Scratch wound assay.

<p>Monolayer was wounded by manual scratch with a pipette tip. Solutions containing different HA at 0.2% in culture medium with 2.5% of FCS were distributed at Day 0 and cultures were kept at 37°C for 24 hours (Day1). A: control, B: HMW-HA, C: MMW-HA, D: LMW-HA. Histogram above represents width of the wound measured for each wound at D0 and D1. Results are expressed as a ratio of D1 on D0. <b>Wound healing area</b>: Results are expressed as a ratio of D1 on D0 after image analysis; % of wound area represents the ration of wound area at D1/wound area at D0. We show that only MMW-HA induced a significant wound healing compared to control and other HA (***: p<0.001 compared to culture medium, n = 3). The wound healing factor is three times higher compared to control.</p

P2X7 receptor activation.

<p>Cells were pre-incubated or not with a specific inhibitor of P2X7 (BBG) at 20 µM for 20 minutes before incubation with HMW-HA or MMW-HA or LMW-HA at 0.2% for 24-hours. P2X7 activation was evaluated by YO-PRO-1 dye uptake. A: cell culture medium, B: positive control, C: HMW-HA without specific inhibitor of P2X7 (BBG), D: HMW-HA with specific inhibitor of P2X7 (BBG), E: MMW-HA without specific inhibitor of P2X7 (BBG), F: MMW-HA with specific inhibitor of P2X7 (BBG), G: FMW-HA without specific inhibitor of P2X7 (BBG), H: LMW-HA with specific inhibitor of P2X7 (BBG). Incubation with MMW-HA induces slight increase in P2X7 activation compared to control and other HA (n = 18). Moreover preincubation of cell with specific inhibitor of P2X7 (BBG) inhibits MMW-HA effect on P2X7 receptor.</p

Cell viability.

<p>Cells were preincubated or not with specific inhibitor of P2X7 (BBG) at 20 µM for 20 minutes before incubation with MMW-HA at 0.2% for 24-hours. Cell viability was evaluated by neutral red test after incubation with MMW-HA. Grey: without specific inhibitor of P2X7 (BBG), black: with specific inhibitor of P2X7 (BBG). Slight activation of P2X7 we observe with MMW-HA has no effect on cell viability (n = 18).</p

CD44 protein expression.

<p>CD44 expression was evaluated using flow cytometry after incubation with HMW-HA or MMW-HA or LMW-HA at 0.2% for 24 hours. Results showed a very slight effect on CD44 expression level after MMW-HA incubation. A:control, B: HMW-HA, C: MMW-HA, D: LMW-HA (n = 3). Results show that CD44 expression is not modified with HA regardless the molecular weight.</p

Summary diagram.

<p>MMW-HA induces wound healing via P2X7 basal activation.</p

Cell proliferation.

<p>Cells were cultured in medium with 2.5% FCS containing HA HMW-HA or MMW-HA or LMW-HA at 0.2%. Proliferation was analyzed by counting cells during 6 days. circle: cell culture medium, triangle: HMW-HA, square: MMW-HA, lozenge: LMW-HA. Our results show that from day 5 only MMW-HA induced a significative increase on cell proliferation compared to control, HMW-HA and LMW-HA have no effect on cell proliferation (***: p<0.001 compared to culture medium, n = 3).</p

Scratch wound assay.

<p>Monolayer was wounded by manual scratch with a pipette tip and cells were preincubated with BBG at 20 µM for 20minutes before HA incubation. Different solutions containing different HA at 0.2% in culture medium with 2.5% of FCS are distributed (Day0) and culture was kept at 37°C for 24 hours (Day1). A: control without specific inhibitor of P2X7 (BBG), B: MMW-HA with without specific inhibitor of P2X7 (BBG), C: MMW-HA with without specific inhibitor of P2X7 (BBG). % of wound area represents the ration of wound area at D1/wound area at D0. We show that cell preincubation with BBG blocks beneficial effect of MMH-HA on wound healing (***: p<0.001 compared to culture medium, n = 3).</p

Chromatin condensation, P2X7 receptor activation and caspase-3 activity in oxaliplatin-treated C57BL/6 mice.

<p>Mice were repeatedly injected i.p. with 7 mg/kg oxaliplatin (OXA) at days 1, 2, 5 and 6 (28 mg/kg cumulated dose; n = 10). Mitochondrial activity was evaluated by determining mitochondrial membrane potential (A) using JC-1 test and mitochondrial levels of negatively charged phospholipids (B) using nonyl acridine orange test. Chromatin condensation (C) was evaluated using Hoechst 33342 test and P2X7 receptor activation (D) using YOPRO-1 test. The apoTarget^TM Caspase-3 Protease assay was used for the <i>in vitro</i> determination of caspase-3 proteolytic activity (E) in lysates of brain mitochondrial homogenates as described by the manufacturer's instructions. Values are the mean ± S.E.M. expressed as percentage of the control (n = 8). *: statistically different (p<0.05) from the mean values in control mice.</p

Cytokines and PGE2 release in oxaliplatin-treated cells after acetaminophen, ibuprofen or N-acetyl cysteine treatment.

<p>SH-SY5Y cells and RAW 264.7 cells were exposed 24 h to oxaliplatin (OXA) (200 µM) with a 30-min pre-treatment either with acetaminophen (AAP, 50 µM), ibuprofen (IBU, 1 µM) or N-acetyl cysteine (NAC, 1 mM). SH-SY5Y cell supernatant was collected and TNF-α, IL-1β and IL-6 levels (pg/mL) were assessed using ELISA kits according to the manufacturer's instructions. Prostaglandin E2 (PGE2) levels were determined in RAW 264.7 supernatant using a PGE2 Enzyme-Immuno-Assay kit. Values are the mean ± S.E.M. levels in pg/ml, five different assays per group. Significance of differences: OXA alone versus control, *p<0.05; AAP, or IBU, or NAC versus OXA alone: ^$ p<0.05.</p

Oxidative stress and mitochondrial activity in oxaliplatin-treated C57BL/6 mice.

<p>Mice were repeatedly injected i.p. with 7 mg/kg oxaliplatin (OXA) at days 1, 2, 5 and 6 (28 mg/kg cumulated dose; n = 10). Oxidative stress was evaluated by ROS production using dihydroethidium (A) and DCF-DA (B) tests and NO content (C) by the Griess reaction. Values are the mean ± S.E.M. expressed as percentage of the control (n = 8). *: statistically different (p<0.05) from the mean values in control mice.</p