:00 a.m. on the 1 st , 3 rd , 5 th , and 7 th day after CFA injection.The behavioral pain test was performed by an investigator blinded to the experimental conditions.To examine mechanical allodynia, we used the up-and-down method to record the paw withdrawal threshold (PWT) [21].For thermal hyperalgesia, the Hargreaves method was used, as described previously [22].The paw withdrawal latency (PWL) was determined as the time from the beginning of radiant heat application to mouse paw withdrawal.To prevent paw damage, a cutoff time of 20 s was implemented.Each mouse underwent three tests, with at least 5 min between each test.

Evans blue staining
Each mouse was injected with 200 μL of 2% Evans blue dye (#E2129, Sigma-Aldrich, USA) through the tail vein; 3 h later, ice-cold phosphate-buffered saline (PBS) was injected into the heart to remove the dye from the blood vessels.During profound anesthesia, the spinal cord segments of the mice were removed, homogenized, and placed in 0.5 mL of a formamide solution to dissolve the Evans blue stain.After incubating at 55 ℃ for 48 h, the sample was centrifuged for 12,000 × g for 30 min at room temperature.The absorbance of the supernatant was determined at 632 nm.The Evans blue content in spinal cord tissue was determined using the standard curve.

SiRNA screening and intrathecal delivery
Four siRNAs targeting mouse PAD4 mRNA and a scrambled control siRNA were provided by GenePharma (Shanghai, China).The nucleotide sequences of the PAD4 siRNA and scrambled siRNA are listed in Table 1.SiRNA was transfected into PC12 cells with liposome 3000 (Invitrogen, USA).Quantitative polymerase chain reaction and western blotting were used to detect the interference efficiency of PAD4 siRNA.PAD4 siRNA2, 3, and 4 inhibited the expression of PAD4 protein by 68.7%, 54%, and 53.9%, respectively.
In vivo analysis revealed that intrathecal injection of PAD4 siRNA4 significantly inhibited PAD4 protein abundance in the spinal cord.Accordingly, the PAD4 siRNA4 was selected for subsequent experiments.
Intrathecal catheterization was conducted as described previously [21].In brief, five days before CFA surgery, a PE-10 catheter (OD, 0.6 mm; ID, 0.28 mm) was inserted into the subarachnoid space of mice through the L5-L6 intervertebral space.PAD4 siRNA or scrambled siRNA (5 μg/10 μL) was administered into the subarachnoid space every other day following CFA administration.
After the mice were anesthetized with pentobarbital, a PE10 catheter connected to a Hamilton syringe was introduced into the mice's subarachnoid space.Successful catheter entry into the subarachnoid space was indicated by a sudden tail movement.Subsequently, the virus solution (5 μL, 5 × 10 12 vg/mL) was slowly injected into the subarachnoid space, and the catheter remained in situ for a minimum of 3 min to prevent drug outflow after injection.The CFA model was established at least three weeks after virus injection.

Reactive oxygen species (ROS) production assay
After anesthesia, mice were intracardially perfused with ice-cold PBS to remove the blood.Next, the L3-L5 spinal cord was dissected to prepare a single-cell suspension.The isolated cells were diluted to 1 × 10 7 cells/ mL and seeded into a 24-well plate.The cells were then incubated with DCFH-DA (10 μM, 50101ES01, Yeasen Biotechnology, China) for 30 min at 37 °C.After washing the cells thrice with serum-free medium, they were suspended in PBS and analyzed by flow cytometry.

Statistical analyses
All data were presented as the mean ± standard error of the mean (SEM).Two-way repeated-measures analysis of variance (ANOVA) followed by Bonferroni's post-hoc test was used to analyze pain behavior data.One-way ANOVA was used to analyze western blot, immunofluorescence, ELISA, and ROS assay data.The unpaired Student's t-test was used to analyze the differences between two groups.Differences were considered statistically significant at P < 0.05.All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, USA).

Blood-spinal cord barrier (BSCB) permeability and neutrophil infiltration increase in the spinal cord of CFA mice
We first established an inflammatory pain model by injecting CFA unilaterally into the hind paws of mice (Fig. 1A).Behavioral testing suggested that, compared to sham-treated mice, CFA mice exhibited significant mechanical and thermal allodynia on Day 1 post-operation, which lasted until Day 7 of testing (Fig. 1B, C).Then we observed increased neutrophil infiltration in the spinal cords of CFA mice from Day 3 post-operation compared to the sham group (Fig. 1H).
Considering that the BSCB protects against neutrophil infiltration into the spinal cord parenchyma under physiological conditions, we assessed the integrity of the BSCB in CFA mice.The amount of Evans blue stain in the spinal cord of mice increased significantly from Day 3 to Day 7 post-CFA injection (Fig. 1D).Western blot results further revealed that the abundance of tight junction proteins, including ZO-1 and occludin were significantly down-regulated (Fig. 1E-G).This evidence suggests that the impaired BSCB promotes recruitment of circulating neutrophils to the spinal cord during inflammatory pain development.

NETs accumulate in the spinal cord of CFA mice, along with neuroinflammation
We then detected NETs released by neutrophils in the spinal cord of CFA mice.As shown in Fig. 1I and J, the expression of PAD4, an indispensable enzyme for NETs generation, was dramatically elevated in the spinal cord of CFA-injected mice.Western blot and immunofluorescence further showed that the number of NETs (citH3 + MPO + ) in the spinal dorsal horn of CFA mice was higher than that in the sham group on Days 5 and 7 post-operation (Fig. 1K-M).
Considering previous studies suggesting that NETs aggravate neuroinflammation [29], we also assessed changes in astrocytes, microglia, and proinflammatory factors within the spinal cords of CFA mice.The number of astrocytes and their branches increased, indicating considerable activation (Fig. 2A, C).Similarly, microglia were activated, characterized by enlargement of the cell body and an increase in their number (Fig. 2B, D).In addition, ELISA results showed that IL-1β, IL-6, and TNF-α levels were notably elevated in the spinal cords of mice injected with CFA (Fig. 2E-G).These findings indicate that NETs are produced in the spinal cords of CFA mice, accompanied by neuroinflammation.

Inhibition of NETs alleviates allodynia and neuroinflammation in CFA mice
To verify the role of NETs in inflammatory pain, we first intrathecally injected PAD4 siRNA into CFA mice to inhibit NETs generation (Fig. 3A).Compared with the scrambled siRNA group, the mechanical and thermal allodynia of CFA mice in the PAD4 siRNA group were significantly alleviated (Fig. 3B, C).Both western blot and immunofluorescence results verified the successful knockdown of PAD4 in the spinal cord of CFAtreated mice (Fig. 3D, E, and H).Western blot and immunofluorescence revealed significant reductions in MPO and citH3 levels in the spinal cord of CFA mice (Fig. 3D, F, G, I).
Consistently, CI-amidine, a specific inhibitor of PAD4 (Fig. 3J), alleviated mechanical and thermal allodynia in CFA mice and inhibited NETs formation (Fig. 3K-O).Moreover, CI-amidine treatment reduced the number of activated astrocytes and microglia compared to the vehicle group, along with decreased levels of proinflammatory factors IL-1β, IL-6, and TNF-α (Fig. 4A-G).These results confirm the role of spinal cord neutrophils and NETs in the aggravation of neuroinflammation and inflammatory pain.

FMD attenuates neuroinflammation and alleviates inflammatory pain by inhibiting NETs formation
FMD exerts robust neuroprotective effects through promoting mitochondria biogenesis, reducing oxidative stress and neuroinflammation [30].We then explored whether FMD alleviated neuroinflammation via inhibiting NETs production by neutrophils (Fig. 5A).The PWT and PWL of FMD-treated CFA mice were significantly higher than those of CFA mice fed with a normal diet, an effect similar to the analgesic effect of CI-amidine (Fig. 5B, C).Moreover, the combined application of FMD and CI-amidine had a synergistic analgesic effect, whereas the analgesic effect of FMD was reversed by the NETs inducer PMA (Fig. 5B, C).Western blot and immunofluorescence analyses of spinal cord samples showed that FMD reduced MPO and citH3 levels (important NETs components) compared to the control group (Fig. 5D-F).Moreover, compared to the FMD group, the expressions of MPO and citH3 in the FMD + CI-amidine group were significantly more decreased, whereas pretreatment with PMA significantly inhibited the FMD-induced NETs reduction (Fig. 5D-F).These data suggest that FMD relieves allodynia, in part by inhibiting NETs production.
We also explored the effects of FMD on neuroinflammation in the spinal cords of CFA mice.Compared to the normal diet + vehicle group, the numbers of activated astrocytes and microglia in the spinal dorsal horns of mice were significantly decreased in the FMD and FMD + CI-amidine groups, so were the IL-1β, IL-6, and TNF-α levels (Fig. 6A-G).However, compared to FMD alone, pretreatment with PMA significantly reversed the effects of FMD on reducing neuroinflammation (Fig. 6A-G).These results suggest that FMD relieves inflammatory pain by reducing neuroinflammation, mediated by inhibition of NETs formation.

MAO-B plays a vital role in inducing NETs formation and promoting neuroinflammation
We then further investigated the mechanisms underlying the FMD-induced inhibition of NETs production.Recent studies have shown that 5-HIAA/GPR35 is a key pathway promoting neutrophil migration to inflammatory sites [27] and that ROS are key factors stimulating neutrophils to produce NETs [31].Meanwhile, MAO-B is not only a key enzyme that metabolizes serotonin (5-HT) to 5-HIAA but also a major inducer of ROS [27,32].Accordingly, we evaluated MAO-B expression in the spinal dorsal horn of CFA mice and found that it increased continuously following CFA administration (Fig. 7A).To determine which cell type expressed the largest amount of MAO-B following CFA treatment, we assessed the cellular localization of MAO-B using immunofluorescence staining.MAO-B was largely expressed by astrocytes (MAO-B + GFAP + ) and neurons (MAO-B + NeuN + ; Fig. 7D, E).Additionally, minor colocalization (MAO-B + IBA1 + ) was detected in microglia (Additional file 1).Similar changes were also observed in the 5-HIAA/GPR35 pathway (Fig. 7B, C).Flow cytometry results showed that ROS levels in the spinal cord of mice treated with CFA were markedly higher than those in the sham group (Fig. 7F, G).

MAO-B inhibition attenuates neuroinflammation and alleviates inflammatory pain by impairing NETs formation
Next, we injected the MAO-B inhibitor selegiline together with the NETs inhibitor CI-amidine or the NETs inducer PMA into CFA mice (Fig. 8A).Compared to the vehicle-treated group, selegiline increased the PWT and PWL of CFA mice; this effect was enhanced by coadministration with CI-amidine.However, PMA reversed the analgesic effect of selegiline (Fig. 8B, C).Consistent with these findings, PMA also abolished the selegiline-induced reduction in MPO and citH3 expression (Fig. 8D-F).Similarly, immunofluorescence and ELISA results showed that selegiline alone and in combination with CI-amidine significantly reduced neuroinflammation in the spinal cords of mice treated with CFA, as characterized by decreases in the numbers of activated astrocytes and microglia and in the levels of IL-1β, IL-6, and TNF-α.However, PMA reversed the anti-neuroinflammatory effects of selegiline (Fig. 9A-G).These results suggest that MAO-B/5-HIAA/GPR35 and MAO-B/ROS signaling pathways exacerbate neuroinflammation, as well as mechanical and thermal allodynia, via induction of NETs formation and may be vital downstream targets of FMD-induced inhibition of NETs.

Specific knockdown of neuron and astrocyte MAO-B alleviates inflammatory pain
To further determine the role of astrocyte and neuron MAO-B in pain, we transfected spinal cord astrocytes and neurons with RNA interference viruses driven by gfaABC1D and hSyn promoters to knock down MAO-B (Fig. 10A, F).The western blot and double immunofluorescence staining results confirmed the cell type-specific knockdown of MAO-B (Fig. 10 B, C,  G, H).Behavioral results showed that specific knockdown of astrocyte and neuron MAO-B alleviated mechanical and thermal allodynia in CFA mice.However, intraperitoneal injection of the MAO-B inhibitor (selegiline) elicited a more effective analgesic effect than specific MAO-B knockdown in neurons or astrocytes (Fig. 10 D, E, I, J).Hence, it is suggested that astrocyte and neuron MAO-B have equally important roles in regulating hyperalgesia.Accordingly, selegiline was employed in subsequent experiments to inhibit MAO-B expression.

MAO-B/5-HIAA/GPR35 and MAO-B/ROS signaling pathways are essential for FMD-induced NETs reduction and pain relief
To further investigate whether FMD relieves NETsrelated neuroinflammation and pain through the MAO-B/5-HIAA/GPR35 and MAO-B/ROS pathways, we compared CFA mice exposed to FMD, selegiline, or FMD combined with selegiline (Fig. 11A).Compared to CFA mice with a normal diet and vehicle treatment, the PWT and PWL in the selegiline and FMD groups were increased.Moreover, FMD and selegiline exhibited synergistic analgesic effects (Fig. 11B, C).Western blot results indicated that the expression of MAO-B in the selegiline and FMD groups was markedly decreased; combined FMD and selegiline significantly decreased these levels compared with either treatment alone (Fig. 11D).Immunofluorescence colocalization results showed that MAO-B expression in astrocytes and neurons in the spinal dorsal horn of mice treated with CFA was significantly decreased by FMD and selegiline treatment (Fig. 11H and Additional file 2).Compared to the control group, the ROS and 5-HIAA levels in the spinal cords of CFA mice treated with FMD or selegiline alone were significantly decreased, while the combined treatment was more effective (Fig. 11E-G).
Considering that NETs formation requires recruitment of neutrophils to the spinal cord and NETs induction, we supplemented FMD-treated mice with MAO-B downstream product 5-HIAA and NETs inducer PMA to investigate whether the analgesic effect of FMD was blocked (Fig. 11A).The analgesic effect of FMD was blocked by the supplementation of 5-HIAA and PMA (Fig, 11B, C).Consistent with the behavioral data, western blotting showed that GPR35, MPO, and citH3 levels substantially decreased in the FMD and FMD + selegiline groups; however, the FMD-induced GPR35 and NETs reduction was abolished by 5-HIAA and PMA co-treatment (Fig. 11I-K).These data suggest that the MAO-B/5-HIAA/GPR35 and MAO-B/ROS pathways are essential for FMD-induced NETs reduction and pain relief.

MAO-B/5-HIAA/GPR35 and MAO-B/ROS pathways are essential for FMD-induced inhibition of neuroinflammation
Finally, we explored the role of MAO-B in the inhibition of FMD-induced neuroinflammation.Both FMD alone and in combination with selegiline significantly reduced the numbers of activated astrocytes and microglia (Fig. 12A-D).However, the combined treatment had no synergistic effect, while the combined application of 5-HIAA and PMA blocked the FMD effect.Similarly, FMD alone or in combination with selegiline decreased the levels of IL-1β, IL-6, and TNF-α; however, this effect was eliminated by 5-HIAA and PMA co-pretreatment (Fig. 12E-G).These data suggest that the MAO-B/5-HIAA/GPR35 and MAO-B/ROS pathways are essential for FMD-induced inhibition of neuroinflammation.

Discussion
Chronic pain is common and not only exerts great unpleasant physiological effects on patients but also leads to emotional disorders, as well as huge social and economic burdens [33].As it is widely recognized that chronicity of neuroinflammation is the primary cause of chronic pain [4], early intervention for acute pain is effective in preventing persistent pain [2].Our results indicate that NETs generated by neutrophils in the spinal cord promote the development of acute pain by exacerbating neuroinflammation.FMD effectively inhibited the progress of pain and enhanced the analgesic effects of NETs inhibitors.Furthermore, FMD inhibited the formation of NETs and NETs-related neuroinflammation by downregulating the MAO-B/5-HIAA/GPR35 and MAO-B/ ROS pathways in astrocytes and neurons within the spinal cords of mice.In particular, MAO-B is a potential mediator of neuron and astrocyte-neutrophil crosstalk (Fig. 13).
There is widespread interest in the communication between immune and nerve cells and its role in pain.In neuropathic pain models, the apparent infiltration of immune cells into the CNS is attributed to disruption of the blood-brain barrier or an increase in its permeability [34][35][36].Neutrophils infiltrating the CNS elicit neuroinflammation and central sensitization by releasing proinflammatory mediators [4].Consistently, we observed an increase in BSCB permeability and a significant augmentation of neutrophil infiltration in the spinal cords of mice in the inflammatory pain model.Upon activation, neutrophils release NETs, which participate in the progression of several neurological diseases, including multiple sclerosis, Alzheimer's disease, and ischemic stroke through inducing neuroinflammation and neurotoxicity [37,38].Here, we found that large amounts of NETs (citH3 + MPO + ) accumulated in the spinal dorsal horn during the acute and subacute phases of inflammatory pain (Days 1-7).Following administration of PAD4 siRNA or CIamidine to inhibit NETs formation, mechanical and thermal allodynia in CFA mice were alleviated, indicating that NETs in the spinal cord participated in pain progression.Neuroinflammation, distinguished by immune cell infiltration, glial cell activation, and the generation of inflammatory mediators in the CNS, is crucial in the onset and persistence of pain [4,39].NETs inhibitors exert anti-inflammatory and microglia-inhibiting effects in animal models of multiple sclerosis and neonatal hypoxia-ischemia [40,41].Consistently, we found that astrocytes and microglia in the spinal cords of CFA mice were significantly activated, accompanied by marked elevation of IL-1β, IL-6, and TNF-α.These responses were completely inhibited by the NETs inhibitor CI-amidine.This suggests that NETs promote the development of inflammatory pain, at least in part, by aggravating neuroinflammation.
Increasing evidence suggests that fasting holds significant promise in treating diverse chronic conditions, especially neurological disorders [30].Intermittent fasting confers neuroprotection in rats with intracerebral hemorrhage by attenuating excessive neuroinflammatory responses and neurological dysfunction, as well as inhibiting neuronal apoptosis [42].Furthermore, while several clinical studies have shown the effectiveness of caloric restriction in alleviating osteoarthritis-related pain, there is currently no consensus on the shortterm and long-term impacts of fasting on pain, and the underlying mechanism(s) remain unclear [18,43,44].Here, we demonstrated that FMD generates broad positive effects in CFA mice, including alleviation of neuroinflammation, inhibition of neutrophil release of NETs, and attenuation of inflammatory pain.The safety of FMD has also been evaluated in other models, showing no significant adverse effects, thus suggesting its potential clinical utility in the treatment of inflammatory pain [14,45].Nevertheless, further research is warranted to evaluate the analgesic effects of FMD in diverse populations, encompassing different age groups and sexes.A previous study reported that long-term caloric restriction had an antinociceptive effect in only adult mice, while this effect was weak or even absent in aging mice [46].Boccella et al. reported that a short-term starvation protocol effectively reduced chronic pain onset more in female than male mice, which was attributed to the liver-produced ketone body, β-hydroxybutyrate (BHB) [47].The analgesic effects of fasting and BHB on chronic pain resulting from nerve injury are mediated through the BHB receptor GPR109A (also known as hydroxycarboxylic acid receptor 2 (HCAR2)) [47].GPR109A has garnered increasing attention in recent years as a crucial target for the regulation of neuroinflammation and pain [48,49].However, whether FMD-mediated inhibition of neuroinflammation and inflammatory pain is dependent on BHB/GPR109A in male mice remains to be further explored.Recent studies indicate that HCAR2 has neuroprotective and anti-inflammatory properties, achieved through the promotion of microglia transformation to an antiinflammatory phenotype or by inhibiting microglia activation and the release of pro-inflammatory factors [50,51].HCAR2 regulates the phenotypic transition of microglia/macrophages towards the protective phenotype, promoting effective phagocytosis of myelin debris and remyelination [52,53].The regulatory effects of HCAR2 on microglia and other immune cells in various physiological conditions suggest the potential role of HCAR2 in the neuroimmune mechanisms underlying pain modulation during FMD.
Based on these results, we further investigated the mechanism by which FMD inhibits NETs formation.A recent study showed that 5-HIAA, a metabolite of 5-HT, promotes the rapid recruitment of neutrophils to inflammatory sites by binding to GPR35 receptors on neutrophils [27].Moreover, GPR35 in the dorsal root ganglion and spinal cord is an important therapeutic target for neuropathic and inflammatory pain [54,55].ROS is a key inducer of NETs production by neutrophils and is involved in many types of pain [31,56,57].Interestingly, MAO-B is not only a key enzyme that promotes the metabolism of 5-HT to 5-HIAA [27] but also a primary source of ROS production [32,58].As expected, the expression of MAO-B and its downstream products 5-HIAA, as well as ROS were significantly upregulated in the spinal dorsal corn of CFA mice.Specific knockdown of MAO-B in astrocytes and neurons or pharmacological inhibition of MAO-B expression effectively relieved inflammatory pain.Furthermore, the inducer of NETs, PMA, reversed the analgesic and anti-inflammatory effects of selegiline.Therefore, we conclude that MAO-B promotes pain development by inducing NETs formation.Consistent with a previous finding that fasting inhibits MAO activity in the white adipose tissue of rats [59], we found that FMD pretreatment inhibited the expression of MAO-B in neurons and astrocytes.Finally, through a rescue experiment, we proved that the MAO-B/5-HIAA/GPR35 and MAO-B/ROS signaling pathways are required for FMD-induced NETs reduction and pain relief.
In addition, we found that MAO-B may function as a mediator for the crosstalk between nerve and immune cells.Indeed, complex bidirectional communication occurs between the nervous and immune systems [60].Upon nerve tissue injury, immune cells such as T cells, macrophages, and neutrophils are recruited by nerve cells to the affected tissue, leading to neuroinflammation [4,61].In the current study, immunofluorescence colocalization staining showed that after CFA administration, astrocytes and neurons in the spinal dorsal horn expressed large amounts of MAO-B, whereas microglia expressed less of this enzyme.However, specific knockdown of neuron and astrocyte MAO-B alleviates inflammatory pain.Following FMD or treatment with an MAO-B inhibitor, the numbers of MAO-B-expressing astrocytes and neurons in the spinal cords of CFA mice were significantly decreased.These results suggest that in the mouse model of CFAinduced inflammatory pain, MAO-B expressed by astrocytes and neurons promotes neutrophil migration to the spinal cord and the subsequent release of NETs, which further promotes the activation of astrocytes and microglia, leading to a cascade amplification of inflammation.FMD blocks this amplification progress by inhibiting MAO-B.The recent reports support our results, which show that astrocytes respond quickly after injury, secrete many soluble molecules, and recruit neutrophils and other immune cells to aggravate inflammation and worsen functional recovery [62,63].However, considering the FMD-mediated inhibition of NETs formation, the complex mechanism of crosstalk between the nervous and immune systems requires further elucidation, which are issues we plan to focus on in the future.
Our study has several limitations.Although our results reveal the contribution of crosstalk between neutrophils and central immune cells (microglia and astrocytes) to the development of inflammatory pain in male C57 mice, it should not be disregarded that the neuroimmune mechanisms involved in abnormal pain differ between males and females [64,65].Recent evidence suggests that hyperalgesia in male mice depends on the regulation of microglia [66].Specifically, tolllike receptor 4 and purinergic P2X4 receptors, which are selectively expressed on microglia, are essential for mediating hyperalgesia in male but not female mice [67].Moreover, Vegeto et al. found that estrogen inhibits the pro-inflammatory phenotypic transformation of microglia induced by lipopolysaccharide [68].Female mice achieve hyperalgesia preferentially through T cells and switch to glial cell-dependent pathway when adaptive immune cells are deficient [66].Recent evidence shows that the level of T cell markers in the spinal cord of CD-1 mice is significantly higher for female than male mice 7 days after nerve injury [66].Female T-cells have an inherent tendency to proliferate and produce higher levels of interferon-γ, while exhibiting lower levels of IL-17A compared to male T-cells, which was associated with androgen, particularly testosterone [69].Although both cytokines have been implicated in pain, T-cells infiltrating the spinal cord following nerve injury mainly exhibit the Th1 subtype (secreting interferon-γ) [70].Hence, infiltrating T-cells in the spinal cord of male mice might have a limited role in mediating abnormal pain, potentially indicating the involvement of alternative immune pathways such as microglia [66].These results reveal significant sex disparities in neuroimmune mechanisms concerning chronic pain, yet a precise comprehension of neuroimmune regulation in both males and females remains elusive.Our research necessitates further validation in female mice.

Conclusions
In summary, the current findings demonstrate that NETs released by neutrophils in the spinal cord participate in the development of acute pain.FMD prevents  the development of acute pain by inhibiting NETs formation and neuroinflammation.We also found that MAO-B and its downstream 5-HIAA/GPR35 and ROS pathways are important FMD targets that inhibit NETs formation and relieve neuroinflammation.Hence, this study identified new targets for pain prevention and treatment.

Fig. 13
Fig. 13 Model showing the mechanism of FMD inhibiting NETs and relieving pain.NETs released by neutrophils in the spinal cord promote the development of pain by aggravating neuroinflammation.FMD inhibits both NETs formation and NETs-related neuroinflammation by downregulating MAO-B/5-HIAA/GPR35 and MAO-B/ROS pathways of astrocytes and neurons in the spinal cords of mice.FMD: fasting-mimicking diet