| Objective and background:Intracerebral hemorrhage(ICH)accounts for 10-15% of all stroke,making it the second most common type of stroke,and is an important public health problem leading to high rates of death and disability in adults.The diffusion of thrombin released from hematoma into the brain parenchyma following ICH has been established as a critical factor in ICH’ neuropathological effects.Furthermore,Thrombin induces secondary brain injury by activating microglia,which subsequently release pro-inflammation and anti-inflammation agents into the surrounding brain tissue,and then promotes or inhibits secondary injury following ICH.Long non-coding RNAs(lncRNAs)are demonstrated to be involved in central nervous system(CNS)disorders,and can also regulate activation of microglia.However,the role of lncRNAs in post-ICH injury and repair are poor understood,especially in thrombin induced microglial activation following ICH.This study shows a distinct lncRNAs and m RNAs expression pattern in vitro thrombin cytoxicity model,and then determine the effects of most significantly differentially expressed lncRNA on the thrombin-induced microglial activation and polarization Methods:The cytotoxic injury model of intracerebral hemorrhage(ICH)was made by using thrombin-treated Microglia in vitro,which were divided into model group and control group(n=3well).Total RNA was extracted from Microglia after treated with Thrombin24 hours and sequenced by gene chip technology,then RT-q PCR was used to verify the gene expression levels in the in vitro and ICH animal models,and to screen the differentially expressed genes.Gene ontology(GO)and the Kyoto Encyclopedia of Gene and Genomes(KEGG)pathway analyses were performed to determine biofunctions and signaling pathways.The BV2 cell line were treated with thrombin for 24 hours after silencing the most significantly differential expressed lncRNA.The effect of lncRNA on the expression of M1 / M2 polarized molecular marker in BV2 cells was detected by immunostaining.Results:(1)A total of 127 differentially expressed lncRNAs(64 upregulated;63downregulated)and differentially expressed m RNAs 1427 m RNAs(762 upregulated;665 downregulated)were identified in both thrombin treating cell group and control group.(2)The results of GO analysis shows that the major relevant three biological processes to differentially expressed m RNAs were as follows: cytosolic transport,regulation of small GTPase mediated signal transduction and dendrite development.The most three relevant cellular components were as follows: cell leading edge,lamellipodium and cell-substrate junction.The most three relevant molecular function were as follows: protein N-terminus binding,“catalytic activity,acting on DNA” and GTPase binding.Those m RNAs function through major three pathways were MAPK signaling pathway,transcriptional misregulation in cancer and TNF signaling pathway.(3)Through high-throughput screening combined with RT-q PCR,some lncRNAs with obvious differential expression and consistent up-regulation in both in vivo and in vitro models were found.The lncRNA with the most significant difference in expression was TCONS＿00145741(P <0.01).(4)After treatment with thrombin for 24 h,it was found that thrombin promoted the expression of microglia M1 microglia marker(CD86)(P <0.01).When TCONS＿00145741 was silenced and then stimulated by thrombin,CD86 expression was down-regulated(P <0.01)than The non-silent group,and the expression of CD86 in the other two lncRNA silent groups was not significantly affected.(5)After treatment with thrombin for 24 h,it was found that thrombin does not affect the expression of microglia M2 microglia marker(CD206),but silencing TCONS＿00145741 of microglia and then stimulated by thrombin,expression of CD206 was Significantly increased(P <0.05);Conclusion:After ICH,Thrombin treatment for 24 h resulted in M1 type polarization of activated Microglia and differential expression of Lnc RNA and m RNA.The expression of TCONS＿00145741 was up-regulated consistently in both ICH models in vivo and in vitro,and the difference was most obvious,when the expression of TCONS＿00145741 was silenced,the thrombin induced microglia polarization was reversed,which inhibited the M1 phenotype polarization and promoted the M2 phenotype polarization,and finally enhanced the injury resistance of microglia.In addition,these abnormally expressed genes may also become new therapeutic targets for tissue damage and dysfunction after ICH. |
