Results

Behavioral data coding of the rats’ social behaviors was conducted using BORIS software, focusing on key parameters such as the duration and frequency of social interactions and percent social interaction per duration (Figure 2), time spent immobile and its frequency during the test as well as grooming behavior frequency and locomotion duration (Figure 3). As shown in Figure 2, social interactions within the social chamber were analyzed. Significant differences in time spent interacting with the novel rat were observed between the control, VPA, and LPS groups (F (4, 25) = 31.09, p<0.0001; Table 1, F (4, 25) = 13.59, p<0.0001; Table 2). Both the VPA-0 and LPS-0 groups exhibited significantly reduced social interaction compared to the control group (p≤0.01 for VPA-0 vs. control; p≤0.05 for LPS-0 vs. Control).

Figure 2:: Social interaction duration and frequency in control and experimental groups. Saline (n=6), VPA (n=6; autism model), VPA-AR (n=6; artichoke treatment), VPA-AR.PRO (n=6; artichoke + probiotic), VPA-AR-AR (n=6; protective), LPS (n=6; autism model), LPS-AR (n=6; artichoke treatment), LPS-AR.PRO.ω3 (n=6; artichoke + probiotic + omega-3), and LPS-AR-AR (n=6; protective). Data are presented as mean ± SEM. One-way ANOVA, (**** p≤0.0001, *** p≤0.001, ** p≤0.01, * p≤0.05). A, social interaction total duration of VPA; B, social interaction frequency of VPA; C, percentage of time spent in the social chamber relative to total test duration of VPA; D, social interaction total duration of LPS; E, social interaction frequency of LPS and F, percentage of time spent in the social chamber relative to total test duration of LPS; VPA, valproic acid; AR, artichoke; Pro, probiotics; ω3, omega3; m LPS, lipopolysaccharide.

Figure 3:: Locomotion total duration, immobility duration and grooming frequency in control and experimental groups. Saline (n=6), VPA (n=6; autism model), VPA-AR (n=6; artichoke treatment), VPA-AR.PRO (n=6; artichoke + probiotic), VPA-AR-AR (n=6; protective), LPS (n=6; autism model), LPS-AR (n=6; artichoke treatment), LPS-AR.PRO.ω3 (n=6; artichoke + probiotic + omega-3), and LPS-AR-AR (n=6; protective). Data are expressed as mean ± SEM. One-way ANOVA significance: ****p≤0.0001, ***p≤0.001, **p≤0.01, *p≤0.05). A, locomotion total duration of VPA and B, locomotion total duration of LPS; C, immobility total duration of VPA; D, immobility total duration of LPS; E, grooming frequency of VPA; F, grooming frequency of LPS; VPA, valproic acid; AR, artichoke; Pro, probiotics; ω3, omega3; LPS, lipopolysaccharide.

Parameters | Groups | N | Mean ± S.E. | Median | F (DFn, DFd) | P value
Social interaction duration (sec) | Control | 6 | 132.05 ± 27.76b | 149.89 | 31.094 (4, 25) | <0.001
 | VPA-0 | 6 | 34.71 ± 2.97a | 35.19 |  | 
 | VPA-AR | 6 | 40.62 ± 18.19a | 39.23 |  | 
 | VPA-AR.PRO | 6 | 151.29 ± 4.39b | 150.91 |  | 
 | VPA.AR-AR | 6 | 255.93 ± 14.23c | 248.65 |  | 
Social interaction frequency | Control | 6 | 12.00 ± 2.44b | 14.00 | 4.876 (4, 25) | 0.005
 | VPA-0 | 6 | 4.67 ± 0.21a | 5.00 |  | 
 | VPA-AR | 6 | 5.83 ± 2.61ab | 5.50 |  | 
 | VPA-AR.PRO | 6 | 12.00 ± 0.68b | 12.50 |  | 
 | VPA.AR-AR | 6 | 11.83 ± 0.83b | 12.00 |  | 
% time in social champer | Control | 6 | 56.06 ± 13.50a | 54.97 | 1.132 (4, 25) | 0.364
 | VPA-0 | 6 | 36.46 ± 6.89a | 32.35 |  | 
 | VPA-AR | 6 | 38.90 ± 17.15a | 26.86 |  | 
 | VPA-AR.PRO | 6 | 56.58 ± 4.11a | 57.38 |  | 
 | VPA.AR-AR | 6 | 60.58 ± 3.04a | 62.41 |  | 
Locomotion duration (sec) | Control | 6 | 198.17 ± 38.35ab | 189.60 | 5.304 (4, 25) | 0.003
 | VPA-0 | 6 | 314.85 ± 20.69c | 307.90 |  | 
 | VPA-AR | 6 | 157.47 ± 41.40a | 167.83 |  | 
 | VPA-AR.PRO | 6 | 280.42 ± 17.90bc | 284.54 |  | 
 | VPA.AR-AR | 6 | 203.47 ± 2.51abc | 202.96 |  | 
Immobility duration (sec) | Control | 6 | 0.80 ± 0.51ab | 0.00 | 25.471 (4, 25) | <0.001
 | VPA-0 | 6 | 12.49 ± 0.83c | 12.40 |  | 
 | VPA-AR | 6 | 3.44 ± 1.25ab | 3.88 |  | 
 | VPA-AR.PRO | 6 | 4.66 ± 1.53b | 6.21 |  | 
 | VPA.AR-AR | 6 | 0.00 ± 0.00a | 0.00 |  | 
Grooming frequency | Control | 6 | 8.67 ± 0.33ab | 8.50 | 9.224 (4, 25) | <0.001
 | VPA-0 | 6 | 17.17 ± 1.68c | 17.50 |  | 
 | VPA-AR | 6 | 13.67 ± 2.22bc | 14.50 |  | 
 | VPA-AR.PRO | 6 | 7.83 ± 0.40a | 8.00 |  | 
 | VPA.AR-AR | 6 | 7.83 ± 1.25a | 7.00 |  | 

Parameters | Groups | N | Mean ± S.E. | Median | F (DFn, DFd) | P value
Social interaction duration (sec) | Control | 6 | 132.05 ± 27.76b | 149.89 | 13.587 (4, 25) | <0.001
 | LPS-0 | 6 | 50.22 ± 10.25a | 57.04 |  | 
 | LPS-AR | 6 | 251.65 ± 23.77c | 252.44 |  | 
 | LPS-AR.PRO.ω3 | 6 | 120.21 ± 14.35ab | 112.15 |  | 
 | LPS.AR-AR | 6 | 137.06 ± 16.81b | 125.44 |  | 
Social interaction frequency | Control | 6 | 12.00 ± 2.44b | 14.00 | 4.396 (4, 25) | 0.008
 | LPS-0 | 6 | 5.33 ± 1.09a | 6.00 |  | 
 | LPS-AR | 6 | 13.00 ± 0.97b | 13.00 |  | 
 | LPS-AR.PRO.ω3 | 6 | 10.33 ± 1.17ab | 9.50 |  | 
 | LPS.AR-AR | 6 | 11.67 ± 0.99 b | 11.50 |  | 
% time in social champer | Control | 6 | 56.06 ± 13.50a | 54.97 | 1.286 (4, 25) | 0.302
 | LPS-0 | 6 | 54.46 ± 12.85a | 50.24 |  | 
 | LPS-AR | 6 | 74.56 ± 8.99a | 72.84 |  | 
 | LPS-AR.PRO.ω3 | 6 | 46.75 ± 2.04a | 48.31 |  | 
 | LPS.AR-AR | 6 | 65.73 ± 4.49a | 60.74 |  | 
Locomotion duration (sec) | Control | 6 | 198.17 ± 38.35ab | 189.60 | 5.616 (4, 25) | 0.002
 | LPS-0 | 6 | 87.11 ± 16.92a | 92.07 |  | 
 | LPS-AR | 6 | 231.81 ± 35.88b | 246.92 |  | 
 | LPS-AR.PRO.ω3 | 6 | 239.05 ± 26.75b | 212.74 |  | 
 | LPS.AR-AR | 6 | 264.73 ± 23.57b | 267.07 |  | 
Immobility duration (sec) | Control | 6 | 0.80 ± 0.51a | 0.00 | 17.836 (4, 25) | <0.001
 | LPS-0 | 6 | 19.90 ± 3.11b | 17.56 |  | 
 | LPS-AR | 6 | 2.92 ± 1.08 a | 3.28 |  | 
 | LPS-AR.PRO.ω3 | 6 | 4.58 ± 2.03a | 3.50 |  | 
 | LPS.AR-AR | 6 | 2.18 ± 1.42a | 0.00 |  | 
Grooming frequency | Control | 6 | 8.67 ± 0.33a | 8.50 | 17.561 (4, 25) | <0.001
 | LPS-0 | 6 | 13.00 ± 0.89b | 12.50 |  | 
 | LPS-AR | 6 | 6.67 ± 0.62a | 7.00 |  | 
 | LPS-AR.PRO.ω3 | 6 | 5.67 ± 0.49a | 5.50 |  | 
 | LPS.AR-AR | 6 | 5.67 ± 1.09a | 5.50 |  | 

Furthermore, the VPA-AR group showed a significant reduction in interaction time compared to the control group (p≤0.001), whereas the LPS-AR group demonstrated a significant increase in social interaction duration relative to the LPS-0 group (p≤0.0001). Notably, the LPS-AR group also exhibited significantly higher social interaction levels than the control group (p≤0.01).

The VPA-AR.PRO group showed a significant improvement in social interaction compared to both VPA-0 and VPA-AR (p≤0.001 for both comparisons). Among the VPA-treated groups, the protective group VPA-AR.AR showed the most substantial improvement, with interaction times significantly higher than VPA-0 (p≤0.0001), VPA-AR (p≤0.0001), and VPA-AR.PRO (p≤0.01). Similarly, the LPS-AR.AR protective group showed a significant increase in interaction time compared to LPS-0 (p≤0.05) were LPS-AR.PRO.ω3 does not show a significant difference with control or LPS-0 groups.

In terms of social interaction frequency (F (4, 25) = 4.876, p=0.0048; Table 1, F (4, 25) = 4.396, p=0.0079; Table 2), both VPA-0 and LPS-0 groups exhibited significantly reduced interaction frequency compared to the control group (p≤0.05 for both). The VPA-AR group does not show a significant difference with control or VPA-0 groups were VPA-AR.PRO group showed a significant increase in frequency relative to VPA-0 (p≤0.05). Additionally, VPA-AR.AR protective group demonstrated a significantly higher frequency of social interaction compared to VPA-0 (p<0.05). The LPS-AR group showed a significant increase in interaction frequency compared to LPS-0 (p≤0.05). While LPS-AR.PRO.ω3 and LPS-AR.AR group does not show a significant difference with control or LPS-0 groups. In addition, Figure 2 illustrates the percentage of time spent in the social chamber across the experimental groups. In panels C (F (4, 25) = 1.132, p=0.3642, Table 1) and F (F (4, 25) = 1.286, p=0.3023, Table 2) did not reveal statistically significant differences between groups, although a non-significant trend toward reduced social preference was observed in the VPA-exposed model.

Figure 3 illustrates the locomotion duration, duration of immobility and grooming frequency behaviors across the experimental groups. In panels A and B, of Figure 3, presents data on locomotion duration, The VPA-0 group (F (4, 25) = 5.304, p=0.0031, Table 1) shows a statistically significant hyperactivity (p≤0.05) in locomotion compared to the control. While LPS -0 group seems to cause hypoactivity. With artichoke treatment in VPA-AR group significantly drops (F (4, 25) = 5.616, p=0.0023, Table 2) locomotion (p≤0.01) compared to the VPA-0 group, while combination treatment with probiotics reduced hyperactivity compared to VPA-0, but remains notably more active than the control group. Meanwhile in the protected group (VPA-AR.AR) shows stable results, with locomotion levels almost matching the control. In the other hand, LPS-AR, LPS-AR.PRO.ω3, and LPS.AR-AR significantly increased locomotion (p≤0.05 and p≤0.01) compared to the LPS-0 group.

In panels C (F (4, 25) = 25.47, p<0.0001, Table 1) and D (F (4, 25) = 17.84, p<0.0001, Table 2) both the VPA-0 and LPS-0 groups exhibited significant increase in immobility duration compared to the control group (p≤0.0001). Treatment with artichoke extract (VPA-AR and LPS-AR) significantly reduced immobility duration compared to their respective untreated groups (p≤0.0001). Similarly, the VPA-AR.PRO and LPS-AR.PRO.ω3 groups also showed significantly reduced immobility compared to the VPA-0 and LPS-0 groups (p≤0.0001).

Notably, the protective groups (VPA-AR.AR and LPS-AR.AR) displayed near-complete normalization of activity, with immobility durations comparable to the control group and significantly lower than both VPA-0 and LPS-0 (p≤0.0001). Figure 3 also presents data on grooming behavior, including frequency. In panels E (F (4, 25) = 9.224, p=0.0001, Table 1) and F (F (4, 25) = 17.56, p<0.0001, Table 2), the VPA-0 and LPS-0 groups exhibited a significant increase in grooming frequency compared to the control group (p≤0.05), indicating elevated repetitive behavior.

The VPA-AR intervention significantly reduced grooming frequency compared to VPA-0 (p≤0.05), normalizing it to levels comparable to the control group. Both the VPA-AR.PRO and VPA-AR.AR groups further reduced grooming frequency beyond control levels (p≤0.001 vs. VPA-0). Similarly, the LPS-AR treatment significantly reduced grooming frequency compared to LPS-0 (p≤0.001). The LPS-AR.PRO.ω3 intervention normalized grooming frequency to control levels (p≤0.001 vs. LPS-0), while the protective group LPS-AR.AR reduced it even further – significantly below both the LPS-0 and control levels (p≤0.001).