Drugs and Chemicals
5FUra and SM were provided from Sigma, St. Louis, MO (USA) and Merck Company (Germany), respectively. Polyoxyl 40 hydrogenated castor oil, glyceryl monooleate and polyethylene glycol 400 (PEG-400) were purchased from Sigma Aldrich. Total Antioxidant Capacity (TAC) and Malondialdehyde (MDA) kits were prepared from Teb Pajohan Razi Company, Iran. Tween 80 was obtained from Kavosh Gostar Daru, Iran. The cDNA synthesis and RNA extraction kits were obtained from Yekta Tajhiz Azma and Sinacolon companies, Tehran, Iran, respectively.
Preparing silymarin-loaded nanoemulsion (SMN)
SMN was formed in an oil phase of PEG 400, PEG-40 hydrogenated castor oil, and GMO (1 : 8 : 1) spontaneously, so an amount of 50 mg of SM was added to 10 gr of the oil phase. SM, surfactant, oil, and co-surfactant were stirred at 100 rpm for 2 hours. More sonication was applied for 1 hour by a bath sonicator (Elmasonic Med 60) to conduct mixing process. Deionized water, then, was added to the oil phase in the ratio of 5: 1 and then stirred mildly to obtain nanoemulsion.
Animals and treatment
Thirty adult male Wistar rats (180±20 g) were housed in wire-bottomed individually cages (approval No:IR.MUBABOL.REC.1400.213) at a controlled and standard temperature (23±2°C) and humidity (60%±5%), and in a 12/12 h light/dark cycle, with food and water available ad libitum based on the protocols of the Research Council of Babol University of Medical Sciences.
Intraperitoneal (IP) injections of animals were conducted for 14 days. The rats were randomly assigned to 6 groups:
1. Control (Crl) group (n =5): IP administration of normal saline.
2. The 5FUra group (n =5): a single IP administration of 5FUra (100 mg.kg-1).
3. 5FUra +SMN group: a single IP administration of 5FUra (100 mg.kg-1) and SMN (5 mg.kg-1) (n=5).
4. SMN group: a single IP administration of SMN (5 mg.kg-1) (n=5).
5. 5FUra+SM group: a single IP administration of 5FUra (100 mg.kg-1) and SM (5 mg.kg-1) (n=5).
6. SM group: a single IP administration of SM (5 mg.kg-1) (n=5).
Macroscopic histopathological analysis
All rats were placed in the animal house on the first day without any treatment to adapt to their environment. Between the fourth and the ninth day of the injection, the severity of oral ulcerations was investigated and this severity was graded based on the following grading system.
For macroscopic analysis, the alterations in the tongue tissue during 6 days were investigated. Damage severity in a totally blind manner for macroscopic analysis, erosion, vasodilatation, erythema, and epithelial ulceration was assessed as follows:
Score 0: completely healthy without injury with no vasodilatation or erosion in the surface area
Score 1: the presence of erythema, despite no surface erosion
Score 1.5: the presence of vasodilation, existing surface erosion, and severe erythema
Score 2: the presence of focal ulcers in one or more mucosal faces, but not surpassing 25% of the surface area, vasodilatation, and severe erythema
Score 2.5: the presence of cumulative ulcers at around half of the surface area
Score 3: the presence of cumulative ulcers at around 75% of the surface area
The body weight mensuration
The rats in all groups were weighed on the 1st, 5th, 9th, and 13th of the treatment period to compare weight changes between these days.
The preparation of serum and tongue, esophageal, and intestinal tissues
After performing different treatments, the experimental animals were anesthetized with ketamine/xylazine (IP) at a dose of 75/25 mg.kg-1, and then 2 cc of blood were obtained from the heart to assess biochemical parameters: MDA and TAC. After weighing, the tongue, esophageal, and intestinal tissues were removed through the incision for histopathological staining.
Thiobarbituric acid reactive substance (TBARS) assay
Shortly, following animal anesthetizing and blood sampling, all the serum samples were stored at -80 °C until measuring the biochemical agents. The TBARS was carried out to assess the levels of oxidative stress in the sera. To measure TBARS levels, after adding 1 mL of serum to 2 mL of TBARS reagent, they were heated at 100 °C for 60 min. Thereafter, samples were placed in an ice bath for 10 min and a 10 min centrifuging was performed at 2500 rpm. Then, thiobarbituric acid and MDA will bind with each other to produce a red component. Elisa reader at 535 nm was used to assess the light absorbance.
Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was employed to evaluate antioxidant power in serum samples. After animal anesthetizing, 1.5 mL of prepared FRAP was added to tubes included with sera, and incubation was performed at 5 °C for 37 min. Subsequent to incubating, 51 μL of the sample was added to the tubes and blended well. After re-incubation of mixtures for 15 min at 37 °C, the antioxidant power of the samples were measured by reducing the ferric ion (Fe3 +) to the ferro one (Fe2 +). Thereafter, the blue color intensity was evaluated by an Elisa reader at 593 nm.
Histopathological assessment
After animal anesthetizing with ketamine/xylazine (at a dose of 75/25 mg.kg-1, IP) and after removing their tongue, esophageal, and intestinal tissues, they were prepared for histological and molecular studies. The tongue, esophageal, and intestinal tissue samples were immediately gathered. After being inflated with mild PBS, they were fixed in 4% paraformaldehyde for 24 hours, placed in paraffin, and then sections (5 µm) were provided by microtome (model Leitz 1512, Germany). A mean of 3 fields was taken into consideration for each slide.
To evaluate the histopathological degeneration in the above-mentioned tissues, H & E staining was used. Three animals per group were available, then 7 slides per animals were provided, 3 fields were chosen in each slide, and inflammation level was calculated. Histological analysis was conducted using image J software.
Briefly, tissue specimens of the samples were placed in 100% alcohol for 5 min, then placed in 96% alcohol for 5 min, stained with hematoxylin for another 5 min, rinsed with running water for 5 min as well, stained with eosin for 15 seconds, immersed in distilled water for decolorization, placed at ethanol 70% for 15 seconds, ethanol 95% for 30 seconds, absolute ethanol for 1 min, and xylene for 5 min and eventually pasted with entellan. Evaluation of histology, inflammation of cells, and morphological deformation in the tongue, esophageal, and intestinal tissues was conducted using H&E staining. These tissues were assessed under a microscope (Olympus BX61VS, Japan), then, the results were analyzed using Image J software.
RNA extraction and real-time polymerase chain reaction (RT- PCR)
To assess TNF-α and IL-2 levels in the tongue, esophagus, and intestinal tissues, the total RNA was immediately isolated based on the instructions of the manufacturer (Pars Tous, Mashhad, Iran), then the extracted RNAs were transferred to -20°C overnight and then to -80 °C until the cDNA synthesis. Synthesizing of cDNA was performed based on the instructions of the manufacturer (Pars Tous, Mashhad, Iran). qRT-PCR was performed by an ABI Step One Plus RT- PCR System (Applied Biosystem, USA) with the primer sets for TNF-α and IL-2 as target genes and a housekeeping gene (GAPDH). An amount of 10 μL of RT- PCR reaction mixture contains cDNA (1 μL), SYBR-Green (6.25 μL) (Amplicon high Rox master mix, Denmark), nuclease-free water (2.25 μL), and 0.25 μL of 10 pmol of each primer (Robin Teb Gostar, Tehran, Iran). Based on kit instructions, the steps of reverse transcription were conducted at 25°C for 10 min, then 37°C for 60 min, and then 85°C for 5 min. The PCR was conducted by keeping the temperature at 95°C for 15 min, afterwards, at 95°C for 40 cycles of 15s, at 62°C for 30 s, and then at 72°C for 30 s followed by melting curve temperature steps. The TNF-α and IL-2 primers are illustrated in Table 1.
Statistical analysis
GraphPad Prism software version 8 was employed to analyze all data. The parametric variables were represented as mean ± SEM. Analysis was conducted using one-way analysis of variance (ANOVA) and two-way ANOVA and a Tukey post-test. Two-way ANOVA was employed for histopathological scoring changes between the groups. The statistical significance criterion was a p-value of less than 0.05.