| Ischemic cerebrovascular disease is characterized with high incidence, mortality and morbidity, which already seriously affects people’s health and life quality. It also brings heavy burdens to society and families. Neurons apoptosis caused by ischemia-reperfusion(I/R) is one of the major causes of brain damage. The mitochondrial ATP sensitive potassium channel(KATP) open agent diazoxide(DZX) can decrease apoptosis rate of nerve cells and protect neurons. However the protection mechanism of DZX is still not clear. So the purpose of this study is to explore the protective rolesof DZX on neurons with cellular and molecular biology technologiesObjectives 1. To study the relationship between the gene expression of KATP and neuron damage, and to explore the related gene expression and its downstream molecules. 2. To explore the protective roles of DZX on hippocampal neurons with oxygen glucose deprivation exposure. 3. Using thread embolism method to build a rat cerebral ischemia reperfusion model, and explore the roles of DZX on ischemia-reperfusion injury of rat. 4. To explore whether some long non-coding RNAs involves in the expression of caspase3, caspase12, and Bax regulated by DZX.Methods 1. We used bioinformatics methods to determine the differentially expressed genes of mitochondrial ATP sensitive potassium channel in cerebral ischemia reperfusion model. And we treated the hippocampal brain slices by oxygen and glucose deprivation, then we used patch clamp to detect current changes of KATP before and after DZX treatment. 2. We cultured hippocampal neuron cells and hippocampal slices in vitro, and used oxygen-glucose-deprivation method to treat the neuron cells and hippocampal slices. The immunocytochemistry, MTT, LDH, and TUNEL assay were used to determine the extent of injuries in vitro models. Flow cytometry and RT-PCR were used to detect the protective role of DZX on damaged hippocampal neurons and slices. 3. We used line embolism method to estabolish the ischemia-reperfusion model, and using brain stereotaxic lateral ventricle injection for DZX treatment, the infarct volume was determined by TTC staining. Neurological score and water maze test were used to detect the memory protection effects of oxygen-glucose deprived nerve. TTC staining, HE staining, TUNEL assay were used to explore the neuroprotective activity of DZX. 4. The lnc RNA microarray was used to analysize and found the differentially expressed genes regulated by DZX, and RT-PCR was used to verify these differentially expressed genes, then lcn RNA s66184 was found. Finally we used RT-PCR to verify the regulation relationships between DZX and lnc RNA S66184 in oxygen-glucose deprived neurons and hippocampal slices.Results 1. We forecasted the differentially expressed genes between cortex and striatum of cerebral ischemia reperfusion by analyzing GSE23160 gene chip, and we found 7 common differentially expressed genes in cortex and striatum of cerebral ischemia reperfusion. Twenty-four hours after surgery, the expressions of PR84, GJB2, TLR2, GPR84, GJB2 in striatum were increased along with the increased degree of damage, the expression of DSCR1, CDKN1 A reached highest level after 8 hours, then decreased with the increased postoperative time. Moreover, the patch clamp results revealed that the electric current density of OGD group(93.2±5.4) was higher than normal group(53.3±5.3), the electric current density of OGD+DZX group was back to normal level(53.2±3.0), while the electric current density of OGD+DZX+TG group was significantly increased(66.9±11.1), it lower than OGD group and a little higher than DZX+OGD group, which suggested 5-HD was able to inhibit the protective role of DZX, and DZX could suppress ERS pathway. 2. We divided the rat hippocampal neurons and hippocampal brain slices into OGD, DZX+OGD, DZX+5-HD+OGD, DZX+OGD+TG groups. The MTT and LDH assay showed that the death of hippocampal neurons increased with the increase in oxygen-glucose deprivation time. The apoptotic rate of OGD group was significantly higher than normal group(p<0.05). However the apoptotic rate of OGD+DZX group was lower than OGD group(p<0.05), and it was similar with OGD+DZX+5-HD. These results suggested that DZX was able to resist the ERS induced hippocampus neuron cell apoptosis. 3. In the histological experiments, we used TTC staining for analysis of infarct volume, H&E staining for histological characterization, and TUNEL assay for apoptosis. The neurological function results showed the score of I/R group(5.86 ± 0.63) was significantly higher than sham group, while the score of DZX+I/R group was dramatically lower(2.63 ± 0.31) than I/R group. Interestingly, the score of DZX +5-HD+I/R increased again(5.12 ± 0.51). Moreover, the water maze results showed that the incubation period of I/R group was longer than sham group(p<0.05), the incubation period was obviously reduced after treated with DZX. In addition, the treatment of DZX+5-HD can significantly prolong the incubation period. Two and three days after surgery, the incubation period of all group decreased, and the incubation period of I/R group was obviously longer than sham and DZX treated group. Furthermore, the standing time of rats in sham group was 33.12±3.34 s, the I/R group was 18.56±3.56 s, the DZX+I/R group was 24.23±4.45 s, the DZX +5-HD+I/R group was 16.45±5.67 s. This result further demonstrated the protective role of DZX on neuron damage.4. The lnc RNA microarray results between the sham group and I/R group showed that 12 lnc RNAs were up-regulated. Then we detected the expression of these lnc RNAs in brain tissues of NC, I/R, I/R+DZX, IR+DZX+5-HD groups, respectively. We found that the expression of lnc RNA S66184 was closely related to DZX treatment. In order to explore whether DZX regulated the expression of apoptotic-related genes through lnc RNA S66184, we exposed cultured hippocampal neurons to DZX, we found the expression of lnc RNA S66184 markedly reduced. And in oxygen glucose deprived hippocampal neurons: 1. the regulating effects of DZX on apoptotic-related genes expression were consistent with the results in animal model. 2. The regulating effect of DZX on the lnc RNA S66184 expression was also consistent with the results in animal model. 3. The regulating effects of DZX on apoptotic-related gene expressions were decreased when the oxygen glucose deprived hippocampal neuron cells were co-treated with DZX and lnc RNA S66184. The results was similar with 5-HD treatment. In this part, we demonstrated that DZX regulated caspase3, caspase12, and Bax expression via lnc RNA S66184, and therefore exerted its protective role.Conclusion 1. KATP is one of the key targets in protection against the ischemic cerebrovascular disease. DZX, the KATP open agent, was able to affect the electric current density of neuron and the expression of sub-gene of KATP 2. DZX was able to resist the ERS induced hippocampus neuron apoptosis. 3. DZX was able to affect the histological characterization brain, cells apoptosis, neurological function, learning and memory function. The in vivo results further demonstrated the protective role of DZX on ischemic cerebrovascular disease. 4. DZX was able to regulate the expression of caspase3, caspase12, Bax by lnc RNA S66184 and therefore exerted its protective roles.The full text conclusionCollectively, according to the experimental data, we have reason to speculate the mitochondrial ATP sensitive potassium channel(KATP) open agent diazoxide(DZX) was able to inhibit the ERS reaction caused by brain ischemic injury, probably through lnc RNA S66184 to regulate the expression of caspase3, caspase12, and Bax. Thus, it has a significant inhibitory effect on the occurrence and development of brain ischemic injury. |
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