Morin improves postoperative cognitive dysfunction by mediating the miR-138-5p/SIRT1 axis to regulate ferroptosis methods
Aim. Evidence-backed execution summary for Morin improves postoperative cognitive dysfunction by mediating the miR-138-5p/SIRT1 axis to regulate ferroptosis methods from Morin improves postoperative cognitive dysfunction by mediating the miR-138-5p/SIRT1 axis to regulate ferroptosis.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Novel location recognition (NLR) and novel object recognition (NOR) assay
reagent used in the protocol.
- Use
- \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\%\:\mathrm{T(Novel)}\mathrm{=}\frac{\mathrm{T(Novel)}\text{}\text{}}{\mathrm{T(Total)}...
Novel location recognition (NLR) and novel object recognition (NOR) assay
reagent used in the protocol.
- Use
- T (Novel): Time spent exploring the novel location or novel object.
Novel location recognition (NLR) and novel object recognition (NOR) assay
reagent used in the protocol.
- Use
- T (Total): Total exploration time, calculated as: T (Total) = T (Novel) + T (Familiar).
Novel location recognition (NLR) and novel object recognition (NOR) assay
reagent used in the protocol.
- Use
- T (Familiar): Time spent exploring the familiar location or familiar object.
Targeting validation of miR-138-5p and SIRT1
reagent used in the protocol.
- Use
- As indicated in Fig. A, the perfect base pairing was observed between the 3'UTR (wt) of SIRT1 mRNA and the seed sequence of miR-138-5p. However, SIRT1-3'UTR (mu) did not have a complementary pairing with miR-138-5p. Dual-luciferase reporter assay shows that mmu-miR-138-5p mimic has a targeted inhib...
Protective effect of Morin on POCD mice
reagent used in the protocol.
- Use
- After identifying the potential mechanism of POCD, we selected Morin (Fig. A), an active natural compound with neuroprotective and cognitive dysfunction-improving properties,, to validate the involvement of the miR-138-5p/SIRT1 axis in POCD. As shown in Fig. B, compared with the sham group, the expression of miR-1...
Novel location recognition (NLR) and novel object recognition (NOR) assay
reagent used in the protocol.
- Use
- 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.
Novel location recognition (NLR) and novel object recognition (NOR) assay
reagent used in the protocol.
- Use
- The percentage of exploration time is used to quantitatively represent the animal's interest in a novel location or object during the test. It is calculated as follows:
Protective effect of Morin on POCD mice
After identifying the potential mechanism of POCD, we selected Morin (Fig. A), an active natural compound with neuroprotective and cognitive dysfunction-improving properties,, to validate the involvement of the miR-138-5p/SIRT1 axis in POCD. As shown in Fig. B, compared with the sham group, the expression of miR-1...
- Use
- After identifying the potential mechanism of POCD, we selected Morin (Fig. A), an active natural compound with neuroprotective and cognitive dysfunction-improving properties,, to validate the involvement of the miR-138-5p/SIRT1 axis in POCD. As shown in Fig. B, compared with the sham group, the expression of miR-1...
Protective effect of Morin on POCD mice
Fig. 3 Protective effect of Morin on cognitive dysfunction in POCD mice. ( A ) Chemical structure of Morin. ( B ) Effect of Morin on miR-138-5p. ( C and D ) Effect of Morin on SIRT1 protein. Effect of Morin on spontaneous alternation rate in Y Maze ( E ), novel location recognition ( F ), and novel object recognitio...
- Use
- Fig. 3 Protective effect of Morin on cognitive dysfunction in POCD mice. ( A ) Chemical structure of Morin. ( B ) Effect of Morin on miR-138-5p. ( C and D ) Effect of Morin on SIRT1 protein. Effect of Morin on spontaneous alternation rate in Y Maze ( E ), novel location recognition ( F ), and novel object recognitio...
Morin promotes cognitive recovery in POCD by inhibiting miR-138-5p
Morin has been previously identified to inhibit miR-138-5p in the mouse hippocampus, and the miR-138-5p agomir was introduced to block the efficacy of Morin. As shown in Fig. A, in Y Maze, agomir NC did not affect the effect of Morin. However, the spontaneous alternation rate of Morin-treated POCD mice was red...
- Use
- Morin has been previously identified to inhibit miR-138-5p in the mouse hippocampus, and the miR-138-5p agomir was introduced to block the efficacy of Morin. As shown in Fig. A, in Y Maze, agomir NC did not affect the effect of Morin. However, the spontaneous alternation rate of Morin-treated POCD mice was red...
Morin promotes cognitive recovery in POCD by inhibiting miR-138-5p
Fig. 4 Effect of Morin on cognitive function in POCD via miR-138-5p. Effect of Morin inhibiting miR-138-5p on spontaneous alternation rate in Y Maze ( A ), novel location recognition ( B ), and novel object recognition ( C ) in POCD mice. Levels of GSH ( D ), MDA ( E ), and Iron ( F ) in hippocampal tissue of POCD m...
- Use
- Fig. 4 Effect of Morin on cognitive function in POCD via miR-138-5p. Effect of Morin inhibiting miR-138-5p on spontaneous alternation rate in Y Maze ( A ), novel location recognition ( B ), and novel object recognition ( C ) in POCD mice. Levels of GSH ( D ), MDA ( E ), and Iron ( F ) in hippocampal tissue of POCD m...
In vitro cell experiments verified the effect of Morin on ferroptosis through miR-138-5p/SIRT1
Fig. 6 Effects of Morin on oxidative stress in hippocampal cells via miR-138-5p. ( A ) Expression of miR-138-5p in HT22 hippocampal cells. ( B ) GSH content in HT22 cells. ( C ) MDA content in HT22 cells. ( D ) Fe 2+ level. ( E ) Representative flow cytometry images of ROS. ( F ) ROS statistical results. * P < 0.05,...
- Use
- Fig. 6 Effects of Morin on oxidative stress in hippocampal cells via miR-138-5p. ( A ) Expression of miR-138-5p in HT22 hippocampal cells. ( B ) GSH content in HT22 cells. ( C ) MDA content in HT22 cells. ( D ) Fe 2+ level. ( E ) Representative flow cytometry images of ROS. ( F ) ROS statistical results. * P < 0.05,...
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...
- Use
- 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...
Novel location recognition (NLR) and novel object recognition (NOR) assay
The experiment utilized the Panlab SMART 3.0 system for image acquisition and video analysis. A square open-field box (40 × 40 × 40 cm) was selected as the testing arena. On postoperative day 4, mice were acclimated to the testing room for 1 h and then placed into the empt...
- Use
- The experiment utilized the Panlab SMART 3.0 system for image acquisition and video analysis. A square open-field box (40 × 40 × 40 cm) was selected as the testing arena. On postoperative day 4, mice were acclimated to the testing room for 1 h and then placed into the empt...
Transcriptome sequencing of miRNAs
The Sham and OP groups each had 3 mice. All mice in the OP group were confirmed to have POCD by cognitive assessment tests before sequencing. The extraction, quality check, and library construction of total RNA from mouse hippocampal tissue were done by Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Tota...
- Use
- The Sham and OP groups each had 3 mice. All mice in the OP group were confirmed to have POCD by cognitive assessment tests before sequencing. The extraction, quality check, and library construction of total RNA from mouse hippocampal tissue were done by Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Tota...
Statistics and analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- SPSS 24.0 software was used for data statistics and analysis. The data were expressed as mean ± standard deviation ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setl...
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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).
Measurement outputs
What raw and processed outputs should exist?
As indicated in Fig. A, the perfect base pairing was observed between the 3'UTR (wt) of SIRT1 mRNA and the seed sequence of miR-138-5p. However, SIRT1-3'UTR (m...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Fig. 2 Expression and targeting relationship of miR-138-5p and SIRT1 in the hippocampus of POCD mice. ( A ) Cloned into a luciferase reporter vector to generate the WT or Mut SI...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
After identifying the potential mechanism of POCD, we selected Morin (Fig. A), an active natural compound with neuroprotective and cognitive dysfunction-improving properties,,...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Based on the fact that Morin regulates miR-138-5p to affect iron ions and mitochondrial ultrastructure, we then studied Morin's regulation of p53, SLC7A11, and GPX4 throug...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Fig.
from paperScoring or quantification
Quantify the primary readouts for this experiment: As indicated in Fig. A, the perfect base pairing was observed between the 3'UTR (wt) of SIRT1 mRNA and the seed sequence of miR-138-5p. However, SIRT1-3'UTR (m...; Fig. 2 Expression and targeting relationship of miR-138-5p and SIRT1 in the hippocampus of POCD mice. ( A ) Cloned into a luciferase reporter vector to generate the WT or Mut SI...; After identifying the potential mechanism of POCD, we selected Morin (Fig. A), an active natural compound with neuroprotective and cognitive dysfunction-improving properties,,...; Based on the fact that Morin regulates miR-138-5p to affect iron ions and mitochondrial ultrastructure, we then studied Morin's regulation of p53, SLC7A11, and GPX4 throug....
from paperStatistical comparison
Fig. 6 Effects of Morin on oxidative stress in hippocampal cells via miR-138-5p. ( A ) Expression of miR-138-5p in HT22 hippocampal cells. ( B ) GSH content in HT22 cells. ( C )...; The Sham and OP groups each had 3 mice. All mice in the OP group were confirmed to have POCD by cognitive assessment tests before sequencing. The extraction, quality check, and...; SPSS 24.0 software was used for data statistics and analysis. The data were expressed as mean ± standard deviation ( \documentclass[12pt]{minimal} \usepackage{a...
from paperReporting output
Report representative outputs alongside summary comparisons for As indicated in Fig. A, the perfect base pairing was observed between the 3'UTR (wt) of SIRT1 mRNA and the seed sequence of miR-138-5p. However, SIRT1-3'UTR (m..., Fig. 2 Expression and targeting relationship of miR-138-5p and SIRT1 in the hippocampus of POCD mice. ( A ) Cloned into a luciferase reporter vector to generate the WT or Mut SI..., After identifying the potential mechanism of POCD, we selected Morin (Fig. A), an active natural compound with neuroprotective and cognitive dysfunction-improving properties,,..., Based on the fact that Morin regulates miR-138-5p to affect iron ions and mitochondrial ultrastructure, we then studied Morin's regulation of p53, SLC7A11, and GPX4 throug....
inferred from protocolStructured statistical methods
Fig. 6 Effects of Morin on oxidative stress in hippocampal cells via miR-138-5p. ( A ) Expression of miR-138-5p in HT22 hippocampal cells. ( B ) GSH content in HT22 cells. ( C )...; The Sham and OP groups each had 3 mice. All mice in the OP group were confirmed to have POCD by cognitive assessment tests before sequencing. The extraction, quality check, and...; SPSS 24.0 software was used for data statistics and analysis. The data were expressed as mean ± standard deviation ( \documentclass[12pt]{minimal} \usepackage{a...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
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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