Redox and bioenergetic modulation by plumbagin attenuated neuropathic allodynia in mice with diet-induced diabetes

Youya Fan; Yangui Wang; Wugeng Cui; Benling Li; Xin Zhao

doi:10.1038/s41387-026-00450-w

. 2026 Jul 7;16:42. doi: 10.1038/s41387-026-00450-w

Redox and bioenergetic modulation by plumbagin attenuated neuropathic allodynia in mice with diet-induced diabetes

Youya Fan ^1,^#, Yangui Wang ^2,^#, Wugeng Cui ¹, Benling Li ³, Xin Zhao ^1,^✉

PMCID: PMC13624258 PMID: 42414251

Abstract

Background

Painful diabetic neuropathy represents a substantial health burden, afflicting approximately 30% of individuals with diabetes. Currently available neuropathic pain medications exhibit only limited efficacy, are associated with significant adverse effects, and often result in poor patient adherence. The need for more effective neuropathic pain treatments in diabetic patients remains critical. This study aimed to evaluate the potential pain-relieving effects of plumbagin, a naturally occurring naphthoquinone, in a mouse model of persistent neuropathic pain associated with diet-induced type 2 diabetes.

Methods

Male C57BL/6 J mice were fed a high-fat diet for eight weeks to induce type 2 diabetes. Pain-like behaviors were assessed using the von Frey test for mechanical allodynia and the acetone test for cold allodynia. The underlying mechanisms related to redox status and bioenergetics were explored using both in vivo and in vitro approaches.

Results

Chronic administration of plumbagin not only alleviated established mechanical and cold allodynia in mice with type 2 diabetes but also delayed the onset of neuropathic pain under a preventive regimen at lower doses. While plumbagin did not impact on metabolic characteristics of diabetic mice, it corrected mitochondrial bioenergetic deficits in dorsal root ganglion neurons, alleviated oxidative stress in pain-related tissues, and enhanced both sciatic nerve conduction velocity and blood flow. Importantly, the analgesic effects of plumbagin were found to be modulated through pharmacological manipulation of mitochondrial bioenergetic function and redox balance.

Conclusions

These findings demonstrate the analgesic activity of plumbagin in mice with diet-induced type 2 diabetes, an effect that is mechanistically linked to its capacity to enhance mitochondrial bioenergetics and attenuate oxidative stress.

Subject terms: Preclinical research, Diseases of the nervous system

Introduction

Diabetic neuropathy represents a predominant complication of diabetes mellitus, with painful diabetic neuropathy (PDN) in the extremities being a particularly debilitating manifestation [1]. Affecting approximately 30% of diabetic patients, this painful condition substantially diminishes quality of life, increases economic burden, and impairs social functioning [2]. Given the rising global prevalence of type 2 diabetes, the incidence of PDN is anticipated to increase correspondingly [3]. The pathogenesis of this neuropathic pain condition involves disruption of the peripheral and central somatosensory nervous systems, which in turn induces hypersensitivity of primary afferent nociceptors. The resultant clinical phenotype is typically characterized by a triad of symptoms: allodynia (pain arising from normally innocuous stimulation), hyperalgesia (enhanced pain responsiveness to noxious stimuli), and spontaneous pain in the absence of an external trigger. Although the underlying mechanisms remain incompletely understood, preclinical evidence strongly implicates oxidative stress and mitochondrial dysfunction as critical pathogenic drivers [4, 5]. Accordingly, compounds that positively modulate mitochondrial function and mitigate oxidative injury hold therapeutic promise for the prevention and treatment of persistent neuropathic pain [6–9].

Current therapeutic strategy for PDN differs fundamentally from that of acute nociceptive pain, relying not on direct nociceptive suppression but on repurposed centrally acting agents from antidepressant and antiepileptic classes [10]. These include tricyclic antidepressants (e.g., amitriptyline), serotonin-noradrenaline reuptake inhibitors (e.g., duloxetine), and α2δ-1 voltage-gated calcium channel blockers (e.g., gabapentin). Notwithstanding their widespread utilization in clinical practice, these therapies are frequently associated with central adverse effects, the development of analgesic tolerance, risk of addiction, and modest overall efficacy-factors that collectively limit their therapeutic benefits [11]. Moreover, these agents provide only symptomatic relief, as they neither address the underlying etiology nor arrest disease progression. Consequently, there is an unprecedented demand for alternative neuropathic pain therapies that offer a more favorable efficacy-to-risk profile. Natural products and herbal medicine-derived compounds represent a promising reservoir of novel therapeutic candidates.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, chemical structure shown in Fig. 1A) is a natural yellow pigment found in the plants of the Plumbaginaceae, Droseraceae, Ancistrocladaceae, and Dioncophyllaceae families [12]. It has been documented that plumbagin possesses a plethora of pharmacological and potential therapeutic activities, including anti-inflammatory, anti-tumor, anti-bacterial, hepatoprotective and neuroprotective properties [13–17], among others. Although scattered evidence seems to show the pain-alleviating activities of plumbagin [18], it remains unknown whether it is capable of quenching diabetic neuropathic pain. Of particular relevance, previous studies have demonstrated antidepressant-like effects of plumbagin in experimental animals [19, 20], lending credibility to the present investigation into its potential efficacy in reducing diabetic neuropathic pain.

The present work was thus undertaken with two main objectives: first, to evaluate the pain-alleviating efficacy of plumbagin in a mouse model of painful diabetic neuropathy resulting from high-fat diet (HFD)-induced type 2 diabetes; and second, to explore the underlying mechanisms, with a particular focus on its mitochondrion-protective and antioxidant potentials. This mechanistic focus is predicated on the established roles of mitochondrial dysfunction and oxidative stress as key pathogenic drivers in the development of PDN [4, 5], and reports showing the antioxidant and mitochondrial-protective properties of plumbagin [13, 14, 20, 21].

Methods

Animals

All animal experimentation was performed using male C57BL/6 J mice obtained at six weeks of age from the Laboratory Animal Center of the Chinese Academy of Sciences. Upon arrival, animals were housed in groups of five to six per cage under specific pathogen-free conditions, maintained on a 12-hour light/dark cycle (lights on at 07:00), with food and water available ad libitum. The housing environment was strictly controlled, with ambient temperature maintained at 22 ± 0.5 °C, relative humidity at 50 ± 2%, and ventilation at 15 air changes per hour. To minimize experimental bias, mice were randomly allocated to treatment groups prior to the initiation of experiments, and all outcome assessments were performed by personnel blinded to group assignment. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Ningbo University (Approval No. 11164) and carried out in strict accordance with the guidelines set forth in the European Communities Council Directive (86/609/EEC). Daily monitoring of animal welfare was conducted by qualified veterinary staff, and every effort was made to minimize animal suffering and the total number used.

Induction of type 2 diabetes

The induction of insulin resistance via a high-fat diet (HFD) is a widely employed and validated method for establishing animal models of type 2 diabetes. In the present study, beginning at 7 weeks of age, mice designated for the diabetic group were fed a HFD comprising 60 kcal% fat (Wuxi Fanbo Biotechnology, China), whereas control animals were maintained on a regular diet (RD) consisting of 10 kcal% fat. A range of metabolic parameters (including body weight, blood glucose, food intake, glucose tolerance, and insulin levels) were monitored on a weekly basis commencing 4–5 weeks after the initiation of the dietary intervention. For the intraperitoneal glucose tolerance test (GTT), a glucose bolus of 2 g/kg body weight was administered via i.p. injection. Blood glucose concentrations were determined from tail vein blood samples collected immediately before glucose injection (baseline) and at 15, 30, 60, and 120 min thereafter. The threshold for diagnosing diabetes was predefined as a blood glucose level exceeding two standard deviations (SD) above the mean value of the RD-fed control group. Only those HFD-fed animals that surpassed this established criterion (representing 87.6% of the original HFD cohort) were considered diabetic and selected for inclusion in subsequent experimental procedures.

Drugs

The drugs utilized in this study included plumbagin, tert-butyl hydroperoxide (t-BOOH; an ROS donor), phenyl-N-tert-butylnitrone (PBN; an ROS scavenger), oligomycin (OMC), and coenzyme Q10 (CoQ10). Plumbagin (purity: 99.3%) was obtained from ChemFaces, while all other compounds were purchased from Sigma-Aldrich.

Experimental design and pharmacological treatment

Experiment 1: Delaying effect of plumbagin on the development of diabetic neuropathic pain. The experimental timeline for the preventive regimen of plumbagin (administered orally via gavage at a volume of 10 mL/kg) is depicted in Fig. 1A. At 5 weeks following the initiation of high-fat diet (HFD) feeding, mice (including both healthy control and diabetic groups) commenced a three-week course of once-daily oral treatment with either plumbagin or vehicle, spanning from day 35 to day 55 post-HFD initiation. Behavioral testing for pain-like phenotypes was performed on designated days, with all assessments conducted 2 h prior to the scheduled administration of plumbagin or vehicle (i.e., at 9:00 AM), to minimize any acute drug effects on behavioral readouts. Following the cessation of treatment on day 55, behavioral monitoring continued in all groups to assess the lastingness of any pharmacological effects.

Experiment 2: Therapeutic effect of plumbagin on diabetic neuropathic pain. As illustrated in Fig. 2A, therapeutic administration of plumbagin (oral gavage; 10 ml/kg) commenced eight weeks following HFD initiation. At this time point, diabetic mice had developed stable pain hypersensitivity, characterized by significant mechanical and cold allodynia. In the chronic treatment protocol, plumbagin was administered once daily (at 11:00 AM) for two weeks (i.e., from day 56 to day 69) to both healthy control and HFD-diabetic mice. The nociceptive behaviors were measured 2 h prior to plumbagin or vehicle administration (at 9:00 AM) as indicated. Following the 2-week treatment, the mechanism of action was probed via pharmacological manipulations. Healthy control or HFD-diabetic mice were co-administered (i.p., 10 ml/kg) with an agent targeting either mitochondrial bioenergetics (OMC or CoQ10) or redox status (t-BOOH or PBN). Doses of OMC, CoQ10, PBN and t-BOOH were initially chosen based on previous studies [22–24], with tiny adjustments made to confirm the absence of intrinsic effects upon pain-related behaviors in healthy control animals. The co-administration regimen varied according to the agent being tested. For repetitive co-administered with plumbagin, OMC and CoQ10 were injected intraperitoneally (10 ml/kg) once daily for five days (from day 69 to day 73), administered 30 min after plumbagin. For single co-administration with plumbagin, PBN and t-BOOH were injected intraperitoneally (10 ml/kg) 30 min after plumbagin administration on day 69.

Behavioral Tests

Mechanical allodynia was quantified using the von Frey test, while thermal (cold) allodynia was assessed via the acetone test. Beginning two weeks prior to behavioral testing, animals underwent acclimatization sessions lasting 30 min every other day. On any designated test day, mice were further habituated to the testing environment for at least 20 min immediately preceding the onset of testing. For evaluation of the acute analgesic effects of plumbagin, nociceptive responses to mechanical and cold stimuli were recorded at 1, 2, 3, and 4 h following a single administration of plumbagin on day 56. For chronic plumbagin treatment (both prophylactic and therapeutic dosing schedules), behavioral tests were conducted 2 h prior to plumbagin administration every 2–3 days throughout the full treatment duration (as illustrated in Fig. 1A and Fig. 2A), in accordance with protocols established in previous studies [25, 26].

For the von Frey test, animals were individually placed in transparent acrylic enclosures on an elevated wire-mesh floor and allowed to acclimate for 20 min. A graded series of calibrated von Frey monofilaments (Stoelting, USA), ranging from 0.07 g to 8 g of incremental bending force, was applied perpendicularly to the plantar surface of the hind paw. Each filament was pressed until it buckled, ensuring a consistent force application. Beginning with the 0.07 g filament, stimuli were delivered in ascending order. A positive response was defined as a brisk, immediate paw withdrawal. For each filament, five applications were performed, and the mechanical withdrawal threshold was determined as the minimum force that elicited a positive response in at least three of the five trials [25]. Results are presented as mechanical threshold (g) or percentages of the maximum possible effect (%MPE), and the %MPE is calculated as (G1 – G0) × 100 / (G2 – G0), where G0 is the minimum of the paw-mechanical threshold in the diabetes-vehicle cohort, G1 is the respective test value, and G2 is the maximum of the paw-mechanical threshold in the control (non-diabetes) cohort.

For the acetone test, cold-evoked nociception was assessed as previously delineated [27]. Mice were placed in acrylic chambers on an elevated wire-mesh floor. A 20-μl droplet of acetone was gently applied to the plantar surface of the hind paw to induce evaporative cooling. Nociceptive behaviors (including licking, shaking, and lifting of the stimulated hind paw) were timed over a 1 min observation period immediately following application. The results from three assays (at least a 5 min interval) were collected and quantified as data, which are presented as duration of nociception (in seconds) or percentages of the maximum possible effect (%MPE). The %MPE is calculated as (D1 – D0) × 100 / (D2 – D0), where D0 is the maximum of the nociception time in the diabetes-vehicle cohort, D1 is the respective test value, and D2 is the minimum of the nociception time in the control (non-diabetes) cohort.

Biochemical assays

Following a 2-week regimen of plumbagin or vehicle treatment (day 69), mice were euthanized by rapid decapitation. The L4–L5 dorsal root ganglia (DRG), the corresponding spinal cord segments, and the sciatic nerve (proximal to its trifurcation) were swiftly harvested on an ice-chilled glass plate. All tissue samples were stored at

-70°C pending biochemical analysis.

To measure lipid peroxidation, sciatic nerve, DRG, and spinal cord tissues were homogenized in 1.35% KCl (1:6, w/v) containing 1.5 mM phenylmethylsulphonyl fluoride (PMSF). The homogenate was centrifuged at 1600 × g for 15 min, and the supernatant was collected. After adding 15% trichloroacetic acid, the mixture was centrifuged at 1500 × g for 3 min. The resulting supernatant was reacted with 0.7% thiobarbituric acid (TBA), and samples and standards were heated at 100 °C for 15 min. Malondialdehyde (MDA) was quantified by absorbance at 535 nm. Results were normalized to protein concentration and expressed as µM/mg protein.

To measure catalase activity, the tissues (sciatic nerve, DRG, and spinal cord) were homogenized in 0.25 M sucrose (1:10, w/v). The homogenate was centrifuged at 1000 × g for 15 min to remove debris; the supernatant was then centrifuged at 12,000 × g for 15 min at 4 °C. Catalase activity was assayed by monitoring the decrease in absorbance at 245 nm using 50 µl supernatant in a 3 ml reaction mixture containing 0.05 M phosphate buffer and 1.6 M H₂O₂. Results were expressed as H₂O₂ decomposed per mg protein per minute.

Hydrogen peroxide was quantified based on its stoichiometric oxidation of Fe²⁺ to Fe³⁺, which complexes with xylenol orange to yield a colored product measured by absorbance. The tissues (sciatic nerve, DRG, and spinal cord) were homogenized in 500 mM phosphate buffer containing 10 mM sodium azide, centrifuged (15,000 × g, 10 min, 4°C), and the supernatant collected. A 100 μl reaction solution was added to 50 μl supernatant and incubated for 20 min at room temperature. Absorbance was read at 560 nm, and H₂O₂ concentration was calculated from a standard curve.

Mitochondrial complex I activity was assessed as rotenone-sensitive NADH–cytochrome c reductase activity, and complex IV activity as cytochrome c oxidase activity. Krebs cycle enzyme activity was evaluated by measuring citrate synthase activity. All assays were performed on the same DRG homogenates described above, using a temperature-controlled visible spectrophotometer (Ultrospec 2100 UV) running Biochrom Swift II software (Biopharmacia Biotech), as previously reported [28].

Measurement of nerve conduction velocity

Sensory nerve conduction velocity (SNCV) and motor nerve conduction velocity (MNCV) were assessed in mice anesthetized with diazepam (2.5 mg/kg) and pentobarbital (12.5 mg/kg) in 0.9% saline. Core body temperature was maintained at 37 °C throughout the procedure. The sciatic nerve was surgically exposed and stimulated with needle electrodes positioned at the sciatic notch and the Achilles tendon (1-6 V, 0.05 ms). Electromyographic (EMG) signals were recorded, and SNCV and MNCV were calculated by dividing the distance between the two stimulation sites by the peak-to-peak latency of the H-wave and M-wave, respectively. All recordings were performed in triplicate, and the median value for each animal was used for analysis.

Measurement of sciatic nerve blood flow

Nerve blood flow (NBF) was measured using LASER Doppler system (Perimed, Järfälla, Sweden). The sciatic nerve was surgically exposed and carefully separated from adjacent tissues. The laser probe was then placed in direct contact with an avascular region of the nerve trunk, avoiding any visible epineurial and perineurial vessels. Blood flux was continuously recorded from the same site for 15 min, and values were expressed in arbitrary perfusion units (PU).

Sensory neuron isolation and culture

DRGs from healthy control and diabetic mice were dissociated as previously described [29]. Briefly, L4-L5 DRGs were excised, trimmed of excess connective tissue, and initially placed in serum-free Ham’s F10 medium at room temperature. The ganglia were then enzymatically dissociated by sequential incubation in 1 ml of 2% collagenase for 30 min and 1 ml of 1.5% trypsin for 60 min at room temperature. The resulting cell suspension was centrifuged at 1500× g for 3 min, and the pellet was gently triturated with a fire-polished glass pipette in F10 medium to release dissociated cells. The cell suspension was layered onto a 10-ml iso-osmotic Percoll gradient and centrifuged at 1200 × g for 10 min. The cell pellet was resuspended in fresh F10 medium and passed through a 40 µm nylon mesh to obtain a single-cell suspension. The filtered cells were collected by centrifugation and resuspended in Ham’s F10 maintenance medium supplemented with 6.2 mM glucose. Isolated neurons were seeded at 4000–8000 cells per well onto 96-well plates pre-coated with poly-ornithine and laminin and cultured in maintenance medium for 12 h before being subjected to mitochondrial bioenergetic assays.

Measurement of mitochondrial bioenergetics in cultured DRG neurons

Neuronal bioenergetics of DRG cultures were assessed using an XF96 Analyzer (Seahorse Bioscience, Billerica, MA, USA) by real-time monitoring of the oxygen consumption rate (OCR) [30]. One hour before the assay, the culture medium was replaced with unbuffered DMEM (Dulbecco’s modified Eagle’s medium, pH 7.4) supplemented with 10 mM D-glucose and 2 mM pyruvate. Cells were seeded at a density of 2500–6000 cells per well to ensure a linear OCR response. The XF96 protocol consisted of repeated cycles of 3 min mixing, 2 min delay (equilibration), and 4 min measurement phases. After recording basal OCR, oligomycin (1 μM), carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP, 1 μM), and rotenone plus antimycin A (1 μM each) were sequentially injected through the ports of the Seahorse Flux cartridge. These measurements enabled the calculation of key mitochondrial parameters: basal respiration, ATP-linked respiration, maximal respiration, proton leak (non-ATP-linked oxygen consumption), and non-mitochondrial respiration [31]. All OCR values were normalized to total protein content per well, with protein concentrations being quantified using a bicinchoninic acid (BCA) assay with bovine serum albumin as the standard (linear range 20-2000 ug/ml).

Statistical analysis

All data are presented as the mean ± standard deviation (SD). Sample size was determined based on a priori power analysis and our pilot studies. The Shapiro–Wilk test and Levene’s test were used to assess normality and homogeneity of variance, respectively. For data that met the normality assumption, a mixed-design analysis of variance (ANOVA) or a two-way ANOVA was applied as appropriate. In the mixed-design ANOVA, diet (regular vs. high-fat) and treatment (vehicle vs. plumbagin) were included as between-subjects factors, and time was treated as a within-subjects factor; post hoc comparisons were performed using Tukey’s test when justified. For the two-way ANOVA, the Duncan multiple range test was used for post hoc comparisons. Data that violated the normality assumption (i.e., the von Frey test data) were analyzed using the Kruskal–Wallis test, followed by Dunn’s test when appropriate. A P-value < 0.05 was considered statistically significant.

Results

Prophylactic plumbagin therapy decelerated the development of neuropathic allodynia in HFD-diabetic mice

In Experiment 1, the effect of prophylactic plumbagin treatment against the development of neuropathic pain was evaluated. On day 35 (i.e., 5 weeks after the initiation of either standard chow or a high-fat diet), healthy control and HFD-diabetic mice began once-daily oral administration of plumbagin (2–18 mg/kg) for three weeks. Of note, the prophylactic dose range was approximately 2.5-fold lower than that used for therapeutic intervention (5–45 mg/kg). Consistent with the expected progression of diabetic neuropathy, vehicle-treated HFD-diabetic mice developed significant mechanical allodynia in the von Frey test (F₈,₂₉₆ = 11.4, p < 0.01; Fig. 1B, left panel) and cold allodynia in the acetone test (F₈,₂₉₆ = 5.6, p < 0.01; Fig. 1C, left panel). By contrast, HFD-diabetic mice receiving 18 mg/kg plumbagin were protected from mechanical and cold allodynia throughout the three-week treatment course (Fig. 1B, C), indicating a prophylactic action against painful diabetic neuropathy. The anti-allodynic effects of plumbagin were dose-dependent (Fig. 1B, right panel for the von-Frey test; Fig. 1C, right panel for the acetone test). Plumbagin treatment was discontinued on day 55. Approximately one week after treatment withdrawal, the previously protected HFD-diabetic mice exhibited a pronounced emergence of mechanical and cold allodynia, confirming that the prophylactic effect was plumbagin-dependent. These data demonstrate that prophylactic administration of plumbagin 18 mg/kg effectively prevents the onset of neuropathic allodynia in a mouse model of HFD-induced diabetes.

Analgesic effects of plumbagin on neuropathic allodynia in HFD-diabetic mice

In Experiment 2, we evaluated the therapeutic efficacy of plumbagin against established mechanical and cold hypersensitivity in HFD-diabetic mice. As anticipated, vehicle treated HFD-diabetic mice exhibited persistent mechanical and cold allodynia throughout the experimental period. Following two weeks of treatment, plumbagin 45 mg/kg significantly ameliorated the pronounced mechanical allodynia (F_6,278 = 15.1, p < 0.01; Fig. 2B, left panel) and cold allodynia (F_6,278 = 6.8, p < 0.01; Fig. 2C, left panel) in HFD-diabetic mice, restoring both mechanical and cold hypersensitivities to physiological levels. These anti-nociceptive effects are dose-dependent, achieving statistical significance at doses of 15 and 45 mg/kg (Fig. 2B, C, right panel). The therapeutic effect of plumbagin persisted for 3–4 days following treatment discontinuation, and after that neuropathic allodynia re-emerged. In the acute treatment session conducted on day 56, plumbagin 45 mg/kg did not alleviate mechanical and cold allodynia in HFD-diabetic mice (Supplementary Fig. S1).

Effects of plumbagin on metabolic features in control and diabetic mice

As shown in Fig. 3, HFD-diabetic mice exhibited progressively worsening metabolic profiles relative to control mice, as reflected by significant increases in body weight (week 5 to week 10 post-HFD initiation, Fig. 3A), one-day food intake (from week 6 to week 10, Fig. 3B), blood glucose (from week 7 to week 10, Fig. 3C), intraperitoneal glucose tolerance (from week 7 to week 10, Fig. 3D) and insulin (from week 7 to week 10, Fig. 3E). Following two weeks of treatment, plumbagin did not significantly alter these metabolic parameters in either control or HFD-diabetic mice.

Mitochondrial bioenergetic profiles were aberrant in DRG neurons from HFD-diabetic mice and were normalized by plumbagin therapy

As expected, DRG neurons of HFD-diabetic mice exhibited impaired mitochondrial bioenergetic features relative to healthy controls (Fig. 4A), characterized by significantly reduced maximal respiratory capacity (MRC), spare respiratory capacity (SRC), and respiratory control ratio (RCR). Following two weeks of treatment, plumbagin dose-dependently (5–45 mg/kg) corrected these mitochondrial bioenergetic deficits in DRG neurons of HFD-diabetic mice (Fig. 4B). Specifically, plumbagin administration resulted in (i) increased MRC (by 176.7% at 45 mg/kg; Fig. 4C), (ii) increased SRC (by 273.5% at 45 mg/kg; Fig. 4D), (iii) increased RCR (by 170.3% at 45 mg/kg; Fig. 4E), and (iv) normalization of the percentages of SRC, ATP turnover, proton leak, and non-mitochondrial OCR (with SRC shifting from 50.6% to 76.4%, ATP turnover from 23.8% to 9.2%, proton leak from 13.3% to 5.3%, and non-mitochondrial OCR from 12.3% to 9.1%; Fig. 4F).

Plumbagin therapy improved mitochondrial enzyme activities in HFD-diabetic mice

As shown in Table 1, the activities of several key mitochondrial enzymes (including NADH–cytochrome c reductase, cytochrome c oxidase, and citrate synthase) were significantly lower in HFD-diabetic mice than in healthy controls. A 2-week plumbagin treatment dose-dependently (5–45 mg/kg) restored these enzyme activities in HFD-diabetic mice, without exerting effect on the same enzyme activities in the control group.

Table 1.

Mitochondrial enzyme activities following two weeks of plumbagin (Plu) or vehicle treatment in control and high fat diet (HFD) mice.

Enzyme (μmol/min/g protein)	Control mice		HFD mice
	Vehicle	Plu-45 mg/kg	Vehicle	Plu-5 mg/kg	Plu-15 mg/kg	Plu-45 mg/kg
NADH-cytochrome c reductase	59.62 ± 6.70	58.78 ± 7.96	34.62 ± 5.09^##	36.06 ± 6.70	47.78 ± 5.27^**	56.92 ± 7.31^**
Cytochrome c oxidase	485.36 ± 41.84	492.55 ± 37.06	339.48 ± 30.30^##	348.71 ± 34.05	418.38 ± 28.63^**	476.28 ± 37.52^**
Citrate synthase	186.25 ± 18.33	182.70 ± 12.66	128.65 ± 13.73^##	124.69 ± 11.57	152.26 ± 16.72	179.83 ± 20.61^**

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Values were expressed as mean values ± SEM of 9–11 mice. ^##p < 0.01, compared with vehicle treated control mice; ^**p < 0.01, compared with vehicle treated diabetic mice.

Antioxidant effects of plumbagin on catalase activity, reactive oxygen species and lipid peroxidation in tissues of dorsal root ganglion (DRG), spinal cord and sciatic nerve of HFD-diabetic mice

Persistent hyperglycemia is a major driver of diabetic complications, such as painful diabetic neuropathy. We therefore investigated whether plumbagin treatment could modulate key markers of oxidative stress, including catalase activity, reactive oxygen species (ROS) levels, and lipid peroxidation in HFD-diabetic mice. As shown in Fig. 5, HFD-diabetic mice exhibited significantly elevated oxidative stress, evidenced by suppressed catalase activity (Fig. 5A) and increased ROS production (Fig. 5B) and lipid peroxidation (Fig. 5C) in the dorsal root ganglia, sciatic nerve, and spinal cord. Repeated administration of plumbagin dose-dependently ameliorated these aberrant oxidative stress markers in HFD-diabetic mice, without significantly affecting these profiles in healthy controls. Specifically, compared with vehicle-treated HFD-diabetic mice, plumbagin-treated HFD-diabetic mice showed increased catalase activity (Fig. 5A) and reduced ROS levels (Fig. 5B) and lipid peroxidation (Fig. 5C) across all three tissues examined.

Fig. 5 — Catalase activity (A), reactive oxygen species (ROS) levels (B), and lipid peroxidation (C) were assessed in the sciatic nerve (left panels), DRG (middle panels), and spinal cord (right panels). Compared with healthy controls, HFD-diabetic mice exhibited significantly reduced catalase activity (A) and elevated levels of ROS (B) and lipid peroxidation (C) across all three tissues. Two-week treatment with plumbagin (5–45 mg/kg, p.o.) dose-dependently normalized these pathological indices-restoring catalase activity and suppressing both ROS production and lipid peroxidation-while producing no discernible effects in control animals. Data are expressed as mean ± SD, assessed by two-way ANOVA followed by Duncan test. ^*p < 0.05 and ^**p < 0.01 denotes statistical difference from vehicle, and ^##p < 0.01 denotes statistical difference from control. Plu: plumbagin, HFD: high fat diet.

The pain-like behavior correlates with mitochondrial bioenergetic profiles and oxidative parameters in diabetic mice

Pearson’s correlation analysis examined associations between pain-like behaviors and indices of mitochondrial bioenergetics and oxidative stress. The analysis reveals that, in both vehicle- and plumbagin-treated diabetic mice, the reduced duration of cold nociception in the acetone test was significantly correlated with enhanced mitochondrial bioenergetics (Fig. 6A-C) and attenuated oxidative stress (Fig. 6D, E). These findings suggest that the analgesic effects of plumbagin are associated with its capacity to enhance mitochondrial bioenergetics and reduce oxidative stress.

Fig. 6 — For panels (A) through (E), the x-axis represents the nociceptive response in the acetone test (cold allodynia). The y-axes correspond to maximal respiratory capacity (MRC; A), spare respiratory capacity (SRC; B), respiratory control ratio (RCR; C), catalase activity (D), and reactive oxygen species (ROS) levels (E). Plu: plumbagin, HFD: high fat diet.

Plumbagin antinociception was abolished by mitochondrial poison oligomycin, but potentiated by mito-protective agent CoQ10

Given that plumbagin improved mitochondrial bioenergetics in DRG neurons of HFD-diabetic mice, we next sought to establish a causal link between this effect and its analgesic action. Using a pharmacological approach, HFD-diabetic mice and healthy controls were co-administered either the mitochondrial ATP synthase inhibitor oligomycin or the mito-protective agent CoQ10, following 2 weeks of plumbagin or vehicle treatment. As shown in Fig. 7, repeated co-administration of oligomycin 0.5 mg/kg completely abolished the pain-alleviating effect of plumbagin in HFD-diabetic mice (the von-Frey test: F_6,278 = 12.6, p < 0.01, Fig. 7A, left panel; the acetone test: F_6,278 = 6.1, p < 0.01, Fig. 7B, left panel), whereas acute administration had no such effect (Supplementary Fig. S2). Notably, oligomycin did not alter nocifensive sensitivity to mechanical or cold stimuli in either vehicle-treated HFD-diabetic mice or healthy controls. Alone, neither coenzyme Q10 (CoQ10, 15 mg/kg) nor an ineffective dose of plumbagin (5 mg/kg) altered sensory sensitivity in the von Frey test (Fig. 7A, right panel) and the acetone test (Fig. 7B, right panel) in either healthy controls or HFD-diabetic mice. However, their co-administration produced significant pain-alleviating effects in HFD-diabetic mice (the von-Frey test: F_6,278 = 8.6, p < 0.01, Fig. 7A, right panel; the acetone test: F_6,278 = 12.7, p < 0.01, Fig. 7B, right panel), indicating that CoQ10, a mitochondrial protectant, potentiated the analgesic effect of a low dose of plumbagin. These results suggest that the pain-alleviating effects of plumbagin could be modulated by pharmacologically targeting mitochondrial bioenergetics.

Fig. 7 — Following a two-week treatment course of plumbagin (on day 69), mice received repeated co-administration of either oligomycin or CoQ10 with plumbagin for five consecutive days. The antiallodynic effect of plumbagin (45 mg/kg, p.o.) in HFD-diabetic mice was abolished by repeated co-administration of oligomycin (0.5 mg/kg), as assessed by the von Frey test (A, left panel) and the acetone test (B, left panel). Administration of CoQ10 (15 mg/kg) or a sub-effective dose of plumbagin (5 mg/kg) alone failed to produce any antinociceptive effect in either control or HFD-diabetic mice (A and B, right panels). In contrast, combined treatment with CoQ10 (15 mg/kg) and sub-effective plumbagin (5 mg/kg) elicited significant antiallodynic effects in HFD-diabetic mice in both the von Frey test (A, right panel) and the acetone test (B, right panel). Data are expressed as mean ± SD (n = 9–11 per group), evaluated by Kruskal-Wallis test for the von Frey data, and multifactor ANOVA followed by Tukey’s test and two-way ANOVA followed by Duncan test for the acetone data. ^**p < 0.01 denotes statistical difference from cohort of diabetes treated with vehicle. Plu: plumbagin, HFD: high fat diet.

Plumbagin antinociception was counteracted by acute co-treatment with the ROS donor tert-butyl hydroperoxide (t-BOOH), but potentiated by the ROS scavenger phenyl-N-tert-butylnitrone (PBN)

Mitochondria are a major source of ROS generated during oxidative phosphorylation. Given the mitochondrial-protective and antioxidant properties of plumbagin, we tested whether its analgesic efficacy could be modulated by co-administration with the ROS donor t-BOOH or the ROS scavenger PBN. As shown in Fig. 8A, C, acute t-BOOH dose-dependently antagonized plumbagin’s pain-alleviating effects in HFD-diabetic mice (the von-Frey test: F_2,120 = 5.7, p < 0.01, Fig. 8A; the acetone test: F_2,120 = 8.8, p < 0.01, Fig. 8C), while having no effect on nociception in healthy controls or vehicle-treated HFD-diabetic mice. In contrast, PBN potentiated plumbagin’s analgesic action. Alone, PBN (10 mg/kg) or a sub-effective dose of plumbagin (5 mg/kg) did not affect nociceptive sensitivity in the von Frey test (Fig. 8B) or the acetone test (Fig. 8D) in for both healthy controls and HFD-diabetic mice. However, co-administration of them induced a significant antiallodynic effect in HFD-diabetic mice (the von-Frey test: F_1,43 = 4.8, p < 0.01, Fig. 8B; the acetone test: F_1,43 = 14.9, p < 0.01, Fig. 8D). These results suggest that plumbagin’s analgesic effects are primarily mediated through its antioxidant capacity, specifically the attenuation of ROS levels.

Fig. 8 — A Acute co-administration of t-BOOH (0.5 and 1 mg/kg, i.p.) dose-dependently reversed the antiallodynic effect of plumbagin (45 mg/kg) on mechanical allodynia in HFD-diabetic mice, without altering mechanical thresholds in healthy controls. B In the von Frey test, neither a sub-effective dose of plumbagin (5 mg/kg) nor PBN (10 mg/kg) alone exerted any antinociceptive effect in control or HFD-diabetic mice; however, their combination produced a significantly antiallodynic effect in HFD-diabetic animals. C Acute t-BOOH (0.5 and 1 mg/kg, i.p.) dose-dependently abolished the antiallodynic effect of plumbagin (45 mg/kg) on cold allodynia in HFD-diabetic mice, with no effect on nociceptive responses in controls. D In the acetone test, neither plumbagin (5 mg/kg) nor PBN (10 mg/kg) alone induced any antinociceptive effect in either group, whereas combined treatment elicited a pronounced antiallodynic effect in HFD-diabetic mice. Data are expressed as mean ± SD (n = 9–11 per group), evaluated by Kruskal-Wallis test for the von Frey data, and multifactor ANOVA followed by Tukey’s test and two-way ANOVA followed by Duncan test for the acetone data. ^**p < 0.01 denotes statistical difference from control, and ^##p < 0.01 denotes statistical difference from vehicle. Plu: plumbagin, HFD: high fat diet.

Plumbagin ameliorated nerve conduction defect in HFD-diabetic mice

Motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) were assessed electrophysiologically in healthy controls and HFD-diabetic mice at designated time points (days 0, 56, 63, and 70). As shown in Fig. 9, HFD-diabetic mice exhibited decreased MNCV (Fig. 9A) and SNCV (Fig. 9B) compared with healthy controls. Notably, repeated plumbagin treatment dose-dependently ameliorated both deficits (MNCV: F_3,52 = 18.4, p < 0.01, Fig. 9A; SNCV: F_3,52 = 11.7, p < 0.01, Fig. 9B) in HFD-diabetic mice (restoring them to near-physiological levels at 45 mg/kg), while producing no significant effects in healthy controls.

Fig. 9 — A Plumbagin treatment dose-dependently restored the impaired motor nerve conduction velocity (MNCV) in HFD-diabetic mice, while exerting no effect on MNCV in healthy controls (left panels, time-course profiles; right panels, dose-response relationships). B Similarly, plumbagin therapy significantly improved the reduced sensory nerve conduction velocity (SNCV) in HFD-diabetic mice without altering SNCV in control animals (left panels, time-course profiles; right panels, dose-response relationships). C Furthermore, repeated plumbagin dosing markedly augmented the compromised sciatic nerve blood flow (NBF) in diabetic mice, with no observable changes in control littermates (left panels, time-course profiles; right panels, dose-response relationships). Data are expressed as mean ± SD (n = 9–10 per group), evaluated by Kruskal-Wallis test for the von Frey data, and multifactor ANOVA followed by Tukey’s test and two-way ANOVA followed by Duncan test for the acetone data. ^**p < 0.01 denotes statistical difference from cohort of diabetes treated with vehicle. Plu: plumbagin, HFD: high fat diet.

Plumbagin improved nerve blood flow in HFD-diabetic mice

We further evaluated the effect of plumbagin on sciatic nerve blood flow (NBF) throughout the treatment period. As expected, HFD-diabetic mice exhibited a significant reduction in sciatic NBF on days 56, 63, and 70 compared with healthy controls. Repeated plumbagin treatment dose-dependently increased sciatic NBF in HFD-diabetic mice, with the 45 mg/kg dose restoring it to near-physiological levels (F_3,52 = 13.0, p < 0.01, Fig. 9C).

Effect of plumbagin on hepatic and renal functions and hematological variables

Repetitive plumbagin treatment (5, 15, and 45 mg/kg) affected neither hepatic and renal function (Supplementary Table. 1) nor hematological parameters (Supplementary Table. 2) in either healthy controls or HFD-diabetic mice.

Discussion

We report that the natural naphthoquinone plumbagin possesses significant therapeutic potential for painful diabetic neuropathy. Repeated oral plumbagin treatment reversed established mechanical and cold allodynia in HFD-diabetic mice, while prophylactic low-dose administration prevented pain onset, indicative of disease-modifying activity. Notably, these effects were dissociated from glycemic changes and were instead mechanistically linked to the direct modulation of cellular bioenergetics and redox homeostasis.

To our best knowledge, it is the first report to reveal the pain-relieving property of plumbagin in the context of type 2 diabetes. Whether its analgesic efficacy can extend to type 1 diabetes deserves further studies. Its pain-relieving effects here exhibit several characteristics. First, plumbagin attenuated nociceptive hyperactivity in HFD-induced diabetic mice without affecting baseline nociception in healthy controls. Such disease-state-dependent effect implies that plumbagin targets pathological drivers of pain, a property that substantially increases its potential for clinical translation. Second, the analgesic efficacy of plumbagin exhibited clear time dependence. A single administration proved ineffective, whereas sustained treatment over approximately two weeks of repeated dosing was necessary to achieve maximal therapeutic effect in HFD-diabetic mice. This time-dependent profile suggests that plumbagin alleviates pain by counteracting maladaptive neural plasticity associated with diabetes. Third, the pain-alleviating effect of plumbagin was durable. It remained measurable 22 h after dosing and persisted for approximately three days (therapeutic regimen) or seven to eight days (preemptive regimen) post-treatment, despite showing gradual decay over time. Finally, the preventive plumbagin doses (2, 6 and 18 mg/kg) are 2.5-bold lower than therapeutic ones (5, 15 and 45 mg/kg). This discrepancy may be explained by its relatively long elimination half-life (T_1/2, around 17 h) [32], the nature of lipophilicity, which should lead to slow elimination, and the wider time course window for preventive treatment (3 weeks) compared to that for therapeutic one (2 weeks). In addition to its pain-alleviating effects, plumbagin also improved nerve conduction velocity and nerve blood flow in HFD-diabetic mice. These findings suggest a potential neuroprotective activity and future studies should be considered to assess structural damage and recovery with plumbagin treatment.

After determining the therapeutic efficacy, an important question rose as whether plumbagin treatment is safe in the present animal study. The selected plumbagin doses (2, 6, 18 mg/kg for preventive treatment and 5, 15, 45 mg/kg for therapeutic treatment) and the once-daily regimen are manly based on our pilot study and previous reports with respect to its therapeutic efficacies [18, 20, 33] and pharmacokinetic profile [32]. At the maximal dose of 45 mg/kg in the present study, plumbagin did not show any toxicity-related effects on body weight, food intake, blood glucose level, hepatic and renal functions, and hematological profiles during the entire period of treatment. Consistently, oral administration of plumbagin 250 mg/kg in the diet for 3 months did not affect food and water intake, and consequently did not alter weight gain of C57BL mice [34]. Likewise, a two-week treatment with plumbagin 50 mg/kg (p.o.) in rats (an equivalent dose of 71.5 mg/kg in mice appropriately) did not influence body weight, cardiac function and hemodynamic parameters [33]. These results indicate that the natural origin of plumbagin may confer it relatively safe profiles, but more information especially clinical data should be pursued.

Mitochondrial dysfunction is a well-established pathogenic factor in diabetic complications, including refractory neuropathic pain [4, 28]. Mutations in key mitochondrial genes, for instance, are known to cause the painful neuropathy seen in Charcot-Marie-Tooth disease [35]. As the main endogenous source of ROS, mitochondria are highly susceptible to oxidative damage. This vulnerability is intensified in diabetes, where antioxidant defenses in the central and peripheral nervous systems are commonly compromised [36]. Given the reported antioxidant activity of plumbagin, we investigated its mechanism of action, with particular emphasis on potential beneficial effects against mitochondrial dysfunction and redox imbalance. To this end, we characterized mitochondrial bioenergetics in DRG neurons, which were suppressed in HFD-diabetic mice. Consistent with previous findings [6, 28], impaired mitochondrial bioenergetics (reduced MRC, SRC, and RCR) was evident in diabetic DRG neurons. Repeated plumbagin treatment dose-dependently reversed these deficits and restored the activities of cytochrome c oxidase, NADH-cytochrome c reductase, and citrate synthase in DRG homogenates. These observations demonstrate that plumbagin can alleviate diabetes-induced mitochondrial dysfunction, supporting the therapeutic potential of mitochondria-targeting agents in persistent neuropathic pain [6, 7].

Mitochondrial dysfunction drives ROS overproduction, thereby disrupting redox homeostasis and amplifying oxidative damage. In both type 1 and type 2 diabetes, excessive production of oxidants, including lipid peroxides and ROS, overwhelms endogenous antioxidant capacity and results in widespread tissue injury [37, 38]. Importantly, several antioxidant compounds have been reported to ameliorate painful diabetic neuropathy [8, 9]. Given that plumbagin exhibits significant antioxidant activity and attenuates ROS production [13, 17, 34], we sought to determine whether its therapeutic efficacy against painful diabetic neuropathy is mediated through this antioxidant mechanism. We then assessed oxidative stress profiles in the DRG, spinal cord, and sciatic nerve by determining ROS generation, lipid peroxidation, and catalase activity. In line with earlier findings, significantly higher levels of oxidative stress were observed in the neural tissues of HFD-diabetic mice relative to healthy controls. Chronic plumbagin administration dose-dependently attenuated these elevated oxidative stress levels, with a maximum effect observed at the 45 mg/kg dose. These findings imply that the therapeutic effect of plumbagin in painful diabetic neuropathy is attributable, at least in part, to the reduction of oxidative stress, a major pathophysiological contributor to the disease. Interestingly, plumbagin may also display potential pro-oxidant activities in some pathophysiological microenvironment [39, 40]. This discrepancy may be ascribed to some factors such as dose (concentration), redox state, pH value and enzyme activities.

Therefore, it is imperative to interrogate whether the restoration of mitochondrial function and redox homeostasis by plumbagin constitutes the primary mechanism driving its therapeutic efficacy, or these molecular improvements merely arise as a secondary consequence of pain alleviation. To address this, we first examined the relationship between the pain-like behaviors and the indices of mitochondrial bioenergetics and redox status in HFD-diabetic animals. The correlation analysis reveals that plumbagin-induced analgesia correlates well with the enhancement of mitochondrial bioenergetics and the amelioration of oxidative stress. Furthermore, we sought to determine whether the analgesic efficacy of plumbagin could be modified by pharmacological modulating mitochondrial bioenergetics and redox status. Our results demonstrate that the analgesic effect of plumbagin was abrogated by repetitive co-administration with the mitochondrial inhibitor oligomycin and by acute co-administration with the pro-oxidant t-BOOH. Conversely, its efficacy was potentiated by repetitive co-treatment with the mitochondrial protectant CoQ10 and by acute co-treatment with the ROS scavenger PBN. These results provide pharmacological evidence demonstrating that the mitochondria-protective and antioxidant properties of plumbagin are causally linked to its analgesic actions against diabetic neuropathic pain. Of note, oligomycin and CoQ10 required a repeated co-administration process to affect plumbagin analgesia, while t-BOOH and PBN produced immediate actions. This temporal discrepancy suggests that the improvement of mitochondrial bioenergetics may serve as an upstream target, with diminished ROS generation being a downstream or end-point molecular event. Whether the mitochondrial alterations are due to direct neuronal effects by plumbagin deserves further studies.

There are several technical considerations before conclusions could be drawn. First, only male mice were used in this study. Given that possible sex-specific differences in nociceptive processing, female mice should be included in future studies. Second, a positive control of existing neuropathic pain treatments like gabapentin and amitriptyline should be considered in further work to titrate plumbagin efficacy. Third, oligomycin is a toxic mitochondrial inhibitor with broad and non-specific effects. Similarly, t-BOOH is a strong systemic oxidant that may affect multiple signaling pathway in vivo. Mechanistic validation concerning the specificity of the two agents should be performed in vitro models.

Conclusion

Our findings demonstrate that plumbagin significantly attenuated neuropathic allodynia in a mouse model of type 2 diabetes. This analgesic effect can also be achieved through prophylactic plumbagin administration at comparatively low doses. Plumbagin treatment additionally ameliorated mitochondrial dysfunction and suppressed exacerbated oxidative stress, thereby targeting the principal pathophysiological drivers of painful diabetic neuropathy. Mechanistic investigations further reveal that the pain-relieving actions of plumbagin are causally linked to its capacity to preserve mitochondrial functions and counteract oxidative stress.

Supplementary information

Supplemental Figure 1^{(263.8KB, pdf)}

Supplemental Figure 2^{(267.7KB, pdf)}

Supplemental Table 1^{(42.5KB, doc)}

Supplemental Table 2^{(43.5KB, doc)}

Author contributions

YF: Methodology, Investigation, Writing—original draft. YW: Methodology, Investigation. WC: Formal analysis, Validation. BL: Methodology, Software. XZ: Project administration, Funding acquisition, Validation, Supervision, Writing—review and editing. All authors agree to be accountable for all aspects of this work ensuring integrity and accuracy.

Funding

This study was sponsored by Ningbo Natural Science Foundation (2024J017, 2024J282), NMNSF (2021J110), Medical Discipline Construction Program of Shanghai Pudong New Area Health Commission (the Key Disciplines Program PWRd2024-05), Pudong New Area People’s Hospital Pilot Talent Development Program (PRYLH202601) and K. C. Wong Magna Fund in Ningbo University.

Data availability

The data generated or analyzed in this study are available upon reasonable request from the corresponding author.

Competing interests

The authors declare no competing interests.

Ethical approval

All animal care and procedures were approved by the Ningbo University Committee on Animal Care and Use (approval process number 11164) and adhere to the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Youya Fan, Yangui Wang.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41387-026-00450-w.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figure 1^{(263.8KB, pdf)}

Supplemental Figure 2^{(267.7KB, pdf)}

Supplemental Table 1^{(42.5KB, doc)}

Supplemental Table 2^{(43.5KB, doc)}

Data Availability Statement

The data generated or analyzed in this study are available upon reasonable request from the corresponding author.

[CR1] 1.Staudt MD, Prabhala T, Sheldon BL, Quaranta N, Zakher M, Bhullar R, et al. Current strategies for the management of painful diabetic neuropathy. J Diab Sci Technol. 2022;16:341–52. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR2] 2.Alam U, Sloan G, Tesfaye S. Treating pain in diabetic neuropathy: current and developmental drugs. Drugs. 2020;80:363–84. [DOI] [PubMed] [Google Scholar]

[CR3] 3.Cheng YJ, Kanaya AM, Araneta MRG, Saydah SH, Kahn HS, Gregg EW, et al. Prevalence of diabetes by race and ethnicity in the United States, 2011-2016. JAMA. 2019;322:2389–98. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR4] 4.Fernyhough P, Roy Chowdhury SK, Schmidt RE. Mitochondrial stress and the pathogenesis of diabetic neuropathy. Expert Rev Endocrinol Metab. 2010;5:39–49. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR5] 5.Finnerup NB, Kuner R, Jensen TS. Neuropathic pain: from mechanisms to treatment. Physiol Rev. 2021;101:259–301. [DOI] [PubMed] [Google Scholar]

[CR6] 6.Ma J, Farmer KL, Pan P, Urban MJ, Zhao H, Blagg BS, et al. Heat shock protein 70 is necessary to improve mitochondrial bioenergetics and reverse diabetic sensory neuropathy following KU-32 therapy. J Pharm Exp Ther. 2014;348:281–92. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR7] 7.McCormick B, Lowes DA, Colvin L, Torsney C, Galley HF. MitoVitE, a mitochondria-targeted antioxidant, limits paclitaxel-induced oxidative stress and mitochondrial damage in vitro, and paclitaxel-induced mechanical hypersensitivity in a rat pain model. Br J Anaesth. 2016;117:659–66. [DOI] [PubMed] [Google Scholar]

[CR8] 8.Comelli F, Bettoni I, Colombo A, Fumagalli P, Giagnoni G, Costa B. Rimonabant, a cannabinoid CB1 receptor antagonist, attenuates mechanical allodynia and counteracts oxidative stress and nerve growth factor deficit in diabetic mice. Eur J Pharm. 2010;637:62–69. [DOI] [PubMed] [Google Scholar]

[CR9] 9.Zhang YP, Eber A, Yuan Y, Yang Z, Rodriguez Y, Levitt RC, et al. Prophylactic and antinociceptive effects of coenzyme Q10 on diabetic neuropathic pain in a mouse model of type 1 diabetes. Anesthesiology. 2013;118:945–54. [DOI] [PubMed] [Google Scholar]

[CR10] 10.Finnerup NB, Attal N, Haroutounian S, McNicol E, Baron R, Dworkin RH, et al. Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis. Lancet Neurol. 2015;14:162–73. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Redox and bioenergetic modulation by plumbagin attenuated neuropathic allodynia in mice with diet-induced diabetes

Youya Fan

Yangui Wang

Wugeng Cui

Benling Li

Xin Zhao

Abstract

Background

Methods

Results

Conclusions

Introduction

Fig. 1. Effect of prophylactic plumbagin treatment on the development of mechanical and cold allodynia in HFD-diabetic mice.

Methods

Animals

Induction of type 2 diabetes

Drugs

Experimental design and pharmacological treatment

Fig. 2. Effect of repeated plumbagin treatment on established mechanical and cold allodynia in HFD-diabetic mice.

Behavioral Tests

Biochemical assays

Measurement of nerve conduction velocity

Measurement of sciatic nerve blood flow

Sensory neuron isolation and culture

Measurement of mitochondrial bioenergetics in cultured DRG neurons

Statistical analysis

Results

Prophylactic plumbagin therapy decelerated the development of neuropathic allodynia in HFD-diabetic mice

Analgesic effects of plumbagin on neuropathic allodynia in HFD-diabetic mice

Effects of plumbagin on metabolic features in control and diabetic mice

Fig. 3. Temporal profiles of metabolic parameters in control and high-fat diet (HFD)-diabetic mice in response to HFD and plumbagin (Plu) treatment.

Mitochondrial bioenergetic profiles were aberrant in DRG neurons from HFD-diabetic mice and were normalized by plumbagin therapy

Fig. 4. Plumbagin treatment restores mitochondrial bioenergetic function in dorsal root ganglion (DRG) neurons from HFD-diabetic mice.

Plumbagin therapy improved mitochondrial enzyme activities in HFD-diabetic mice

Table 1.

Antioxidant effects of plumbagin on catalase activity, reactive oxygen species and lipid peroxidation in tissues of dorsal root ganglion (DRG), spinal cord and sciatic nerve of HFD-diabetic mice

Fig. 5. Plumbagin treatment dose-dependently attenuates oxidative stress in the dorsal root ganglion (DRG), sciatic nerve, and spinal cord of HFD-diabetic mice.

The pain-like behavior correlates with mitochondrial bioenergetic profiles and oxidative parameters in diabetic mice

Fig. 6. Correlation analysis between pain-like behavior and mitochondrial bioenergetic profiles or oxidative parameters in HFD-diabetic mice.

Plumbagin antinociception was abolished by mitochondrial poison oligomycin, but potentiated by mito-protective agent CoQ10

Fig. 7. Modulation of plumbagin-induced antinociception by repeated co-administration of the mitochondrial ATP synthesis inhibitor oligomycin (OMC) and the mitochondrial protectant coenzyme Q10 (CoQ10).

Plumbagin antinociception was counteracted by acute co-treatment with the ROS donor tert-butyl hydroperoxide (t-BOOH), but potentiated by the ROS scavenger phenyl-N-tert-butylnitrone (PBN)

Fig. 8. Effects of acute co-administration of the ROS donor tert-butyl hydroperoxide (t-BOOH) or the ROS scavenger phenyl-N-tert-butylnitrone (PBN) on the pain-relieving effects of plumbagin.

Plumbagin ameliorated nerve conduction defect in HFD-diabetic mice

Fig. 9. Repeated administration of plumbagin (5, 15, and 45 mg/kg) ameliorated nerve conduction deficits and enhanced nerve blood flow in HFD-diabetic mice.

Plumbagin improved nerve blood flow in HFD-diabetic mice

Effect of plumbagin on hepatic and renal functions and hematological variables

Discussion

Conclusion

Supplementary information

Author contributions

Funding

Data availability

Competing interests

Ethical approval

Footnotes

Supplementary information

References

Associated Data

Supplementary Materials

Data Availability Statement

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