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. 2020 Jul 18;41(7):1563–1577. doi: 10.1007/s10571-020-00923-1

SIRT1 Activation by Polydatin Alleviates Oxidative Damage and Elevates Mitochondrial Biogenesis in Experimental Diabetic Neuropathy

Preethi Bheereddy 1, Veera Ganesh Yerra 2, Anil Kumar Kalvala 1, Bhoomika Sherkhane 1, Ashutosh Kumar 1,3,✉
PMCID: PMC11448605  PMID: 32683581

Abstract

Mitochondrial dysfunction has been implicated as a one of the major factors linked to the development of painful diabetic neuropathy (DN). Several studies have demonstrated that sirtuin (SIRT1) activation recuperates nerve function by activating mitochondrial biogenesis. Polydatin, a resveratrol glycoside, has been explored to improve mitochondrial function via SIRT1 activation. However, the neuroprotective effects of polydatin in DN remain elusive. In this study, polydatin (25 and 50 mg/kg, oral) was administered for last 2 weeks of 8-week study to diabetic Sprague–Dawley rats weighing 250–300 g (post 6-weeks of streptozotocin 55 mg/kg, intraperitoneal). Treatment with polydatin significantly attenuated mechanical and thermal hyperalgesia in diabetic rats. Treated diabetic rats also showed improvement in motor/sensory nerve conduction velocities and nerve blood flow. For in vitro studies, Neuro2a cells were exposed to high-glucose (30 mM) condition to simulate short-term hyperglycemia. Polydatin was evaluated for its role in SIRT1 and Nrf2 activation at a dose of 5, 10, and 20 µM concentrations. Polydatin exposure normalized the mitochondrial superoxides, membrane potentials and improved neurite outgrowth in high-glucose-exposed Neuro2a cells. Increased SIRT1 activation by polydatin resulted in peroxisome proliferator activated receptor-gamma coactivator-1α (PGC-1α) directed mitochondrial biogenesis. SIRT1 activation also facilitated Nrf2-directed antioxidant signaling. Study results inferred that decline in mitochondrial biogenesis and oxidative function in diabetic rats and high-glucose-exposed Neuro2a cells, could be counteracted by polydatin administration, postulated via enhancing SIRT1 and Nrf2 axis.

Electronic supplementary material

The online version of this article (10.1007/s10571-020-00923-1) contains supplementary material, which is available to authorized users.

Keywords: Polydatin, Mitochondrial biogenesis, Oxidative stress, SIRT1, Nrf2

Introduction

Diabetic neuropathy (DN), a major longstanding, hyperglycemia-induced complication is affecting 50–60% of diabetic patients globally (Yerra et al. 2017; Tesfaye et al. 2010). This complication of diabetes is marked by gradual and slow diminution of nerve fibers leading to loss of sensation, foot ulceration, and to limb amputation in advance stages (Tesfaye and Selvarajah 2012). Currently, the specific mechanisms involved in the development of DN are not clearly understood. Moreover, the treatment options in addition to strict glucose control by anti-diabetic drugs include anticonvulsants (pregabalin) and antidepressants (duloxetine, tapentadol) (Callaghan et al. 2012). Evidences have shown glycemic control to delay the progression of diabetes but no impact on long-term diabetic complications as described in the metabolic memory concept (Yerra and Kumar 2017; Testa et al. 2017).

There are several reports which have postulated involvement of reactive oxygen species (Du et al.) in triggering the major pathophysiological pathways implicated in DN (Sandireddy et al. 2014; Vincent et al. 2005). Hyperglycemia-induced electron leakage from mitochondria increases shuttling of energy equivalents (NADH and FADH2) through electron transport chain (ETC) and results in generation of error prone ROS/superoxides which oxidize proteins, lipids, and nucleic acids (Rolo and Palmeira 2006; Cadenas et al. 1977; Li et al. 2015). These effects could further get aggravated with compromised antioxidant defense mechanisms including superoxide dismutase (SOD), NADPH dehydrogenase quinone 1 (NQO-1), and heme oxygenase-1 (HO-1). Mitochondrial structures are vulnerable to oxidative damage, and its dysfunction could further result in a futile ETC cycle with reduced ATP and increased ROS generation (Vincent and Feldman 2004; Russell et al. 2002). In order to maintain functional homeostasis, new mitochondria need to be generated (biogenesis) to replace damaged or aged mitochondria. Recent investigations in our laboratory and by others have shown defective mitochondrial biogenesis to underlie in the pathogenesis of diabetic complications (Vincent and Feldman 2004; Kalvala et al. 2020a). Hence, therapeutic strategies aiming at simultaneous stimulating of antioxidant defense machinery and mitochondrial biogenesis could be beneficial for the treatment of diabetic complications.

The silent information regulator-1 (sirtuin, SIRT1), a nicotinamide adenosine dinucleotide (NAD+) dependent deacetylase acts as a bioenergetic sensor which respond to changes in energy status of cell (NAD+: NADH ratio) (North and Verdin 2004). The SIRT1 enzyme deacetylates histone proteins and transcription factors initiating downstream processes of various cellular functions including mitochondrial biogenesis and antioxidant defense mechanism (North et al. 2003; Yerra et al. 2017). The activity of SIRT1 to deacetylate peroxisome proliferator activated receptor-gamma coactivator-1α (PGC-1 α) and induce healthy mitochondrial generation have been previously reported (Nemoto et al. 2005). Studies have also shown that SIRT1 deacetylate nuclear factor-erythroid 2- related factor (Nrf2) which in turn activates transcription of genes coding antioxidant enzymes (NQO-1, HO-1, SOD) (Huang et al. 2017; Gong et al. 2017). Hence, the SIRT1 activators could be considered as a potential treatment option for the management of DN.

Polydatin, a stilbenoid glucoside, is a major resveratrol derivative with known anti-inflammatory, antioxidant, and neuroprotective properties (Lanzilli et al. 2012; Ji et al. 2012). It is also documented that polydatin potentially activates SIRT1 and Nrf2 (Ma et al. 2016; Gao et al. 2016). This preclinical, randomized, and interventional study was conducted to assess if polydatin has any neuroprotective effects in in vivo and in vitro DN models and if so, whether such effects might involve SIRT1-directed mitochondrial biogenesis and Nrf2-mediated antioxidant protection.

Materials and Methods

Drugs and Chemicals

Unless specified, all the chemicals were obtained from Sigma-Aldrich Ltd, USA.

Mouse Neuroblastoma Cells

Mouse neuroblastoma cells (Neuro2a) were procured from National Centre for Cell Science Pune, India, and cultured in minimum essential medium (MEM). The medium was supplemented with 5.5 mM glucose and 10% fetal bovine serum, 1% streptomycin/penicillin, and 2 mM glutamine. The cultured Neuro2a cells were maintained at 37 °C temperature and 95% air humidity and 5% CO2. As established previously, high-glucose condition (30 mM glucose concentration) was stimulated by adding 25 mM glucose to MEM for simulating short-term hyperglycemia in the cultured neuronal cells (Aghdam et al. 2013).

Methods

MTT Assay for Cell Viability

Neuro2a cells were cultured in 96-well plate at 10,000 cells/well density for a period of 24 h in CO2 incubator. After incubation, cells were given a polydatin treatment of serial concentrations (6.25 μM–200 μM) and incubated for more 24 h. Later, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; 5 mg in 10 ml of MEM) was added into the wells and maintained for 4 h at 37 °C. The medium was later removed and crystals were solubilized using dimethyl sulfoxide (DMSO). Absorbance was measured at 570 nm.

Assessment of Neurite Outgrowth

Neuro2a cells were cultured in a 12-well plate at 5000 cells/well density for 24 h. After 24-h incubation, cells were exposed to 30 mM glucose and polydatin (10 and 20 µM). Five fields with approximately 100–250 cells/well were selected randomly and examined under phase contrast microscope (Nikon ECLIPSE, Ti-U, Japan). The Image J software (NIH, USA) was used to measure the length of neurite outgrowths in 30 cells of each chosen field. The number of neurite outgrowths/axon like extensions which are double or more than the diameter of cell body were noted. The percentage of neurite-bearing cells is calculated by dividing the number of neurite-bearing cells by the total number of cells and then multiplied by 100 (Phan et al. 2013).

DCFDA Staining to Measure Intracellular ROS

Intracellular ROS levels were measured using a non-fluorescent probe, H2DCF-DA (2′,7′-dichlorodihydrofluorescein diacetate, Sigma-Aldrich), which gives green fluorescence in the presence of ROS. Neuro2a cells were cultured in 24-well plate at 4000 cells/well density for 24 h. Subsequently, cells were treated with 30 mM glucose and 10 and 20 µM polydatin for 6 h. The cells were then treated with 10 μM DCFDA stain (Sigma-Aldrich) for 15 min and fluorescence images were captured using a Nikon 80i fluorescence microscope (Bass et al. 1983).

Detection of Mitochondrial Superoxides Using MitoSOX Staining

MitoSOX Red reagent is a fluorogenic dye specifically designed to target the mitochondria in cells under live condition. Oxidation of MitoSOX reagent by superoxide yields red fluorescence. Therefore, the intensity of red fluorescence produced is directly proportional to the mitochondrial superoxides. Briefly, 24-well plate was seeded with Neuro2a cells (4000 cells/well) with MEM for 24 h. These cells were then exposed to 30 mM glucose, and 10 and 20 µM polydatin for 6 h. The cells were then incubated with 5 μM MitoSOX for 10 min at 37 °C. This step is followed by washing the cells twice with phosphate buffer saline (PBS) and processed for imaging under a fluorescence microscope (Massicot et al. 2013).

Detection of Mitochondrial Membrane Potential (Δψm) Using JC-1 Stain

Briefly, JC-1 stain exists in monomeric and dimeric form. Dimeric form exhibits orange fluorescence indicates the aggregated form of JC-1 (negatively polarized mitochondria) and green color is that of monomeric form (depolarized mitochondria). Neuro2a cells were seeded in a 6-well plate (4 × 104 cells/well) for 24 h. Post 24 h, 30 mM glucose, and 10 and 20 µM polydatin treatment was given for 6 h. After 6 h of incubation, these cells were treated with freshly prepared JC-1 (1 μg/ml) in PBS for 20 min. Thereafter, the cells were washed with PBS and observed under a fluorescent microscope for red color fluorescence (Massicot et al. 2013).

Western Blotting in Neuro2a Cells

In brief, Neuro2a cells were exposed to 30 mM glucose and treated with 5, 10, and 20 µM polydatin. After 24-h incubation, proteins were isolated using RIPA reagent. The cellular lysates were subjected to centrifugation at 10,000 RPM for 8 min at 4 °C. Supernatant was collected and total protein content estimated using bicinchonic acid assay (BCA) kit. Equal amount of proteins across the groups were resolved using SDS–PAGE (10%) gel electrophoresis. Separated proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane. After successful transfer of proteins on PVDF membrane, blocking was done using 3% bovine serum albumin (BSA). Blocking was followed by addition of primary rabbit monoclonal antibodies, Nrf2, SIRT1, TFAM, PGC-1α, NRF1, SOD2, and NQO-1 (1:1000; Cell Signaling Technology, USA) and incubated for overnight at 4 °C. The following day, TBST was used to wash the membranes which were subsequently incubated with HRP conjugated secondary mouse/rabbit antibodies (1:20,000; Cell Signaling Technology, USA). Fusion-FX chemiluminescence imager (Vilber Lourmat, Germany) was used to visualize the bound antibodies. Image J software (NIH, USA) was used to quantify relative band densities.

Real Time-Polymerase Chain Reaction

A 6-well plate was used to culture Neuro2a cells (4 × 105 cells/well) overnight. After overnight incubation, 30 mM glucose and polydatin treatment (10 and 20 µM) was given to the cells and incubated for 48 h. After incubation, total RNA was isolated by RNeasy Mini kit (Qiagen, USA). Verso cDNA synthesis kit (Thermo Scientific) was used to produce cDNA using reverse transcription from 2 μg total RNA. Primers express software (version 3.0.17) was used to design the primers. The sequences of primers used for RT-PCR are as follows: mouse TFAM: forward, 5′-CCAGTTAGGTCTCAAAGCCT-3′ and reverse, 5′-CAGTCTCCGTTCCAGTTCTT-3′; mouse NRF2: forward, 5′-GAGTCCTGACACTGTGACAA-3′ and reverse, 5′-CTGAATTGCACCATCCACAG-3. RT-PCR was performed on the Light Cycler (AB applied biosystem, USA) using SYBR green master mix. The following reaction components: 2 μl of primer mix (1 µl forward primer, 1 µl reverse primer), 1 μl of cDNA, 10 μl of 2 × SYBR green master mix (Invitrogen, Life Technologies), and nuclease free double distilled water (to a final volume of 20 μl) were prepared to indicated concentrations. RT-PCR was performed on the cDNA samples in triplicate. Briefly, samples were first denatured (5 min; 95 °C). Further, 4 cycles of 15 s each were performed at 94 °C followed by annealing for 20 s at 47 °C and extension for 25 s at 72 °C. Comparative Ct (ΔCt) method was used to assess relative gene expression and normalized to β-actin.

Immunofluorescence Analysis

Briefly, 30 mM glucose was added to the Neuro2a cells and then treated with 20 µM polydatin for 24 h. MitoTracker® Deep Red FM dye (200 nM; Molecular Probes, Invitrogen) was used to stain the cells for 30–45 min. Cells were given PBS washing and fixed in 4% paraformaldehyde (prepared in PBS) for 5 min at room temperature. 0.5% Triton- × 100 was used to permeabilize the cells for 10 min. Later, cells were washed and subjected to blocking using 3% BSA for 1 h at room temperature. Thereafter, primary antibody solution (1:100) of anti-TFAM antibody (rabbit polyclonal, Cell Signaling Technology) in PBS was added to the cells. A day later the cells were given three washings of PBS and then FITC conjugated anti-rabbit secondary antibody solution (1:100) was added and kept for 2 h in the dark. The cells were then PBS washed, air-dried, and mounted with DAPI (Invitrogen) on a glass slide. Finally, SP8 version of Leica confocal microscope (Germany) was used to perform confocal imaging of the slides.

Animals and Experimental Design

Healthy male Sprague–Dawley (SD) rats weighing 250–300 g were fed standard diet and water ad libitum. Plastic cages were used to house the rats. Temperature was maintained at 24 ± 1 °C and humidity at 55 ± 5% with 12 h light–dark cycle. All the experiments were compliant with the regulations of Institutional Animal Ethics Committee (IAEC)-NIPER-Hyderabad, India (Protocol No: NIP/8/2016/PC/213).

Streptozotocin (STZ; 55 mg/kg; intraperitoneal dose) was administered to SD rats to induce diabetes. After 48 h, the rats with plasma glucose levels of ≥ 250 mg/dl were considered as diabetic and selected for the study. The selected rats were randomized (1:1:1:1:1) to one of the five groups: non-diabetic rats (ND), ND treated with polydatin 50 mg/kg (ND + PD-50 mg/kg), diabetic control (STZ-D), diabetic rats treated with polydatin 25 mg/kg (STZ-D + PD-25 mg/kg) and diabetic rats treated with polydatin 50 mg/kg (STZ-D + PD-50 mg/kg). The experimental design and dosage regimen was selected based on the previous reports (Kumar et al. 2012). Polydatin suspension was made in 0.5% sodium carboxy methyl cellulose (CMC) and was administrated to rats (25 and 50 mg/kg, oral).

3.10 Functional Parameters

Motor Nerve Conduction Velocity and Sensory Nerve Conduction Velocity

Power lab 4/35(AD Instruments, Australia) was used to assess motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) in posterior sciatic-tibial conduction system. Isoflurane gaseous anesthesia system was used to anaesthetize the rats. Needle electrodes were inserted at the sciatic notch and Achilles tendon and a three-volt stimulus was applied on the sciatic nerve. Bipolar surface receiving electrodes were placed near the interosseous foot muscle of rat. After the stimulations, both M-wave and H-wave reflex latencies were measured. The physical distance (between the stimulation electrode and receiving electrodes) was divided by the time latencies of M and H wave, respectively, to obtain MNCV and SNCV in m/s (Yerra and Kumar 2017).

Nerve Blood Flow

LASER Doppler system (Moor Instruments, UK) was used to measure the nerve blood flow (NBF) to the sciatic nerves (Kumar et al. 2012). Briefly, rats were anaesthetized and left flank was exposed to visualize the sciatic trunk and made free from the surrounding tissue. Normal saline was applied to exposed site to avoid dehydration of the exposed nerve. Then tip of the laser probe was placed in contact with sciatic trunk. The NBF was reported in arbitrary perfusion units by measuring the flux for 10 min for each rat.

3.11 Behavioral Parameters

Thermal Hyperalgesia

Rats were acclimatized to the room prior to the experiment and stimulated with both hot (45 °C) and cold (10 °C) stimuli. The time taken by rat, i.e., tail flick response latency was recorded as endpoint response. Cutoff time of 15 s was kept in both tests. A total of five readings were taken at an interval of 10 min (Narenjkar et al. 2011) for each rat.

Mechanical Hyperalgesia

Randall sellitto callipers (IITC Life Sciences, USA) were used to measure the mechanical hyperalgesia (Sandireddy et al. 2016). The force causing paw withdrawal was noted down in grams. A total of five readings were taken with an interval of 5 min. Mechanical hyperalgesia was also assessed using Vonfrey aesthesiometer and Hargreaves test.

Western Blotting Analysis

Protein lysates were prepared by homogenizing sciatic nerves with tissue protein extraction reagent containing phosphatase and protease inhibitors (1:100). SDS-PAGE was used to separate proteins across all groups. Volumes containing equal amount of proteins were used. Electrophoresis was followed by blotting as mentioned above in Neuro2a cells.

Statistical Analysis

All the results are expressed as mean ± standard error mean (SEM). The comparisons between the different rat groups were made with “Bonferroni's Multiple Comparison Test”. The intergroup differences was defined by one way analysis of variance (ANOVA) using the Graph Pad Prism. Results with p < 0.05 were considered as statistically significant.

Results

In Vitro Results

Polydatin has found to produce significant neurotoxicity at concentrations greater than 25 µM (IC50: 119 µM). From the MTT results obtained and based on the available studies on polydatin, we have chosen 10 and 20 μM doses of polydatin to assess its neuroprotective potential in high-glucose-exposed Neuro2a cells which simulate short-term hyperglycemic condition (Supplementary Fig S1).

Polydatin Treatment Alleviated Oxidative Stress and Mitochondrial Dysfunction in Short-Term Hyperglycemic Neuro2a Cells

Non-fluorescent DCFDA probe gives green fluorescence in the presence of ROS. We observed increased green fluorescence in high-glucose-exposed Neuro2a cells (indicating increased ROS production) compared to normal Neuro2a. Polydatin co-treatment at 10 and 20 μM concentration reduced ROS generation as shown in Fig. 1 (first panel).

Fig. 1.

Fig. 1

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Effect of high-glucose-exposure and polydatin treatment on intracellular ROS generation, mitochondrial superoxide generation and mitochondrial membrane potential: First panel Images showing DCF fluorescence ROS generation in Neuro2a cells under untreated control, high-glucose and polydatin treatment conditions. Second panel Fluorescent microscopic images showing MITOSOX fluorescence in different groups of Neuro2a cells. Third panel: Fluorescent microscopic images showing the mitochondrial membrane potential (JC-1 fluorescence) in different groups of Neuro2a cells. NC normal Neuro2a cells, PD-20 normal Neuro2a cells incubated with polydatin at 20 µM, HG high-glucose (30 mM) exposed Neuro2a cells, HG + PD-10 and HG + PD-20 high glucose-insulted cells co-incubated with polydatin at 10 and 20 μM, respectively

MITOSOX gives red fluorescence in the presence of mitochondrial specific superoxides. High-glucose (30 mM) exposed Neuro2a cells significantly enhanced the production of mitochondrial specific superoxide as evidenced by elevated red fluorescence. The elevated red fluorescence was subsequently reduced by both doses of polydatin (10 and 20 μM) treatment (second panel).

In JC straining, the orange fluorescence indicated the aggregated form of JC-1 (negatively polarized mitochondria) and the green color indicated monomeric form (depolarized mitochondria). Increased mitochondrial membrane potential was observed in hyperglycemia-induced Neuro2a cells as evidenced by intensive formation of JC-1 monomers (green color) in cytosol. Treatment with polydatin (10 and 20 µM) restored negative membrane potential of mitochondria (third panel) (Fig. 1).

Treatment with Polydatin Augmented Neuritogenesis in Short-Term Hyperglycemic Neuro2a Cells

We examined whether reduced mitochondrial superoxide generation by polydatin results in increased ATP production or not. We examined these effects by indirect means of assessing the neuritogenesis which is a high energy demanding (ATP) process. Our studies have clearly indicated that exposure to 30 mM glucose for 24 h reduced percent neurite-bearing cells (15.9 ± 0.84 vs. 34.06 ± 1.24, p < 0.01) and neurite length (53.3 ± 1.45 μM vs. 84.66 ± 0.98 μM, p < 0.01) as compared to normal Neuro2a cells. Mean neurite length (p < 0.001) and mean neurite-bearing cells significantly increased (p < 0.00 ≈ three folds) with polydatin treatment when compared to high -glucose-exposed Neuro2a cells indicating increased ATP availability under polydatin treated conditions (Fig. 2).

Fig. 2.

Fig. 2

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Effect of polydatin on neuritogenesis in high-glucose-exposed Neuro2a cells: Morphological images of Neuro2a cells showing neurite extensions in different groups, graphical representation of average neurite length (μM) and percentage neurite-bearing cells. Values are expressed as mean ± SEM (n = 5). NC normal Neuro2a cells, PD-20 normal Neuro2a cells incubated with polydatin at 20 µM, HG high-glucose (30 mM) exposed Neuro2a cells, HG + PD-10 and HG + PD-20 high-glucose-insulted cells co-incubated with polydatin at 10 and 20 μM, respectively. ^^p < 0.01 vs. NC, ***p < 0.001 vs. HG

Polydatin Treatment Elevated the Nrf2-Mediated Antioxidant Defenses in High-Glucose-Exposed Neuro2a Cells for 24 h

To examine the mechanism responsible for reduced oxidative stress by polydatin, we carried out immunoblotting studies in Neuro2a cells lysates. We have evaluated Nrf2 expression, since it is a unique transcription factor responsible for antioxidant enzymes production like SOD2, NQO-1 and HO-1. We found that high-glucose exposure resulted in substantial decrease in the level of Nrf2 (p < 0.001) than in the normal Neuro2a cells. As a result, high-glucose exposure also impaired downstream antioxidant defense enzyme levels such as NQO1 (p < 0.001) and SOD2 (p < 0.05). Co- polydatin treatment significantly increased Nrf2 expression (p < 0.001, at 5, 10, and 20 μM concentration) and its downstream effector proteins, NQO1 (p < 0.001 at 5, 10 ,and 20 μM concentration) and SOD2 (p < 0.01 at 10 μM and p < 0.001 at 20 μM concentration) under high-glucose conditions (Fig. 3).

Fig. 3.

Fig. 3

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Effect of polydatin on Nrf2-mediated antioxidant defense and SIRT1-mediated mitochondrial biogenesis in high-glucose-insulted Neuro2a cells: Immunoblots showing the expression pattern of Nrf2, NQO1 and SOD proteins in Neuro2a cells. Graphical images showing the densitometric analysis of respective immunoblots. Values are expressed as mean ± S.E.M. NC normal Neuro2a cells, PD-20 normal Neuro2a cells incubated with polydatin at 20 µM, HG high-glucose (30 mM) exposed Neuro2a cells, HG + PD-5, HG + PD-10 and HG + PD-20 high-glucose-insulted cells co-incubated with polydatin at 5, 10 and 20 μM, respectively. ^p < 0.05, ^^p < 0.01, ^^^p < 0.001 vs. NC, *p < 0.05, **p < 0.01, ***p < 0.001 vs. HG

Polydatin Promoted Mitochondriogenesis in Short-Term Hyperglycemic Neuro2a Cells by SIRT1 Activation

Finally, we have assessed the effect of polydatin on mitochondrial biogenesis by conducting immunoblotting and immunofluorescence studies. High-glucose condition leads to reduced SIRT1 expression (p < 0.05). Polydatin treatment at 20 μM concentration significantly (p < 0.001) improved SIRT1 expression. High-glucose exposure also leads to decreased expression of PGC-1α (p < 0.01), NRF1 (p < 0.01), and TFAM (p < 0.001) proteins. Simultaneous polydatin treatment significantly increased the expression of PGC-1α (p < 0.01), NRF-1 (p < 0.001), and TFAM (p < 0.05) proteins at 20 μM dose (Fig. 4a). These results were further validated by studying gene expression changes by qRT-PCR. Short-term hyperglycemia-induced Neuro2a cells significantly reduced mRNA expression of TFAM and nuclear respiratory factor 2 (NRF2) (p < 0.05) in Neuro2a cells when compared to their expression at normal level. Polydatin exposure especially at 20 µM significantly elevated the expression of mRNA levels of TFAM (p < 0.001) and NRF2 (p < 0.001) (Fig. 4b). We have finally confirmed polydatin-induced mitochondriogenesis by studying the colocalization of TFAM with mitochondria using immunofluorescence analysis. High-glucose-insulted Neuro2a cells showed a substantial reduction in TFAM expression and its colocalization with mitotracker when compared to untreated control. Polydatin co-treatment with glucose stimulus leads to significant increase in TFAM expression as well as its colocalization with mitotracker as indicated by increased yellow color (indicates the degree of colocalization) in treatment conditions (Fig. 4c).

Fig. 4.

Fig. 4

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Effect of polydatin on SIRT1-mediated mitochondrial biogenesis in high-glucose-insulted Neuro2a cells: a Immunoblots showing the expression pattern of SIRT1, PGC-1α, NRF-1 and TFAM proteins in the cellular lysates of Neuro2a cells. Graphical images showing the densitometric analysis of respective immunoblots. b Graphical images showing mRNA expression of NRF2 and TFAM analyzed by RT-PCR. Values are expressed as mean ± S.E.M. c Confocal images representing mitochondrial number (Mitotracker red) and TFAM (FITC) immunolocalization in Neuro2a cells. DAPI (blue color) indicated the nucleus. The yellow color in merged image indicates the colocalization of TFAM with mitochondria. Results are expressed as mean ± SEM (n = 3). NC:normal Neuro2a cells, PD-20 normal Neuro2a cells incubated with polydatin at 20 µM, HG high-glucose (30 mM) exposed Neuro2a cells, HG + PD-5, HG + PD-10 and HG + PD-20 high-glucose-insulted cells co-incubated with polydatin at 5, 10 and 20 μM, respectively. ^p < 0.05, ^^p < 0.01, ^^^p < 0.001 vs. NC, *p < 0.05, **p < 0.01, ***p < 0.001 vs. HG

In Vivo results

Single dose of STZ administration resulted in elevated levels of plasma glucose and gradually lowered body weights in the SD rats. Polydatin treatment at two dose level (25 and 50 mg/kg, oral) for last 2 weeks of total 8 week study has no significant effect on plasma glucose levels and body weights of SD rats.

Polydatin Administration Reversed the Nerve Functional Defects Associated with Chronic Diabetic Rats

Eight weeks STZ-induced diabetic rats displayed significant reduction in MNCV (37.33 ± 2.6 m/s vs. 62.33 ± 4.80 m/s, p < 0.01) and SNCV (34.60 ± 2.6 m/s vs. 55.66 ± 1.76 m/s, p < 0.05) when compared to the healthy control rats. Diabetic rats also showed compromised NBF (38.16 ± 2.84 PU vs. 98.93 ± 2.14 PU, p < 0.05) when compared to the control group rats. Polydatin-treated diabetic rats significantly improved MNCV (63.73 ± 2.19 PU vs. 37.33 ± 2.60 PU, p < 0.01) at 50 mg/kg, SNCV at both 25 mg/kg (48.23 ± 5.2 PU vs. 34.60 ± 2.6 PU, p < 0.01) and 50 mg/kg (62.18 ± 5.2 PU vs. 34.60 ± 2.6 PU, p < 0.001), and NBF (p < 0.05) at 50 mg/kg (Fig. 5a). Eight-week-old diabetic rats displayed the features of thermal and mechanical hyperalgesia as evident by reduced tail and paw withdrawal latencies toward thermal and mechanical stimuli. Polydatin treatment for 2 weeks at both dose level (25 mg/kg and 50 mg/kg) significantly improved tail and paw withdrawal thresholds in diabetic rats (Fig. 5b).

Fig. 5.

Fig. 5

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Effect of polydatin treatment on nerve functional changes and neuropathic pain in STZ-induced rats: a Graphical depictions representing the effect of polydatin administration on motor and sensory nerve conduction velocities (MNCV and SNCV) and neuronal blood flow (NBF). Values are expressed as mean ± SEM (n = 6). b Graphical depictions representing the effect of polydatin administration on thermal (hot immersion test, cold immersion test, Hargreaves test) and mechanical (Vonfrey test and Randal selitto test) stimuli. Values are expressed as mean ± SEM (n = 6). ND non-diabetic, ND + PD-50 non-diabetic rats treated with 50 mg/kg polydatin, STZ diabetic, STZ + PD25 and STZ + PD-50 are diabetic rats treated with polydatin at 25 and 50 mg/kg, respectively. ^p < 0.05, ^^p < 0.01, ^^^p < 0.001 vs. ND and *p < 0.05, **p < 0.01, ***p < 0.001 vs. STZ

Polydatin Treatment Elevated the Nrf2-Mediated Antioxidant Defenses in STZ-Induced Diabetic Neuropathy

Similar to in vitro studies, chronic diabetes in STZ-induced diabetic rats significantly reduced expression of antioxidant defense enzymes such as NQO-1 (p < 0.001), HO-1 (p < 0.01) and SOD2 (p < 0.05) proteins in peripheral nerves when compared to their levels in age matched healthy counterparts. Polydatin treatment at 50 mg/kg for 2 weeks significantly elevated the expression of these antioxidant enzymes NQO-1 (p < 0.001), HO-1 (p < 0.001) and SOD2 (p < 0.01) in sciatic nerve lysates when compared to the diabetic rats (Fig. 6).

Fig. 6.

Fig. 6

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Effect of polydatin on Nrf2-mediated antioxidant defense in peripheral nerves of STZ-induced rats: Immunoblots showing the expression patterns of NQO-1, HO-1, SOD and β-actin protein in sciatic nerve lysates of normal and STZ-induced rats. Graphical representations indicate densitometric analysis for respective western blots.). Values are expressed as mean ± S.E.M (n = 6). ND non-diabetic, ND + PD-50 non-diabetic rats treated with 50 mg/kg polydatin STZ diabetic, STZ + PD-50 diabetic rats treated with polydatin 50 mg/kg. ^p < 0.05, p < 0.01, ^^^p < 0.001 vs. ND, **p < 0.01, ***p < 0.001 vs. STZ

Polydatin Promoted Mitochondriogenesis in Peripheral Nerves of STZ-Induced Diabetic Rats by SIRT1 Activation

To obtain an in vivo evidence for mitochondrial biogenesis improvement by polydatin, we carried out immunoblotting experiments in the protein lysates prepared from sciatic nerve of STZ-induced diabetic rats. Diabetic rats showed a profound reduction in SIRT1 (p < 0.001) protein expression as compared to the normal rats. This reduction in SIRT1 significantly brought down the levels of PGC-1α (p < 0.001), NRF2 (p < 0.001) and TFAM (p < 0.001) proteins in the sciatic nerves of STZ-induced diabetic rats when compared to the normal control rats. Polydatin treatment reversed these effects by significantly restoring the expressions of SIRT1, PGC-1α (p < 0.001), NRF2 (p < 0.001) and TFAM (p < 0.001) (Fig. 7). Administration of polydatin to normal control rats (perse) has shown neutral effect in altering these endogenous protein expressions when compared to healthy untreated rats.

Fig. 7.

Fig. 7

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Effect of polydatin on SIRT1-mediated mitochondrial biogenesis in peripheral nerves of STZ-induced rats: Immunoblots indicating the expression of SIRT1, PGC-1α, NRF2, TFAM and β-actin protein in sciatic nerve lysates of normal and STZ-induced rats. Graphical representations indicate densitometric analysis for respective western blots. Values are expressed as mean ± S.E.M (n = 3). ND non-diabetic, ND + PD-50 non-diabetic rats treated with 50 mg/kg polydatin, STZ diabetic, STZ + PD-50 diabetic rats treated with polydatin 50 mg/kg. ^p < 0.05, p < 0.01, ^^^p < 0.001 vs. ND, **p < 0.01, ***p < 0.001 vs. STZ

Discussion

Mitochondrial dysfunction and associated oxidative stress plays a crucial role in the pathophysiology of short- and long-term DN. Several phytochemical molecules have previously shown to ameliorate DN in both in vivo and in vitro DN models by virtue of their effects on oxidative stress (Yerra et al. 2017; Sandireddy et al. 2016; Vincent et al. 2005). The SIRT1 enzyme activation is known to have various health benefits in several metabolic diseases and chronic age related diseases (Imai and Guarente 2014; Kalvala et al. 2020b). Phytochemical modulators of SIRT1 have shown promising therapeutic outcomes by augmenting mitochondrial biogenesis and abrogating the oxidative stress in target tissues (Lagouge et al. 2006). Polydatin is known to possess SIRT1 modulatory activity and Nrf2 activation efficacy in various disease models (Zeng et al. 2015; Chen et al. 2016). In our study, we have assessed if polydatin possess any neuroprotective effects in DN and whether such effect is exerted through its SIRT1 and Nrf2 modulatory potential in both in vivo and in vitro DN models.

We have identified that polydatin treatment reversed the high-glucose-induced mitochondrial dysfunction and ROS generation by promoting SIRT1-mediated mitochondrial biogenesis and Nrf2-mediated antioxidant signaling, respectively, as evidenced from the study results. Findings from this study confirm that deficits in mitochondrial biogenesis have a vital role in the pathogenesis of DN and SIRT1 activation reversed this condition by inducing mitochondrial biogenesis. These findings are in line with our previous reports (Yerra and Kumar 2017; Kalvala et al. 2020b). The current study stands as a preliminary indication that polydatin may be a SIRT1 inducer in experimental DN.

The diffusion-mediated process of glucose entry in neuron induces high susceptibility to hyperglycaemic stress (Vannucci et al. 1997). Further, neurons are equipped with mild antioxidant defense systems increasing their susceptible to hyperglycemic damage (Friedman 2011). In either of the short- or long-term hyperglycemic condition, increased glycolysis and TCA cycle results in generation of elevated levels of reducing equivalents like NADH, FADH2 (Du et al. 2000). These reducing equivalents undergo oxidation in ETC, yielding an electrochemical gradient across the inner mitochondrial membrane which is involved in ATP production (Kalvala et al. 2019). However, the electron transfer associated with increased electron leakage generating higher levels of superoxide can form highly toxic reactive free radicals (Nishikawa et al. 2000). These toxic radicals might cause DNA damage and alter the membrane potential of mitochondria and in turn induce neuronal apoptosis. In the present study, short-term high-glucose exposure in Neuro2a cells has shown increased levels of mitochondrial superoxide’s and depolarization of mitochondrial membrane, the features of mitochondrial dysfunction. Polydatin treatment attenuated these high-glucose-induced superoxide generation and mitochondrial membrane depolarisation as evidenced by reduced superoxide generation. These effects of polydatin have clearly indicated its mitoprotective effects probing us to evaluate its mechanistic basis further.

It has been reported earlier that diminished Nrf2-mediated antioxidant enzymes cause an elevation in oxidative stress which is one of the factor involved in the pathogenesis of DN (Xu et al. 2012; Negi et al. 2011). In this study, densitometric analysis of western blots clearly revealed that Nrf2 expression and its downstream effector enzyme levels HO-1, NQO1 were reduced in both in vitro and in vivo hyperglycemic condition. Consistent to our study findings, few reports have indicated short- and long-term hyperglycemic condition to expedite the ROS production by downregulating the antioxidant defensive mechanisms (Nrf2-ARE axis) (Yu et al. 2006; Kim and Vaziri 2009). In the present study, polydatin treated diabetic rats and polydatin treated high-glucose exposed Neuro2a cells have shown increased expression of antioxidant enzymes like HO-1, NQO-1 and SOD2. Supporting evidence also articulated polydatin to enhance Nrf2-ARE pathway in rat mesangial cells (AGE) (Huang et al. 2015). These results clearly indicate the antioxidant effects of polydatin, which might have contributed for its therapeutic potential against neuronal damage associated with metabolic excess in experimental DN.

Neurons require continuous supply of ATP for sprouting through which they maintain connectivity with the neighboring cells for transmission of chemical signals (Chang and Reynolds 2006). Several studies indicate short- and long-term hyperglycemic condition leading to bioenergetic deprivation of neurons through reduced mitochondrial ATP production (Malone et al. 2008; Kalvala et al. 2020b) and any reduction in energy production would result in compromised dendritic arbouring and other neuroplastic changes. We also observed that metabolic excess in Neuro2a cells to have severely compromised neuritogenesis than normal control cells, validating the metabolic excess-mediated bioenergetic deficits in the pathogenesis of DN. In polydatin treated cells (10 and 20 µM), there was considerable increase in the length of neurite and as well in the percentage neurite-bearing cells when compared to the 30 mM glucose exposed cells for 24 h. These findings clearly suggest that polydatin may have neuroprotective effects against high-glucose-induced neurotoxicity by monitoring the cellular homeostasis in terms of balancing oxidative stress and mitochondrial function.

Polydatin treatment significantly promoted the SIRT1 levels in both sciatic nerves of diabetic rats and in short-term high-glucose exposed Neuro2a cells. Increased SIRT1 expression in turn resulted in activation and up regulation of PGC-1α and are considered as a master regulator of mitochondrial biogenesis (Fernandez-Marcos and Auwerx 2011). SIRT1 is a transcriptional coactivator promoting the transcriptional activation of nuclear respiratory factors (NRF1, NRF2) which are involved in the synthesis of respiratory chain components and mitochondrial transcription factors (TFAM, TFB1M) (Li et al. 2013). These mitochondrial transcription factors in turn might initiate mitochondrial DNA replication increasing mitochondrial mass (Fernandez-Marcos and Auwerx 2011). Polydatin treatment in diabetic rats and short-term high-glucose exposed Neuro2a cells has shown significant rise in the protein level of PGC-1α and TFAM. These results evidence that SIRT1 activation by polydatin resulted in increased PGC-1α levels in nerves which might lead to PGC-1α-mediated mitochondrial biogenesis. Moreover, polydatin administration significantly attenuated high-glucose-induced mitochondrial biogenetic deficits via promoting the gene expression of TFAM and NRF2, the transcription factors essential for mitochondrial biogenesis (Li et al. 2017). Several experimental studies supported SIRT1 deacetylation of Nrf2 and its dissociation from KEAP1 complex in the cytoplasm. The dissociated Nrf2 translocates into the nucleus at its promoter region and binds with its co-transcription factors regulating the expressions of antioxidant defensive enzymes (Tamaki et al. 2014). Based on the present study results and reported literature, we can say that polydatin reduces neuropathic pain via boosting mitochondrial function by Sirt1-Nrf2-PGC-1α axis (Fig. 8).

Fig. 8.

Fig. 8

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Probable mechanism of neuroprotection by polydatin in experimental diabetic neuropathy: Hyperglycemia driven metabolic flux in glycolysis and TCA cycle results in increased NADH generation, inactivating the enzymatic activity of SIRT1 which further inhibiting PGC-1α (master regulator)-mediated mitochondrial biogenesis. SIRT1 activation by polydatin deacetylates PGC-1α facilitating its transcription and NRF1-mediated mitochondrial biogenesis. Activated SIRT1 also deacetylates and promotes the transcription of Nrf2, facilitating antioxidant enzyme production which can neutralize elevated levels of ROS in hyperglycemic condition. On the other hand, polydatin can also directly activates Nrf2 signaling. ARE advanced glycation end products, ETC electron transport chain, HO-1 heme oxygenase-1, NRF1 nuclear respiratory factor-1, Nrf2 nuclear factor-erythroid 2- related factor, NQO-1 NADPH dehydrogenase quinone 1, PGC-1α peroxisome proliferator activated receptor-gamma coactivator-1α, ROS reactive oxygen species, SIRT1 silence information regulator 2 homologue 1, SOD superoxide dismutase, TFAM Mitochondrial DNA transcription factor A

Our study results demonstrated increased oxidative burden and mitochondrial dysfunction by SIRT1 inactivation and their association with short- and long-term hyperglycemia to underlie the pathogenesis of neuronal dysfunction in experimental DN. Results from in vitro findings have proven polydatin 10 and 20 µM to increase the neurite outgrowth, attenuate high-glucose-mediated ROS generation, and revert mitochondrial membrane potential. In vivo studies revealed 2 weeks polydatin 25 and 50 mg/kg administration to restore behavioral and functional deficits in STZ-induced diabetic rats. Polydatin treatment in both short-term hyperglycemic Neuro2a cells and STZ-induced long-term diabetic rats increased the levels of proteins responsible for mitochondrial biogenesis and antioxidant defense mechanisms. These observations imply that polydatin can be used as an adjuvant therapy against the various manifestations associated with DN.

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Acknowledgements

Authors sincerely acknowledge the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India for providing financial support in carrying out this work.

Author contributions

MS Preethi Bheereddy (Department of Pharmacology and Toxicology, NIPER-HYDERABAD, Telangana, India-500037), performed studies, evaluated data, prepared figures and wrote manuscript; Dr Veera Ganesh Yerra (Postdoctoral Fellow at Keenan Research Centre for Biomedical Sciences, St. Michael's Hospital, Toronto, Canada), performed studies, evaluated data, prepared figures and wrote manuscript; Mr Anil Kumar Kalvala (Department of Pharmacology and Toxicology, NIPER-HYDERABAD, Telangana, India-500037), performed studies, evaluated data, prepared figures and wrote manuscript; Ms Bhoomika S (Department of Pharmacology and Toxicology, NIPER-HYDERABAD, Telangana, India-500037), performed studies, evaluated data, prepared figures and reviewed manuscript; Dr Ashutosh Kumar (Department of Pharmacology and Toxicology, NIPER-HYDERABAD, Balanagar, Telangana, India-500037), designed this work, evaluated data and wrote and reviewed manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.

Research Involving Human and Animals Participants

This article does not contain any studies with human participants performed by any of the authors. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted. (Institutional Animal Ethics Committee (IAEC)—NIPER-Hyderabad, India + Protocol No: NIP/8/2016/PC/213).

Footnotes

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