Materials and methods

Animal model and ethics statement

All experimental procedures were carried out in the Center for Experimental Surgery and Animals Lab, Prince Naif Health Research Center (PNHRC), King Khalid University Hospital (KKUH), Riyadh, Saudia Arabia. The experimental procedure was pre-approved by the Ethical Committee of Bioethics at King Saud University (KSU), Ref Number: SE-24-33.

All animals hosted in polypropylene cages in an environmentally controlled clean air room, with a temperature of (25 °C ± 1 °C) 12 h light/12 h dark cycle and a relative humidity of 50 ± 5 %.

Sixteen healthy female Wistar albino rats (8 ± 1 weeks old, weighting 250 ± 15 g), were mated overnight with male rats in the proportion of 2:1, and the day spermatozoa were detected in the vaginal smear, was designated as the first day of gestation (Figure 1).

Figure 1:: Diagrammatic scheme of the animal experiments.

The pregnant females were randomly assigned into fife experimental groups as follows:

Group.1: 2 Control females were kept on normal standard diet and injected with physiological saline.

Group.2: 5 Females were kept on normal standard diet and received a single intraperitoneal (IP) injection of 600 mg/kg sodium valproate on day 12.5 after conception.

Group.3: 2 Females were kept on normal standard diet and received oral administration of 400 mg/Kg of artichoke daily from the 1st day of gestation until the date of birth. Also, they received a single IP injection of 600 mg/kg VPA on day 12.5 after conception.

Group.4: 5 Females were kept on normal standard diet and received a single IP injection of 100 µg/kg LPS on day 9.5 after conception.

Group.5: 2 Females were kept on normal standard diet and received oral administration of 400 mg/Kg of artichoke daily from the 1st day of gestation until the date of birth. Also, they received a single IP injection of 100 µg/kg LPS on day 9.5 after conception.

Females were housed individually and allowed to raise their own litters, and the experiments were carried out on male offspring as follows:

Group.1.1: 6 male puppies received normal saline orally after day 7 of birth (control).

Group.2.1: 6 male puppies were used as valproate-exposed rats (VPA-0).

Group.2.2: 6 male puppies were received 400 mg/Kg of artichoke orally after day 7 of birth (VPA-AR).

Group.2.3: 6 male puppies were received a mix of 400 mg/Kg of artichoke and mixture of probiotics after day 7 of birth (VPA-AR.Pro).

Group.3.1: 6 male puppies were received 400 mg/Kg of artichoke orally after day 7 of birth (VPA.AR-AR).

Group.4.1: 6 male puppies were used as LPS-exposed rats (LPS-0)

Group.4.2: 6 male puppies were received 400 mg/Kg of artichoke orally after day 7 of birth (LPS-AR).

Group.4.3: 6 male puppies were received a mix of 400 mg/Kg of artichoke and mixture of probiotics with omega −3 after day 7 of birth (LPS-AR.Pro.ω3).

Group.5.1: 6 male puppies were received 400 mg/Kg of artichoke orally after day 7 of birth (LPS.AR-AR).

At the end of the experiment, a three-chamber social test was performed with offspring beginning at 4 weeks of age.

Drugs & dosage

Sodium valproate (VPA)

VPA salt was obtained from Sigma-Aldrich (Catalog No. P4543). It was dissolved in normal saline (0.9 % NaCl) at a concentration of 250 mg/ml as described by Schneideret al., (2005). Exposure to VPA around embryonic day 12 (E12) is associated with the highest susceptibility for ASD development [35]. While the commonly used VPA dose in animal models of ASD is 600 mg/kg, it is important to note that clinical doses of VPA in humans range from approximately 3 to 55 mg/kg, corresponding to a plasma concentration of ∼100 μg/mL. Thus, the dose used in animal studies is typically 10–20 times higher than therapeutic levels in humans [8], 36].

Lipopolysaccharide (LPS)

LPS (from Escherichia coli, serotype 055: B5, Solarbio, Beijing, China) was dissolved in saline solution at a concentration of 100 µg/kg and administered to pregnant dams (I.P. in a volume of 2 mL/kg at GD 9.5) in order to induce some autistic-like behavioral changes in the rat offspring as previously reported [37], 38].

Extract from cynara scolymus L. (Artichoke)

The shade-dried powdered artichoke (1 Kg) was extracted with 96 % ethanol (4 × 1 L), using Soxhlet equipment for 3 h at 80 °C. All the extracts were combined and filtered through the Whatman paper No 1. The filtrate was then freed from the solvent under reduced pressure at ± 40 °C temperature using rotavapor, resulting in a dark green extract (101.7 g). The extract was placed in the hood to ensure complete ethanol evaporation. The final dry ethanolic extract was stored at 4 °C until further use. Artichoke extract was diluted in distilled water at a final concentration of 400 mg/kg. The dose of artichoke used in this study was determined based on previous studies [33], 34], 39].

Mixture of probiotics

Infant probiotics (LoveBug Nutrition, NY) obtained from iHerb, are a mixture of healthy bacteria, including Bifidobacterium infants, Bifidobacterium lactis and Lactobacillus rhamnosus GG, with the concentration of 1 billion CFU per 1.5 g [40].

Omega-3 polyunsaturated fatty acids (or omega-3 PUFAs, n-3 PUFAs)

Omega-3 Premium Fish Oil (from California Gold Nutrition, USA) obtained from iHerb, was orally given at a dose of 200 mg/kg body weight/day [41].

Evaluation of social behavior

Social behavior, an important feature of ASD, was assessed using the three-chamber social test, a commonly used test to evaluate social impairments in animal models of autism [42]. A 120 cm × 40 cm × 40 cm clear rectangular plexiglass box divided into three chambers with walls and 15 cm × 15 cm doors with removable slides to allow the animals to pass through. The three-chamber box was cleaned with 70 % ethanol, dried with paper towels, and then let to air dry between trails. The animals were transferred to the testing room 1 h before the test for acclimatization. Animals were picked from the cage and placed individually in the center chamber and allowed to explore for 5 min while the two doorways of the box were open. Following this habituation period, an age, weigh and sex-matched stranger rat (conspecific) to the testing rats was placed in one of two perforated holding containers that were located on either side of the box. The subject rat was allowed to explore all three chambers freely for 10 min. The test was recorded using an HD camcorder. Videos were later analyzed to code behaviour using BORIS 7.9.16 software [43]. Social interaction time was quantified by measuring the focal animal’s orientation towards and investigating the containing the conspecific, evidenced by sniffing or rearing. Time spent in each chamber was analyzed, and the percentage of time spent in the social chamber (containing the conspecific rat) relative to the non-social chamber (containing the empty holding container) and the center chamber were calculated. Grooming was analyzed for total duration and the location where it was performed, comparing time spent in the stimulus-containing chamber vs. the empty chamber. Time spent immobile (bouts of no movements) was recorded. To assess activity, locomotion was analyzed for total duration.

Statistical analysis

Behavioral data were analyzed using one-way ANOVA by GraphPad Prism 10. Ink, software to evaluate the effects of VPA and LPS on social interaction, grooming, immobility and locomotion in experimental following groups (n=6 per group): Control, VPA (valproic acid; autism model group), VPA-AR (artichoke treatment group), VPA-AR.PRO (artichoke and probiotic combination treatment group), VPA.AR-AR (artichoke protective treatment group), LPS (lipopolysaccharide; autism model group), LPS-AR (artichoke treatment group), LPS-AR.PRO.ω3 (artichoke, probiotic, and omega-3 combination treatment group), and LPS.AR-AR (artichoke protective treatment group). Tukey’s post hoc tests were used for pairwise comparisons, with p≤0.05 considered significant. Data are presented as mean +/− standard errors of the mean. Significant differences are indicated as follows: ****p≤0.0001, ***p≤0.001, *p≤0.01, p≤0.05.