Mechanism Of MTOR/Hif-1α Involvement In Early Brain Injury Formation After Subarachnoid Hemorrhage By Regulating The Activation Of M1 Microglia

Objective:This study aimed to explore the effect of the mammalian target of rapamycin(m TOR)/ hypoxia-inducible factor-1α(Hif-1α)pathway on the glycolytic metabolic pathway,M1 microglial activation,and early brain injury(EBI)severity by constructing an in vivo model of Subarachnoid hemorrhage(SAH).Methods:In the present study,After the construction of the rat SAH model by using the internal carotid artery puncture method,firstly,chemical reagents were used to intervene in the expression of the m TOR,brain edema was observed by the dry and wet weight method;The severity of BBB damage was observed by EB curve method;The expression of the key enzymes of glycolysis(HK2/PKM2),M1 microglial marker inducible nitric oxide synthase(i NOS)and Hif-1α were observed by western blot and RT-PCR;Lactate levels were measured by a lactate kit;Co-localization of M1 microglia marker(Iba-1)with HK2 and PKM2 by immunofluorescence assay,respectively.Secondly,after inhibiting the expression of Hif-1α,the degree of brain edema and blood-brain barrier damage were observed;the expression of key glycolytic enzymes(HK2/PKM2)and M1 microglial marker(i NOS)was observed by western blot and RT-PCR;Lactate levels were detected by a lactate kit.In addition,all rats were scored for neurological function before death,and EBI severity was assessed based on cerebral edema,BBB disruption and neurologicalfunction score.SAH bleeding score of less than 7 indicated that the modeling was unsuccessful and supplemented by new rats.Results:1.After SAH,the upregulation of m TOR resulted in a significant increase in the expression of glycolytic key enzymes(HK2/PKM2),glycolytic products(lactate),and M1 microglia markers(i NOS),and exacerbated the degree of brain edema and blood-brain barrier disruption,while the downregulation of m TOR had the opposite effect.2.After SAH,activating m TOR remarkably enhanced the number of HK2-Iba-1 and PKM2-Iba-1 double-positive cells,while inhibition had the contrary effect.3.After SAH,the down-regulation of mTOR significantly decreased the expression of Hif-1α,whereas up-regulation had the opposite effect.4.After SAH,inhibiting Hif-1α could significantly reduce the expression of key glycolysis enzymes(HK2/PKM2),glycolysis products(lactate),and M1 microglial marker(i NOS),and reduced brain edema and blood-brain barrier damage.Conclusion:The m TOR/Hif-1α axis can be involved in the formation of EBI after SAH by regulating the activation of glycolysis-dependent M1-type microglia,which in turn mediates the onset of the inflammatory cascade response.