| IntroductionIntracerebral hemorrhage(ICH)is an acute and spontaneous process of cerebrovascular rupture and blood flowing into the brain parenchyma,ventricle or subarachnoid.As a subtype of stroke,it harms to human health seriously,with characteristics of high incidence,high mortality and high disability.The brain damage caused by ICH,includes primary hemotoma compression and secondary injury resulted from hematoma decomposition products.Studies have demonstrated that secondary injury after ICH is the critical factor in the prognosis of patients with ICH.The mechanism of secondary injury after ICH is complicated,including acute inflammatory reaction,local free oxygen release,edema effect,apoptosis,autophagy and so on.Secondary inflammation after ICH is considered to be the most important mechanism of secondary injury after ICH recently,but the detail of its mechanism of action has not been known clearly.Microglia plays an important role in the initial inflammatory response after spontaneous ICH.As an important inflammatory effective cells in the nerve system,microglia is activated firstly,after ICH,and releases a series of inflammatory mediators and biological activity factors:such as TNF-?,IL-1?,IL-6,IL-8,and so on,through the process of cell morphological and physiological changes,which lead to occurrence of inflammatory response after ICH and result in further secondary injury.MicroRNAs(miRNAa,miRs)are a group of small non-coding RNAs with a length of 18-25 nucleotides,that can accumulate in cells and down-regulate the expression of target genes,by binding the special sequence on the 3' untranslated region(3' UTR)of mRNA,and have effect of regulating gene transcription.MiRNAs play a very important role in maintain the biological activity of cells.Studies showed that miRNAs are expressed abnormally in the prehemotoma tissue and blood of patients with ICH and demonstrated that miRNAs are related to pathophysiological changes and prognosis after ICH,and the development of inflammation closely.Objective:Constructing of microglia inflammatory model and rat ICH model.Regulating the expression of miRs by adenovirus transfection.Exploring the effect of miR-21 and miR-146a on secondary inflammatory reaction of microglia in vitro and in vivo and further analyzing the their protective effect on nerve cells.Methods:(1)In vitro experiments:We induced microglia inflammatory reaction by lipoprotein(LPS)to construct microglia inflammatory model.Compared with negative expression adenovirus group,we measured the expression level of miR-21 and miR-146a in microglia by PCR,the expression level of IL-1?,IL,6,IL-8,MMP-9,IRAK1,TIMP3 and TNF-? by ELISA,after miR-21 and miR-146a overexpression adenovirus and negative control adenovirus transfect microglia.Then we analyzed the relationship between the expression level of miR-21 and miR-146a and inflammatory factors.(2)In vivo experiment:We conducted rat model of ICH.Compared with negative expression adenovirus group,we measured the expression level of miR-21 and miR-146a in perihematomal tissue by PCR,the expression level of IL-1?,IL-6,IL-8,MMP-9,IRAK1,TIMP3 and TNF-a by ELISA,after miR-21 and miR-146a overexpression adenovirus and negative control adenovirus were injected into rat ICH model.Then we analyzed the relationship between the expression level of miR-21 and miR-146a and inflammatory factors.(3)The histopathological changes of brain tissue after hemorrhage were observed by HE staining.Cell apoptosis in brain tissue after injecting miR-21 and miR-146a overexpression adenovirus were observed by TUNEL and DAPI double fluorescent staining.Results:(1)The expression of IL-1?,IL-8 and TNFa was significantly increased under the action of LPS in microglia.The inflammatory response in microglia is most significant,when LPS at 10?/ml dose,the role of 24 hours.(2)The expression level of miR-21 in microglia cells infected by miR-21 overexpression was 2.17 fold and miR-146a was 3.52 fold than that infected by negative control adenovirus.The relative expression level of miR-21 in microglia cells treated with LPS and infected by miR-21 overexpression adenovirus was 1.53 fold and miR-146a was 2.10 fold than cell infected by negative control adenovirus and treated with LPS.(3)The expression level of IL-1?,IL-6,IL-8,IRAK1,MMP-9 and TNF-? was up-regulated and the expression level of TIMP3 was down-regulated(p<0.001)significantly after being treated with LPS.Overexpression of miR-21 or miR-146a decreased the expression level of IL-1?,IL-6,IL-8,IRAK1,MMP-9 and TNF-a and increased the expression level of TIMP3 significantly(p<0.001),compared with that of negative control cells.(4)The expression level of L-1?,IL-6,IRAKI and TNF-a in the perihematomal tissue was higher than that in the contralateral basal ganglia significantly(p<0.001),in rat intracerebral hemorrhage model.The expression of IL-1?,IL-6,IRAK1 peaked at12h,and began to decrease after 24h after ICH.The expression of IL-8 and TNF-a peaked at 24h,and began to decrease after 72h after ICH.The expression of miR-21 began to decrease at 12h and decreased at 24h and 72h significantly.(5)The expression level of miR-21 and miR-146a in the perihematomal tissue was significantly increased after ICH and injection with miR-21/miR-146a overexpression adenovirus(p<0.001).The relative expression level of IL-1?,IL-6,IL-8,IRAK1,MMP-9 and TNF-a in perihematomal tissue(Model)were up-regulated(p<0.05)and TIMP3 levels down-regulated significantly(p<0.01)after ICH or ICH and injection negative control adenovirus.The expression level of IL-1?,IL-6,IL-8,IRAK1,MMP-9 and TNF-a of the perihematomal tissue after ICH and injection miR-21 or miR-146a overexpression adenovirus decreased significantly(p<0.01)and the expression level of TIMP3 increased(p<0.01)compared with that of negative control.(6)TUNEL positive cells had no significant difference in normal brain tissue between miR-21 group,miR-146a group and negative control group,but decreased in miR-21 and miR-146a groups in the perihematomal tissue compared with negative control group.Conclusion:MiR-21 and miR-146a could induce the inflammatory response of microglia induced by LPS,and also reduce the inflammatory response of brain tissue in rat ICH model.Therefore,this study proved that miR-21 and miR-146a can reduce the inflammatory response of microglia and have the protective effect on nerve cells after ICH in vitro and vivo experiments,that provided a new theoretical basis for the treatment of secondary inflammatory injury after ICH in human. |
PDF Full Text Request