Silicified curcumin microspheres as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of silicified curcumin microspheres, and the silicified curcumin microspheres prepared on the basis of the method. The particle size of the prepared silicified curcumin microspheres is about 50nm, the silicified curcumin microspheres are rich in calcium and silicon elements, and when the silicified curcumin microspheres are used for administration, curcumin in the silicified curcumin microspheres can be effectively released and utilized within 14 days, so that the bioavailability of curcumin is improved, and the demand of continuous administration is met; in addition, the silicified curcumin can obviously inhibit the increase of the thickness of a blood vessel wall in a high-glucose environment, reduce vascular fibrosis, obviously improve ejection fraction reduction and myocardial cell apoptosis caused by adriamycin, effectively improve the quality and / or improve myocardial damage; meanwhile, the silicified curcumin does not cause any damage to organs such as liver, spleen, lung and kidney in the administration process, and has good biological safety.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically, to a silicified curcumin microsphere and its preparation method and application. Background Art

[0002] Currently, approximately 537 million people worldwide suffer from diabetes, accounting for 10.5% of the world's population. At the same time, the number of newly diagnosed cancer cases globally reaches approximately 20 million per year, indicating that a significant portion of these cancer patients also have diabetes. However, existing studies have shown that chemotherapeutic drugs (such as doxorubicin) can induce cardiotoxicity, manifested as heart failure, arrhythmia, and ischemic heart disease; in addition, diabetes causes vascular remodeling, which cannot be effectively solved by taking hypoglycemic drugs.

[0003] It is worth noting that currently, more than half of the deaths of diabetic patients are attributed to cardiovascular diseases caused by vascular remodeling. Therefore, when diabetic patients develop cancer and receive chemotherapy simultaneously, their cardiovascular risk is further escalated. In view of this, there is an urgent need to explore specialized treatment strategies for cardiovascular diseases in this unique and complex patient population, aiming to reduce mortality and improve their quality of life.

[0004] However, the disease characteristics of diabetic cancer patients receiving chemotherapy are extremely complex and affected by multiple factors. On the one hand, high glucose levels stimulate endothelial cells, prompting them to release a large amount of inflammatory factors. These inflammatory factors not only guide the adhesion and infiltration of inflammatory cells into the blood vessel wall but also trigger inflammatory responses in other cells within the blood vessel wall, further leading to vascular remodeling; on the other hand, chemotherapeutic drugs directly attack cardiomyocytes, inducing a large number of cell apoptoses and ultimately resulting in cardiac dysfunction. Given the close connection between the vascular network and the heart, the coexistence of the above two situations is likely to trigger a vicious cycle of continuous cardiovascular damage. In this case, traditional cardiovascular treatment regimens alone may have limited effectiveness.

[0005] With the advancement of science, a variety of valuable medicinal natural products have been successfully extracted from natural plants of herbal medicine (NPHM). These chemicals can precisely regulate physiological processes and effectively treat a spectrum of diseases. Among them, curcumin (Cur), a valuable phenolic compound derived from the rhizomes of turmeric and other ginger plants, is well-known for its effective antioxidant and anti-inflammatory properties and has shown significant efficacy in treating inflammatory diseases such as myocarditis, osteoarthritis, and periodontitis. Despite the high medicinal value of Cur, it mainly faces the following challenges in clinical applications: (1) The poor solubility of Cur in water severely limits its use; (2) The low bioavailability of Cur, with more than 75% of orally administered Cur being excreted without being absorbed by the intestine; (3) Currently, organic solvents such as dimethyl sulfoxide and ethanol are often used or an alkaline environment is created to improve the solubility of Cur. However, due to the toxicity of organic solvents or the corrosiveness of chemical bases (such as sodium hydroxide and potassium hydroxide), the current dissolution methods of Cur have low biosafety.

[0006] Therefore, there is an urgent need for a new Cur delivery method to improve the therapeutic effect of Cur while ensuring biosafety. Summary of the Invention

[0007] The object of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a silicated curcumin microsphere, its preparation method, and application.

[0008] The first object of the present invention is to provide a preparation method of a silicated curcumin solution.

[0009] The second object of the present invention is to provide the silicated curcumin solution prepared by the above preparation method.

[0010] The third object of the present invention is to provide a preparation method of a silicated curcumin microsphere.

[0011] The fourth object of the present invention is to provide the silicated curcumin microsphere prepared by the above preparation method.

[0012] The fifth object of the present invention is to provide the application of the above silicated curcumin solution and / or the above silicated curcumin microsphere in the preparation of a drug for simultaneously treating diabetic vascular remodeling and / or myocardial injury.

[0013] To achieve the above object, the present invention is realized through the following solutions:

[0014] A preparation method of a silicated curcumin solution includes the following steps:

[0015] S1. Place the calcium silicate bioceramics in distilled water, PBS, ECM basal medium, DMEM basal medium or physiological saline at a final concentration of 100 - 200 mg / mL, soak them thoroughly, and then perform solid-liquid separation to obtain Si liquid;

[0016] S2. Mix the Si liquid obtained in step S1 with curcumin at a ratio of 10 - 50 mL: 5 - 50 mg thoroughly, perform solid-liquid separation, and collect the liquid to obtain a silicified curcumin solution.

[0017] Preferably, in step S1, the calcium silicate bioceramics are placed in distilled water, PBS, ECM basal medium, DMEM basal medium or physiological saline at a final concentration of 200 mg / mL and soaked thoroughly.

[0018] More preferably, the concentration of calcium silicate in the calcium silicate bioceramics is 100 - 150 μg / mg.

[0019] Even more preferably, the concentration of calcium silicate in the calcium silicate bioceramics is 120 μg / mg.

[0020] Preferably, in step S1, the thorough soaking is for 24 h.

[0021] Preferably, in step S2, the Si liquid obtained in step S1 is mixed with curcumin at a ratio of 50 mL: 20 mg thoroughly.

[0022] More preferably, the thorough mixing is specifically: placing it on a constant temperature vibrating sieve and stirring evenly at 60 - 120 rpm.

[0023] Even more preferably, the temperature of the constant temperature vibrating sieve is 37 °C.

[0024] Preferably, in step S2, the solid-liquid separation is filtration.

[0025] More preferably, the filtration is through a 0.22 μm filter.

[0026] The present invention also requests protection for the silicified curcumin solution prepared by the above preparation method.

[0027] The present invention also requests protection for a preparation method of silicified curcumin microspheres, including the following steps:

[0028] S11. Dissolve cetyltrimethylammonium bromide and ammonium fluoride in water thoroughly, and place them at 25 - 37 °C to mix evenly to obtain solution 1;

[0029] Among them, the final concentration of cetyltrimethylammonium bromide is 1 - 5 mg / mL, and the final concentration of ammonium fluoride is 1 - 12 mg / mL;

[0030] S12. Dissolve calcium nitrate in absolute ethanol at a ratio of 0.05 - 1 g: 1 - 18 mL, and then mix it evenly with tetraethyl orthosilicate at a volume ratio of 0.1 - 1: 9 - 18 to obtain Solution 2;

[0031] S13. Mix Solution 2 obtained in Step S12 and Solution 1 obtained in Step S1 at a volume ratio of 1 - 10: 100 - 500, and react for 1 - 6 h. After sufficient cooling, perform solid-liquid separation, collect the solid, freeze-dry it, and calcine it to obtain silicon microspheres;

[0032] S14. Disperse the silicon microspheres obtained in Step S13 in a curcumin solution at a ratio of 0.1 - 1 g: 100 - 500 mL. After sufficient mixing, perform solid-liquid separation, collect the solid, and freeze-dry it to obtain siliconized curcumin microspheres;

[0033] Among them, the concentration of curcumin in the curcumin solution is 0.2 - 2 mg / mL.

[0034] Preferably, in Step S11, the final concentration of cetyltrimethylammonium bromide is 3.64 mg / mL, and the final concentration of ammonium fluoride is 6 mg / mL.

[0035] Preferably, the sufficient mixing in Step S11 is: stir at 80 °C for 1 h.

[0036] Preferably, in Step S12, dissolve calcium nitrate in absolute ethanol at a ratio of 0.5 g: 1 mL.

[0037] Preferably, in Step S12, mix it evenly with tetraethyl orthosilicate at a volume ratio of 1: 9 to obtain Solution 2.

[0038] Preferably, in Step S13, mix Solution 2 obtained in Step S12 and Solution 1 obtained in Step S1 at a volume ratio of 9: 500.

[0039] Preferably, before freeze-drying and calcining in Step S13, the solid needs to be washed.

[0040] More preferably, wash it alternately with absolute ethanol and water.

[0041] Preferably, the solid-liquid separation in Step S13 is specifically: centrifuge at 8000 rpm for 25 min.

[0042] Preferably, the calcining in Step S13 is: calcine at 500 - 1000 °C for 3 - 12 h.

[0043] More preferably, calcine at 600 °C for 6 h.

[0044] Preferably, in Step S14, disperse the silicon microspheres in a curcumin solution at a ratio of 0.2 g: 100 mL.

[0045] More preferably, the concentration of curcumin in the curcumin solution is 2 mg / mL.

[0046] Further preferably, the solvent of the curcumin solution is ethanol.

[0047] Preferably, the sufficient mixing in step S14 is stirring for 48 h.

[0048] Preferably, the solid-liquid separation in step S14 is centrifuging at 8000 rpm for 5 min.

[0049] Preferably, before the lyophilization in step S14, the collected solid needs to be washed.

[0050] More preferably, wash with ethanol three times.

[0051] The present invention also claims the silicified curcumin microspheres prepared by any of the above preparation methods.

[0052] The present invention also claims the application of the above silicified curcumin solution and / or the above silicified curcumin microspheres in the preparation of drugs for simultaneously treating diabetic vascular remodeling and / or myocardial injury.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The present invention provides a preparation method of silicified curcumin microspheres, and silicified curcumin microspheres are prepared based on the method. The prepared silicified curcumin microspheres have a particle size of about 50 nm, and the silicified curcumin microspheres are rich in calcium and silicon elements. When the silicified curcumin microspheres are used for administration, curcumin therein can be effectively released and utilized within 14 days, realizing the improvement of the bioavailability of curcumin and the need for continuous administration; and the silicified curcumin can significantly inhibit the increase in the vascular wall thickness in a high-glucose environment, reduce vascular fibrosis, and can significantly improve the decrease in ejection fraction and cardiomyocyte apoptosis caused by doxorubicin, effectively improving myocardial injury; at the same time, the silicified curcumin does not cause any damage to organs such as the liver, spleen, lungs, and kidneys during the administration process, and has good biosafety. Description of the Drawings

[0055] Figure 1 It is a characterization test result diagram of the silicified curcumin solution in Example 1; A is an observation diagram of the silicified curcumin solution; B is the test result of an ultraviolet spectrophotometer; C is the solubility test result;

[0056] Figure 2 It is a characterization result diagram of the silicified curcumin microspheres in Example 2; A is the observation result diagram of a scanning electron microscope; B is the release concentration result diagram of Cur;

[0057] Figure 3 Graphs of inflammatory factor detection and staining observation results in Example 3; A is the photo observation result graph of diabetic mice and control group mice; B is the fasting body weight change curve graph of each group of mice; C is the blood glucose content change curve graph of each group of mice; D is the IL-1β expression level result graph of each group of mice; E is the IL-6 expression level result graph of each group of mice; F is the TNF-α expression level result graph of each group of mice; G is the H&E staining, Masson staining and Victoria blue staining observation result graph of the aorta of each group of mice; H is the measurement result graph of the intima-media thickness of the aorta of each group of mice; is the measurement result graph of the collagen fiber area of each group of mice; J is the measurement result graph of the elastic fiber area of each group of mice;

[0058] Figure 4 Graphs of vascular adhesion factor detection results in Example 3; A is the immunohistochemical staining result graph of ICAM-1 and VCAM-1 in each group of mice; B is the Western blot result graph of ICAM-1 in each group of mice; C is the quantitative detection result graph of ICAM-1 in each group of mice; D is the Western-blot result graph of VCAM-1 in each group of mice; E is the quantitative detection result graph of VCAM-1 in each group of mice; F is the CD68 staining result graph of each group of mice;

[0059] Figure 5 Graphs of the effects of silicified curcumin microspheres on myocardial injury in cardiomyopathy mice in Example 3; A is the body weight change curve graph of each group of mice; B is the M-mode echocardiogram of the left ventricle of each group of mice; C is the left ventricular ejection fraction result graph of each group of mice; D is the left ventricular shortening fraction result graph of each group of mice; E is the ratio of heart weight to tibia length result graph of each group of mice; F is the Masson staining observation graph of the heart tissue sections of each group of mice; G is the calculation result graph of the positive area of Masson staining of the heart tissue sections of each group of mice; H is the TUNEL staining result graph of the heart tissue sections of each group of mice; I is the calculation result graph of TUNEL-positive cells in the heart tissue sections of each group of mice; Figure 3 In J is the CK-MB concentration detection result graph of each group of mice; K is the cTnT concentration detection result graph of each group of mice; L is the LDH concentration detection result graph of each group of mice;

[0060] Figure 6Results graph of the effect of silicified curcumin on HUVECs cells in Example 4; A is the Western blot result graph of ICAM-1 in each group of cells; B is the quantitative detection result graph of ICAM-1 in each group of cells; C is the Western blot result graph of VCAM-1 in each group of cells; D is the quantitative detection result graph of VCAM-1 in each group of cells; E is the RNA expression result graph of ICAM-1 in each group of cells; F is the RNA expression result graph of VCAM-1 in each group of cells; G is the ROS fluorescence level result graph of each group of cells; H is the ROS level result graph of each group of cells; I is the Western blot result graph of HO-1 in each group of cells, Figure 4 J in it is the quantitative detection result graph of HO-1 in each group of cells; K is the Western blot result graph of Nrf2 in each group of cells; L is the quantitative detection result graph of Nrf2 in each group of cells; M is the Nrf2 protein cell distribution result graph of each cell well;

[0061] Figure 7 Results graph of the detection of inflammatory factors in each group of cells in Example 4; A is the detection result graph of IL-6 in each group of cells; B is the detection result graph of IL-1β in each group of cells; C is the detection result graph of TNF-α in each group of cells;

[0062] Figure 8 Results graph of the test on the effect of cell adhesion in Example 4; A is the observation result graph under an inverted fluorescence microscope; B is the statistical graph of fluorescence signal results;

[0063] Figure 9 Results graph of the test on the effect of silicified curcumin on smooth muscle cells in Example 4; A is the scratch graph of each group of cells; B is the cell migration rate result graph of each group of cells; C is the Western blot result graph of α-SMA in each group of cells; D is the quantitative detection result graph of α-SMA in each group of cells; E is the expression level result graph of IL-1β in each group of cells; F is the expression level result graph of IL-6 in each group of cells; G is the Western blot result graph of HO-1 in each group of cells; H is the quantitative detection result graph of HO-1 in each group of cells; I is the Western blot result graph of Nrf2 in each group of cells; J is the quantitative detection result graph of Nrf2 in each group of cells;

[0064] Figure 10Results of the effect of silicified curcumin on cardiomyocytes in Example 4; A shows the results of ROS fluorescence levels in cells of each group; B shows the results of ROS levels in cells of each group; C shows the results of cell viability assays in cells of each group; D shows the results of cell apoptosis rates in cells of each group; E shows the Western blot results of HO-1 in cells of each group; F shows the Western blot results of Nrf2 in cells of each group; G shows the quantitative detection results of HO-1 in cells of each group; H shows the quantitative detection results of Nrf2 in cells of each group; I shows the results of Nrf2 distribution in cells of each group; J shows the quantitative results of Nrf2 distribution in cells of each group;

[0065] Figure 11 Results of the effect of silicified curcumin on mice with mixed diseases in Example 5; A shows the staining results of aortic blood vessels in mice of each group; B shows the results of aortic intima-media thickness in mice of each group; C shows the results of collagen fiber area in mice of each group; D shows the results of elastic fiber area in mice of each group; E shows the staining results of ICAM-1 in mice of each group; F shows the staining results of VCAM-1 in mice of each group; G shows the relative expression level results of ICAM-1 protein in mice of each group; H shows the relative expression level of VCAM-1 protein in mice of each group; I shows the M-mode echocardiogram of the left ventricle in mice of each group; J shows the results of left ventricular ejection fraction in mice of each group; K shows the results of left ventricular shortening fraction in mice of each group; L shows the Masson staining results of heart sections in mice of each group; M shows the percentage of positive area of Masson staining in heart sections of mice of each group; N shows the results of the ratio of heart weight to tibia length in mice of each group; O shows the TUNEL staining results of heart sections in mice of each group; P shows the results of TUNEL positive cell count in heart sections of mice of each group;

[0066] Figure 12 Results of the quantitative detection of inflammatory factors in mice of each group in Example 6; A shows the quantitative detection results of CK-MB in mice of each group; B shows the quantitative detection results of IL-1β in mice of each group; C shows the quantitative detection results of TNF-α in mice of each group; D shows the quantitative detection results of IL-6 in mice of each group;

[0067] Figure 13 Results of the observation of liver staining in mice of each group in Example 6. Detailed implementation manners

[0068] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0069] All animal experiments involved in the embodiments of the present invention have been approved by the Ethics Committee of the Tenth Affiliated Hospital of Southern Medical University.

[0070] Example 1 Preparation method of silicified curcumin

[0071] I. Experimental method

[0072] 1. Preparation method

[0073] A preparation method of silicified curcumin includes the following steps;

[0074] S1. Calcium silicate bioceramics (containing 120 μg / mg of calcium silicate) are respectively immersed in physiological saline at a final concentration of 200 mg / mL for 24 h, and during the immersion process, they are placed on a constant temperature shaker at 37 °C and stirred at a speed of 120 rpm; after the immersion ends, they are centrifuged at a speed of 4000 rpm for 20 min to separate the solid and liquid to obtain Si liquid;

[0075] S2. The Si liquid obtained in step S1 and curcumin (Cur, CAS number: 458-37-7) are fully mixed in a ratio of 50 mL:20 mg, and then filtered through a 0.22 μm filter, and the liquid is collected to obtain a silicified curcumin solution.

[0076] 2. Characterization of silicified curcumin

[0077] The absorption peak intensity of the silicified curcumin solution obtained in step 1 is measured by an ultraviolet-visible spectrophotometer, and it is compared with the solubility of curcumin in DMSO to obtain the solubility of curcumin in the silicified curcumin solution.

[0078] II. Experimental results

[0079] The characterization test result diagram of the silicified curcumin solution is as Figure 1 shown, Figure 1 A in Figure 1 is the observation diagram of the silicified curcumin solution, Figure 1 B in

[0080] is the test result of the ultraviolet spectrophotometer, and

[0081] C in

[0082] is the solubility test result.

[0083] 1. Preparation method

[0084] A preparation method of curcumin-silica microspheres, comprising the following steps:

[0085] S11. Add 1.82 g of cetyltrimethylammonium bromide (CTAB) and 3.00 g of ammonium fluoride (NH4F) into a beaker containing 500 mL of deionized water, place it at 80 °C and stir for 1 h to obtain Solution 1;

[0086] S12. Add 0.5 of calcium nitrate into 1 mL of absolute ethanol, completely dissolve it by ultrasonic treatment, and then uniformly mix it with tetraethyl orthosilicate according to a volume ratio of 1:9 to obtain Solution 2;

[0087] S13. Use a 10 mL syringe to drop Solution 2 into Solution 1 according to a volume ratio of 9:500, after fully reacting for 4 h, cool it to 25 °C, then centrifuge at 8000 rpm for 25 min, collect the solid and wash it alternately with absolute ethanol and deionized water, after washing, freeze-dry it and calcine it in a muffle furnace at 600 °C for 6 h to obtain silica microspheres;

[0088] S14. Disperse 0.2 g of the silica microspheres prepared in step S13 into 100 mL of a curcumin alcohol solution (Cur concentration is 2 mg / mL), stir it fully for 48 h, then centrifuge at 8000 rpm for 5 min, collect the solid and wash it with alcohol 3 times, then freeze-dry it for 24 h to obtain curcumin-silica microspheres.

[0089] 2. Characterization of curcumin-silica microspheres

[0090] Use a scanning electron microscope (SEM, S-4800, Hitachi, Japan) to observe the morphology of the curcumin-silica microspheres prepared in step 1; observe the microstructure of the curcumin-silica microspheres through a transmission electron microscope (TEM, JEM-2100F, JEOL, Japan); use ICP-AES (Thermo Fisher X Series 2, USA) to measure the release concentration of SiO3 2- ions in the curcumin-silica microspheres, and use an ultraviolet-visible spectrophotometer to measure the release concentration of Cur.

[0091] II. Experimental results

[0092] The characterization result diagrams of the curcumin-silica microspheres are as Figure 2 shown, Figure 2 A in it is the observation result diagram by scanning electron microscope, Figure 2 B in it is the result diagram of the release concentration of Cur.

[0093] Si / Cur composite microspheres were prepared. Scanning electron microscopy (SEM) showed that the particle size of these Si / Cur microspheres was about 50 nm, and they were rich in Ca and Si elements. The drug release curve indicated that curcumin was effectively released within 14 days along with the release of Si ions, meeting the requirement of sustained release( Figure 2 ).

[0094] Effect of silicified curcumin microspheres on mice in Example 3

[0095] I. Effect of silicified curcumin microspheres on vascular remodeling in diabetic mice

[0096] 1. Experimental method

[0097] Db / db type diabetic mice with a body weight of 50 - 55 g, 8 weeks old and homozygous mutation of the leptin receptor gene (purchased from Changzhou Cavens Experimental Animal Co., Ltd., Jiangsu Province) were selected as diabetic mice, and normal blood glucose C57BL / 6j mice were used as control mice. To ensure that there were no significant differences in fasting blood glucose and body weight among the mice before the experiment, five different experimental groups were set up as follows:

[0098] Diabetic mouse group (Control group): According to the dosage of 8 μL / g, normal saline was intravenously injected into db / db type diabetic mice once a day, and the food and water sources of the mice were ensured to be sufficient.

[0099] The difference between the silicified curcumin microsphere intervention group (Si / Cur group) and the Control group was that normal saline was replaced with a 31.25 μg / mL silicified curcumin microsphere solution, and the other treatments were the same.

[0100] The difference between the Si intervention group (Si group) and the Control group was that normal saline was replaced with a 1.875 μg / mL Si microsphere solution, and the other treatments were the same.

[0101] The difference between the curcumin solution intervention group (Cur group) and the Control group was that normal saline was replaced with a 6.25 μg / mL curcumin solution, and the other treatments were the same.

[0102] The difference between the normal mouse group (Sham group) and the Control group was that db / db type diabetic mice were replaced with C57BL / 6j mice, and the other treatments were exactly the same.

[0103] (1) Detection of inflammatory factors and staining observation

[0104] Before the experiment, diabetic mice (Control group) and control mice (Sham group) were photographed and observed respectively. Then, after obtaining the mice in each group according to the above method, the fasting body weight and blood glucose content of the mice in each group were measured every two weeks. After 8 weeks of treatment, the mice were fully anesthetized and dissected. The whole blood of the mice was taken and allowed to clot naturally at 25°C for 40 min, then placed in a centrifuge pre-cooled to 4°C and centrifuged at 3000 rpm for 25 min. The supernatant was extracted to obtain the serum of the mice in each group, and the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α in the mice in each group were quantitatively detected by ELISA; and the organs and blood vessels from the aortic root to the common iliac artery of the mice in each group were taken and fixed with 4% (w / w) paraformaldehyde for 24 h, and then observed by H&E staining, Masson staining, and Victoria blue staining respectively; at the same time, quantitative analysis of the aortic intima-media thickness, collagen fiber area, and elastic fiber area of the mice in each group was performed.

[0105] And the expression levels of IL-1β, IL-6, and TNF-α, the staining observation results, the aortic intima-media thickness, the collagen fiber area, and the elastic fiber area of the untreated Control group mice and Sham group mice were used as baseline values.

[0106] (2) Detection of vascular adhesion factors

[0107] After pre-cooling the homogenizer to 4°C, it was mixed and homogenized according to the volume ratio of RIPA: protease inhibitor: phosphatase inhibitor of 100:1:1 to obtain a lysis buffer.

[0108] After treating and dissecting the mice according to step (1), the blood vessels from the aortic root to the common iliac artery of the mice in each group were obtained. After cutting the blood vessels into pieces, they were mixed with the lysis buffer at a ratio of 1 mg: 10 mL, and then 2 small magnetic beads were added and homogenized 3 - 5 times, 60 s each time, until the tissue was thoroughly ground. Then, it was centrifuged at 12000 rpm for 15 min, and the supernatant was collected. The supernatant was mixed with the loading buffer at a volume ratio of 1:4 and heat-treated at 100°C for 10 min, and then Western blot was performed to detect ICAM-1 protein and VCAM-1 protein (using β-actin as the internal reference protein), and the relative expression levels of ICAM-1 protein and VCAM-1 protein in the mice in each group were quantitatively detected (using β-actin as the internal reference protein).

[0109] And the blood vessels from the aortic root to the common iliac artery of the mice in each group were observed by ICAM-1 staining, VCAM-1 staining, and CD68 staining.

[0110] The staining process of ICAM-1 staining is as follows:

[0111] (1) Deparaffinization of paraffin sections: Immerse the sections successively in environment-friendly deparaffinizing solution I for 10 min - environment-friendly deparaffinizing solution II for 10 min - environment-friendly deparaffinizing solution III for 10 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - distilled water rinse. (2) Antigen retrieval: Gently rinse the sections with PBS 3 times, 3 minutes each time. Drop the working solution of proteinase K (20 μg / mL) on the sections, incubate at 37 °C for 15 minutes, then rinse the sections with PBS 3 times, 3 minutes each time. (3) Block endogenous peroxidase: Immerse the sections in 3% (w / w) hydrogen peroxide solution, incubate at 25 °C in the dark for 25 min, place the slides in PBS (pH 7.4) and wash them on a shaker for 3 times, 5 minutes each time. (4) Serum blocking: Drop 3% (w / w) BSA in the immunohistochemical circle to evenly cover the tissue, block at 25 °C for 30 min. (Use rabbit serum to block when the primary antibody is from goat, and use BSA to block for other sources). (5) Add primary antibody: After removing the liquid, drop the ICAM-1 primary antibody (GB11106, 1:1000) on the sections, place the sections flat in a wet box and incubate at 4 °C for 12 h. (6) Add secondary antibody: Place the slides in PBS (pH 7.4) and wash them on a shaker for 3 times, 5 minutes each time. After gently shaking off the liquid on the sections, drop the secondary antibody (HRP-labeled) corresponding to the primary antibody in the circle to cover the tissue, incubate at 25 °C for 50 min. (7) DAB color development: Place the slides in PBS (pH 7.4) and wash them on a shaker for 3 times, 5 minutes each time. After gently shaking off the liquid on the sections, drop the freshly prepared DAB color development solution in the circle, control the color development time under the microscope, the positive is brownish yellow, and rinse the sections with tap water to terminate the color development. (8) Counterstain the cell nucleus: Counterstain with hematoxylin for 3 min, wash with tap water, differentiate with hematoxylin differentiating solution for a few seconds, rinse with tap water, blue with hematoxylin blueing solution, and rinse with running water. (9) Dehydration and mounting: Immerse the sections successively in 75% alcohol for 5 min - 85% alcohol for 5 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - n-butanol for 5 min - xylene I for 5 min. After dehydration and clearing, take out the sections and air-dry them, then mount them with mounting medium. (10) Microscopic examination: Place them under a white light microscope for result interpretation.

[0112] The difference between the staining process of VCAM-1 and that of ICAM staining lies in: replacing the ICAM-1 primary antibody with the VCAM-1 primary antibody (GB113498, 1:800), and replacing the secondary antibody with the secondary antibody (HRP-labeled) corresponding to the primary antibody.

[0113] The difference between the staining process of CD68 and that of ICAM staining lies in: replacing the ICAM-1 primary antibody with the CD68 primary antibody (GB113109, 1:200), and replacing the secondary antibody with the secondary antibody (HRP-labeled) corresponding to the primary antibody.

[0114] 2. Experimental results

[0115] (1) The results of inflammatory factor detection and staining observation are shown in Figure 3 as follows Figure 3 In A of , the photographed observation results of diabetic mice and control group mice are shown; Figure 3 In B of , the fasting body weight change curve of mice in each group is shown; Figure 3 In C of , the blood glucose content change curve of mice in each group is shown; Figure 3 In D of , the IL-1β expression level results of mice in each group are shown; Figure 3 In E of , the IL-6 expression level results of mice in each group are shown; Figure 3 In F of , the TNF-α expression level results of mice in each group are shown; Figure 3 In G of , the observation results of H&E staining, Masson staining and Victoria blue staining of the aorta of mice in each group are shown; Figure 3 In H of , the measurement results of the intima-media thickness of the aorta of mice in each group are shown; Figure 3 In I of , the measurement results of the collagen fiber area of mice in each group are shown; Figure 3 In J of , the measurement results of the elastic fiber area of mice in each group are shown.

[0116] The results show that: Diabetic mice (Control group) showed a larger body size and obvious obesity compared with normal blood glucose mice (Sham group); During the 8-week treatment period, compared with the mice in the Sham group, the mice in the Control group always had higher body weight and blood glucose levels. Compared with the Control group, Si group and Cur group, the mice in the Si / Cur group showed a slight decrease in blood glucose at the 6th week and a significant decrease in blood glucose at the 8th week.

[0117] Compared with the mice in the Sham group, the expression levels of inflammatory factors IL-1β, IL-6 and TNF-α in the blood of the mice in the Control group increased, and continued to increase with time; And compared with the mice in the Sham group, the expression levels of inflammatory factors in the Si group, Cur group and Si / Cur group also continued to increase with time, but the expression levels of IL-1β, IL-6 and TNF-α in the mice in the Si / Cur group were significantly lower than those of the mice in other groups except the Sham group, indicating that the silicified curcumin microspheres prepared in Example 2 can effectively reduce the expression levels of inflammatory factors in diabetic mice.

[0118] And the images of H&E staining, Masson staining, and Victoria blue staining of the aorta of each group of mice showed that there was no significant difference in the vascular wall thickness of the mice in the Sham group and the Control group without treatment. As the treatment time increased, the vascular wall of the mice in the Control group significantly thickened and proliferated, protruding into the lumen, and vacuolated foam was observed in the vascular wall. In the Si / Cur group and the Si group, the thickening of the vascular wall of the mice was significantly inhibited, and the vascular wall thickness of the mice in the Si / Cur group was significantly closer to that of the mice in the Sham group. At the same time, the measurement results of the intima-media thickness, collagen fiber area, and elastic fiber area of the aorta of each group of mice further showed that the vascular fibrosis of the vascular wall thickness of the mice in the Si / Cur group was closer to that of the mice in the Sham group, indicating that the silicified curcumin microspheres prepared in Example 2 could effectively reduce the vascular wall thickening, fibrosis, and elastic fiber degradation in diabetic mice.

[0119] (2) The detection results of vascular adhesion factors are as Figure 4 shown, Figure 4 In A of Figure 4 are the immunohistochemical staining results of ICAM-1 and VCAM-1 of each group of mice, Figure 4 In B of Figure 4 are the Western blot results of ICAM-1 of each group of mice, Figure 4 In C of Figure 4 are the quantitative detection results of ICAM-1 of each group of mice,

[0120] The results showed that: at the time of no treatment (Day0), there was no significant difference in the expression levels of ICAM-1 and VCAM-1 between the mice in the Control group and the Sham group. As the treatment time increased, the expression levels of both ICAM-1 and VCAM-1 in the mice in the Control group increased significantly. In the Si group and the Si / Cur group, the increase in the expression levels of ICAM-1 and VCAM-1 in the mice was significantly inhibited, especially in the Si / Cur group, where the expression levels of ICAM-1 and VCAM-1 in the mice were significantly closer to those in the Sham group.

[0121] And at the time of no treatment, there was no significant difference in the proportion of CD68-positive cells in the vascular wall between the mice in the Control group and the Sham group. As the treatment time increased, the proportion of CD68-positive cells in the vascular wall of the mice in the Control group increased significantly, while the proportion of CD68-positive cells in the vascular wall of the mice in the Si / Cur group decreased significantly and was closer to that in the Sham group.

[0122] It is illustrated that the curcumin-silicon microspheres prepared in Example 2 can effectively treat diabetic vascular remodeling.

[0123] II. Effects of curcumin-silicon microspheres on myocardial injury in doxorubicin-induced cardiomyopathy mice

[0124] 1. Experimental method

[0125] Eight-week-old male db / db type diabetic mice with homozygous mutation of the leptin receptor gene (purchased from Changzhou Cavens Experimental Animal Co., Ltd., Jiangsu Province) were selected as diabetic mice. The diabetic mice were intraperitoneally injected with doxorubicin hydrochloride (DOX, MCE, HY-15142) at a dosage of 5 mg / kg, and the administration frequency was once every 5 days for 5 times to obtain DIC mice. Four different experimental groups were set up as follows:

[0126] Control group 1: Saline was intravenously injected into DIC mice at a dosage of 8 μL / g once a day, and the food and water sources of the mice were ensured to be sufficient.

[0127] The difference between Si / Cur group 1 and Control group 1 is that the saline was replaced with a 31.25 μg / mL curcumin-silicon microsphere solution, and the other treatments were the same.

[0128] The difference between Si group 1 and Control group 1 is that the saline was replaced with a 1.875 μg / mL silicon microsphere solution, and the other treatments were the same.

[0129] The difference between Cur group 1 and Control group 1 is that the saline was replaced with a 6.25 μg / mL curcumin solution, and the other treatments were the same.

[0130] The difference between Sham group 1 and Control group 1 is that the acute DIC mice were replaced with C57BL / 6j mice, and the other treatments were exactly the same.

[0131] After obtaining the mice in each group according to the above method, the mice in each group were continuously treated for 25 days. After the treatment on the 25th day, the mice in each group were anesthetized with 5% isoflurane, and echocardiography was performed on the mice in each group using a Vevo 2100 system equipped with a 30 mhz sensor to obtain the M-mode echocardiogram of the left ventricle of the mice in each group, and the left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) of the mice in each group were calculated.

[0132] Subsequently, each group of mice was euthanized, and samples of each group of mice were collected. Through median laparotomy, the remaining blood cells in cardiomyocytes were retrogradely perfused with normal saline. Then, the hearts in the thoracic cavities of the mice were removed and blotted dry. The cardiac tumors were weighed, and at the same time, the tibia lengths (TL) of each group of mice were measured, and the heart weight-tibia length ratio (HW / TL) of each group of mice was calculated.

[0133] Subsequently, the heart tissues of each group of mice were fixed with 4% paraformaldehyde, then paraffin-embedded, and sectioned at 5 mm to obtain heart sections of each group of mice. The heart sections of each group of mice were stained with Masson to obtain the Masson staining results, and the percentage of the positive area of Masson staining was calculated using Image J software. The positive area of Masson staining of the mice in the Sham group 1 was used as the standard for fold change. The heart sections of each group of mice were stained with TUNEL to obtain the TUNEL staining results, and the TUNEL-positive cell count in the heart sections of each group of mice was calculated using Image J software. The TUNEL-positive cell count in the heart sections of the mice in the Sham group 1 was used as the standard for fold change.

[0134] After each group of mice was euthanized, the whole blood of each group of mice was collected and allowed to clot naturally at 25 °C for 40 min, then placed in a centrifuge at 4 °C and centrifuged at 3000 rpm for 25 min. The supernatant was collected and the concentrations of creatine kinase-mb isoenzyme (CK-MB), serum cardiac troponin T (cTnT), and lactate dehydrogenase (LDH) were quantitatively detected using ELISA.

[0135] 2. Experimental Results

[0136] The results of the effect of siliconized curcumin microspheres on myocardial injury in cardiomyopathic mice are shown as Figure 5 shown, Figure 5 in which A is the curve graph of the body weight change of each group of mice, Figure 5 in which B is the M-mode echocardiogram of the left ventricle of each group of mice, Figure 5 in which C is the result graph of the left ventricular ejection fraction of each group of mice, Figure 5 in which D is the result graph of the left ventricular shortening fraction of each group of mice, Figure 5 in which E is the result graph of the heart weight-tibia length ratio of each group of mice, Figure 5 in which F is the observation graph of Masson staining of the heart tissue sections of each group of mice, Figure 5 in which G is the calculation result graph of the positive area of Masson staining of the heart tissue sections of each group of mice, Figure 5 in which H is the result graph of TUNEL staining of the heart tissue sections of each group of mice, Figure 5 in which I is the calculation result graph of TUNEL-positive cells in the heart tissue sections of each group of mice;Figure 5 In J, the graph shows the detection results of CK-MB concentration in each group of mice. Figure 5 In K, the graph shows the detection results of cTnT concentration in each group of mice. Figure 5 In L, the graph shows the detection results of LDH concentration in each group of mice.

[0137] The results showed that: The body weight of the mice in Control group 1 (i.e., the mice with doxorubicin-induced myocardial injury) decreased significantly with the increase of treatment time. Although the body weight of the mice in Si group 1 and Cur group 1 increased compared with that in Control group 1, the increase was not obvious. However, the body weight of the mice in Si / Cur group 1 increased significantly compared with that of the mice in Control group 1, and was significantly closer to that in Sham group 1.

[0138] The left ventricular systolic function of the mice in Control group 1 decreased significantly. The left ventricular systolic function of the mice in Si group 1 and Cur group 1 had certain improvement compared with that in Control group 1, but there was no statistical significance. However, the left ventricular systolic function of the mice in Si / Cur group 1 improved significantly, and there was no obvious difference compared with that in Sham group 1.

[0139] The ratio of heart weight to tibia length of the mice in Control group 1 decreased significantly, indicating that the heart weight of the mice in Control group 1 decreased significantly due to myocardial injury. This phenomenon was not significantly improved in Si group 1 and Cur group 1. However, the ratio of heart weight to tibia length of the mice in Si / Cur group 1 increased significantly compared with that in Control group 1, and there was no significant difference compared with that in Sham group 1.

[0140] And the results of Masson staining showed that: Obvious myocardial fibrosis appeared in the heart tissue of the mice in Control group 1, while the degree of myocardial fibrosis in the heart of the mice in Si / Cur group 1 was significantly reduced and was close to that of the mice in Sham group 1; The results of TUNEL staining showed that: The apoptosis of myocardial cells in the mice in Si / Cur group 1 was significantly lower than that in Control group 1, Si group 1 and Cur group 1, and there was no significant difference compared with that in Sham group 1. At the same time, the concentrations of myocardial injury markers (CK-MB, cTnT and LDH) in the mice in Si / Cur group 1 were significantly reduced compared with those in Control group 1.

[0141] Explanation: The prepared curcumin-silica microspheres using Example 2 can effectively inhibit myocardial injury caused by the chemotherapeutic drug doxorubicin (DOX).

[0142] Example 4 Effects of curcumin-silica on cells

[0143] I. Effects of curcumin-silica on human umbilical vein endothelial cells (HUVECs)

[0144] 1. Experimental method

[0145] (1) After digesting human umbilical vein endothelial cells (HUVECs) with 0.25% (w / w) trypsin, they were resuspended in high-glucose medium (supplemented with 50 mmol / L glucose), counted, and a cell suspension of 20 cells / μL was obtained. Then, 100 μL of the cell suspension was inoculated into a 96-well cell plate at 2000 cells / well. After incubating in a 37°C cell culture incubator for 24 h, the following cell groups were set up respectively:

[0146] Control group 2: 100 μL of endothelial cell basal medium (ECM) was added to the cell wells inoculated with HUVECs for culture.

[0147] The difference between Si / Cur group 2 and Control group 2 is that the endothelial cell basal medium contains 31.25 μg / mL of the siliconized curcumin microspheres prepared in Example 2.

[0148] The difference between Si group 2 and Control group 2 is that the endothelial cell basal medium contains 120 μg / mL of the silicon microspheres prepared in Example 2.

[0149] The difference between Cur group 2 and Control group 2 is that the endothelial cell basal medium contains 240 μg / mL of curcumin.

[0150] Blank group: HUVECs were inoculated into a 96-well cell plate at 2000 cells / well, and 100 μL of endothelial cell basal medium was added for culture.

[0151] After culturing the cells in each group at 37°C for 48 h, the experimental cells in each group were obtained.

[0152] After digesting and resuspending the cells in each group, they were inoculated into a culture dish with a diameter of 35 mm. After culturing for 24 h, DMEM medium containing 5% (v / v) fetal bovine serum and 0.2% (v / v) growth factor was introduced for culture for 24 h. Then, the medium was replaced with fresh medium and cultured for another 24 h. Subsequently, Western blot protein electrophoresis was used to detect ICAM-1, VCAM-1, HO-1, and Nrf2 proteins in HUVECs (using β-actin as the internal reference protein), and quantitative detection was carried out (using β-actin as the internal reference protein).

[0153] Dilute DCFH-DA to 10 μmol / L with serum-free medium to obtain a DCFH-DA solution. Then, add the DCFH-DA solution to each group of experimental cells at 1 mL / well to completely cover the cells. Incubate the cells in an incubator at 37 °C for 20 min, then wash the cells three times with serum-free medium. Next, place the cells under a fluorescence microscope to observe the cells and take pictures of the ROS fluorescence level, and use ImagePro Plus 6 software to quantify the fluorescence intensity of HUVECs cells in each group to obtain the reactive oxygen species (ROS) level of each group of cells.

[0154] Next, select the fourth-generation HUVECs cells with good growth. After digestion and resuspension, inoculate them into a 24-well plate (with ECM cell culture medium) with a coverslip, and then place them in an incubator at 37 °C to allow the cells to adhere to the coverslip for 24 h.

[0155] Si / Cur well: Introduce a 31.25 μg / mL silicified curcumin microsphere solution at 500 μL / well and maintain it for 48 h (replace the silicified curcumin solution every 24 h to avoid PBS washing). Then, use a pipette to remove the cell culture medium in the well and wash the cells 2 times with PBS. Next, add 4% paraformaldehyde at 500 μL / well and fix it at 25 °C for 15 min. Then, remove the paraformaldehyde. Next, dilute the Nrf2 monoclonal antibody with an antibody diluent at a volume ratio of 1:500 to obtain the diluted antibody. Then, add the diluted antibody at 200 μL / well and incubate it at 4 °C for 12 h. Then, add DAPI reagent at 200 μL / well and incubate it in the dark at 25 °C for 6 min. After washing with PBST, use an anti-fading mounting medium to mount the coverslip and observe the distribution of Nrf2 protein by taking pictures under a fluorescence microscope.

[0156] Blank well: Human umbilical vein endothelial cells (HUVECs) cultured in normal medium.

[0157] Control well: Human umbilical vein endothelial cells (HUVECs) cultured in high-glucose medium.

[0158] For each group of cells, use an ELISA kit to detect the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α in each group of cells.

[0159] (2) Test for the effect on cell adhesion

[0160] THP-1 cells were cultured in RPMI-1640 medium at 37°C and 5% CO2, and fetal bovine serum and 1% (v / v) double antibody (penicillin-streptomycin) were added at a final concentration of 10%. Healthy THP-1 cells were then centrifuged and resuspended in a 1 mL centrifuge tube, and 1 μL of calcein AM was added to each 1 mL of cell suspension. After incubation at 37°C for 30 minutes, the cells were centrifuged and resuspended, and washed 3 times with serum-free RPMI-1640 to obtain a THP-1 cell suspension.

[0161] Then, the HUVECs cells were washed twice with PBS, added to the THP-1 cell suspension, incubated at 37°C for 2 h, and then observed and counted using an inverted fluorescence microscope.

[0162] 2. Experimental results

[0163] (1) The effect of silicified curcumin on HUVECs cells is shown in the figure below. Figure 6 As shown, Figure 6 A in the figure is the Western blot result of ICAM-1 in each group of cells. Figure 6 B in the figure is the quantitative detection result of ICAM-1 in each group of cells. Figure 6 C in the figure is the Western blot result of VCAM-1 in each group of cells. Figure 6 D in the figure is the quantitative detection result of VCAM-1 in each group of cells. Figure 6 E in the figure is the result of RNA expression of ICAM-1 in each group of cells. Figure 6 F in the figure is the result of RNA expression of VCAM-1 in each group of cells. Figure 6 G in the figure is the ROS fluorescence level result diagram of each group of cells. Figure 6 H in the figure is the ROS level result diagram of each group of cells. Figure 6 I in the figure is the Western blot result of HO-1 in each group of cells. Figure 6 Middle J is the quantitative detection result of HO-1 in each group of cells. Figure 6 K in the figure is the Western blot result of Nrf2 in each group of cells. Figure 6 L in the figure is the quantitative detection result of Nrf2 in each group of cells. Figure 6 M in the figure is the Nrf2 protein cell distribution result diagram of each cell well.

[0164] The results showed that under high glucose concentration conditions, the RNA expression levels of the adhesion molecules ICAM-1 and VCAM-1 at the protein level of HUVECs cells in Control group 2 were significantly increased compared with those in Blank group, while the increased expression levels of the above adhesion molecules in Si / Cur group 2, Si group 2 and Cur group 2 were all inhibited, and Si / Cur group 2 showed the most significant inhibitory effect.

[0165] In addition, the increased levels of ICAM-1 and VCAM-1 in HUVECs cells in Control group 2 triggered by high concentrations of glucose further induced oxidative stress response in HUVECs cells, leading to a massive accumulation of intracellular reactive oxygen species (ROS). The ROS levels in Si / Cur group 2, Si group 2 and Cur group 2 were reduced to varying degrees compared with those in Control group 2. At the same time, there was no significant difference between the ROS level in Si / Cur group 2 and that in Blank group.

[0166] At the same time, the detection results of HO-1 protein and Nrf2 protein showed that under high glucose concentration conditions, the expression levels of HO-1 and Nrf2 in Control group 2 were increased compared with those in Blank group, while the expression levels of HO-1 and Nrf2 in Si / Cur group 2, Si group 2 and Cur group 2 were further increased compared with those in Blank group, and the increase in Si / Cur group 2 was the most significant, indicating that Si / Cur group 2, Si group 2 and Cur group 2 all exerted antioxidant and anti-inflammatory effects by regulating the HO-1 and Nrf2 pathways, and the silicified curcumin microspheres prepared in Example 2 had the best antioxidant and anti-inflammatory effects.

[0167] The results of inflammatory factor detection in each group of cells are shown in the figure Figure 7 As shown, Figure 7 A in the figure is the IL-6 detection result of each group of cells. Figure 7 B in the figure is the IL-1β detection result of each group of cells. Figure 7 C in the figure shows the TNF-α detection results of each group of cells.

[0168] The results showed that under high glucose concentration conditions, the expression levels of inflammatory factors IL-1β, IL-6 and TNF-α in Control group 2 were significantly increased compared with those in Blank group, while the expression levels of these inflammatory factors in Si / Cur group 2, Si group 2 and Cur group 2 decreased to varying degrees, and the expression level of inflammatory factors in Si / Cur group 2 was significantly closer to that in Blank group, indicating that the siliconized curcumin microspheres prepared in Example 2 have excellent anti-inflammatory effects in cells.

[0169] (2) Test results on cell adhesion Figure 8 As shown,Figure 8 In [A] is the observation result diagram under an inverted fluorescence microscope, Figure 8 and in [B] is the statistical chart of fluorescence signal results.

[0170] The results showed that: under high glucose concentration conditions, the adhesion of THP-1 cells and HUVECs cells increased significantly, manifested as significantly higher fluorescence signal values in the fluorescence images, while the adhesion of THP-1 cells and HUVECs cells in the group treated with the silicified curcumin microspheres prepared in Example 2 was significantly inhibited and was closer to normal cells.

[0171] II. Effects of silicified curcumin on smooth muscle cells (SMCs)

[0172] 1. Experimental method

[0173] Healthy adult rats were selected and anesthetized. Using sterile surgical instruments, the aorta or other tissues rich in smooth muscle of the rats were taken out, and the tissues were placed in ice-cold sterile PBS to maintain cell viability. Under sterile conditions, the tissues were rinsed repeatedly with PBS to remove blood and impurities. The adventitia and intima of the tissues were stripped using ophthalmic scissors and forceps, and the middle membrane layer (the layer where smooth muscle cells are located) was retained. The stripped middle membrane layer tissues were cut into small pieces, and the tissue pieces were placed in collagenase digestion solution and placed in a thermostatic oscillator for digestion. The digestion temperature was 37 °C, and the digestion time depended on the activity of collagenase, usually 1-2 hours.

[0174] During the digestion process, the digestion degree was checked regularly. When the tissue became transparent and the structure became loose, the digestion was terminated. After digestion, it was rinsed with PBS, and the digestion solution was filtered through a cell sieve to remove undigested tissue fragments. The filtered cell suspension was centrifuged at 1000 rpm for 5 minutes to collect cell pellets. The cell pellets were resuspended in fresh smooth muscle cell medium to obtain a cell suspension, and the cell suspension was inoculated into a pretreated culture dish and cultured in a cell culture incubator. The culture conditions were 37 °C and 5% CO2. SMCs were inoculated into 96-well cell plates at 1000 cells / well. After incubation at 37 °C for 2 h, the following cell groups were set up respectively:

[0175] Control group 3: 100 μL of endothelial cell basal medium (ECM) was added to the cell wells inoculated with SMCs cells for culture.

[0176] The difference between the Si / Cur group 3 and the Control group 3 was that: the endothelial cell basal medium contained 1.875 μg / mL of the silicified curcumin microspheres prepared in Example 2.

[0177] The difference between Si group 3 and Control group 3 is that the endothelial cell basal medium contains 120 μg / mL of the silicon microspheres prepared in Example 2.

[0178] The difference between Cur group 3 and Control group 3 is that the endothelial cell basal medium contains 240 μg / mL of curcumin.

[0179] Blank group 1: SMCs cells were seeded at 2000 cells / well in a 96-well cell culture plate, and 100 μL of endothelial cell basal medium was added for culture.

[0180] After culturing the cells in each group at 37 °C for 48 h, the experimental cells in each group were obtained.

[0181] After the cells in each group covered the bottom of the cell wells, a sterilized ruler was placed on the culture dish and ensured to be vertically aligned with the marked line. Then, a scratch was made, and the original medium was replaced with DMEM medium containing 1% (v / v) fetal bovine serum concentration, and then cultured for another 48 h (the special intervention medium was replaced every 24 h to avoid PBS washing). The culture dishes before and after culture were photographed and observed respectively to obtain the scratch images of the cells in each group, and the scratch area of the cells in each group was measured using ImageJ software to calculate the cell migration rate.

[0182] The cells in each group were digested and resuspended respectively, and seeded in a culture dish with a diameter of 35 mm. After culturing for 24 h, DMEM medium containing 5% (v / v) fetal bovine serum and 0.2% (v / v) growth factor was introduced for culture for 24 h. Then, the medium was replaced with fresh medium and cultured for another 24 h. Then, Western blot protein electrophoresis was used to detect α-SMA, HO-1, and Nrf2 proteins in HUVECs cells (using β-actin as the internal reference protein), and quantitative detection was performed (using β-actin as the internal reference protein).

[0183] And according to the method shown in Step 1, the expression levels of inflammatory factors IL-1β and IL-6 in the cells of each group were detected.

[0184] 2. Experimental results

[0185] The test result graph of the effect of siliconized curcumin on smooth muscle cells is as Figure 9 shown, Figure 9 A in Figure 9 is the scratch image of the cells in each group, Figure 9 B in Figure 9 is the result graph of the cell migration rate of the cells in each group, Figure 9The graph of the expression level of IL-1β in each group of cells is shown in E, Figure 9 The graph of the expression level of IL-6 in each group of cells is shown in F, Figure 9 The Western blot result graph of HO-1 in each group of cells is shown in G, Figure 9 The quantitative detection result graph of HO-1 in each group of cells is shown in H, Figure 9 The Western blot result graph of Nrf2 in each group of cells is shown in I, Figure 9 The quantitative detection result graph of Nrf2 in each group of cells is shown in J.

[0186] The results showed that: compared with Blank group 1, the SMCs in Control group 3 showed significantly enhanced migration ability. However, the SMCs in Si / Cur group 3, Si group 3 and Cur group 3 showed a significant decrease in cell migration ability compared with Control group 3, and there was no significant difference between them and Blank group 1. At the same time, the expression of α-SMA protein in Control group 3 was significantly reduced compared with Blank group 1, while the reduction of α-SMA protein expression in Si / Cur group 3 was significantly inhibited and close to that in Blank group 1.

[0187] At the same time, the expressions of inflammatory factors IL-1β and IL-6 in Control group 3 were both significantly increased, while the increase of inflammatory factors in Si / Cur group 3 was significantly inhibited, indicating that the silica-curcumin microspheres prepared in Example 2 have excellent anti-inflammatory effects. And the effects on HO-1 protein and Nrf2 protein are consistent with those in HUVECs cells, both indicating that the silica-curcumin microspheres prepared in Example 2 have excellent antioxidant and anti-inflammatory properties.

[0188] III. Effects of Silica-Curcumin on Cardiomyocytes

[0189] 1. Experimental Method

[0190] Newborn SD rats aged 1-2 days were obtained from the Experimental Animal Center of Southern Medical University, and primary neonatal rat cardiomyocytes (NRCMs) were isolated from them. The NRCMs were randomly divided into the following experimental groups:

[0191] Control group 4: The NRCMs were seeded in 96-well plates, incubated with 0.5 μM DOX for 24 h, and then cultured with endothelial cell basal medium.

[0192] The difference between Si / Cur group 4 and Control group 4 is that the endothelial cell basal medium was replaced with the silica-curcumin microspheres prepared in Example 2 containing 1.875 μg / mL.

[0193] The difference between Si group 4 and Control group 4 is that the endothelial cell basal medium contains 120 μg / mL of the silicon microspheres prepared in Example 2.

[0194] The difference between Cur group 4 and Control group 4 is that the endothelial cell basal medium contains 240 μg / mL of curcumin.

[0195] Blank group 2: NRCMs cells were seeded in a 96-well cell plate and cultured with 100 μL of endothelial cell basal medium.

[0196] After treating the cells in each group according to the above-mentioned groups for 24 h, the cell viability of the cells in each group was detected by the CCK8 assay method, and the apoptosis of NRCMs in each group of cardiomyocytes was detected by flow cytometry, and the apoptosis rate was calculated.

[0197] Then, the ROS fluorescence levels of the cells in each group were observed and the reactive oxygen species levels in the cells in each group were detected according to the experimental method shown in Step 1; the HO-1 and Nrf2 proteins in the cells in each group were detected according to the method shown in Step 1 to obtain the Western blot images and quantitative detection results of HO-1 and Nrf2 proteins in the cells in each group, and at the same time, the distribution and quantitative results of Nrf2 in the cells in each group were detected.

[0198] 2. Experimental results

[0199] The results of the effect of siliconized curcumin on cardiomyocytes are shown in Figure 10 as follows, Figure 10 in which A is the result graph of the ROS fluorescence levels of the cells in each group, Figure 10 in which B is the result graph of the ROS levels of the cells in each group, Figure 10 in which C is the result of the cell viability determination of the cells in each group, Figure 10 in which D is the result of the cell apoptosis rate of the cells in each group, Figure 10 in which E is the Western blot result graph of HO-1 in the cells in each group, Figure 10 in which F is the Western blot result graph of Nrf2 in the cells in each group, Figure 10 in which G is the quantitative detection result graph of HO-1 in the cells in each group, Figure 10 in which H is the quantitative detection result graph of Nrf2 in the cells in each group, Figure 10 in which I is the result graph of the distribution of Nrf2 in the cells in each group, Figure 10 in which J is the quantitative result graph of the distribution of Nrf2 in the cells in each group.

[0200] Results showed that after induction with DOX, the level of reactive oxygen species (ROS) in NRCMs cells increased significantly and ultimately induced apoptosis, which significantly reduced the cell viability and increased the apoptosis rate of cardiomyocytes; while after intervention with the silica-curcumin microspheres prepared in Example 2, the increase in ROS level in NRCMs cells was effectively prevented, the cells maintained a relatively high viability, and the apoptosis rate was reduced.

[0201] Meanwhile, the effects on HO-1 protein and Nrf2 protein were consistent with those in HUVECs cells, indicating that the silica-curcumin microspheres prepared in Example 2 had excellent antioxidant and anti-inflammatory properties.

[0202] In summary, the silica-curcumin prepared in Example 2 not only had excellent antioxidant and anti-inflammatory effects, but also could reduce the ROS level in cells, avoid the decrease of cell viability and reduce the apoptosis rate.

[0203] Effect of silica-curcumin in Example 5 on mice with a mixed disease of diabetic vascular remodeling and doxorubicin cardiomyopathy

[0204] I. Experimental methods

[0205] Establishment of mice with mixed diseases: SPF-grade C57 mice weighing between 18 and 22 g were intraperitoneally injected with 40 mg / kg of 1% streptozotocin (STZ). After 72 hours, they were fasted again for 4 hours, and blood was taken from the tail to measure fasting blood glucose. If the blood glucose concentration > 11 mmol / L and symptoms of polydipsia, polyphagia, and polyuria appeared, the diabetes induction was successful. After successful induction of diabetes, the mice in the model group received an injection of 15 mg / kg of doxorubicin to obtain mice with mixed diseases.

[0206] The established mice with mixed diseases were grouped and treated as follows:

[0207] Control group 5: The mice with mixed diseases were intravenously injected with normal saline at a dose of 8 μL / g once a day.

[0208] The difference between the Si / Cur group 5 and the Control group 5 was that normal saline was replaced with a 100 μg / mL silica-curcumin microsphere solution, and the other treatments were the same.

[0209] The difference between the Si group 5 and the Control group 5 was that normal saline was replaced with a 1.875 μg / mL silicon microsphere solution.

[0210] The difference between the Sham group 2 and the Control group 5 was that the mice with mixed diseases were replaced with healthy mice, and the other treatments were the same.

[0211] After obtaining the mice in each group according to the above method, the mice in each group were continuously treated for 25 days, and then the following indexes were detected in the mice in each group according to the experimental method shown in Example 3:

[0212] (1) Observation result diagrams of H&E staining, Masson staining and Victoria blue staining of organs and blood vessels from the aortic root to the common iliac artery in each group of mice; (2) Aortic intima-media thickness, collagen fiber area and elastic fiber area in each group of mice; (3) ICAM-1 staining and VCAM-1 staining result diagrams from the aortic root to the common iliac artery in each group of mice; (4) Relative expression levels of ICAM-1 protein and VCAM-1 protein in each group of mice (using β-actin as the internal reference protein); (5) M-mode echocardiogram of the left ventricle in each group of mice; (6) Left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) in each group of mice; (7) Masson staining result diagrams of heart sections in each group of mice and the percentage of positive Masson staining area in myocardial sections of each group of mice; (8) Ratio of heart weight to tibia length (HW / TL) in each group of mice; (9) TUNEL staining result diagrams of heart sections in each group of mice and the results of TUNEL positive cell counting.

[0213] 2. Experimental results

[0214] The result diagrams of the effect of silicified curcumin on mice with mixed diseases are as Figure 11 shown, Figure 11 in which A in Figure 11 is the staining result diagram of the aortic blood vessels in each group of mice, Figure 11 B in Figure 11 is the result diagram of the aortic intima-media thickness in each group of mice, Figure 11 C in Figure 11 is the result diagram of the collagen fiber area in each group of mice, Figure 11 D in Figure 11 is the result diagram of the elastic fiber area in each group of mice, Figure 11 E in Figure 11 is the ICAM-1 staining result diagram in each group of mice, Figure 11 F in Figure 11 is the VCAM-1 staining result diagram in each group of mice, Figure 11 G in Figure 11The N in it is the result graph of the ratio of heart weight to tibia length of each group of mice. Figure 11 The O in it is the result graph of TUNEL staining of heart sections of each group of mice. Figure 11 The P in it is the result graph of the count of TUNEL-positive cells in heart sections of each group of mice.

[0215] The results showed that in mice with both diabetes and adriamycin-induced myocardial injury, the silica-curcumin microspheres prepared in Example 2 could effectively inhibit the increase in the thickness of the vascular wall (intima-media) of mice, and effectively improve the degree of collagen fibrosis and the problem of reduced elastic fibers in the blood vessels of mice.

[0216] Meanwhile, the silica-curcumin microspheres prepared in Example 2 could significantly inhibit the expression of the vascular intima inflammatory factors ICAM-1 and VCAM-1. At the same time, it could also improve adriamycin-induced myocardial injury (including the decrease in ejection fraction and shortening fraction), inhibit the increase in cardiac fibrosis, and inhibit the apoptosis of myocardial cells in mice. And there was no significant difference in the ratio of heart weight to tibia length of the mice administered with the silica-curcumin microspheres prepared in Example 2 compared with healthy mice.

[0217] Biological safety test of silica-curcumin microspheres in Example 6

[0218] I. Experimental method

[0219] The establishment of mixed-disease mice was the same as that in Example 5.

[0220] The established mixed-disease mice were grouped and treated as follows:

[0221] Control group 5: The mixed-disease mice were intravenously injected with normal saline at a dosage of 8 μL / g once a day.

[0222] The difference between Si / Cur group 5 and Control group 5 was that normal saline was replaced with a 100 μg / mL silica-curcumin microsphere solution, and the other treatments were the same.

[0223] The difference between Si group 5 and Control group 5 was that normal saline was replaced with a 1.875 μg / mL silicon microsphere solution.

[0224] The difference between Sham group 2 and Control group 5 was that the mixed-disease mice were replaced with healthy mice, and the other treatments were the same.

[0225] After obtaining the mice of each group according to the above method, each group of mice was continuously treated for 25 days. Then, according to the experimental method shown in Example 3, the following indexes of each group of mice were detected:

[0226] (1) The levels of myocardial creatine kinase isoenzyme MB (CK-MB), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the serum of mice were measured by ELISA method.

[0227] (2) H&E staining of the liver, spleen, lung, and kidney of each group of mice was performed to evaluate the safety of major organs.

[0228] II. Experimental Results

[0229] The quantitative detection results of inflammatory factors in each group of mice are as Figure 12 shown. Figure 12 In A of Figure 12 are the quantitative detection results of CK-MB in each group of mice, Figure 12 in B of Figure 12 are the quantitative detection results of IL-1β in each group of mice,

[0230] The results showed that there was no significant difference in the levels of inflammatory factors in the mice administered with the silicified curcumin microspheres prepared in Example 2 compared with healthy mice.

[0231] The observation results of liver staining of each group of mice are as Figure 13 shown. The results showed that there was no significant difference in the important organs (liver, spleen, lung, and kidney) of the mice administered with the silicified curcumin microspheres prepared in Example 2 compared with healthy mice, indicating that the silicified curcumin microspheres did not cause damage to the organs of mice and had good biosafety.

[0232] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description and ideas. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A preparation method of a curcumin silicate solution, characterized in that, The following steps are involved: S1. Soak the calcium silicate bioceramic in distilled water, PBS, ECM basal culture medium, DMEM basal culture medium or physiological saline at a final concentration of 100-200 mg / mL, and separate the solid and liquid to obtain Si liquid; S2. After the Si liquid obtained in step S1 is fully mixed with curcumin in a ratio of 10-50 mL: 5-50 mg, the liquid is collected by solid-liquid separation to obtain a siliconized curcumin solution.

2. The preparation method according to claim 1, wherein The calcium silicate bioceramic described in step S1 is fully immersed in distilled water, PBS, ECM basal culture medium, DMEM basal culture medium or physiological saline at a final concentration of 200 mg / mL.

3. The preparation method according to claim 1, wherein In step S2, the Si liquid obtained in step S1 and curcumin are fully mixed according to 10-50 mL: 5-50 mg.

4. The silicified curcumin solution prepared by the preparation method according to any one of claims 1 to 3.

5. A preparation method of curcumin silicate microspheres, characterized in that, The following steps are involved: S11. Dissolve cetyltrimethylammonium bromide and ammonium fluoride in water and mix well at 25 to 37° C. to obtain solution 1; The final concentration of hexadecyltrimethylammonium bromide is 1 to 5 mg / mL, and the final concentration of ammonium fluoride is 1 to 12 mg / mL; S12. Calcium nitrate is fully dissolved in anhydrous ethanol at a volume ratio of 0.05-1 g: 1-18 mL, and then mixed with tetraethyl orthosilicate at a volume ratio of 0.1-1: 9-18 to obtain solution 2; S13. The solution 2 obtained in step S12 and the solution 1 obtained in step S11 are mixed in a volume ratio of 1 to 10:100 to 500, and reacted for 1 to 6 hours. After sufficient cooling, the solid and liquid are separated, and the solid is collected, freeze-dried and calcined to obtain silicon microspheres; S14. The silicon microspheres obtained in step S13 are dispersed into the curcumin solution at a ratio of 0.1 to 1 g: 100 to 500 mL, mixed thoroughly, separated into solid and liquid, and the solid is collected and freeze-dried to obtain siliconized curcumin microspheres; The concentration of curcumin in the curcumin solution is 0.2-2 mg / mL.

6. The preparation method according to claim 5, characterized in that, In step S11, the final concentration of the hexadecyltrimethylammonium bromide is 3.64 mg / mL, and the final concentration of the ammonium fluoride is 6 mg / mL.

7. The preparation method according to claim 5, characterized in that, In step S14, the silicon microspheres are dispersed into the curcumin solution at a ratio of 0.2 g:100 mL.

8. The preparation method according to claim 7, characterized in that, The concentration of curcumin in the curcumin solution is 2 mg / mL.

9. Siliconized curcumin microspheres prepared by the preparation method according to any one of claims 5 to 8.

10. Use of the siliconized curcumin solution according to claim 4 and / or the siliconized curcumin microspheres according to claim 9 in the preparation of a drug for simultaneously treating diabetic vascular remodeling and / or myocardial injury.

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