| ObjectiveThis experiment is on the basis of li-pilocarpine induced epilepsy model,To investigate whether miR-9 is involved in the occurrence and development of TLE process;MiR-9 whether to participate in the TPM inhibition of temporal lobe epilepsy rat hippocampal neuron apoptosis process;TPM is by adjusting the ratio of bcl-2 protein and bax protein to inhibit the apoptosis of neuron.Methods1.Experimental animals and grouping:Healthy adult female and male rats were randomly divided into four groups:1) control group(n=20),2) TLE group(n=20),3) low dose TPM group (n=20),4) high dose TPM group (n=20).2.The preparation of epilepsy model and intervention:Using the li-pilocarpine induced epilepsy model,model animals need to achieve the level of IV above, and continue more than 50 minutes.For control group and TLE group need to gavage normal saline 40mg/kg everyday.For TL group need to gavage TPM 40mg/kg everyday.For TH group need to gavage TPM 80mg/kg everyday.3.Marerials and experiment measurement:Through the analysis of the chip and real-time quantitative PCR detected the differential expression of miR-9 in the above four groups of rats hippocampus;Through the western blot and IHC detected the change of the ratio of bcl-2 protein and bax protein of the above each hippocampus;Through HE staining investigated the cell morphology change of each group rat hippocampus.Resultsl.The results of miRNAs chip analysis:Compared with control group, TLE group had 12 miRNAs (miR-207,-204,-466,-511,-2964,-3581,-764,-214,-210,-351,-466,-298) were up-regulated, and 28 miRNAs (miR-9,-101,-136,-872,-154,-153,-382,-362,-377,-337,-380,-218,-219,-31,-383,-499,-33,-34,-376,-300,-19,-592,-186,-488,-598,-30,-582,-708) were down-regulated. In the TL and TH group 12 up-regulated miRNAs were down-regulated, and the 28 down-regulated miRNAs were up-regulated. Of these, miR-9 was one of the greater change in regulation.2.The results of real time quantitative PCR:Compared with control group, miR-9 was down-regulated obviously in the TLE group(P<0.05). Compared with TLE group, miR-9 was up-regulated obviously in the TH group (P<0.05). All of the other groups had no difference(P>0.05).3.The results of Western blot:Bax was significantly up-regulated in the TLE group(P<0.01), but down-regulated in TPM-treated groups(P<0.05), especially in the high dose group(P<0.05).Bcl-2 was significantly down-regulated in the TLE group(P<0.05), but up-regulated in TPM-treated groups, especially in the high dose group(P<0.05). Bcl-2/bax was significantly down-regulated in the TLE group(P<0.01), compared with TLE group, Bcl-2/bax was up-regulated in TPM-treated groups, especially in the high dose group(P<0.01).4. The results of Immunohistochemical:Compared with control group, bax+was significantly increased in the TLE group(P<0.01), but reduced in TPM-treated groups(P<0.05), especially in the high dose group(P<0.05). Compared with control group, bcl-2+ slightly reduced in the TLE group(P>0.05), but up-regulated in TPM-treated groups obviously(P<0.01); The both of TL group and TH group has no obvious difference(P>0.05). Compared with TLE group, bcl-2+/bax+was significantly increased in the control group(P<0.01); compared with TLE group, bcl-2+/bax+ was obviously up-regulated in TPM-treated groups (P<0.05); especially in the high dose group(P<0.05).5.The results of HE stain:The control group had more number of cells, the neat cell morphology and the clear order. Hower, all of the TLE,TL,TH groups had the decrease in the number of cell, the not neat cell morphology, the clearance increases obviously, disordered arrangement, the disappear of the partial nucleus. TLE group was more worse than TPM-treated group; TH group was more improve than TL group.Conclusions1) MiR-9 may regulate the protein of bcl-2 and bax to participate in the apoptosis pathway of TLE.2) TPM could regulate the protein of bcl-2 and bax to inhibit the apoptosis in hippocampus of TLE. |
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