Research method of influence of aerobic exercise on ferroptosis of spontaneously hypertensive rats

Abstract

The application discloses a research method for the influence of aerobic exercise on ferroptosis of spontaneously hypertensive rats, and relates to the technical field of medical research; and comprises the following steps: S1: experimental animals and grouping; animal selection 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: aerobic exercise intervention; the research method verifies the assumption that aerobic exercise may inhibit ferroptosis and hinder the further development of chronic kidney disease by activating the systemχc-GPX4-GSH pathway of hypertensive kidney cells, provides a reference for promoting further in-depth scientific research of subsequent follow-up of the field subject, and has a positive practical value and significance in exploring new pathogenesis and treatment strategies of hypertension.

Description

Technical Field

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

[0002] Objective: This study found that exercise training can alter the expression levels of ferroptosis genes in diseases such as cerebral hemorrhage in rats, myocardial injury in mice on a high-fat diet, brain I / R injury, pulmonary fibrosis tissue, and skeletal muscle disorder in mice. However, it remains unclear whether exercise training can act on ferroptosis in hypertensive renal cells; the mechanism by which exercise training alleviates spontaneously hypertensive chronic kidney disease, especially in the regulation of iron ion levels in renal cell damage, has not yet been systematically studied.

[0003] Hypertension is a recognized factor in kidney damage and the progression of chronic kidney disease (CKD), therefore CKD patients have a high cardiovascular risk, and better blood pressure control is crucial. A meta-analysis observed that aerobic exercise could better control SBP levels in the short and medium term, although the difference was not statistically significant. Studies by Cao Shuyuan et al. have shown that regular aerobic exercise can reduce kidney damage in spontaneously hypertensive rats, and that moderate-intensity aerobic exercise had a more significant effect on reducing kidney damage than low-intensity aerobic exercise.

[0004] While the above studies have yielded relevant conclusions, they have not conducted a systematic study on the regulation of iron ion levels in renal cell damage. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a research method for studying the effects of aerobic exercise on ferroptosis in spontaneously hypertensive rats.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0008] S1: Laboratory animals and grouping;

[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 duration and intensity: Exercise intervention was conducted using an experimental treadmill, including:

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

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

[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 underwent exercise intervention;

[0015] S3: Cellular experiments;

[0016] Cell culture: Human renal tubular epithelial cells (HK-2) were 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%, they were synchronized with serum-free medium for 12 hours and then divided into four groups for different treatments: normal control group, hypertension group, hypertension group with exercise mimic intervention, and hypertension group with exercise mimic intervention after cell transfection.

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

[0018] S4: Detection;

[0019] Basic physiological indicators: heart rate and blood pressure were measured using the tail-cloak method, and weight was recorded using an electronic scale;

[0020] Kidney function related indicators: Detection of kidney function related indicators in serum;

[0021] Ferric death-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 ANOVA was used for comparisons between parameter groups, and LSD test was used for multiple comparisons.

[0024] Preferably, in S1, specifically:

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

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

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

[0028] All rats will be placed in separate air-conditioned rooms with a constant temperature of 22±2℃. The room will have a light cycle of 12 hours of light and 12 hours of darkness, with the lights turned on at 7:00 am and turned off at 7:00 pm 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, with an incline of 0 and a speed of 13-16 m / min. The formal exercise training lasts for 8 weeks, 5 days a week, 60 minutes a day, with an incline of 0 and a speed of 16 m / min in the first and second weeks, increasing by 1 m / min every two weeks thereafter, reaching 19 m / min in the 7th and 8th weeks.

[0030] Preferably, in step S4, during the blood pressure test, a smart non-invasive blood pressure monitor - Softron BP-2010 series blood pressure monitor - is used to perform heart rate and blood pressure tests using the tail-cuff method.

[0031] Preferably, in step S4, when detecting kidney function-related indicators:

[0032] Creatinine detection: The level of creatinine in serum is measured using a kit;

[0033] Urea nitrogen testing: Urea nitrogen levels are measured using a kit;

[0034] Urinary microalbumin test: The level of microalbumin in urine is measured using a kit.

[0035] Preferably, in step S4, the detection of ferroptosis-related indicators specifically includes:

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

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

[0038] MDA assay: The MDA content is determined using a kit;

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

[0040] SLC7A11, GSH, and GPX4 expression levels were detected using Western blotting in rat kidney tissue cells.

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

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

[0043] Normality test of data: The experimental results were statistically analyzed and processed using SPSS 20.0 statistical software. First, the normality of the data was tested. If the data did not conform to a normal distribution, non-parametric test methods were used for analysis.

[0044] One-way ANOVA: One-way ANOVA is used to compare parameters among groups.

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

[0046] Multiple comparison tests: LSD test was used for pairwise 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 this invention are as follows:

[0049] 1. In terms of theoretical innovation: While the idea that exercise training can treat some diseases by improving ferroptosis has been scientifically proven, the regulation of iron ion levels in renal cell damage and its efficacy in treating hypertensive nephropathy have not been systematically studied. Therefore, this study verifies the hypothesis that aerobic exercise may inhibit ferroptosis and hinder the further development of chronic kidney disease by activating the systemχc- / GPX4 / GSH pathway in hypertensive renal cells. This provides 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 for hypertension.

[0050] 2. In terms of application innovation, this invention verifies and further clarifies the application of the AMPK activator AICAR in cellular experiments to simulate exercise. Its innovation lies in its ability to simulate the regulatory effects of exercise on cellular metabolic pathways without requiring actual exercise. This makes AICAR an ideal tool for studying metabolic regulatory pathways using cell models, especially in areas where actual exercise studies are difficult, such as muscle atrophy and obesity. Furthermore, it provides technical support for research in areas such as drug screening and treatment of related diseases. This research method compares the effects of AICAR and aerobic exercise on improving ferroptosis in renal cells through experiments, demonstrating that AICAR has a similar effect to exercise training. Promoting the widespread application of AICAR in cellular experiments to simulate exercise will have positive practical value and significance for similar scientific research. Attached Figure Description

[0051] Figure 1 This is an experimental technical roadmap for a method proposed in this invention to study the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats. Detailed Implementation

[0052] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0053] Example 1:

[0054] A research method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats was demonstrated through two parts: aerobic exercise training in rats and cellular experiments. Specifically, the method included:

[0055] Aerobic exercise training for rats:

[0056] Wistar Kyoto rats (WKY, n=6) and 8-week-old male SHRs (n=18) were randomly divided into 5 groups: a quiet WKY control group (WKY-S, n=6), a quiet SHR control group (SHR-S, n=6), an exercise group (SHR-E, n=6), and an exercise-medication group (SHR-ED, n=6). All rats were housed in designated cages with free access to food and water. They were kept in a separate air-conditioned room (22±2℃) with a 12-hour light-12-hour darkness cycle, with lights on at 7:00 AM and off at 7:00 PM. Based on literature review and previous research on exercise programs, moderate-intensity aerobic exercise was selected as the intervention method for the exercise group. A moderate-intensity aerobic exercise prescription for rats was developed according to animal exercise guidelines, cardiovascular research training protocols, and previous research results.

[0057] SHR Moderate Aerobic Intensity Exercise Prescription:

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

[0059] Exercise intensity: Moderate intensity (approximately 55% of maximum oxygen uptake);

[0060] Exercise mode and speed changes: An experimental treadmill was used for treadmill exercise intervention: 5 days a week, 60 min / day, with an incline of 0. The speed was 16 m / min in the first 2 weeks, and increased by 1 m / min every two weeks, reaching 19 m / min in the 7th and 8th weeks.

[0061] Supplementary notes on exercise intervention for drug groups: 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] Precautions: To eliminate the influence of other environmental factors on the results, the WKY-S group rats were exposed to the same experimental environment during exercise training, but did not undergo treadmill exercise. Specific details are as follows:

[0063]

[0064] Cellular experiments:

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

[0066] HK-2 cell lines were cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37°C and 5% CO2. When cell confluence reached approximately 70%, the cells were synchronized with serum-free medium for 12 hours, and then divided into four groups for different treatments: normal control group (HK-2 group, control group), hypertension group (HK-2+Ang II group), hypertension group with exercise mimic intervention (HK-2+Ang II+AICAR group, 2mM AICAR), and hypertension group with exercise mimic intervention after cell transfection (HK-2+Ang II+SLC7A11-siRNA+AICAR group, 2mM AICAR). After culturing for another 16 hours, proteins or RNA were extracted for relevant detection.

[0067] The cells were treated as follows: a normal control group (HK-2 group, control group), a hypertension group (HK-2+Ang II group), a hypertension group treated with exercise mimicry (HK-2+Ang II+AICAR group, 2mM AICAR), and a hypertension group treated with exercise mimicry after cell transfection (HK-2+Ang II+SLC7A11-siRNA+AICAR group, 2mM AICAR). After culturing for another 16 hours, proteins or RNA were extracted for relevant detection.

[0068] detection indicators

[0069] Basic physiological indicators of rats: body weight, blood pressure, and heart rate. The Softron BP-2010 series smart non-invasive blood pressure monitor uses the tail-cuff method for heart rate and blood pressure testing, and an electronic scale for weight recording.

[0070] Kidney function related indicators: Serum creatinine, blood urea nitrogen, and urinary microalbumin were detected using a kit.

[0071] Ferric mortality related indicators:

[0072] The expression levels of free Fe2+, PTGS2, MDA, and ROS in rat kidney tissue cells were detected using a kit.

[0073] The expression levels of SLC7A11, GSH, and GPX4 in rat kidney tissue cells were detected by Western blotting.

[0074] Mitochondrial morphology was observed using transmission electron microscopy.

[0075] Statistical processing

[0076] The experimental results were statistically analyzed and processed using SPSS 20.0 statistical software. All data are expressed as mean ± standard deviation (x±s). One-way ANOVA was used for comparisons between parameter groups, and LSD test was used for multiple comparisons. The significance level was set at α = 0.05.

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

[0078] Methodology used in this study:

[0079] In terms of theoretical innovation: The idea that exercise training can treat some diseases by improving ferroptosis has been scientifically proven. However, the regulation of iron ion levels in renal cell damage and its efficacy in treating hypertensive nephropathy have not been systematically studied. Therefore, this study validates the hypothesis that aerobic exercise may inhibit ferroptosis and hinder the further development of chronic kidney disease by activating the systemχc- / GPX4 / GSH pathway in hypertensive renal cells. This provides 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 for hypertension.

[0080] In terms of application innovation: This study validates and further clarifies the application of the AMPK activator AICAR in cellular experiments to simulate exercise. Its innovation lies in its ability to simulate the regulatory effects of exercise on cellular metabolic pathways without requiring actual exercise. This makes AICAR an ideal tool for studying metabolic regulatory pathways using cell models, especially in areas where actual exercise studies are difficult, such as muscle atrophy and obesity. Furthermore, it provides technical support for research in areas such as drug screening and treatment of related diseases. This research method compares the effects of AICAR and aerobic exercise on improving ferroptosis in renal cells through experiments, demonstrating that AICAR has a similar effect to exercise training. Promoting the widespread application of AICAR in cellular experiments to simulate exercise will have positive practical value and significance for similar scientific research.

[0081] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats, characterized in that, Includes the following steps: S1: Laboratory animals and grouping; Animal selection and grouping: Normal WKY rats and spontaneously hypertensive SHR rats were randomly divided into: quiet WKY control group, quiet SHR control group, exercise group and exercise drug group; S2: Aerobic exercise intervention; Exercise duration and intensity: Exercise intervention was conducted using an experimental treadmill, including: Adaptation period: slope is 0, speed is 13-16 m / min; Formal training period: The slope is 0, the speed is 16m / min in the first 1-2 weeks, and then increases by 1m / min every two weeks thereafter, with the speed reaching 19m / min in the 7th-8th weeks; Among them, the SHR rats in the exercise drug group were given Erastin, a ferroptosis inducer, at 10 mg / kg / day for 20 consecutive days, followed by exercise intervention. S3: Cellular experiments; Cell culture: Human renal tubular epithelial cells HK-2 were 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%, they were synchronized with serum-free medium for 12 hours and then divided into four groups for different treatments: normal control group, hypertension group, hypertension group with exercise mimic intervention, and hypertension group with exercise mimic intervention after cell transfection. The normal control group was: HK-2 group; The hypertension group was the HK-2+Ang II group; The hypertension intervention group using exercise analogues was the HK-2+Ang II+AICAR group; The hypertension group treated with exercise mimics after cell transfection was the HK-2+AngⅡ+SLC7A11-siRNA+AICAR group; Experimental treatment: After culturing for another 16 hours, protein or RNA was extracted for detection; S4: Detection; Basic physiological indicators: heart rate and blood pressure were measured using the tail-cloak method, and weight was recorded using an electronic scale; Kidney function related indicators: Detection of kidney function related indicators in serum; Ferric death-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 ANOVA was used for comparisons between parameter groups, and LSD test was used for multiple comparisons. In step S4, when detecting kidney function-related indicators: Creatinine detection: The level of creatinine in serum is measured using a kit; Urea nitrogen testing: Urea nitrogen levels are measured using a kit; Urinary microalbumin detection: The level of microalbumin in urine is measured using a kit; In S4, the detection of ferroptosis-related indicators specifically includes: Free Fe 2+ Detection: Free Fe in rat kidney tissue cells was detected using a kit. 2+ The content; PTGS2 assay: The expression level of PTGS2 was determined using a kit; MDA assay: The MDA content is determined using a kit; ROS detection: ROS levels are detected using a kit; SLC7A11, GSH, and GPX4 expression levels were detected using Western blotting in rat kidney tissue cells. Mitochondrial morphology observation: Transmission electron microscopy was used to observe changes in mitochondrial morphology.

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

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 methodology also includes environmental control, specifically: All rats will be placed in separate air-conditioned rooms with a constant temperature of 22±2℃. The room will have a light cycle of 12 hours of light and 12 hours of darkness, with the lights turned on at 7:00 am and turned off at 7:00 pm 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 was used for exercise intervention. The adaptation period was 1 week, 3 days a week, 10 to 20 minutes a day, with an incline of 0 and a speed of 13 to 16 m / min. The formal exercise training lasted for 8 weeks, 5 days a week, 60 minutes a day, with an incline of 0 and a speed of 16 m / min in the first and second weeks, increasing by 1 m / min every two weeks thereafter, reaching 19 m / min in the 7th and 8th weeks.

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

6. The method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats according to claim 1, characterized in that, In S5, data analysis includes: Normality test of data: The experimental results were statistically analyzed and processed using SPSS 20.0 statistical software package. First, the normality of the data was tested; if the data did not conform to a normal distribution, nonparametric test methods were used for analysis. One-way ANOVA: One-way ANOVA is used to compare parameters among groups.

7. A method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats according to claim 6, characterized in that, In S5, data analysis also includes: Multiple comparison tests: The LSD test is used to compare the differences between groups.

8. A method for studying the effect of aerobic exercise on ferroptosis in spontaneously hypertensive rats according to claim 7, characterized in that, In S5, data analysis also includes: Pearson correlation analysis: The correlation analysis between variables was performed using the Pearson correlation analysis method.

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