Morin improves postoperative cognitive dysfunction by mediating the miR-138-5p/SIRT1 axis to regulate ferroptosis methods
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Novel location recognition (NLR) and novel object recognition (NOR) assay
For NOR task, 1 h after the NLR, object B was replaced with a novel object (C). Mice were tested for 10 min, and the time spent exploring the novel object (C) and the familiar object at the location (A') was recorded.
Methods and materials
8 weeks old C57BL/6J mice without specific pathogens (SPF) were purchased from the Experimental Animal Center of Guizhou Medical University (SPF (Beijing) biotechnology co., Ltd., Beijing, China), body weight 18-22 g, and animal license number: SCXK (jing) 2019-0010. The animals were housed in an SPF-grade laboratory at the Experimental Animal Center of Guizhou Medical University and fed and watered ad libitum. All experimental protocols of the present study were approved by the Animal Ethics Committee of Guizhou Medical University (license number: SCXK (qian) 2018-0001) (IRB number: 2100557). All authors complied with the ARRIVE guidelines. All treatments and experimental procedures were performed in accordance with the National Institutes of Health guidelines and were approved by the Animal Ethics Committee of Guizhou Medical University (license number: SCXK (qian) 2018-0...
Methods and materials
The mice were randomly divided into the sham group, OP group, OP + morin group, OP + morin + agomir NC group, and OP + morin + miR-138-5p agomir group, with 6 mice in each group. OP (POCD) group: The cardiac surgery animal model in the OP group was established by continuous ligation of the left anterior descending coronary artery (LAD) for 45 min and then removing the ligature for blood reperfusion. The mice were maintained under general anesthesia by continuous inhalation of a mixture of 1-2% sevoflurane and 0.5 L/min oxygen. A skin incision and purse-string suture were made on the left anterior chest of C57 mice. The pectoralis major and pectoralis minor muscles were separated with hemostatic forceps and the 3rd to 4th ribs were spread to expose the heart. The left anterior descending coronary artery was qui...
Y maze
The Y maze apparatus is a spatial recognition memory test consisting of three identical arms (45 × 13 cm, with 45 cm high walls) that were intersected at 120° around a central triangle. On the third day after surgery, the mice were placed in the Y maze experiment room for 1 h of adaptation. On the fourth day after surgery, the mice were placed at the end of arm A and allowed to freely explore arms A, B, and C for 5 min. The order and total number of arms entered were recorded. The criterion for mice to enter each arm was that all four limbs entered. Successful alternation was defined as entering consecutive arms (such as ABC, ACB, BCA, BAC, CAB, and CBA) in sequence, and the number of alternations was counted. The alternation rate was calculated using the following formula: alternation rate = [number of successful alternations/(total number of...
Flow cytometry for ROS
HT22 cells were inoculated in 6-well plates, and 1 × 10 6 cells were taken in each group and washed twice with PBS. The cells were loaded into the probe to make 500 µL suspension cells containing 5 mol/L DCFH-DA (S0033S, Beyotime), and the reaction was carried out at 37 °C for 30 min protected from light, and washed twice with PBS. The FITC channel was chosen to measure the variation of ROS in each group, and the change in ROS levels was quantified based on the optical density values of the cells detected by the FITC channel (CytoFLEX, Beckman Coulter Life Sciences).
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For NOR task, 1 h after the NLR, object B was replaced with a novel object (C). Mice were tested for 10 min, and the time spent exploring the novel object (C) and the familiar object at the location (A') was recorded.
8 weeks old C57BL/6J mice without specific pathogens (SPF) were purchased from the Experimental Animal Center of Guizhou Medical University (SPF (Beijing) biotechnology co., Ltd., Beijing, China), body weight 18-22 g, and animal license number: SCXK (jing) 2019-0010. The animals were housed in an SPF-grade laboratory at the Experimental Animal Center of Guizhou Medical University and fed and watered ad libitum. All experimental protocols of the present study were approved by the Animal Ethics Committee of Guizhou Medical University (license number: SCXK (qian) 2018-0001) (IRB number: 2100557). All authors complied with the ARRIVE guidelines. All treatments and experimental procedures were performed in accordance with the National Institutes of Health guidelines and were approved by the Animal Ethics Committee of Guizhou Medical University (license number: SCXK (qian) 2018-0001) (IRB number: 2100557).
The mice were randomly divided into the sham group, OP group, OP + morin group, OP + morin + agomir NC group, and OP + morin + miR-138-5p agomir group, with 6 mice in each group. OP (POCD) group: The cardiac surgery animal model in the OP group was established by continuous ligation of the left anterior descending coronary artery (LAD) for 45 min and then removing the ligature for blood reperfusion. The mice were maintained under general anesthesia by continuous inhalation of a mixture of 1-2% sevoflurane and 0.5 L/min oxygen. A skin incision and purse-string suture were made on the left anterior chest of C57 mice. The pectoralis major and pectoralis minor muscles were separated with hemostatic forceps and the 3rd to 4th ribs were spread to expose the heart. The left anterior descending coronary artery was quickly ligated, and then the heart was repositioned, sutured, and a purse-string suture was made. After the operation, the mice were placed on a warming blanket, and waited for them to wake up. After 45 min, the slipknot was loosened for myocardial reperfusion to complete the model-making process...
The Y maze apparatus is a spatial recognition memory test consisting of three identical arms (45 × 13 cm, with 45 cm high walls) that were intersected at 120° around a central triangle. On the third day after surgery, the mice were placed in the Y maze experiment room for 1 h of adaptation. On the fourth day after surgery, the mice were placed at the end of arm A and allowed to freely explore arms A, B, and C for 5 min. The order and total number of arms entered were recorded. The criterion for mice to enter each arm was that all four limbs entered. Successful alternation was defined as entering consecutive arms (such as ABC, ACB, BCA, BAC, CAB, and CBA) in sequence, and the number of alternations was counted. The alternation rate was calculated using the following formula: alternation rate = [number of successful alternations/(total number of entries into the arm-2)] × 100%. After each trial, the maze was treated with 75% alcohol before the next trial.
HT22 cells were inoculated in 6-well plates, and 1 × 10 6 cells were taken in each group and washed twice with PBS. The cells were loaded into the probe to make 500 µL suspension cells containing 5 mol/L DCFH-DA (S0033S, Beyotime), and the reaction was carried out at 37 °C for 30 min protected from light, and washed twice with PBS. The FITC channel was chosen to measure the variation of ROS in each group, and the change in ROS levels was quantified based on the optical density values of the cells detected by the FITC channel (CytoFLEX, Beckman Coulter Life Sciences).
Machine-readable layer
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