Effect of Vitis vinifera hydroalcoholic extract against oxaliplatin neurotoxicity: in vitro and in vivo evidence

Oxaliplatin treatment is associated with the development of a dose-limiting painful neuropathy impairing patient’s quality of life. Since oxidative unbalance is a relevant mechanism of oxaliplatin neurotoxicity, we assessed the potential antioxidant properties of Vitis vinifera extract in reducing oxaliplatin-induced neuropathy as a valuable therapeutic opportunity. A hydroalcoholic extract of Vitis vinifera red leaf was characterized and tested in primary rat astrocyte cells treated with oxaliplatin (100 μM). Oxaliplatin lethality in the human adenocarcinoma cell line HT-29 was evaluated in the absence and presence of the extract. In vivo, pain hypersensitivity was measured in a rat model of neuropathy induced by oxaliplatin and ex vivo molecular targets of redox balance were studied. Vitis vinifera extract (50 μg mL−1, 4 h incubation) significantly reduced the oxaliplatin-dependent superoxide anion increase and lipid peroxidation in rat astrocytes but did not interfere with the mortality elicited by oxaliplatin in HT-29 cancer cells. In oxaliplatin-treated rats, a repeated daily administration of the Vitis vinifera extract (300 mg kg−1, p.o.) significantly prevented mechanical and thermal hypersensitivity to noxious and non noxious stimuli. mRNA and protein levels of Nrf2 were normalized in spinal cord and DRGs. Moreover, in the spinal cord, the extract significantly decreased the activation of astrocytes. Vitis vinifera reduced oxidative damages and relieved pain without influencing oxaliplatin anti-cancer activity.

ESI source Q-ToF (Agilent 6545). The UHPLC is equipped with a vacuum degasser, a binary pump, a Peltier thermostated autosampler at 10°C, a Peltier thermostated column compartment.

Phenols total, expressed as gallic acid
The grinded sample (0.3 g) is extracted with 100 ml of ethanol 50 % (vol/vol) by means of ultrasound at 35° C. After 30 minutes the samples is centrifuged 10 minutes at 4000 rpm. The supernatant is collected in a 100 ml volumetric flask ad it is made up to volume by ethanol 50 % (vol/vol). The resulting solution (0.5 ml) is introduced in a 100 ml volumetric flask containing ultrapure water (50 ml), then Folin-Ciocalteu reagent (5 ml) and Calcium carbonate 20% solution (10 ml) are added, respectively. The volume is made up to 100 ml by ultrapure water. After two hours the absorbance of the solution is read at 760 nm, using water as reference solution.
Using a gallic acid calibration curve (y= 158.42x+0.0068) with concentration ranging from 0.002 mg/ml to 0.004 mg/ml, the total phenols percentage expressed as gallic acid was determined with the following calculation: % of total phenol (as gallic acid) = [(A-a)*F1/(b*p)], where A is the sample absorbance at 760nm; p is the sample weight in grams; a is the intercept of calibration curve; b is the slope of calibration curve; F1 is the Dilution factor, calculated as follows: Ve*Vc/V*10, where Ve is the volume of the initial extract; Vc is the volume of the colorimetric reaction; V is the volume of the initial extract used for the colorimetric reaction.

Anthocyanins total, expressed as delphynidin chloride
The grinded sample (0.025 g) is extracted with a concentrated hydrochloric acid methanol solution 2% (vol/vol) (25 ml) by means of ultrasound at 35° C. After 30 minutes the samples is centrifuged 10 minutes at 4000 rpm. The supernatant is collected in a 50 ml volumetric flask. The extraction is repeated on the residue in the same condition, then the volume is made up to volume (50 ml) using the same solvent. The resulting solution is diluted 1:25 with the same solvent, after which the absorbance of the sample is read at 540 nm, using concentrated hydrochloric acid methanol solution 2% (vol/vol) as reference solution. Knowing that the delphynidin chloride A1%, 1 cm is 1020, the total anthocyanins percentage expressed as delphynidin chloride is calculated using the following calculation: % of total anthocyanins (as delphynidin chloride)= (A*F*V)/(1020*p), where A is the sample absorbance at 540nm; F is the Dilution factor, V is the volume of the sample solution, p is the sample weight in grams.

Hydrosoluble Polysaccharides >20KDa
The grinded sample (1.0 g) is extracted with 25 ml of ultra pure water by means of ultrasound at 35° C. After 30 minutes the samples is centrifuged 10 minutes at 4000 rpm. The supernatant is collected in a 50 ml volumetric flask. The extraction is repeated on the residue in the same condition, then the volume is made up to volume (50 ml) using the same solvent. The sample (5 ml) is dialyzed at room temperature, using a dialysis membrane of 3.5 KDa pore cut-off. After three days the sample retentate is collected and made up to volume (10 ml) with water and used to perform the polysaccharides analysis by a HPLC-RID method according the following conditions: Dextran 25000 Da was used as external standard, that was used after solubilization in a 0.02% NaN3 water solution. The concentration of polysaccharides was calculated by means of a calibration curve between the range of 2-0.12 mg/ml.  Elaborations were performed by means of "Qualitative analysis" program and by "Find-by-Formula" algorithm. Database searching: The data file is loaded into "Qualitative analysis" then "Find-by-Formula" is run on the low channel against an MS/MS library. Find-by-Formula returns possible precursor formulas found as well as their product ions in the library. Using the list of product ions, "Qualitative analysis" extracts EICs from the high channel and aligns them with an EIC of the precursor. A coelution score is calculated and compounds which pass the threshold (user-set at 80) are retained. Data Analysis. The high resolution mass spectrometry chromatographic fingerprint profile was obtained (figure 1, BPC, base peak chromatogram). After matching with the Aboca's natural compounds library and using the ion "find by formula" algorithm, focusing on anthocyanosides the molecules found are reported in table 1. Astrocytes was starved in serum-free DMEM overnight before all treatments. Protein homogenate concentrations were measured by bicinchoninc acid (BCA; Sigma-Aldrich, Italy) assay.

Cell viability assay
Cell viability was evaluated by the reduction of 3-(4,5-dimethylthiozol-2-yl)-2,5diphenyltetrazolium bromide (MTT; Sigma-Aldrich, Italy) as an index of mitochondrial compartment functionality. After the 24 or 48 h incubation, 1 mg mL -1 MTT in serum-free DMEM without phenol red was added into each well and incubated for 30 minutes at 37 °C. After washing, the formazan crystals were dissolved in 150 µL dimethyl sulfoxide. The absorbance was measured at 550 nm. Experiments were performed in quadruplicate on at least three different cell batches.

SOD-inhibitable superoxide anion production evaluated by cytochrome c assay
The supernatants were collected, and the optical density was spectrophotometrically measured at 550 nm. After subtracting the non-specific absorbance, the SOD-inhibitable O 2 -. amount was calculated by using an extinction coefficient of 2.1 . 10 4 M -1 cm -1 and expressed as µM/mg proteins/4 hours. The 4h incubation interval was chosen on the basis of preliminary experiments which showed poor reliability for longer cytochrome c exposure to the cellular environment 18 .

Lipid peroxidation (thiobarbituric acid-reactive substances)
Thiobarbituric acid-reactive substances (TBARS) assay was assessed as an index of lipid peroxidation. The

Paw pressure test
The nociceptive threshold in the rat was determined with an analgesimeter (Ugo Basile, Varese, Italy) according to the method described by 63 . Briefly, a constantly increasing pressure was applied to a small area of the dorsal surface of the hind paw using a blunt conical mechanical probe. Mechanical pressure was increased until vocalization or a withdrawal reflex occurred while rats were lightly restrained. Vocalization or withdrawal reflex thresholds were expressed in grams. These limits assured a more precise determination of mechanical withdrawal threshold in experiments aimed to determine the effect of treatments. An arbitrary cut-off value of 100 g was adopted. The data were collected by an observer who was blinded to the protocol.

Von Frey Test
The animals were placed in 20 × 20 cm plexiglas boxes equipped with a metallic meshy floor, 20 cm above the bench. A habituation of 15 minutes was allowed before the test. An electronic Von Frey hair unit (Ugo Basile, Varese, Italy) was used: the withdrawal threshold was evaluated by applying force ranging from 0 to 50 grams with a 0.2 gram accuracy. Punctuate stimulus was delivered to the mid-plantar area of each anterior paw from below the meshy floor through a plastic tip and the withdrawal threshold was automatically displayed on the screen. Paw sensitivity threshold was defined as the minimum pressure required to elicit a robust and immediate withdrawal reflex of the paw. Voluntary movements associated with locomotion were not taken as a withdrawal response. Stimuli were applied on each anterior paw with an interval of 5 seconds.
The measure was repeated 5 times and the final value was obtained by averaging the 5 measures 64 .

Cold plate test
The animals were placed in a stainless box (12 cm × 20 cm × 10 cm) with a cold plate as floor. The temperature of the cold plate was kept constant at 4°C ± 1°C. Pain-related behaviours (i.e. lifting and licking of the hind paw) were observed and the time (s) of the first sign was recorded. The cutoff time of the latency of paw lifting or licking was set at 60 s.
Then, the suspension was sonicated on ice using three 10-second bursts at high intensity with a 10second cooling period between each burst. After centrifugation (13000 ×g for 15 minutes at 4°C) aliquots containing 20 µg total protein underwent to western blot analysis. Blots were incubated overnight at 4° with a rat anti-Nrf2 (C-20) antibody (1 : 1000; SantaCruz Biotechnology, USA).
After being washed with PBS containing 0.1% Tween, the nitrocellulose membrane was incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antisera (1:5000) and left for 1 h at room temperature. Blots were then extensively washed and developed using enhanced chemiluminescence detection system (Pierce, Milan, Italy) and signal intensity (pixels/mm2) quantified (ImageJ, NIH). Exposition and developing time used was standardized for all the blots. Densitometric analysis was performed using the "ImageJ" analysis software, and results were normalized vs GAPDH expression measured by a specific antibody (1 : 1000; Cellsignal, USA) as internal control.

mRNA level analysis
The sciatic nerve, L4-L5 DRGs and spinal cord of SD rats (day 21) were collected as described above. mRNA was extracted using TRI -Reagent© (Sigma Aldrich, Milan, Italy). cDNA was obtained using the iScript cDNA Synthesis Kit® (Bio Rad, Milan, Italy) according to the manufacturer's protocol. Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) mRNA (GenBank accession number: NM_031789.2) was amplified using the following rat gene specific primers: Images were acquired using a motorized Leica DM6000 B microscope equipped with a DFC350FX camera (Leica, Mannheim, Germany). Morphologic characteristics of microglia and astrocytes were assessed by inspection of at least 3 fields (40X 0.75NA objective) in the dorsal horn areas per section. The full-specimen thicknesses were acquired as z-stack series, deconvolved using Huygens Professional software (SVI, Hilversun, The Netherlands), and displayed using ImageJ software.
For the dorsal horns in each rat we obtained a single optical density value by averaging the 2 sides, and these values were compared to the homologous average values from the vehicle-treated animals.
Quantitative analysis of GFAP and Iba1-positive cells was performed by collecting at least 3 independent fields through a 20X 0.5NA objective. GFAP-positive cells were counted using the "cell counter" plugin of ImageJ, whereas Iba1-positive cells were quantified by means of the automatic thresholding and segmentation features of ImageJ.