| Objective: Ischemic is inevitably in the process of organ transplantation, reperfusion will bring organ of the secondary injury, which is the contradiction of various clinical treatment methods. The complex mechanism makes the further research in the bottleneck. ALDH2 is an enzyme that plays an pivotal role in human body, its protection role in a variety of organs during IR has been confirmed. And in the role of kidney IR needs to be explored. Studies showed that mi R-146 a could protect heart and small intestine from IR injury through regulation of ALDH2 transcription. Therefore, we hope to reveal the role of ALDH2 in kidney IR and regulatory role of mi R-146 a in ALDH2 regulation and its possible regulatory mechanism. Providing the theory basis for the further study of ALDH2 in the role of the kidney I/R.Methods: For in vivo experiments, male C57BL/6 J mice were used, aging 8 to 10 weeks. Kidney ischemia-reperfusion injury model were made by clip bilateral kidney renal pedicle for 30 mins. Blood and kidneys were harvested in certain points. Renal pathological damage were observed through HE stain. Blood creatinine was detected after harvest. Expression of ALDH2 and mi R-146 a m RNAs were analysed by RT-PCR system. Detection ALDH2 protein was applied by Western Blot. For in vitro experiments, male C57BL/6 J mice were used, aging 8 to 10 weeks. Renal tubular epithelial cells and peritoneal macrophages were separated and purificated.I/R injury was analoged by using paraffin oil. Oxidative stress was analoged by using10 u M H2O2. RNA were extracted by using Trizol reagent. ALDH2 and mi R-146 a m RNAs were analysed by RT-PCR system.Results: After reperfusion, the expression of ALDH2 significantly increased, restored to control level at 12 h. mi R-146 a significant increased at 3 hours after IR, and after 72 hours of IR, mi R-146 a expression is still 3.7±0.7 times of the control group. 1h after IR, protein of ALDH 2 significantly decreased and fell to the lowest at 3 h, then gradually increases. 12 h protein levels are significantly lower than the control group, reach the peak at 24 h, then gradually decline. TEC: After reperfusion, the expression of ALDH2 significantly increased, the ALDH2 expression is still significantly higher than the control group after 24 hours. 3 h after IR, mi R-146 a expression significantly decreased; then gradually increase, 24 hours after IR, mi R-146 a expression significantly increased, is about 4.2±0.8 times of the control group. ALDH2 expression significantly increased after H2O2 stimulation; Within 6 h after H2O2 stimulation, mi R-146 a expression has no obvious change and rapidly increase at 24 hours, the expression level is about 2.7±0.7 times of the control group. MΦ: After reperfusion, the expression of ALDH2 significantly increased, the ALDH2 expression is still significantly higher than the control group after 24 hours. mi R-146 a expression level has no significant change after IR. ALDH2 expression significantly increased after 6h of H2O2 stimulation; mi R-146 a expression significantly increased right after H2O2 stimulation.Conclusion: Our results suggest that in the model of kidney IR, ALDH2 m RNA expression increase rapidly after IR, and ALDH2 protein decreased in the early stage of IR, then gradually increase; mi R-146 a m RNA expression increase rapidly after IR. In vitroexperiments of TEC and MΦ show that the ALDH2 expression rapidly increase after I/R and H2O2 stimulation, but mi R-146 a expression decreased in the early stage of IR and gradually increase after 6h, which is different with in vivo experiment results. mi R-146 a is expected to become the early diagnostic biomarker of renal IR injury. |
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