Amomum villosum extract alleviates diabetic neuropathy via phosphoinositide 3-kinase/AKT-mediated antioxidative and anti-apoptotic effects

Abstract

BACKGROUND

Diabetic peripheral neuropathy (DPN) affects nearly half of patients with diabetes and is projected to increase substantially as the diabetes prevalence rises globally. Current treatments remain largely symptomatic with limited efficacy in halting disease progression. Amomum villosum Lour. (AVL), a traditional Chinese medicinal herb with anti-inflammatory and antioxidant properties, represents a potential new therapeutic candidate for DPN management.

AIM

To explore the therapeutic effects of the aqueous extract of AVL on DPN in rats and its underlying molecular mechanisms.

METHODS

A type 1 diabetic rat model was induced by streptozotocin. Pain thresholds were assessed using paw withdrawal threshold and paw withdrawal latency. Primary dorsal root ganglion (DRG) neurons were extracted and cultured to detect indicators related to oxidative stress, inflammatory response, and apoptosis. The therapeutic effects of AVL on DPN rats were evaluated using Western blotting, quantitative PCR, enzyme-linked immunosorbent assay, reactive oxygen species (ROS) content detection, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and flow cytometry. The molecular mechanisms of AVL in inhibiting oxidative stress and apoptosis via the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway were also explored.

RESULTS

AVL treatment significantly counteracted the elevated blood glucose and reduced body weight in diabetic rats. The mechanical and thermal pain thresholds were significantly increased, indicating AVL's analgesic effects. In the diabetes mellitus (DM) + AVL group, the expression of inflammatory cytokines [tumor necrosis factor alpha, interleukin 6 (IL-6), IL-1β] and malondialdehyde content in DRG tissue were lower than those in the DM + vehicle group. However, compared with the DM + vehicle group, the glutathione level was increased in the DM + AVL group. Consistently, immunofluorescence showed that the fluorescence intensity representing ROS in primary DRG neurons was also significantly reduced compared with the DM + vehicle group. In addition, flow cytometry and TUNEL assays revealed that AVL treatment markedly decreased the apoptosis rate of DRG neurons, as evidenced by downregulated caspase-3 and B-cell lymphoma 2 (Bcl-2)-associated X protein expression and upregulated Bcl-2 expression, indicating that AVL also inhibits DRG neuronal apoptosis. Further mechanistic studies demonstrated that AVL treatment activated the PI3K/AKT signaling pathway in DRG neurons. However, intervention with the PI3K inhibitor LY294002 significantly reversed the therapeutic effects of AVL on DNP rats. Mechanistically, AVL activated the PI3K/AKT signaling pathway, suppressing oxidative stress and apoptosis in the DRG tissue of DNP rats.

CONCLUSION

AVL alleviates DNP in rats by activating the PI3K/AKT signaling pathway, inhibiting oxidative stress and apoptosis and reducing inflammatory responses. This study provides strong experimental evidence for the application of AVL in DNP treatment and offers new ideas and directions for the development of DNP treatments based on natural medicines.

Key Words: Amomum villosum Lour.; Diabetic peripheral neuropathy; phosphoinositide 3-kinase/AKT; Oxidative stress; Apoptosis

Core Tip: Diabetic peripheral neuropathy (DPN) lacks effective therapies beyond symptomatic relief. This study demonstrates that the aqueous extract of Amomum villosum Lour. (AVL) significantly alleviates DPN in rats by activating the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. AVL reduced oxidative stress, inflammation, and neuronal apoptosis in dorsal root ganglion tissues, thereby improving pain thresholds. Inhibition of PI3K/AKT abrogated these protective effects. These findings highlight AVL as a promising natural medicine with multi-targeted neuroprotective properties and provide mechanistic evidence for its potential application in DPN treatment.

INTRODUCTION

Diabetic peripheral neuropathy (DPN) affects nearly half of patients with diabetes at various stages of the disease course[1,2]. It is anticipated that 1.3 billion people worldwide would have diabetes by 2050, and the incidence and healthcare burden associated with DPN will correspondingly escalate. However, compared to diabetic nephropathy and retinopathy, clinical research and therapeutic development for DPN have historically received less attention[3].

The pathogenesis of DPN is highly complex, involving various metabolic and molecular abnormalities. Chronic hyperglycemia and insulin resistance induce oxidative stress, inflammatory responses, mitochondrial dysfunction, and microvascular damage, collectively promoting nerve injury[4]. Excessive reactive oxygen species (ROS) generated under hyperglycemic conditions cause oxidative stress, resulting in neuronal injury and apoptosis[5-7]. Simultaneously, chronic low-grade inflammation exacerbates neurovascular damage[8]. Oxidative stress and inflammation are considered critical factors in DPN pathogenesis[9-13]. Due to its multifactorial etiology, current therapies remain largely unable to arrest or reverse DPN progression[14]. Analgesics that are frequently used, including anticonvulsants (pregabalin, gabapentin) and antidepressants (duloxetine), only partially relieve symptoms and have side effects like nausea, dizziness, and somnolence[15]. Even widely used antioxidants like alpha-lipoic acid show modest symptomatic relief with uncertain long-term efficacy. Consequently, developing novel therapeutic strategies targeting multiple pathological mechanisms of DPN remains a significant unmet need.

In recent years, natural products have gained considerable attention for DPN management. Furthermore, natural compounds demonstrate higher safety profiles and fewer side effects based on extensive historical clinical use[16]. Herbal extracts rich in flavonoids, phenols, and alkaloids have demonstrated efficacy in reducing oxidative nerve injury and inflammation-mediated neuropathic pain. These findings suggest that natural medicines could offer effective, low-side-effect therapeutic options for DPN.

Amomum villosum Lour. (AVL), a traditional medicinal herb and spice in Chinese medicine, has a long clinical history treating indigestion, rheumatoid arthritis, and neuralgia[17-21]. Modern pharmacological studies have shown that AVL contains flavonoids, phenolic acids, and volatile oils with pronounced anti-inflammatory, antioxidant, and analgesic properties[22-25]. Recent research further isolated antioxidant aromatic compounds and anti-inflammatory active constituents from AVL fruit. Liang et al[24] identified aromatic compounds capable of scavenging free radicals, whereas Xu et al[26] reported significant anti-inflammatory effects from AVL-derived phenolic and diterpene compounds. These multifunctional pharmacological properties highlight AVL’s potential in neuroprotection and analgesia. However, studies investigating AVL’s role specifically in DPN remain superficial, and its precise mechanisms are unclear.

Based on these considerations, we hypothesize that AVL ameliorates DPN pathology by simultaneously suppressing oxidative stress, inflammation, and neuronal apoptosis. Using a type-1 diabetic rat model caused by streptozotocin (STZ) and primary cultured dorsal root ganglion (DRG) neurons in vitro, we systematically evaluated AVL’s impact on pain behavior and neuronal injury markers, focusing particularly on its neuroprotective action. The phosphoinositide 3-kinase (PI3K)/AKT pathway is critical for neuronal survival and metabolism, mediating processes such as axonal growth, myelination, and neuronal survival[27-29]. Diabetes-induced insulin resistance impairs the neurotrophic function of the PI3K/AKT pathway, thereby restricting nerve regeneration and increasing neuronal susceptibility to injury. Hence, activation of the PI3K/AKT pathway could mitigate hyperglycemia-induced neuronal damage. This study elucidates AVL’s therapeutic effects and multi-layered action mechanisms on DPN, providing experimental evidence and novel insights into natural product-based DPN management.

MATERIALS AND METHODS

Experimental animals

We purchased healthy, 6- to 8-week-old male Sprague-Dawley rats (specific pathogen-free grade, 180-220 g) from Hunan Silex Laboratory Animal Co., Ltd. (Permit No. SCXK (Xiang) 2019-0004). This study was approved by the Medical Research Animal Ethics Committee.

Type 1 diabetic rat model

The type 1 diabetic rat model was established according to previously described methods[30]. Before the experiment, the rats were acclimated to the laboratory environment for 1 week. Rats were allocated to groups by the random number table method. The sample size was determined according to the method described by Jin et al[31] (n = 8 per group). Following a 16-hour fast, type 1 diabetes was induced by intraperitoneal injections of STZ (Sigma-Aldrich, St. Louis, MO, United States) at a dose of 60 mg/kg in 0.1 M cold citrate buffer (pH = 4.5) for seven consecutive days. Control rats received an equal volume of sterile saline. To confirm successful diabetes induction, tail vein blood glucose levels were measured weekly using a glucometer. Rats with blood glucose levels exceeding 16.7 mmol/L after STZ injection were confirmed as diabetic. Body weight was also recorded weekly throughout the experiment.

Drug administration and experimental design

For behavioral tests, rats were allocated to five groups (n = 8 per group): Diabetes mellitus (DM) + AVL (low dose, 300 mg/kg/day), DM + AVL (medium dose, 450 mg/kg/day), DM + AVL (high dose, 600 mg/kg/day), DM + vehicle (sterile saline), and control. The control group received an equivalent volume of sterile saline intraperitoneal injection. The extraction of AVL and dose selection were performed according to previously described methods[25,32]. Briefly, dried AVL fruits were extracted with distilled water at 100 °C for 2 hours, filtered, and concentrated under reduced pressure. The extract was analyzed by high-pressure liquid chromatography, and the main bioactive components, including bornyl acetate and borneol, were identified and quantified to ensure batch-to-batch consistency and quality control. The type 1 DM (T1DM) rat model was established in week 1. Starting from week 2, AVL was administered intraperitoneal once daily until the end of the experiment. The total study duration was 5 weeks. For molecular analyses, rats were assigned to three groups: DM + AVL (high dose, 600 mg/kg/day), DM + vehicle, and control. For in vitro experiments, primary DRG neurons were divided into four treatment groups: High glucose (HG) + LY294002, HG + AVL (500 µg/mL), HG + vehicle [sterile phosphate-buffered saline (PBS)], and normal glucose (NG). Controls received an equal volume of sterile PBS. Where indicated, cells were pre-incubated with 0.5 μmol/L LY294002 for 2 hours before exposure to HG (50 mmol/L) plus AVL. The NG group was incubated with 5.5 mmol/L glucose.

Paw withdrawal threshold test

The mechanical sensitivity to stimuli was assessed using von Frey nylon filaments, with the withdrawal response of the paw being observed. Before the test, rats were placed in transparent plastic cages with wire mesh bottoms to acclimate for 30 minutes. The left hind paw was stimulated with von Frey nylon filaments, starting from 1 g and increasing up to 16 g. Stimulation was stopped when the paw bent and the response was maintained for about 6-8 seconds. Paw withdrawal threshold (PWT) was determined by recording the minimum force that caused the paw withdrawal response. Each rat was tested three times at 5-minute intervals, and the average was taken as the final result. Testing was performed once weekly throughout the experiment. All behavioral tests were performed by an experimenter blinded to the experimental groups to ensure objective data collection.

Paw withdrawal latency test

Testing was performed once weekly, on the same day as the PWT test. After the three PWT tests were completed, the paw withdrawal latency (PWL) test was started after 5 minutes. The heat sensitivity was assessed using the hot plate method, with PWL serving as the indicator of thermal pain threshold. The hot plate consisted of a 25 cm × 25 cm metal plate placed inside a glass casing. Each rat was acclimated to the hot plate apparatus for 10 minutes without heating before the test. The hot plate temperature was set at 54 °C, with the timer set to 30 seconds maximum (to avoid tissue damage). The hind paw was gently placed on the hot plate, and the latency of pain threshold was measured by observing the time until the paw shook, withdrew, or licked. The average of three trials was recorded, with a 15-minute interval between each trial.

Extraction and culture of primary DRG neurons

Before the experiment, rats were anesthetized via intraperitoneal injection of pentobarbital sodium at 30 mg/kg, then euthanized by cervical dislocation. The spine was carefully separated to remove the spinal cord, which was then washed in pre-cooled Hank’s balanced salt solution (HBSS). The L4-L5 segments of the DRG were separated using micro forceps and micro scissors and placed in a culture dish containing HBSS. The separated DRG was digested in HBSS containing 0.25% trypsin-EDTA at 37 °C for 15 minutes. After digestion, the process was terminated with Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 culture medium containing 10% fetal bovine serum (FBS). The outer membrane of the DRG was gently peeled off with forceps, and the tissue blocks were transferred to a centrifuge tube containing the culture medium. The tissue blocks were gently pipetted to disperse them into a single-cell suspension. The cell suspension was filtered through a 70 μm cell strainer to remove undissociated tissue blocks. Using a hemocytometer, the cell concentration was adjusted to 1 × 10⁶ cell/mL. The cell suspension was seeded into culture dishes or plates pre-coated with poly-L-lysine, with 100 μL of cell suspension per well, resulting in approximately 10⁵ cells per well. This seeding density has been validated as optimal for primary DRG neuron cultures and is suitable for subsequent Western blot analysis, ROS detection, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays[33]. The cells were cultured in a 37 °C, 5% carbon dioxide incubator with DMEM/F12 culture medium containing 10% FBS and 1% penicillin-streptomycin, and the culture medium was changed every 2 days. The expression of the neuronal-specific marker neuronal nuclei (NeuN) was detected by immunofluorescence staining to confirm cell purity and ensure the extracted cells were primarily DRG neurons (the proportion of NeuN positivity reaches over 95%).

For the DNP group, after the cells were cultured to 70%-80% confluence, they were treated with 50 mmol/L HG for 24 hours to simulate a hyperglycemic environment, while the control group was treated with NG concentration (5.5 mmol/L). AVL (500 μg/mL) was added to specific groups according to experimental needs, and the control group was treated with an equal volume of sterile PBS. The treated cells were used for subsequent experimental analyses.

Western blot analysis

The DRG tissues or cells were lysed with RIPA lysis buffer containing protease and phosphatase inhibitors to obtain equal amounts of cell lysates. These lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% skim milk diluted in Tris-Buffered Saline with Tween-20 (TBST) buffer for 1 hour at room temperature, and then washed three times with TBST. Then they were incubated overnight at 4 °C with specific primary antibodies, including superoxide dismutase 1 (SOD-1) (1:1000, 10269-1-AP; Proteintech, Rosemont, IL, United States), caspase-3 (1:2000, 19677-1-AP; Proteintech), B-cell lymphoma 2 (Bcl-2)-associated X protein (BAX) (1:1000, 50599-2-Ig; Proteintech), Bcl-2 (1:1000, 26593-1-AP; Proteintech), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000, 10494-1-AP; Proteintech). The next day, the membranes were washed three times with TBST and incubated at room temperature for 1 hour with secondary antibodies (1:10000, SA00001-2; Proteintech). After washing three times with TBST, the membranes were developed with enhanced chemiluminescent reagent (PK10001; Proteintech). The band intensity was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, United States) and the protein expression levels were normalized to GAPDH. Each experiment was repeated at least three times to ensure reliability of the results.

Quantitative PCR

Total RNA was extracted from the DRG tissue of rats in each group using Trizol reagent (Invitrogen, Carlsbad, CA, United States). cDNA was synthesized according to the instructions of the PrimeScript RT Master Mix (TaKaRa, Shiga, Japan). SYBR Green qPCR Master Mix was used to detect the abundance of mRNA.

Primer sequences: Tumor necrosis factor alpha (TNF-α): F: 5'-CCACCACGCTCTTCTGTCTAC-3', R: 5'-AGGGTCTGGGCCATAGAACT T-3'. Interleukin 6 (IL-6): F: 5'-TGATGCACTTGCAGAAAACA-3', R: 5'- CCAGAGGAAATTTTCAATAGGC-3'. IL-1β: F: 5'-TGTGAAATGCCACCTTTTGA-3', R: 5'-GGTCAAAGGTTTGGAAGCAG-3'. GAPDH: F: 5'-CCCACTAACATCAAATGGGG-3', R: 5'-CCTTCCACAATGCCAAAGTT-3'. The relative quantitative analysis was performed using the 2^-ΔΔCt method to calculate the mRNA expression levels of the inflammatory factors.

Enzyme-linked immunosorbent assay

Malondialdehyde (MDA) and glutathione (GSH) levels in the DRG tissue were detected using MDA and GSH enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, United States) according to the manufacturer’s instructions.

ROS content detection

After the experiment, the ROS content in primary DRG neurons was detected using a ROS detection kit (CA1410; Solarbio, Beijing, China). Briefly, DRG neurons were incubated with 10 μM DCFH-DA at 37 °C for 10 minutes. The cells were washed three times with PBS to remove unbound dye. The cells were then observed and imaged using an inverted fluorescence microscope. The fluorescence intensity was proportional to the intracellular ROS content. The average fluorescence intensity was measured using ImageJ software to compare differences between groups. Each sample was tested at least three times to ensure reliability of the results.

TUNEL assay

The cells were washed three times with PBS for 5 minutes each. The apoptosis of DRG neurons was detected according to the instructions of the Click-It^TM Plus TUNEL Detection Kit (Thermo Fisher Scientific, Waltham, MA, United States). The nuclei of DRG neurons were labeled with rabbit anti-NeuN antibody (1:1000; Abcam, Cambridge, MA, United States). Neurons with green TUNEL fluorescence signals and positive NeuN were considered apoptotic neurons, and the proportion of apoptotic neurons in all neurons was further calculated.

Flow cytometry detection of apoptosis

Neurons were freshly extracted from the DRG tissue of rats in each group and adjusted to a concentration of 1 × 10⁶ cells/mL in cold PBS. For each sample, 100 μL cell suspension was transferred to a flow cytometry tube, to which 5 μL Annexin V-FITC and 5 μL propidium iodide (PI, V13242; Thermo Fisher Scientific) were added and gently mixed. The samples were incubated at room temperature in the dark for 15 minutes to allow binding of the fluorescent markers to apoptotic and necrotic cells. After incubation, 400 μL of binding buffer was added to each sample to optimize the detection conditions. Apoptosis rate was measured using a flow cytometer (BD FACSCanto II; BD Biosciences, Franklin Lakes, NJ, United States), with at least 10000 events collected per sample. The proportion of apoptotic cells (Annexin V-FITC positive/PI-negative for early apoptosis and Annexin V-FITC positive/PI-positive for late apoptosis) was analyzed using FlowJo software (version 10.6.1; Ashland, OR, United States). Results are presented as the percentage of total apoptotic cells (early plus late apoptosis).

Statistical analyses

Graphing and statistical analyses were performed using GraphPad Prism 9.5.0. All data are presented as the mean ± SD. For repeated measurements of blood glucose level, body weight, PWT and PWL, two-way repeated-measures analysis of variance (ANOVA) followed by Tukey’s post-hoc test was applied. For other datasets involving three or more groups, one-way ANOVA was used. When homogeneity of variance was met, Tukey’s post-hoc test was conducted; otherwise, Welch’s ANOVA followed by Dunnett’s T3 post-hoc test was employed. P < 0.05 was considered statistically significant.

RESULTS

AVL treatment counteracts hyperglycemia and weight loss in diabetic rats

The fasting blood glucose levels were measured using blood samples obtained from the tail vein 72 hours following the intraperitoneal injection of STZ. The results showed that in the first week after STZ induction, the fasting blood glucose levels of rats in the model group (DM + vehicle group) exceeded 16.67 mmol/L (Figure 1A), and the body weight gradually decreased (Figure 1B). This indicated that the STZ-induced DM (T1DM) rat model was successfully established. Compared with the DM + vehicle group, the rate of increase in blood glucose levels in rats treated with AVL was significantly reduced (Figure 1A), and the body weight was significantly increased (Figure 1B). From week 3 onward, the rats' fasting blood glucose declined progressively with time, whereas their body weight showed the opposite trend. This therapeutic effect of AVL was dose-dependent. The improvements in the DM + AVL (600 mg/kg) group were significantly greater than those in the DM + AVL (300 mg/kg) and DM + AVL (450 mg/kg) groups. This suggests that AVL can alleviate the hyperglycemia in STZ-induced diabetic rats and promote weight gain.

Figure 1 Amomum villosum Lour. treatment counteracted hyperglycemia and weight loss in diabetic rats. A type 1 diabetic rat model was established by intraperitoneal injection of 60 mg/kg streptozotocin for seven consecutive days in week 1, and the control group was injected with an equal volume of sterile saline. Starting from week 2, Amomum villosum Lour. (AVL) was administered intraperitoneally once daily until the end of the experiment. The total study duration was 5 weeks. Blood glucose and body weight were recorded regularly. A: Blood glucose level; B: Rat body weight. ^aP < 0.05 diabetes mellitus (DM) + vehicle group vs control group.

AVL treatment increases mechanical and thermal pain thresholds in diabetic rats

In diabetic mice, nerve damage can lead to changes in pain perception thresholds and sensitivity. After STZ induction, considerable allodynia was indicated by the considerable reduction in PWT and PWL (Figure 2A and B). From week 3 onward, AVL treatment reversed the decline in both PWT and PWL, and the elevation was significantly greater in the DM + AVL (600 mg/kg) group than in the DM + AVL (300 mg/kg) and DM + AVL (450 mg/kg) groups as the experiment progressed. Therefore, we chose the high dose (600 mg/kg/day) of AVL for subsequent experiments.

Figure 2 Diabetic rats treated with Amomum villosum Lour. had higher mechanical and thermal pain thresholds. A: Mechanical sensitivity of rats was assessed by stimulating with von Frey nylon filaments, with the withdrawal response of the paw being observed; B: Thermal sensitivity was assessed using the hot plate method, with paw withdrawal latency serving as the indicator of thermal pain threshold. ^aP < 0.05 diabetes mellitus (DM) + vehicle group vs control group. AVL: Amomum villosum Lour.

AVL treatment reduces inflammation and oxidative stress in DRG neurons of diabetic rats

Inflammatory reactions are made worse by disturbances in oxidative stress homeostasis[33-35]. The DRG tissue of the DM group had higher mRNA levels of inflammatory factors, including TNF-α, IL-6, and IL-1β, according to quantitative PCR data (Figure 3A-C). To assess the effect of AVL on oxidative stress, the levels of MDA and GSH in the DRG were detected. The DRG of the DM group showed a considerable increase in biochemical levels of MDA and a decrease in GSH. Following treatment with AVL, this effect was reversed (Figure 3D and E). The DM group's DRG tissue showed lower SOD-1 protein levels compared to the control group (Figure 3F). To further investigate the role of AVL in the oxidative stress of neurons under diabetic conditions, primary DRG neurons were treated with 50 mmol/L HG to simulate the in vivo hyperglycemic environment. Compared with the group treated with NG concentration (5.5 mmol/L), HG treatment led to the accumulation of ROS in DRG neurons, along with decreased SOD-1 levels (Figure 3G), which complemented the observations made in vivo (Figure 3D-F). Even under HG conditions, SOD-1 protein levels were increased by AVL intervention. In summary, these findings indicate that AVL can reduce inflammation and oxidative stress levels in DRG neurons under chronic hyperglycemic conditions.

Figure 3 Amomum villosum Lour. treatment reduced inflammation and oxidative stress in the dorsal root ganglion neurons of diabetic rats. The type 1 diabetes mellitus (DM) rat model was established in week 1. Starting from week 2, Amomum villosum Lour. (AVL; 600 mg/kg/day) was administered intraperitoneally once daily until the end of the experiment. The total study duration was 5 weeks. A-C: MRNA expression levels of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-1β in dorsal root ganglion (DRG) tissue were analyzed by quantitative PCR; D and E: Levels of malondialdehyde (MDA) and glutathione (GSH) in DRG tissue were detected by the enzyme-linked immunosorbent assay; F: Protein levels of superoxide dismutase 1 (SOD-1) in DRG tissue were analyzed by Western blotting; G: For 24 hours, normal glucose (NG; 5.5 mmol/L) and high glucose (HG; 50 mmol/L) were administered as pretreatments to primary DRG neurons, and AVL treatment was added in the last 12 hours. The protein levels of SOD-1 in primary DRG neurons were detected by Western blotting. ^aP < 0.05.

AVL treatment reduces the apoptosis of DRG neurons induced by hyperglycemia in vivo and in vitro

Hyperglycemia-induced apoptosis of sensory neurons in the DRG is a typical pathological change in the DM group, and excessive activation of oxidative stress also promotes the occurrence of neuronal apoptosis. AVL treatment significantly inhibited apoptosis of DRG neurons (Figure 4A). By contrast, Bcl-2 protein expression levels increased in the AVL-treated group, whereas caspase-3 and BAX expression levels decreased (Figure 4B). In addition, AVL effectively alleviated the apoptosis of neurons induced by HG in vitro, as evidenced by the decreased proportion of TUNEL-positive neurons (Figure 4C). The above results indicate that AVL treatment can effectively reduce the apoptosis of DRG neurons in the STZ-induced diabetic rat model.

Figure 4 Amomum villosum Lour. treatment reduced the apoptosis of dorsal root ganglion neurons induced by hyperglycemia in vivo and in vitro. A: Apoptosis levels in rat dorsal root ganglion (DRG) tissue were analyzed by flow cytometry; B: Protein expression levels of caspase-3, B-cell lymphoma 2 (Bcl-2)-associated X protein (BAX), and Bcl-2 in rat DRG tissue were detected by Western blotting; C: Primary DRG neurons were pre-treated with 5.5 mmol/L and 50 mmol/L glucose for 24 hours, and Amomum villosum Lour. (AVL) treatment was added in the last 12 hours. Primary cultured DRG neurons were labeled with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) (green) and nuclei were labeled with neuronal nuclei (NeuN) (blue) on the left, and the right figure shows the percentage of healthy neurons (non-apoptotic). ^aP < 0.05. DM: Diabetes mellitus; HG: High glucose; NG: Normal glucose; PI: Propidium Iodide.

AVL treatment inhibits oxidative stress and apoptosis in DRG of diabetic rats

Our study further explored the molecular mechanisms of AVL in improving STZ-induced diabetes. Consequently, we observed alterations in the PI3K/AKT signaling pathway in rats with diabetes. PI3K [phosphorylated PI3K (p-PI3K)] and AKT (p-AKT) phosphorylated protein expression was markedly lower in the DM group, whereas their expression was higher in the AVL treatment group (Figure 5A). The p-PI3K and p-AKT protein levels in primary DRG neurons treated with HG were higher in the HG + AVL group than in the HG group (Figure 5B). In contrast to the HG + AVL therapy group, we discovered that the accumulation of ROS in primary DRG neurons increased, and the proportion of apoptotic cells increased in the HG + AVL + LY294002 intervention group (Figure 5C and D).

Figure 5 Amomum villosum Lour. treatment inhibited oxidative stress and apoptosis in the dorsal root ganglion of diabetic rats by activating the phosphoinositide 3-kinase/AKT signaling pathway. A: Protein levels of phosphorylated phosphoinositide 3-kinase (p-PI3K) and phosphorylated AKT (p-AKT) in dorsal root ganglion (DRG) were analyzed by Western blotting; B: Protein levels of p-PI3K and p-AKT in primary DRG neurons were analyzed by Western blotting; C: Primary DRG neurons were pre-treated with 0.5 μM LY294002 for 2 hours, and then incubated with high glucose (HG) and Amomum villosum Lour. (AVL). The reactive oxygen species levels in primary DRG neurons were analyzed by immunofluorescence; D: Apoptosis proportion of primary DRG neurons was analyzed by flow cytometry. ^aP < 0.05; DM: Diabetes mellitus; NG: Normal glucose; NS: Not significant; PI: Propidium iodide.

DISCUSSION

DPN is challenging to treat. Current treatments mostly provide symptomatic relief with frequent side effects. Due to their multi-target advantages, natural products have emerged as a focal point in research. This study shows that AVL extract activates the PI3K/AKT signaling pathway. It reduces oxidative stress damage in DRG tissue of diabetic rats, lowers pro-inflammatory cytokine expression, and inhibits neuronal apoptosis. These effects are closely related to the regulation of Bcl-2 family of proteins.

The PI3K/AKT pathway regulates cellular resistance to stress and cell survival, promoting AKT phosphorylation and modulating apoptotic factors[36-39]. Activated AKT suppresses pro-apoptotic factors (e.g., glycogen synthase kinase-3 beta) and enhances anti-apoptotic proteins (e.g., Bcl-2), reducing neuronal apoptosis. AKT activation also enhances antioxidant defenses, reducing ROS accumulation. In the peripheral nervous system, AKT signaling is crucial for Schwann cell differentiation, myelination, and axonal regeneration. Conversely, diabetes-induced insulin signaling impairment decreases PI3K/AKT activity, weakening neuronal resistance to damage, contributing significantly to DPN pathogenesis[40]. In this study, p-PI3K and p-AKT levels were notably lower in diabetic DRG tissues compared to controls, whereas AVL treatment restored the expression. Importantly, inhibiting PI3K signaling with LY294002 in vitro reversed AVL’s antioxidative and anti-apoptotic effects, confirming that AVL acts via PI3K/AKT activation. Based on previous reports, AVL triggers the PI3K/AKT cascade and appears to relieve DPN damage through two linked arms. First, the pathway can transcriptionally boost the antioxidant master nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream enzymes (heme oxygenase-1, SOD), cutting off ROS generation at the source. We did not directly measure Nrf2 activation or nuclear translocation, yet our data showed that AVL lowered oxidative stress in diabetic DRG via PI3K/AKT. Most importantly, AVL extract exerts anti-inflammatory effects by suppressing the nuclear factor kappa B/Nrf2/NLR family pyrin domain containing 3 axis in lipopolysaccharide-stimulated macrophages[26]. Future studies should apply Nrf2-specific inhibitors or track Nrf2 nuclear import to confirm whether AVL acts through a PI3K/AKT-Nrf2 module. Second, AKT can raise pro-survival Bcl-2 and shut down the mitochondrial apoptotic program. In line with this, AVL markedly increased Bcl-2 and decreased caspase-3 and BAX, consistent with PI3K/AKT activation (Figure 6).

Figure 6 Schematic diagram illustrating Amomum villosum Lour.’s mechanism in alleviating diabetic peripheral neuropathy. Under hyperglycemic/diabetic conditions, excessive reactive oxygen species (ROS) generation induces oxidative stress, triggering neuronal apoptosis and subsequently leading to neuronal injury and sensory dysfunction (red arrows). Amomum villosum Lour. (AVL) activates the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, thereby upregulating nuclear factor erythroid 2-related factor 2-mediated antioxidant defense and cell survival factors like B-cell lymphoma 2 (Bcl-2). This action inhibits ROS accumulation and neuronal apoptosis, alleviating diabetes-induced neural damage (green arrows).

The findings of this study are consistent with recent trends investigating mechanisms of natural product interventions in DPN. Numerous plant-derived natural compounds have similarly demonstrated potential in improving DPN through combined antioxidative and anti-inflammatory effects[40]. Likewise, daidzein, a soybean isoflavone, reportedly improves PN and enhances sensory nerve conduction in diabetic rats[41]. It should be noted that our assessment of the DPN phenotype was based primarily on functional and molecular read-outs. In future work, it will be particularly interesting to determine how AVL affects sciatic-nerve conduction velocity, myelin architecture, and intra-epidermal nerve-fiber density in DNP rats. The traditional Chinese medicine compound, Qiying granules, demonstrate preclinical efficacy by inhibiting endoplasmic reticulum stress and neuronal apoptosis[12], suggesting that multi-component herbal formulations can simultaneously target multiple pathways in DPN pathogenesis. Another noteworthy mechanism involves autophagy regulation. Gypenosides derived from Gynostemma pentaphyllum have been shown to alleviate DPN by inhibiting thioredoxin-interacting protein, thus activating the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway and enhancing neuronal autophagy[36]. This observation aligns with our hypothesis that AVL’s activation of the PI3K/AKT pathway may subsequently influence downstream mTOR signaling and autophagy. Regarding antioxidant and anti-apoptotic actions, a recent study reported that arctigenin, a lignan isolated from Arctium lappa, ameliorated nerve dysfunction in STZ-induced diabetic mice by mitigating oxidative damage, reducing neuronal apoptosis, and promoting autophagy[42]. The action of arctigenin closely resembles our findings, further reinforcing the critical importance of combined antioxidative and anti-apoptotic strategies in managing DPN.

Moreover, although not plant-derived, emerging physical and nutritional interventions also target oxidative stress. For example, low-intensity pulsed ultrasound reportedly alleviated diabetic neuropathy in mice through antioxidative mechanisms[9], and probiotic supplementation reduced oxidative stress and alleviated neuropathic pain in rat models of sciatic nerve injury[34]. Collectively, these studies underscore that multi-targeted intervention strategies, especially those focusing on reducing oxidative stress and inflammation while inhibiting neuronal apoptosis, have become prominent research directions in DPN treatment over the past 5 years[43]. Compared to conventional single-target drugs, natural products often concurrently modulate multiple pathological processes, resulting in synergistic therapeutic outcomes. The mechanism identified in our study, demonstrating that AVL's activation of the PI3K/AKT pathway leads to inhibition of oxidative damage and neuronal apoptosis, provides additional evidence supporting this therapeutic concept.

However, there were also some limitations. First, the study period was short. The 6-week experimental cycle was not sufficient to fully evaluate the long-term efficacy and potential side effects of AVL. We also only chose male mouse models for the experiment, and we failed to consider the differences in the incidence and pathogenesis of the disease between males and females in the real world. The impact of sex differences on the results needs to be thoroughly analyzed by researchers in future studies. In addition, the STZ-induced type 1 diabetes rat model used in this study differs from human DPN (which is more common in type 2 diabetes) in terms of pathological mechanisms and clinical manifestations, limiting the clinical relevance of the research results. At the same time, AVL contains complex components, and the specific active components and their synergistic or antagonistic effects are still unclear. Importantly, although this study preliminarily indicates that the PI3K/AKT pathway is involved in the protective effect of AVL, as evidenced by pharmacological inhibitor experiments, genetic-level functional verification (such as gene knockout or overexpression studies) and direct detection of downstream effector molecules such as Nrf2 were not performed due to laboratory constraints. Therefore, the precise causal relationship between AVL and this pathway, as well as its complete downstream network, still require confirmation through subsequent studies. Future research can focus on evaluating the efficacy and safety of AVL in the treatment of human DPN to more comprehensively assess the application value of AVL aqueous extract in the treatment of DPN. Finally, it is worth noting that AVL lowers blood glucose and increases body weight, which may indirectly improve pain thresholds in DPN rats. However, AVL has also been observed to directly alleviate apoptosis and oxidative stress in DRG neurons. Whether the beneficial effects of AVL on DPN result from direct neuroprotection or from secondary improvements in systemic metabolism remains an important question for future investigation.

CONCLUSION

This study experimentally investigated the therapeutic effects and molecular mechanisms of AVL extract on DPN. The results demonstrated that the extract significantly ameliorated mechanical and thermal hyperalgesia in diabetic rats, suppressed apoptosis of DRG neurons, and attenuated oxidative stress within DRG tissue. These protective effects were closely linked to activation of the PI3K/AKT signaling pathway. The findings provide theoretical support for multi-target therapeutic strategies against DPN and open a new avenue for applying natural medicines in neuropathy management. Given the favorable safety profile of AVL extract, this work offers an important reference for future development of natural-product-based DPN therapies.