Gastrodin alleviates bortezomib-induced peripheral neuropathy by inhibiting NF-κB/NLRP3 pathway-mediated microglial inflammation

Abstract

Background

Multiple chemotherapeutic agents exhibit neurotoxicity. For example, bortezomib (BTZ)-induced peripheral neuropathy (BIPN) is characterized by sensory abnormalities and pain, and effective treatment strategies are currently lacking. This study aimed to investigate the alleviating effects of gastrodin (GAS) on BIPN and its potential mechanisms.

Methods

Behavioral tests were used to assess changes in pain thresholds across all groups of mice. Hematoxylin-eosin staining, transmission electron microscopy, and immunofluorescence were used to evaluate peripheral nerve injury and the activation of spinal glial cells. ELISA was used to measure the levels of inflammatory cytokines. Proteins associated with the NF-κB/NLRP3 pathway were examined using Western blot.

Results

GAS significantly improved thermal and mechanical hyperalgesia in BIPN mice. Moreover, BTZ can cause loss of intraepidermal nerve fibers, microstructural damage to the dorsal root ganglia, a disorganized arrangement of sciatic nerve fibers, and demyelination, all of which were effectively reversed by GAS treatment. Further investigation revealed that GAS significantly suppressed the upregulation of IBA-1 and GFAP in the spinal cord of BIPN mice, and the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were concurrently reduced. IBA-1/IL-1β and IBA-1/TNF-α double-labeled positive cells were significantly increased in BIPN mice, and GAS intervention reduced the number of these double-labeled cells. In addition, GAS significantly inhibited aberrant NF-κB signaling and upregulation of NLRP3 inflammasome-related proteins of BIPN mice.

Conclusions

GAS may alleviate BIPN by suppressing microglia-mediated neuroinflammatory responses, and the NF-κB/NLRP3 inflammasome signaling pathway appears to be involved in this process.

INTRODUCTION

Multiple myeloma (MM) is a relatively common hematologic malignancy that often involves multiple organ systems, has a high mortality rate, and tends to recur frequently [1]. Bortezomib (BTZ), a proteasome inhibitor, serves as the drug of choice for first-line induction therapy in MM and has markedly improved patient survival [2]. However, BTZ exhibits considerable neurotoxicity, damaging nerve fibers and inducing peripheral neuropathy through multiple pathways [3]. bortezomib-induced peripheral neuropathy (BIPN) primarily manifests as pain, numbness, and a burning sensation in the distal extremities, with an incidence reaching 40%–60% [4]. Currently, there are no effective preventive or therapeutic measures for BIPN. Consequently, some BIPN-affected patients are forced to modify treatment regimens, reduce dosages, or discontinue therapy prematurely, significantly compromising the survival benefits of anticancer treatment [5]. Therefore, investigating the pathogenesis of BIPN and developing effective therapeutic strategies is clinically imperative.

Peripheral nerve injuries often cause chronic neuropathic pain, which is characterized by significant proliferation of spinal glial cells. The neuroinflammatory response is a major contributor to both the initiation and maintenance of neuropathic pain [6]. Although BTZ cannot directly target the central nervous system, evidence indicates that its accumulation in the dorsal root ganglia (DRG) induces central glial activation, glutamate metabolic imbalance, and inflammatory responses [3,7]. Previous studies have demonstrated that multiple pro-inflammatory cytokines exhibit significant upregulation in the spinal cord of mice after BTZ treatment [8]. However, their origin remains unclear. A study has shown that 1 week after BTZ treatment, the NF-κB pathway is activated in DRG neurons of mice, accompanied by peak mRNA expression of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 [9]. Notably, NF-κB promotes NLRP3 expression through transcriptional activation. NLRP3 interacts with caspase-1 via the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) to assemble the inflammasome, thereby facilitating pro-inflammatory cytokine release and amplifying the inflammatory response [10]. Furthermore, BTZ induces STAT3-dependent histone acetylation-mediated NLRP3 upregulation in DRG. This pathological alteration correlates with mechanical allodynia development [11]. During peripheral nerve injury or inflammatory conditions, sensory neurons in the DRG upregulate colony-stimulating factor 1 (CSF1), which binds to CSF1 receptors on spinal dorsal horn microglia, thereby promoting their proliferation and activation [12]. It is important to note that spinal microglia are closely associated with inflammatory responses [13], and their role in BIPN warrants further attention.

As the resident immune cells of the central nervous system, microglia are critically involved in chronic pain and nerve injury [14]. Upon activation, microglia secrete various inflammatory mediators and pain-modulating substances, causing alterations in the glial microenvironment and resulting in neurotoxicity, which may be associated with the occurrence of central sensitization in chronic pain [15]. Among the critical pathways of inflammation, microglial NLRP3 inflammasome promotes IL-1β maturation via caspase-1 activation and recognizes danger-associated molecular patterns in response to different types of stimuli [16]. It has been shown that IL-1β produced by microglia is neurotoxic to motor and sensory neurons and closely linked to the onset of various neurological disorders [17]. BTZ has been reported to induce microglial activation [18]; however, its precise role in BIPN remains unclear, especially since interventions targeting the NF-κB/NLRP3 pathway are still limited.

Compounds derived from traditional Chinese medicine exhibit a favorable safety profile and do not interfere with the antitumor activity of chemotherapeutic agents [19]. However, the literature on employing traditional Chinese medicine to mitigate or prevent BIPN is relatively sparse. Gastrodin (GAS), an active molecule from Gastrodia elata rhizomes, possesses anti-inflammatory and antitumor properties and exhibits neuroprotective effects [20]. Increasing evidence suggests that GAS has the potential to treat neuropathic pain. GAS has been reported to effectively treat chronic inflammatory pain in mice, with dose-dependent elevations in mechanical and thermal nociceptive thresholds [21]. Recently, GAS was shown to alleviate migraine-like pain, which provides supporting evidence for its analgesic effects [22]. In a lipopolysaccharide-induced inflammation model, microglial activation was significantly inhibited by GAS, an effect closely associated with inhibition of NLRP3 inflammasome assembly [23]. Another study demonstrated that GAS can inhibit NF-κB signaling, thereby exerting neuroprotective effects [24]. However, the therapeutic potential of GAS in BIPN has not yet been reported.

This study aims to use an animal model to investigate whether GAS can alleviate BIPN by reducing glia-mediated neuroinflammation, and to examine whether the NF-κB/NLRP3 pathway is involved in this process.

MATERIALS AND METHODS

1. Animals and grouping

Because the modeling period was relatively long, this study used only wild-type male C57BL/6 mice (8–10 weeks old) to minimize the potential effects of hormonal fluctuations associated with the estrous cycle in females. The results and conclusions of this study are therefore applicable only to male mice. Experimental animals were obtained from Hunan SJA Laboratory Animal Company, maintained under specific-pathogen-free conditions at Zunyi Medical University, and all procedures were approved by the Ethics Committee of The Affiliated Hospital of Zunyi Medical University (License No: SYXK [Qian] 2021-0004). The experimental groups were as follows: the BIPN model group (BTZ), the BIPN model with GAS treatment group (BTZ + GAS), the GAS-only treatment group (GAS), and the control group (control).

2. Model establishment

To establish the BIPN model, mice in the BTZ group were given BTZ (Qilu Pharmaceutical Co., Ltd.) via tail vein injection (0.8 mg/kg, twice weekly) over a period of 4 weeks [25]. In the BTZ + GAS group, GAS (purity ≥ 98%, Solarbio Science & Technology Co., Ltd.) was administered intraperitoneally (100 mg/kg, once daily) during the same 4-week period [26]. Animals in the GAS group were treated only with intraperitoneal GAS at the identical dosage used in the BTZ + GAS group. The control animals were injected via the tail vein with saline in volumes equivalent to those used for BTZ administration. Successful BIPN model establishment was defined by a significant reduction in behavioral pain thresholds relative to baseline values in mice.

3. Observation of body weight changes

With the first dose administration defined as Day 1 (D1), mouse body weights were measured at baseline (D0) and subsequently at predetermined intervals up to D28 during the dosing period, while mortality was monitored across all groups.

4. Behavioral tests

Von Frey filaments (Aesthesio; Danmic Global) were applied to the central plantar surface of the mice to assess mechanical thresholds using the ascending stimulus method, starting at 0.008 g. Each filament was bent to 1 cm and held for 3–5 seconds to observe nociceptive responses such as paw licking or withdrawal. If no response occurred, the stimulus intensity was gradually increased until a response was elicited. Each test was repeated 5 times, and if reactions were observed in ≥ 3 trials, the corresponding filament force (g) was recorded as the mechanical threshold. For thermal testing, the radiant heat intensity of the thermal pain analyzer (IITC Life Science Inc.) was set to 40 arbitrary units (AU), with a maximum exposure time of 20 seconds. The central plantar area was irradiated, and upon observing paw licking or withdrawal, the light source was immediately turned off, and the response time was recorded. Each mouse was tested 6 times (3 times per hind paw), with a 5-minute interval between each measurement, and the mean values were considered for both the mechanical and thermal pain thresholds [21].

5. Transmission electron microscopy

Transmission electron microscopy (TEM) was used to evaluate ultrastructural damage in the DRG and sciatic nerves. Following anesthesia, phosphate-buffered saline (PBS) was transcardially perfused through the mice, followed by 4% paraformaldehyde. The study collected sciatic nerve and dorsal root ganglion tissues and immediately fixed them in glutaraldehyde. Subsequently, the authors fixed the samples with osmium tetroxide, dehydrated them through a graded acetone series, and embedded them in resin to prepare ultrathin sections. The sections underwent staining with uranyl acetate and lead citrate. Images were acquired using a transmission electron microscope (JEM-1400FLASH; JEOL Ltd.) at magnifications of ×2,000, ×10,000, and ×20,000. For each mouse, three fields containing approximately 40–60 myelinated axons in total were randomly selected, and the G-ratio of each axon was measured by an investigator blinded to the experimental groups. The sciatic nerve G-ratio is described as the proportion of axonal diameter relative to the full diameter of the myelinated fiber [27].

6. Histopathological evaluation

Histopathological observation and evaluation of the mouse sciatic nerve were performed using hematoxylin and eosin (H&E) staining. Fresh sciatic nerve tissues were immersed in 4% paraformaldehyde, processed through dehydration, and embedded in paraffin. Following 1 hour of dewaxing, the sections underwent rehydration, hematoxylin staining (10 minutes), subsequent differentiation, and counterstaining with eosin (2 minutes). Afterward, they were dehydrated in graded ethanol and sealed with neutral resin. Microscopic images were obtained using a light microscope (BA210Digital; Motic). Histological scoring was performed by an investigator blinded to the experimental groups using a previously established peripheral nerve inflammation scale [28].

7. Immunofluorescence staining

Immunofluorescence staining was performed to examine glial activation in the spinal cord and to quantify intraepidermal nerve fiber density (IENFD). Tissue samples, including L4–L6 regions of the spinal cord and plantar skin from the right hind paw, were collected from mice, dehydrated in 30% sucrose after fixation, and then embedded in optimal cutting temperature and sectioned using a cryostat (CM1950; Leica). After PBS washing and blocking with 1% bovine serum albumin for 2 hours, primary antibodies were then applied individually and incubated overnight at 4°C: IBA-1 (019-19741, 1:500; Wako), GFAP (D1F4Q, 1:500; Cell Signaling), PGP9.5 (ab108986, 1:500; Abcam), IL-1β (B122, 1:250; Santa Cruz), and TNF-α (ab1793, 1:250; Abcam). After washing with PBS the next day, the anti-rabbit (A11008, 1:300; ThermoFisher) and anti-mouse (A-11012, 1:300; ThermoFisher) IgG secondary antibodies were dropped onto the sections and kept at room temperature in the dark for 2 hours. Following PBS washes, the researchers captured images with a fluorescence microscope (BX61WI; Olympus). Fluorescence intensity was quantified using ImageJ software (version 1.53; National Institutes of Health). To assess the expression of IBA-1 and GFAP, standardized regions of interest (ROIs) were outlined in images of the spinal dorsal horn, and the percentage of fluorescence-positive area within each ROI was calculated in ImageJ. The total number of cells in each ROI was quantified using ImageJ, while double-positive cells (appearing as yellow in merged images) were manually identified and counted by a laboratory technician blinded to the experimental design. All experiments were performed with six independent biological replicates per group. The analysis of IENFD was performed according to a previous study [29].

Quantitative analysis of fluorescence intensity and area was performed using ImageJ software (version 1.53). For each image, the same exposure settings were applied during acquisition, and identical threshold values were set across all groups to ensure comparability. The fluorescence area percentage was calculated as the ratio of the immunopositive area to the total area of the spinal dorsal horn ROI.

8. ELISA detection of inflammatory cytokines in the spinal dorsal horn

ELISA kits (Jonlnbio) were used to measure the levels of inflammatory cytokines, including IL-1β, TNF-α, IL-10, and IL-6. Cytokine levels were normalized to total protein content determined by the bicinchoninic acid (BCA) assay. Each well received 100 μL of standards and samples, and the plates were maintained at 37°C for 1 hour. After washing, biotinylated antibodies were added and incubated for 1 hour. Following washing, enzyme conjugates were added, and the colorimetric reaction was developed and stopped with a stop solution. Optical density values were then measured.

9. Western blot

The expression levels of key proteins in the NF-κB/NLRP3 inflammasome signaling pathway were analyzed using western blotting. Spinal cord tissues were lysed in RIPA buffer containing phenylmethylsulfonyl fluoride, homogenized by sonication, and centrifuged to collect the supernatant. Protein concentrations were determined using the BCA assay. Equal amounts of protein were subjected to electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking at room temperature for 2 hour, membranes were incubated overnight at 4°C with primary antibodies: p-NF-κB p65 (Ser536, 1:1000; Signalway), NLRP3 (SC06-23, 1:1500; HUABIO), ASC (HY-P80548; MCE), NF-κB p65 (L8F6, 1:500; Cell Signaling), caspase-1 (SU40-07, 1:80,000; HUABIO), cleaved-caspase-1 (Asp296, 1:1000; Affinity), and GAPDH (60004-1-Ig, 1:50000; Proteintech). On the following day, membranes were washed and exposed to secondary antibodies (SA00001-1; SA00001-2, 1:5000; Proteintech) for 1 hour at room temperature, and signals were subsequently detected using ECL chemiluminescent solution (BMU102; Abbkine). The band intensities were quantified using ImageJ software, and the expression levels of target proteins were normalized to GAPDH. For phosphorylated proteins, the ratios of phosphorylated to total protein were calculated. All experiments were performed with three independent biological replicates per group.

10. Statistical analysis

Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, Inc.). All measurement data are presented as the mean ± standard error of the mean. The Shapiro–Wilk test was used to assess data normality. Unless otherwise specified, all datasets in this study followed a normal distribution. One-way analysis of variance (ANOVA) was applied for multiple-group comparisons, and unpaired t-tests were used for two-group comparisons. Behavioral data involving repeated measurements were analyzed using two-way repeated-measures ANOVA. Tukey’s post hoc test was conducted for multiple comparisons between groups. P value < 0.05 was considered statistically significant.

RESULTS

1. GAS improves BTZ-induced hyperalgesia

A reduction in mechanical and thermal pain thresholds following BTZ administration indicated successful establishment of the BIPN model, and the monitoring time points are shown in Fig. 1. Before drug administration, there were no differences in thermal or mechanical pain thresholds among the groups of mice. Starting from day 14, the thermal and mechanical pain thresholds were significantly reduced in the BTZ group (mean difference [MD]: –2.68, 95% confidence interval [CI]: –3.93 to –1.43, P < 0.001; MD: –0.31, 95% CI: –0.48 to –0.13, P < 0.001 vs. control). The BTZ + GAS group showed an upward trend in thermal and mechanical pain thresholds, with a statistically significant difference observed on day 21 (MD: 1.63, 95% CI: 0.63 to 2.64, P = 0.001; MD: 0.20, 95% CI: 0.10 to 0.30, P < 0.001 vs. BTZ group) (Fig. 2A, B). Meanwhile, body weight was recorded at each time point of BTZ administration. Before drug administration, body weight did not differ among the groups. From day 15 onward, the body weight of the BTZ group decreased significantly (MD: –2.44, 95% CI: –4.47 to –0.41, P = 0.016 vs. control). In contrast, the body weight of the BTZ + GAS group increased significantly after day 25 (MD: 1.78, 95% CI: 0.50 to 3.06, P = 0.005 vs. BTZ group) (Fig. 2C). Additionally, no obvious changes were observed in the thermal and mechanical pain thresholds or body weight of the GAS group. Overall, treatment with GAS increased the thermal and mechanical pain thresholds and prevented body-weight loss in BIPN mice.

Fig. 1.

Time points of drug administration and behavioral testing. Red arrows indicate BTZ injection days, and blue squares indicate behavioral testing days (mechanical and thermal thresholds). BTZ: bortezomib, GAS: gastrodin.

Fig. 2.

Pain intensity evaluation and body weight changes in each group of mice. (A) Changes in thermal pain thresholds in each group of mice. (B) Changes in mechanical pain thresholds in each group of mice. (C) Changes in body weight in each group of mice. Data are presented as mean ± standard deviation. BTZ: bortezomib, GAS: gastrodin. BTZ group vs. Control group: *P < 0.05, **P < 0.01, ***P < 0.001; BTZ + GAS group vs. BTZ group: #P < 0.05, ##P < 0.01, ###P < 0.001, n = 10 per group.

2. GAS alleviates BTZ-induced peripheral nerve injury

On Day 28, tissue specimens were harvested from mice in each group for subsequent experimental analysis. Fluorescence staining of skin nerve fibers showed that BTZ significantly reduced the density of nerve fibers (MD: –7.17, 95% CI: –12.16 to –2.17, P = 0.004 vs. control), while GAS intervention attenuated the loss of nerve fibers (MD: 5.00, 95% CI: 0.01 to 9.99, P < 0.050 vs. BTZ group) (Fig. 3A, F). The ultrastructural changes in the DRG and sciatic nerves were examined using TEM to evaluate the model and verify the intervention effects of GAS. The results showed that BTZ induced mitochondrial swelling with vacuolation in neurons and satellite glial cells of the DRG; in the sciatic nerves, the myelin lamellae were loosely arranged, with demyelination observed in some areas. GAS intervention markedly alleviated the aforementioned pathological changes, as indicated by the mitochondrial morphology in neurons and satellite glial cells tending toward normal, with only occasional vacuolation and relatively intact and clearly defined cristae; in the sciatic nerve, only localized areas exhibited mild loosening of the myelin lamellae, and demyelination was reduced (Fig. 3B–D). Additionally, H&E staining of the sciatic nerve revealed disorganized nerve fiber arrangement and abnormal Schwann cell distribution in the BTZ group. After GAS intervention, only mild swelling of a few nerve fibers was observed, with relatively regular arrangement and reduced inflammatory cell infiltration (Fig. 3E). To quantify the extent of pathological changes, G-ratio and nerve histological scores were assessed (see MATERIALS AND METHODS). The BTZ group exhibited a higher G-ratio (0.72 ± 0.06) and sciatic nerve histological score (2.50 ± 0.55), whereas both indices were significantly reduced in the BTZ + GAS group (G-ratio: 0.56 ± 0.05; histological score: 1.50 ± 0.55) (Fig. 3G, H). In addition, the results also indicated that administration of GAS alone at 100 mg/kg did not cause peripheral nerve damage.

Fig. 3.

GAS alleviates BTZ-induced peripheral nerve injury. (A) Immunostaining images of nerve fibers in the plantar skin of mice (red arrows indicate intraepidermal nerve fibers), scale bar = 50 μm. (B) Representative electron microscopy image of DRG neurons, scale bar = 2 μm, n = 3 per group. Red arrows indicate mitochondrial swelling with vacuolation. (C) Representative electron microscopy image of DRG satellite glial cells, scale bar = 1 μm, n = 3 per group. Red arrows indicate mitochondrial swelling with vacuolation. (D) Image of the sciatic nerve under TEM, scale bar = 1 μm and 2 μm. (E) H&E-stained images of sciatic nerve fibers (×400). White arrows indicate loose and disorganized sciatic nerve fibers with axonal deformation. (F) IENFD analysis, n = 6 per group. (G) G-ratio of sciatic nerve samples in each group was measured under TEM, n = 3 per group. (H) Sciatic nerve injury scores of mice in each group, n = 6 per group. Data are presented as mean ± standard deviation. BTZ: bortezomib, GAS: gastrodin, IENFD: intraepidermal nerve fiber density. *P < 0.05, **P < 0.01, ***P < 0.001.

3. GAS inhibits BTZ-induced glial activation and associated inflammatory responses

To further clarify whether spinal glial cells were involved in the analgesic and neuroprotective effects of GAS in BIPN mice, the expression of IBA-1 and GFAP were assessed by immunofluorescence (Fig. 4A, B). The results showed that the BTZ group exhibited significantly higher fluorescence area ratios of IBA-1+ and GFAP+ (MD: 2.02, 95% CI: 1.60 to 2.44; MD: 3.77, 95% CI: 2.96 to 4.58, P < 0.001 vs. control). Following GAS intervention, the fluorescence area ratios of both markers were significantly reduced (MD: –0.68, 95% CI: –1.10 to –0.26, P < 0.001; MD: –2.30, 95% CI: –3.11 to –1.49, P < 0.001 vs. BTZ group). In addition, no significant differences were observed between the GAS group and the control group (Fig. 4C, D). Glia-mediated inflammatory responses are associated with neural injury and neuropathic pain. Subsequently, ELISA was used to detect the expression of inflammation-related factors of the spinal cord. The results showed that BTZ significantly increased the expression levels of IL-1β, TNF-α, and IL-6 (MD: 55.35, 95% CI: 38.82 to 71.88; MD: 239.70, 95% CI: 121.70 to 357.80; MD: 23.33, 95% CI: 16.34 to 30.31, P < 0.001 vs. control), while IL-10 levels tended to decline but did not reach statistical significance (MD: –10.35, 95% CI: –28.65 to 7.95, P = 0.375 vs. control). Following GAS intervention, these proinflammatory cytokines significantly decreased (MD: –49.27, 95% CI: –65.79 to –32.74, P < 0.001 vs. BTZ group for IL-1β; MD: –387.90, 95% CI: –505.90 to –269.80, P < 0.001 vs. BTZ group for TNF-α; MD: –12.21, 95% CI: –19.20 to –5.23, P = 0.001 vs. BTZ group for IL-6), whereas IL-10 expression was significantly upregulated (MD: 23.40, 95% CI: 5.11 to 41.70, P = 0.012 vs. BTZ group) (Fig. 4E–H).

Fig. 4.

GAS alleviates BTZ-induced glial inflammatory responses. (A) Fluorescence-stained images of IBA-1 in the spinal dorsal horn of mice from each group, scale bar = 100 µm. (B) Fluorescence-stained images of GFAP in the spinal dorsal horn of mice from each group, scale bar = 100 µm. (C, D) Comparison of IBA-1+ and GFAP+ fluorescence proportion in the spinal dorsal horn of mice in each group, n = 6 per group. (E–H) Expression levels of TNF-α, IL-6, IL-1β, and IL-10 in the spinal dorsal horn of mice in each group, n = 4 per group. Data are presented as mean ± standard deviation. BTZ: bortezomib, GAS: gastrodin, DH: dorsal horn, TNF: tumor necrosis factor, IL: interleukin. *P < 0.05, **P < 0.01, ***P < 0.001, ns: P > 0.05.

4. GAS alleviates BIPN-associated neuroinflammation by inhibiting spinal microglial activation

The authors subsequently performed immunofluorescence co-localization analysis to clarify the source of inflammatory factors. The results indicated that IL-1β and TNF-α were primarily co-localized with IBA-1 rather than GFAP (Fig. 5A–D). Furthermore, BTZ significantly elevated the proportion of IBA-1/IL-1β and IBA-1/TNF-α double-positive cells (MD: 38.72, 95% CI: 22.03 to 55.41; MD: 22.79, 95% CI: 14.82 to 30.75, P < 0.001 vs. control). Following GAS treatment, a significant reduction in the proportion of double-positive cells was observed (MD: –19.96, 95% CI: –36.65 to –3.27, P = 0.016 vs. BTZ group for IBA-1+ + IL-1β+; MD: –10.96, 95% CI: –18.93 to –2.99, P = 0.005 vs. BTZ group for IBA-1+ + TNF-α+) (Fig. 5E, F). This result expands the authors’ previous finding that the ameliorative effect of GAS on BIPN may be related to the suppression of microglial inflammation.

Fig. 5.

GAS alleviates BIPN-associated neuroinflammation by inhibiting spinal microglial activation. (A) Immunofluorescence co-staining images of IBA-1 and GFAP (green) with IL-1β (red), scale bar = 100 µm. (B) Immunofluorescence co-staining images of IBA-1 and GFAP (green) with TNF-α (red), scale bar = 100 µm. (C, D) Statistical analysis of the co-localization proportion of IBA-1 and GFAP with TNF-α and IL-1β in mice of each group, n = 6 per group. (E, F) Co-localization analysis of IBA-1 with IL-1β and TNF-α in the spinal dorsal horn of mice in each group, n = 6 per group. Data are presented as mean ± standard deviation. BTZ: bortezomib, GAS: gastrodin, TNF: tumor necrosis factor, IL: interleukin. *P < 0.05, **P < 0.01, ***P < 0.001.

5. GAS alleviates BIPN by modulating the NF-κB/NLRP3 signaling pathway

To further elucidate the mechanism through which GAS alleviates BIPN, western blot analysis was employed to detect changes in protein expression. The results indicated a significant increase in p-NF-κB p65, NLRP3, ASC, and cleaved caspase-1 expression in BIPN mice (MD: 0.51, 95% CI: 0.18 to 0.85, P = 0.005 vs. control for p-NF-κB p65/NF-κB p65; MD: 0.30, 95% CI: 0.07 to 0.51, P = 0.010 vs. control for NLRP3/GAPDH; MD: 0.27, 95% CI: 0.05 to 0.48, P = 0.017 vs. control for ASC/GAPDH; MD: 0.42, 95% CI: 0.13 to 0.71, P = 0.007 vs. control for cleaved-caspase-1/pro-caspase-1). The relative expression levels of the above proteins were significantly decreased following GAS treatment (MD: –0.35, 95% CI: –0.69 to –0.02, P = 0.038 vs. BTZ group for p-NF-κB p65/NF-κB p65; MD: –0.27, 95% CI: –0.49 to –0.05, P = 0.017 vs. BTZ group for NLRP3/GAPDH; MD: –0.33, 95% CI: –0.55 to –0.12, P = 0.005 vs. BTZ group for ASC/GAPDH; MD: –0.38, 95% CI: –0.67 to –0.09, P = 0.013 vs. BTZ group for cleaved-caspase-1/pro-caspase-1). Moreover, administration of GAS alone at 100 mg/kg did not alter the expression of the above proteins (Fig. 6). These findings suggest that the NF-κB/NLRP3 inflammasome pathway may be involved in the neuroprotective effect of GAS against BTZ-induced neuroinflammation.

Fig. 6.

NF-κB/NLRP3 pathway-associated protein expression in each group of mice. (A) Protein bands of NLRP3, NF-κB p65, p-NF-κB p65, pro-caspase-1, cleaved-caspase-1, ASC, and GAPDH in the spinal dorsal horn of mice in each group. (B–E) Bar graphs showing the relative expression levels of p-NF-κB p65, NLRP3, ASC, and cleaved caspase-1 proteins in the spinal dorsal horn of mice from each group, n = 3 per group. Data are presented as mean ± standard deviation. BTZ: bortezomib, GAS: gastrodin, ASC: apoptosis-associated speck-like protein containing a CARD. *P < 0.05, **P < 0.01.

DISCUSSION

The primary clinical manifestations of BIPN are spontaneous pain and sensory abnormalities attributable to neuroinflammation [30]. The results of this study demonstrated that BIPN mice developed hyperalgesia, indicating the successful establishment of the model [25]. Approximately 60% of mice exhibited reductions in mechanical and thermal pain thresholds following BTZ administration, whereas GAS treatment significantly attenuated the reductions in pain thresholds. Furthermore, it was observed that GAS mitigated the loss of plantar skin nerve fibers in BIPN mice, an effect that may be related to the attenuation of hyperalgesia [31]. Studies have shown that BTZ readily accumulates in DRG neurons, thereby directly exposing neuronal cells to neurotoxic injury [7,32]. The authors observed that BTZ caused abnormal changes in the cellular morphology and structure of mouse DRG neurons, with numerous swollen mitochondria seen in both neurons and satellite cells. These ultrastructural alterations in the DRG were partially reversed after GAS intervention. This morphological improvement implies a possible mitochondrial-stabilizing effect that may help preserve cellular energy homeostasis and prevent secondary inflammatory cascades. Meregalli et al. [33] previously found that BTZ treatment led to slowed sciatic nerve conduction velocity in mice, suggesting that the neuropathy induced by BTZ may involve axonal injury. Sun et al. [34] recently reported that the structure of the sciatic nerve was disorganized and damaged in BIPN mice. The authors further observed a reduction in overall myelin sheath thickness in the sciatic nerves of mice after BTZ administration, along with disorganized nerve fiber alignment and inflammatory cell infiltration. GAS treatment attenuated sciatic nerve demyelination and promoted orderly nerve fiber arrangement. These findings indicate that GAS effectively attenuates BTZ-induced hyperalgesia and peripheral nerve injury, thereby exerting a neuroprotective effect in BIPN.

Peripheral injury signals are conveyed via the DRG to the spinal dorsal horn, where activated glial cells trigger the production of inflammatory mediators involved in pain transmission and central sensitization [35]. It has been reported that chemotherapy-induced peripheral neuropathy is often accompanied by reactive changes in spinal glial cells. For instance, in a preclinical model of vincristine-induced neuropathy, spinal glial cells were markedly activated, together with upregulation of TNF-α, MCP-1, and CCL2 [36]. A recent study reported that BTZ can induce abnormal activation and increased numbers of microglia in the mouse brain, but spinal glial responses were not clarified [18]. The results of this study showed that BIPN mice exhibited upregulated expression of IBA-1 and GFAP in the spinal dorsal horn, accompanied by an increase in cell body size. This result is partially consistent with previous reports [37,38]. In other neurological disease models, the neuroprotective mechanism of GAS involves glial cells [24,39]. It was confirmed that abnormal glial cell reactivity within the spinal dorsal horn of BIPN mice is suppressed by GAS. Abnormal glial cell activation can trigger neuroinflammatory responses [40]. The results of the present study demonstrated a significant upregulation of IL-1β, IL-6, and TNF-α in the spinal dorsal horn of BIPN mice, whereas GAS suppressed the expression of these cytokines and markedly enhanced IL-10 expression. Microglia–neuron interactions are known to contribute to central sensitization through the release of cytokines such as IL-1β and TNF-α, which enhance neuronal excitability [41]. Subsequently, the authors further identified that the spinal neuroinflammatory response associated with BTZ was primarily mediated by microglia. BTZ treatment markedly increased IBA-1/IL-1β and IBA-1/TNF-α double-positive cells, whereas GAS significantly reduced their numbers. These findings suggest that the reduction of these mediators following GAS treatment may indirectly modulate microglia–neuron communication and attenuate pain transmission.

Peripheral nerve diseases often lead to neuropathic pain, and the NLRP3 inflammasome is thought to participate in this process [42]. The results of the present study revealed that phosphorylated NF-κB p65 and critical components of the NLRP3 inflammasome were significantly upregulated in the spinal dorsal horn of BIPN mice, whereas these proteins were significantly downregulated following GAS treatment. A previous study reported that vincristine-induced peripheral neuropathy is driven by NLRP3 activation [43], which is consistent with the findings of this study. Another study demonstrated that the alleviating effect of GAS on vincristine-induced peripheral neuropathy may be associated with the suppression of inflammatory responses mediated by the CX3CL1/CX3CR1–p38 MAPK signaling pathway [44]. These findings indicate that, depending on the specific type of chemotherapy-induced peripheral neuropathy model, GAS may act on multiple neuroinflammatory targets. Notably, reactive oxygen species generation and oxidative stress are key triggers of NLRP3 inflammasome activation, and these mechanisms may also participate in the protective effects of GAS against BIPN [10].

From a clinical perspective, GAS has been investigated in humans for several neurological disorders and has generally demonstrated good tolerability in both randomized controlled and observational studies, supporting its feasibility for further translational development [45]. In the authors’ animal experiments, mice treated with GAS at 100 mg/kg showed no apparent adverse effects. However, the safety profile in MM patients receiving BTZ remains to be determined. Disease-related immunometabolic alterations and chemotherapy-induced organ vulnerability may influence the pharmacokinetics, pharmacodynamic response, and overall tolerability of GAS in this population.

In summary, this study found that GAS can suppress reactive changes of microglia in the spinal dorsal horn of BIPN mice, leading to reduced neuroinflammation and thereby effectively alleviating BTZ-induced neurological injury. The underlying mechanism may involve modulation of the NF-κB/NLRP3 pathway. These results offer new perspectives on BIPN pathogenesis and indicate that GAS has potential as a therapeutic agent for its treatment.

This study has several limitations. Firstly, the authors did not employ causal interventions such as pharmacologic inhibition or genetic knockdown/depletion of microglial or NF-κB/NLRP3 signaling components. Secondly, the observation period was limited to a 4-week dosing window, without post-treatment washout or long-term follow-up to determine the durability or reversibility of the effects. Thirdly, only male C57BL/6 mice were included, which precludes assessment of potential sex or strain-dependent differences. Finally, although these findings demonstrate the neuroprotective effect of GAS against BIPN under otherwise normal physiological conditions, they do not fully recapitulate the complex tumor–host interactions, systemic inflammation, or metabolic alterations that occur in MM models or in patients receiving BTZ therapy.