Application of spirulina in preparation of products for treating diabetes-related diseases

Abstract

The invention discloses an application of spirulina (SP) in preparation of a product for treating diabetes-related diseases. According to the application disclosed by the invention, spirulina is found to be capable of alleviating oxidative stress in cells, promoting macrophages to be converted from a proinflammatory M1 type to an anti-inflammatory M2 type, remodeling redox balance and inhibiting an inflammatory microenvironment. More importantly, it is found that spirulina can lower the level of advanced glycation end products (AGEs) to inhibit the glycosylation process, and healing of diabetic wounds and bone defects is accelerated. In addition, the invention also finds that the spirulina can promote proliferation, migration and vascularization of vascular endothelial cells in a high-glucose and high-fat microenvironment, and further accelerate healing of bone defects. On the basis, the invention provides application of spirulina in preparation of products for treating diabetes-related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of spirulina in the preparation of products for treating diabetes-related diseases. BACKGROUND

[0002] Diabetes is a chronic metabolic disease characterized by high blood sugar, caused by insulin secretion defects or dysfunction. Common diabetes-related diseases include diabetic skin wounds and diabetic bone defects. The commonality of the two is that under the high glycotoxicity of diabetes, a large amount of advanced glycation end products (AGEs) will accumulate at diabetic skin wounds and bone defects, triggering a series of adverse reactions, including chronic inflammation and vascular damage, leading to difficulty in healing of diabetic wounds and bone defects.

[0003] In addition, the high glycotoxicity at diabetic wounds and bone defects also causes M1→M2 conversion disorder of macrophages, resulting in accumulation of M1 type macrophages at skin wounds and bone defects, forming excessive inflammatory reactions characterized by oxidative stress, enhanced proteolysis and cell damage, leading to difficulty in healing of diabetic wounds and repair of bone defects.

[0004] Although there are currently a variety of oral hypoglycemic drugs that can effectively control the blood sugar status of diabetic patients, these drugs cannot directly improve the microenvironment at diabetic wounds and bone defects, and have limited therapeutic effect on the two diseases. SUMMARY

[0005] The purpose of the present application is to solve the problem of limited therapeutic effect of existing hypoglycemic drugs on diabetic skin wounds and diabetic bone defects, and to provide the application of spirulina (SP) in the preparation of products for treating diabetes-related diseases.

[0006] In order to achieve the above purpose, the present application provides the application of spirulina in the preparation of products for treating diabetes-related diseases, including diabetic wounds.

[0007] Optionally, the above-mentioned products include at least one of spirulina cell solution, spirulina hydrogel, and spirulina patch, and the spirulina hydrogel is made of spirulina cells and methacrylated gelatin.

[0008] Optionally, the preparation method of the spirulina hydrogel is as follows: The methacrylated gelatin is added to the photoinitiator solution to obtain a hydrogel solution; the spirulina cells are added to the hydrogel solution, and the spirulina hydrogel is obtained after blue-violet light irradiation and solidification; the concentration of methacrylated gelatin in the hydrogel solution is 0.1 g / mL ~ 0.5 g / mL, and the concentration of the photoinitiator solution is 0.0025 g / mL ~ 0.005 g / mL.

[0009] Optionally, the concentration of the above-mentioned Spirulina cells in the hydrogel is 1×10⁻⁶. 4 cells / mL ~ 1×10 8 cells / mL.

[0010] Optionally, the diabetes-related disease includes diabetic bone defects, and the product includes a spirulina scaffold.

[0011] Optionally, the above-mentioned spirulina scaffold is made of palladium-engineered spirulina cells and methacrylated gelatin.

[0012] Optionally, the method for preparing the palladium-engineered Spirulina cells described above is as follows: Sodium chloropalladium and branched polyethyleneimine solution were mixed, and sodium borohydride was added as a reducing agent to obtain palladium nanoparticles modified with branched polyethyleneimine; the branched polyethyleneimine-modified palladium nanoparticles were added to a spirulina cell solution and mixed to obtain palladium-engineered spirulina cells.

[0013] Compared with the prior art, the beneficial effects of the present invention are: This invention reveals that spirulina can alleviate intracellular oxidative stress, promote the conversion of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type, reshape the redox balance, and inhibit the inflammatory microenvironment. More importantly, this invention finds that spirulina can downregulate the level of advanced glycation end products (AGEs) to inhibit the glycation process and accelerate the healing of diabetic wounds and bone defects.

[0014] Furthermore, this invention also found that spirulina can promote the proliferation, migration, and angiogenesis of vascular endothelial cells in a high-sugar microenvironment, thereby further accelerating the healing of diabetic bone defects.

[0015] Based on this, the present invention provides an application of spirulina in the preparation of products for treating diabetes-related diseases. Attached Figure Description

[0016] Figure 1 This is a diagram showing the results of Example 1 of the present invention, which shows that Spirulina can promote the proliferation and migration of fibroblasts; A shows the AGEs fluorescence staining results of mouse fibroblasts after 24 h of high-sugar, high-fat stimulation; B is Figure 1 The relative fluorescence intensity of AGEs in A; C represents the CCK8 experiment results of mouse fibroblasts after 24 h, 48 h, and 72 h of high-sugar and high-fat stimulation; D shows the Transwell experiment results of mouse fibroblasts after 24 hours of high-sugar, high-fat stimulation; E is Figure 1The number of migrating cells of mouse fibroblasts in D.

[0017] Figure 2 Figure for the result that spirulina can induce macrophages to be reprogrammed into M2 type in embodiment 2 of the present application; A is the fluorescence staining result of ROS of macrophages after 24 hours of high-sugar high-fat stimulation; B is Figure 2 A is the relative fluorescence intensity of ROS; C is the fluorescence staining result of CD86 and CD206 of macrophages treated by spirulina after 24 hours of high-sugar high-fat stimulation; D is Figure 2 C is the relative fluorescence intensity of CD86 and CD206.

[0018] Figure 3 Figure for the result that spirulina can promote the healing of diabetic wounds of diabetic rats in embodiment 3 of the present application; A is a representative image of wounds and wound healing rate at 0 day, 3 days, 7 days and 14 days; B is Figure 3 A is the quantitative analysis of wound healing rate; C is the quantitative analysis of granulation tissue thickness of wounds at 0 day, 3 days, 7 days and 14 days; D is the quantitative analysis of epidermis thickness of wounds at 0 day, 3 days, 7 days and 14 days; E is the quantitative analysis of the number of dermal appendages of wounds at 0 day, 3 days, 7 days and 14 days; F is the quantitative analysis of the proportion of collagen of wounds at 0 day, 3 days, 7 days and 14 days.

[0019] Figure 4 Figure for the result of the effect of palladium engineered modified spirulina cells on the proliferation, migration and tube formation of vascular endothelial cells in embodiment 5 of the present application; A is the result of the proliferation experiment of human umbilical vein endothelial cells HUVEC after high-sugar high-fat stimulation; B is the result of the migration experiment of human umbilical vein endothelial cells HUVEC after high-sugar high-fat stimulation; C is the result of the tube formation experiment of human umbilical vein endothelial cells HUVEC after high-sugar high-fat stimulation.

[0020] Figure 5 Figure for the result of the angiogenesis experiment of the diabetic bone defect model in embodiment 5 of the present application; A is the photoacoustic imaging diagram of diabetic skull defect rats in each group at different time points; B is the quantitative analysis diagram of blood oxygen saturation (SO2) of diabetic skull defect rats in each group at different time points; C is the quantitative analysis diagram of hemoglobin (HBT) of each group of diabetic skull defect rats at different time points; D is the quantitative analysis diagram of blood flow area of each group of diabetic skull defect rats at different time points. DETAILED DESCRIPTION

[0021] The technical solutions of the present application are further described below in combination with the drawings and examples.

[0022] Spirulina (SP) is a microalgae that is commercially cultivated as a food worldwide and has been widely used in preclinical studies for oral treatment of tumors, intestinal diseases, and anemia. Recent studies have further expanded its application range, including for magnetic driving and drug transport by intravenous injection, confirming its biocompatibility and safety as an intravenous injection. However, to date, there has been no report of the application of spirulina in products related to diabetes-related diseases.

[0023] The present application found that SP can reduce intracellular oxidative stress, promote the conversion of macrophages from pro-inflammatory M1 type to anti-inflammatory M2 type, reshape the redox balance and inhibit the inflammatory microenvironment. More importantly, the present application found that SP can down-regulate the level of advanced glycation end products (AGEs) to inhibit the glycation process and accelerate the healing of diabetic wounds and bone defects. In addition, the present application also found that SP can promote the proliferation, migration and angiogenesis of vascular endothelial cells in a high-glucose microenvironment, further accelerating the healing of bone defects.

[0024] Based on the above, the present application provides the use of SP in the preparation of products for treating diabetes-related diseases. The present application is further described below in combination with specific examples, and the spirulina used in each example is purchased from Shanghai Guangyu Biotechnology Co., Ltd.

[0025] Example 1: Spirulina can promote fibroblast proliferation and migration Fibroblasts play a core role in diabetic wound healing, and their dysfunction is one of the key mechanisms that leads to the difficulty of healing diabetic wounds. The high glucose toxicity at the site of diabetic wounds can lead to the accumulation of AGEs in fibroblasts, inhibiting the proliferation and migration of fibroblasts, and leading to the difficulty of healing diabetic wounds. The following experiments were conducted in this embodiment to find that spirulina can promote fibroblast proliferation and migration, and promote diabetic wound healing.

[0026] 1. AGEs fluorescence experiment Normal growing L929 mouse fibroblasts were inoculated in a 96-well plate at 5x10 3 Each group of L929 mouse fibroblasts was divided into 3 groups with 3 replicates each, and the groups were as follows: (1) The NC group was cultured with complete culture medium; (2) HFHS group was cultured with high glucose and high fat complete medium added with glucose (55 mM) and palmitic acid (0.12 mM); (3) HFHS+SP group was cultured with high glucose and high fat complete medium (HFHS) added with glucose (55 mM) and palmitic acid (0.12 mM), and co-cultured with spirulina (concentration of 1×10 6 cell / mL).

[0027] After 24 h of culture, AGEs and DAPI fluorescence images were taken, as shown in Figure 1 A and B, AGEs accumulated in L929 mouse fibroblasts under high glucose stimulation, and this accumulation was inhibited after spirulina treatment.

[0028] 2. CCK8 experiment Normal growing L929 mouse fibroblasts were inoculated in 96-well plates, 5×10 3 cells per well, and cultured at 37°C. The L929 mouse fibroblasts were divided into 5 groups, each with 6 replicates, as follows: (1) NC group was cultured with complete medium; (2) HFHS group was cultured with high glucose and high fat complete medium (HFHS) added with glucose (55 mM) and palmitic acid (0.12 mM); (3) HFHS+SP (1×10 6 ) group was cultured with high glucose and high fat complete medium (HFHS) added with glucose (55 mM) and palmitic acid (0.12 mM), and co-cultured with spirulina (concentration of 1×10 6 cell / mL); (4) HFHS+SP (5×10 6 ) group was cultured with high glucose and high fat complete medium (HFHS) added with glucose (55 mM) and palmitic acid (0.12 mM), and co-cultured with spirulina (concentration of 5×10 6 cell / mL); (5) HFHS+SP (1×10 7 ) group was cultured with high glucose and high fat complete medium (HFHS) added with glucose (55 mM) and palmitic acid (0.12 mM), and co-cultured with spirulina (concentration of 1×10 7 cell / mL).

[0029] CCK8 experiment was performed after 24 h, 48 h, and 72 h of culture, and absorbance was detected by microplate reader at 450 nm. As shown in Figure 1As shown in Figure C, under a high-sugar, high-fat environment, the proliferation of fibroblasts in L929 mice was enhanced after treatment with Spirulina.

[0030] 3. Transwell experiment Normally growing L929 mouse fibroblasts were collected at a density of 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 200 μL of serum-free medium in the upper chamber of a 24-well Transwell chamber. Subsequently, 500 μL of Spirulina SP at different concentrations (1 × 10⁻⁶) was added to the lower chamber. 6 5×10 6 1×10 7 The culture medium (cells / mL) was incubated at 37°C for 24 h. L929 cells in the upper chamber were fixed with 1 mL of 4% paraformaldehyde for 15 min. The fixative was discarded, and the cells were washed twice with PBS. Subsequently, 1 mL of 0.5% crystal violet was added to each well, and staining was performed for 20 min. The chondrocytes were washed three times with PBS. Images were taken, and cells were counted at 200x magnification.

[0031] like Figure 1 As shown in D and E, under a high-sugar, high-fat environment, the migration ability of L929 mouse fibroblasts was enhanced after treatment with Spirulina.

[0032] Example 2: Spirulina can induce macrophages to reprogram into the M2 type. During normal wound healing, macrophages undergo a sophisticated “M1→M2” phenotypic transition: in the early stage of wound healing (1-3 days), monocytes infiltrate and differentiate into M1 macrophages; in the later stage of wound healing (from about the 4th day onwards), M1 macrophages transform into M2 macrophages, which play an anti-inflammatory and tissue repair role.

[0033] The hyperglycemic environment of diabetes severely disrupts this normal process, leading to excessive production of reactive oxygen species (ROS) in macrophages, inhibiting the conversion of M1 macrophages to M2 macrophages, resulting in a persistent dominance of M1 macrophages and a severe deficiency of M2 macrophages, thus hindering the achievement of anti-inflammatory and tissue repair functions.

[0034] This embodiment uses the following experiments to find that Spirulina can induce macrophages to reprogram into the M2 type, promoting wound healing in diabetic patients.

[0035] 1. ROS fluorescence experiment Normally growing Raw264.7 macrophages were seeded into 6-well plates at a density of 5 × 10⁶ cells per well. 3 Raw264.7 macrophages were cultured at 37°C. The macrophages were divided into 5 groups, with 4 replicates per group: (1) The NC group was cultured in a complete culture medium; (2) HFHS group was cultured with high glucose and high fat complete medium (HFHS) added with glucose (55 mM) and palmitic acid (0.12 mM); (3) HFHS+SP (1x10 6 ) group was cultured with high glucose and high fat complete medium (HFHS) added with glucose (55 mM) and palmitic acid (0.12 mM), and co-cultured with spirulina (concentration of 1x10 6 cell / mL); (4) HFHS+SP (5x10 6 ) group was cultured with high glucose and high fat complete medium (HFHS) added with glucose (55 mM) and palmitic acid (0.12 mM), and co-cultured with spirulina (concentration of 5x10 6 cell / mL); (5) HFHS+SP (1x10 7 ) group was cultured with high glucose and high fat complete medium (HFHS) added with glucose (55 mM) and palmitic acid (0.12 mM), and co-cultured with spirulina (concentration of 1x10 7 cell / mL).

[0036] After 24 h of culture, the cells were washed with sterile PBS buffer (pH = 7.4), and then 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) solution was added and incubated for 20 min. Then the cells in the 6-well plate were observed and photographed under an inverted fluorescence microscope. As shown in Figs. A and B, ROS accumulated in Raw264.7 macrophages under high-fat high-sugar stimulation, and this accumulation was inhibited after spirulina treatment. Figure 2

[0037] 2. M2 macrophage reprogramming experiment Normal growing Raw264.7 macrophages were inoculated in 6-well plates at 5x10 3 cells per well, and cultured at 37°C. The Raw264.7 macrophages were divided into 3 groups with 3 repeats in each group, which were: (1) NC group was cultured with complete medium; (2) HFHS group was cultured with high glucose and high fat complete medium added with glucose (55 mM) and palmitic acid (0.12 mM); (3) HFHS+SP group was cultured with high glucose and high fat complete medium (HFHS) added with glucose (55 mM) and palmitic acid (0.12 mM), and co-cultured with spirulina (concentration of 1x10 6 cell / mL).

[0038] ​After 24 h of culture, the cells were washed with PBS for 3 times, then fixed in 4% paraformaldehyde for 20 min, washed with PBS for 3 times again, 0.5% Triton-X-100 was added to the plate and incubated at room temperature for 10 min, the cells were blocked with 5% bovine serum albumin (BSA) for 30 min, then incubated with primary antibodies CD86 (for labeling M1 macrophages) and CD206 (for labeling M2 macrophages) at 37°C for 2 h in the dark, followed by incubation with corresponding fluorescent secondary antibodies for 1 h at room temperature, the cells were stained with DAPI for 10 min, and photographed using a fluorescence confocal microscope.

[0039] As shown in C and D of FIG. 6, under high-sugar high-fat stimulation, Raw264.7 macrophages differentiated into pro-inflammatory M1 macrophages, while after spirulina treatment, the differentiation of M1 macrophages was inhibited and the differentiation of anti-inflammatory M2 macrophages was promoted. Figure 2 As shown in C and D of FIG. 6, under high-sugar high-fat stimulation, Raw264.7 macrophages differentiated into pro-inflammatory M1 macrophages, while after spirulina treatment, the differentiation of M1 macrophages was inhibited and the differentiation of anti-inflammatory M2 macrophages was promoted.

[0040] Example 3: Spirulina can promote diabetic wound healing in diabetic rats The following experimental findings were made in this example: spirulina can promote diabetic wound healing in diabetic rats.

[0041] Diabetic rat model was constructed: all SD rats were fed with high-fat and high-sugar diet for four weeks. After the end of feeding, the rats were fasted for 12 hours, and then injected with 65 mg / kg body weight of streptozotocin STZ solution (dissolved in phosphate buffer, pH 4.5) through intraperitoneal injection. One week after injection, blood was collected from the tail vein, and blood glucose level was measured. Rats with blood glucose level ≥16 mmol / L were considered as diabetic rats.

[0042] The above diabetic rats were shaved and anesthetized on their backs, and a biopsy punch was used to make a wound of the same diameter on each side of their backs in a sterile operating table. Each rat was individually raised and prevented from bacterial infection during the period. The rats were divided into 3 groups, 4 rats in each group, respectively: (1) Saline group: wound smeared with saline; (2) GelMA-gel group: wound covered with GelMA hydrogel, which was prepared as follows: Methacrylated gelatin (GelMA) was added to a photoinitiator

lithium phenyl (2,4,6-trimethylbenzoyl) phosphate salt

[0043] (3) SP-gel group: wound covered with spirulina hydrogel, which was prepared as follows: Methacrylated gelatin was added into the solution of photoinitiator

lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate salt

[0044] The wound diameter was measured regularly and photographed, as shown in Figure 3 Compared with the Saline group and the GelMA-gel group, the wound of the SP-gel group was shallow and small after spirulina treatment, indicating that spirulina can promote the healing of diabetic wounds.

[0045] Example 4: Preparation method of spirulina scaffold The present embodiment provides a spirulina scaffold, which is prepared by the following method: Step 1: A branched polyethyleneimine solution (PEI, molecular weight Mw ~ 25,000 g / mol) of 1 mg / mL was prepared, and 2 mL of a sodium tetrachloropalladate (Na2PdCl4) solution (1 mM) was slowly added thereto, and the mixture was stirred for 30 minutes to complex PEI with Na2PdCl4, thereby obtaining a PEI-Na2PdCl4 solution.

[0046] The above complexation of PEI with Na2PdCl4 is achieved by the amine group of PEI and the Pd 2+ in Na2PdCl4.

[0047] Step 2: A sodium borohydride (NaBH4) solution (10 mM) of 10 mL was prepared under ice bath conditions, and it was added dropwise into the PEI-Na2PdCl4 solution, and the mixture was continuously stirred at 1000 rpm for 1-2 hours, so that NaBH4 reduced Pd 2+ to Pd nanoparticles, thereby obtaining branched polyethyleneimine-modified palladium nanoparticles (PEI-Pd).

[0048] During the above reduction process of NaBH4, PEI is adsorbed on the surface of Pd nanoparticles, PEI is positively charged, and there is an electrostatic repulsion effect between molecules, which can prevent Pd nanoparticles from aggregating, thereby obtaining uniformly dispersed Pd nanoparticles.

[0049] Step 3: The PEI-Pd particles were washed with ultrapure water and dispersed into 10 mL of a spirulina cell solution, and the mixture was shaken at room temperature for 4 hours. Since the surface of PEI is positively charged and the surface of spirulina is negatively charged, electrostatic interaction can enable PEI to bind to spirulina cells, thereby obtaining spirulina cells modified by palladium engineering (SP-Pd cells).

[0050] Step 4, dissolve the methacrylated gelatin in 0.25% (w / v) light initiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) solution in a 60-70 ℃ water bath under light shielding condition, shake well, then filter immediately with a 0.22 μm sterile filter (to prevent the hydrogel solution from gelling at low temperature) to obtain a hydrogel solution.

[0051] The mass-volume ratio of the methacrylated gelatin and the LAP solution is 1 g: 10 mL.

[0052] Step 5, mix the palladium-engineered spirulina cells with the hydrogel solution to obtain a 3D bioprinting ink. Load the 3D bioprinting ink into the low-temperature cartridge of a 3D printer, print layer by layer, irradiate 405 nm blue-violet light for 30 s after the end of each layer to crosslink and solidify, and obtain a spirulina scaffold after the whole solidification.

[0053] The concentration of the spirulina cells in the 3D bioprinting ink is 1×10 7 cell / ml.

[0054] The spirulina scaffold provided in the embodiment can release the palladium-engineered spirulina cells slowly as the hydrogel degrades, and the spirulina cells have enhanced repair effect on diabetic bone defects after being modified by palladium nanoparticles, and can better complete the repair of diabetic bone defects.

[0055] The spirulina scaffold provided in the embodiment takes palladium and spirulina cells as the main functional components, and takes collagen, gelatin, hyaluronic acid, chitosan and other natural hydrogels as the main matrix components. These materials all have good biocompatibility, and the spirulina scaffold prepared therefrom will not produce allergic and toxic reactions after being implanted into a living body.

[0056] Example 5 Palladium-engineered spirulina cells promote vascularization under high-sugar high-fat environment 1. Proliferation, migration and angiogenesis of human umbilical vein endothelial cells in vitro The proliferation, migration and tube formation ability of vascular endothelial cells are the core driving force for bone defect repair, and all of them promote angiogenesis to provide a suitable microenvironment for osteoblasts.

[0057] Take human umbilical vein endothelial cells HUVEC growing normally, inoculate 5×10 3 4 cells per well in a 96-well plate, and culture at 37℃. Divide the human umbilical vein endothelial cells into 4 groups, each with 4 replicates, and they are: (1) The Con group is cultured with complete culture medium; (2) DM group is cultured with high-sugar high-fat complete medium (HFHS) added with glucose (25.5 mM) and palmitic acid (0.2 mM); (3) SP group is cultured with high-sugar high-fat complete medium (HFHS) added with glucose (25.5 mM) and palmitic acid (0.2 mM), and co-cultured with spirulina (the concentration of spirulina is 1×10 6 cell / mL); (4) SPP group is cultured with high-sugar high-fat complete medium (HFHS) added with glucose (25.5 mM) and palmitic acid (0.2 mM), and co-cultured with palladium-engineered spirulina (the concentration of palladium-engineered spirulina is 1×10 6 cell / mL).

[0058] After culturing the cells in each group for 1, 3, 5 and 7 days, CCK8 experiment was performed, and the absorbance was detected by microplate reader at 450 nm. Figure 4 As shown in FIG. 1A, under the stimulation of high-sugar high-fat, the proliferation ability of human umbilical vein endothelial cells was enhanced after treatment with palladium-engineered spirulina cells.

[0059] Scratch experiment was performed on the cells in each group, and the scratch migration area at 12 h and 24 h was calculated by ImageJ software. Figure 4 As shown in FIG. 1B, under the stimulation of high-sugar high-fat, the migration ability of human umbilical vein endothelial cells was enhanced after treatment with palladium-engineered spirulina cells.

[0060] Tube formation experiment was performed on the cells in each group, and microscopic observation and photography were performed after 1 day. Figure 4 As shown in FIG. 1C, under the stimulation of high-sugar high-fat, the tube formation ability of human umbilical vein endothelial cells was enhanced after treatment with palladium-engineered spirulina cells.

[0061] 2. Vascularization of diabetic bone defect model in vivo 75 male SD rats (8 weeks old, weighing 250-280 g) were randomly divided into 5 groups with 5 replicates in each group: (1) Con group: normal feeding, to construct a skull defect model; (2) DM group: to construct a diabetic rat model by the method of Example 3, and then to construct a skull defect model; (3) Gel group: to construct a diabetic rat model by the method of Example 3, to construct a skull defect model, and then to implant a GelMA scaffold at the bone defect site. The preparation method of the GelMA scaffold is as follows: GelMA is added into a LAP photoinitiator solution to obtain a hydrogel solution; the hydrogel solution is loaded into a 3D printer for layer-by-layer printing, and a GelMA scaffold is obtained after blue-violet light irradiation and solidification.

[0062] (4) SP@Gel group: A diabetic rat model was constructed using the method in Example 3, and a skull defect model was constructed. Then, SP@Gel scaffolds were implanted at the bone defect sites. The difference between the SP@Gel scaffold and the GelMA scaffold mentioned above is that Spirulina cells were added to the SP@Gel scaffold.

[0063] (5) SPP@Gel group: A diabetic rat model was constructed using the method in Example 3, and a skull defect model was constructed. Then, an SPP@Gel scaffold was implanted at the bone defect site. The difference between the SPP@Gel scaffold and the GelMA scaffold is that palladium-engineered Spirulina cells were added to the SPP@Gel scaffold.

[0064] The above-mentioned method for constructing the diabetic rat model of skull defects is as follows: diabetic rats are anesthetized by intraperitoneal injection of sodium pentobarbital, a midline incision is made along the skull with a scalpel, and a circular defect with a diameter of 5 mm is created in the center of the skull with a 5 mm trephine saw.

[0065] Regular photoacoustic / ultrasound imaging was performed to assess blood flow, oxygen saturation, and hemoglobin levels within the skull defect area. Imaging was performed using the VEVO LAZR photoacoustic imaging system (Fujifilm VisualSonics, Japan) at 4, 8, and 12 weeks postoperatively. Figure 5 As shown, Figure 5 In A, the first row at each time point is the ultrasound image, the second row (blue and red) represents the intensity of the photoacoustic signal; the higher the signal, the higher the blood oxygen saturation (SO2) and hemoglobin (HBT); and the third row (red) represents blood flow. Figure 5 B, C, and D are Figure 5 The quantitative results of various vascular properties in A show that the SPP@Gel scaffold can effectively enhance vascular regeneration at diabetic bone defects and help repair bone defects.

[0066] In summary, this invention reveals that spirulina can alleviate intracellular oxidative stress, promote the conversion of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type, reshape redox balance, and inhibit the inflammatory microenvironment. More importantly, this invention finds that spirulina can downregulate the level of advanced glycation end products (AGEs) to inhibit glycation, thereby accelerating the healing of diabetic wounds and bone defects. Furthermore, this invention also finds that spirulina can promote the proliferation, migration, and angiogenesis of vascular endothelial cells in a high-glycemic microenvironment, further accelerating the healing of bone defects. Based on these findings, this invention provides an application of spirulina in the preparation of products for treating diabetes-related diseases.

[0067] While the application has been described in detail and with reference to specific preferred embodiments thereof, it will be apparent to one skilled in the art that various modifications and alternatives can be employed without departing from the spirit and scope of the application. Accordingly, the scope of the application should be determined by the appended claims and their equivalents.

Claims

1. Application of Spirulina in the preparation of products for the treatment of diabetes-related diseases.

2. The application as described in claim 1, characterized in that, The diabetes-related conditions include: diabetic wounds.

3. The application as described in claim 2, characterized in that, The product includes at least one of spirulina cell solution, spirulina hydrogel, and spirulina patch.

4. The application as described in claim 3, characterized in that, The spirulina hydrogel is made from spirulina cells and methacrylated gelatin.

5. The application as described in claim 3, characterized in that, The preparation method of the spirulina hydrogel is as follows: Methacrylated gelatin was added to a photoinitiator solution to obtain a hydrogel solution; Spirulina cells were added to the hydrogel solution, and the solution was cured by blue-violet light to obtain the Spirulina hydrogel; the concentration of methacrylated gelatin in the hydrogel solution was 0.1 g / mL to 0.5 g / mL, and the concentration of the photoinitiator solution was 0.0025 g / mL to 0.005 g / mL.

6. The application as described in claim 5, characterized in that, The concentration of the spirulina cells in the hydrogel was 1×10⁻⁶. 4 cells / mL ~ 1×10 8 cells / mL.

7. The application as described in claim 1, characterized in that, The diabetes-related diseases mentioned include: diabetic bone defects.

8. The application as described in claim 7, characterized in that, The product includes a spirulina support.

9. The application as described in claim 8, characterized in that, The spirulina scaffold is made of palladium-engineered spirulina cells and methacrylated gelatin.

10. The application as described in claim 9, characterized in that, The method for preparing palladium-engineered Spirulina cells is as follows: Sodium chloropalladium and branched polyethyleneimine solution were mixed, and sodium borohydride was added as a reducing agent to obtain palladium nanoparticles modified with branched polyethyleneimine; the branched polyethyleneimine-modified palladium nanoparticles were added to a spirulina cell solution and mixed to obtain palladium-engineered spirulina cells.

Images