Total glucosides of paeony ameliorates chemotherapy-induced neuropathic pain by suppressing microglia pyroptosis through the inhibition of KAT2A-mediated p38 pathway activation and succinylation

Abstract

Chemotherapy-induced neuropathic pain (CINP) is a prevalent side effect of chemotherapy. Total glucosides of paeony (TGP) have been shown to be effective in pain management. This study aimed to investigate the efficacy and mechanism of TGP in alleviating CINP. Sprague–Dawley rats were treated with oxaliplatin to establish CINP models, and BV2 microglia were exposed to lipopolysaccharides (LPS) to induce pyroptosis. The impact of TGP on CINP was assessed by measuring mechanical withdrawal threshold (MWT), cold pain threshold (CPT), and thermal pain threshold (TPT), as well as inflammatory factor levels. Pyroptosis was evaluated using flow cytometry, lactate dehydrogenase (LDH) release, and pyroptosis marker levels. Quantitative real-time PCR and molecular docking were employed to identify TGP targets, while phospho-kinase arrays, western blotting, and co-immunoprecipitation were used to elucidate the mechanism. Results indicated that TGP increased MWT, CPT, and TPT and inhibited inflammatory factor release in CINP rats. Furthermore, TGP suppressed LPS-induced pyroptosis and downregulated KAT2A expression in BV2 cells; this suppression was reversed by KAT2A overexpression. Mechanistically, KAT2A overexpression activated the p38 pathway and promoted p38 succinylation at K295. KAT2A knockdown inhibited pyroptosis in LPS-induced BV2 cells, an effect that was reversed by the p38 activator metformin. Additionally, the improvements in MWT, CPT, TPT, and inflammatory factor levels observed in CINP rats treated with TGP were negated by KAT2A overexpression. In conclusion, TGP alleviated CINP by suppressing microglial pyroptosis through inhibition of the KAT2A-mediated p38 pathway activation and succinylation. This study provides insights into a potential new therapeutic approach for CINP.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-83207-8.

Introduction

Chemotherapy-induced neuropathic pain (CINP) is a prevalent side effect of chemotherapeutic agents, including platinum-based drugs, taxanes, and proteasome inhibitors. For example, oxaliplatin (ASLB) a platinum-based chemotherapeutic widely used to treat colorectal and ovarian cancers, induces CINP in over 90% of patients undergoing clinical treatment^1–3. CINP can manifest during or persist after chemotherapy, potentially developing into chronic neuropathy following repeated treatments^4–6. Characterized by symmetric paresthesia in the limbs, symptoms include slowness, numbness, tingling, and abnormal pain, often exacerbated by thermal stimuli⁷. CINP significantly impairs quality of life and may necessitate the interruption of chemotherapy, thereby increasing patient mortality. Although several drugs and strategies are employed to manage CINP, their therapeutic efficacy is limited, and they often come with adverse effects^8,9. Consequently, there is a need to develop novel therapeutic agents for CINP.

Total glucosides of paeony (TGP) are an effective ingredient derived from the roots of Paeonia species, a traditional Chinese medicine. TGP is a mixture primarily composed of paeoniflorin, which constitutes more than 90% of its total content. Modern pharmacological studies have demonstrated that TGP possesses potent anti-inflammatory and immunomodulatory properties and is utilized in the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus^10–12. Additionally, traditional Chinese medicine regards TGP as an efficacious remedy for pain. Contemporary medical research has shown that TGP alleviates abdominal pain in colitis patients and low back pain and peripheral joint pain in those with ankylosing spondylitis^13,14. However, the potential of TGP to alleviate CINP remains unclear.

Microglia, as immune cells of the CNS, play a crucial role in regulating various types of pain. Activated spinal microglia exacerbate pain by releasing bioactive molecules such as interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-α), and nitric oxide (NO)¹⁵. Recent studies have highlighted the role of microglial pyroptosis in the development of neurological disorders, including Alzheimer’s disease, spinal cord injury, and stroke^16–18. Pyroptosis is a gasdermin-mediated form of programmed cell death characterized by the release of pro-inflammatory factors¹⁹. Given that microglial pyroptosis is a hallmark of neuroinflammation and that its inhibition has been shown to alleviate mechanical allodynia^20,21, the potential involvement of microglial pyroptosis in CINP warrants investigation. We hypothesize that microglial pyroptosis may represent a therapeutic target for CINP.

Succinylation is a recently identified post-translational modification involving the covalent attachment of a succinyl group to a lysine residue of a substrate protein, either enzymatically or non-enzymatically²². Succinylation levels are primarily regulated by succinyl donors, succinyltransferases, and desuccinylases, and are implicated in various physiological activities and diseases, including cancers and metabolic disorders^23,24. Recent evidence suggests that inhibiting succinylation may suppress pyroptosis²⁵. However, the role of succinylation in mediating microglial pyroptosis remains unexplored.

In this study, we evaluated the pain behavior in CINP rats to assess the efficacy of TGP and investigated the mechanisms underlying the improvement of CINP by assessing microglial pyroptosis levels. This study aims to provide a potential new therapeutic option for CINP and offer a novel theoretical basis for its clinical management.

Materials and methods

Animal study

The study was approved by the Ethics Committee of The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. All procedures were conducted in accordance with relevant guidelines and regulations, and reporting adhered to the ARRIVE guidelines.

Sprague-Dawley rats (180–220 g) were housed at 21–23 °C with a relative humidity of 60–70% and a 12-hour light/dark cycle, and were provided with food and water ad libitum. The rats were randomly divided into six groups (n = 6): sham group, CINP group, CINP + 0.09 g/kg TGP group, CINP + 0.18 g/kg TGP group, CINP + TGP + LV-NC group, and CINP + TGP + LV-KAT2A group. To establish the CINP rat model, ASLB (Qilu Pharmaceutical, Shandong, China) was dissolved in 5% glucose to a concentration of 1 mg/mL, and rats were intraperitoneally (i.p.) injected with 2 mg/kg once daily for five consecutive days. The sham group received an equivalent volume of saline. Rats treated with ASLB also received TGP (Liwah Pharmaceutical, Zhejiang, China) via oral gavage (0.09 and 0.18 g/kg) twice daily for 15 consecutive days. To investigate the in vivo function of KAT2A, rats treated with 0.18 g/kg TGP were injected with lentiviral vectors carrying either negative control (NC) or KAT2A overexpressing plasmids via the tail vein 30 min before ASLB administration. Following gavage, rats were anesthetized with 0.1% sodium pentobarbital, and blood was collected from the abdominal aorta. Serum was isolated by centrifugation to measure the levels of TNF-α, IL-1β, and IL-6.

Behavioral tests

Mechanical withdrawal threshold (MWT) was assessed using von Frey hairs (VFH). Stimulation of the mid-left hind paw of rats was started at 0.6 g VFH stimulus strength. If the rat did not retract the foot, the fibre wire was replaced with a larger one until the rat retracted the foot, and the stimulation intensity of this fibre wire was the effective intensity when the foot was retracted 3 times in 5 stimulation sessions. The lowest effective intensity was the MWT.

Cold pain threshold (CPT) was measured according to the following experiments. The tails of rats were immersed in a water bath at 4 °C at 0, 3, 6, 9, 12, and 15 d of TGP treatment until the tails were removed, and the tail immersion time was recorded. The test was performed at 5 min intervals and repeated three times, and the mean value was calculated and recorded, with a cut-off time of 15 s. The test was performed at 5 min intervals and repeated three times.

Thermal pain threshold (TPT) was assessed at 0, 3, 6, 9, 12, and 15 d of TGP treatment. The temperature of the hot plate apparatus was set at 50 °C. After the temperature was constant, the rats were placed on the hot plate apparatus with an organic glass cover and acclimatised for 1 min. The temperature of the hot plate apparatus was set at 50 °C. After the temperature was constant, the rats were placed on the hot plate apparatus with an organic glass cover and acclimatised for 1 min. The time of the first foot licking was recorded, and the rat was tested every 5 min, repeated 3 times, and the mean value of the record was calculated, with a cut-off time of 60 s.

Cell culture and treatment

BV2 microglial cells were obtained from Procell (Wuhan, China). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO₂.Cells were treated with 1 µg/mL lipopolysaccharide (LPS) for 6 h to construct an in vitro inflammation model. To evaluate the effect of TGP on pyroptosis, cells were treated with 12.5, 25, 50, and 100 µM TGP for 24 h. The chemical structure of TGP is presented in Fig. 1A.

Fig. 1.

TGP improved pain behaviors in CINP rats and the inhibited the release of inflammatory factors. (A) The chemical structure of TGP. (B–D) The pain behaviors of CINP rats were assessed by the levels of MWT, CPT and TPT. (E) The levels of TNF-α, IL-1β and IL-6 were measured using ELISA kits.

Cell transfection

The plasmid KAT2A overexpressing plasmid (pcDNA3.1-KAT2A), empty vector pcDNA3.1-negative control (NC), short hairpin RNA targeting KAT2A (sh-KAT2A) and sh-NC were constructed by GenePharma (Shanghai, China). BV2 cells were seeded in 6-well plates at a density of 1 × 10⁵ cells per well and incubated overnight to reach approximately 70–80% confluence. For each transfection, 2 µg of plasmid DNA and 5 µL of Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) were used. The plasmid DNA and Lipofectamine 2000 reagent were diluted separately in 100 µL of Opti-MEM Reduced Serum Medium (Gibco, Thermo Scientific, Waltham, MA, USA) and incubated for 5 min at room temperature. The diluted plasmid DNA and Lipofectamine 2000 reagent were then mixed gently and incubated for an additional 20 min at room temperature to form the transfection complexes. The transfection complexes were added dropwise to the wells containing the cells, and the plates were gently swirled to ensure even distribution. Cells were harvested 48 h post-transfection for further analysis.

Measurement of cell viability

Cell viability of BV2 cells in different groups was assessed using a cell counting kit-8 (CCK8; Beyotime, Shanghai, China). Cells were seeded in 96-well plates (100 µL, 5 × 10³ cells) and stimulated by different concentrations of TGP for 24 h. Subsequently, CCK8 solution (10 µL) was added to each well for 1 h incubation. Finally, the absorbance was measured using a microplate reader at 450 nm.

Flow cytometry

Cell death of BV2 cells was evaluated using a FLICA 660 Caspase-1 (YVAD) assay kit (Immunochemistry technologies, Davis, CA, USA). Briefly, BV2 cells were seeded in 6-well plates at a density of 1 × 10⁵ cells per well and allowed to adhere overnight. The FLICA 660 working solution was prepared by diluting the FLICA reagent 1:30 in the provided apoptosis wash buffer. The cells were then incubated with 1 mL of the FLICA 660 working solution for 1 h at 37 °C in a humidified CO₂ incubator. To mark cells with membrane pores, the cells were stained with 1 µg/mL propidium iodide (PI) in PBS for 15 min at room temperature in the dark. The cells were analyzed via flow cytometry (BD Biosciences, San Jose, CA, USA).

Western blot

Total protein of BV2 cells was isolated by RIPA buffer and quantified using a BCA kit (Beyotime). The protein samples were loaded onto SDS-PAGE for electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder. After washed by TBST solution, the membranes were incubated with primary antibodies overnight at 4 °C and then with secondary antibodies (1: 10,000, ab205718, Abcam, Cambridge, UK) for 2 h incubation. Blots were visualized using the ECL reagent (Beyotime). The following antibodies were used for the experiments: anti-N-GSDMD (ab215203, 1/1000, Abcam), anti-Caspase-1 (ab207802, 1/1000, Abcam), anti-p38 (ab170099, 1/1000, Abcam), anti-p-p38 (ab178867, 1/1000, Abcam), anti-GAPDH (ab181602, 1/10,000, Abcam) and anti-succinyllysine (1: 2000, PTM-401, PTM BIO, Hangzhou, China).

Measurement of LDH release

The LDH release was detected using a LDH release assay kit (Beyotime). BV2 cells were seeded in a 96-well plate at a density of 1 × 10⁴ cells per well and incubated overnight to reach approximately 80% confluence. To each well, 60 µL of the LDH working solution was added, and the plate was protected from light. The mixture was then incubated for 30 min at room temperature. Following the incubation, 50 µL of the stop solution provided in the kit was added to each well to terminate the reaction. The absorbance of each well was measured at 490 nm using a microplate reader.

ELISA

The levels of TNF-α, IL-1β, IL-6 and IL-18 was measured using ELISA assay kits (Beyotime). Cells were centrifuged at 500×g for 5 min to get supernatant. The supernatant and the serums of rats were used for the experiments and the experiments were conducted following the manufacturer’s protocol. The absorbance was measured at A450.

Quantitative real-time PCR (qPCR)

Total RNA of BV2 cells were isolated by Trizol reagent (Invitrogen). The concentration and purity of the isolated RNA were determined by spectrophotometric analysis at 260 nm and 280 nm, respectively, using a NanoDrop spectrophotometer (Thermo Scientific). Reverse transcription was performed to convert the isolated RNA into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). qPCR was performed using an AceQ qPCR SYBR Green master mix (Vazyme, Nanjing, China) on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The relative expression of each gene was calculated by 2^−ΔΔCt, and GAPDH was used as the internal control for normalization. The sequences of qPCR primers were as follows: KAT2A, 5ʹ-AACCTGAGCGAGTTGTGCC-3ʹ (forward) and 5ʹ-GCCGGTTAATCTCGTCCTCTG-3ʹ (reverse); KAT3B, 5ʹ-AGCCAAGCGGCCTAAACTC-3ʹ (forward) and 5ʹ-CGCCACCATTGGTTAGTCCC-3ʹ (reverse); SIRT5, 5ʹ-CCAGTTGTGTTGTAGACGAAAGC-3ʹ (forward) and 5ʹ-CCAGTTGTGTTGTAGACGAAAGC-3ʹ (reverse); SIRT7, 5ʹ-GCACTTGGTTGTCTACACGG-3ʹ (forward) and 5ʹ-TGTCCATACTCCATTAGGACCC-3ʹ (reverse); CPT1A, 5ʹ-TGGCATCATCACTGGTGTGTT-3ʹ (forward) and 5ʹ-GTCTAGGGTCCGATTGATCTTTG-3ʹ (reverse).

Molecular docking

The 3D structure of KAT2A was obtained from the UniProt database. The structure was then processed and geometrically optimized using the Maestro 11.9 platform (Schrödinger, LLC, New York, NY, USA). The molecular structure of TGP was retrieved from the PubChem database and was formatted and energy-minimized using Chem3D (PerkinElmer, Waltham, MA, USA). Molecular docking was performed using the Glide module in the Schrödinger Maestro software. The KAT2A protein structure was prepared using the Protein Preparation Wizard module. After protein preparation, ligand preparation, grid generation and molecular docking, the final docking results were analyzed based on the docking scores and the interaction profiles between the TGP ligand and the KAT2A protein.

Phospho-kinase array

The signaling pathways affected by KAT2A in BV2 cells were identified using a phospho-kinase array (Full Moon BioSystems, San Francisco, CA, USA). Briefly, BV2 cells were lysed in the provided extraction buffer, and the isolated protein samples were then biotinylated using the provided biotinylation reagents. The antibody microarray slides were blocked with the blocking buffer for 45 min to minimize non-specific binding. Following blocking, the slides were incubated with the biotin-labeled protein samples overnight at 4 °C with gentle agitation. After incubation, Cy3-streptavidin was added to the slides, and they were incubated for 1 h in the dark to detect the biotinylated proteins. The slides were then washed again with the wash buffer to remove excess Cy3-streptavidin. Fluorescence of the conjugation-labeled proteins on the antibody-arrayed slides was detected using a microarray scanner (Axon Instruments, Molecular Devices, San Jose, CA, USA). The resulting images were analyzed using GenePix Pro 6.0 software (Molecular Devices, San Jose, CA, USA) to quantify the fluorescence intensities and identify the phosphorylated kinases.

Co-immunoprecipitation (co-IP) and IP

Co-IP and IP assays were performed to evaluate the interaction between KAT2A and p38 and to determine the succinylation level of p38, respectively. These experiments utilized an IP kit with protein A + G magnetic beads (Beyotime). Cells were lysed with lysis buffer and then centrifuged at 4 °C and 13,000 × g for 5 min to obtain the supernatant. Protein A + G magnetic beads were pre-incubated with specific antibodies (anti-Flag, anti-IgG, anti-HA, or anti-p38) for 1 h at room temperature to allow antibody binding. Subsequently, the samples were incubated with the prepared magnetic beads overnight at 4 °C. The antigen-antibody complexes were eluted using acid elution buffer. Protein levels were quantified by Western blot analysis.

Bioinformatic analysis

The succinylation sites of KAT2A were predicted using the GPSuc database (http://kurata14.bio.kyutech.ac.jp/GPSuc/index.php).

Statistical analysis

All data were analyzed using SPSS 22.0 software. The comparison between two or more groups was performed by student’s t-test or one-way analysis of variance (ANOVA). Results were expressed as mean ± standard deviation of at least three replicates. P < 0.05 was recognized as statistically significant.

Results

TGP improves pain behaviors and inhibited the levels of inflammatory factors in CINP rats

To investigate the effect of TGP on pain behaviors in CINP rats, we measured the MWT, CPT, and TPT before and after TGP treatment. Compared with the sham group, MWT, CPT, and TPT were significantly decreased in CINP rats. Treatment with both 0.09 g/kg and 0.18 g/kg TGP increased the MWT, CPT, and TPT in CINP rats, with 0.18 g/kg TGP showing a more pronounced effect (Fig. 1B–D). Furthermore, we assessed the levels of several inflammatory factors in different groups of rats. The results indicated that the levels of TNF-α, IL-1β, and IL-6 were elevated in CINP rats, and these increases were partially reversed by TGP treatment. Again, 0.18 g/kg TGP exhibited a superior effect (Fig. 1E). In conclusion, our findings demonstrated that TGP improved pain behaviors and reduced the levels of inflammatory factors in CINP rats, with 0.18 g/kg TGP being more efficacious. Therefore, 0.18 g/kg TGP was selected for subsequent experiments.

TGP inhibites LPS-induced pyroptosis in LPS-induced BV2 cells

To evaluate the effect of TGP on pyroptosis in LPS-induced BV2 cells, we first assessed cell viability across different concentrations of TGP. Our results indicated that cell viability was significantly reduced by 100 µM TGP (Fig. 2A), suggesting cytotoxicity at this concentration. Therefore, we selected lower concentrations of TGP for subsequent experiments. We observed that LPS treatment significantly decreased cell viability, whereas treatment with 25 and 50 µM TGP significantly restored cell viability (Fig. 2B). Furthermore, LPS induced pyroptosis in BV2 cells, as evidenced by increased levels of pyroptosis markers GSDMD and Caspase-1, which were effectively attenuated by 25 and 50 µM TGP (Fig. 2C–E). Additionally, LPS increased the release of LDH and the levels of IL-1β and IL-18, effects that were significantly inhibited by TGP, with 50 µM TGP exhibiting the most potent effect (Fig. 2F–H). Collectively, these findings indicate that TGP significantly inhibits LPS-induced pyroptosis in BV2 cells, and 50 µM TGP was chosen for further experiments.

Fig. 2.

TGP inhibited LPS-induced pyroptosis in LPS-induced BV2 cells. (A,B) Cell viability of BV2 cells was assessed using a CCK-8 kit. (C,D) Pyroptosis of BV2 cells was evaluated by flow cytometry. (E) The protein levels of GSDMD and Caspase-1 were measured by western blot. (F) LDH release of BV2 cells was measured using a LDH release kit. (G,H) The levels of IL-1β and IL-18 were assessed by ELISA kits. NS no significance.

TGP inhibits the expression of KAT2A in LPS-induced BV2 cells

To elucidate the target of TGP in LPS-induced BV2 cells, we measured the expression of several succinyltransferases before and after TGP treatment. Our results indicated that LPS increased the expression of KAT2A and KAT3B but did not affect the expression of SIRT5, SIRT7, or CPT1A. Importantly, TGP significantly downregulated the expression of KAT2A in LPS-treated BV2 cells, whereas the expression of KAT3B remained unchanged (Fig. 3A). Molecular docking analysis was performed to examine the binding of TGP to KAT2A, revealing a strong interaction and high binding affinity (Fig. 3B–D). Collectively, these findings suggested that TGP inhibited the expression of KAT2A in LPS-induced BV2 cells.

Fig. 3.

TGP inhibited the expression of KAT2A in LPS-induced BV2 cells. (A) The expression of succinylases KAT2A, KAT3B, SIRT5, SIRT7 and CAPT1 in BV2 cells were measured by qPCR. (B) The 3D structure of molecular docking between KAT2A and TGP. (C) The electrostatic surface of KAT2A. (D) The detail binding mode of KAT2A and TGP. NS no significance.

KAT2A overexpression restores pyroptosis in LPS-induced BV2 cells inhibited by TGP

To verify the role of KAT2A in pyroptosis, BV2 cells were transfected with pcDNA3.1-KAT2A plasmids, resulting in a significant increase in KAT2A expression (Fig. 4A). Furthermore, the enhanced cell viability of LPS-induced BV2 cells treated with TGP was significantly reduced by KAT2A overexpression (Fig. 4B). Overexpression of KAT2A also reversed the TGP-suppressed pyroptosis rate in LPS-induced BV2 cells (Fig. 4C,D). The protein levels of pyroptosis markers GSDMD and Caspase-1, which were reduced by TGP treatment, were restored by KAT2A overexpression (Fig. 4E). Additionally, measurements of LDH release and the levels of IL-1β and IL-18 showed that the levels of these pro-inflammatory indicators, which were inhibited by TGP in LPS-induced BV2 cells, were significantly increased upon KAT2A overexpression (Fig. 4F–H). Together, these results indicated that KAT2A overexpression reversed the inhibition of pyroptosis in LPS-induced BV2 cells treated with TGP.

Fig. 4.

KAT2A overexpression restored pyroptosis in LPS-induced BV2 cells inhibited by TGP. (A) The expression of KAT2A was assessed by qPCR. (B) Cell viability of BV2 cells was assessed using a CCK-8 kit. (C,D) Pyroptosis of BV2 cells was evaluated by flow cytometry. (E) The protein levels of GSDMD and Caspase-1 were measured by western blot. (F) LDH release of BV2 cells was measured using a LDH release kit. (G,H) The levels of IL-1β and IL-18 were assessed by ELISA kits.

KAT2A activates p38 pathway and promotes p38 succinylation at K295 site

To investigate the mechanism by which KAT2A mediates pyroptosis, we performed a phospho-kinase array to identify the pathways affected by KAT2A overexpression in BV2 cells. The results indicated that KAT2A increased the phosphorylation of p38, suggesting that KAT2A overexpression may activate the p38 pathway (Fig. 5A). Further analysis confirmed that KAT2A overexpression upregulated the phosphorylation levels of p38 protein (Fig. 5B). Co-IP assays demonstrated an interaction between KAT2A and p38 (Fig. 5C). Several potential succinylation sites were predicted, and the top three sites are shown in Fig. 5D. Mutagenesis of these sites (K53, K66, and K295) to arginine (R) revealed that only the K295 mutation inhibited the phosphorylation of p38 and the succinylation levels of p38 (Fig. 5E). These findings collectively suggested that KAT2A activated the p38 pathway and promotes p38 succinylation at the K295 site.

Fig. 5.

KAT2A activates p38 pathway and promotes p38 succinylation at K295 site. (A) The pathways was identified using a phospho-kinase array in BV2 cells affected by KAT2A knockdown. (B) The protein levels of p38 were assessed by western blot. (C) The interaction between KAT2A and p38 was evaluated by co-IP. (D) The potential succinylation site was predicted by GPSuc database. (E) The protein and the succinylation levels of p38 were measured by western blot.

p38 activator metformin restores pyroptosis inhibited by KAT2A knockdown in LPS-induced BV2 cells

Given that KAT2A activates the p38 pathway, we conducted rescue experiments to verify this mechanism. KAT2A knockdown significantly reduced the expression of KAT2A in BV2 cells (Fig. 6A). LPS treatment decreased cell viability, which was rescued by KAT2A knockdown. This effect was reversed by the p38 activator metformin (Fig. 6B). Moreover, KAT2A knockdown inhibited LPS-enhanced pyroptosis, an effect that was restored by metformin treatment (Fig. 6C, D). The protein levels of GSDMD and Caspase-1, which were downregulated by KAT2A knockdown in LPS-induced BV2 cells, were partially restored by metformin (Fig. 6E). Additionally, KAT2A knockdown suppressed the increased levels of LDH release, IL-1β, and IL-18 in LPS-treated cells, and this suppression was partially reversed by metformin (Fig. 6F–H). Taken together, these results demonstrated that the p38 activator metformin restored pyroptosis inhibited by KAT2A knockdown in LPS-induced BV2 cells.

Fig. 6.

Metformin restored pyroptosis inhibited by KAT2A knockdown in LPS-induced BV2 cells. (A) The expression of KAT2A was assessed by qPCR. (B) Cell viability of BV2 cells was assessed using a CCK-8 kit. (C,D) Pyroptosis of BV2 cells was evaluated by flow cytometry. (E) The protein levels of GSDMD and Caspase-1 were measured by western blot. (F) LDH release of BV2 cells was measured using a LDH release kit. (G,H) The levels of IL-1β and IL-18 were assessed by ELISA kits.

KAT2A overexpression reverses pain behavior improved by TGP in CINP rat model

To confirm the functional role of KAT2A, we conducted in vivo experiments. The results indicated that the improvements in MWT, CPT, and TPT observed in CINP rats treated with TGP were reversed by KAT2A overexpression (Fig. 7A–C). Furthermore, the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6, which were suppressed by TGP in the CINP rat model, were partially restored by KAT2A overexpression (Fig. 7D). In conclusion, our findings demonstrated that KAT2A overexpression reversed the improvement in pain behavior mediated by TGP in CINP rats.

Fig. 7.

KAT2A overexpression reversed pain behavior improved by TGP in CINP rat model. (A–C) The pain behaviors of CINP rats were assessed by the levels of MWT, CPT and TPT. (D) The levels of TNF-α, IL-1β and IL-6 were measured using ELISA kits.

Discussion

With the rising incidence of cancer, the prevalence of chemotherapy-induced neuropathic pain (CINP) attributed to alkylating agents like ASLB is also increasing. Given the limitations of current therapeutic options for CINP and the restricted availability of suitable medications for cancer patients, traditional Chinese medicine (TCM) has emerged as a promising approach for managing CINP. For instance, Wu et al.²⁶ demonstrated that puerarin reduces mechanical allodynia and thermal hyperalgesia in paclitaxel-treated rats, and repeated administration of puerarin can prevent the development of paclitaxel-induced peripheral neuropathic pain. Similarly, Yi et al.²⁷ reported that the aqueous extract of Forsythia viridissima fruits significantly alleviates ASLB-induced mechanical hypersensitivity and prevents the onset of mechanical hyperalgesia. Zhang et al.²⁸ found that curcumin increases the MWT and repairs injured spinal cord neurons in CINP rats, along with inhibiting the levels of TNF-α, IL-1β, and IL-6. These findings suggested the potential of TCM in treating CINP. Paeonia lactiflora Pallas, a widely used TCM for pain management, contains the compound TGP, which has shown efficacy in pain relief. Wang et al.¹⁴ have shown that TGP improves the lumbar pain index and peripheral joint pain index in patients with ankylosing spondylitis. Additionally, TGP alleviates abdominal pain in patients with ulcerative colitis¹³. In this study, we investigated the effect of TGP on CINP and found that TGP significantly increased the MWT, CPT, and TPT, as well as inhibited the release of inflammatory factors such as TNF-α, IL-1β, and IL-6 in CINP rats. These results indicated that TGP was effective in improving CINP in vivo.

Numerous studies have demonstrated that microglial pyroptosis mediates multiple neurological disorders. The activation of microglial pyroptosis and NLRP3 inflammasome complexes promotes neuroinflammation, contributing to the progression of diseases such as Alzheimer’s disease and spinal cord injury^16,29. Recent research has indicated that targeting microglial pyroptosis can effectively alleviate pain. For example, Hua et al.²¹ showed that exosomes derived from human umbilical cord mesenchymal stem cells alleviate mechanical allodynia and thermal hyperalgesia in inflammatory pain models by inhibiting microglial pyroptosis. Gao et al.²⁰ demonstrated that overexpression of miR-99b-3p attenuates mechanical allodynia and neuroinflammation through the suppression of microglial pyroptosis. These findings highlighted the role of microglial pyroptosis in the progression of pain. However, the function of microglial pyroptosis in CINP remains unclear. In this study, we confirmed that TGP inhibited pyroptosis in LPS-induced BV2 cells, as evidenced by the reduction in the protein levels of pyroptosis markers GSDMD and Caspase-1, as well as decreases in LDH release and the levels of IL-1β and IL-18. Collectively, these results demonstrated that TGP inhibited LPS-induced pyroptosis in BV2 microglial cells.

KAT2A is a succinyltransferase that binds to oxoglutarate dehydrogenase to catalyze the conversion of α-ketoglutarate (α-KG) to succinyl-CoA, playing a critical role in various biological processes and in the development of cancers. For example, KAT2A has been shown to promote the progression of pancreatic ductal adenocarcinoma (PDAC) by enhancing glycolysis, cell proliferation, and the migration and invasion of PDAC cells³⁰. Ye et al.³¹ demonstrated that KAT2A-mediated succinylation of Notch1 promotes the proliferation and differentiation of dental pulp stem cells. However, whether KAT2A mediates pyroptosis has not been reported yet. In the present study, we identified for the first time that LPS increased the expression of KAT2A in BV2 cells, an effect that was reversed by TGP. Additionally, overexpression of KAT2A promoted pyroptosis in LPS-induced BV2 cells, counteracting the inhibitory effects of TGP. Furthermore, KAT2A overexpression reversed the improvements in pain behaviors achieved by TGP and increased the levels of inflammatory factors that were otherwise suppressed by TGP in a CINP rat model. These findings revealed the relationship between KAT2A and pyroptosis and suggested a role for KAT2A in CINP.

Additionally, our results demonstrated that overexpression of KAT2A activated the p38 pathway and promoted p38 succinylation at the K295 site in BV2 cells. p38 is a member of the mitogen-activated protein kinase (MAPK) family, which is involved in numerous signaling cascades and plays a crucial role in multiple intracellular activities, including cell differentiation, proliferation, inflammation, and carcinogenesis³². Accumulating evidence suggests that the p38 pathway is activated in microglial cells, thereby mediating the progression of pain, and inhibition of phosphorylated p38 alleviates neuropathic pain³³. Li et al.³⁴ demonstrated that paeonol relieves neuropathic pain by modulating microglial M1 and M2 polarization via the RhoA/p38 signaling pathway. Additionally, several studies reveal that p38 pathway-mediated pyroptosis is involved in many diseases. For instance, Huo et al.³⁵ confirmed that dibutyl phthalate exposure leads to NLRP3-mediated pyroptosis of hepatocytes via the p38MAPK/NF-κB signaling pathway, contributing to liver fibrosis. Wang et al.³⁵ revealed that IL-35 inhibits the p38 pathway to suppress bronchial epithelial cell pyroptosis, thereby reducing asthma injury. In this study, we verified the role of the p38 pathway in pyroptosis and found that metformin restored pyroptosis inhibited by KAT2A knockdown in LPS-induced BV2 cells by suppressing the p38 pathway. Collectively, these findings demonstrated that KAT2A overexpression promoted pyroptosis through activation of the p38 pathway.

In conclusion, this study revealed that TGP improved CINP by suppressing microglial pyroptosis through the inhibition of KAT2A-mediated p38 pathway activation and succinylation. These results may provide a novel and effective therapeutic strategy for the treatment of CINP.

Electronic Supplementary Material

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Author contributions

ST and BM conceived the study; RC and JH conducted the experiments; YZ, YL, JZ, ZP, HY, QW and YC analyzed the data; ST and BM ware major contributors in writing the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by Guizhou Provincial Health Commission Science and Technology Fund Project (gzwkj2022-122).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval and consent to participate

The study was approved by the Ethics Committee of The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. All methods were performed in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.