A method for establishing a systemic amyloidosis model and application thereof

Abstract

The application relates to a method for establishing a systemic amyloidosis model and application, a systemic amyloidosis model is established by injecting amyloidosis lysozyme into the tail vein of a mouse to induce amyloidosis of the mouse tissue, the systemic amyloidosis model obtained can simulate the pathological state of systemic amyloidosis in the body, is used for activity screening of amyloidosis prevention and treatment drugs, and has a good application prospect in the treatment and prevention of amyloidosis.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a method and application for establishing a systemic amyloidosis model. Background Technology

[0002] Amyloidosis is a chronic clinical syndrome caused by various factors that lead to the misfolding and gradual aggregation of amyloid precursor proteins, forming insoluble fibers that deposit between tissues and organs, causing gradual functional decline. The disease can be classified into systemic (multi-organ involvement) and localized (affecting only one organ) based on the location of the lesions.

[0003] The kidneys, as a vital site for the metabolism of numerous substances, are also one of the organs most commonly affected by amyloidosis. Renal amyloidosis is more common in middle-aged and elderly patients, with the main clinical manifestations being proteinuria or nephrotic syndrome, eventually progressing to end-stage renal disease. The mesangial area of ​​the glomerulus, rich in protein, is the most prone site for amyloid deposition. The mesangial area, composed of mesangial cells and their produced mesangial matrix, has multiple functions including support, contraction, phagocytosis, and secretion. Glomerular amyloidosis initially manifests as mesangial cell damage and thickening of the mesangial matrix; amyloid deposits can be observed in the glomerular mesangial area under both light and electron microscopy.

[0004] Hepatic amyloidosis is caused by the accumulation of misfolded proteins in the liver, leading to a series of liver injuries, including hepatocyte and tissue inflammation and subsequent liver failure. Amyloid A (AA) is an acute-phase reactant produced by the liver, with serum amyloid A (SAA) as its precursor. SAA is a typical amyloid protein in the liver and plays a crucial role in the initiation and maintenance of inflammation. SAA induces neutrophil proliferation at sites of inflammation and induces the secretion of various inflammatory cytokines, including TNF-α and IL-6. Alterations in SAA expression play a pathogenic role in hepatic amyloid deposition. Sustained elevation of SAA is a prerequisite for the development of hepatic amyloidosis.

[0005] Besides the most common liver and kidney amyloidosis, the heart, spleen, blood vessels, and skin are also common organs or tissues affected by systemic amyloidosis. Amyloid protein deposition in heart tissue can cause left ventricular hypertrophy, impaired diastolic function, and increased ventricular stiffness, leading to impaired cardiac systolic function. In the end-stage of the disease, the patient's left ventricular ejection fraction decreases significantly, manifesting as heart failure. In the spleen, amyloid protein often deposits in the splenic white pulp and blood vessels, leading to splenomegaly and calcification. Early splenic amyloidosis is usually benign, but long-term progression increases the risk of splenic hemorrhage and rupture.

[0006] Egg white lysozyme is one of the most widely studied proteins in amyloidosis research. When incubated in vitro, it forms amyloid fibrils. Furthermore, egg white lysozyme shares a high degree of homology with human lysozyme, making it a frequent model protein for in vivo and in vitro studies of amyloidosis.

[0007] There are currently no reports of amyloid lysozyme (LYSO-6) inducing secondary systemic amyloidosis in multiple organs and cells in vivo or in vitro. Summary of the Invention

[0008] The purpose of this invention is to overcome the technical problems existing in the prior art, provide a method for establishing a systematic amyloidosis biological model, and simultaneously provide its application.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of this invention provides a method for establishing a systemic amyloidosis model, which involves first stimulating mice with drugs to induce inflammation, during which amyloidosis lysozyme LYSO-6 is injected into the tail vein to obtain a systemic amyloidosis model.

[0011] As a further improvement of the present invention, the method for stimulating inflammation in mice is as follows: administer 0.15-0.25 mL of AgNO3 with a concentration of 1.5-2.5% subcutaneously to the back for 13-15 consecutive days to stimulate inflammation.

[0012] As a further improvement of the present invention, 0.05-0.15 mL of amyloid lysozyme LYSO-6 with a final concentration of 0.5-1.5 mg / mL was injected via the tail vein on days 7-9 and 13-15, respectively.

[0013] As a further improvement of the present invention, the mice were selected from 7-week-old male C57BL / 6 mice.

[0014] As a further improvement of the present invention, the amyloid lesion tissue includes heart tissue, liver tissue, spleen tissue, kidney tissue, blood vessels, and skin tissue.

[0015] As a further improvement of the present invention, the establishment of the systemic amyloidosis model takes 48-52 days.

[0016] As a further improvement of the present invention, the preparation method of amyloidosis lysozyme LYSO-6 is as follows:

[0017] S1: Add an appropriate amount of egg white lysozyme powder to a hydrochloric acid solution with a pH of 2, to a final concentration of 1 mM. Let it stand at 4°C for 8-10 hours to allow it to fully dissolve. After it is completely dissolved, filter it through a 0.22-0.45 μm filter membrane.

[0018] S2: Place the lysozyme incubation solution in a constant temperature shaker. Set the shaker to 65±2℃ and 50 rpm for 6 consecutive days, and store at 3~5℃.

[0019] As a further improvement of the present invention, 7-week-old male C57BL / 6 mice were acclimatized for one week. Starting from day 8, 0.20 mL of 2% AgNO3 was subcutaneously administered to the back for 14 consecutive days to stimulate inflammation. On day 8 and day 15, 0.10 mL of amyloidosis lysozyme LYSO-6 was injected via the tail vein to obtain a systemic amyloidosis model. On day 50, tissue samples were collected from the mice.

[0020] A second aspect of the present invention provides a systematic amyloidosis model obtained by the method described above.

[0021] A third aspect of the present invention provides the application of the systemic amyloidosis model obtained by the method described above in screening drugs for the treatment or prevention of amyloidosis.

[0022] The beneficial effects of adopting the above technical solution are as follows:

[0023] This invention induces amyloidosis in mouse tissues and organs by intravenous injection of amyloidosis lysozyme via the tail vein, thus obtaining a systemic amyloidosis model. The systemic amyloidosis model obtained by this invention can simulate the pathological state of systemic amyloidosis in vivo and can be used for activity screening of drugs for the prevention and treatment of amyloidosis, showing promising application prospects in the treatment and prevention of amyloidosis. Attached Figure Description

[0024] Figure 1 These are the HE staining results of heart organ tissue sections from the control group and model group in Example 1 of this invention;

[0025] Figure 2 These are the Congo red staining results of heart organ tissue sections from the control group and model group in Example 1 of this invention;

[0026] Figure 3 These are the HE staining results of liver tissue sections from the control group and model group in Example 1 of this invention;

[0027] Figure 4 These are the Congo red staining results of liver tissue sections from the control group and model group in Example 1 of this invention;

[0028] Figure 5 These are the HE staining results of spleen tissue sections from the control group and model group in Example 1 of this invention;

[0029] Figure 6These are the Congo red staining results of spleen organ tissue sections from the control group and model group in Example 1 of this invention;

[0030] Figure 7 These are the HE staining results of kidney organ tissue sections from the control group and model group in Example 1 of this invention;

[0031] Figure 8 These are the Congo red staining results of kidney organ tissue sections from the control group and model group in Example 1 of this invention;

[0032] Figure 9 These are the HE staining results of blood vessel (abdominal aorta) sections from mice in the control group and model group in Example 1 of this invention; A: Normal group (200×); B: Model group (200×); C: Normal group (500×); D: Model group (500×);

[0033] Figure 10 These are the Congo red staining results of blood vessel (abdominal aorta) sections from mice in the control group and model group in Example 1 of this invention; A: Normal group (bright field); B: Model group (bright field); C: Normal group (dark field); D: Model group (dark field) (200×). Detailed Implementation

[0034] To make the objectives, technical solutions and advantages of the present invention clearer, the invention will be clearly and completely described below in conjunction with specific embodiments. Unless otherwise specified, the reagents and methods used in this embodiment are conventional.

[0035] The PBS buffer used in this example was prepared as follows: 1.2g of Na2HPO4 powder and 0.2g of KH2PO4 powder were dissolved in ddH2O and the volume was adjusted to 1000mL.

[0036] The method for preparing a hydrochloric acid solution with a pH of 2 is as follows: Under the monitoring of a pH meter, adjust the pH of triple-distilled water to 2.0 with concentrated hydrochloric acid.

[0037] The preparation method of 2% AgNO3 solution is as follows: Dissolve 0.5g AgNO3 powder in 25mL distilled water, and filter it through a 0.22μm filter membrane before use.

[0038] Preparation Example 1: Preparation of amyloidosis lysozyme LYSO-6

[0039] LYSO incubation solution: Weigh an appropriate amount of LYSO powder and add it to a hydrochloric acid solution with a pH of 2, to a final concentration of 1×10⁻⁶. -3 M, let stand at 4℃ overnight to allow it to fully dissolve. After it is completely dissolved, filter it once through a 0.22μm filter membrane.

[0040] Preparation of amyloid lysozyme LYSO-6: The LYSO incubation solution was placed in a constant temperature shaker and the shaker was set at 65℃ and 50 rpm for 6 consecutive days. The samples were stored at 4℃.

[0041] Example 1: Method for establishing a mouse model of secondary systemic amyloidosis

[0042] I. Experimental Methods

[0043] 1. Construction of mouse animal model

[0044] Seven-week-old male C57BL / 6 mice were acclimatized for one week and randomly divided into two groups (n=4): a control group and a model group. Starting on day 8, the model group received subcutaneous administration of 0.2 mL of 2% AgNO3 to the back for 14 consecutive days to stimulate inflammation. On days 8 and 15, they were injected via the tail vein with 0.1 mL of LYSO-6 to a final concentration of 1 mg / mL. The control group received an equal volume of carrier solvent via the tail vein. On day 50, tissue samples were collected from the liver, kidneys, spleen, heart, blood vessels, and skin of the mice.

[0045] 2. Histopathological observation of various tissues

[0046] (1) Paraffin slice preparation

[0047] Fixation and sampling: Each tissue was taken and fixed in 4% paraformaldehyde fixative for 24 hours. After removal, the tissue at the target site was trimmed with a scalpel.

[0048] Dehydration and paraffin infiltration: The tissue was placed in a dehydrator for alcohol gradient dehydration. The dehydration solvent and time settings were as follows: 75% ethanol solution for 4 hours, 85% ethanol solution for 2 hours, 90% ethanol solution for 2 hours, 95% ethanol solution for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, 65℃ melted paraffin I for 1 hour, 65℃ melted paraffin II for 1 hour, and 65℃ melted paraffin III for 1 hour.

[0049] Paraffin embedding: The paraffin-impregnated tissue is embedded in an embedding machine. Before the paraffin solidifies, the tissue is removed from the dehydration box and placed into an embedding frame, which is then cooled on a -20°C freezing stage. After the paraffin solidifies, the paraffin block is removed from the embedding frame and trimmed.

[0050] Sectioning: Prepare 5μm thick sections from the paraffin block of tissue. Dry the sections in an oven at 65℃ for 6 hours and store them in a refrigerator at 4℃.

[0051] (2) HE staining of tissue

[0052] Baking: Place the slices in a 65℃ oven and bake for 2 hours.

[0053] Dewaxing: Place the sections in xylene I and II for 15 min each, anhydrous ethanol for 10 min, 90% ethanol solution, 80% ethanol solution, and 70% ethanol solution for 5 min each, and distilled water for 5 min.

[0054] Hematoxylin staining: Immerse the dewaxed sections in hematoxylin staining solution for 3 minutes to stain the cell nuclei.

[0055] Washing: Rinse the tissue sections with running water for 5 minutes and then perform bluing.

[0056] Differentiation with 1% hydrochloric acid and ethanol: The sections were placed in 1% hydrochloric acid and ethanol for 30 seconds for differentiation.

[0057] Washing: Rinse the differentiated sections with tap water for 10 minutes.

[0058] Eosin staining: Place the section in eosin staining solution for 1 minute to stain the cytoplasm.

[0059] Washing: Rinse the slices with tap water for 5 minutes.

[0060] Gradient ethanol dehydration: The sections were placed in 70% ethanol solution, 80% ethanol solution and 90% ethanol solution for 30 seconds each, and then placed in anhydrous ethanol I and anhydrous ethanol II for 2 minutes each.

[0061] Clearing: Dehydrated tissue sections were placed in xylene I and xylene II for 3 min each.

[0062] Mounting: Remove the slide from xylene and mount it with neutral resin glue.

[0063] Images were taken under a bright-field microscope and then analyzed using image analysis techniques.

[0064] (3) Tissue Congo Red (CR) Staining

[0065] Baking: Place the slices in a 65℃ oven and bake for 2 hours.

[0066] Dewaxing: Place the sections in xylene I and xylene II for 15 min each, anhydrous ethanol for 10 min, 90% ethanol solution, 80% ethanol solution, and 70% ethanol solution for 5 min each, and distilled water for 5 min.

[0067] Hematoxylin staining: Immerse the dewaxed sections in hematoxylin staining solution for 3 minutes to stain the cell nuclei.

[0068] Washing: Rinse the tissue sections with running water for 5 minutes and then perform bluing.

[0069] Differentiation with 1% hydrochloric acid and ethanol: The sections were placed in 1% hydrochloric acid and ethanol for 30 seconds for differentiation.

[0070] Washing: Rinse the differentiated sections with tap water for 10 minutes.

[0071] CR staining: The stained sections were immersed in CR staining solution and stained in the dark for 4 hours.

[0072] Washing: Rinse the CR-stained tissue sections with running tap water for 4 minutes to remove the staining solution floating on the surface.

[0073] Alkaline ethanol differentiation: The sections were placed in alkaline ethanol differentiation solution for 10 seconds for differentiation.

[0074] Gradient ethanol dehydration: Tissue sections were placed in 70% ethanol solution, 80% ethanol solution and 90% ethanol solution for 30 seconds each, and then placed in anhydrous ethanol I and anhydrous ethanol II for 2 minutes each for dehydration.

[0075] Clearing: After dehydration, the tissue sections were placed in xylene I and xylene II for 3 minutes each to clear the tissue sections.

[0076] Mounting: Remove the transparent section from xylene and mount it with neutral resin glue.

[0077] The images were taken under a polarizing microscope and then analyzed.

[0078] II. Experimental Results

[0079] 1. Mouse heart staining results

[0080] (1) HE staining

[0081] Figure 1 Staining results showed that the myocardial fibers in the control group were neatly and tightly arranged, while the myocardial fibers in the model group had larger gaps and were arranged disordered. In HE staining, amyloid protein was deposited as eosinophilic homogeneous unstructured clumps. Under light microscopy, amyloid material was visible adhering to the blood vessel walls in the model group. Compared with the control group, the arteriolar walls were significantly thickened and the lumen was narrowed.

[0082] (2) Congo red staining

[0083] Figure 2 Staining results showed that Congo red stained sections of the heart from the model group mice contained uniformly stained orange-red material under a light microscope; apple-green birefringence was observed under a polarized light microscope, indicating the presence of amyloid protein in the heart of the model group mice, with its main deposition in the myocardial interstitium. No amyloid protein deposition was observed in the heart of the control group.

[0084] 2. Mouse liver staining results

[0085] (1) HE staining

[0086] Figure 3The staining results showed that the cells in the liver lobules of the blank group had normal morphology, the cells around the central vein were neatly arranged, and the hepatocyte cords were arranged radially outward. Compared with the blank group, some hepatocytes in the model group were swollen, ballooned, lost their normal hexagonal shape, the cytoplasm was finely net-like, and the hepatocyte cords were disordered.

[0087] (2) Congo red staining

[0088] Figure 4 Staining results showed that in the model group, uniformly red-stained clumps of material were visible in the central vein under a light microscope. Furthermore, this material exhibited apple-green birefringence under a polarizing microscope, indicating that amyloid material was mainly deposited in the walls of the central vein. No amyloid protein deposition was observed in the liver of the control group.

[0089] 3. Staining results of mouse spleen

[0090] (1) HE staining

[0091] Figure 5 Staining results showed proliferation in the peripheral zone of the white pulp in the model group. Since lymphocytes mature in the peripheral zone and then migrate to the central zone, the deposition of amyloid protein in the peripheral zone affects lymphocyte development, resulting in a decrease in central lymphocytes and an increase in small lymphocytes in the peripheral zone. Furthermore, compared to the control group, the cells in the white pulp of the model group were enlarged and loosely distributed.

[0092] (2) Congo red staining

[0093] Figure 6 The staining results showed that orange-red substances were present at the junction of the white and red pulp in the spleen of the model group, and apple-green birefringence could be observed under polarized light. No orange-red substances or apple-green birefringence were observed in the spleen of the control group.

[0094] 4. Mouse kidney staining results

[0095] (1) HE staining

[0096] Figure 7 The staining results showed that the glomerular cells in the blank group were full and normal in morphology, the capillary lumen was open, and there was no deposition or proliferation in the mesangial area. Compared with the blank group, the model group had amyloid material in the glomerular vascular plexus, the glomeruli were ischemic and shrunken, and capillary adhesion, closure and mesangial proliferation were also visible.

[0097] (2) Congo red staining

[0098] Figure 8 Staining results showed that amyloid protein was mainly deposited in the glomerular mesangial area and capillary walls. Under a regular optical microscope, the glomeruli were stained dark reddish-brown, and under polarized light, the glomeruli showed apple-green birefringence. No such phenomena were observed in the control group.

[0099] 5. Staining results of mouse blood vessels (abdominal aorta)

[0100] (1) HE staining

[0101] Figure 9 HE staining results of the abdominal aorta of mice showed that the abdominal aorta wall of the blank control group was intact and the elastic fibers were neat and wavy; in the model group, the elastic fibers became disordered and some places were broken.

[0102] (2) Congo red staining

[0103] Figure 10 Staining results showed that after Congo red staining, the elastic fibers of the abdominal aorta appeared orange-red, while the amyloid fibers appeared reddish-brown. In the control group, the elastic fibers were intact and exhibited a graceful wavy shape; in the model group, the elastic fibers were broken, the overall structure became disordered, and local deposits of reddish-brown material were visible. Under polarized light, the control group showed virtually no apple-green birefringence, while the model group showed clearly yellow-green birefringence in the vessel wall gaps, indicating the presence of amyloid fiber deposits in the model group.

[0104] III. Experimental Conclusions

[0105] HE staining and Congo red staining of mouse tissue sections revealed that lesions occurred in the heart, liver, spleen, kidney, blood vessels and skin of mice after LYSO-6 modeling, with changes in cell morphology. After Congo red staining, apple green birefringence was visible under a polarized light microscope, indicating that amyloid deposition occurred in multiple organs of the model group mice, proving that a mouse model of secondary systemic amyloidosis was successfully established.

[0106] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for establishing a model of systemic amyloidosis, characterized by, Mice were first stimulated to produce inflammation with drugs, and during this period, amyloidosis lysozyme LYSO-6 was injected into the tail vein to obtain a systemic amyloidosis model. The systemic amyloidosis model is a secondary systemic amyloidosis model; The preparation method of the amyloid lysozyme LYSO-6 is as follows: S1: Add an appropriate amount of egg white lysozyme powder to a hydrochloric acid solution with a pH of 2, to a final concentration of 1 mM. Let it stand at 4°C for 8-10 hours to allow it to fully dissolve. After it is completely dissolved, filter it through a 0.22-0.45 μm filter membrane. S2: Place the lysozyme incubation solution in a constant temperature shaker. Set the shaker to 65±2℃ and 50 rpm for 6 consecutive days, and store at 3~5℃.

2. The method of claim 1, wherein the model is a systemic amyloidosis model. The method to stimulate inflammation in mice was to administer 0.15-0.25 mL of 1.5-2.5% AgNO3 subcutaneously to the back for 13-15 consecutive days to stimulate inflammation.

3. The method of claim 1, wherein the model is a systemic amyloidosis model. Amyloid lysozyme LYSO-6 was administered via tail vein injection at 0.05-0.15 mL on days 7-9 and 13-15, with a final concentration of 0.5-1.5 mg / mL.

4. The method of claim 1, wherein the model is a systemic amyloidosis model. The mice were selected from 7-week-old male C57BL / 6 mice.

5. The method of claim 1, wherein the model is a systemic amyloidosis model. Amyloidosis tissues include heart tissue, liver tissue, spleen tissue, kidney tissue, blood vessels, and skin tissue.

6. The method of claim 1, wherein the model is a systemic amyloidosis model. The establishment of a systemic amyloidosis model takes 48-52 days.

7. The method of claim 1, wherein the model is a systemic amyloidosis model. Seven-week-old male C57BL / 6 mice were acclimatized for one week. Starting on day 8, 0.20 mL of 2% AgNO3 was administered subcutaneously to the back for 14 consecutive days to stimulate inflammation. On days 8 and 15, 0.10 mL of amyloidosis lysozyme LYSO-6 was injected via the tail vein to obtain a systemic amyloidosis model. Tissue samples were collected from the mice on day 50.

8. The use of a systemic amyloidosis model obtained by the method of any one of claims 1 to 7 in screening drugs for the treatment or prevention of amyloidosis.

Images