| [Objective] Objective to establish the model of hypobaric hypoxia injury in rats and the animal injury model under the protection of Potentilla anserine. Using gene chip, miRNA microarray, suspended chip and RT-PCR technology, study the changes of gene expression profiling, miRNA and cytokines in rat cerebral cortex, which are caused by hypoxia and the protective effect of potentilla anserine, and the analysis of their internal mechanisms are performed followed by identifying the appropriate target genes to explore the relationship among them and their effects in hypobaric hypoxia damage.[Methods] Study on the mechanism of hypoxic injury:the rats were randomly divided into normal control group,3000m hypoxia group and7000m hypoxia group. Totally3groups, each group had8rats. Study on the protective effect of Potentilla: the rats were randomly divided into normal control group,7000m hypoxia group and7000m hypoxia Potentilla group. Totally3groups, each group had8rats. In advance, intragastric administration with Potentilla anserine was performed on hypoxia Potentilla group one week and other two groups were treated with equivalent normal saline. Using Hypobaric hypoxia cabin to establish acute high altitude hypoxia model.The morphological changes of cerebral cortex neurons after hypobaric hypoxia injury were studied by H.E and toluidine blue stain. Water concentration in brain and a series of biochemical indicators (SOD, MDA) were determinated with physical and biochemistry method individually.Using Gene Chip, miRNA microarray to study the changes of expressed genes, using qRT-PCR detected the levels of Tac1, Mtla and Dynlt3, using suspended chip technology detected monocyte chemotactic factor-1(MCP-1), interleukin-1β(IL-1β) and vascular endothelial growth factor (VEGF) levels.[Result]1.By conventional H.E staining we can see the cerebral cortex nerve cells of the control group cone-shaped. Uniform size, Neat, the ratio of nucleus and cytoplasm is large, nucleus are in the middle of the cell, which are circular or oval-shaped, and light blue purple, The nucleolus is clearly visible.Nevertheless, in hypobaric hypoxia3000m and7000m groups, the cytoplasm is pale red the brain cortex cells are mainly necrosis and apoptosis,layer numbers of the pyramidal cell is decreased, sparse arrangement Irregular with the altitude increased damage gradually increased. We can see most of the cells shrinkage deformation deep cytoplasm staining, Nuclear fragmentation and nucleus dissolved, cell debris and apoptotic bodies are full of the mesenchyme.The hypoxia Potentilla group is relatively light degree of injury mainly caused by cell edema, we can see part of the nerve cell body swelling, the cytoplasm irregular lightly stained the nucleus enlargement, lightly stained or disappear most of the nerve cell body shrink nucleus solid shrunk to triangle or poly gon nucleols disappeared cytoplasm deeply stained.2.Toluidine blue staining:microscopic observation, after toluidine blue staining in rat cerebral cortex we can see, the neuronal nuclear of control group is light blue, neurons arranged rules, the cytoplasm lightly stained, a lot of dark blue coarse granular nissl body can be seen, the nucleolus is visible;3000m hypoxia group Neurons were comparison rules, Part of the axons fracture or disappear Nissl body reduced or disappeared;7000m hypoxia group, neurons were scattered, cell shrink deformation, cytoplasm deeply stained, Nissl body disappeared, axonotmesis; the hypoxia Potentilla group, Neurons were comparison rules, Part of the axons fracture or disappear.3.Brain water content detection:Hypobaric hypoxia in brain water content is gradually increased with increasing altitude, Compared with the control group, there were significant differences (P<0.01). compared with the7000m hypoxia potentilla group,7000m hypoxia group was increased significantly (P<0.01)4.Biochemical detection:The hypobaric hypoxia group of brain tissues SOD activity gradually decreased with altitude increases, Compared with the control group were significantly different (P<0.01). Compared with the7000m hypoxia potentilla group,7000m hypoxia group was significantly decreased (P<0.01)5.3000m Gene chip detection:Study on the mechanism of hypoxic injury: hypoxia group consisted of215different expressed genes, of which29were up-regulated186were down-regulated. Up-regulated genes were Tacl, Rgs9, Serpinb6a, Adora2a, Penkl. Down-regulated genes were Siah1a, Acvrl, Btbdl, Cir, Abi1. The raised most obvious gene is Tacl.7000m hypoxia group consisted of205differentially expressed genes,of which21were up-regulated184were down-regulated. Up-regulated genes were Mtla, Cml3, Wfdc1, Tfpi, Vwf down-regulated genes were Zfp238, Atad1, Leprotl1, Tpm4, Fxrl. The raised most obvious gene is Mtla two sets of differentially expressed genes the common expressed genes were119, of which6were up-regulated,113were down-regulated。Up-regulated genes were Tac1, Ephx1, Cml3, Olr606, Dusp7. Down-regulated genes were Acvrl, Btbd1, Leprotl1, Uncl19, Canx.Study on the protective effect of Potentilla:7000m hypoxia potentilla group and control group consisted of232Common gene expression,of which30were up-regulated202were down-regulated up-regulated genes were TAC1, RGS9, ADORA2A, UPP1, GNG7down-regulated genes were SFRS15, STX12, CS, ASCL1, SSG1.7000m hypoxia potentilla group and hypoxia group consisted of37Common gene expression. Two sets of differentially expressed genes coexpressed genes were1712were up-regulated,5were down-regulated.Up-regulated genes were Dynlt3, Scn4b, Rgs9, Adora2a, Gpr88Down-regulated genes were Nov, Sfxn5, Wdfy1, Col1a2, Prkce.6. MiRNA chip detection:Study on the mechanism of hypoxic injury:7000m hypoxia group consisted of10different expressed miRNA, of which5were up-regulated,5were down-regulated. Up-regulated miRNA were rno-miR-125b-5p, rno-miR-127, rno-miR-7a, rno-miR-7b, rno-miR-99a. Down-regulated miRNA were rno-miR-32*, rno-miR-466b, rno-miR-466d, rno-miR-92a-2*, rno-miR-92b*. The target gene were predicted by Targetscan and miRBase software, down-regulated target gene were13, There are Dicerl, Klhl24, St3gal6, Gpc4, Bmpr2. Up-regulated target gene were3, they are Dusp7, Tfpi, Btg2. Among them, the most significant gene is Tfpi (ratio=2.358224)Study on the protective effect of Potentilla:7000m hypoxia potentilla group consisted of9different expressed miRNA, of which4were up-regulated,5were down-regulated. Up-regulated miRNA were rno-miR-92b*, rno-miR-92a-2*, rno-miR-326*, rno-miR-125b-3p. down-regulated miRNA were rno-miR-345-5p, rno-miR-668, rno-miR-363*, rno-miR-29b, rno-miR-219-5p。The target gene were predicted by Targetscan and miRBase software, up-regulated target gene were2, There are Plp1(ratio=1.518752) and Mog (ratio=1.568031)7.RT-PCR:Tacl showed low expression in control group. compared with the control groups,3000m hypoxia group and7000m hypoxia groups Tacl expression were significantly increased (P<0.05), and with the increase of altitude increased. The control group Mtla showed low expression; compared with control group,3000m hypoxia group was not statistically significant.7000m hypoxia group expression was significantly increased (P<0.05) Compared with the control group and hypoxia grouphypoxia potentilla group Dynlt3expression were significantly increased.8. Suspension Array Detection:Study on the mechanism of hypoxic injury:compared with the control groups,3000m hypoxia group MCP-1, IL-1β, VEGF do not have obvious change,7000m hypoxia group, the expression level was significantly increased (P<0.01)Study on the protective effect of Potentilla:compared with the control groups7000m hypoxia potentilla group and hypoxia group MCP-1, IL-1β and VEGF expression level was significantly increased (P<0.01)[Conclusion]1. Tac1is more sensitive to hypoxia, the expression of genes in mild and severe hypoxia are up-regulated, Mtla react slow to hypoxia, Mtla under mild hypoxia gene expression levels do not change obvious, and under severe hypoxia the levels are significantly increase.2. Different hypoxic conditions different miRNA expression, by regulating target gene miRNA affect the expression of cytokines and the life processes of the body.3. The rat in hypoxia potentilla group Dynlt3gene is significantly increased. Dynlt3is probably one of the major gene involved in the protective effect of potentilla anserine.4. Potentilla anserina could increase the activity of cerebral cortical cells, and could reverse the changes in cytokine levels caused by hypoxia injury. |
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