(M0930-04-26) Sodium Butyrate Decreases VCAM-1 Expression by Inhibiting Stat3/GATA6 Signaling Pathway in Alzheimer’s disease

Purpose: Alzheimer’s disease (AD) is a progressive neurodegenerative disease that causes cognitive decline and neuronal loss. Deposition of amyloid beta (Aβ) peptides in the brain is one of the major pathological hallmarks of AD. Around 80% of AD patients have vascular dysfunction and manifest cerebrovascular inflammation, which is emerging as a core pathological feature of AD that can potentially be detected before the onset of cognitive changes. Increased expression of vascular cell adhesion molecule (VCAM)-1, a cerebrovascular inflammatory marker expressed on the blood-brain barrier endothelial cells contributes to cerebrovascular inflammation, which may, in part, be regulated by soluble Aβ exposure to the BBB endothelium. It was recently discovered that altered gut microbiota, which may alter the composition of bacterial metabolites in the plasma, is an early risk factor for AD pathogenesis. For instance, reduction in the levels bacterial metabolites like short chain fatty acids (SCFA), especially sodium butyrate (NaB), can increase intestinal permeability, which promotes the release of TNF alpha, an inflammatory cytokine into the bloodstream and promote cerebrovascular inflammation. However, the effect and mechanism underlying the anti-inflammatory properties of SCFAs and how they alleviate cerebrovascular inflammation following Aβ exposure remain unknown. In the present study, we have tested the hypothesis that soluble Aβ42 exposure promotes an increase in VCAM-1 expression. We report that NaB, by inhibition of HDAC1/2, significantly suppressed Aβ42 induced Stat3 and GATA6 expression and subsequent VCAM1 expression through a Stat3-GATA6 axis (Fig 1).

Methods: In this study, we aimed to investigate the effect of soluble Aβ42 on the expression of VCAM-1, an inflammatory marker, in human cerebral microvascular endothelial cells (hCMEC/D3) with and without pre-treatment with NaB. The polarized endothelial monolayer was pretreated with NaB (10 mM) for 24 hrs. followed by treatment with Aβ42 (1 mg/mL) for 3 hrs. Immunocytochemistry was conducted to investigate VCAM-1 expression and the samples were analyzed using confocal microscopy. To understand the underlying mechanisms driving observed changes in VCAM-1 expression, we investigated the alterations in the expression of phosphorylated Stat3 (Tyr 705) and GATA6 in hCMEC/D3 cells using western blot. Subsequently, we examined the contributions of Stat3 and GATA6 in regulating the expression of VCAM-1 by pretreating hCMEC/D3 cells with an inhibitor for Stat3 (S3I-201) or knocking out GATA6 using siRNA. Then changes in VCAM-1 expression following treatment with soluble Aβ42 were investigated using immunocytochemistry and the samples were imaged by confocal microscopy.

Results: Findings of the current study indicate that exposure to soluble Aβ42 resulted in an increase in VCAM-1 expression. However, the addition of butyrate was found to inhibit the increase in VCAM-1 expression induced by soluble Aβ42 in hCMEC/D3 cell monolayers. Furthermore, exposure to soluble Aβ42 resulted in the activation of the inflammatory signaling pathway, which was characterized by significant increase in the expressions of GATA6 and phosphorylated Stat3 (Tyr 705), as observed in Figure 2 and 3. Furthermore, the decrease in VCAM-1 expression observed after pre-treatment with NaB was associated with a reduction in the phosphorylation of Stat3 (Tyr 705) triggered by soluble Aβ42 (Figure 3). Finally, inhibiting Stat3 using S3I-201 inhibitor or knocking out GATA6 using siRNA reduced the soluble Aβ42-mediated increase in VCAM-1 expression.

Conclusion: In summary, the current study demonstrates that Aβ42 increased the expression of VCAM-1 on the BBB endothelium. SCFAs such as NaB decrease Aβ42 mediated increase in VCAM-1. Furthermore, NaB may act on Stat3/GATA6 signaling pathway to reduce Aβ42-mediated VCAM-1 expression.

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NaB decreases VCAM-1 expression via the Stat3/GATA6 signaling pathway.

Soluble Aβ42 increases GATA6 expression in hCEMC/D3 cell monolayers.

NaB decreases Aβ42 mediated increases in pStat3 expression.