Ameliorative Effect Of Hypericin On Valproic Acid-induced Autism-like Behavior In Mice And Its Molecular Mechanism

The core symptoms of autism spectrum disorder(ASD)are impaired social skills,high levels of repetitive interests and behaviors.The prevalence of autism is high,typically between 1 and 2 percent,and is increasing.The causes of autism are complex and are believed to be a combination of genetic and environmental factors.The prefrontal lobe is closely related to higher neural functions such as social cognition and emotion.Studies have shown that the structure and function of neurons in the medial prefrontal cortex(mPFC)are abnormal in patients with autism and animal models,and the level of oxidative stress is high,suggesting that the prefrontal cortex plays a very important role in the pathogenesis of autism.Valproic acid(VPA)has been widely used clinically to fight epilepsy and has a mood stabilizing effect.Clinical studies have found that pregnant women who use VPA are 4.42 percent more likely to have a child with autism after birth.At present,VPA exposure during pregnancy is a commonly used method to establish autism models.Autism models constructed by this method have advantages of good construction validity and surface validity and high stability.Hypericin(HY)is a kind of naphthalene dianthranone compound,which has antioxidant effect,antidepressant effect,antiviral and other pharmacological effects.HY has long been used in the study of depression in Europe.Some studies have shown that HY also has therapeutic effect on Alzheimer's disease model mice,exerting neuroprotective effect both in vivo and in vitro.Recent studies suggest that HY can prevent maternal isolation induced autistic behavior in offspring rats.Based on HY's rich potential pharmacological value,antioxidant effect,neuroprotective effect,and preventive effect on autistic behavior in offspring caused by mother-parent isolation,we speculated that HY could improve the autistic behavior induced by VPA in mice.In this study,HY was used to treat VPA-induced mouse autism model to observe whether the mice's autism-like behavior was improved,and to explore the molecular mechanisms.Then,VPA-induced cell injury model was used to verify its possible molecular mechanism in vitro.Firstly,a mouse model of VPA-induced autism was established.From the 14th day after birth,male VPA mice were given 50 mg/kg HYL for 14 days,and the behavioral test was used to detect whether HY improved the autism-like behavior of VPA mice.Then oxidative stress levels(ROS level,MDA content,CAT and SOD activity)and phosphorylation levels of ERK1/2 and Akt in the mPFC tissues of male VPA mice were detected to explore the molecular mechanism of HY improving autism-like behavior in VPA mice from these two aspects.Finally,a VPA-induced SH-SY5Y cell injury model was established.The cell activity,oxidative stress levels and phosphorylation levels of ERK1/2 and Akt were detected by HY intervention,and the molecular mechanism of HY action was verified in vitro.Results:1.Establish a VPA-induced autism model in mice:A single injection of 500 mg/kg VPA into the abdominal cavity of the mouse on 12.5 days of gestation was performed to conduct behavioral tests on its offspring male mice.The results showed that there were obvious social communication obstacles and repetitive stereotyped behaviors,which proved that the VPA-induced mouse autism model was successfully established.2.HY improved the social interaction ability and repetitive behavior of VPA autism mice,which may be achieved by reducing the oxidative stress level of mPFC in VPA mice.(1)Behavioral test to detect whether HY can improve the autism-like behavior of VPA mice.The results of social interaction ability test showed that after HY treatment,mice's ability to communicate and their social preferences were improved(P<0.05,P<0.05),and the number and time of social interaction were increased in different degrees.Repeated stereotyped behavior test results showed that after HY treatment,VPA mice had a burying of beads and the grooming times were significantly decreased(P<0.05,P<0.01).(2)The oxidative stress level of mPFC and the phosphorylation level of ERK1/2 and Akt in VPA mice were detected to explore the molecular mechanism of HY improving autism-like behavior in VPA mice.The results of oxidative stress level test showed that after HY treatment,ROS level and MDA content in mPFC of VPA mice were significantly decreased(P<0.05,P<0.05),SOD activity was significantly increased(P<0.01),indicating that HY significantly increased the antioxidant capacity of mPFC of VPA mice.Westen blot results showed that there were no significant changes in ERK1/2 and Akt phosphorylation levels in mPFC of VPA mice(P>0.05,P>0.05).3.HY had a protective effect on VPA-induced SH-SY5Y cell injury.(1)Screening VPA concentration suitable for subsequent studies.The results of MTT colorimetric analysis and oxidative stress level test showed that the damage rate of cells and the change degree of oxidative stress level under 2 mmol/L VPA treatment were suitable for further research.(2)To detect the cytotoxicity of HY and screen out the concentration of HY suitable for subsequent studies.The results of MTT colorimetric analysis showed that there was no cytotoxicity of HY in the concentration range of 0.05-5 ?mol/L,and screening HY at 2 ?mol/L was the best concentration for subsequent studies.(3)To explore the molecular mechanism of the protective effect of HY on VPA-induced SH-SY5Y cells.SH-SY5Y cells were pretreated with 2?mol/L HY for 12 h,and then treated with 2 mmol/L VPA.After treatment,oxidative stress level of cells was detected by kit method.The phosphorylation levels of ERK1/2 and Akt were detected by Western blot.The results showed that compared with VPA+HY group,ROS level and MDA content in cells were significantly decreased(P<0.05,P<0.05),SOD activity was significantly increased(P<0.01),and HY had protective effect on oxidative stress induced by VPA.Western blot analysis showed that HY significantly improved the decrease of ERK1/2 phosphorylation induced by VPA(P<0.001).Conclusion:1.Hypericin improves social interaction and repetitive behavior in VPA-induced loneliness mice.2.HY may play a neuroprotective role by reducing the oxidative stress level of mPFC in VPA mice,and ultimately improve the autism-like behavior of VPA mice.The changes of ERK1/2 phosphorylation were probably related to the effect of HY on the autism-like behavior of VPA mice.However,the changes of ERK1/2 phosphorylation level may be different in different stages of mouse development.In this study,the ERK1/2 phosphorylation level of mPFC in VPA mice did not significantly change.Therefore,whether the improvement effect of hypericin on the autism-like behavior of VPA mice is related to the changes of ERK1/2 phosphorylation level?We also need to find new conditions and methods for exploration.