Method for researching influence of aerobic exercise on spontaneous hypertensive rat ferroptosis

Abstract

The invention discloses a method for researching the influence of aerobic exercise on spontaneous hypertensive rat ferroptosis, and relates to the technical field of medical research. Comprising the following steps: S1, experimental animals and grouping; normal rats WKY and spontaneously hypertensive rats SHR are randomly divided into a quiet WKY control group, a quiet SHR control group, an exercise group and an exercise drug group; s2, performing aerobic exercise intervention. The research method provided by the invention verifies the assumption that aerobic movement possibly inhibits ferroptosis and hinders further development of chronic kidney diseases by activating a hypertensive kidney cell systemxc- / GPX4 / GSH pathway, and provides reference for promoting subsequent further in-depth scientific research of the subject in the field. The method has positive practical value and significance in the aspect of discussing new pathogenesis and treatment strategy of hypertension.

Description

Technical Field

[0001] The present invention relates to the field of medical research technology, and in particular to a method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats. Background Art

[0002] Objective: The study found that exercise training can change the expression levels of ferroptosis genes in rats with cerebral hemorrhage, myocardial injury in mice fed a high-fat diet, brain I / R injury, pulmonary fibrosis, and skeletal muscle disorders in mice. However, it is still unclear whether exercise training can affect ferroptosis in hypertensive kidney cells; the mechanism by which exercise training alleviates spontaneous hypertensive chronic kidney disease, especially in the regulation of iron ion levels in kidney cell damage, has not been systematically studied.

[0003] Hypertension is a recognized factor in kidney damage and CKD progression, so CKD patients have a high cardiovascular risk, and better control of blood pressure is very important. A meta-analysis study observed that aerobic exercise can better control SBP levels in the short and medium term, although no significant difference was achieved. Cao Shuyuan et al. showed that regular aerobic exercise can reduce renal damage in spontaneously hypertensive rats, and moderate-intensity aerobic exercise is more effective in reducing renal damage than low-intensity aerobic exercise.

[0004] Although the above studies have reached corresponding conclusions, they have not conducted systematic research on the regulation of iron ion levels in renal cell damage. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a research method for the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats, comprising the following steps:

[0008] S1: Experimental animals and groups;

[0009] Animal selection and grouping: Normal rats (WKY) and spontaneously hypertensive rats (SHR) were randomly divided into: quiet WKY control group, quiet SHR control group, exercise group and exercise drug group;

[0010] S2: aerobic exercise intervention;

[0011] Exercise time and intensity: The experimental treadmill was used for exercise intervention, including:

[0012] Adaptation period: slope is 0, speed is 13-16m / min;

[0013] Formal exercise training period: slope is 0, speed is 16m / min in the first 2 weeks, then increases by 1m / min every two weeks, and speed is 19m / min in the 7th to 8th week;

[0014] Among them: the SHR rats in the exercise drug group were injected with the ferroptosis inducer Erastin (10 mg / kg / day) for 20 consecutive days and then exercise intervention;

[0015] S3: cytological experiments;

[0016] Cell culture: Human renal tubular epithelial cells (HK-2) were cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37°C and 5% CO2 in an incubator. When the cell confluence reached about 70%, the cells were synchronized with serum-free medium for 12 hours and then divided into four groups for different treatments: normal control group, hypertension group, exercise mimic intervention hypertension group, and cell transfection and exercise mimic intervention hypertension group.

[0017] Experimental treatment: After culturing for 16 hours, protein or RNA was extracted for relevant detection;

[0018] S4: Detection;

[0019] Basic physiological indicators: testing heart rate and blood pressure with tail cuff method, and electronic scale recording weight;

[0020] Renal function related indicators: Detection of renal function related indicators in serum;

[0021] Ferroptosis-related indicators: detection of free Fe in rat kidney tissue cells 2+ , PTGS2, MDA, and ROS expression levels;

[0022] S5: Data analysis;

[0023] The experimental results were statistically analyzed and processed; one-way analysis of variance was used for comparison of parameters among groups, and LSD test was used for multiple comparisons.

[0024] Preferably, in said S1, specifically:

[0025] S11: Animal selection: Wistar Kyoto rats (WKY) and 8-week-old male spontaneously hypertensive rats (SHR) were selected, a total of 24 rats;

[0026] S12: Random grouping: 24 rats were randomly divided into four groups, specifically: 6 rats in a quiet WKY control group, 6 rats in a quiet SHR control group, 6 rats in an exercise group, and 6 rats in an exercise drug group.

[0027] Preferably: the research method further includes environmental control, specifically:

[0028] All rats will be housed in separate air-conditioned rooms with a constant temperature of 22±2°C; the light cycle in the room is a 12-hour light and 12-hour dark cycle, with lights on at 7:00 a.m. and off at 7:00 p.m. every day.

[0029] Preferably: in S2, an experimental treadmill is used for exercise intervention, the adaptation period is 1 week, 3 days a week, 10-20 minutes a day, the slope is 0, and the speed is 13-16 m / min; the formal exercise training lasts for 8 weeks, 5 days a week, 60 minutes a day, the slope is 0, and the speed is 16 m / min in the first to second weeks, and then increases by 1 m / min every two weeks to reach 19 m / min in the seventh to eighth weeks.

[0030] Preferably, in S4, during the blood pressure test, an intelligent non-invasive blood pressure meter - Softron BP-2010 series blood pressure meter is used to perform tail cuff method heart rate and blood pressure tests.

[0031] Preferably: in said S4, when detecting the renal function related indexes:

[0032] Creatinine test: Use a kit to measure the creatinine level in serum;

[0033] Urea nitrogen detection: Use a kit to detect urea nitrogen levels;

[0034] Urine microalbumin test: Use a kit to measure the level of microalbumin in urine.

[0035] Preferably, in said S4, when detecting the ferroptosis-related indicators, it specifically includes:

[0036] Free Fe2+ detection: The free Fe2+ content in rat kidney tissue cells was detected using a kit;

[0037] PTGS2 detection: The expression level of PTGS2 was determined using a kit;

[0038] MDA detection: MDA content was determined by a kit;

[0039] ROS detection: ROS levels were detected using a kit;

[0040] Detection of SLC7A11, GSH, and GPX4 expression levels: Western Blot was used to detect the expression levels of SLC7A11, GSH, and GPX4 in the intracellular pathways of rat kidney tissue;

[0041] Mitochondrial morphology observation: Transmission electron microscopy was used to observe the changes in mitochondrial morphology.

[0042] Preferably: in S5, the data analysis includes:

[0043] Data normality test: SPSS20.0 statistical software package was used to perform statistical analysis and processing on the experimental results. First, the normal distribution test of the data was performed; if the data did not conform to the normal distribution, non-parametric test methods were used for analysis;

[0044] One-way analysis of variance: One-way analysis of variance was used to compare the parameters among the groups.

[0045] Preferably: in S5, the data analysis further comprises:

[0046] Multiple comparison test: LSD test was used to compare the differences between groups;

[0047] Pearson correlation analysis: The correlation analysis between variables was performed using the Pearson correlation analysis method.

[0048] The beneficial effects of the present invention are:

[0049] 1. Theoretical innovation of the present invention: At present, the view that exercise training can treat some diseases by improving ferroptosis has been scientifically proven, but there has been no systematic study on the regulation of iron ion levels in kidney cell damage and whether it has an effect on hypertensive nephropathy. Therefore, this research method verifies the idea that aerobic exercise may inhibit ferroptosis and hinder the further development of chronic kidney disease by activating the system χc- / GPX4 / GSH pathway of hypertensive kidney cells, providing a reference for further in-depth scientific research in this field, and has positive practical value and significance in exploring new pathogenesis and treatment strategies of hypertension.

[0050] 2. The invention in terms of application innovation: Verify and further clarify the application of AMPK activator AICAR for simulating exercise in cell experiments. Its innovation is that it can simulate the regulatory effect of exercise on cellular metabolic pathways without actually exercising. This makes AICAR an ideal tool for studying metabolic regulatory pathways with the help of cell models, especially in areas where actual exercise research is difficult, such as muscle atrophy, obesity, etc. In addition, it provides technical support for research in the fields of drug screening and treatment of related diseases. Through experiments, this research method compares AICAR with aerobic exercise in improving the effect of renal cell ferroptosis, demonstrates that AICAR has a similar effect to exercise training, and promotes AICAR to be widely used in cell experiments to simulate exercise, which will have positive practical value and significance for similar scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is an experimental technical roadmap for a research method proposed in the present invention for the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0053] Embodiment 1:

[0054] A research method for the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats is demonstrated through two parts: aerobic exercise training of rats and cytological experiments, specifically including:

[0055] Aerobic exercise training in rats:

[0056] Wistar Kyoto rats (WKY, n = 6) and 8-week-old male SHR (n = 18) were selected and randomly divided into 5 groups: quiet WKY control group (WKY-S, n = 6), quiet SHR control group (SHR-S, n = 6), exercise group (SHR-E, n = 6), and exercise drug group (SHR-ED, n = 6). All the above rats were placed in separate cages with free access to food and water. They were kept in a separate air-conditioned room (22 ± 2 ℃) with a 12-hour light and 12-hour dark cycle, with lights on at 7:00 am and off at 7:00 pm. Through literature review and combined with the previous exercise program research foundation of the research group, moderate-intensity aerobic exercise was selected as the intervention method of the exercise group. The moderate-intensity aerobic exercise prescription for rats was formulated according to the animal exercise guidelines and cardiovascular research training program and previous research results.

[0057] Moderate aerobic intensity exercise prescription for SHR:

[0058] Exercise time: Adaptation period 1 week: 3 days a week, 10-20 min / d, slope 0, 13-16 m / min; formal exercise training lasts 8 weeks;

[0059] Exercise intensity: moderate intensity (about 55% of maximum oxygen uptake);

[0060] Exercise mode and speed changes: Experimental treadmill was used for treadmill exercise intervention: 5 days a week, 60 minutes / day, slope of 0, speed of 16m / min in the first 2 weeks, speed increased by 1m / min every two weeks, and 19m / min in the 7th to 8th week;

[0061] Supplementary explanation of exercise intervention in the drug group: The SHR rats in the exercise drug group were injected with the ferroptosis inducer Erastin (10 mg / kg / day; MedChem Express) for 20 consecutive days before exercise intervention;

[0062] Note: In order to eliminate the influence of other environmental factors on the results, rats in the WKY-S group were exposed to the same experimental environment during exercise training, but did not perform treadmill exercise. The details are as follows:

[0063]

[0064] Cytological experiments:

[0065] Human renal tubular epithelial cells (HK-2) culture and grouping:

[0066] HK-2 cell line was cultured in DMEM / F12 medium containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2. When the cell confluence reached about 70%, it was synchronized with serum-free medium for 12 hours and then divided into 4 groups for different treatments, namely normal control group (HK-2 group, control group), hypertension group (HK-2+AngⅡ group), exercise mimic intervention hypertension group (HK-2+AngⅡ+AICAR group, 2mM AICAR), and cell transfection and exercise mimic intervention hypertension group (HK-2+AngⅡ+SLC7A11-siRNA+AICAR group, 2mM AICAR). After culturing for 16 hours, protein or RNA was extracted for related detection.

[0067] Treatments were as follows: normal control group (HK-2 group, control group), hypertension group (HK-2+AngⅡ group), exercise mimetic intervention hypertension group (HK-2+AngⅡ+AICAR group, 2mM AICAR), and exercise mimetic intervention hypertension group after cell transfection (HK-2+AngⅡ+SLC7A11-siRNA+AICAR group, 2mM AICAR). After 16 hours of continuous culture, protein or RNA was extracted for related detection.

[0068] Detection indicators

[0069] Basic physiological indicators of rats: body weight, blood pressure, heart rate. Intelligent non-invasive sphygmomanometer-Softron BP-2010 series sphygmomanometer, tail cuff method for heart rate and blood pressure testing, electronic scale to record body weight.

[0070] Renal function-related indicators: Use kits to detect serum creatinine, urea nitrogen, and urine microalbumin.

[0071] Ferroptosis related indicators:

[0072] The kit was used to detect the expression levels of free Fe2+, PTGS2, MDA, and ROS in rat kidney tissue cells;

[0073] The expression levels of SLC7A11, GSH, and GPX4 in the intracellular pathways of rat kidney tissue were detected by western Blot;

[0074] The mitochondrial morphology was observed using transmission electron microscopy.

[0075] Statistical processing

[0076] SPSS 20.0 statistical software package was used to perform statistical analysis and processing of the experimental results. All data were expressed as x±s. One-way analysis of variance was used for comparison of each parameter group, and LSD test was used for multiple comparisons. The significance level was set at α=0.05.

[0077] Experimental technology roadmap, such as Figure 1 As shown;

[0078] Methods of this study:

[0079] In terms of theoretical innovation: At present, the view that exercise training can treat some diseases by improving ferroptosis has been scientifically proven, but there has been no systematic research on the regulation of iron ion levels in kidney cell damage and whether it is effective for hypertensive nephropathy. Therefore, this research method verifies the idea that aerobic exercise may inhibit ferroptosis and hinder the further development of chronic kidney disease by activating the system χc- / GPX4 / GSH pathway of hypertensive kidney cells, providing a reference for further in-depth scientific research in this field, and has positive practical value and significance in exploring new pathogenesis and treatment strategies of hypertension;

[0080] In terms of application innovation: verify and further clarify the application of AMPK activator AICAR in cell experiments to simulate exercise. Its innovation lies in the fact that it can simulate the regulatory effects of exercise on cellular metabolic pathways without actually exercising. This makes AICAR an ideal tool for studying metabolic regulatory pathways with the help of cell models, especially in areas where actual exercise research is difficult, such as muscle atrophy and obesity. In addition, it provides technical support for research in the fields of drug screening and treatment of related diseases. Through experiments, this research method compares AICAR with aerobic exercise in improving the effects of renal cell ferroptosis, demonstrating that AICAR has a similar effect to exercise training, and promoting AICAR to be widely used in cell experiments to simulate exercise, which will have positive practical value and significance for similar scientific research.

[0081] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats, characterized in that: The steps include: S1: Experimental animals and groups; Animal selection and grouping: Normal rats WKY and spontaneously hypertensive rats SHR were randomly divided into: quiet WKY control group, quiet SHR control group, exercise group and exercise drug group; S2: aerobic exercise intervention; Exercise time and intensity: The experimental treadmill was used for exercise intervention, including: Adaptation period: slope is 0, speed is 13-16m / min; Formal exercise training period: slope is 0, speed is 16m / min in the first 2 weeks, then increases by 1m / min every two weeks, and speed is 19m / min in the 7th to 8th week; Among them: the SHR rats in the exercise drug group were injected with the ferroptosis inducer Erastin at 10 mg / kg / day for 20 consecutive days before exercise intervention; S3: cytological experiments; Cell culture: Human renal tubular epithelial cells HK-2 were cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37°C and 5% CO2 in an incubator. When the cell confluence reached about 70%, the cells were synchronized with serum-free medium for 12 hours and then divided into four groups for different treatments: normal control group, hypertension group, exercise mimic intervention hypertension group, and cell transfection and exercise mimic intervention hypertension group. Experimental treatment: After culturing for 16 hours, protein or RNA was extracted for detection; S4: Detection; Basic physiological indicators: testing heart rate and blood pressure with tail cuff method, and electronic scale recording weight; Renal function related indicators: Detection of renal function related indicators in serum; Ferroptosis-related indicators: detection of free Fe in rat kidney tissue cells 2+ , PTGS2, MDA, and ROS expression levels; S5: Data analysis; The experimental results were statistically analyzed and processed; one-way analysis of variance was used for comparison of parameters among groups, and LSD test was used for multiple comparisons.

2. The method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats according to claim 1, characterized in that: In the S1, specifically: S11: Animal selection: Wistar Kyoto rats (WKY) and 8-week-old male spontaneously hypertensive rats (SHR) were selected, a total of 24 rats; S12: Random grouping: 24 rats were randomly divided into four groups, specifically: 6 rats in a quiet WKY control group, 6 rats in a quiet SHR control group, 6 rats in an exercise group, and 6 rats in an exercise drug group.

3. The method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats according to claim 1, characterized in that: The research method also includes environmental control, specifically: All rats will be housed in separate air-conditioned rooms with a constant temperature of 22±2°C; the light cycle in the room is a 12-hour light and 12-hour dark cycle, with lights on at 7:00 a.m. and off at 7:00 p.m. every day.

4. The method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats according to claim 1, characterized in that: In S2, an experimental treadmill is used for exercise intervention, with an adaptation period of 1 week, 3 days a week, 10 to 20 minutes a day, a slope of 0, and a speed of 13 to 16 m / min; the formal exercise training lasts for 8 weeks, 5 days a week, 60 minutes a day, with a slope of 0 and a speed of 16 m / min in the first to second weeks, and then increases by 1 m / min every two weeks to reach 19 m / min in the seventh to eighth weeks.

5. The method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats according to claim 1, characterized in that: In S4, during the blood pressure test, an intelligent non-invasive blood pressure monitor, Softron BP-2010 series blood pressure monitor, was used to perform tail cuff method heart rate and blood pressure tests.

6. The method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats according to claim 5, characterized in that: In said S4, when detecting the renal function related indexes: Creatinine test: Use a kit to measure the creatinine level in serum; Urea nitrogen detection: Use a kit to detect urea nitrogen levels; Urine microalbumin test: Use a kit to measure the level of microalbumin in urine.

7. The method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats according to claim 6, characterized in that: In said S4, when detecting the ferroptosis-related indicators, it specifically includes: Free Fe2+ detection: The free Fe2+ content in rat kidney tissue cells was detected using a kit; PTGS2 detection: The expression level of PTGS2 was determined using a kit; MDA detection: MDA content was determined by a kit; ROS detection: ROS levels were detected using a kit; Detection of SLC7A11, GSH, and GPX4 expression levels: Western Blot was used to detect the expression levels of SLC7A11, GSH, and GPX4 in the intracellular pathways of rat kidney tissue; Mitochondrial morphology observation: Transmission electron microscopy was used to observe the changes in mitochondrial morphology.

8. The method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats according to claim 1, characterized in that: In S5, the data analysis includes: Data normality test: SPSS20.0 statistical software package was used to perform statistical analysis and processing on the experimental results. First, the normal distribution test of the data was performed; if the data did not conform to the normal distribution, non-parametric test methods were used for analysis; One-way analysis of variance: One-way analysis of variance was used to compare the parameters among the groups.

9. The method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats according to claim 8, characterized in that: In S5, the data analysis further includes: Multiple comparison test: LSD test was used to compare the differences between groups.

10. The method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats according to claim 9, characterized in that: In S5, the data analysis further includes: Pearson correlation analysis: The correlation analysis between variables was performed using the Pearson correlation analysis method.

Images