Methyl Ferulic Acid Alleviates Neuropathic Pain by Inhibiting Nox4-induced Ferroptosis in Dorsal Root Ganglia Neurons in Rats

Neuropathic pain is a disease that has become one of the major public health problems and a global burden. Nox4-induced oxidative stress can lead to ferroptosis and neuropathic pain. Methyl ferulic acid (MFA) can inhibit the Nox4-induced oxidative stress. This study aimed to estimate whether methyl ferulic acid alleviates neuropathic pain by inhibiting the expression of Nox4 and its induction of ferroptosis. Adult male Sprague–Dawley rats were subjected to spared nerve injury (SNI) model to induce neuropathic pain. After the establishment of the model, methyl ferulic acid was given 14 days by gavage. Nox4 overexpression was induced by microinjection of the AAV-Nox4 vector. All groups measured paw mechanical withdrawal threshold (PMWT), paw thermal withdrawal latency (PTWL), and paw withdrawal cold duration (PWCD). The expression of Nox4, ACSL4, GPX4, and ROS was investigated by Western blot and immunofluorescence staining. The changes in iron content were detected by a tissue iron kit. The morphological changes in mitochondria were observed by transmission electron microscopy. In the SNI group, the paw mechanical withdrawal threshold, the paw withdrawal cold duration decreased, the paw thermal withdrawal latency did not change, the Nox4, ACSL4, ROS, and iron content increased, the GPX4 decreased, and the number of abnormal mitochondria increased. Methyl ferulic acid can increase PMWT and PWCD but does not affect PTWL. Methyl ferulic acid can inhibit Nox4 protein expression. Meanwhile, ferroptosis-related protein ACSL4 expression was decreased, GPX4 expression was increased, ROS, iron content and abnormal mitochondrial number were decreased. By overexpressing Nox4, the PMWT, PWCD, and ferroptosis of rats were more severe than those of the SNI group, but they could be reversed after treatment with methyl ferulic acid. In conclusion, methyl ferulic acid can alleviate neuropathic pain, which is related to Nox4-induced ferroptosis.


Introduction
Neuropathic pain, owing to a lesion or disease, is directly affecting the somatosensory system. Based on the position of the lesion or disease, neuropathic pain can be categorized into two types including peripheral and central neuropathic pain [1]. The epidemiological investigation report shows that the global incidence rate of neuropathic pain is approximately 7 ~ 10%. Pain is an extremely unpleasant experience that can lead to people's anxiety and depression and can also seriously affect people's quality of life [2][3][4]. Therefore, finding new effective targets in neuropathic pain is critically important [5].
In recent years, growing researches have demonstrated that neuropathic pain has a strong correlation with ferroptosis [6][7][8][9]. Ferroptosis is a novel iron-dependent cell death that is significantly inconsistent with apoptosis. In essence, Tielong Liu and Ruixue Wang are authors have contributed equally to this work it is a regulated kind of cell death resulting from lethal lipid peroxidation. Excessive accumulation of oxidizing substances or reduction of reducing substances can lead to cell ferroptosis. Its main feature is mitochondrial damage and an iron-dependent accumulation in lipid reactive oxygen species (ROS). When increasing ROS exceeds the redox content kept by glutathione (GSH) and phospholipid hydroperoxides using GSH as a substrate, ferroptosis occurs [10].
Intracellular iron overload promotes GSH consumption and lipid peroxidation, and oxidative stress reduces the mRNA and protein expression levels of glutathione peroxidase 4 (GPX4). In addition, it also increases the mRNA and protein expression of acyl-CoA synthetase long-chain family member 4 (ACSL4). In addition, iron overload promotes lipid peroxidation and mitochondrial ROS production and reduces mitochondrial membrane potential (MMP), which is consistent with ferroptosis [11]. GPX4 plays so vital a role in regulating ferroptosis, and GSH is an essential cofactor of GPX4. GPX4 is inactivated after GSH consumption, which leads to an increase in cytosolic ROS, thus leading to ferroptosis [12]. Doll researches manifests that ACSL4 is the key regulator of ferroptosis sensitivity and mainly promotes ferroptosis by binding polyunsaturated fatty acids to cellular phospholipids, particularly phosphatidylethanolamine [13]. In a neuropathic pain rat model, the expression level of the landmark protein GPX4 which is correlated with ferroptosis, reduced, the level of ACSL4 increased, and iron content increased correspondingly [14].
NADPH oxidases (Noxs) are a family of enzymes that produce ROS. NADPH oxidase 4 (Nox4) is the main source of ROS production in seven types of Nox isoforms (Nox1, Nox2, Nox3, Nox4, Nox5, dual oxidase (DUOX)1, and DUOX2) [15,16]. Nox4 is a key factor that leads to mitochondrial metabolic damage and ferroptosis by increasing the production of mitochondrial ROS (mtROS) and mitochondrial fragmentation, inhibiting mitochondrial respiration and ATP production. In addition, Nox4 can activate oxidative stress-induced lipid peroxidation in cells and inhibit cellular antioxidant processes, promoting ferroptosis [17,18]. Miao disclosed that Nox4 was high in a rat neuropathic pain model, and sensitivity of pain thresholds like paw mechanical withdrawal threshold and paw withdrawal cold duration was increased. After Nox4 was inhibited, the oxidative stress products were also correspondingly reduced, indicating that Nox4-induced oxidative stress may be partly responsible for neuropathic pain [19]. However, whether ferroptosis is caused by Nox4 in neuropathic pain has not been systematically studied.
Methyl ferulic acid (MFA), a kind of organic acid, originates in traditional Chinese medicine that has antioxidative stress, anti-inflammatory, and antiapoptotic effects. After using MFA in some diseases, it can be observed that the oxidative stress products Nox4 and ROS are reduced, and the degree of damage is reduced [20][21][22][23]. Thus, this study aimed to determine whether Nox4-induced ferroptosis pathway in the DRG exists in neuropathic pain and whether MFA alleviates neuropathic pain caused by SNI by inhibiting the Nox4-induced ferroptosis pathway in the DRG of rats.

Animals and Grouping
Animal experiments consistented with the requirements of the People's Republic of China on the Administration of Laboratory Animals. Male 10 ~ 12-week-old SD rats (200-250 g) were bought from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Laboratory animal license No. SCXK 20,190,003). Rats were kept in a box with 24 ± 2℃ and humidity of 40-50% and subjected to light/dark cycles for 12 h each. They could freely obtain enough water and food. All protocols complied with the Animal Research Committee by Shihezi University (Protocol A2021-074-01).
Modeling: The rats were selected at random to the sham group and the SNI group. The behavioral tests were carried out on the first day before the operation and 1 day, 3 days, 7 days, 14 days, and 21 days after the operation. The dorsal root ganglion (DRG) tissue was taken for subsequent experiments. Drug administration: rats were randomized into six groups: sham group, SNI group, SNI + MFA 5 mg/kg group, SNI + MFA 10 mg/kg group, SNI + MFA 20 mg/kg group and SNI + DMSO group. MFA (3,4-dimethoxycinnamic acid) was purchased from Sigma Chemical Co. (Sigma #D133809-25G) and was dissolved in dimethyl sulfoxide (DMSO) (Sigma #20-139). MFA of different concentrations was given to rats by gavage from the first day after the operation, once a day for 2 weeks. All groups were taken on the 14th day after the operation, and the behavioral tests were carried out on the 1st day before the operation and 1 day, 3 days, 7 days, and 14 days after the operation (Fig. 1a). In vitro virus injection: The rats were selected at random into six groups: sham group, SNI group, SNI + AAV-Nox4 group, SNI + AAV-NC group, SNI + AAV-Nox4 + MFA 10 mg/kg group, and SNI + AAV-NC + MFA 10 mg/kg group. The virus was injected into the dorsal root ganglia (DRG) 4 weeks before the SNI modeling. The samples were taken from each group on the 14th day after the SNI operation, and the behavioral tests were conducted on the 1st day before the operation and 1 day, 3 days, 7 days, and 14 days after the operation (Fig. 1b). The animals were grouped blind, and the researchers did not know the grouping information.

Preparation of the SNI RAT MODEL
According to previous studies, SNI was used to prepare neuropathic pain model [24]. The Rats were anesthetized with mask inhalation of 2% isoflurane (RWD, R510-22) for surgery. The rats were disinfected routinely and laid with sterile sheets. The skin was cut along the left lower limb sciatic nerve to take apart the biceps femoris and expose the three branches of sciatic nerve. The common peroneal nerve and tibial nerve were ligated with a 4-0 wire and cut with scissors, and both of them are closely connected with removal of the distal nerve ends by approximately 3 mm. The nearby sural nerve was well preserved. After the operation, washing wounds with sterile saline cautiously and closed in layers. In the sham group, the sciatic nerve was exposed and isolated, and then sutured.

Behavioral Assays
Each behavioral test was conducted by two researchers in a double-blind manner. Each researcher measured three times. After the test, the average value of the two researchers' measurement results was taken as the corresponding pain threshold.

Paw Mechanical Withdrawal Threshold (PMWT)
The PMWT of rats was measured according to the method of "up and down" reported by Chaplan [25]. The rats were put on a metal net shaded with transparent plexiglass. The indoor temperature is suitable and the environment is quiet. After being adapted to the environment for at least 30 min, plenty of standardized von Frey fiber filaments were used to stimulate the center of the plantar of the hind limbs of the rats until they were bent into an S-shape, and the stimulation lasted for 6-8 s. The rats were observed to determine whether there was foot contraction or licking reaction throughout the time of stimulation or when the von Frey fiber filaments were removed. If the rat had a rapid foot contraction or licking reaction, it was recorded as a positive reaction " × ". In contrast, it is a negative reaction "O." Substitute the formula to calculate the 50% foot retraction threshold = (10 [X f + kδ] )/10,000 where X f is the log value of the strength (2.00 g) of the von Frey fiber applied for the first time, δ is the average value of the difference between the log values of the strength of adjacent von Frey fibers (0.224), and k is the look-up value of the positive and negative reaction types.

Paw Thermal Withdrawal Latency (PTWL)
PTWL was assessed as mentioned by the protocol [26]. An analgesia meter (Ugo basil, Stoelting, IL, USA) was prepared to gauge PTWL. The rats were put above the glass plate of the analgesia meter and permitted to acclimatize for 30 min ahead of testing. The parameters of the analgesia meter were adjusted to 53℃ and the interruption time was 30 s to prevent scalding of the rats. A radiant heat source was focused under the glass floor beneath the hind paws. When the pain prompts the rat to lift the hind foot, the instrument automatically takes the time. This time is the paw thermal pain threshold. During the measurement, the excrement on the glass plate was wiped in time to avoid affecting the results.

Paw Withdrawal Cold Duration (PWCD)
The PWCD was evaluated on the basis of the protocol described previously [27]. The rats were put on a glass plate with a Plexiglas cover and permitted to acclimatize for 30 min. We put dry ice into a 3-ml syringe with the tip cut open and compress the ice. The tip of the compressed b Dorsal root ganglion AAV virus injection and MFA gavage treatment dry ice particles was extended out of the edge of the syringe to test the hind paws of rats in a completely resting state. The time from dry ice contacting the glass plate to the rats leaving the cooled glass plate was recorded.

AAV Generation and AAV Microinjections
HBAAV-hSyn-r-Nox4-3xflag-ZsGreen viruses were donations from Hanbio Biotechnology Co. Ltd. (Shanghai, China). Gene ID: NM -053524.1, the length of CDS: 1727 bp, sources: rat, serotype: II, titer: 1.8 × 10 12 . According to previously published methods, adeno-associated virus (AAV) was used to overexpress Nox4 and was microinjected into the left L4-5 DRGs [28]. After rats were anesthetized, fixed, disinfected, and spreadsheets, the intervertebral foramen was carefully separated and exposed in layers. The L 4-5 DRGs can be discovered by removing the laminar bone. Virus injection was performed using a stereotactic device (Beijing Zhongshi Dichuang Technology Development Co., Ltd.). AAV (2 µL) was slowly injected into the DRG within 5 min, and then the glass pipette was placed in situ for at least 5 min. After injection, the wound was sutured and then the rats were put on a constant temperature heating pad for recovery and finally placed in a separate cage.

ROS Determination
ROS were measured using a fluorescent probe (BB-470516 Bestbio). Fresh tissues (within 1 h after surgical resection) were taken, and 10-20-um thick frozen sections were made without fixation. Cleaning solution was added to the frozen section and allowed to stand for 3-5 min. Then, the cleaning solution was removed, and we added the staining probe working solution. The sections were cultured at 37 °C for 20-60 min. Staining solution was removed, and coverslips were added after cleaned by PBS. Finally, we observed the sections with the use of a confocal laser scanning microscope (LSM710; Carl Zeiss AG, Oberkochen, Germany).

Transmission Electron Microscopy
After the rats were killed quickly, the DRGs of the operation side were taken and cut into 1-mm 2 sections. Tissue specimens were prepared by prefixation, postfixation, block staining, gradient dehydration, and embedding. Ultrastructural organelles were inspected with the use of a transmission electron microscope (FEI Tecnai G2 F30).

Iron Content Assay
The iron content of the rat DRG was detected by the colorimetric method (A039-2-1, Nanjing, China) at 520 nm. The L 4-6 DRG was accurately weighed on the model side of the rats, and 9 times the volume of normal saline was added according to the ratio of weight (g): volume (mL) = 1:9. Samples were mechanically homogenized under ice water bath conditions, rotated at 2500 rpm, and centrifuged for 10 min. The supernatant was collected to determine absorbance OD value. Calculation formula: tissue iron content = (A measurement -Ablank/Astandard -A blank ) × C standard ÷ CPR, C standard is 55.847 g/mol, CPR is the protein concentration of tissue homogenate. The iron content of the DRG is calculated by substituting the measurement results into the calculation formula.

Statistical Analysis
Statistical analyses were carried out using GraphPad Prism 9.4.0 (GraphPad Software, La Jolla, CA, USA). Before further statistical analysis, the normal distribution hypothesis of the test data and the homogeneity of variance were examined. we analyzed PMWT, PTWL, and PWCD with the use of repeated-measures analysis of variance, and conducted multiple comparisons between groups each time with the help of Bonferroni's post hoc tests. Student's t test and oneway analysis of variance (ANOVA) were used to test the significance of data with normal distribution. Non-normally distributed data were analyzed with the use of a nonparametric Mann-Whitney U test. The level of significance for all outcome was 0.05.

The Neuropathic Pain Model was Successfully Established, Nox4 Expression Increased, and Ferroptosis Occurred on DRG Neurons of SNI Model Rats
We found no markedly differences in PMWT, PWCD, or PTWL between the rats in each group on the day before surgery (P > 0.05). By contrast with the sham group, the SNI group produced significant mechanical and cold hypersensitivities in rats throughout the 21 days after surgery (P < 0.001). No markedly correlation was found in PTWL between the SNI group and the sham group (P > 0.05) (Fig. 2a, b, c). These results revealed that our neuropathic pain model was definitively built.
In addition, we also observed Nox4 protein and ferroptosis markers ACSL4 and GPX4 expression changes. IF and WB analyses illustrated weak, cytoplasmic Nox4 and ACSL4 expression in DRG neurons before surgery. In contrast, the expression of GPX4 was strong in the cytoplasm of DRG neurons. In contrast with that in the sham group, Nox4 and ACSL4 accumulated after SNI injury (P < 0.001); however, the expression of GPX4 gradually decreased after SNI injury (P < 0.001) (Fig. 2d-m). In addition, IF demonstrated that the expression of ROS gradually increased with time after SNI injury (P < 0.001) (Fig. 2n, o). These findings indicate that Nox4 expression is increased in neuropathic pain and that ferroptosis-related proteins are activated.

Nox4 Can Be Expressed in Large, Medium, and Small Neurons of the DRG in SD Rats
DRG neurons played an important role in pain transduction. Therefore, we continued to explore whether Nox4 was expressed in DRG neurons. Immunofluorescence double staining experiments manifested to us that Nox4 protein colocalized with CGRP, IB4, and NF-200 (Fig. 3a). The percentages of CGRP-, IB4-, and NF-200-positive neurons relative to the percentage of Nox4-positive cells were 22.80 ± 5.919%, 38.63 ± 10.86%, and 29.15 ± 9.482%, respectively (Fig. 3b). The results demonstrated that Nox4 was mainly positioned in A-and C-type neurons in the DRG. The neuronal diameter size ranges of CGRP, IB4, and NF-200 were 19.61 ± 2.812, 25.94 ± 4.182, and 44.59 ± 4.272, respectively (Fig. 3c). These results suggest that Nox4 may be related to neuropathic pain.

MFA Inhibited the Increase in Nox4 Expression and Ferroptosis Induced by SNI Injury and Increased the Threshold of PMWT and PWCD
To evaluate the role of MFA in neuropathic pain, SNI injured rats were given intragastric treatment for 14 days. First of all, before building the SNI model, the basic values of the pain threshold of all experimental rats were precisely measured. The difference in PMWT, PTWL, or PWCD among the groups has no marked correlation (P > 0.05). After SNI injury, PMWT and PWCD in the SNI group were decreased in contrast to the sham group (P < 0.001). Then, the PMWT and PWCD of the SNI + MFA 5 mg/kg, SNI + MFA 10 mg/ kg, and SNI + MFA 20 mg/kg groups rats were substantially accumulated in contrast with those from the SNI-vehicle and SNI groups (P < 0.001). No markedly correlation happened in analgesic effect between the SNI + MFA 20 mg/ kg and SNI + MFA 10 mg/kg groups (P > 0.05). No clear relationship occurred in PTWL among the group. (P > 0.05) (Fig. 4a, b, c).
Next, we continue to observe the effects of MFA on Nox4, ferroptosis-related proteins ACSL4, GPX4, and reactive oxygen ROS. After MFA injection, L 4-6 DRG neurons were collected on day 14 of SNI. IF and WB analysis illustrated that, compared with SNI + MFA 5 mg/kg, SNI + MFA 10 mg/kg, and SNI + MFA 20 mg/kg groups, Nox4, ACSL4, and ROS expressions were significantly lower, and GPX4 expression was markedly higher than that in the SNI group (P < 0.001). There was no substantial correlation between the SNI + vehicle and SNI groups (P > 0.05). Among them, there was no marked correlation in the treatment effect between the SNI + MFA 10 mg/kg group and the SNI + MFA 20 mg/kg group (P > 0.05), but a difference occurred in the treatment effect compared with the SNI + MFA 5 mg/kg group (P < 0.05) (Fig. 4d-o). Fig. 2 The SNI model can cause neuropathic pain and ferroptosis. a, b, c The PMWT, PWCD, and PTWL values to stimulation on days 0, 1, 3, 7, 14, and 21 after sham or operation SNI (n = 9 per group). d Western blot analysis of ferroptosis-related proteins Nox4, ACSL4, and GPX4, GAPDH was used as an internal control (n = 6 per group). e, f, g Quantitative analysis of Nox4, ACSL4, and GPX4 expression levels. h, j, l The expression of Nox4, ACSL4, and GPX4 in different groups in the immunofluorescence experiment. Green fluorescence is the target protein, red fluorescence is the nucleus. Scale bar = 50 µm (n = 6 per group). n The expression of ROS in different groups in the immunofluorescence experiment. Red fluorescence is the ROS. Scale bar = 50 µm (n = 6 per group). i, k, m, o Quantitative analysis of Nox4, ACSL4, GPX4, and ROS expression levels. Data are shown as the mean ± SD.*P < 0.05, **P < 0.01, ***P < 0.001, and. ns P > 0.05 The results we got suggest that MFA may suppress the expression of Nox4 and alleviate ferroptosis and neuropathic pain.

MFA can Reverse Ferroptosis and Neuropathic Pain Caused by Nox4 Overexpression
To further verify that MFA can attenuate neuropathic pain by inhibiting Nox4 to alleviate ferroptosis, in vivo overexpression of Nox4 was accomplished by stereotactic injection of AAV-Nox4 into the DRG. First, the Nox4 transfection range and transfection efficiency were evaluated by immunofluorescence, Western blotting, and qRT-PCR. We established that AAV-Nox4 rats exhibited increased Nox4 protein expression in contrast with AAV-NC rats 28 days after injection (P < 0.001) (Fig. 5).
Next, for a more in-depth analysis of the effect of Nox4 overexpression on neuropathic pain, we initially injected AAV-Nox4, established SNI injury models, and administered MFA intervention. In the SNI model, we observed that the thresholds of PMWT and PWCD were significantly increased by overexpressing Nox4 (P < 0.001) and were reversed after gavage treatment with MFA (P > 0.05). There was no substantial correlation in PTWL between all groups (P > 0.05) (Fig. 6a, b, c). Through IF and WB analysis, in contrast with the SNI + AAV-Nox4 group, the expression of Nox4 was markedly higher than that in the SNI + AAV-NC group and the SNI group (P < 0.01). At the same time, we also found that in comparison with the SNI + AAV-NC group and the SNI group, ACSL4 and ROS were correspondingly higher, and GPX4 was correspondingly lower (P < 0.05). These results that Nox4 may cause ferroptosis in neuropathic pain. In addition, we observed that compared with the SNI + AAV-Nox4 + MFA 10 mg/kg group, the expression of the ferroptosis-related protein ACSL4 Fig. 4 MFA can alleviate neuropathic pain and inhibit ferroptosis. a, b, c Effects of different concentrations of MFA on PMWT, PWCD, and PTWL of SNI group. *** P < 0.001, SNI + MFA20mg/ kg vs. SNI group; ### P < 0.001, SNI + MFA10mg/kg vs. SNI group; &&& P < 0.001, SNI + MFA5mg/kg vs. SNI group; ++ P < 0.01, +++ P < 0.001, SNI + MFA20mg/kg vs. SNI + MFA5 mg/kg group; @@@ P < 0.001, SNI + MFA10mg/kg vs. SNI + MFA5mg/kg group (n = 9 per group). d Western blot analysis of ferroptosis-related proteins Nox4, ACSL4, and GPX4, GAPDH was used as an internal control (n = 6 per group). e, f, g Quantitative analysis of Nox4, ACSL4, and GPX4 expression levels. h, j, l The expression of Nox4, ACSL4, and GPX4 in different groups in the immunofluorescence experiment. Green fluorescence is the target protein, red fluorescence is the nucleus. Scale bar = 50 µm (n = 6 per group). n The expression of ROS in different groups in the immunofluorescence experiment. red fluorescence is the ROS. Scale bar = 50 µm (n = 6 per group). i, k, m, o Quantitative analysis of Nox4, ACSL4, GPX4, and ROS expression levels. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and. ns P > 0.05 ◂ Fig. 5 DRG injection of AAV virus overexpressed Nox4. a, c Immunofluorescence showed that Nox4 was overexpressed 28 days after DRG injection of AAV-Nox4. Scale bar = 50 µm. b, d Western blot analysis showed that the expression of Nox4 in DRG increased 28 days after AAV-Nox4 microinjection. e Quantitative RT-PCR detection showed that the mRNA transcription of Nox4 in DRG of AAV-Nox4 group was increased. ***P < 0.001 was lower, GPX4 was higher, and ROS was also lower than those in the SNI + AAV-Nox4 group (P < 0.001) (Fig. 6d-o). Therefore, MFA may reverse neuropathic pain when Nox4 expression was inhibited and its induction of ferroptosis in DRG neurons.

MFA Protects Against SNI-induced Morphological Changes in Mitochondria in DRG Neurons
The ultrastructure of DRG was examined by transmission electron microscopy on the 1st, 3rd, 7th, 14th, and 21st days after SNI injury. The mitochondria in DRG neurons illustrated changes characteristic of ferroptosis, including mitochondrial membrane thickening, mitochondrial shrinkage, and mitochondrial sputum disappearance (Fig. 7a). In addition, the percentage of mitochondria with abnormal morphology was higher in the SNI group than before surgery (P < 0.01) (Fig. 7b). To further analyze the changes in mitochondria that were related to Nox4 overexpression, in vivo overexpression of Nox4 was carried out with the use of stereotactic administration of AAV-Nox4 RNAi into the DRG. We found that the percentage of mitochondria with morphological abnormalities in the SNI group was lower than that in the SNI + AAV-Nox4 group (P < 0.05). Finally, we found that compared with that in the SNI group and SNI + AAV-Nox4 group, the percentage of mitochondria with morphological abnormalities in the SNI + AAV-Nox4 + MFA 10 mg/kg group was decreased (P < 0.001) (Fig. 7c-f).

Iron Accumulation Induced by SNI was Suppressed by MFA
As contrasted with the preoperative values, the iron content in the DRG of rats increased significantly on day 14 after SNI injury (P < 0.01) (Fig. 7g). Thus, iron accumulation happened during SNI in the neuropathic pain model. Treatment with MFA substantially reduced iron concentrations in SNI rats in opposition to the SNI + vehicle and SNI groups (P < 0.05) (Fig. 7h). After microinjection of the AAV virus into the DRG, the iron content of the SNI + AAV-Nox4 group was higher than that of the SNI group and SNI + AAV-NC group (P < 0.05). However, after MFA treatment, the iron content of the SNI + AAV-Nox4 + MFA 10 mg/kg group was markedly lower than that of the SNI + AAV-Nox4 group (P < 0.001) (Fig. 7i).

Discussion
At present, neuropathic pain affects more than 1 million people worldwide. Due to its complex mechanism, it cannot be effectively treated at present. Thus, it is critically vital to explore new technologies or develop effective drugs [29]. Researches showed that inhibiting oxidative stress in dorsal root ganglion can diminish neuropathic pain [30,31]. MFA has an obvious effect on antioxidative stress [32]. Therefore, this study aimed to prove that MFA can alleviate neuropathic pain.
In this study, we successfully established a neuropathic pain SNI model. We observed that PMWT and PWCD significantly decreased from 1 day after the operation and continued to the end of the experiment on day 21 day. In related studies, it was reported that the neuropathic pain caused by SNI lasted longer [33]. However, there was no significant change in PTWL, which was in agreement with the results of Xu et al. [34]. After MFA treatment, we observed that PMWT and PWCD were significantly improved compared with the SNI group, confirming that MFA may alleviate neuropathic pain.
We further explored the molecular mechanism by which MFA alleviates pain. Studies have shown that Nox4 overexpression can increase the level of oxidative stress in the spinal cord and DRG and cause neuropathic pain [35]. In this study, Nox4 was expressed in large, medium, and small DRG neurons in the immunofluorescence double labeling experiment. In addition, IF and WB analysis implied that the Nox4 level in the DRG on the affected side of rats was significantly increased 14-21 days after SNI. However, the increase in Nox4 protein was reversed after MFA administration. This change was parallel to the results of behavioral PMWT and PWCD. This result suggests that MFA may alleviate neuropathic pain by inhibiting Nox4 protein in the DRG.
The results prescribe that ferroptosis is one of the important mechanisms involved in neuropathic pain. ACSL4 and GPX4 are ferroptosis marker proteins. During ferroptosis, ACSL4 expression increases, and GPX4 has the opposite effect [14]. On top of that, ROS increase and iron accumulation also play a key role in ferroptosis [36,37]. Nox4 can Fig. 6 The pain threshold of rats injected with AAV-Nox4 by DRG was significantly decreased and ferroptosis was aggravated after SNI injury, but it was reversed after MFA treatment. a, b, c Changes in pain behavior in all group. *** P < 0.001, SNI + AAV-Nox4 + MFA10mg/kg vs. SNI group; ### P < 0.001 SNI + AAV-NC + MFA 10 mg/kg vs. SNI group; &&& P < 0.001, SNI + AAV-Nox4 vs. Sham group; +++ P < 0.001, SNI + AAV-NC vs. Sham group (n = 9 per group). d Western blot analysis of ferroptosis-related proteins Nox4, ACSL4 and GPX4, GAPDH was used as an internal control (n = 6 per group). e, f, g Quantitative analysis of Nox4, ACSL4, and GPX4 expression levels. h, j, l The expression of Nox4, ACSL4, and GPX4 in different groups in the immunofluorescence experiment. Green fluorescence is the target protein, red fluorescence is the nucleus. Scale bar = 50 µm (n = 6 per group). n The expression of ROS in different groups in the immunofluorescence experiment. Red fluorescence is the ROS. Scale bar = 50 µm (n = 6 per group). i, k, m, o Quantitative analysis of Nox4, ACSL4, GPX4, and ROS expression levels. Data are shown as the mean ± SD.*P < 0.05, **P < 0.01, ***P < 0.001, and. ns P > 0.05 ◂ Fig. 7 Changes in mitochondrial morphology and iron content between all groups. a, c, e Electron micrographs of dorsal root ganglia. The yellow boxes highlight the representative mitochondrial ultrastructure in each group. b, d, f percentages of mitochondria with morphological abnormalities. g, h, i change of iron content.*P < 0.05, **P < 0.01, ***P < 0.001, and. ns P > 0.05 increase ROS to cause oxidative stress and ferroptosis in cells [17,38]. The inhibitory effect of MFA on Nox4 may reduce ferroptosis and alleviate neuropathic pain. In this study, IF and WB analysis showed that ACSL4 expression was significantly higher and GPX4 expression was significantly lower in the SNI injury group than in the sham group. The ROS fluorescence probe also showed that ROS increased. At the same time, the iron concentration was significantly elevated. In addition, transmission electron microscopy of DRG on the affected side showed that the mitochondrial volume was reduced, the membrane density was increased, and the mitochondrial cristae disappeared. These data suggest that ferroptosis occurred on the DRG of the affected side after SNI in rats. These changes were accompanied by Nox4 overexpression and increased sensitivity to PMWT and PWCD. At 14 days after MFA administration, we perceived that the expression of ACSL4 and GPX4 was reversed, and the ROS and iron contents were reduced. In addition, electron microscopy showed that the damaged mitochondria were reduced. Therefore, Nox4 overexpression on DRG neurons may cause ferroptosis and neuropathic pain. MFA may alleviate neuropathic pain by inhibiting Nox4 to alleviate ferroptosis.
To further verify that MFA regulates ferroptosis and alleviates neuropathic pain by regulating Nox4 expression in the DRG, an adeno-associated virus vector and stereotactic microinjection were used to increase Nox4 expression in the DRG. The results demonstrated the Nox4 in the SNI + AAV-Nox4 group was markedly higher than that in the SNI + AAV-NC group. Meanwhile, through IF and WB analysis, we declared that ACSL4 increased, GPX4 decreased, and ROS and iron ions increased. In addition, electron microscopy showed that mitochondrial damage increased. These results indicated that Nox4 overexpression in the DRG causes ferroptosis. In addition, PMWT and PWCD thresholds also decreased. Therefore, ferroptosis caused by increased Nox4 leads to neuropathic pain. Next, we treated the SNI + AAV-Nox4 group with MFA. The results showed that Nox4 expression was decreased, ferroptosis was alleviated, and PMWT and PWCD thresholds were increased. Therefore, we confirmed that MFA can inhibit Nox4-induced ferroptosis in DRG neurons and alleviate neuropathic pain in rats.
There are still some deficiencies in this study. We only focused on the fact that MFA can alleviate neuropathic pain by inhibiting Nox4 expression on DRG neurons, but its specific mechanism was not discussed. Nox4 can be expressed on the mitochondrial membrane and can produce ROS [39]. How does MFA combine with Nox4 to produce inhibition? We will solve this problem in future research. In addition, we only confirmed the existence of cell ferroptosis in the DRG in neuropathic pain, but the underlying mechanism of ferroptosis leading to neuropathic pain has not been discussed, so further research is needed to solve these problems.

Conclusions
To sum up, we demonstrated that Nox4 overexpression in the DRG can cause ferroptosis and can lead to neuropathic pain and that MFA can alleviate neuropathic pain by inhibiting the increase in Nox4 in the DRG. This may provide a new drug choice for neuropathic pain.