ORIGINAL ARTICLE
Melatonin prevents mitochondrial dysfunction and promotes neuroprotection by inducing autophagy during oxaliplatin-evoked peripheral neuropathy
Abstract
Oxaliplatin, an organoplatinum compound, is used in the treatment of colorectal cancer, but its clinical use can be limited due to the development of peripheral neuropathy. Whilst mitochondrial dysfunction has been implicated as a major pathomechanism for oxaliplatin-induced neurotoxicity, the prevention of autophagy may also aggravate neuronal cell death. Melatonin, a well-known mitoprotectant and autophagy inducer, was used to examine its neuroprotective role in oxaliplatin-induced peripheral neuropathy (OIPN). Melatonin prevented the loss of mitochondrial membrane potential (Ψm) and promoted neuritogenesis in oxaliplatin-challenged neuro-2a cells. It did not interfere with the cytotoxic activity of oxaliplatin in human colon cancer cell line, HT-29. Melatonin treatment significantly alleviated oxaliplatin-induced pain behavior and neuropathic deficits in rats. It also ameliorated nitro-oxidative stress mediated by oxaliplatin, thus prevented nitrosylation of proteins and loss of antioxidant enzymes, and therefore, it improved mitochondrial electron transport chain function and maintained cellular bioenergetics by improving the ATP levels. The protective effects of melatonin were attributed to preventing oxaliplatin-induced neuronal apoptosis by increasing the autophagy pathway (via LC3A/3B) in peripheral nerves and dorsal root ganglion (DRG). Hence, it preserved the epidermal nerve fiber density in oxaliplatin-induced neuropathic rats. Taken together, we provide detailed molecular mechanisms for the neuroprotective effect of melatonin and suggest it has translational potential for oxaliplatin-induced neuropathy.1 Introduction
Oxaliplatin, a platinum-based chemotherapeutic agent, is used as the standard of care treatment for metastatic colorectal cancer. However, clinical use of oxaliplatin may be limited due to the development of severe peripheral neuropathy.[1] The neurotoxicity of oxaliplatin differs from other chemotherapeutic agents as it induces an initial rapid onset of cold-induced distal dysesthesia (acute neuropathy) followed by sensory and motor nerve dysfunction with long-term treatment (chronic neuropathy).[2] The incidence of neuropathy after oxaliplatin therapy ranges from 80% to 90% and increases with the cumulative dose (>540-850 mg/m2). It is characterized by a glove-and-stocking distribution sensory loss, paresthesia, dysesthesia, and allodynia.[3] Although the pathomechanisms underlying oxaliplatin-induced neuropathy remain unclear, altered Na+-K+ channel activity due to mitochondrial dysfunction and bioenergetic crisis are involved in the acute neuropathy,[4, 5] and neurotoxicity due to the accumulation of platinum adducts in dorsal root ganglion (DRG) is implicated in the chronic neuropathy.[6] Current therapeutic options are limited to drugs approved for the relief of neuropathic pain and have no impact on the underlying neuronal damage of chemotherapy-induced peripheral neuropathy (CIPN).[7] Therapies to limit platinum compound-induced neurotoxicity are being actively sought whilst maintaining their anticancer activity.[8, 9]Oxidative/nitrosative stress in peripheral nerves and DRG may be an important underlying and interconnected pathomechanism with mitochondrial dysfunction, inflammation, and apoptosis leading to neurodegeneration. The mitotoxicity hypothesis posits that oxaliplatin causes mitochondrial injury with swelling and vacuolation leading to abnormal spontaneous discharges and compartment degeneration in somatosensory primary afferent neurons.[10, 11] Whilst several antioxidant treatments for chemotherapy-induced neuropathy have been tested and include n-acetylcysteine, n-acetyl carnitine, glutathione, alpha-lipoic acid, and vitamin E,[8, 12] but have been shown to have limited efficacy,[13] due to their inability to modify redox signaling pathways.[14] Hence, therapeutic interventions that simultaneously prevent oxidative/nitrosative stress associated with mitochondrial dysfunction and maintain the bioenergetic status of the neuron may have therapeutic potential in the treatment of oxaliplatin-induced neuropathy.
Melatonin, is synthesized by a variety of cells, but is principally secreted by the pineal gland, and is known for maintaining circadian rhythmicity and regulating aging, reproductive functions, and antioxidant activities.[15-17] Recent findings suggest that melatonin is synthesized in the mitochondria of eukaryotic cells, so that it would certainly possess a potent capacity to protect the organelle from toxic oxygen derivatives.[18] Melatonin appears to exhibit its antioxidant actions by (i) directly scavenging the free radicals,[19] (ii) stimulation of antioxidative enzymes, (iii) increasing the efficiency of mitochondrial oxidative phosphorylation and electron transport chain, and (iv) augmenting the efficiency of other antioxidants.[16] It has been also shown to have pleiotropic effects with neuroprotective properties in preclinical and clinical studies.[20, 21] Its potential as a neuroprotective agent may be based on its potent antioxidant activity and capacity to cross the lipid membrane with accumulation in nerve cells.[21] Melatonin has been shown to affect cell proliferation after nerve injury, maintain mitochondrial homeostasis, enhance autophagy, and possess anti-inflammatory properties.[22-25] It has been also shown to inhibit autophagy in certain conditions such as ischemia/IR-mediated stress. Here melatonin inhibits the nonselective autophagy and prevents the cell death and organ failure.[23] When melatonin induces selective autophagy which helps in organelle homeostasis and cytoprotection.[26, 27] By virtue of its multiple contributions in cellular protection, melatonin has been explored as a pharmacological agent in diabetic neuropathy and taxane-induced neuropathy.[28, 29] It has been well demonstrated that oxaliplatin-induced neuropathy is associated with oxidative/nitrosative stress along with mitochondrial dysfunction and neuronal apoptosis.[30, 31] The accumulation of oxidative damage proteins and lipids may abrogate constitutive autophagy leading to an accumulation of dysfunctional mitochondria and neuronal apoptosis.[23, 32, 33]
Therefore, the current study was designed to assess the following: (i) oxidative/nitrosative stress and mitochondrial dysfunction in relation to perturbations in neuronal autophagy; (ii) if so, whether melatonin could modulate mitochondrial dysfunction and autophagy to prevent or attenuate oxaliplatin-evoked pain; and (iii) to explore the protective mechanisms of melatonin against oxaliplatin-induced peripheral neurotoxicity in the peripheral sciatic nerve and DRG of rats, with oxaliplatin-induced peripheral neuropathy.
2 Materials and Methods
2.1 Drugs and chemicals
All chemicals including melatonin and culture medium were procured from Sigma-Aldrich Co, USA, unless until specified. Oxaliplatin was a gift from Astron pharmaceuticals (Ahmedabad, India). Cell culture consumables were provided by Tarsons, India. Fetal bovine serum (certified, US origin) purchased from Gibco (life technologies). Fluorescent probes, MitoSox and JC-1, were obtained from Invitrogen, California, USA. 2′,7′-dichlorofluorescein diacetate (DCFDA), MITOISO 1, and Complex IV Assay kits were also procured from Sigma-Aldrich Co, USA. Complex I, complex II, and ATP assay kits were obtained from Abcam, Cambridge, UK. Tissue protein extraction reagent (T-PER) was purchased from Thermo Scientific, USA. Immunohistochemistry (IHC) detection kit brought from PathnSitu Biotechnologies Pvt Ltd. Hyderabad, India. Isoflurane was obtained from Raman and Weil Pvt. Ltd. (Mumbai, India).2.2 In vitro methods
2.2.1 Neuronal cell culture
The Neuro-2a (N2a) cell line was procured from NCCS, Pune, India. Human colon cancer cell line HT-29 was obtained from the American Type Culture Collection (Rockville, MD, USA) and was grown in MEM and high-glucose DMEM medium, respectively, supplemented with 10% fetal bovine serum, glutamine (2 mmol/L), streptomycin/penicillin (1%) at 37°C, in a humidified atmosphere of 95% air and 5% CO2. The culture media was replaced every other day, and when confluent, the cells were subcultured or seeded into T25 or multiwell plates as per the experimental requirement.2.2.2 Cell viability assay (MTT assay) in HT-29 cells
HT-29 cells were plated at a density of 1×105 cells per well into 96-well microplate wells, and after 24 hour, cells were replaced with low-serum medium (0.5% FBS). Cells were treated with different concentrations of oxaliplatin (0-50 μmol) and also in the combination of melatonin (15 and 30 μmol). After 48 hour of incubation, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) solution (5 mg/mL in MEM) was added to the plate, and then, cells were incubated for 4 hour at 37°C. Cells were washed, and the formazan crystals were solubilized using 100 μL dimethylsulfoxide. The absorbance was measured at 570 nm using a multimode reader (Spectramax M4, USA).[34]2.2.3 Measurement of intracellular reactive oxygen species (ROS) and mitochondrial superoxide anion (O2−)
Oxaliplatin-induced generation of intracellular ROS including mitochondrial O2− was determined by 2′,7′-dichlorofluorescein diacetate (DCFDA) and MitoSox staining, respectively. The N2a cells were seeded in 96-well plate (black plate) and, after 24 hour of incubation, were exposed to drug solutions with indicated concentrations for 6 and 12 hour. Cells were incubated with 10 μmol of DCFDA for 20 minutes and 5 μmol MitoSox Red for 30 minutes at 37°C in Hanks solution (HBSS, CaCl2 140 mg/dL, MgCl2-6H2O 10 mg/dL, and MgSO4-7H2O 10 mg/dL). DCFDA, a nonfluorescent polar probe, in the presence of intracellular ROS, gets oxidized and produces a high intensity of green fluorescence.[35] The fluorescent signal λexc = 485 nm; λem = 535 nm is proportional to ROS production, whereas the fluorescent signal at λexc = 530 nm; λem = 580 nm is proportional to mitochondrial superoxide production.[36] Fluorescent microscopic images were captured using a fluorescence microscope (Nikon 80i, Japan). The fluorescent signal was measured by multiplate reader (Spectramax M4, USA).2.2.4 Assessment of neurite outgrowth
N2a cells were used as neurodegeneration model to demonstrate oxaliplatin-induced neurite degeneration.[37] Cells were seeded at 1×104 cells/cm2 onto a 12-well plate and used for experiments on the following day. Cells were induced for neurite growth by adding nerve growth factor (NGF) (50 ng/mL). Then, they were exposed to drug solutions with indicated concentrations and neurite outgrowth was measured after 24 hour of treatment. Morphometric analysis was performed on digitalized images of live cells taken under phase-contrast illumination using an inverted microscope (NIKON, USA). The neurite length was measured by software (Image J 1.36; Wayne Rasband, National Institutes of Health, MD, USA) tracing the length of the distance between the cell periphery and the tip of the longest neurite for each cell in a field. Data from the 6 wells with 10 fields in each were pooled. Neurite outgrowth is expressed as average neurite length (μm) and the percentage of cells bearing neurites (%).[38]2.2.5 Measurement of mitochondrial membrane potential by JC-1 staining
JC-1 is DIOC2 (3,3′-diethyloxacarbocyanine iodide), a Molecular probe (Life Technologies-Invitrogen, Saint Aubin, France), which is a cationic and lipophilic dual fluorescence dye, was utilized to determine variations in the mitochondrial transmembrane potential (∆ψm).[39] It freely diffuses into cellular cytosol, selectively accumulates, and forms aggregates in the healthy mitochondria with active membrane potential emitting red fluorescence (detected at 590 nm). When the mitochondria lose membrane potential, it remains as monomers in the cytosol and emits green fluorescence (detected at 530 nm). Cells were seeded in a 24-well plate at a density of 2×104 cells/cm2 and incubated for 24 hour. Cells were exposed to drug solutions with indicated concentrations for 24 hour followed by JC-1 (1 μg/mL) for 20 minutes. Cells were washed with phosphate-buffered saline (PBS-pH 7.4) twice, and the fluorescence intensity was measured at 590 nm.2.3 In vivo methods
2.3.1 Experimental animals
Male Sprague Dawley rats weighing 200-250 g were procured from the National Institute of Nutrition (NIN), Hyderabad, India. They were housed as three per cage, had free access to food and water ad libitum, maintaining a constant temperature of 23°C±1°C, relative humidity 55%±10% and 12:12 hour light and dark cycle during the experiments. The animal study protocols were approved by the Institutional Animal Ethics Committee constituted for the purpose of control and supervision of experimental animals (CPCSEA) by the ministry of Environment and Forests, Government of India. Animals were naive to intervention treatment and experimentation at the initiation of all studies. They were acclimatized for 1 week, and baseline measurements were taken prior to starting the experiments. All behavioral measures were obtained after acclimatizing the animals to the experimental environment and also by a researcher who was blinded to the assigned groups to avoid bias.2.3.2 Experimental design and Animal treatment
Rats were randomly allocated into five groups consisting of 10 animals each and assigned to normal control (NC), oxaliplatin control (OC), and three treated groups. To establish oxaliplatin-induced peripheral neuropathy (OIPN), the OC rats were injected with oxaliplatin (4 mg/kg, ip in 5% dextrose solution) given twice a week for 4 weeks in a total of nine injections so that total cumulative dose is 36 mg/m2 that corresponds to >1184 mg/m2 which simulates the clinical cumulative oxaliplatin dose causing chronic neuropathy;[14] 3 and 10 mg/kg of melatonin was selected as low and high doses, dissolved in absolute ethanol and then diluted with normal saline in which the final concentration of the ethanol was 5%.[40] The third and fourth groups consist of OC rats treated with melatonin at 3 (OC+M3) and 10 mg/kg, ip (OC+M10). The fifth group were normal rats receiving melatonin alone at 10 mg/kg, ip (M10). After 28 days of treatment, animals were euthanized with CO2 anesthesia, and immediately, the sciatic nerves and DRG were collected and fixed in 10% neutral buffered formalin for immunohistochemistry studies. For biochemical and protein expression assessments, supernatants of homogenized nerves were used according to the requirement of the protocols of different estimations. Total protein content was estimated using protein assay kit (Bio-Rad, USA) with bovine serum albumin (BSA) as a standard.2.3.3 Functional assessment (Nerve conduction velocity)
The conduction velocity was measured by Power Lab 8sp system by anaesthetizing animals in a mixture of 4% isoflurane and oxygen. The sciatic nerve was stimulated with 3 V proximally at the sciatic notch and distally at the ankle using bipolar needle electrode. Receiving electrodes were placed on the muscle of foot. The latencies of muscle action potential were recorded via bipolar surface electrodes from the foot muscle of the hind paw. Negative M-Wave deflection and H-reflex latencies were measured, and the related motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) were calculated, respectively, using the distance between the stimulation points and latencies with LabChart software. The results were expressed in m/s.[41]2.3.4 Behavioral assessment
2.3.4.1 Thermal hyperalgesia (cold plate test)
Thermal hyperalgesia of the paw cold (4°C±1°C) stimuli was performed using Eddy's hot/cold plate (Ugo Basile Biological Research Apparatus, Varese, Italy). The latency of the first escaping sign of paw flicking or paw licking was considered as the index of the pain threshold. The cutoff time was kept at 60 seconds to avoid paw damage. The average of six consecutive readings was taken and given as the paw withdrawal latency.[30]2.3.4.2 Cold chemical allodynia (Acetone spray test)
The test was performed by spraying 100 μL of acetone onto the surface of the paw of the rat by placing it on top of a mesh with grids without touching the paw skin. The response of the rat was noted for 20 seconds, and the response was graded on a 4-point scale: 0, no response; 1, quick withdrawal or flicking the paw; 2, prolonged withdrawal or repeated flicking; and 3, repeated flicking of the paw with licking of the paw. With a time gap of 5 minutes, acetone was applied thrice onto the hind paw and the individual scores noted in 20-seconds intervals were added to obtain a single cumulative score with a minimal score of 0 and a maximum score of 9.[42]2.3.4.3 Mechanical allodynia and hyperalgesia
Mechanical allodynia and hyperalgesia were measured using Von Frey hairs (Samitek, USA; 1, 2, 4, 6, 8, 10, 15, and 20 g)[43] and Randall-Selitto apparatus (IITC, Life Sciences, USA).[44] To measure hyperalgesia, pressure was applied on the surface of both paws with a 5-minutes interval between consecutive readings. The paw withdrawal responses to the corresponding pressure (g) were recorded. The cut-off pressure was 250 g to avoid paw damage. Von Frey hairs for allodynia assessment were applied perpendicular to the plantar surface of the rat paw placed in transparent perspex boxes on a mesh surface from a lower pressure to higher pressure. Sufficient force was applied to bend the filaments slightly for 2 to 3-seconds, and a sudden withdrawal of paw with a paw licking sign was recorded as a positive response. The test was carried out three times for each animal with a 5-minutes interval and the average pressure (g) of the monofilament which evoked the paw reflex was considered as the paw withdrawal threshold.2.3.5 Estimation of oxidative/nitrosative stress markers
Sciatic nerves were homogenized using phosphate buffer (pH-7.4). The supernatant was collected after centrifugation at 10 000 g and 4°C for 15 minutes and were then used for the determination of protein content, malondialdehyde (MDA), nitrite, and glutathione (GSH) levels. The level of lipid peroxidation was assessed by measuring “Thiobarbituric Acid Reactive Substances” (TBARS).[45] MDA levels measured at 532 nm were expressed as μmol/mg protein. Nitrite levels were calculated at 540 nm using the Griess reagent, and total nitrite levels were expressed as nmol/mg of protein after protein normalization.[46] Glutathione (GSH) levels were estimated using 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB/Ellman's reagent) at 405 nm.[47] The total quantity of GSH was calculated by means of a calibration curve, normalized to the protein concentration, and expressed as μmol/mg.2.3.6 Assessment of mitochondrial functions
2.3.6.1 Determination of mitochondrial membrane potential (Ψm)
Mitochondria were isolated from the sciatic nerve using the MITOISO1 Mitochondrial Isolation Kit (Sigma, St. Louis, MO, USA), and Ψm was determined using the JC-1 dye according to manufacturer's instructions. The fluorescence of a 2-mL reaction mixture was measured at an excitation wavelength of 490 nm and an emission wavelength of 590 nm using a multiplate reader (Spectramax M4, USA). The fluorescence observed at 590 nm shows the extent of formation of J-aggregates which indicates the Ψm.[48, 49]2.3.6.2 Mitochondrial complex enzyme activity assays
The complex I and complex II enzyme activities of isolated mitochondria were measured using microplate immunocapture assay kits (Abcam, UK) according to the manufacturer's protocols. The complex I enzyme is immunocaptured in the wells of the microplate, and its activity was determined by following the oxidation of NADH to nicotinamide adenine dinucleotide and the simultaneous reduction of a dye, which gives increased absorbance at 450 nm. The complex II enzyme activity is assessed by coupling the production of ubiquinol to the reduction in the dye DCPIP (2,6-dichlorophenolindophenol), which leads to a decreased absorbance at 600 nm. The cytochrome c reducing capacity was measured in 96-well format based on a method described by Kramer et al., with few modifications.[50] The isolated mitochondrial fraction was added to an assay solution containing required proportions of sodium succinate, KH2PO4, rotenone, KCN, and oxidized cytochrome c (Sigma, USA), pH 7.5. The cytochrome c reduction capability of complex II + III is assessed by measuring its increase in absorbance at 550 nm using Spectramax M4, USA, in kinetic mode at room temperature and the complex I, complex II, and complex III enzyme activities were expressed in mOD/min/mg of protein, whereas complex IV activity was measured using Cytochrome c Oxidase assay kit (Sigma-Aldrich, St. Louis, MO). It is a colorimetric assay based on the observation, that is, decrease in the absorbance of ferricytochrome c at 550 nm due to its oxidation by cytochrome c oxidase. The activity of cytochrome c oxidase is expressed in Units/mg/min of the protein.[51]2.3.7 Measurement of ATP levels
The total ATP levels were estimated at 570 nm using a colorimetric-based assay kit (Abcam, UK) according to the manufacturer's instructions. The total quantity of ATP was calculated by means of a calibration curve (Spectramax M4, USA), normalized to the protein concentration, and expressed as μmol/gm protein.2.3.8 Immunohistochemical analysis and quantification of intra-epidermal nerve fiber (IENF) density
Sciatic nerves and DRG sections were rehydrated and incubated with primary antibodies nitrotyrosine (Novus biological, USA, 1:200 dilution), LC3A/3B, cleaved caspase-3, SOD2 (Cell Signaling Technology, Beverly, MA, USA, 1:200 dilution), whereas the footpad samples of 8-μm sections were rehydrated and incubated with an antibody to PGP 9.5 (1:200, Abcam, UK). Immunohistochemistry was performed according to a protocol of Poly Excel HRP/DAB Detection System (PathnSitu Biotechnologies Pvt Ltd, Hyderabad, India).[52] Microsections were washed, counterstained with hematoxylin, and observed under a light OPTIKA microscope (Model no. B-293). Images were captured at 400× magnification, and brown profiles were considered as positive. The immunohistochemical scores were given as per the following scheme (1, very low intensity; 2, low intensity; 3, moderate intensity; and 4, high intensity). Images were captured at 1000× magnification (under oil immersion), and nerve fibers (PGP 9.5 immunoreactive profiles) crossing the dermo-epidermal junction were quantified relative to the length of tissue examined.[53] At least 6 sections per group were counted and expressed as an average number of nerve fibers per mm.2.3.9 Western blotting analysis
Sciatic nerve samples were homogenized in T-PER (Thermo scientific, USA) containing 1% protease cocktail inhibitor, and clear supernatants were estimated for protein. An equal amount of protein was loaded separated on SDS polyacrylamide gel; the separated proteins were blotted or transferred onto polyvinyl difluoride (PVDF) membrane followed by blocking with blocking solution (3% BSA) for 1 hour. Then, the membrane was incubated overnight with primary antibodies to Bcl-2, Bax, Cyt c, Caspase-3, LC3A/3B, beclin, Atg7, Atg5, Atg3, SOD2, β-actin (1:1000, Cell Signaling Technology, Beverly, MA, USA) and complex I, complex II, ATP Synthase (1:200, Abcam, UK). The washed membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000) and visualized by an enhanced chemiluminescence. The relative band densities were quantified by densitometry using analyzing software (Image J 1.36; Wayne Rasband, National Institutes of Health, MD, USA). Equal loading of protein was confirmed by measuring β-actin expression.2.3.10 TUNEL assay
Longitudinal sections of sciatic nerves were subjected to terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to observe oxaliplatin-induced neuronal apoptosis. The 3′ end of fragmented DNA was labeled with fluorescein isothiocyanate (FITC) according to manufacturer's protocol (Calbiochem, CA, USA). Sections were then mounted with nuclear counterstain 4′,6-diamidino-2-phenylindole (DAPI), and images were obtained using a fluorescent microscope (Nikon DC 300F). The total number of cells and TUNEL-positive cells was counted from at least six sections per group, and TUNEL-positive cells were expressed as the percentage of total cells.[52]2.4 Data analysis
The data obtained were expressed as the mean±standard error of mean (SEM) for n animals. The data from the behavioral results were statistically analyzed by two-way analysis of variance (ANOVA), and end-point data were statistically analyzed by one-way analysis of variance using the GraphPad Prism Version-5.0 software (GraphPad software, San Diego, CA, USA). Follow-up analysis was made with “Bonferroni's Multiple Comparison Test.” A probability level of P values <.05 was considered as statistically significant.3 Results
3.1 Melatonin does not compromise the in vitro antitumor activity of oxaliplatin in human colon cancer cells, HT-29
To assess the possible interaction between the chemotherapeutic activity of oxaliplatin and melatonin treatment, we measured the viability of colon cancer cell line HT-29. After 48 hour of treatment, the observed IC50 of oxaliplatin (0-50 μmol) alone was 4.98±1.12 μmol whilst in combination with melatonin at 15 and 30 μmol showed an IC50 of 4.42±1.3 μmol and 4.26±2.3 μmol, respectively. Therefore, co-treatment of melatonin did not alter oxaliplatin-induced lethality and did not compromise the anticancer properties of oxaliplatin against human colon cancer cells, HT-29.3.2 Melatonin prevents oxidative stress, loss of Ψm, and improves neuritogenesis against oxaliplatin-induced toxicity in N2a cells
Intracellular ROS was measured in N2a cells using DCFDA and mitochondrial superoxide levels using MitoSox staining, and both were increased significantly (P<.001) compared to the untreated control cells (Figure 1A). Melatonin treatment at 15 and 30 μmol significantly (P<.001) reduced intracellular ROS and mitochondrial superoxide levels, as indicated by the reduced mean fluorescence intensity (Figure 1B).
3.3 General toxicity
The general toxicity of oxaliplatin in vivo was monitored on a daily basis, and body weights were measured once weekly. The body weights of the oxaliplatin injected animals were reduced nonsignificantly during the first week, but this was normalized later and weights were improved gradually (Figure 3A). No deterioration in the general health status of the animals was observed, although, oxaliplatin-administered animals appeared weaker and less active compared to control animals. There was no mortality observed during the study.3.4 Melatonin prevents oxaliplatin-induced functional deficits and blocks neuropathic pain
Treatment with oxaliplatin, there was a significant reduction in SNCV (P<.01) and a slight effect on MNCV. Rats treated with melatonin demonstrated a significant (P<.05) prevention of oxaliplatin-induced lowering of conduction velocities (Table 1). There was no significant change in sensory thresholds among any of the groups after the 1st week of oxaliplatin, but there was a significant change after 3rd and 4th weeks (P<.001) with a reduction in mechanical thresholds and paw withdrawal latencies compared to the normal control group. Melatonin treatment significantly (P<.001) increased the paw withdrawal latencies to cold stimuli (Figure 3B). It also increased the paw withdrawal threshold to Von Frey fibers and Randall-Selitto analgesiometer (Figure 3D and Figure 3E, respectively). The mean score of allodynia in response to acetone spray significantly (P<.001) decreased with melatonin compared to oxaliplatin rats by the 28th day (Figure 3C). Melatonin alone treated animals showed no significant (P>.05) functional and behavioral changes compared to control rats.| Parameter | NC | OC | OC+M3 | OC+M10 | M10 |
|---|---|---|---|---|---|
| |||||
| MNCV (m/s) | 61.7±1.2 | 55.8±2.4 | 62.2±2.1 | 60.0±3.6 | 60.3±3.1 |
| SNCV (m/s) | 59.7±1.9 | 45.5±2.1^^ | 53.3±3.5 | 55.7± 3.0* | 58.7±1.8 |
| MDA (μmol/mg protein) | 0.45±0.09 | 2.45±0.32^^ | 1.43±0.16* | 1.03±0.30** | 0.52±0.10 |
| Nitrite (nmol/mg protein) | 8.61±1.16 | 29.86±3.23^^ | 20.68±2.41* | 17.56±1.93** | 9.62±0.86 |
| GSH (μmol/mg protein) | 42.17±2.96 | 19.67±2.01^^ | 27.91±1.84 | 37.18±3.91** | 44.29±3.82 |
3.5 Melatonin abrogates oxaliplatin-mediated oxidative/nitrosative stress
Oxaliplatin administration caused a significant increase in the levels of MDA and nitrite, whereas it decreased the levels of GSH in the sciatic nerve. Four weeks treatment with melatonin (10 mg/kg, ip) significantly (P<.01) attenuated these changes (Table 1). The immunohistochemical analysis also revealed a significant increase (P<.001) in the expression of nitrotyrosine (Figure 4A) and decrease (P<.01) in the levels of MnSOD (Figure 4B) and in the sciatic nerve and DRG in rats with OIPN. Melatonin treatment significantly (P<.001 at 10 mg/kg) inhibited this effect by abrogating protein nitrotyrosylation and improving the levels of MnSOD. Melatonin alone did not show any significant changes when compared to untreated normal control rats.3.6 Melatonin prevents mitochondrial dysfunction and improves bioenergetic status in oxaliplatin-induced neuropathic rats
Oxaliplatin significantly (P<.01) affected the Ψm with a decrease in the aggregation of JC-1 dye inside mitochondria compared to normal rats (Figure 5A (a)). The rate of mitochondrial complex I and complex II enzyme activities in sciatic nerves of oxaliplatin injected rats was significantly (P<.01) (Figure 5A (b and c)) lower than complexes III and IV (P<.05) (Figure 5A (d and e)) when compared to normal control rats. Due to oxaliplatin-induced mitochondrial dysfunction, ATP production was also significantly (P<.01) impaired in comparison with normal control rats (Figure 5A (f)). These oxaliplatin-induced mitochondrial functional alterations were significantly improved by melatonin treatment. Melatonin at a low dose (3 mg/kg, ip) did not show any effect on complexes III and IV but significantly improved complex I, complex III, and complex IV activities at the high dose (P<.01) than complex II activity (P<.05). Melatonin treatment significantly (P<.01) preserved the Ψm and restored ATP levels. Rats treated with melatonin alone showed a slight increase in mitochondrial function, but this was not significantly different to normal control rats. The expression of complexes I and II, ATP synthase, and MnSOD were significantly (P<.001) decreased in oxaliplatin-treated rats compared to normal control rats (Figure 5B). Melatonin treatment significantly (P<.001 at 10 mg/kg, ip) increased the levels of complexes I and II, ATP synthase, and MnSOD and significantly (P<.001 at 10 mg/kg, ip)3.7 Melatonin induces autophagy and inhibits apoptosis
The levels of autophagy proteins were evaluated in sciatic nerve and DRG. The basal levels of LC3A/3B were significantly decreased (P<.05) in the sciatic nerve and DRG, indicating inhibition of autophagy in oxaliplatin-administered rats compared to control rats (Figure 6A). The expression levels of proteins that aid autophagy (LC3A/3B-I and II, beclin, Atg 3, Atg 5, and Atg 7) were decreased in sciatic nerve samples of oxaliplatin-treated rats indicating impaired autophagy when compared to control rats (Figure 6B). Melatonin treatment significantly (P<.01 at 10 mg/kg, ip) attenuated these changes and increased basal autophagy proteins.3.8 Melatonin prevents the loss of IENF in the oxaliplatin-induced neuropathic rats
The intra-epidermal nerve fiber density in the hind paw of oxaliplatin-treated rats was also significantly (P<.001) decreased. Melatonin significantly (P<.001) prevented oxaliplatin-induced loss of intra-epidermal nerve fibers (Figure 9).4 Discussion
Given the pathological role of oxidative/nitrosative stress-mediated mitochondrial dysfunction and perturbations in autophagy in peripheral neuropathy, studies that explore pharmacological agents that may promote autophagy and maintain mitochondrial homeostasis have garnered much interest.[9, 10, 22] Herein, our study reveals in vitro and in vivo neuroprotective mechanisms of melatonin against oxaliplatin-induced neurotoxicity. The results suggest that melatonin efficiently prevents mitochondrial dysfunction and neuronal apoptosis by virtue of its antioxidant effects and has the ability to improve autophagy in oxaliplatin-induced neurotoxicity. Mechanistically, the beneficial effects of melatonin largely relied on its actions in preventing oxidative stress, mitochondrial dysfunction, and apoptosis leading to the induction of autophagy and preserved cell quality.[23, 25] To the best of our knowledge, our study is the first to describe the neuroprotective effects of melatonin against oxaliplatin-induced neurotoxicity, by reversing mitochondrial dysfunction and promoting autophagy.Previous reports have shown that markers of oxidative/nitrosative stress including increased lipid peroxidation, nitrite levels, oxidatively damaged proteins, and DNA are increased in the systemic circulation, peripheral nerves, and spinal cord of oxaliplatin-induced neuropathic rats.[54, 55] The increased levels of nitrite and superoxide result in inactivation of antioxidant enzymes like MnSOD,[56] loss of glutathione, enhanced pro-nociceptive glutamatergic signaling,[57] activation of ion channels like TRPA1,[58] damage to mitochondrial proteins (especially complex I and complex II),[31] and autophagy protein,[59] leading to accumulation of oxidatively damaged organelles and peripheral neuronal damage with sensitization.[60] Oxidative stress-mediated abnormalities in mitochondrial structure and function in peripheral nerve fibers and DRG have been postulated as a key underpinning mechanism and appeared to be correlated directly to pain behavior.[9, 61]
Although the therapeutic benefits of antioxidants and agents that improve mitochondrial function for the treatment/prevention of OIPN have been shown in multiple preclinical and clinical studies, none of the agents have been approved for the treatment.[13] Hence, it is worth exploring the underlying mechanisms to enable the development of novel therapeutic agents that may treat oxaliplatin-evoked neuropathic pain.
In the present study, we found that oxaliplatin induces ROS and mitochondrial superoxide in N2a cells and significantly inhibits NGF-induced neuritogenesis.[62] We also found that the oxaliplatin administration in vivo increased lipid peroxidation, nitrite level along with decreased levels of GSH and MnSOD in the peripheral sciatic nerve. It was further confirmed by increased levels of nitrosylated proteins and decreased MnSOD levels in the peripheral sciatic nerve and DRG of rats treated with oxaliplatin in agreement with previous reports.[56] The other key finding is that sciatic nerves in rats with oxaliplatin-evoked neuropathy had reduced the activity of mitochondrial enzyme complexes I and II associated with the loss of Ψm (in vitro and in vivo) and impaired production of ATP levels. This indicates that oxaliplatin induces mitochondrial dysfunction and increased pro-apoptotic proteins like caspase-3, Bax, Cyt c, and TUNEL-positive cells in the sciatic nerve leads to caspase-dependent neuronal apoptosis. This was confirmed by the expression of cleaved caspase-3 in sciatic nerve and DRG of oxaliplatin-administered rats and in according with previous reports.[6, 63, 64]
Finally, the mitochondria become both source and target of oxidative/nitrosative stress, sustaining the vicious cycle.[65] Oxidatively damaged mitochondria are unable to maintain the energy demands of the cell leading to an increased production of free radicals and decreased ATP production.[66] Autophagy, a cellular housekeeping process is essential for the removal of damaged proteins and organelles which help in preserving cellular homeostasis and prevent the apoptotic program.[67] Although physiological levels of free radicals may facilitate the autophagy process, their release can result in accumulation of oxidized and nitrated proteins which suppresses autophagy and promotes apoptosis in neurons via activation of various stress signaling pathways such as MAPK and PARP-dependent pathways.[68] Several reports have shown the role of these pathways in oxaliplatin-induced neuropathy and demonstrated that low levels of autophagy and upregulation of apoptosis in neuronal cells cause neurodegeneration.[69] Hence, we studied the expression of LC3A/3B, beclin-1, Atg3, Atg5, Atg7 that aid in the clearance of damaged organelles through autophagy[70] in peripheral sciatic nerve samples and confirmed their downregulation in oxaliplatin-induced neuropathic rats. These findings strongly suggest that abrogation of the autophagy process precedes neuronal apoptosis. The experimental evidence also supports the mitotoxicity hypothesis where oxaliplatin causes a functional impairment of peripheral nerve mitochondria and chronic axonal energy deficits thus become the primary cause of neuropathy. Indeed these abnormalities are related to the loss of IENF in the plantar region of hind paw skin, explaining oxaliplatin-induced cold hyperalgesia and mechanical allodynia.[41]
We selected a pharmacological antioxidant, melatonin with a potential to induce autophagy and suppress apoptosis, as a therapeutic candidate for the treatment of OIPN. It also has a unique feature of acting as an anti-apoptotic agent in normal cells but inducing detrimental effects in tumor cells.[71] Furthermore, melatonin did not compromise the anticancer activity of oxaliplatin. Melatonin is believed to suppress apoptosis by enhancing autophagy by virtue of (i) scavenges excess free radicals, (ii) increases glutathione content that maintains the integrity of mitochondrial permeability transition pore (mPTP),[72] (iii) enhances mitochondrial complex enzyme activities.[23]
In the present study, melatonin treatment in vitro enhanced neurite outgrowth and suppressed the levels of ROS and superoxide indicating its capability to increase mitochondrial activity.[73] Melatonin treatment also attenuated oxidative/nitrosative stress and improved mitochondrial functions in the peripheral sciatic nerves and DRG of oxaliplatin-induced neuropathic rats. Treatment with melatonin enhanced autophagy and abrogated oxaliplatin-induced neuronal apoptosis helping to maintain mitochondrial homeostasis and cell survival evidenced by an enhanced expression of the mitochondrial proteins complex I, complex II, ATP synthase, and MnSOD in the sciatic nerves of oxaliplatin-treated rats.
In conjunction with the biochemical and protein expression studies, melatonin treatment also prevented oxaliplatin-induced loss of IENF density in the hind paw. This is relevant because the intra-epidermal terminals are regions of high energy requirement and are packed with mitochondria.[9] Oxaliplatin-induced mitochondrial dysfunction and consequent energy deficiency have been implicated in IENF degeneration and abnormal spontaneous discharge of sensory nerve fibers[31] and oxaliplatin-evoked pain behavior.[61] Melatonin treatment significantly attenuated the mechanical allodynia and hyperalgesia as well as loss of nerve fibers responsible for the hyperexcitability.[74] It also inhibited oxaliplatin-induced cold allodynia which may be related to its beneficial effects on the loss of Aδ fibers that cause cold allodynia.[75] No changes were observed in the motor nerve conduction velocities during oxaliplatin administration, which may be due to the fact that the main pathology occured in DRG and primary sensory neurons of the sciatic nerve. Indeed, melatonin treatment normalized sensory nerve conduction velocities in rats administered oxaliplatin. Reassuringly, rats treated with melatonin alone did not show any significant alterations in the behavioral or biochemical parameters when compared to untreated normal control group rats.
Taken together, our study provides novel insights into the mechanisms underlying oxaliplatin-induced neurotoxicity and shows that melatonin has beneficial roles in preventing oxaliplatin-induced mitochondrial dysfunction and apoptosis by enhancing cell survival through autophagy. Our experimental findings provide a robust foundation for clinical studies exploring the role of melatonin in oxaliplatin-induced neuropathy.
TESTIMONY OF HOW I GOT CURED FROM (HERPES SIMPLEX VIRUS) BY THE GREAT HEALER DOCTOR EHBOS
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