Research method for reducing atherosclerosis risk mechanism

Abstract

The invention discloses a research method for reducing atherosclerosis risk mechanism, and belongs to the technical field of medicine application. Selecting laboratory mice, feeding the laboratory mice in a 12-hour light-dark period environment, replacing the laboratory mice with high-phosphorus feed, continuously feeding the laboratory mice, monitoring the serum phosphorus level, and grouping the mice which reach the standard; respectively preparing experimental drugs for each group of mice through calcium carbonate, calcium acetate, sevelamer and a composite drug, administering the experimental drugs to the mice in the group, and monitoring the body weight and the serum phosphorus level and adjusting the administration dosage during the period; dynamic monitoring is alternately carried out during administration, and the dynamic monitoring comprises metabolism cage monitoring and blood fat and inflammation detection; collecting blood, aorta and kidney samples after administration; and atherosclerotic plaques and kidney tissues are detected. The invention verifies whether the phosphorus-binding agent phosphorus-reducing drug can reduce the risk of atherosclerosis of hyperphosphatemia patients by reducing the serum phosphorus level, improving blood lipid metabolism, reducing inflammatory response, improving vascular functions and the like, and provides a theoretical basis for clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical application technology, specifically relating to a research method for a mechanism to reduce the risk of developing atherosclerosis. Background Technology

[0002] Hyperphosphatemia is a common complication in maintenance hemodialysis patients and is associated with various adverse outcomes, including cardiovascular disease and increased mortality. Currently, the most commonly used phosphorus-lowering drugs in clinical practice are phosphate binders, including calcium-containing phosphate binders, aluminum-containing phosphate binders, non-calcium, non-metallic phosphate binders, lanthanum carbonate non-calcium phosphate binders, sucrose ferric hydroxide, and tenapanol.

[0003] While existing phosphate binders have shown some efficacy in lowering serum phosphorus, they still have some drawbacks and limitations. For example, long-term use of calcium-containing phosphate binders may lead to hypercalcemia and vascular calcification, increasing the risk of cardiovascular events. Long-term use of aluminum-containing phosphate binders may cause aluminum poisoning, leading to adverse reactions such as bone disease, nervous system damage, and anemia. Although non-calcium, non-metallic phosphate binders are relatively safe, they have other side effects; for example, sevelamer carbonate may cause gastrointestinal adverse reactions. Although phosphate-lowering drugs such as lanthanum carbonate are well-tolerated, their long-term effects on atherosclerosis still require further investigation.

[0004] Atherosclerosis is a common complication in maintenance hemodialysis patients and is closely related to hyperphosphatemia. However, current research on whether phosphate-binding drugs can reduce the risk of atherosclerosis in maintenance hemodialysis patients with hyperphosphatemia remains insufficient. Exploring the impact of phosphate-binding drugs on the risk of atherosclerosis can provide stronger theoretical support for clinical treatment, optimize hyperphosphatemia management strategies, reduce the incidence of atherosclerosis, and alleviate the current predicament. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a research method for reducing the risk of developing atherosclerosis.

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

[0007] This invention provides a research method for reducing the risk of atherosclerosis, comprising the following steps:

[0008] S1. Select experimental mice and feed them in a 12-hour light-dark cycle environment for 1 week, then switch to a high-phosphorus diet and continue feeding for 1 week. Then monitor the blood phosphorus level and divide the mice that meet the standard into 4 groups.

[0009] S2. Experimental drugs were prepared for each group of mice using calcium carbonate, calcium acetate, sevelamer, and compound drugs, and administered to mice in the group for 8 weeks. During this period, body weight and blood phosphorus levels were monitored and the dosage was adjusted accordingly.

[0010] S3. Dynamic monitoring was conducted intermittently during drug administration, including metabolic cage monitoring, blood lipid and inflammation detection.

[0011] S4. Collect blood, aortic, and kidney samples after the drug administration is completed;

[0012] S5. Perform atherosclerotic plaque and kidney tissue examination.

[0013] Furthermore, in step S2, there are 4 groups of experimental drugs. The first group is prepared into a 10% suspension using calcium carbonate, and the dosage is 1g / kg / d.

[0014] Group 2 was prepared as a 10% suspension using calcium acetate, and the dosage was 1 g / kg / d.

[0015] Group 3 was prepared as a 10% suspension using sevelamer, and the dosage was 1 g / kg / day.

[0016] Group 4 was prepared by mixing calcium carbonate and calcium acetate at a ratio of 1 g / kg / d to form a suspension.

[0017] Furthermore, in step S2, the method for detecting blood phosphorus levels is as follows: blood is collected from the tail vein, the serum is separated by centrifugation, and stored at -80°C. The serum phosphorus level is detected using a biochemical analyzer, and the phosphorus content in the serum is determined using a phosphorus reagent kit.

[0018] Furthermore, in step S3, when detecting blood lipids, total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol are detected; when detecting inflammation, interleukin, C-reactive protein, and tumor necrosis factor-α are detected.

[0019] Further, in step S5, the detection method for the atherosclerotic plaque is as follows: aortic tissue is collected and fixed in 4% paraformaldehyde, then embedded in OCT embedding medium to prepare frozen sections. The sections are fixed in glacial acetic acid and stained with Oil Red O staining solution at room temperature for 15 minutes. They are then rapidly differentiated with 60% isopropanol until the sections turn pink. The cell nuclei are counterstained with hematoxylin and rinsed with running water for 5 minutes. Finally, the sections are mounted with neutral resin and observed and calculated using an optical microscope.

[0020] Further, in step S5, the kidney tissue is detected by collecting kidney tissue and fixing it in 4% paraformaldehyde, then embedding the kidney tissue in paraffin to make sections, dewaxing the sections in xylene and graded ethanol, staining the cell nuclei with hematoxylin, then differentiating them, then staining the cytoplasm with eosin, then dehydrating them with graded ethanol, mounting them with neutral resin, and then observing and calculating them with an optical microscope.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention verifies whether phosphate-binding drugs can reduce the risk of atherosclerosis in patients with hyperphosphatemia by lowering blood phosphorus levels, improving lipid metabolism, reducing inflammatory response, and improving vascular function, thus providing a theoretical basis for clinical treatment.

[0023] Other advantages, objectives, and features of the invention will be set forth in the following description and will be apparent to those skilled in the art in some respects, or may be learned by practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0024] To make the purpose, technical solution, and beneficial effects of the invention clearer, the following figures are provided for illustration:

[0025] Figure 1 This is a flowchart of the present invention;

[0026] Figure 2 This is a flowchart of the intervention process for grouping according to the present invention;

[0027] Figure 3 This is a flowchart of the termination experiment of the present invention;

[0028] Figure 4 This is a diagram showing the drug validation results of the present invention. Detailed Implementation

[0029] like Figures 1-4 As shown, this invention provides a research method for reducing the risk of developing atherosclerosis.

[0030] Example 1

[0031] S1. Eight-week-old male rats were selected and fed a normal diet for one week. The diet was then replaced with a high-phosphorus diet (phosphorus content 2%) for another week. Starting from the third week, serum phosphorus levels were monitored. Rats with serum phosphorus ≥7.5 mg / dL for two consecutive weeks were selected as experimental rats and divided into four groups.

[0032] S2. Prepare a 10% suspension using calcium carbonate at a dose of 1 g / kg / d; prepare a 10% suspension using calcium acetate at a dose of 1 g / kg / d; prepare a 10% suspension using sevelamer at a dose of 1 g / kg / d; prepare a mixed drug by mixing calcium carbonate and calcium acetate at a dose of 1 g / kg / d; administer the above four drugs to the four groups of mice by gavage every morning from week 4 to week 11. Weigh the mice weekly and adjust the dosage according to their body weight. Then, collect 1 mL of blood from the tail vein, centrifuge at 3000 rpm for 10 min to separate the serum, store at -80℃, and detect the serum phosphorus level using a biochemical analyzer. Prepare the reagent using a phosphorus reagent kit, dilute the serum sample 1:10, add 10 μL of the diluted serum to a reaction vessel, add 200 μL of phosphorus reagent, mix well, and measure the absorbance at a wavelength of 340 nm. Calculate the phosphorus content in the sample according to the standard curve.

[0033] At week S3, week 6, and week 9, rats were placed in metabolic cages 12 hours in advance, and urine and feces were collected within 24 hours. Food intake and water intake were recorded. Urine samples were centrifuged at 3000 rpm for 10 min, and the supernatant was collected and stored at -80℃. Reagents were prepared using a phosphorus reagent kit. 10 μL of urine was added to the reaction vessel, followed by 200 μL of phosphorus reagent. After mixing, the absorbance was measured at a wavelength of 340 nm. The phosphorus content in the sample was calculated based on the standard curve.

[0034] Blood samples were collected in weeks 7 and 10 to measure blood lipids (total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol) and to assess inflammation (interleukin, C-reactive protein, and tumor necrosis factor-α) to evaluate changes in the risk of atherosclerosis.

[0035] S4. In week 12, blood, aorta, kidney and other tissue samples were collected for pathological examination. Aortic oil red O staining was used to assess atherosclerotic plaques, and kidney tissue was examined to assess the effects of the drug on the kidneys.

[0036] S5. Establish a database to record blood phosphorus levels, urinary phosphorus excretion, blood lipid indicators, inflammatory indicators, and histological scores. Perform intergroup comparisons and correlation analysis using statistical methods, and finally visualize the results.

[0037] Example 1: The method for detecting total cholesterol is as follows: blood is collected from the tail vein, the serum is separated by centrifugation and stored at -80℃. After preparing the reagent using the TC kit, 10 μL of serum sample is added to the reaction vessel, and then 200 μL of TC reagent is added. After mixing, the absorbance is measured at a wavelength of 500 nm and the result is calculated.

[0038] The method for detecting triglycerides is as follows: the reagent is prepared using a TG kit. 10 μL of serum is added to a reaction vessel, followed by 200 μL of TG reagent. After mixing, the absorbance is measured at a wavelength of 500 nm. The TG content in the sample is calculated based on the standard curve.

[0039] The method for detecting low-density lipoprotein cholesterol is as follows: prepare the reagent using an LDL-C kit, add 10 μL of serum sample to a reaction vessel, add 200 μL of LDL-C reagent, mix well, and measure the absorbance at a wavelength of 500 nm. Calculate the LDL-C content based on the standard curve.

[0040] The method for detecting high-density lipoprotein cholesterol is as follows: prepare the reagent using an HDL-C kit, add 10 μL of serum to a reaction vessel, add 200 μL of HDL-C reagent, mix well, and measure the absorbance at a wavelength of 500 nm. Calculate the HDL-C content based on the standard curve.

[0041] The method for detecting interleukins is as follows: using an IL-6 ELISA kit, 100 μL of serum sample is added to the reaction plate, along with 100 μL of biotin-labeled antibody. The plate is incubated at 37°C for 2 hours, followed by 100 μL of enzyme-labeled secondary antibody. The plate is then incubated at 37°C for 1 hour, then 100 μL of substrate solution is added. The plate is then incubated at room temperature for 10-15 minutes. Finally, 50 μL of stop solution is added to terminate the reaction, and the results are calculated.

[0042] The detection method for C-reactive protein is as follows: use a CRP kit to prepare the reagent, add 10 μL of serum sample to the reaction vessel, add 200 μL of CRP reagent, mix well, and measure the absorbance at a wavelength of 546 nm. Calculate the CRP content according to the standard curve.

[0043] The detection method for tumor necrosis factor-α is as follows: 100 μL of serum sample is added to the reaction plate, followed by 100 μL of biotin-labeled antibody. The mixture is incubated at 37°C for 2 hours, then 100 μL of enzyme-labeled secondary antibody is added and incubated at 37°C for 1 hour. Next, 100 μL of substrate solution is added and the mixture is incubated at room temperature for 10-15 minutes. Finally, 50 μL of stop solution is added to terminate the reaction, and the TNF-α content is calculated based on the standard curve.

[0044] The detection method for atherosclerotic plaques is as follows: aortic tissue is collected and fixed in 4% paraformaldehyde, then embedded in OCT embedding medium to prepare frozen sections. The sections are fixed in glacial acetic acid and stained with Oil Red O staining solution at room temperature for 15 minutes. They are then rapidly differentiated with 60% isopropanol until the sections turn pink. The cell nuclei are counterstained with hematoxylin and rinsed with running water for 5 minutes. Finally, the sections are mounted with neutral resin and observed and calculated using an optical microscope.

[0045] The kidney tissue was collected and fixed in 4% paraformaldehyde. The kidney tissue was then embedded in paraffin to prepare sections. The sections were dewaxed in xylene and graded ethanol, and the cell nuclei were stained with hematoxylin. The sections were then differentiated, and the cytoplasm was stained with eosin. The sections were then dehydrated with graded ethanol, mounted with neutral resin, and observed and calculated using an optical microscope.

[0046] In conclusion, the results for each indicator of the four groups of drugs in this invention are shown in Table 1:

[0047] Table 1

[0048]

[0049]

[0050] The pathological changes of the four groups of drugs in this invention are shown in Table 2:

[0051] Table 2

[0052] Pathological changes calcium carbonate group calcium acetate group Sveram Group Compound calcium group Renal tubular calcification (0-3) 2.1±0.4 1.9±0.3 0.7±0.2* 1.2±0.3# Interstitial fibrosis (0-3) 1.8±0.3 1.6±0.3 0.5±0.1* 0.9±0.2# Inflammatory cell infiltration (0-3) 2.0±0.5 1.7±0.4 0.6±0.2* 1.1±0.3#

[0053] like Figure 4 As shown, the verification results of this invention have revealed that phosphorus-lowering drugs of the phosphorus binding class can significantly reduce blood phosphorus levels.

[0054] Phosphate-binding drugs can improve lipid metabolism, reduce TC, TG, and LDL-C levels, and increase HDL-C levels.

[0055] Phosphate-binding drugs can reduce inflammatory responses and lower levels of IL-6, CRP, and TNF-α.

[0056] Phosphate-binding drugs can improve vascular function, increase aortic elasticity, and improve endothelial-dependent vasodilation.

[0057] Phosphate-binding drugs can reduce the formation of atherosclerotic plaques in the aorta.

[0058] This invention verifies that phosphate-binding drugs can reduce the risk of atherosclerosis in patients with maintenance hemodialysis and hyperphosphatemia by lowering blood phosphorus levels, improving lipid metabolism, reducing inflammatory response, and improving vascular function, thus providing a theoretical basis for clinical treatment.

[0059] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A research method for understanding the mechanisms by which the risk of developing atherosclerosis is reduced, characterized by: Includes the following steps, S1. Select experimental mice and feed them in a 12-hour light-dark cycle environment for 1 week, then switch to a high-phosphorus diet for another week, monitor blood phosphorus levels, and divide the mice that meet the standards into 4 groups. S2. Experimental drugs were prepared for each group of mice using calcium carbonate, calcium acetate, sevelamer, and compound drugs, and administered to mice in the group for 8 weeks. During this period, body weight and blood phosphorus levels were monitored and the dosage was adjusted accordingly. S3. Dynamic monitoring was conducted intermittently during drug administration, including metabolic cage monitoring, blood lipid and inflammation detection. S4. Collect blood, aortic, and kidney samples after the drug administration is completed; S5. Perform atherosclerotic plaque and kidney tissue examination.

2. The research method for reducing the risk of atherosclerosis according to claim 1, characterized in that: In step S2, there are 4 groups of experimental drugs. The first group is prepared into a 10% suspension using calcium carbonate, and the dosage is 1g / kg / d. The second group is prepared into a 10% suspension using calcium acetate, and the dosage is 1g / kg / d. Group 3 was prepared as a 10% suspension using sevelamer, and the dosage was 1 g / kg / day. Group 4 was prepared by mixing calcium carbonate and calcium acetate at a ratio of 1 g / kg / d to form a suspension.

3. The research method for reducing the risk of atherosclerosis according to claim 2, characterized in that: In step S2, the method for detecting blood phosphorus levels is as follows: blood is collected from the tail vein, the serum is separated by centrifugation, and stored at -80°C. The serum phosphorus level is detected by a biochemical analyzer and the phosphorus content in the serum is determined by a phosphorus reagent kit.

4. The research method for reducing the risk of atherosclerosis according to claim 1, characterized in that: In step S3, when detecting blood lipids, total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol are detected; when detecting inflammation, interleukin, C-reactive protein, and tumor necrosis factor-α are detected.

5. A research method for reducing the risk of atherosclerosis according to claim 1: In step S5, the atherosclerotic plaque is detected by collecting aortic tissue and fixing it in 4% paraformaldehyde, then embedding it in OCT embedding medium to prepare frozen sections, fixing the sections in glacial acetic acid, adding Oil Red O staining solution and staining at room temperature for 15 minutes, rapidly differentiating with 60% isopropanol until the sections are pink, counterstaining the cell nuclei with hematoxylin and rinsing with running water for 5 minutes, finally mounting with neutral resin, and observing and calculating using an optical microscope.

6. A research method for reducing the risk of atherosclerosis according to claim 5: In step S5, the kidney tissue is detected by collecting kidney tissue, fixing it in 4% paraformaldehyde, embedding the kidney tissue in paraffin to make sections, dewaxing the sections in xylene and graded ethanol, staining the cell nuclei with hematoxylin, differentiating them, staining the cytoplasm with eosin, dehydrating them with graded ethanol, mounting them with neutral resin, and observing and calculating them with an optical microscope.

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