JMT is composed of twelve botanical drugs which are as follows: Semen Cuscutae, Fructus Ligustri Lucidi, Herba Ecliptae, Herba Prunella Vulgaris, Semen Litchi, Scorpio, Ramulus Cinnamoml, Rhizoma Corydalis, Semen Persicae, Senmen Cassiae, Radix et Rhizoma Asari, and Hirudo at a fixed ratio of 10: 10: 10: 10: 30: 3: 10: 10: 10: 30: 3: 3 (w/w), respectively (Sun et al., 2019). These botanical drugs were purchased from Beijing Tong Ren Tang Pharm Co., Ltd (Beijing, China) and authenticated by Prof. Xiaochun Liang according to the Chinese Pharmacopoeia (2015 Edition). Detailed information and voucher numbers for the botanical drugs are presented in Supplementary Table S1. The voucher specimens (No. JMT-17A-JMT-17L) were submitted to the Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Beijing, China. Alpha-lipoic acid (ALA) was purchased from Shandong Qidu Pharmaceutical Co., Ltd. (1.45mmol/tablet; Zibo City, Shandong, China, Lot No. H20100152) and was used as a positive control.

50 mg of JMT powder was extracted in the mixture of 400 μL methanol and 100 μL water. Ultra-high performance liquid chromatography/mass spectrometry (UPLC/MS) analysis was performed on a 1290 UPLC system (Agilent Technologies, Santa Clara, CA, United States) with an ACQUITY UPLC C18 column (Waters Corp, Milford, MA). The flow rate was 0.4 ml/min and the sample injection volume was 5 μL. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The linear elution gradient program was as follows: 0–3.5 min, 95–85% A; 3.5–6 min, 85–70% A; 6–6.5, 70–70% A; 6.5–12 min, 70–30% A; 12–12.5 min, 30–30% A; 12.5–18 min, 30–0% A; 18–25 min, 0–0% A; 25–26 min, 0–95% A; 26–30 min, 95–95% A. The MS data was acquired by Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, United States) with a mass range of 100–1500 m/z.

Male Sprague-Dawley rats weighing 180–200 g were acquired from Vital River Laboratory Animal Technology (Beijing, China). The diabetic model was induced by a single intraperitoneal injection of streptozotocin (0.21 mmol/kg in citrate buffer; Sigma-Aldrich, St Louis, MO, United States), while control rats received a vehicle control of citrate buffer in equal volume. Blood glucose levels were measured 72 h after STZ administration using the glucometer (Abbott Molecular, Abbott Park, IL, United States). Rats with blood glucose levels ≥16.7 mmol/L were considered diabetic, and control rats with blood glucose levels <7.0 mmol/L were classified as normal control rats.

The rats were randomly divided into four groups (n = 8) as follows: 1) Con (normal control rats that only received distilled water); 2) DM (diabetic rats that also received distilled water); 3) DM + JMT (diabetic rats that received 0.88 g/kg/d JMT, 10-fold the dosage for a human adult); 4) DM + ALA (diabetic rats that received 0.48 mmol/kg/d ALA). We chose the JMT dosage which showed the best neuroprotective effects in STZ-induced diabetic rats based on our previous studies (Song et al., 2019; Song et al., 2021). ALA dosage chosen was based on published reports outlining STZ-induced diabetic rat models (Lee et al., 2018; Sadeghiyan et al., 2019). JMT powder and ALA tablets were suspended in distilled water, mixed thoroughly, and gavaged once daily for 12 weeks. Body weight and blood glucose levels were monitored throughout the treatment period.

After the treatment period ended, rats were anesthetized with sodium pentobarbital (0.24 mmol/kg, i.p.). Blood samples were collected from the carotid arteries, and were centrifuged at 4,000 r/min (10 min) for serum preparation. The serum was stored at -80 °C. The left L4-L6 DRGs were removed and rapidly frozen at -80 °C for molecular detection techniques, and the right side was harvested and fixed in 10% phosphate-buffered formalin for histological examination. The experiments were approved by the Institutional Animal Care and Use Committee of Peking Union Medical College Hospital (No. XHDW-2017–005), and all procedures were performed in accordance with the Principles of Laboratory Animal Care (NIH).

After 12 weeks of treatment, mechanical withdrawal threshold (MWT) in response to mechanical stimuli was used to assess mechanical allodynia using Von Frey filaments (IITC Life Science, Woodland Hills, CA, United States) as described previously (Sun et al., 2019). Rats were individually placed in a clear plastic cage with mesh at the bottom. After a 15-min acclimation period, the hind paw was vertically stimulated by an electronic Von Frey probe, and the force was recorded in grams when the paw was withdrawn. Tail-flick latency (TFL) in response to heat-water was used to evaluate thermal hyperalgesia. The rats were wrapped in a towel and placed on the apparatus. The distal 5 cm of their tails were immersed in a hot-water bath (50 ± 0.5°C) and the tail withdrawal reflex was recorded in seconds (cut-off time 12 s) using a stopwatch (Yadlapalli et al., 2016). These tests were performed at 10 a.m. with three repeat measures for each rat within a 5-min interval.

Serum IL-1β levels were measured, using a commercially available ELISA kit (eBioscience, San Diego, CA, United States), according to the manufacturer’s instructions. Optical density at 450 nm was measured with a plate reader, and sample values were calculated from standard curve analysis.

The DRG tissues were processed, embedded in paraffin, and cut into 4 μm sections for histological analysis. After dewaxing and hydration, sections were stained with hematoxylin and 25% eosin for hematoxylin and eosin (H&E) staining. For Nissl’s staining, sections were stained with toluidine blue solution (Solarbio, Beijing, China) for 30 min at 50°C, followed by 95% ethanol to remove excess staining. Images were scanned and exported using a NanoZoomer Digital Slide Scanner (Hamamatsu Photonics, Hamamatsu, Shizuoka, Japan) at ×400 magnification.

Following dewaxing and hydration, the sections were treated with hot citric acid buffer (pH 6.0) to repair antigens, incubated in 3% H₂O₂ and blocked with 10% goat serum. The sections were then incubated with the following primary antibodies: NLRP3 and IL-1β at 4°C overnight, followed by the appropriate HRP-conjugated secondary antibodies (Zhongshan Golden Bridge Biotechnology, Beijing, China). Detailed information on the antibodies is shown in Supplementary Table S2. After DAB staining, the sections were counterstained with hematoxylin, cleared in xylene, and mounted. Images were captured using a NanoZoomer Digital Slide Scanner (Hamamatsu Photonics, Hamamatsu, Shizuoka, Japan) at ×400 magnification. Quantitative analysis was carried out with NIH ImageJ software.

Total RNA was extracted from DRG tissues using Tissue RNA Purification Kit Plus (ES Science, Shanghai, China) according to the manufacturer’s protocol, and total RNA concentrations were tested using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). Reverse transcription of the RNA (≤1 μg) was performed in a final volume of 20 μL (Absin Bioscience Inc., Shanghai, China). qRT-PCR was performed in duplicate in a Roche LightCycler 480 real-time PCR detection system using SYBR Green (Absin Bioscience Inc., Shanghai, China). The program was run with the following conditions: 2 min at 95°C, followed by 40 cycles of 15 s at 95°C, 25 s at 60°C and 30 s at 72°C. Relative mRNA levels were determined using the 2^−△△CT method. Results were expressed as relative mRNA expression in the treated groups compared to the normal control group. The primers for NLRP3, ASC, caspase-1, IL-1β, GSDMD and β-actin were synthesized by TSINGKE Biological Technology (Beijing, China), and the primer sequences are displayed in Supplementary Table S3.

Total proteins from L4-L6 DRG were extracted with RIPA lysis buffer and determined using a BCA protein assay kit (Beyotime, Shanghai, China). Equal sized 30 μg proteins were separated in 10% SDS-PAGE (Beyotime, Shanghai, China) and transferred to PVDF membranes (Merck Millipore, Billerica, MA, United States). After blocking with 5% fat-free milk, the membranes were incubated with following primary antibodies: NLRP3, caspase-1, IL-1β, GSDMD and β-actin overnight at 4°C. Detailed information on the antibodies is presented in Supplementary Table S2. The corresponding secondary antibodies were probed after washing the membranes. Final results were visualized using an ECL kit (Merck Millipore, Billerica, MA, United States). The relative protein levels were analyzed with NIH ImageJ software.

GraphPad Prism 7.0 software (La Jolla, CA, United States) was used for statistical analysis. Data were presented as means ± standard error of the mean (SEM). The results between multiple groups were compared using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. The correlations between two variables were assessed using Pearson correlation analysis. p < 0.05 was considered statistically significant.

At 12 weeks after STZ injection, blood glucose levels were significantly increased in diabetic rats (Figure 1A), and body weights decreased by about 50% in diabetic rats compared with the Con group (Figures 1B,C). While JMT and ALA treatment had hypoglycemic and weight gaining trends, there was no significant difference in either body weights or blood glucose levels between the DM group and the JMT or ALA-treated DM groups (p > 0.05).

MWT and TFL levels were used to evaluate the effects of JMT on mechanical allodynia and thermal hyperalgesia in diabetic rats. Compared to normal rats, diabetic rats had decreased MWT and TFL levels at 12 weeks after STZ injection. While JMT and ALA treatment partly reversed diabetes-induced decreases in MWT and TFL, there was no significant difference between the JMT and ALA groups (p > 0.05) (Figures 2A,B). In a word, both JMT and ALA alleviated mechanical allodynia and thermal hyperalgesia in diabetic rats, with ALA showing no predominance over JMT.

H&E and Nissl’s staining was used to assess the effects of JMT on DRG morphological injury in diabetic rats. According to H&E staining (Figure 2C), the cells were divided into two groups based on size, appearance, and histochemical reactions. Type A cells were larger and their cytoplasm occupied the entire cell, creating a light background. The nuclei of these neurons were generally lightly stained, with one large and less intensely stained central nucleolus. In contrast, type B cells had darker, more heterogeneously stained cytoplasm than type A. Type B cells were also usually smaller and had more evident vacuolation. Their nuclei were characterized by more basophilic and smaller chromatin condensations (Palanivelu et al., 2018). We found that type B cells were more abundant in the DM group compared to the Con group. Both the JMT and ALT-treated DM groups showed a reduction in Type B cells and an increase in type A cells with a dramatic reduction in vacuolation. Type A cells appeared to be more prevalent in the JMT group than the ALA group.

According to Nissl’s staining (Figure 2C), DRG neurons in the Con group showed clear structures with numerous Nissl’s bodies in the cytoplasm and nucleoli. The nuclei were large and round, and the nucleoli were in the middle and clearly visible. The Nissl bodies in the DRG of diabetic rats broke down and gradually disappeared, and even loss of neurons with vacuolar-like degeneration, the nuclei shifted and the nucleoli blurred, suggesting peripheral nerve damage. The morphological damage and Nissl’s body loss in DRG neurons improved in the JMT and ALA-treated DM groups. However, JMT showed no better effect on the neurological morphology of DRG than ALA.

NLRP3 inflammasome consists of the receptor protein NLRP3, the adaptor protein apoptosis-associated speck-like protein (ASC), and the effector protein caspase-1, which response to caspase-1 activation and IL-1β maturation (Elliott and Sutterwala, 2015). We determined the cellular distribution of NLRP3 in DRG with immunohistochemistry (IHC). We found that NLRP3 was primarily distributed in the cytoplasm of type B DRG neurons, with increased expression in the DM group compared to the Con group. JMT treatment markedly attenuated NLRP3 expression in rats with diabetes, with no significant difference between the JMT and ALA-treated DM groups (p > 0.05) (Figures 3A,B). These findings were consistent with our qRT-PCR and western blot data (Figures 4A, 5A–B, ). We found elevated ASC and caspase-1 mRNA via qRT-PCR, and increased activated caspase-1 protein levels and lower pro-caspase-1 levels in diabetic rats compared to normal rats, indicating that NLRP3 inflammasome was activated and caspase-1 was cleaved in the DNP animal model. These changes were evidently restored by both JMT and ALA treatment, with no differences between the two drug groups (p > 0.05) (Figures 4B,C, 5A, C–D). Together, these data suggest that JMT can reduce NLRP3 inflammasome activation in DRG tissues under chronic hyperglycemic conditions.

GSDMD fragments were cleaved by activated caspase-1, allowing them to form membrane pores, which resulted in pyroptosis and IL-1β secretion and ultimately triggered an inflammatory cascade (Shi et al., 2015; Liu et al., 2016). Given this, we checked GSDMD and IL-1β expression in our animal model. IHC results showed that IL-1β was mainly distributed in the cytoplasm of DRG neurons and in satellite glial cells (SGCs), with increased expression in SGCs than in DRG (Figures 3A,C). Quantitative analysis showed that IL-1β and GSDMD mRNA and protein levels were increased in diabetic rats compared to normal control rats, and that JMT and ALA treatment partly normalized the mRNA and protein levels. However, there was no evident difference between the JMT and ALA groups (p > 0.05) (Figures 4D,E, 5A, 5E,F). Moreover, the maturation and secretion of IL-1β displayed a similar trend compared with GSDMD (Figure 5G).

We found that MWT levels were negatively correlated with NLRP3 (r = −0.675, Figure 6A), and IL-1β (r = −0.672, Figure 6B) expression, and that TFL levels were also negatively correlated with NLRP3 (r = −0.668, Figure 6C), and IL-1β (r = −0.664, Figure 6D) expression. Moreover, we found that NLRP3 expression positively correlated with IL-1β (r = 0.664, Figure 6E) expression. We confirmed these results with qRT-PCR and western bolt analysis, indicating that JMT improved DNP development by decreasing the NLRP3/IL-1β pathway.