Experimental test method for treating stress injury based on danshen

Abstract

The invention belongs to the technical field of pressure sore treatment, and discloses an experimental test method for treating stress injury based on danshen emodin, which comprises the following specific steps: step 1, preparing experimental materials, selecting a healthy SD (Sprague Dawley) rat, and preparing a clean and sterile magnet and a shaver; the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 1 part of isoflurane, 1 part of iodophor, 1 part of Vaseline, 1 part of Rhapontigenin and 1 part of resveratrol. The method comprises the following steps: modeling a stress injury model for an SD (Sprague Dawley) rat so as to simulate a stress injury disease, then respectively smearing three medicines, i.e., Danjie emodin, resveratrol and vaseline, tracking the healing condition of a stress injury wound, and finally performing ELISA (Enzyme-Linked Immunosorbent Assay) and Hamp; according to the present invention, the detection analysis is performed through the dyeing E, and the results show that the rheum emodin has good treatment effect on the stress injury, can reduce the expression of the proinflammatory factor, can promote the wound healing, has characteristics of environmental protection and low cost, and provides the new research direction for the treatment of the stress injury.

Description

Technical Field

[0001] This invention belongs to the field of pressure ulcer treatment technology, specifically an experimental testing method based on the treatment of pressure injuries using emodin. Background Technology

[0002] Pressure injuries, commonly known as pressure ulcers or bedsores, refer to localized damage to skin tissue caused by strong or prolonged pressure, which leads to persistent ischemia, hypoxia, and malnutrition, resulting in tissue ulceration and necrosis. They often occur at bony prominences and are clinically manifested as local tissue damage, intact epidermis, or open ulcers.

[0003] Pressure injuries are chronic, difficult-to-heal wounds, and their treatment is quite complex. Modern medicine offers a variety of treatment methods for pressure injuries, mainly including local debridement, physical therapy, wound dressings, vacuum-assisted closure (VSD) techniques, and surgery. For these wounds, there are various dressings based on the concept of moist healing, as well as topical medications to promote wound healing, such as epidermal growth factor and basic fibroblast growth factor. Traditional Chinese medicine considers pressure injuries to be mostly a case of "deficiency of the root and excess of the branch," and in the treatment process, external treatment is the primary method, supplemented by internal treatment. External treatments are based on the principle of removing necrotic tissue and promoting tissue regeneration, mainly including single-drug therapy, compound Chinese medicine preparations, acupuncture, and moxibustion. Traditional Chinese medicine (TCM) treatments include single-herb therapies such as dragon's blood scorpion, cactus, aloe vera, and purple gromwell root. Internal treatments are based on a holistic approach and syndrome differentiation, using herbs such as angelica, rehmannia root, schisandra fruit, ginseng, and astragalus root. While Western medicine offers diverse treatments, the complex pathogenesis and difficulty in healing pressure injuries limit modern medical treatments. TCM treatments struggle to inhibit bacterial growth and address wound drainage issues, and the most commonly used herbs for pressure injuries are limited to a few, such as dragon's blood scorpion, Shengji Hongyu ointment, and purple gromwell root, making the approach somewhat limited. Therefore, it is necessary to explore a more economical, effective treatment method with fewer side effects. Summary of the Invention

[0004] The purpose of this invention is to provide an experimental testing method for treating pressure injuries based on emodin, in order to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: an experimental testing method for treating pressure injuries based on emodin from danshen leaves, the specific steps of which are as follows:

[0006] Step 1: Preparation of Experimental Materials

[0007] Select healthy SD rats, and prepare clean and sterile magnets, razors, and sufficient amounts of isoflurane, povidone-iodine, Vaseline, rhapontigenin, and resveratrol.

[0008] Step Two: Drug Preparation

[0009] Using Vaseline as a dressing, rhapontigenin, resveratrol, and Vaseline were prepared separately.

[0010] Step 3: Establish a pressure injury model

[0011] After anesthetizing SD rats with isoflurane, the skin and hair on the back of the SD rats were shaved off with a razor. Then, the hairless area on the back of the SD rats was disinfected with povidone-iodine. After disinfection, the skin on the back of the SD rats was clamped with a magnet and IR circulation was performed to complete the modeling of the pressure injury model.

[0012] Step 4: Apply medication

[0013] After the pressure injury model was established, rhapontigenin, resveratrol and Vaseline were applied to the wound, and the wound condition was recorded by taking pictures until the wound met the wound healing criteria.

[0014] Step 5: Skin tissue sampling

[0015] Once the wounds had healed to the required standard, the SD rats were euthanized using an overdose anesthesia method, and then skin, liver, spleen, and kidney tissue samples were collected from the back wound.

[0016] Step Six: Experimental Testing

[0017] SD rat samples were analyzed using ELISA and H&E staining to observe the actual healing of the skin tissue on the back of the rat.

[0018] As a preferred technical solution of the present invention, the SD rats mentioned in step one are selected as female individuals aged six weeks, and are adapted to being individually fed in cages for one week at a temperature of 19.0±2.1℃, a humidity of 40-50%RH, and a light-dark cycle of 12h. During this period, they are fed and have free access to food, water, and activity.

[0019] As a preferred technical solution of the present invention, the magnets described in steps one and three have the following specifications: diameter 20mm, thickness 10mm, and when using the magnets to clamp the skin on the back of the SD rat in step three, the clamping time is 12 hours and the release time is 12 hours.

[0020] As a preferred technical solution of the present invention, the time and number of IR cycles in step three are as follows: three ischemia-reperfusion cycles are performed within three days.

[0021] As a preferred embodiment of the present invention, when applying the medication in step four, the dosage is a thin 30 μM application; the route of administration is to apply Vaseline as a dressing; the frequency of administration is daily application; and the administration cycle is 21 days.

[0022] As a preferred technical solution of the present invention, the wound healing standard in step four is: granulation tissue completely covers the wound, and the wound healing rate reaches 90%.

[0023] As a preferred embodiment of the present invention, the method for preparing the rhapontigenin drug in step two is as follows: a certain amount of rhapontigenin powder is dissolved in 1 mL of dimethyl sulfoxide (DMSO) to prepare a 20 mM stock solution; then 7 μL of the stock solution is weighed and dissolved in heated petrolatum, and quickly stirred to prepare a 30 μM petrolatum ointment; the resveratrol drug in step two... The preparation method is as follows: Take a certain amount of resveratrol powder and dissolve it in 1 mL of dimethyl sulfoxide (DMSO) to prepare a 20 mM stock solution. Then, weigh 7 μL of the stock solution and dissolve it in heated petroleum jelly. Stir quickly to prepare a 30 μM petroleum jelly ointment. The preparation method of the petroleum jelly drug mentioned in step two is as follows: Weigh 7 μL of dimethyl sulfoxide (DMSO) stock solution and dissolve it in heated petroleum jelly. Stir quickly to prepare a 30 μM petroleum jelly ointment.

[0024] As a preferred technical solution of the present invention, the ELISA experimental detection method described in step six is ​​as follows: Taking IL-1β as an example: 1. Set up standard wells, blank wells and sample wells respectively; add 100μL of serially diluted standard to the standard well, add 100μL of standard & sample diluent to the blank well, and add 100μL of the sample to be tested to the remaining wells;

[0025] 2. Discard the liquid in the wells without washing; add 100 μL of biotinylated antibody working solution to each well, cover the plate with a membrane, and incubate at 37°C for 1 hour.

[0026] 3. Shake off the liquid in the wells and pat dry on clean absorbent paper; add 350 μL of washing buffer to each well, soak for 1 minute, aspirate or shake off the liquid in the microplate, and pat dry; repeat this washing step 3 times.

[0027] 4. Add 100 μL of HRP enzyme conjugate working solution to each well, cover the microplate with a membrane, and incubate at 37°C for 30 minutes;

[0028] 5. Shake off all the liquid in the hole and wash the plate 5 times, using the same method as step 3.

[0029] 6. Add 90 pL of substrate solution (TMB) to each well, cover the microplate with a membrane, and incubate at 37°C in the dark for 15 minutes;

[0030] 7. Add 50 μL of stop solution to each well to terminate the reaction;

[0031] 8. Immediately measure the optical density (OD value) of each well using an enzyme-labeled enzyme at a wavelength of only 450 nm.

[0032] As a preferred technical solution of the present invention, the H&E staining test method described in step six is ​​as follows: A section of skin tissue sample from a wound on the back of an SD rat is sequentially immersed in xylene twice for 10 minutes each time; then sequentially immersed in 100%, 95%, 80%, and 70% ethanol for 5 minutes each time, followed by rinsing twice with distilled water; then immersed in hematoxylin stain for 5-10 minutes; after rinsing with water, it is immersed in 1% hydrochloric acid ethanol to remove excess staining; rinsed with running water for 10 minutes to achieve blue staining; then immersed in eosin stain for 1-3 minutes; subsequently immersed in 70%, 80%, 95%, and 100% ethanol for 2 minutes each time; then immersed in xylene twice for 5 minutes each time; finally, neutral resin is added, a coverslip is placed on top, and after drying, the tissue morphology is observed under a microscope.

[0033] The beneficial effects of the present invention are as follows:

[0034] This invention simulates pressure injury symptoms by establishing a pressure injury model in SD rats. Then, by applying three drugs—danye rhein, resveratrol, and petrolatum—and tracking the healing of pressure injury wounds, the results were analyzed using ELISA and H&E staining. The results showed that danye rhein has a good therapeutic effect on pressure injury, reducing the expression of pro-inflammatory factors and promoting wound healing. Furthermore, danye rhein is environmentally friendly and low-cost, providing a new research direction for the treatment of pressure injury. Attached Figure Description

[0035] Figure 1 This is a real-world illustration of wound healing according to the present invention.

[0036] Figure 2 This is an image of the wound healing trace of the present invention;

[0037] Figure 3 This is a bar chart showing the healing rate versus time of the present invention.

[0038] Figure 4 This is a bar chart showing the healing rate versus drug efficacy of this invention.

[0039] Figure 5 This is the ELISA detection diagram of the present invention;

[0040] Figure 6 This is a Vaseline H&E staining diagram from the present invention.

[0041] Figure 7 This is a H&E staining image of resveratrol from the present invention.

[0042] Figure 8 This is an H&E staining image of rhapontigenin from this invention. Detailed Implementation

[0043] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0044] like Figures 1 to 8 As shown in the figure, this invention provides an experimental testing method for treating pressure injuries based on emodin, the specific steps of which are as follows:

[0045] Step 1: Preparation of Experimental Materials

[0046] Select healthy SD rats, and prepare clean and sterile magnets, razors, and sufficient amounts of isoflurane, povidone-iodine, Vaseline, rhapontigenin, and resveratrol.

[0047] Step Two: Drug Preparation

[0048] Using Vaseline as a dressing, rhapontigenin, resveratrol, and Vaseline were prepared separately.

[0049] Step 3: Establish a pressure injury model

[0050] After anesthetizing SD rats with isoflurane, the skin and hair on the back of the SD rats were shaved off with a razor. Then, the hairless area on the back of the SD rats was disinfected with povidone-iodine. After disinfection, the skin on the back of the SD rats was clamped with a magnet and IR circulation was performed to complete the modeling of the pressure injury model.

[0051] Step 4: Apply medication

[0052] After the pressure injury model was established, rhapontigenin, resveratrol and Vaseline were applied to the wound, and the wound condition was recorded by taking pictures until the wound met the wound healing criteria.

[0053] Step 5: Skin tissue sampling

[0054] Once the wounds had healed to the required standard, the SD rats were euthanized using an overdose anesthesia method, and then skin, liver, spleen, and kidney tissue samples were collected from the back wound.

[0055] Step Six: Experimental Testing

[0056] SD rat samples were analyzed using ELISA and H&E staining to observe the actual healing status of the skin tissue on the back of the wound.

[0057]

[0058] ALT, AST, TBIL, and DBIL are all indicators for evaluating liver function in rats, while CREA is an indicator for evaluating kidney function in rats. These results indicate that the drug rhapontigenin did not damage liver or kidney function during the treatment of a rat model of stress injury.

[0059] Rhapontigenin is a stilbene compound widely found in nature in various medicinal plants of the Rheum genus, including Rheum palmatum, Rheum chinense, Rheum undulatum, and Rheum palmatum. Its pharmacological activities include antitumor, antioxidant, antibacterial, lipid-lowering, cardiovascular protection, anti-inflammatory, and skin-whitening effects. Based on its anti-inflammatory, antibacterial, and skin-whitening pharmacological effects, it has shown good performance in experimental tests for the treatment of pressure injuries, reducing the expression of pro-inflammatory factors and promoting wound healing, while also being environmentally friendly and low-cost.

[0060] In step one, female SD rats aged six weeks were selected and subjected to adaptive single-cage feeding for one week at a temperature of 19.0±2.1℃, humidity of 40-50%RH, and a light-dark cycle of 12h. During this period, they were fed and allowed free access to food, water, and activity.

[0061] SD rats have blood pressure, heart rate, and metabolic rate that are similar to those of humans, making them suitable for studying stress-related injuries. In addition, SD rats are docile, easy to handle and raise, have a short breeding cycle, produce many offspring, are low in cost, are highly adaptable to the environment, and tolerate roughage, making them suitable for large-scale experiments.

[0062] The magnets used in steps one and three have the following specifications: 20mm in diameter and 10mm in thickness. In step three, when using the magnet to clamp the skin on the back of the SD rat, the clamping time is 12 hours and the release time is 12 hours.

[0063] By controlling the clamping and releasing of the magnets, pressure-induced skin injuries can be simulated, thus providing a basis for subsequent experiments.

[0064] In step three, the IR cycle is performed for three times within three days.

[0065] By performing three ischemia-reperfusion IR cycles within three days, the damage signal can be amplified, the experimental efficiency can be improved, and it is suitable for simulating pressure injury symptoms.

[0066] In step four, when applying the medication topically, the dosage is 30 μM thin layer; the route of administration is to apply Vaseline as a dressing; the frequency of administration is daily application; and the administration cycle is 21 days.

[0067] When applying medication, the application tools need to be cleaned and sterilized. Each type of medication requires a corresponding application tool to avoid contamination.

[0068] The wound healing standard in step four is: granulation tissue completely covers the wound, and the wound healing rate reaches 90%.

[0069] Figure 1 and Figure 2 The study clearly demonstrated the healing of the wound on the back of the SD rats, showing that the wound healing rate reached 90% by day 21.

[0070] The preparation methods for Rhapontigenin in step two are as follows: A certain amount of Rhapontigenin powder is dissolved in 1 mL of dimethyl sulfoxide (DMSO) to prepare a 20 mM stock solution. Then, 7 μL of the stock solution is dissolved in heated petroleum jelly and quickly stirred to prepare a 30 μM petroleum jelly ointment. The preparation methods for Resveratrol in step two are as follows: A certain amount of Resveratrol powder is dissolved in 1 mL of dimethyl sulfoxide (DMSO) to prepare a 20 mM stock solution. Then, 7 μL of the stock solution is dissolved in heated petroleum jelly and quickly stirred to prepare a 30 μM petroleum jelly ointment. The preparation methods for Vaseline in step two are as follows: 7 μL of the dimethyl sulfoxide (DMSO) stock solution is dissolved in heated petroleum jelly and quickly stirred to prepare a 30 μM petroleum jelly ointment.

[0071] The dosage of rhapontigenin and resveratrol is calculated as follows: mass (mg) = concentration (mM) × molecular weight (g / mol) × volume (L) × 1000.

[0072] The ELISA assay method in step six is ​​as follows: Taking IL-1β as an example: 1. Set up standard wells, blank wells and sample wells respectively; add 100 μL of serially diluted standard to the standard wells, add 100 μL of standard and sample diluent to the blank wells, and add 100 μL of the sample to be tested to the remaining wells.

[0073] 2. Discard the liquid in the wells without washing; add 100 μL of biotinylated antibody working solution to each well, cover the plate with a membrane, and incubate at 37°C for 1 hour.

[0074] 3. Shake off the liquid in the wells and pat dry on clean absorbent paper; add 350 μL of washing buffer to each well, soak for 1 minute, aspirate or shake off the liquid in the microplate, and pat dry; repeat this washing step 3 times.

[0075] 4. Add 100 μL of HRP enzyme conjugate working solution to each well, cover the microplate with a membrane, and incubate at 37°C for 30 minutes;

[0076] 5. Shake off all the liquid in the hole and wash the plate 5 times, using the same method as step 3.

[0077] 6. Add 90 pL of substrate solution (TMB) to each well, cover the microplate with a membrane, and incubate at 37°C in the dark for 15 minutes;

[0078] 7. Add 50 μL of stop solution to each well to terminate the reaction;

[0079] 8. Immediately measure the optical density (OD value) of each well using an enzyme-labeled enzyme at a wavelength of only 450 nm.

[0080] Figure 5 The image shows a comparison of the expression levels of VEGF-A, IFN-γ, IL-1β, IL-6, and TNF-α in the skin measured by ELISA. The effect of rhapontigenin on the expression levels of IFN-γ and TNF-α in the skin tissue of rats with stress injury (21 days after administration) was also presented. The expression levels of inflammatory cytokines IL-6 and IL-1β, as well as vascular endothelial growth factor (VEGF), were significantly increased in the skin tissue of rats with stress injury. Treatment with rhapontigenin reduced the expression levels of inflammatory cytokines IL-6 and IL-1β, as well as VEGF, in serum. However, treatment with resveratrol (positive control) effectively reduced their expression levels, and the differences were statistically significant.

[0081] The H&E staining experiment in step six is ​​as follows: A section of skin tissue sample from the back wound of an SD rat is sequentially immersed in xylene twice for 10 minutes each time; then sequentially immersed in 100%, 95%, 80%, and 70% ethanol for 5 minutes each time, followed by rinsing twice with distilled water; then immersed in hematoxylin stain for 5-10 minutes; after rinsing with water, it is immersed in 1% hydrochloric acid ethanol to remove excess staining; rinsed with running water for 10 minutes to allow it to return to blue; then immersed in eosin stain for 1-3 minutes; subsequently immersed in 70%, 80%, 95%, and 100% ethanol for 2 minutes each time; then immersed in xylene twice for 5 minutes each time; finally, neutral resin is added, a coverslip is placed on top, and after drying, the tissue morphology is observed under a microscope.

[0082] from Figure 6 As can be seen from the data, when Vaseline is used, the epidermis shows intact repair with slight thickening (brown arrow); small areas of granulation tissue proliferation and repair are visible in the dermis and subcutaneous tissue (yellow polygon), with proliferating fibroblasts (purple arrow) and fine collagen fibers interwoven, accompanied by a small amount of lymphocyte infiltration (blue arrow), and abundant capillary proliferation (green arrow). A small amount of muscle cell atrophy (orange arrow) and reduced volume are visible in the muscle layer at the edge of the injury; the number of skin appendages such as hair follicles and sebaceous glands in the injured area is significantly reduced, and follicular plugs are occasionally seen (cyan arrow).

[0083] from Figure 7As can be seen from the data, when resveratrol is used, a large amount of necrotic tissue is sloughed off in the skin tissue (orange arrows). The epidermis in the damaged area has not been fully repaired, and there is slight thickening (brown arrows). Hyperkeratosis is common (yellow arrows), and the stratum corneum is thickened. Extensive granulation tissue proliferation and repair are visible in the dermis and subcutaneous tissue (yellow polygons). Proliferating fibroblasts (purple arrows) and fine collagen fibers are interwoven, accompanied by a large amount of granulocyte and lymphocyte infiltration (blue arrows) and a large amount of capillary proliferation (green arrows). The number of skin appendages such as hair follicles and sebaceous glands in the damaged area is significantly reduced.

[0084] from Figure 8 As can be seen from the data, when rhapontigenin was used, the skin tissue showed intact epidermal repair with slight thickening (brown arrow), a small number of epidermal cells with edema (silver arrow), loose and lightly stained cytoplasm, and predominantly hyperkeratosis (yellow arrow), and thickened stratum corneum. In the dermis and subcutaneous tissue, there was extensive granulation tissue proliferation and repair (yellow polygon), with proliferating fibroblasts (purple arrow) and fine collagen fibers interlaced, accompanied by a small amount of lymphocyte infiltration (blue arrow), and a large amount of capillary proliferation (green arrow). At the edge of the injury, a small number of muscle cells showed atrophy (orange arrow) and reduced volume. The number of skin appendages such as hair follicles and sebaceous glands in the injured area was significantly reduced, and follicular plugs were occasionally seen (cyan arrow).

[0085] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0086] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An experimental testing method for treating pressure injuries based on emodin from danshen leaves, characterized in that, The specific steps are as follows: Step 1: Preparation of Experimental Materials Select healthy SD rats, and prepare clean and sterile magnets, razors, and sufficient amounts of isoflurane, povidone-iodine, Vaseline, rhapontigenin, and resveratrol. Step Two: Drug Preparation Using Vaseline as a dressing, rhapontigenin, resveratrol, and Vaseline were prepared separately. Step 3: Establish a pressure injury model After anesthetizing SD rats with isoflurane, the skin and hair on the back of the SD rats were shaved off with a razor. Then, the hairless area on the back of the SD rats was disinfected with povidone-iodine. After disinfection, the skin on the back of the SD rats was clamped with a magnet and IR circulation was performed to complete the modeling of the pressure injury model. Step 4: Apply medication After the pressure injury model was established, rhapontigenin, resveratrol and Vaseline were applied to the wound, and the wound condition was recorded by taking pictures until the wound met the wound healing criteria. Step 5: Skin tissue sampling Once the wounds had healed to the required standard, the SD rats were euthanized using an overdose anesthesia method, and then skin, liver, spleen, and kidney tissue samples were collected from the back wound. Step Six: Experimental Testing SD rat samples were analyzed using ELISA and H&E staining to observe the actual healing of the skin tissue on the back of the rat.

2. The experimental testing method for treating pressure injuries based on emodin according to claim 1, characterized in that: The SD rats mentioned in step one are selected from female individuals aged six weeks. They are then acclimatized and individually fed for one week at a temperature of 19.0±2.1℃, a humidity of 40-50%RH, and a light-dark cycle of 12h. During this period, they are fed and have free access to food, water, and activity.

3. The experimental testing method for treating pressure injuries based on emodin according to claim 1, characterized in that: The magnets described in steps one and three have the following specifications: 20mm in diameter and 10mm in thickness. In step three, when using the magnet to clamp the skin on the back of the SD rat, the clamping time is 12 hours and the release time is 12 hours.

4. The experimental testing method for treating pressure injuries based on emodin according to claim 1, characterized in that: The time and number of IR cycles described in step three are as follows: three ischemia-reperfusion procedures within three days.

5. The experimental testing method for treating pressure injuries based on emodin according to claim 1, characterized in that: When applying the medication as described in step four, the dosage is a thin 30 μM application; the route of administration is to apply the medication using petroleum jelly as a dressing; the frequency of administration is daily application; and the administration cycle is 21 days.

6. The experimental testing method for treating pressure injuries based on emodin according to claim 1, characterized in that: The wound healing standard described in step four is: granulation tissue completely covers the wound, and the wound healing rate reaches 90%.

7. The experimental testing method for treating pressure injuries based on emodin according to claim 1, characterized in that: The preparation method for the rhapontigenin drug mentioned in step two is as follows: a certain amount of rhapontigenin powder is dissolved in 1 mL of dimethyl sulfoxide (DMSO) to prepare a 20 mM stock solution. Then, 7 μL of the stock solution is weighed and dissolved in heated petroleum jelly, and stirred quickly to prepare a 30 μM petroleum jelly ointment. The preparation method for the resveratrol drug mentioned in step two is as follows: a certain amount of resveratrol powder is dissolved in 1 mL of dimethyl sulfoxide (DMSO) to prepare a 20 mM stock solution. Then, 7 μL of the stock solution is weighed and dissolved in heated petroleum jelly, and stirred quickly to prepare a 30 μM petroleum jelly ointment. The preparation method for the petroleum jelly drug mentioned in step two is as follows: 7 μL of dimethyl sulfoxide (DMSO) stock solution is weighed and dissolved in heated petroleum jelly, and stirred quickly to prepare a 30 μM petroleum jelly ointment.

8. The experimental testing method for treating pressure injuries based on emodin according to claim 1, characterized in that: The ELISA experimental detection method described in step six is ​​as follows: Taking IL-1β as an example:

1. Set up standard wells, blank wells and sample wells respectively; add 100 μL of serially diluted standard to the standard wells, add 100 μL of standard & sample diluent to the blank wells, and add 100 μL of the sample to be tested to the remaining wells; 2. Discard the liquid in the wells without washing; add 100 μL of biotinylated antibody working solution to each well, cover the plate with a membrane, and incubate at 37°C for 1 hour.

3. Shake off the liquid in the wells and pat dry on clean absorbent paper; add 350 μL of washing buffer to each well, soak for 1 minute, aspirate or shake off the liquid in the microplate, and pat dry; repeat this washing step 3 times.

4. Add 100 μL of HRP enzyme conjugate working solution to each well, cover the microplate with a membrane, and incubate at 37°C for 30 minutes; 5. Shake off all the liquid in the hole and wash the plate 5 times, using the same method as step 3.

6. Add 90 pL of substrate solution (TMB) to each well, cover the microplate with a membrane, and incubate at 37°C in the dark for 15 minutes; 7. Add 50 μL of stop solution to each well to terminate the reaction; 8. Immediately measure the optical density (OD value) of each well using an enzyme-labeled enzyme at a wavelength of only 450 nm.

9. The experimental testing method for treating pressure injuries based on emodin according to claim 1, characterized in that: The H&E staining test method described in step six is ​​as follows: A section of skin tissue sample from the back wound of an SD rat is sequentially immersed in xylene twice for 10 minutes each time; then sequentially immersed in 100%, 95%, 80%, and 70% ethanol for 5 minutes each time, followed by rinsing twice with distilled water; then immersed in hematoxylin stain for 5-10 minutes; after rinsing with water, it is immersed in 1% hydrochloric acid ethanol to remove excess staining; rinsed with running water for 10 minutes to allow the stain to return to blue; then immersed in eosin stain for 1-3 minutes; subsequently immersed in 70%, 80%, 95%, and 100% ethanol for 2 minutes each time; then immersed in xylene twice for 5 minutes each time; finally, neutral resin is added, a coverslip is placed on top, and after drying, the tissue morphology is observed under a microscope.

Images