Neuroprotective effects of Apigenin on prenatal Valproic acid-induced autism spectrum disorder in rats

Abstract

Valproic acid (VPA) demonstrates teratogenic effects during pregnancy. Prenatal exposure to VPA may result in autism spectrum disorder (ASD) -like phenotypes. Apigenin, a natural flavonoid, has been shown to have neuroprotective impacts due to its antioxidant properties. This study aimed to investigate the protective effects of apigenin in prenatal Valproic acid-induced autism in rats. Female rats (220-240 g, 2-3 months) received a single dose of VPA (600 mg/kg, i.p.) on the 12.5th day of gestational. The male offspring were given oral apigenin (50 mg/kg, p.o.) or the vehicle for 30 days. Behavioral tests, biochemical assessments for oxidative stress markers and pro-inflammatory cytokines were performed. VPA-treated rats exhibited increased anxiety-like behavior, and repetitive behavior. Social interaction was reduced, and detection of the novel object was impaired. Also, VPA-treated rats have shown higher levels of oxidative stress malondialdehyde (MDA) and lower GPX and superoxide dismutase (SOD) levels. Furthermore, IL-6 and TNF-α increased in the prefrotalcortex decreased. On the other hand, apigenin-treated rats restored the cognitive consequences and lowered oxidative stress and inflammation in the prefrotalcortex.

Conclusion: Chronic apigenin treatment restored the behavioral and biochemical abnormalities caused by prenatal VPA exposure.

Keywords: Apigenin; Autism spectrum disorder; Inflammation; Oxidative stress; Rats; Valproic acid.

Fig. 1

Evaluation of social behavior using three chamber apparatus A) Two-way ANOVA analysis showed that prenatal exposed to VPA caused social deficits, as demonstrated significantly less time spent with stranger 1 and more time spent in the center chamber when compared to control rats. B) Treatment, One ANOVA revealed a significant reduce in the sociability index, as the ratio of time spent in stranger 1 side over empty side. C) On day 4, VPA-treated rat spent more time both with the familiar rat and in the center compartment, but less time with the novel rat. D) One ANOVA revealed a significant reduction in the preference index, as the ratio of time spent in novel side over the time spent in familiar side. Data represented as the mean ± SEM (n = 8, males). *p<0.05, **p<0.01, ***p<0.001 when compared to control. +p<0.05, ++p<0.01, +++p<0.001 when compared to VPA-treated rat.

Fig. 2

Evaluation of anxiety-related behavior in using the elevated plus maze (EPM). A) VPA exposed rats spent less time in open arms of the elevated plus maze (B). In the EPM test, VPA-exposed rats showed a significant reduced in the anxiety index, which is the ratio of time spent in open to the total time spent in both open and closed arms. Data represented as the mean ± SEM (n = 8, males). *p<0.05, **p<0.01, ***p<0.001 when compared to control. +p<0.05, ++p<0.01, +++p<0.001 when compared to VPA-treated rat.

Fig. 3

Evaluation of repetitive behavior using the Marbel Burring test. VPA exposure increased the repetitive behaviors in rat. Treatment with apigenin decreased the repetitive behaviors in rat prenatally exposed to VPA. Data represented as the mean ± SEM (n = 8, males). *p<0.05, **p<0.01, ***p<0.001 when compared to control. +p<0.05, ++p<0.01, +++p<0.001 when compared to VPA-treated rats.

Fig. 4

Evaluation of exploratory behavior using novel object recognition test. VPA exposure decreased the exploration behaviors in rat. Treatment with apigenin increased the exploration behaviors in rat prenatally exposed to VPA. Data represented as the mean ± SEM (n = 8, males). *p<0.05, **p<0.01, ***p<0.001 when compared to control. +p<0.05, ++p<0.01, +++p<0.001 when compared to VPA-treated rat.

Fig. 5

Evaluation of oxidative stress markers between groups (n = 3). A: VPA decreased SOD and treatment with apigenin increased SOD in prefrontal cortex. B: VPA decreased GPx in prefrontal cortex and treatment with apigenin increased GPx. C: VPA decreased MDA and Treatment with apigenin decreased MDA in prefrontal cortex. Data represented as the mean ± SEM (n = 8, males). *p<0.05, **p<0.01, ***p<0.001 when compared to control. +p<0.05, ++p<0.01, +++p<0.001 when compared to VPA-treated rat.

Fig. 6

Evaluation of inflammatory markers between groups (n = 3). A: VPA increased IL-1β and treatment with apigenin decreased IL-1β in prefrontal cortex. B: VPA increased TNF-α and treatment with apigenin decreased TNF-α in prefrontal cortex. Data represented as the mean ± SE (n = 8, males). *p<0.05, **p<0.01, ***p<0.001 when compared to control. +p<0.05, ++p<0.01, +++p<0.001 when compared to VPA-treated rat.