| Objective: To observe the effecet of ?-elemene on inhibiting retinal neovascularization through establishment of high-oxygen-induced retinal neovascularization in mice models.On this basis,we compare and test the change of retina micro RNA expression profiles among nomal control group ?model group and treatment group that mice retina treated with ?-elemene through application of fast and efficient mi RNA microarray technology.To explore the molecular mechanism of inhibiting of retinal angiogenesis by ?-elemene.Methods: The seven-days-old C57BL\6J mice were divided into three groups randomly,nomol control group,model group and treatment group.A retinal neovascularization model was established on model group and treatment group,which exposed to hyperoxia and 12 mice of control group were raised in normal air environment.The mice from treatment group treated with intravitreal injections 1ul of?-elemene 12 days after born.The mice were sacrificed in 17 days after born and the eyeballs were enucleated to make retinal stretched preparation.To assess the role of?-elemene inhibited retinal neovascularization,adenosine diphosphate-ase(ADPase)stained retina flat-mounts was performed to assess the retinal vascular profiles,and H&E staining was applied to count the number of new vascular cell nuclei.Total retinal RNA were extracted from three groups,and placed in Ep tube,Respectively.Then add 1ml of Trizol,sample were sent to the Kang Chen Bio-Tech detected by mi RCURYLNA mi RNA microarray,then filter out the significant differences expression micro RNAs in retinal tissue among the three group.According to results of retinal tissue microarray in C57BL\6J7 mice,Select the micro RNAs(mir-23a?mir-22?mir-93 ?mir-106a?mir-19b?mir-190b?mir-34)that were substantially increased in model group,while were substantially decreased in the treatment group.Real-time PCR for detection m RNA expression of VEGE among nomol control group,model group and treatment group,and detect VEGF expression levels of protein by immunohistochemistry.Construction of VEGF untranslated regions(3' UTR),length of 1800 bp,including site that mir-23 a combined with the target gene VEGF.we constructed a luciferase reporter gene p MIR-REPORT-VEGF,and cotransfected into native human embryonic kidney(HEK)-293 cells along with pre-mir-23 a.Then collected the cell lysates to determine luciferase activity to observe mir-23 a inhibition on exogenous target gene after 48 h.b-galactosidase were cotransfected to detect the beta-galactosidase activity to correct for variations in transfection efficiency,while the double-stranded oligonucleotide and empty plasmid was used as a negative control.Microarray analysis was performed using microarray platform Gene Pix Pro 6.0 software,Frequency distribution of signal intensity values in different chips were analyzed by Volacano Plot filtering.if they met all of the following Fold change>1.5 or <0.67 as the difference significant.However,if P < 0.05,datasets was analyzed by MEV and SPSS16.0.Correlation analysis were analyzed using Spearman,significant differences(p<0.05)are indicated.Results:(?)1.Histological examination and counting retinal vascular endothelial cells:Retinal tissue from the mice of each group after 17 days,H&E staining: observed under light microscope and counted prominent endothelial cells within the limiting membrane,that is,the number of new endothelial cells.The number of nuclei from the vascular endothelial cells in the new vessels breaking through the internal limiting membrane in the OIR model group was(30.73±5.11),and nomal control group was(0.23±0.11),treatment group was(12.13±0.19),indicating a significant increase in comparison with control group(P<0.001).2.Adenosine diphosphate-ase(ADPase)stained retina flat-mounts:From the center of the optic nerve radiate the major blood vessels of the retina,until the side of retina in the control group in 17 days after born.The retinal stretched preparation presented more neovascularization in mice from the OIR model group compared with control group in 17 days after born.The retinal neovascularization in treatment group 17 days after born compared with OIR model group was significantly reduced.3.Ultrastructural changes by TEMUltrastructure of the retina show no abnormal changes in nomol control group;TEM results showed the following retinal ultrastructural changes in OIR model group:mitochondrial swelling and cristae disruption in pericytes,decreased membranous disc (photoreceptor cell's outer segment),and widened gap between membranous discs.However,Ultrastructure of Retinal in treatment group tended to return to normal.(?)Identified the altered expression of microRNA in nomol control group,model group,and treatment group1.Compared to the normal group,The expression levels of 54 mi RNAs were determined from the submission 6 copies of a model / normal retinal tissue.,43 micro RNA were downregulated,11 micro RNA expression increased in the model group.There is 3mi RNA down-regulated,including mir-22,mir-23 a,mir-190 b,which significantly different(fold change >5,p <0.05).2.To control model group,The expression levels of 56 mi RNAs were determined from the submission 6 copies of a treatment/ model retinal tissue.44 micro RNAs were upregulated,12 micro RNAs expression decreased.Significantly different(fold change>3,p <0.05)increase were 5 up-regulated(mir-22 ? mir-23 a ? mir-181a-1* ?mir377?mir17*)and 3 down-regulated(mir-872?mir-19 b,mir-26 b).3.Examination of mi RNA expression patterns during disease occurs and the treatment process showed that 43 micro RNAs expression decreased in the model group,in which23 micro RNAs expression levels increased after treatment.In the model group,11 micro RNAs up-regulation,but mi R-19 b,was decreased in the treatment group.Part of the differential expression of micro RNAs were validatied by stem-loop real-time RT-PCR.4.Based on the results of Micro RNA microarray,we selected the 7 micro RNAs of differential expression that detected by real time PCR respectively,including mi R-190b?mi R-93?mi R-106a?mi R-22?mi R-19b?mi R-23a?mi R-34 a,.The results are consistent with the results of microarray.(?)Forecast and identify of target gene on microRNA1.Expression of VEGF protein detected by immunohistochemical stainingControl group(32.17±20.98),model group(144.04±65.72)and treatment group55.56±36.69,which statistically significantly different(p <0.01)between the two.2.In the present study we have devised relative quantification real-time RT-PCR protocols using the intercalating dye SYBR-Green fluorescence for fast detection of expression of VEGF in the retinal tissue in mice among groups.The results demonstrated that the expression of VFGF in model group was upregulated by 5.31 times(P<0.05),comparing with control group.However,the expression VFGF has no significant changes in in treatment group(P>0.05),which compared to model group.3.After p MIR-REPORT-VEGF and mir-23 a cotransfected HEK293 cells,compared with p MIR-REPORT-VEGF and NC,the activity of luciferase was significantly decreased(P<0.05);p MIR-REPORT-VEGF or p MIR-REPORT with the mir-23 a transfected HEK293 cells,the former luciferase activity was inhibited(P <0.05);Moreove,in p MIR-REPORT with the mir-23 a co-transfected with p MIR-REPORT single transfection of the two groups compared to the relative luciferase activity is no significantly different(P> 0.05).Conclusion:1.The successful rate of high-oxygen-induced retinal neovascularization model in mice was 100%,?-elemene plays a significantly role of inhibiting retinal neovascularization in mice.2.Differential expression of microRNAs(mi RNAs)in retinal tissue were detected by mi RCURYLNA microarray technology firstly among the three groups.The results show that 43 mi RNAs decreased and 11 mi RNA increased relative to normal group,in which 3down regulated significantly different(fold change>5,p <0.05),including mir-22 ?mir-23a?mir-190 b.Compared with model group,44 mi RNAs increased and 12 mi RNA decreased,in which 5 upregulated significantly different(fold change>3,p <0.05),including mir-22?mir-23a?mir-181a-1*?mir377?mir17*,3 downregulated including mir-872 ? mir-19 b mir-26b;After analysis we found that mi RNA regulation in the treatment group,but total 23 mi RNAs decreased in the model group,including mir-22,mir-181a-1 *,mir335-5 p,mir433,mir-669 n,mir-190 b,mir-23 a,mir-93 and so on.3.Although VEGF m RNA levels shows no significant changes in the treatment group(P> 0.05)compared with model group,there is a decreased in protein level,indicating that VEGF levels by regulation of translation and post-translation.mir-23 a and VEGF protein levels were negatively correlated,suggesting that mir-23a-mediated decrease in VEGF levels of ?-elemene inhibited retinal neovascularization in mice.Elucidated the initial ?-elemene can be regulatedby micro RNA-23 a inhibition of VEGF signaling pathway retinal neovascularization.4.These experiments show that mir-23 a could inhibit the expression of exogenous VEGF of HEK293 cells in vitro. |
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