Carboxymethylcellulose sodium salt

Carboxymethylcellulose sodium salt (low viscosity), urea, meglumine, TRIS, L-lysine, L-aspartic acid, and L-histidine were bought from Sigma Aldrich (St. Louis, MO, USA). Purified water, which was used for the solvent evaporation, was taken from a MilliQ Millipore filter system (Millipore Co., Bedford, MA, USA).

Female WT (n = 21) and A53T (n = 8) mice aged 12 weeks received rotenone (30 mg/kg) daily via oral gavage for 28 days (between 9.00–10.00 AM AEDT) (Table 1). This dose of oral rotenone and duration of treatment has been shown to induce specific nigrostriatal DA neurodegeneration and marked reduction in endurance time on the rota‐rod in C57bl/6 mice^{26 (link)}. Rotenone was suspended in a solution of 1.25% (v/v) chloroform (Sigma Aldrich, St Louis, USA) and 1% (w/v) carboxymethyl cellulose sodium salt (Sigma Aldrich, St Louis, USA). The total volume of solution delivered was 0.05 mL/10 g of bodyweight. A separate cohort of WT (n = 18) and A53T (n = 6) mice received vehicle solution only (1.25% (v/v) chloroform in 1% (w/v) carboxymethyl cellulose)^{27 (link)}.

Plaque forming unit assays were used to quantify the release of infectious viral particles [23 (link),28 (link)]. Vero cells were seeded the previous day in 48-well culture plates at a density of 3×10⁴ cells per well. Cells were infected by 0.1 mL of ten-fold dilutions of supernatant. Following an incubation of 2 h at 37 °C, 0.2 mL of the culture medium supplemented with 5% fetal bovine serum (FBS) and 0.8% carboxymethylcellulose sodium salt (Sigma-Aldrich, Saint-Quentin-Fallavier, France)) were added, and the incubation was extended for 4 days at 37 °C. Cells were fixed (PFA, 3.7%) and stained with 0.5% crystal violet (Sigma-Aldrich) diluted in 20% ethanol, after the media had been removed. Plaques were counted and expressed as plaque-forming unit per mL (PFU mL⁻¹).

POE extract was suspended in 1% carboxymethylcellulose sodium salt (CMC; Sigma-Aldrich, Milan, Italy) and acutely administered per os (p.o.) in a dose ranging from 10 to 100 mg kg⁻¹. Control animals were treated with vehicle.

Virus infectious titre was quantified using plaque forming unit assay. Vero cells were seeded in 48-well culture plates at a density of 3 × 10⁴ cells per well and incubated overnight at 37 °C to produce confluent monolayers. Ten-fold serial dilutions of supernatant were prepared in duplicate in culture medium and 0.1 mL of each dilution was added to the cells. Plates were incubated for 2 h at 37 °C, then 0.2 mL of culture medium supplemented with 5% foetal bovine serum (FBS) and 0.8% carboxymethylcellulose sodium salt (Sigma-Aldrich, France) were added, followed by a 4 days incubation at 37 °C. Then, media were removed and cells were fixed (PFA, 3.7%) and stained with 0.5% crystal violet (Sigma-Aldrich, France) diluted in 20% ethanol. Plaques were counted and expressed as plaque-forming unit per mL (PFU.mL⁻¹).

The mice were randomly divided into four groups: control (H₂O) n = 3, DSS n = 4, DSS + GED n = 5, and DSS + 5ASA n = 4. Chronic colitis and fibrosis were induced in mice by oral administration of 2.5% (w/v) DSS (MW: 36,000–44,000, purchased from TdB Consultancy, Uppsala, Sweden), solubilized in autoclaved tap water and administered ad libitum for three cycles (5 days DSS, followed by 7 days of tap water). The control group received only tap water. GED-0507-34 Levo (Nogra Pharma Ltd., Dublin, Ireland), a selective agonist of PPAR-γ, was dissolved in a solution containing 0.5% Carboxymethylcellulose sodium salt (Sigma-Aldrich, Darmstadt, Germany) and 1% Tween 80, administrated by oral gavage (100 μL/mouse), at a dose of 30 mg/Kg/day. The 5-aminosalicylic acid (5-ASA) (Pentasa, Ferring Pharmaceuticals, Gentilly, France), the most common anti-inflammatory drug used in IBD treatment, was mixed with standard chows and was administrated daily at a dose of 150 mg/kg. All drugs were administrated at the beginning of the second cycle of DSS (day 12).

Plaque forming unit assay was used to quantify virus infectious particles. Vero cells were seeded the previous day in 24-well culture plates at a density of 7 × 10⁴ cells per well. Cells were infected by 0.15 mL of tenfold dilutions of supernatant. After 2 h of incubation at 37 °C, 0.2 mL of culture medium supplemented with 5% fetal bovine serum (FBS) and 0.8% carboxymethylcellulose sodium salt (Sigma-Aldrich) was added, and the incubation was extended for 96 h at 37 °C. Cells were rinsed with PBS 1X, fixed with PFA 3.7 % in PBS 1X, and stained with 0.5% crystal violet (Sigma-Aldrich, Saint-Quentin-Fallavier, France) diluted in 20% ethanol. Plaques were counted and expressed as plaque-forming unit per mL (PFU/mL)