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Expression of MCT1 in brain tissue of rats with acute permanent cerebral ischemiaObjective: The aim of this study was to observe the expression of MCT1(monocarboxylate transporter 1)in acute permanent cerebral ischemia rats.Methods: 1.Healthy adult male SD rats were randomly divided into the sham group(Sham,n = 10)and the model group(n =50).The model group was further divided into 5 subgroups,and the samples were taken 1 h,3 h,6 h,12 h,and 24 h after the successful modeling.2.Cerebral ischemia model of rats with permanent middle cerebral artery occlusion(p MCAO)was prepared by using suture method,and its incidence was observed by behavioral analysis.3.TTC staining was used to observe the changes of cerebral ischemia in different periods after ischemia.4.Expression of MCT1 in rat brain tissues was detected by confocal laser scanning with immunofluorescence.Double immunofluorescence labeling was applied to detect the co-expression of MCT1 and GFAP astrocytes markers(Glial fibrillary acidic protein,GFAP).5.Western blot was used to detect MCT1 protein expression changes in brain tissues of rats at different time periods after cerebral ischemia.Results: 1.The behavioral changes of the rats after the establishment of the p MCAO model were as follows: rats were not awake within 1 h,and all the rats were awake after 3 h ischemia.While lifting up the tail of rats,it could be seen that the right forelimb of the rats was close to the chest.While laying down the rats,it could be observed that the body of the rats fall to the opposite side,and the phenomenon of turning and rear-ending can be observed.2.TTC staining showed that in the rat model of acute cerebral ischemia,the ischemic area gradually increased with the time extension to 24 h after ischemia.3.Confocal laser detection showed that MCT1 was mainly expressed in capillaries and ependymal cells.When MCT1 was co-labeled with GFAP,MCT1 was only co-expressed at the foot of the astrocytes,close to the microvessels.Compared with the sham group,MCT1 expression was enhanced in both cortical and ependymal cells with prolonged ischemia.4.WB results showed that,with the extension of ischemia time,the expression of MCT1 increased at both the ischemic area and the ischemic penumbra,especially in the ischemic area,and the expression of MCT1 increased abnormally after 1 h ischemia.Conclusions: 1.The cerebral ischemia model of p MCAO rats was successfully established by suture method.2.The expression of MCT1 gradually increases as the ischemia time extends to after 24 h ischemia,indicating that MCT1 plays an important role in the transportation of lactic acid of the brain after cerebral ischemia.Part? Post-transcriptional regulation of MCT1 m RNA expression in acute permanent cerebral ischemia rat brain tissueObjective: The aim of this study was to predict and validate the mi RNAs regulating rat MCT1 under acute permanent cerebral ischemia.Methods: 1.The authoritative mi RNA prediction softwares(Target Scan Human,mi Randa)were used to predict the mi RNAs targeting MCT1(whose gene symbol is Slc16a1)in rats.The mi RNA predicted by both softwares was taken as the target gene,and then input into mi RDB and DIANA TOOLS softwares to predict the target gene reversely.2.Double luciferase reporter gene assay was used to confirm the targeting relationship between Slc16a1 and the predicted mi RNA.3.Total RNA was extracted from the ischemic central area and ischemic penumbral zone of brain tissue at each time period after p MCAO,and then transcribed reversely into mi RNA.QRT-PCR technology was used to detect the expression differences of the screened mi RNA at different time periods,and combined with the MCT1 protein expression changes at each time period of acute cerebral ischemia in rats to further determine the mi RNA regulating Slc16a1.Results: 1.rno-mi R-124-3p was predicted as the mi RNA that might target Slc16a1 in rats by Target Scan Human and mi Randa softwares.When rno-mi R-124-3p was input into mi RDB and DIANA TOOLS softwares for reverse validation,it was predicted that Slc16a1 was indeed one of the targets of rno-mi R-124-3p.2.Dual luciferase reporter gene results showed that the luciferase activity of 293 T cells transfected with rno-mi R-124-3p mimics was nearly 30% lower than that of the control group when using wild-type Slc16a1 vector.However,there was no significant change in luciferase activity when the mutant Slc16a1 vector was co-expressed with rno-mi R-124-3p.The results indicated that Slc16a1 was one of the targets of rno-mi R-124-3p.3.QRT-PCR results showed that the expression of rno-mi R-124-3p in brain tissue at different time periods of p MCAO showed a negative correlation with the protein expression of MCT1 at corresponding time periods,suggesting that rno-mi R-124-3p might be a target mi RNA regulating Slc16a1 in rats.Conclusions: In this part,rno-mi R-124-3p was predict as the mi RNA that could target rat MCT1(whose gene symbol is Slc16a1)by bioinformatics method.Double luciferase reporter gene assay and q RT-PCT results suggested that Slc16a1 was indeed one of the targets of rno-mi R-124-3p in rats.Part ? Role of rno-mi R-124-3p agomir in permanent acute cerebral ischemic injury in ratsObjective: This study was to observe the treatment effect of rno-mi R-124-3p agomir on acute permanent cerebral ischemic injury in rats.Methods: 1.Rats were randomly divided into sham group(n=10),agomir control group(n=30)and agomir group(n=90).The agomir group was randomly divided into three subgroups,with concentrations of a.20 pmol/ l,b.100 pmol/ l,and c.200 pmol/ l.The drugs were administered via the left lateral ventricle.The p MCAO model was performed a week after lateral ventricle administration.2.Western blot was used to detect the expression of MCT1 protein in brain tissue of rats.Expressions of rno-mi R-124-3p and MCT1 m RNA in rat brain tissues were detected by q RT-PCR.TTC staining was used to observe the changes of cerebral ischemia before and after administration of lateral ventricle.Results: 1.Western blot results showed that compared with the model group and agomir control group in the same ischemic period,the protein expression of MCT1 increased after 6 h ischemia and decreased after 24 h ischemia in the rno-mi R-124-3p agomir group after lateral ventricle administration.2.QRT-PCR results showed that,compared with the model group and agomir control group in the same ischemic period,the m RNA expression in the agomir group increased after 6 h ischemia and decreased after 24 h ischemia,both in the ischemic central area and in the ischemic penumbral area.However,the expression of rno-mi R-124-3p in the agomir group increased after 6 h and 24 h ischemia.3.TTC results showed that compared with the model group and agomir control group in the same ischemic period,the ischemic area in agomir group decreased both after 6 h and 24 h ischemia,and the ischemic area decreased more significantly after 24 h ischemia.Conclusions: Mi RNAs post-transcriptionally regulate gene expression by binding to the 3' untranslated region(3' UTR)of target m RNAs through base-pairing and subsequently disable them or inhibit their translation.The expression MCT1 m RNA was negativly regulated by rno-mi R-124-3p agomir in agomir group.The ischemia area was significantly reduced after rno-mi R-124-3p agomir administration,indicating that rno-mi R-124-3p plays a protective role by regulating the expression of MCT1 under cerebral ischemia. |
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