Methylated MFGE8 Promotes Early Brain Injury After Subarachnoid Hemorrhage and Inhibiting Autophagy of Nerve Cell

Abstract

Background: Spontaneous subarachnoid hemorrhage (SAH) is a relatively common clinical hemorrhagic stroke crisis. The important role of early brain injury (EBI) and autophagy in SAH has been increasingly recognized in recent years. This study aims to investigate the function and the undergoing mechanism of MFGE8 in EBI after SAH.

Methods: SAH model was established using C57BL/6 mice, and the SAH cell model was constructed by oxy-hemoglobin (Oxy-Hb) induced BV2 and SH-SY5Y co-culture system. Various methods were used to detect EBI and autophagy after SAH in mouse/cell lines, including mouse neurological function score, wet/dry method, HE and Evans blue staining, etc. The effect on EBI was explored after knockdown or overexpression of key genes DNMT1, MFGE8, and P2X7R. MSP was used to detect the methylation of MFGE8 promoter region, and ChIP was used to detect the binding relationship between DNMT1 and MFGE8 promoter region.

Results: The results showed that the activation of autophagy attenuates EBI in SAH mice. Increased level of DNMT1 and decreased level of MFGE8 were observed in brain tissues of SAH mice. Knockdown of DNMT1 or overexpression of MFGE8 attenuates EBI in mice by promoting autophagy. At the same time, we found that DNMT1 promotes methylation of MFGE8 promoter region and suppresses its protein levels. MFGE8 downregulates P2X7R levels and subsequently activates PI3k/Akt/mTOR axis, promotes autophagy, and attenuates EBI in SAH mice.

Conclusion: DNMT1 promotes the methylation of MFGE8 promoter region and downregulates MFGE8 level; restoring MFGE8 downregulates P2X7R, and promotes autophagy by limiting the activation of PI3k/Akt/mTOR, thus exerting a protective effect on brain tissue of SAH mice.

Highlights: • High expression of DNMT1 and P2X7R and low expression of MFGE8 were observed in SAH mice. • Activation of autophagy reduces EBI after SAH in mice. • DNMT1/MFGE8 reduces autophagy in EBI after SAH in both mouse and cell models. • DNMT1 targets the MFGE8 promoter to upregulate its methylation level. • MFGE8 inhibits P2X7R to activate PI3k/AKT/mTOR pathway and autophagy, thus inhibiting EBI after SAH.

Keywords: Autophagy; DNMT1; MFGE8; Methylation; Spontaneous subarachnoid hemorrhage.