The Effect Of Lactate In Regulating Astrocytes Into Reactive Astrocytes Via NDRG2 Under Oxygen Glucose Deprivation

Astrocytes(ACs),hold the largest number of the glial cells,are thought to be important for the homeostasis in brain tissue.However,the ACs are a kind of sensitive cells,and in response to various types of signaling molecules produced in the process of the central nervous system(CNS)damage,they will proceed reactive astrogliosis into reactive astrocytes(RACs),which are important to the eventual outcome of ischemic stroke.In the CNS,lactate is an important substrate for energy metabolism,which is mainly produced by ACs and playing a dual role of energy source and signaling molecule.Previous studies have shown that lactate content increases significantly after ischemic stroke.However,the effect oflactateon the biological function of ACs as a signal molecule has rarely been reported.N-myc downstream regulatory gene 2(Ndrg2),a specific marker of ACs,is thought to be closely related to the proliferation,survival and apoptosisof ACs,and its expression is up regulated in the process of reactive astrogliosis caused by hypoxia.In this study,based on the observation of the rat middle artery occlusion(MCAO)animal model,we used OGD cell model to explore the mechanism of lactatein regulating astrocytes into reactive astrocytes via NDRG2.It will provide a new idea for the formation of glia scar in the cerebral ischemia area to control damage range and promotethe regeneration of damaged tissues.We successfully established middle artery occlusion(MCAO)rat models and detected the changes in lactate content in ipsilateraland contrallateral tissues.Western blot and immunohistochemistry were used to detect NDRG2 and astrogliosis related protein expression levels in tissues.Primary ACs from newborn rats were separated and purified.OGDadding extra lactate cell culture conditions was establishedto mimic the ischemic stroke pathological environments.Na Cl was added in the control group to instead lactate to balance the osmotic pressure changes resulting from lactate in the experimental group.RT-q PCRwas applied in detecting the gene expression and western blot experiments was applied to detect the protein expression levels.The differentially expressed genes(DEGs)were detected byRNA-seq to explore the effect of lactate and NDRG2 on the biological function of ACs.The results are as follows:(1)The MCAO model of rats was successfully established.The TTC staining results showed that the infarct size increased with time.The above experimental results showed that the damage focal area was large and the edge was clear at 8h.In subsequent experiments,8h was selected for corresponding detection at a single time point.The changes of lactate content and the reactive proliferation of ACs in the brain tissue of the injured brain tissue and the contralateral control tissue were analyzed.The lactate content in the brain tissue of the injured side was increased,and the expression of GFAP,a marker of reactive hyperplasia of ACs,was significantly increased.Western blot results also confirmed that,compared with the contralateral control group,GFAP protein expression in the injured side was significantly up-regulated at 4 and 8 hours after injury,indicating that ischemia injury resulted in significantly increased lactate content and accompanied by reactive hyperplasia of ACs.(2)Under the action of OGD combined with exogenous lactate in vitro,the expression of MARKERS related to ACs reactive hyperplasia such as GFAP and SOX9 was significantly increased under the action of lactate,and the phosphorylation level of key proteins involved in the regulation of the ACs reactive hyperplasia signaling pathway,such as Akt and STAT3,was increased.After MCT1 expression was silenced,the content of intracellular lactate was significantly reduced,and the up-regulation trend of ACs reactive hyperplasia related markers was offset,indicating that the above experiments indicated that under OGD conditions,intracellular lactate played a major role in promoting ACs reactive hyperplasia.(3)Using Western blot 8 hours to 2 hours,4 hours and MCAO model of damage and the contralateral organization NDRG2 protein expression were analyzed,and compared with the contralateral control group,damage side NDRG2 protein expression in damage rise significantly after 8 h,immunohistochemical staining results also show that the damage in brain tissue,NDRG2 positive cell volume increased,its expression had a significantly higher than that of the contralateral quantity and positioning with GFAP logo RACs exist obvious phenomenon;RT-q PCR and Western blot experiments confirmed that the addition of lactate were NDRG2 had no effect on gene expression,and significantly increase the amount of protein expression,that lactate may be through the transcription level of NDRG2 expression of regulation;Molecular docking analysis predicted that lactate could form hydrogen bond interactions with amino acid residues Asp172,Lys176,Asp180,and Asp240 on NDRG2,thus stabilizing the interaction between lactate and NDRG2 protein.Co-ip experiments confirmed that,under the condition of OGD,the content of ubiquitin protein binding NDRG2 in the lactate treatment group significantly decreased,that is,lactate may inhibit the ubiquitination degradation of NDRG2 through direct binding.NDRG2 silencing could not inhibit the expression of RACs markers of reactive hyperplasia under OGD and high lactate treatment,indicating that NDRG2 silencing had little effect on the activation state of RACs.(4)We used RNA-seq to explore the impact of Ndrg2 silencing on the biological functions of RACsunder OGD and high content of lactate treatment conditions.It was found that Ndrg2 silencing raised many gene expressionassociated with inflammation and immunity function;RT-q PCR,Western blot and ELISA confirmed that Ndrg2 silence led to significant increase in Tnfα gene expression,protein expression and secretion.This trend could be reversed by plasmid mediated Ndrg2 overexpression.The results suggested that NDRG2 and Tnfαhad closely relationship.GSEA gene set enrichment results confirmed that under the condition of OGD and lactate treatment conditions,the apoptosis related gene set trended to enrich in Ndrg2 silencing RACs.Western blot conformed that Ndrg2 silencing led to a decrease expression of BCL2,which could be reversed by Ndrg2 overexpression.Annexin V/PI staining results confirmed that Ndrg2 silencingresulted in apoptosis rate increased significantly,and the trend had partially inhibited by TNFα neutralizing antibodies.These results suggested that ACs promoted apoptosis by upregulating TNFα after Ndrg2 silencing.In conclusion,based on in vitro cell models under OGD conditions and MCAO animal models,we can conform that lactate enters into ACs through MCT1 during the transformation from ACs into RACs,and regulates NDRG2 ubiquitination in a directly binding way,in turns,affecting intracellular protein content and playing a corresponding biological role.After NDRG2 silencing,inflammation-related signaling pathways were up-regulated,indicating that NDRG2 deletion can promote inflammatory response,suggesting that the high expression of NDRG2 in RACs plays an important role in controlling the degree of inflammatory response at the site of injury,promoting the repair of damaged tissue.Under OGD conditions,lactate inhibited the apoptosis of RACs by NDRG2-TNFαaxis,indicating that lactate promotsthe survival of RACs by inhibiting its apoptosis,which is of great significance in promoting the formation of glial scar and further preventing the expansion of damage.