Obstructive sleep apnea aggravates neuroinflammation and pyroptosis in early brain injury following subarachnoid hemorrhage via ASC/HIF-1α pathway

Obstructive sleep apnea can worsen the prognosis of subarachnoid hemorrhage.Howeve r,the underlying mechanism remains unclear.In this study,we established a mouse model of subarachnoid hemorrhage using the endovascular perforation method and exposed the mice to intermittent hypoxia for 8 hours daily for 2 consecutive days to simulate sleep apnea.We found that sleep apnea aggravated brain edema,increased hippocampal neuron apoptosis,and worsened neurological function in this mouse model of subarachnoid hemorrhage.Then,we established an in vitro HT-22 cell model of hemin-induced subarachnoid hemorrhage/intermittent hypoxia and found that the cells died,and lactate dehydrogenase release increased,after 48 hours.We further investigated the underlying mechanism and found that sleep apnea increased the expression of hippocampal neuroinflammatory factors interleukin-1β,interleukin-18,inte rleukin-6,nuclear factorκB,pyro ptosis-related protein caspase-1,pro-caspase-1,and NLRP3,promoted the prolife ration of astrocytes,and increased the expression of hypoxia-inducible factor 1αand apoptosis-associated speck-like protein containing a CARD,which are the key proteins in the hypoxia-inducible factor 1α/apoptosis-associated speck-like protein containing a CARD signaling pathway.We also found that knockdown of hypoxia-inducible factor 1αexpression in vitro greatly reduced the damage to HY22 cells.These findings suggest that sleep apnea aggravates early brain injury after subarachnoid hemorrhage by aggravating neuroinflammation and pyroptosis,at least in part through the hypoxia-inducible factor 1α/apoptosis-associated speck-like protein containing a CARD signaling pathway.

Cloning of human XAF1 gene promoter and assay of its transcription activity in a variety of cell lines

To investigate the regulation of tumor sup-pressor XAF1 gene expression in digestive system cancers,we studied XAF1 gene promoter transcription activity and mRNA level in digestive system cancer cell lines(human hepatoma cell line HepG2,human colon cancer cell line LoVo,and human gastric cancer cell line AGS)and nontumor cell lines(human embryonic liver cell line L02(L02 cells)and human embryonic kidney 293 cells[HEK293 cells])as controls.1395-bp-promoter fragment of XAF1 gene was amplified by polymerase chain reaction(PCR)and cloned into pGL3-basic vector and pEGFP-1 vector to assay its promoter transcription activity.The plasmids were transfected into a variety of cell lines by lipofectamine 2000.The promoter transcription activity was determined by dual-luciferase report assay,and enhanced greenfluorescent protein(EGFP)-positive cells were detected byfluorescence microscope.The expression of XAF1 mRNA in HEK293 and L02 were significantly higher than that in any of the three digestive system cancer cell lines.The dual-luciferase reporter assay showed that the promoter transcription activity in digestive system tumor cell lines transfected with pGL3-XAF1p promoter was apparently lower than that of both HEK293 and L02 cells.Expression of greenfluorescent protein(GFP)under the control of XAF1 promoter in the three digestive system cancer cell lines was lower than that of both HEK293 and L02 cells.The activities of pGL3-XAF1p in the three digestive system cancer cell lines after treatment with heat stress were significantly lower than those in the unstressed cells.The results suggested that remarkably down-regulated XAF1 mRNA expression in digestive system cancer cell lines may be due to loss of transcription activity of XAF1 promoter.