Application of EPA (eicosapentaenoic acid) plasmalogen in preparation of product for improving or preventing diabetic nephropathy

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of EPA (eicosapentaenoic acid) plasmalogen in preparation of medicines for improving or preventing diabetic nephropathy, which is characterized by treating diabetic nephropathy mouse models induced by high fat diet and streptozotocin and having insulin resistance and hyperglycemia accompanied by kidney lipid accumulation and glomerular filtration rate reduction, and particularly relates to application of EPA plasmalogen in preparation of medicines for improving or preventing diabetic nephropathy. The treatment effect of the EPA plasmalogen on the diabetic nephropathy is observed; results show that the EPA plasmalogen has obvious effects of improving kidney functions and kidney ectopic lipid deposition on diabetic nephropathy mice induced by high fat diet and streptozotocin, and also has the effects of reducing blood sugar, improving blood fat, reducing insulin resistance and the like. The EPA plasmalogen is used for intervening the diabetic nephropathy for the first time, and an important basis is provided for clinically treating the diabetic nephropathy by using the EPA plasmalogen.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of EPA phospholipids in the preparation of products that improve or prevent diabetic nephropathy. Background Technology

[0002] Diabetic nephropathy (DKD) is a chronic kidney disease caused by diabetes mellitus. Its pathogenesis is complex, primarily manifesting in the early stages as renal lipid deposition, persistently increased albuminuria, and / or a progressive decline in glomerular filtration rate (GFR), ultimately progressing to end-stage renal disease. The pathogenesis of DKD involves pathophysiological processes such as microvascular dysfunction, excessive activation of the renin-angiotensin-aldosterone system (RAAS), destruction of podocyte foot processes, oxidative stress, mitochondrial dysfunction, inflammatory responses, and autophagy. Hyperglycemia, insulin resistance, and metabolic disorders can ultimately lead to kidney damage through various mechanisms. According to the Clinical Practice Guidelines for the Assessment and Management of Chronic Kidney Disease published by Kidney Disease: Improving Global Outcomes (KDIGO), patients with diabetic nephropathy have a poor prognosis and currently lack effective treatments.

[0003] EPA plasmalogen is a unique type of phospholipid with a long-chain enol at the sn-1 position of its glycerol backbone, containing an enyl ether bond. It is widely found in mammalian tissues, accounting for 15-20% of cell membrane phospholipids. Its sn-2 position fatty acid is predominantly EPA (>80%), making it a good source of plasmalogen-type EPA. Animal studies have also demonstrated its oral safety. Previous studies have reported that EPA plasmalogen can effectively prevent, improve, or treat atherosclerosis and effectively reduce the area of ​​atherosclerotic plaques (Chinese Patent CN201810105098.5). Research and drug development surrounding EPA plasmalogen is currently at the forefront of research both domestically and internationally. A series of studies have shown the broad prospects of EPA plasmalogen as a drug target in clinical drug development; however, its application in improving and treating diabetic nephropathy is rarely reported. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide the application of EPA phospholipids in the preparation of products that improve or prevent diabetic nephropathy.

[0005] To achieve the above objectives, this invention provides the application of EPA phospholipids in the preparation of products that improve or prevent diabetic nephropathy. A mouse model of diabetic nephropathy induced by streptozotocin (STZ) combined with a high-fat diet, characterized by insulin resistance and hyperglycemia, accompanied by reduced and fused podocyte foot processes, podocyte lipid deposition, and / or an increased urinary albumin / creatinine ratio, was used to observe the effect of EPA phospholipids on diabetic nephropathy. The results showed that EPA phospholipids can improve renal function and ectopic lipid deposition in diabetic nephropathy mice, and can also lower blood glucose, improve blood lipids, and reduce insulin resistance.

[0006] The products described in this invention include pharmaceuticals, food, and health products.

[0007] Furthermore, the drug dosage form is: granules, capsules, tablets, oral liquid, suspension or emulsion.

[0008] The drug is a drug or drug composition containing EPA phospholipids, and the drug or drug composition is administered orally.

[0009] The drug includes pharmaceutically acceptable excipients.

[0010] The food and health products mentioned refer to those containing EPA phospholipids.

[0011] The application described in this invention includes the prevention of diabetic nephropathy in diabetic patients by taking EPA phospholipids.

[0012] The preparation method of EPA phospholipids of the present invention is as follows: take Icelandic sea cucumber powder, extract with ethanol overnight, filter, and concentrate under reduced pressure to obtain crude lipid extract; after activating silica gel and packing it into a column, equilibrate the silica gel column with chloroform, dissolve the obtained crude lipid extract in chloroform and pour it into the silica gel column, and elute successively with chloroform, chloroform-methanol 9:1, and chloroform-methanol 2:1, collect the chloroform-methanol 2:1 eluent, and concentrate under reduced pressure to obtain EPA phospholipids.

[0013] Compared with the prior art, this invention is the first to use EPA phospholipids to intervene in diabetic nephropathy. EPA phospholipids can improve the morphology of glomerular podocyte foot processes, renal lipid deposition and glomerular filtration rate in diabetic nephropathy mice, restore renal function, reduce blood glucose, improve blood lipids and reduce insulin resistance in mice, providing important evidence for the clinical use of EPA phospholipids to improve diabetic nephropathy. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the body weight and oral glucose tolerance test (OGTT) results of the model group mice at week 4 of the present invention, where A is body weight and B is blood glucose concentration.

[0015] Figure 2 This is a schematic diagram of the experimental results of the effect of EPA phospholipids on body weight and kidney organ coefficient in diabetic nephropathy mice, where A is body weight and B is the ratio of kidney weight to body weight.

[0016] Figure 3 This is a schematic diagram of the experimental results of the effects of EPA phospholipids on blood biochemistry, blood glucose, insulin and other indicators in diabetic nephropathy mice, as involved in this invention. In the diagram, A is the blood glucose concentration in the OGTT test after 20 weeks of intervention, B is the area under the curve of oral glucose tolerance test, C is the fasting blood glucose level, D is the insulin level (FINS), E is the insulin resistance index (HOMA-IR), F is the serum triglyceride (TG) and G is the serum total cholesterol (TC).

[0017] Figure 4 This is a schematic diagram of the experimental results of the effect of EPA acetal phospholipids on renal function in diabetic nephropathy mice, where A is a transmission electron microscope scan of the foot processes of renal podocytes, B is urinary creatinine, C is urinary albumin, D is UACR, E is renal TC, and F is renal TG.

[0018] Figure 5 This is a schematic diagram of the experimental results of the effect of EPA acetal phospholipids on the kidney tissue pathology of diabetic nephropathy mice, as involved in this invention. In the diagram, A is HE staining and B is BODIPY lipid droplet immunofluorescence staining. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0020] Example 1:

[0021] This embodiment relates to a method for preparing EPA phospholipids, specifically as follows: 2.5 kg of dried Icelandic sea cucumber powder was extracted with 50 L of ethanol by stirring, allowed to stand overnight, filtered, and the filtrate was collected and concentrated under reduced pressure to obtain a crude lipid extract; after activating and packing a silica gel column, the silica gel column was equilibrated with chloroform, and the crude lipid extract obtained above was dissolved in chloroform and poured into the silica gel column, and eluted sequentially with chloroform, chloroform-methanol 9:1, and chloroform-methanol 2:1, and the chloroform-methanol 2:1 eluent was collected and concentrated under reduced pressure to obtain EPA phospholipids.

[0022] Example 2:

[0023] This embodiment relates to an experiment on the effects of EPA phosphatidylcholine on streptozotocin (STZ) combined with a high-fat diet in mice with diabetic nephropathy. To verify the effect of EPA phosphatidylcholine on diabetic nephropathy, a diabetic nephropathy mouse model was established using STZ combined with a high-fat diet. The changes in kidney function and tissue structure in mice after intervention with EPA phosphatidylcholine were observed. The specific experimental steps are as follows:

[0024] 1. Establishment and grouping of animal models of diabetic nephropathy

[0025] This embodiment used 50 male C57BL / 6 mice (20±2g) of 8 weeks old SPF grade, purchased from Beijing Vital River Pharmaceuticals, for testing. All animal experiments were conducted in accordance with the "Guidelines of the Animal Experiment Ethics Review Committee of Qingdao University" (Approval No.: 2025-299), and every effort was made to minimize animal suffering. Mice were allowed free access to food and water for one week at a temperature of (24±2)℃ and humidity of (50±5)%. Ten mice were randomly selected as the normal control group (ND group) and fed a normal diet (D12450J) with 10% fat energy ratio; the remaining 40 mice were used as the model group and fed a high-fat diet (D12492) with 60% fat energy ratio. After 4 weeks, body weight was measured, and the body weight of the model group mice was significantly higher than that of the ND group mice. Figure 1 A) A mouse obesity model was successfully established. Subsequently, mice in the model group were intraperitoneally injected with STZ (60 mg / kg) for three days to establish a diabetic mouse model. Both groups of mice underwent an oral glucose tolerance test (OGTT). A blood glucose level greater than 11.1 mmol / L was considered a successful model, and mice that did not successfully establish the model were discarded. The specific OGTT procedure and results are as follows:

[0026] Before fasting blood glucose measurement, mice were fasted overnight for 12 hours. Then, each mouse was administered 50% glucose solution (1 g / kg body weight) by gavage. At 0 min, 30 min, 60 min, 90 min, and 120 min, the tail vein blood glucose concentration was measured using a Roche glucometer, and the area under the curve (AUC) was calculated according to the following formula. Area under the curve (AUC) (mmol·h⁻¹) -1 ·L -1 = 0 h blood glucose level / 2 + 0.5 h blood glucose level + 1 h blood glucose level + 1.5 h blood glucose level + 2 h blood glucose level / 2. The result is as follows: Figure 1 As shown in B. From Figure 1 As shown in B, compared with the ND group, the area under the OGTT curve in the model group was significantly increased, and the difference between the two groups was statistically significant (P < 0.05), indicating that there were abnormalities in insulin secretion and glucose metabolism in the model group. The fasting blood glucose in the model group was greater than 11.1 mmol / L, and the modeling was successful.

[0027] The successfully constructed diabetic mice were further divided into four groups using a random number table: a diabetic nephropathy mouse model group (MOD group), an EPA ethyl ester group (EPA group), an EPA phosphatidylcholine group (EPA-PlsEtn group), and a positive control metformin group (MET group), with 10 mice in each group. The MOD, EPA, EPA-PlsEtn, and MET groups continued to be fed a high-fat diet (D12492), with each group of mice receiving 5 g / day of feed. The EPA group was administered EPA ethyl ester (200 mg / kg body weight, with corn oil as the solvent) by gavage daily. The EPA phosphatidylcholine group was administered EPA phosphatidylcholine (200 mg / kg body weight, with corn oil as the solvent) by gavage daily. Meanwhile, the MET group mice were administered metformin (150 mg / kg body weight) by gavage daily, while the ND, MOD, and MET groups mice were administered the same volume of corn oil by gavage daily until the end of the experiment at week 20.

[0028] 2. Effects of EPA phospholipids on body weight and cardiac organ coefficient in diabetic nephropathy mice.

[0029] The body weight of mice in the ND group, MOD group, EPA group, EPA-PlsEtn group, and MET group was measured weekly, and the results were as follows: Figure 2 As shown in Figure A. Mice in each group (ND, MOD, EPA, EPA-PlsEtn, and MET) were sacrificed at week 20, and their kidneys were weighed. The kidney weight / body weight ratio for each mouse was calculated. The results are shown in Figure A. Figure 2 As shown in B.

[0030] from Figure 2 As shown in Figure A, at week 4, there was no significant difference in baseline body weight among the MOD, EPA, EPA-PlsEtn, and MET groups. However, compared with the ND group, the body weight of these four groups of mice increased significantly during the experiment (P<0.01). After week 20, the body weight of mice in the EPA-PlsEtn and MET groups was significantly lower than that in the MOD group (P<0.01), indicating that EPA phosphatidylcholine effectively reduced the body weight of mice with diabetic nephropathy, meaning that EPA phosphatidylcholine can be used to improve diabetic nephropathy.

[0031] from Figure 2B shows that the kidney weight / body weight ratio in the MOD group was significantly higher than that in the ND group (P < 0.05), indicating that the diabetic nephropathy model was successfully established. The kidney organ coefficients in the EPA group, EPA-PlsEtn group, and MET group were all significantly lower than those in the MOD group (P < 0.01), indicating that EPA-PlsEtn intervention can reduce the kidney weight / body weight ratio in diabetic nephropathy mice. The kidney weight / body weight ratio is associated with pathological glomerular hypertrophy and glomerular volume increase in DKD. Therefore, EPA phospholipids can be used to improve pathological kidney damage in diabetic nephropathy mice.

[0032] 3. Detection of the effects of EPA phospholipids on blood biochemistry, blood glucose, insulin levels, and other indicators in DKD mice.

[0033] Before euthanizing mice at week 20, all mice in each group were fasted overnight for 12 hours. Blood was then collected from the tail vein using a Roche blood glucose meter for an oral glucose tolerance test (OGTT), and the area under the curve (AUC) was calculated using a formula. Three days later, the mice were fasted again and blood was collected from their eyes. Fresh samples were centrifuged at 3000 rpm for 10 minutes in a 4°C centrifuge. The supernatant was collected, stored at 80°C, and the levels of total cholesterol (TC), triglycerides (TG), fasting blood glucose (FBG), and fasting insulin (FINS) were measured. The homeostasis model assessment of insulin resistance (HOMA-IR) was also calculated. The results are as follows: Figure 3 As shown.

[0034] Figure 3 AC shows the results of the OGTT experiment at week 20 for each group of mice. From Figure 3 AC results showed that, compared with the ND group, the MOD group had significantly higher OGTT area under the curve and fasting blood glucose (P < 0.01), indicating that the MOD group mice had abnormal glucose tolerance throughout the experimental period. Compared with the MOD group, the EPA group had a smaller decrease in OGTT area under the curve and a significant decrease in fasting blood glucose, indicating that EPA ethyl ester had a smaller effect on blood glucose in diabetic nephropathy mice. Compared with the MOD group, the EPA phosphatidylcholine group had significantly lower OGTT area under the curve and fasting blood glucose (P < 0.01), indicating that compared with EPA ethyl ester, EPA phosphatidylcholine can increase insulin sensitivity and improve abnormal glucose tolerance.

[0035] Figure 3 D-3E shows the experimental results of fasting insulin (FINS) and insulin resistance index (HOMA-IR) in mice of each group. From Figure 3As shown in D-3E, compared with the MOD group, the fasting insulin (FINS) and insulin resistance index (HOMA-IR) of mice in the EPA group did not change significantly (P > 0.05); the FINS and HOMA-IR indices of mice in the EPA-PlsEtn group were significantly reduced, indicating that compared with EPA ethyl ester, EPA phosphatidylcholine can significantly reduce the insulin resistance index of DKD mice. The increase in insulin resistance index is closely related to the occurrence and development of diabetic nephropathy. Therefore, EPA phosphatidylcholine can be used to improve diabetic nephropathy.

[0036] Figure 3 F-3G shows the experimental results of total cholesterol (TC) and triglyceride (TG) levels in each group of mice. From Figure 3 F-3G analysis showed that, compared with the ND group, the MOD group mice had significantly higher TC and TG levels (P < 0.01); the EPA group mice had significantly lower TG levels than the MOD group, but no significant difference in TC levels; the EPA-PlsEtn group mice had significantly lower TC and TG levels than the MOD group (P < 0.01). This indicates that, compared with EPA ethyl ester intervention, EPA phosphatidylcholine can reduce TC and TG levels in diabetic nephropathy mice.

[0037] 4. Detection of the effect of EPA phospholipids on renal function in diabetic nephropathy mice

[0038] After euthanizing mice at week 20, tissue samples from the same location in the renal cortex were harvested from freshly dissected left kidneys. The tissue was rapidly separated and fixed in 2.5% glutaraldehyde fixative for 24 hours, then placed in a 4°C freezer. After dehydration, curing, and embedding, sections were selected from the glomeruli and prepared for electron microscopy. Transmission microscopy was used to observe the morphology of mouse kidney podocytes. The results are as follows: Figure 4 As shown in Figure A. Figure 4 A shows the morphological results of podocytes in the kidneys of mice in each group. Compared with the ND group, the MOD group showed significantly thickened glomerular basement membrane (GBM) (P < 0.01), widened podocyte foot processes (P < 0.01), and significantly reduced podocyte numbers (P < 0.01), indicating that the filtration barrier structure of the kidneys in the MOD group was altered, accompanied by impaired glomerular filtration function, and the diabetic nephropathy model was successfully established. Compared with the MOD group, the EPA group showed reduced foot process width, but no significant changes in GBM thickness or podocyte number; while the EPA phosphatidylcholine group showed significantly reduced GBM thickening (P < 0.05), significantly reduced podocyte foot process width (P < 0.05), and improved podocyte number loss (P < 0.05), indicating that EPA phosphatidylcholine can significantly improve GBM thickness, restore foot process morphology and number in diabetic nephropathy mice (P < 0.05), and the effect is better than EPA ethyl ester.

[0039] The results of urine creatinine, urine albumin, UACR, and renal total cholesterol (TC) and triglycerides (TG) measurements are as follows: Figure 4 As shown in Figure B-4F, compared with the ND group, the MOD group mice had significantly higher levels of urinary creatinine, urinary albumin, renal TC, and renal TG; the EPA group mice had significantly lower levels of urinary creatinine, urinary albumin, and renal TG than the MOD group (P < 0.05), while UACR and renal TC showed no significant changes (P > 0.05); while the EPA-PlsEtn group mice had significantly lower levels of urinary creatinine, urinary albumin, UACR, renal TC, and renal TG than the MOD group (P < 0.05); the positive control MET group significantly reduced the levels of urinary creatinine, urinary albumin, UACR, and renal TC in mice (P < 0.05), indicating that EPA phosphatidylcholine can effectively improve renal function and reduce ectopic lipid deposition in the kidneys, and that EPA phosphatidylcholine is more effective than EPA ethyl ester.

[0040] These results indicate that EPA phospholipids can significantly improve glomerular filtration rate and renal function in diabetic nephropathy mice.

[0041] 5. Detection of the effect of EPA phospholipids on the histopathology of kidney tissue in diabetic nephropathy mice.

[0042] Freshly dissected kidneys from mice in the ND, MOD, EPA, EPA-PlsEtn, and MET groups, sacrificed at week 20, were harvested from the same area of ​​the renal cortex. The kidneys were rapidly isolated and fixed in 4% paraformaldehyde solution for 24 hours. After routine processing, the sections were embedded in paraffin, sectioned to a thickness of 2 μm, stained with hematoxylin and eosin (HE), embedded in OCT, frozen sectioned to a thickness of 8 μm, and subjected to BODIPY immunofluorescence staining. The sections were then scanned using a digital scanner. Semi-quantitative analysis was performed using Image-Pro plus 6.0 image analysis software. The results are as follows: Figure 5 As shown.

[0043] Figure 5Figure A-5B shows the histopathological results of kidney tissue from mice in the ND, MOD, EPA, EPA-PlsEtn, and MET groups (Figure A: HE staining (×400); Figure B: Glomerular immunofluorescence lipid droplet staining (BODIPY) (×400)). In the ND group, the glomerular basement membrane (GBM) of mice was uniform in thickness, with oval and regularly arranged endothelial cell nuclei, homogeneous chromatin in the tubular epithelial cell nuclei, neatly arranged microvilli on the luminal surface, and no interstitial fibrosis or inflammatory cell infiltration. In the MOD group, the glomerular basement membrane of mice showed homogeneous thickening, increased glomerular mesangial matrix, increased glomerular lipid droplets, dilated and thickened tubules, and irregular arrangement of tubular epithelial cells. Compared with the MOD group, the disordered arrangement of glomerular basement membrane, endothelial cells, and podocyte nuclei in the EPA, EPA phospholipid, and MET groups was improved. BODIPY staining results showed a significant reduction in glomerular lipid droplets in the EPA, EPA phosphatidylcholine, and MET groups, with the EPA phosphatidylcholine group exhibiting the most significant reduction. This indicates that EPA phosphatidylcholine can significantly improve glomerular structure and ectopic lipid deposition in the kidneys of diabetic nephropathy mice, and can be used to improve diabetic nephropathy.

Claims

1. Use of EPA acetal phospholipid in preparation of products for improving or preventing diabetic nephropathy.

2. The use of the EPA acetal plasmalogen according to claim 1 in the preparation of a product for improving or preventing diabetic nephropathy, characterized in that, EPA acetal phospholipid can improve kidney function and ectopic lipid deposition in the kidney of diabetic nephropathy mice, and can also reduce blood glucose, improve blood lipid and reduce insulin resistance.

3. The use of the EPA acetal plasmalogen according to claim 1 in the preparation of a product for improving or preventing diabetic nephropathy, characterized in that, The use includes preventing diabetic nephropathy from occurring in diabetic patients by taking EPA acetal phospholipid.

4. The use of the EPA acetal plasmalogen according to claim 1 in the preparation of a product for improving or preventing diabetic nephropathy, characterized by, The products include drugs, food and health products.

5. The use of EPA acetal phospholipid according to claim 4, for the preparation of a product for the improvement or prevention of diabetic nephropathy, characterized by, The dosage form of the drug is granules, capsules, tablets, powders, oral liquids, suspensions or emulsions.

6. The use of the EPA acetal plasmalogen according to claim 4 in the preparation of a product for improving or preventing diabetic nephropathy, characterized in that, The drug includes pharmaceutically acceptable excipients.

7. The use of the EPA acetal plasmalogen according to claim 4 in the preparation of a product for improving or preventing diabetic nephropathy, characterized in that, The drug is a drug or a pharmaceutical composition containing EPA acetal phospholipid, and the drug or the pharmaceutical composition is administered orally.

8. The use of the EPA acetal plasmalogen according to claim 4 in the preparation of a product for improving or preventing diabetic nephropathy, characterized by, The food and health products refer to food and health products containing EPA acetal phospholipid.

9. The use of the EPA acetal plasmalogen according to claim 1 in the preparation of a product for improving or preventing diabetic nephropathy, characterized by, The preparation method of the EPA acetal phospholipid is as follows: taking dry powder of Cossura isenica, extracting with ethanol overnight, filtering and concentrating under reduced pressure to obtain a crude lipid extract; loading silica gel into a column after activation, equilibrating the silica gel column with chloroform, dissolving the obtained crude lipid extract with chloroform and pouring into the silica gel column, eluting with chloroform, chloroform-methanol 9:1 and chloroform-methanol 2:1 in sequence, collecting the chloroform-methanol 2:1 eluent, and concentrating under reduced pressure to obtain the EPA acetal phospholipid.

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