Foxo1-mediated Inflammatory Response After Cerebral Hemorrhage In Rats

Background:Foxo is a vital transcription factors which exists in eukaryotes. Its biological function has been widely applied in clinic. However, in Foxo family, Foxo1 is the member which has been studied widely. Studies have shown that Foxo1 plays a crucial role in apoptosis, inflammation, and tissue development. In recent years, studies have found that Foxo1 regulates a wide range of inflammation in many tissue types. but its immunoregulation function in brain tissue after ICH in rats was uncertain. Therefore, through regulating the expression of Foxo1, inflammation injury and disease recovery after cerebral hemorrhage has profound clinical significance.Objective:To explore neuroprotective effects of regulating Foxo1 after cerebral hemorrhage in rats and explore the effect of the inflammation factor downstream in related signal transduction pathways.Methods:ICH was induced with a single infusion model of autologous blood which was collected from the arteria femoralis on the operation side. Rats were randomly divided into 7 groups including sham surgery, 1h, 6h, 12 h, 24 h, 48 h, 72 h to explore the most significant time point about expression of Foxo1 after ICH. Then, rats were randomly divided into4 groups including Insi-CX, Insi-CX, Cont-CS and Cont-CX. To explore the most significant brain area about expression of Foxo1 after ICH. After that Rats were randomly divided into 4 groups including non-specific control siRNA and three specific Foxo1 si RNA(siRNA-1285, siRNA-1612 and siRNA-1434)groups to find out siRNA of the strongest inhibitory effect, siRNAs were injected into rat brains 24 h prior to ICH by intracerebroventricular injection. At last Rats were randomly divided into 4 groups including Sham, ICH, ICH+ Foxo1 si RNA and ICH+ nonsiRNA to explore the effects of regulating Foxo1 after cerebral hemorrhage and the inflammation factor downstream. As followed:(1)Proteins were extracted in 1h, 6h, 12 h, 24 h, 48 h and 72 h after ICH. Western blot applied to detect Foxo1 protein expression. And then, proteins were extracted from Insi-CS, Insi-CX, Cont-CS and Cont-CX after the best time point ICH.Western blot and RT- PCR applied to detect Foxo1 protein expression and mRNA levels.(2)Screen for an optimized siRNA sequence targeting Foxo1 in the best time points and brain areas After ICH.(3)Garcia neurological scores and Brain Water Content Assay were evaluated were applied to neurologic deficits and edema. Western blot and RT- PCR applied to detect Foxo1 protein expression and mRNA levels.(4)Western blot applied to detect TLR4, NF-κB, TNF-α, IL-1β and IL-18 protein expression. ELISA applied to detect MPO, IL-1β and IL-18 protein activity.Results:(1)Foxo1 expression peaked at 12 h ICH(P < 0.01), compared with sham, 1 h, 6 h, 24 h, 48 h, and 72 h ICH rats.(2)The highest level of Foxo1 expression and mRNA was in the ipsilateral corpus striatum(P < 0.01), compared with the ipsilateral cortex, or contralateral corpus striatum and cortex.(3)Compared with nonsi RNA group, three pairs of siRNAs targeting Foxo1 significantly suppressed the Foxo1 expression(P < 0.01); the inhibitory effect of siRNA-1612 was the strongest one, with an interference efficiency over 50%.(4)Foxo1 expression levels and mRNA were significantly increased at 12 h post-ICH compared with the sham operation group, and also significantly decreased by knocking down Foxo1 with siRNA(P < 0.01).(5)Neurological symptoms significantly improved 12 h post-ICH and Garcia scores were significantly reduced. Brain water content was also significantly reduced 12 h post-ICH(P < 0.01). In addition, ipsilateral brain water content increased compared with the contralateral side except in the sham operation rats.(6)TLR4, NF-κB, TNF-α, IL-1β, and IL-18 expression levels were significantly decreased by knocking down Foxo1 with siRNA, compared with the 12 h ICH and ICH + non-siRNA groups. MPO, IL-1β and IL-18 activity were significantly decreased by knocking down Foxo1 with siRNA, compared with the 12 h ICH and ICH + non-siRNA groups(P < 0.01).Conclusion:(1)Foxo1 expression peaked at 12 h post-intracerebral hemorrhage and in the ipsilateral corpus striatum; Intracerebroventricular injection of Foxo1 siRNA effectively inhibited Foxo1 mRNA and protein expression and increased neurological function and decreased brain water content.(2)Foxo1 si RNA significantly reduced inflammatory factors release. Inhibition of Foxo1 inhibited the TLR4/ NF-κB pathway after ICH injury. Inhibition of Foxo1 expression may be one of the vital anti-inflammatory mechanism after cerebral hemorrhage.