Research method for promoting healing of diabetic wound by ginger extract hydrogel

Abstract

The invention discloses a research method for promoting healing of diabetic wounds by ginger extract hydrogel, and relates to the technical field of ginger extracts. The method specifically comprises the following steps: step 1, carrying out a biocompatibility experiment on the ginger extract hydrogel; living cells and dead cells are distinguished by adopting a living and dead immunofluorescence staining experiment, and the expression condition of specific protein in the cells is detected by utilizing fluorescence labeling; 2, carrying out an experiment on the influence of the ginger extract hydrogel on the activity of NIH3T3 cells; the CCK8 reagent is used for evaluating the condition of incubating NIH3T3 cells by ginger extracts with different concentrations. According to the research method, experiments show that the ginger extract hydrogel on diabetic mice can relieve wound inflammatory factor secretion, promote increase of anti-inflammatory factor secretion and promote healing of diabetic wounds; the expression of related indexes of fibroblasts is promoted, the collagen deposition is promoted, and the healing of diabetic wounds is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of ginger extracts, and in particular to a research method for promoting diabetic wound healing by ginger extract hydrogels. Background Art

[0002] Wound healing is a complex and orderly biological process, including four classic stages: hemostasis, inflammation, proliferation, and remodeling. It involves different cells that release many cytokines and growth factors. Fibroblasts play a vital role in wound healing. They are one of the main cell types in connective tissue, responsible for the synthesis and secretion of extracellular matrix components (such as collagen and elastin), and are also involved in regulating many aspects of the healing process. Specific functions include: synthesizing collagen, promoting angiogenesis, regulating inflammatory responses, participating in tissue remodeling, and generating scar tissue. In short, fibroblasts coordinate and promote the healing process through a variety of mechanisms during wound healing, and can effectively repair damaged tissues.

[0003] Ginger is one of the oldest known spices and has a variety of bioactive compounds that have been linked to various health benefits. Ginger extract has been shown to have anti-inflammatory, antioxidant, and anti-cancer effects.

[0004] At present, there are 504 patents related to wound dressings in China, but only 10 of them are related to diabetic wound dressings. These 10 patents are mainly aimed at bacterial infection and chronic inflammation of diabetic wounds, and rarely involve regulating the wound microenvironment (fibroblast proliferation) to promote diabetic wound healing. Difficulty in diabetic wound healing is one of the most serious complications of diabetes. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a research method for promoting diabetic wound healing by ginger extract hydrogel.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A research method for promoting diabetic wound healing by ginger extract hydrogel, specifically comprising the following steps:

[0008] Step 1: Conduct biocompatibility experiments on ginger extract hydrogels; use live-dead immunofluorescence staining experiments to distinguish live cells from dead cells, and use fluorescent markers to detect the expression of specific proteins in cells;

[0009] Step 2: Conduct an experiment on the effect of ginger extract hydrogel on the viability of NIH3T3 cells; use CCK8 reagent to evaluate the incubation of NIH3T3 cells with different concentrations of ginger extract;

[0010] Step 3: Conduct an experiment on promoting NIH3T3 proliferation by ginger extract hydrogel; observe the effect of ginger extract hydrogel on promoting fibroblast proliferation by scratch test;

[0011] Step 4: Conduct an experiment on the effect of ginger extract hydrogel on diabetic wounds; use diabetic mice and normal mice as controls to conduct an enzyme-linked immunosorbent assay experiment;

[0012] Step 5: Conduct an experiment on the effect of ginger extract hydrogel on fibroblasts; use quantitative polymerase chain reaction experiment to measure the effect of ginger extract hydrogel on fibroblasts.

[0013] Preferably: the step 1 specifically includes the following steps:

[0014] S11: Cultivate NIH3T3 cells and plate them in 6-well plates, and divide them into: control group, blank hydrogel group, ginger extract hydrogel group;

[0015] S12: After incubation for 24 hours, the culture medium was removed, the cells were rinsed with PBS 2-3 times, and then live-dead cell dye prepared at a dilution of 1:200 was added and incubated for 30 minutes;

[0016] S13: Remove the culture medium containing the dye, add serum-free DMEM, and observe under a fluorescence microscope.

[0017] Further: the step 2 specifically includes the following steps:

[0018] S21: NIH3T3 cells were seeded in a 96-well plate at a cell count of 5000 cells per well with 100 μl added;

[0019] S22: After incubation for 24 h, different concentrations of ginger extract hydrogels were added and incubated for another 24 h;

[0020] S23: Remove the culture medium, add 10 μl of CCK-8 reagent to each well, and mix CCK8 reagent and DMEM;

[0021] S24: Continue incubating in the cell culture incubator for 1 hour;

[0022] S25: Measure absorbance at 450 nm.

[0023] As a preferred embodiment of the present invention: the step three specifically includes the following steps:

[0024] S31: NIH3T3 cells were seeded into 24-well plates at an appropriate density for culture;

[0025] S32: Scratch preparation: Use a sterile pipette tip or a scratcher to gently make a straight line or grid-like wound on the cell monolayer;

[0026] S33: Wash the cells, rinse the cells twice with PBS;

[0027] S34: Replace the culture medium: Replace with fresh culture medium to maintain consistency of experimental conditions;

[0028] S35: Observe the healing of the scratch.

[0029] Based on the above scheme: Step 4 specifically includes the following steps:

[0030] S41: A mouse model was constructed, and 40 C57 male mice were taken, including 30 C57 male mice with established diabetes and 10 normal C57 male mice without any treatment. The C57 male mice with established diabetes were divided into a diabetes model group, a blank hydrogel group, and a ginger extract hydrogel group.

[0031] S42: Enzyme-linked immunosorbent assay experiments were performed on mouse models and statistical analysis was performed to obtain experimental results.

[0032] Preferably: the step five specifically includes the following steps:

[0033] S51: Sample preparation;

[0034] S52: prepare qPCR reaction mixture;

[0035] S53: Set thermal cycling conditions according to the experimental purpose and the qPCR instrument used;

[0036] S54: Load samples and run qPCR;

[0037] S55: Data analysis.

[0038] The beneficial effects of the present invention are:

[0039] This research method was found through experiments: ginger extract hydrogel reduced the secretion of inflammatory factors in the wound of diabetic mice, promoted the secretion of anti-inflammatory factors, and promoted the healing of diabetic wounds; it promoted the expression of fibroblast-related indicators, promoted collagen deposition, and promoted the healing of diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a flow chart of the research method of the ginger extract hydrogel of the present invention for promoting diabetic wound healing;

[0041] Figure 2 This is a result picture taken under a fluorescence microscope of the live-dead immunofluorescence staining experiment of the present invention;

[0042] Figure 3This is a graph showing the absorbance results of the CCK8 reagent of the present invention evaluating NIH3T3 cells incubated with ginger extracts of different concentrations;

[0043] Figure 4 This is a microscopic result of evaluating the effect of ginger extract hydrogel on fibroblast proliferation through a scratch test in the present invention;

[0044] Figure 5 The wound surface ELISA index of the present invention is a bar chart of IL-10;

[0045] Figure 6 The wound surface ELISA index of the present invention is a bar chart of IL-6;

[0046] Figure 7 The wound surface ELISA index of the present invention is a bar chart of TNF-α;

[0047] Figure 8 The wound surface qPCR index of the present invention: a bar chart of TGF-β;

[0048] Fig. 9 This is a bar chart of the wound surface qPCR indicator: COLI. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0050] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0051] A research method for promoting diabetic wound healing by ginger extract hydrogel, such as Figures 1 to 9 As shown, the specific steps include:

[0052] Step 1: Conduct biocompatibility experiments on ginger extract hydrogels; use live-dead immunofluorescence staining experiments to distinguish live cells from dead cells, and use fluorescent markers to detect the expression of specific proteins in cells.

[0053] The specific steps include:

[0054] S11: NIH3T3 (mouse embryonic fibroblast cell line) was cultured and plated in 6-well plates, divided into: control group (CON), blank hydrogel group (Blank hydrogel), ginger extract hydrogel group (Ginger extract hydrogel); after NIH3T3 grew to 50-60% in the culture medium, different hydrogels were added and incubated for 24 hours.

[0055] S12: After incubation for 24 hours, the culture medium was removed, and the cells were rinsed with PBS (phosphate buffer) for 2-3 times. Then, live and dead cell dyes prepared at a dilution of 1:200 were added and incubated for 30 minutes. The live and dead cell dyes include Calcein AM staining solution and PI staining solution.

[0056] Calcein AM Staining Solution is a fast and convenient solution for live cell staining of 3D cultured cell spheroids or organoids. After staining for 10 minutes, very bright green fluorescence staining of live cells can be observed under a fluorescence microscope. PI Staining Solution is a fast and convenient solution for nucleus staining of necrotic cells in 3D cultured cell spheroids or organoids. After staining for 10 minutes, very bright red fluorescence staining of nuclei in necrotic cells can be observed under a fluorescence microscope.

[0057] S13: Remove the culture medium containing the dye, add serum-free DMEM, and observe under a fluorescence microscope.

[0058] Reference Figure 2 ,The experimental results are as follows: ,It can be observed from live-dead immunofluorescence staining that the ,hydrogel has no significant effect on the activity and proliferation ,of fibroblasts after 24h of incubation, and the ginger extract hydrogel has ,good biocompatibility.

[0059] Step 2: Conduct an experiment on the effect of ginger extract hydrogel on the viability of NIH3T3 cells; use CCK8 reagent to evaluate the incubation of NIH3T3 cells with different concentrations of ginger extract.

[0060] The specific steps include:

[0061] S21: NIH3T3 cells were seeded in a 96-well plate at a cell number of 5,000 cells per well by adding 100 μl.

[0062] S22: After incubation for 24 h, different concentrations of ginger extract hydrogel were added and incubated for another 24 h.

[0063] S23: Remove the culture medium, add 10 μl of CCK-8 reagent to each well, and mix the CCK8 reagent and DMEM.

[0064] S24: Continue incubating in the cell culture incubator for 1 hour.

[0065] S25: Measure absorbance at 450 nm.

[0066] Reference Figure 3 ,The experimental results are as follows: CCK8 evaluated the incubation of NIH3T3 cells with different concentrations of ginger extract. The ginger extract hydrogel has the effect of promoting proliferation, and the increase depends on the concentration gradient.

[0067] Step 3: Conduct an experiment on promoting NIH3T3 proliferation by ginger extract hydrogel; observe the effect of ginger extract hydrogel on promoting fibroblast proliferation through a scratch test.

[0068] Specifically include the following steps:

[0069] S31: NIH3T3 cells were seeded into 24-well plates at an appropriate density for culture and divided into: control group (CON), blank hydrogel group (Blank hydrogel), ginger extract hydrogel group (Ginger extract hydrogel), until the cells grew to 80%-90% confluence.

[0070] S32: Scratch preparation: Use a sterile pipette tip or a scratcher to gently make a straight line or grid-like wound on the cell monolayer, ensuring that a uniform scratch can be formed; at the scratch, try to avoid damaging surrounding cells to reduce interference with the data.

[0071] S33: Wash the cells and rinse the cells twice with PBS (phosphate buffered saline) to remove free, non-attached cells.

[0072] S34: Replace culture medium: Replace with fresh culture medium to maintain consistency of experimental conditions.

[0073] S35: Observation of scratch healing: Use a microscope to take images of the scratches, record the initial scratch state (T0), add different hydrogels for incubation, and then take pictures at different time points such as 0, 24, and 48 hours to observe the width of the scratch or the number of migrated cells.

[0074] Reference Figure 4 The experimental results are as follows: Scratch test: At 24h and 48h, ginger extract hydrogel significantly promoted fibroblast proliferation compared with the other two groups

[0075] Step 4: Conduct an experiment on the effect of ginger extract hydrogel on diabetic wounds; use diabetic mice and normal mice as controls to conduct an enzyme-linked immunosorbent assay experiment;

[0076] Specifically include the following steps:

[0077] S41: A mouse model was constructed. 40 C57 male mice were taken, including 30 C57 male mice with established diabetes and 10 normal C57 male mice without any treatment. The C57 male mice with established diabetes were divided into a diabetes model group (DM), a blank hydrogel group (Blank hydrogel), and a ginger extract hydrogel group (Ginger extract hydrogel). The normal C57 male mice were used as a control group (CON).

[0078] After one week of adaptation and culture, the four groups of mice were skinned and wounds with a diameter of 1 cm were established. The four groups were given different treatments: CON group: no treatment; DM group: no treatment; Blank hydrogel group: given blank hydrogel; Ginger extract hydrogel group: given ginger extract hydrogel;

[0079] After 2 weeks of treatment, high concentrations of CO 2 After death from asphyxiation, the wound skin was taken; half of the wound skin was reserved for enzyme-linked immunosorbent assay (ELISA) experiment.

[0080] S42: Enzyme-linked immunosorbent assay experiments were performed on mouse models and statistical analysis was performed to obtain experimental results.

[0081] The ELISA test is a laboratory technique used to detect and quantify antigens or antibodies. The basic steps are as follows:

[0082] 1. Prepare reagents and materials: coating buffer; test sample; enzyme-labeled secondary antibody (secondary antibody conjugated with enzyme, such as horseradish peroxidase HRP or alkaline phosphatase AP); substrate solution (substrate that produces color reaction, such as TMB or OPD); stop solution; washing solution (such as PBS containing Tween 20 solution); standard (for establishing standard curve); microplate reader (for light absorption measurement).

[0083] 2. Coating antigen: dilute the antigen in coating buffer, add the diluted antigen to the pre-numbered wells of the ELISA plate, seal the ELISA plate, place it in a wet box, and incubate at 4°C overnight or at room temperature for 1-2 hours.

[0084] 3. Washing: Discard the liquid in the wells and wash the wells thoroughly with washing solution to remove unadsorbed antigens.

[0085] 4. Blocking: Add blocking solution (such as milk, BSA or blocking buffer) to each well to block non-specific binding sites and incubate at room temperature for 1-2 hours.

[0086] 5. Washing: Wash the ELISA plate again 3-5 times, remove the blocking solution, and add the primary antibody:

[0087] 6. Dilute the sample to be tested (antibody) in diluent.

[0088] 7. Discard the liquid in the wells, wash the wells thoroughly with washing solution to remove unbound primary antibody, and add enzyme-labeled secondary antibody.

[0089] 8. Dilute the enzyme-labeled secondary antibody (secondary antibody bound to an enzyme) in diluent, add it to each well, and incubate to allow the secondary antibody to bind to the primary antibody.

[0090] 9. Add substrate: Add substrate solution to each well, and the enzyme catalyzes the substrate to produce a color reaction.

[0091] 10. Color development: Depending on the substrate used, the ELISA plate will develop color within a certain period of time.

[0092] 11. Stop reaction: Add stop solution to each well to stop the color reaction.

[0093] 12. Determine absorbance: Use an ELISA reader to measure the absorbance (OD value) of each well, usually at a wavelength of 450nm; calculate the concentration of the sample based on the standard curve, perform statistical analysis, and obtain the experimental results.

[0094] Reference Figures 5 to 7 The experimental results are as follows: Ginger extract hydrogel reduced the secretion of inflammatory factors in the wound of diabetic mice, promoted the secretion of anti-inflammatory factors, and promoted the healing of diabetic wounds.

[0095] Step 5: Conduct an experiment on the effect of ginger extract hydrogel on fibroblasts; use a quantitative polymerase chain reaction (qPCR) experiment to measure the effect of ginger extract hydrogel on fibroblasts.

[0096] Quantitative polymerase chain reaction (qPCR) is a molecular biology technique used to quantitatively measure DNA or RNA expression levels; it includes the following steps:

[0097] S51: Sample preparation: Extraction and purification of DNA or RNA samples.

[0098] S52: Prepare the qPCR reaction mixture, including: qPCR Master Mix (including DNA polymerase, dNTPs, MgCl2, etc.); forward and reverse primers; cDNA; sterile water or nuclease-free water.

[0099] S53: Set the thermal cycling conditions according to the experimental purpose and the qPCR instrument used, which usually include: initial denaturation step (95°C, 15-30 seconds); cycling process, including: denaturation (95°C, 3-5 seconds), annealing (depending on the primer Tm value, 5-10 seconds), extension (72°C, 10-30 seconds), melting curve analysis (60-95°C, to determine the specificity of PCR products).

[0100] S54: Load samples and run qPCR: Add the reaction system to the qPCR reaction tube; add it to the qPCR instrument and run the set program.

[0101] S55: Data analysis.

[0102] Reference Figures 8 to 9 The experimental results are as follows: Ginger extract hydrogel promoted the expression of fibroblast-related indicators in diabetic mice, promoted collagen deposition, and promoted the healing of diabetic wounds.

[0103] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A research method for promoting diabetic wound healing by ginger extract hydrogel, characterized in that: The specific steps include: Step 1: Conduct biocompatibility experiments on ginger extract hydrogels; use live-dead immunofluorescence staining experiments to distinguish live cells from dead cells, and use fluorescent markers to detect the expression of specific proteins in cells; Step 2: Conduct an experiment on the effect of ginger extract hydrogel on the viability of NIH3T3 cells; use CCK8 reagent to evaluate the incubation of NIH3T3 cells with different concentrations of ginger extract; Step 3: Conduct an experiment on promoting NIH3T3 proliferation by ginger extract hydrogel; observe the effect of ginger extract hydrogel on promoting fibroblast proliferation by scratch test; Step 4: Conduct an experiment on the effect of ginger extract hydrogel on diabetic wounds; use diabetic mice and normal mice as controls to conduct an enzyme-linked immunosorbent assay experiment; Step 5: Conduct an experiment on the effect of ginger extract hydrogel on fibroblasts; use quantitative polymerase chain reaction experiment to measure the effect of ginger extract hydrogel on fibroblasts.

2. The research method of promoting diabetic wound healing by ginger extract hydrogel according to claim 1, characterized in that: The step 1 specifically includes the following steps: S11: Cultivate NIH3T3 cells and plate them in 6-well plates, and divide them into: control group, blank hydrogel group, ginger extract hydrogel group; S12: After incubation for 24 hours, the culture medium was removed, the cells were rinsed with PBS 2-3 times, and then live-dead cell dye was added at a dilution of 1:200 and incubated for 30 minutes. S13: Remove the culture medium containing the dye, add serum-free DMEM, and observe under a fluorescence microscope.

3. The research method of promoting diabetic wound healing by ginger extract hydrogel according to claim 1, characterized in that: The step 2 specifically includes the following steps: S21: NIH3T3 cells were seeded in a 96-well plate at a cell count of 5000 cells per well with 100 μl added; S22: After incubation for 24 h, different concentrations of ginger extract hydrogels were added and incubated for another 24 h; S23: Remove the culture medium, add 10 μl of CCK-8 reagent to each well, and mix CCK8 reagent and DMEM; S24: Continue incubating in the cell culture incubator for 1 hour; S25: Measure absorbance at 450 nm.

4. The research method of promoting diabetic wound healing by ginger extract hydrogel according to claim 1, characterized in that: The step three specifically includes the following steps: S31: NIH3T3 cells were seeded into 24-well plates at an appropriate density for culture; S32: Scratch preparation: Use a sterile pipette tip or a scratcher to gently make a straight line or grid-like wound on the cell monolayer; S33: Wash the cells, rinse the cells twice with PBS; S34: Replace the culture medium: Replace with fresh culture medium to maintain consistency of experimental conditions; S35: Observe the healing of the scratch.

5. The research method of promoting diabetic wound healing by ginger extract hydrogel according to claim 1, characterized in that: The step 4 specifically includes the following steps: S41: A mouse model was constructed, and 40 C57 male mice were taken, including 30 C57 male mice with established diabetes and 10 normal C57 male mice without any treatment. The C57 male mice with established diabetes were divided into a diabetes model group, a blank hydrogel group, and a ginger extract hydrogel group. S42: Enzyme-linked immunosorbent assay experiments were performed on mouse models and statistical analysis was performed to obtain experimental results.

6. The research method of promoting diabetic wound healing by ginger extract hydrogel according to claim 1, characterized in that: The step five specifically includes the following steps: S51: Sample preparation; S52: prepare qPCR reaction mixture; S53: Set thermal cycling conditions according to the experimental purpose and the qPCR instrument used; S54: Load samples and run qPCR; S55: Data analysis.

Images