A combined medicament and its application in promoting wound healing

Abstract

The present invention provides a combination medicament and its application in promoting wound healing, belonging to the field of biomedical technology. The combination medicament provided by the present invention is composed of humic acid and a Chinese medicine compound preparation, wherein the Chinese medicine compound preparation is composed of four medicines, namely, dragon's blood, lithospermum officinale, borneol, and puffball. The concentration of sodium humate in the combination medicament is 5g / L, and the preparation method of the Chinese medicine compound preparation is to soak 100g lithospermum officinale in 500ml sesame oil for 24h, filter and boil; then grind 6g dragon's blood, 0.75g borneol, and 5g puffball powder into powder, add them into the soaking liquid and mix them evenly. The present invention finds that the combination of sodium humate and the Chinese medicine compound preparation can synergistically promote wound healing.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a combined medicament and its application in promoting wound healing. Background Art

[0002] Pressure injury (PI) is a clinical problem that nursing often faces, and it also occurs frequently in the community and family. PI is one of the most common complications of patients with mobility disorders, chronic diseases and the elderly. It is mainly caused by strong and / or persistent pressure or pressure combined with shear force, which leads to localized injury of the skin and / or soft tissue at the bony prominence, under medical or other devices. The disease is more common in elderly patients who have been bedridden for a long time. The disease progresses rapidly and the treatment cycle is long. It seriously threatens the quality of life and life of elderly bedridden patients, and also increases medical costs. Therefore, as the world enters the aging acceleration lane, the prevention and treatment of elderly PI has become a key and difficult issue in clinical nursing work.

[0003] PI can cause severe pain, infection, bleeding, necrosis, and even death. Delayed healing is the most common complication of PI, occurring in 35.2% of patients with primary PI. Wound healing is a series of continuous, protective, and adaptive complex responses made by the body to repair local tissue loss and cell damage caused by adverse factors. It is divided into four stages: coagulation, inflammation, repair, and remodeling, and requires the integration of biological processes such as cell proliferation and migration, extracellular matrix synthesis and deposition, and angiogenesis.

[0004] At present, sterile dressings are usually chosen for clinical coverage. However, there are many types of new medical dressings, and each dressing has different characteristics and indications. They need to be selected according to the specific conditions of the patient and the wound. How to correctly select the right dressing and decide whether to use a specific dressing increases the difficulty of wound management. So far, only recombinant human PDGF preparations have been approved for the treatment of chronic wounds, but due to various limiting factors such as short half-life, carcinogenic risk and high price, clinical promotion and application have not yet been achieved. In addition, long-term use of antibiotics such as cephalosporins to treat damaged skin areas is prone to adverse reactions such as drug-resistant bacteria and neutropenia. Therefore, it is relatively urgent to find and develop new drugs or new strategies that are efficient, convenient and cost-effective to promote wound healing.

[0005] With the significant increase in research on natural products, the medicinal properties of humic acid (HA) have gradually attracted attention. HA is a multifunctional high-molecular organic acid obtained by biochemical degradation containing a large number of different active groups such as carboxyl, amino and phenolic acid groups. It has the effects of antibacterial and anti-inflammatory, decomposing and promoting tissue regeneration. Chinese medicinal formulae (CMF) is composed of four herbs: dragon's blood, lithospermum officinale, borneol and puffball. Clinical experience has confirmed its effectiveness in the treatment of various traumas. Its effects of reducing swelling and relieving pain, removing blood stasis and stopping bleeding, and promoting tissue regeneration can form complementary advantages with HA. However, there are few basic studies on wound healing under aging conditions using HA combined with CMF, and there is a lack of comparative studies on the effects of HA and CMF on wound healing.

[0006] Aging will induce abnormal iron metabolism. The accumulated free iron will initiate cellular lipid peroxidation through the Fenton reaction, and lipid peroxidation will further stimulate cell ferroptosis. Ferroptosis is an iron-dependent programmed cell death mode characterized by the accumulation of lipid peroxides. It plays an important role in the occurrence and development of various diseases such as tissue ischemia-reperfusion injury, cardiovascular disease, cancer, neuropathy, etc., and is also involved in regulating skin damage and healing processes. It has been found that the iron content in chronic wound exudate increases, but after intervention with iron chelators, iron-driven lipid peroxidation can be eliminated and chronic wound healing can be promoted. A large amount of iron ions and ROS are enriched in aging skin tissue, initiating endothelial cell ferroptosis. At the same time, excessive iron ions occupy the P53 iron ion binding domain, weaken P53-mediated DNA damage repair, and accelerate the aging of the body. It can be seen that endothelial cell ferroptosis caused by aging further accelerates the aging process. The two form a vicious cycle and jointly cause the wound tissue to not heal for a long time. These indicate that ferroptosis is one of the potential mechanisms for delayed wound healing in aging and is expected to become a new therapeutic target for delayed wound healing in elderly patients. However, the effect of CMF+HA treatment on ferroptosis in aging-related pressure injury wounds has not yet been clarified. Summary of the invention

[0007] The purpose of the present invention is to provide a combined medicament and its application in promoting wound healing, thereby providing a new medicament for promoting wound healing with excellent effect.

[0008] To achieve the above objectives, in a first aspect, the present invention provides a combination agent for promoting wound healing in aging individuals, wherein the combination agent is composed of sodium humate and a Chinese medicine compound preparation, wherein the Chinese medicine compound preparation is composed of four medicines: dragon's blood, lithospermum officinale, borneol, and puffball.

[0009] Preferably, the humic acid in the combined agent is sodium humate, and the concentration of the sodium humate is 5 g / L;

[0010] In the combined medicament, the preparation method of each 500ml of the Chinese medicine compound preparation is as follows:

[0011] (1) Soak 100 g of Lithospermum officinale in 500 ml of sesame oil for 24 h, filter and boil;

[0012] (2) 6 g of Sanguisorba officinalis, 0.75 g of borneol, and 5 g of puffball powder are ground into powder, added into the soaking solution and mixed evenly to obtain the Chinese medicine compound preparation.

[0013] Preferably, the method of using the combined medicament is to first apply sodium humate externally at 0.5 ml / cm, and then apply the Chinese medicine compound preparation.

[0014] Preferably, the wound surface is a pressure injury wound surface.

[0015] In a second aspect, the present invention provides an application of a combination agent in the preparation of a drug for promoting wound healing in aging individuals, wherein the combination agent is composed of sodium humate and a Chinese medicine compound preparation, and the Chinese medicine compound preparation is composed of four medicines: dragon's blood, lithospermum officinale, borneol, and puffball.

[0016] Preferably, the concentration of sodium humate in the combined agent is 5 g / L;

[0017] The preparation method of each 500ml Chinese medicine compound preparation is as follows:

[0018] (1) Soak 100 g of Lithospermum officinale in 500 ml of sesame oil for 24 h, filter and boil;

[0019] (2) 6 g of Sanguisorba officinalis, 0.75 g of borneol, and 5 g of puffball powder are ground into powder, added into the soaking solution and mixed evenly to obtain the Chinese medicine compound preparation.

[0020] Preferably, the wound surface is a pressure injury wound surface.

[0021] In the third aspect, the present invention provides an application of a combination agent in the preparation of an inhibitor of ferroptosis in wounds of aging individuals, wherein the combination agent is composed of sodium humate and a Chinese medicine compound preparation, and the Chinese medicine compound preparation is composed of four medicinal materials: dragon's blood, lithospermum officinale, borneol, and puffball.

[0022] Preferably, the concentration of sodium humate in the combined agent is 5 g / L;

[0023] In the combined medicament, the preparation method of each 500ml of the Chinese medicine compound preparation is as follows:

[0024] (1) Soak 100 g of Lithospermum officinale in 500 ml of sesame oil for 24 h, filter and boil;

[0025] (2) 6 g of Sanguisorba officinalis, 0.75 g of borneol, and 5 g of puffball powder are ground into powder, added into the soaking solution and mixed evenly to obtain the Chinese medicine compound preparation.

[0026] Preferably, the wound surface is a pressure injury wound surface.

[0027] In a fourth aspect, the present invention provides an application of a combination agent in the preparation of an agent for promoting the expression of collagen I and collagen III in wound tissue of aging individuals, wherein the combination agent is composed of sodium humate and a Chinese medicine compound preparation, and the Chinese medicine compound preparation is composed of four medicinal materials: dragon's blood, lithospermum officinale, borneol, and puffball.

[0028] Preferably, the concentration of sodium humate in the combined agent is 5 g / L;

[0029] In the combined medicament, the preparation method of each 500ml of the Chinese medicine compound preparation is as follows:

[0030] The preparation method of each 500ml Chinese medicine compound preparation is as follows:

[0031] (1) Soak 100 g of Lithospermum officinale in 500 ml of sesame oil for 24 h, filter and boil;

[0032] (2) 6 g of Sanguisorba officinalis, 0.75 g of borneol, and 5 g of puffball powder are ground into powder, added into the soaking solution and mixed evenly to obtain the Chinese medicine compound preparation.

[0033] Preferably, the wound tissue is pressure injury wound tissue.

[0034] In a fifth aspect, the present invention provides an application of a combined agent in the preparation of a collagen deposition promoter in wound tissue of an aging individual, characterized in that the combined agent is composed of sodium humate and a Chinese medicine compound preparation, and the Chinese medicine compound preparation is composed of four medicines: dragon's blood, lithospermum officinale, borneol, and puffball;

[0035] The concentration of sodium humate in the combined agent is 5 g / L;

[0036] In the combined medicament, the preparation method of each 500ml of the Chinese medicine compound preparation is as follows:

[0037] (1) Soak 100 g of Lithospermum officinale in 500 ml of sesame oil for 24 h, filter and boil;

[0038] (2) 6 g of Sanguisorba officinalis, 0.75 g of borneol, and 5 g of puffball powder are ground into powder, added into the soaking solution and mixed evenly to obtain the Chinese medicine compound preparation.

[0039] Preferably, the wound tissue is pressure injury wound tissue.

[0040] In a sixth aspect, the present invention provides an application of a combined agent in the preparation of a cell proliferation promoter for wounds of aging individuals, wherein the combined agent is composed of sodium humate and a Chinese medicine compound preparation, wherein the Chinese medicine compound preparation is composed of four medicines: dragon's blood, lithospermum officinale, borneol, and puffball;

[0041] Preferably, the concentration of sodium humate in the combined agent is 5 g / L;

[0042] In the combined medicament, the preparation method of each 500ml of the Chinese medicine compound preparation is as follows:

[0043] (1) Soak 100 g of Lithospermum officinale in 500 ml of sesame oil for 24 h, filter and boil;

[0044] (2) 6 g of Sanguisorba officinalis, 0.75 g of borneol, and 5 g of puffball powder are ground into powder, added into the soaking solution and mixed evenly to obtain the Chinese medicine compound preparation.

[0045] Preferably, the wound surface is a pressure injury wound surface.

[0046] In a seventh aspect, the present invention provides an application of a combined agent in the preparation of an angiogenesis promoter for wounds of aging individuals, wherein the combined agent is composed of sodium humate and a Chinese medicine compound preparation, wherein the Chinese medicine compound preparation is composed of four medicines: dragon's blood, lithospermum officinale, borneol, and puffball;

[0047] Preferably, the concentration of sodium humate in the combined agent is 5 g / L;

[0048] In the combined medicament, the preparation method of each 500ml of the Chinese medicine compound preparation is as follows:

[0049] (1) Soak 100 g of Lithospermum officinale in 500 ml of sesame oil for 24 h, filter and boil;

[0050] (2) 6 g of Sanguisorba officinalis, 0.75 g of borneol, and 5 g of puffball powder are ground into powder, added into the soaking solution and mixed evenly to obtain the Chinese medicine compound preparation.

[0051] Preferably, the wound surface is a pressure injury wound surface.

[0052] In an eighth aspect, the present invention provides an application of a combined agent in the preparation of an agent for alleviating inflammation in wound tissue of an aging individual, wherein the combined agent is composed of sodium humate and a Chinese medicine compound preparation, wherein the Chinese medicine compound preparation is composed of four medicines: dragon's blood, lithospermum officinale, borneol, and puffball;

[0053] Preferably, the concentration of sodium humate in the combined agent is 5 g / L;

[0054] In the combined medicament, the preparation method of each 500ml of the Chinese medicine compound preparation is as follows:

[0055] (1) Soak 100 g of Lithospermum officinale in 500 ml of sesame oil for 24 h, filter and boil;

[0056] (2) 6 g of Sanguisorba officinalis, 0.75 g of borneol, and 5 g of puffball powder are ground into powder, added into the soaking solution and mixed evenly to obtain the Chinese medicine compound preparation.

[0057] Preferably, the wound tissue is pressure injury wound tissue.

[0058] In a ninth aspect, the present invention provides a combination agent for inhibiting ferroptosis in wounds of aging individuals, characterized in that the combination agent is composed of sodium humate and a Chinese medicine compound preparation, and the Chinese medicine compound preparation is composed of four medicines: dragon's blood, lithospermum officinale, borneol, and puffball;

[0059] Preferably, the concentration of sodium humate in the combined agent is 5 g / L;

[0060] In the combined medicament, the preparation method of each 500ml of the Chinese medicine compound preparation is as follows:

[0061] (1) Soak 100 g of Lithospermum officinale in 500 ml of sesame oil for 24 h, filter and boil;

[0062] (2) 6 g of Sanguisorba officinalis, 0.75 g of borneol, and 5 g of puffball powder are ground into powder, added into the soaking solution and mixed evenly to obtain the Chinese medicine compound preparation.

[0063] Preferably, the wound surface is a pressure injury wound surface.

[0064] In a tenth aspect, the present invention provides a combination agent for promoting angiogenesis in wounds of aging individuals, characterized in that the combination agent is composed of sodium humate and a Chinese medicine compound preparation, and the Chinese medicine compound preparation is composed of four medicines: dragon's blood, lithospermum officinale, borneol, and puffball;

[0065] Preferably, the concentration of sodium humate in the combined agent is 5 g / L;

[0066] In the combined medicament, the preparation method of each 500ml of the Chinese medicine compound preparation is as follows:

[0067] (1) Soak 100 g of Lithospermum officinale in 500 ml of sesame oil for 24 h, filter and boil;

[0068] (2) 6 g of Sanguisorba officinalis, 0.75 g of borneol, and 5 g of puffball powder are ground into powder, added into the soaking solution and mixed evenly to obtain the Chinese medicine compound preparation.

[0069] Preferably, the wound surface is a pressure injury wound surface.

[0070] The beneficial effects of the present invention are:

[0071] The present invention finds that in the process of wound healing caused by pressure injury in aging individuals, the combined use of sodium humate and a Chinese medicine compound preparation can produce obvious synergistic effects on collagen deposition, collagen expression, inflammation relief, cell proliferation, angiogenesis, and ferroptosis inhibition in wound tissue, thereby promoting wound healing more effectively and quickly. Therefore, sodium humate and a Chinese medicine compound preparation can be used in combination to prepare a better quality wound healing drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Results of the D-gal-induced rat aging model construction; (A) Results of aging-related indicators GLB1, P53, and P16 detected by Western Blot; (B) Results of relative protein levels of GLB1, P53, and P16; * P < 0.05 versus Young;

[0073] Figure 2 Results of CMF+HA synergistically accelerating wound healing in aged rats; (A) Experimental design of animal study. (B) Representative images of wound healing, Scale bars = 12 mm; (C) H&E staining results on days 3, 7, and 14 after injury, Scale bars = 1 mm; (D) Quantification of wound closure rate and scar width of rats treated with control, CMF, HA, and CMF+HA on days 3, 7, and 14; $ P<0.05versus Young-Control, * P<0.05versus Aged-control, # P<0.05versus Aged-CMF, & P < 0.05 versus Aged-HA;

[0074] Figure 3 The results show that CMF+HA synergistically promotes extracellular matrix arrangement and collagen synthesis in aged wounds; (A) Masson trichrome staining results of wound samples from different groups on days 3 and 7 after injury; (B) Western Blot evaluation of extracellular collagen composition of wounds on day 7; (C) Relative protein expression of type I and type III collagen on day 14 after injury, Scale bars = 20 μm; $ P<0.05versus Young-Control, * P<0.05versus Aged-control, # P<0.05versus Aged-CMF, & P < 0.05 versus Aged-HA;

[0075] Figure 4 Results of CMF+HA synergistic treatment to inhibit inflammatory response in aged wounds; (A) Representative immunohistochemical images of IL-10, TNF-α and MCP-1 in wound sites on day 3 after injury; (B) Quantitative analysis of IL-10, TNF-α and MCP-1 expression, Scale bars = 50 μm; $ P<0.05versus Young-Control, * P<0.05versus Aged-control, # P<0.05versus Aged-CMF, & P < 0.05 versus Aged-HA;

[0076] Figure 5 CMF+HA synergistically improves cell proliferation and angiogenesis in aged rats. (A) The image shows the results of Ki67 immunohistochemical staining on day 7; (B) Quantification of the number of Ki67-positive cells in the wound area, Scale bars = 50 μm; (C) Immunofluorescence evaluation of blood vessels stained with PCNA / CD31 on day 7, Scale bars = 100 μm; $ P<0.05versus Young-Control, * P<0.05versus Aged-control, # P<0.05versus Aged-CMF, & P < 0.05 versus Aged-HA;

[0077] Figure 6 The results of CMF+HA synergistically protecting aged wounds by inhibiting ferroptosis. (A) Prussian blue staining results of wound sections obtained from different groups on day 7; (B) quantification results of relative lead intensity; (C) results of ptgs2 mRNA levels determined by RT-qPCR on day 7; (D) results of lipid peroxidation analysis using malondialdehyde; (E) results of quantitative analysis of GSH content in wound tissue on day 7; (F) results of Western Blot detection of Tfr1, Fpn, Slc7a11 and Gpx4; (G) results of normalizing Tfr1, Fpn, Slc7a11 and Gpx4 protein levels to the level of Gapdh; (H) results of quantitative analysis of Tfr1 / Fpn ratio, scale bar = 50 μm; $ P<0.05versus Young-Control, *P<0.05versusAged-control, # P<0.05versus Aged-CMF, & P<0.05versus Aged-HA. DETAILED DESCRIPTION

[0078] The technical solution of the present invention is further illustrated and described in detail below in conjunction with specific embodiments, but the protection scope and implementation methods of the present invention are not limited thereto.

[0079] Example 1: Construction of pressure injury model and wound treatment

[0080] 1. The 7-week-old male SD rats used in this experiment were kept in the animal room of the cardiovascular laboratory of Qilu Hospital of Shandong University. The laboratory temperature was constantly controlled at 23±2℃, the environmental humidity was stable at 50%-70%, the whole animal room was well ventilated and alternating day and night, and the mice had free access to food and water. After 1 week of adaptive feeding, they were randomly divided into a young group and an aging group. The rats in the aging group were intraperitoneally injected with D-gal (100 mg / kg / d) dissolved in normal saline (NS) for 12 weeks to induce the aging model, and the rats in the young group were given the same volume of NS.

[0081] 2. Four pressure injury wounds were prepared at fixed positions on the back of each rat, namely the control hole, CMF hole, HA hole, and CMF+HA hole. In short, the rats were anesthetized by isoflurane inhalation, and the back hair was completely removed. The back skin was gently pulled up and clamped between four circular magnets with a diameter of 12 mm for 12 hours. The magnets were then removed to restore blood flow for 12 hours. This process was regarded as an ischemia-reperfusion cycle. After three IR cycles, debridement was performed, and the necrosis and eschar were removed from the ulcer site. The four pressure injury wounds were established and recorded as day 0 after trauma.

[0082] 3. Use a pipette to adjust the volume at 0.5 ml / cm 2 CMF (soak 200g lithospermum in 500ml sesame oil for 24 hours, filter and boil. 12g Sanguisorba officinalis, 1.5g borneol, 10g puffball powder are ground into powder, added to the soaking solution and mixed evenly) and 10g / L sodium humate were applied topically to CMF wells and HA wells, and the same method was used for CMF+HA wells, first applying 5g / L sodium humate and then applying CMF (soak 100g lithospermum in 500ml sesame oil for 24 hours, filter and boil. 6g Sanguisorba officinalis, 0.75g borneol, 5g puffball powder are ground into powder, added to the soaking solution and mixed evenly), while the control wells were treated with blank solvent once a day for a total of 2 weeks.

[0083] 4. The wound healing was observed and photographed on days 0, 3, 7, and 14 of the experiment. The wound area was calculated using Image J software, and the wound healing rate was calculated as follows: wound healing rate (%) = (A0-At) / A0×100, where A0 is the wound area on day 0 and At is the wound area at a specified time.

[0084] Example 2: Western Blot Detection

[0085] 1. Use RIPA buffer to extract total protein from wound tissue, separate the extracted protein samples into different molecular weights by polyacrylamide gel electrophoresis, and transfer the proteins separated by electrophoresis to PVDF membrane;

[0086] 2. To prevent nonspecific binding, use 5% skim milk as a blocking solution to block unbound sites on the membrane surface;

[0087] 3. After washing the membrane three times with TBST, incubate the membrane with specific primary antibodies overnight at 4°C on a shaker: Anti-β-galactosidase (Proteintech 15518-1-AP), Anti-P53 (Abcam ab26), Anti-p16INK4a (Proteintech10883-1-AP), Anti-CollagenⅠ (Proteintech 66761-1-lg), Anti-CollagenⅢ (Proteintech22734-1-AP), Anti-SLC7A11 (Proteintech 26864-1-AP), Anti-Gpx4 (Abcam ab252833), Anti-Tfr1 (Proteintech 10084-2-AP), Anti-Fpn (Proteintech26601-1-AP), Anti-GAPDH (Proteintech 10494-1-AP);

[0088] 4. On the second day, after removing the strips and washing the membrane, select the corresponding secondary antibody (HRP-labeled goat anti-mouse IgG ZSGB-BIO ZB-2301; goat anti-rabbit IgG ZSGB-BIO ZB-2305) according to the species of the primary antibody and dilute it, and incubate the secondary antibody at room temperature for 1 hour;

[0089] 5. Then, wash away the unbound antibodies to reduce background noise, drop the mixed ECL developer evenly onto the strip, and expose using Amersham Imager 680 (General Electric Company, USA);

[0090] 6. The obtained protein bands were statistically analyzed using Image J.

[0091] Example 3: HE staining

[0092] 1. The wound granulation tissue was fixed with 4% paraformaldehyde for 24-48 hours, then rinsed with running water overnight, dehydrated and made transparent, and then embedded, and uniformly made into 4um paraffin sections;

[0093] 2. Tissue sections were dewaxed to water, followed by hematoxylin staining for 5 minutes, and then rinsed with running water to remove excess stain. 1% hydrochloric acid ethanol was used to rapidly differentiate the sections for about 3 seconds, and then soaked in running water for 3 minutes until the tissue turned blue, and then stained with eosin for 5 minutes, and rinsed with running water to remove floating color;

[0094] 3. After routine sectioning, dehydration and transparency treatment are performed, and then neutral gum is used for sealing;

[0095] 4. Observe the wound tissue healing under a microscope and measure the diameter of the wound bed.

[0096] Example 4: MASSON trichrome staining

[0097] 1. After the tissue sections are dewaxed and hydrated according to the procedure, the sections are first immersed in the mordant solution, incubated in a 60-degree incubator for 1 hour, and rinsed with running water for 10 minutes;

[0098] 2. Then stain with Ponceau fuchsin solution for 10 min and wash twice with distilled water for 5 s each time;

[0099] 3. Then, treat with phosphomolybdic acid solution for about 3 minutes, pour off the upper liquid, and directly add aniline blue dye solution for 2 minutes;

[0100] 4. Finally, wash away the excess staining reagent with a weak acid solution, and continue to add weak acid working solution to cover the slices for 1 minute;

[0101] 5. Dehydrate and transparentize the slices and then seal them. Observe and take pictures under a microscope.

[0102] Example 5: Prussian blue staining

[0103] 1. Dewax the sections routinely, wash with distilled water for 1 minute, soak the sections in Perls staining solution for 20 minutes, and then rinse with distilled water for 2 minutes;

[0104] 2. Then add nuclear fast red staining solution, lightly stain the cell nucleus for 10 minutes, and rinse with tap water for 3 seconds;

[0105] 3. Finally, dehydrate, make transparent and seal the slides, observe and photograph under an optical microscope, and use Image J software to determine the average optical density of iron deposition.

[0106] Example 6: RT-qPCR

[0107] 1. According to the steps of RNA extraction kit (NCM Biotech M5102), total RNA was extracted from rat wound tissue and its content was determined. The total RNA was reverse transcribed into template cDNA using reverse transcription kit (Sparkjade AG0304), and qPCR reaction was performed using 2×SYBRGreen qPCR Mix kit (Sparkjade AH0104);

[0108] 2. SYBR Green was used for QRT-PCR to detect the mRNA expression of PTGS2. The reaction conditions were: pre-deformation at 94°C / 2min, 40 cycles (94°C / 15s, 60°C / 15s, 72°C / 25s)

[0109] 3. Use 2 -ΔΔCq Methods GAPDH was used as an internal reference to calculate the mRNA level.

[0110] Example 7: Immunohistochemical staining

[0111] 1. After the paraffin sections are dewaxed and hydrated, heat repair is performed using sodium citrate antigen repair solution;

[0112] 2.3% hydrogen peroxide solution was used to cover the tissue sections for 10 min to block endogenous peroxidase;

[0113] 3. Add 5% BSA solution to the tissue and let it stand at room temperature for 30 minutes to block it. Discard the blocking solution and add specific primary antibodies (IL-10, TNFA, MCP-1) and incubate in a humidified box at 4 degrees overnight.

[0114] 4. The next day, the slices were removed and allowed to stand for 30 minutes to warm up, and then incubated with secondary antibodies at 37 degrees for 1 hour. Between each step, the slices were slowly rinsed 3 times with phosphate buffered saline, 5 minutes each time.

[0115] 5. Then, add an appropriate amount of freshly prepared DAB working solution to the tissue and observe the color development process under a microscope. After a positive reaction occurs, transfer the slices to distilled water to terminate the color development reaction;

[0116] 6. Stain the nucleus with hematoxylin for 5 minutes, rinse with running water for 30 seconds, differentiate with 1% hydrochloric acid ethanol for 3 seconds, and return to blue with running water for 3 minutes. Finally, dehydrate the tissue sections with gradient ethanol and seal the sections. After collecting images, use Image J for analysis.

[0117] Example 8: Immunohistofluorescence staining

[0118] 1. After the paraffin sections are dewaxed and hydrated, they are placed in sodium citrate antigen retrieval buffer and subjected to antigen retrieval in a microwave oven;

[0119] 2. After slightly drying the liquid on the slices, add 5% BSA to evenly cover the tissue, block at room temperature for 30 minutes, discard the blocking solution, and incubate with a mixed solution of CD31 (1:) and PCNA (1:) primary antibodies at 4 degrees overnight;

[0120] 3. The next day, after standing at room temperature for 30 minutes, add goat anti-rabbit IgG H&L (Alexa Fluor 488) (1:200) and goat anti-mouse IgG H&L (Alexa Fluor 594) (1:200) antibody mixture and incubate at 37 degrees in the dark for 50 minutes;

[0121] 4. Finally, add immunofluorescence anti-fading mounting medium with DAPI and place at room temperature for 8 minutes away from light. Observe the slices under an inverted fluorescence microscope (Cat. No.) and collect images (CD31 is detected as red fluorescence at an excitation wavelength of 520-560nm and an emission wavelength of 570-620nm; PCNA is detected as green fluorescence at an excitation wavelength of 450-500nm and an emission wavelength of 515-565nm).

[0122] Example 9: Detection of MDA content in wound tissue

[0123] 1. Take the frozen wound tissue, weigh it, grind it to prepare tissue homogenate, centrifuge it and take the supernatant, use the malondialdehyde content detection kit to detect the content of lipid peroxidation product MDA in the supernatant, and measure the absorbance of each sample at 532nm and 600nm;

[0124] 2. Calculate ΔA532 = A532 measurement - A532 blank, ΔA600 = A600 measurement - A600 blank, ΔA = ΔA532 - ΔA600, MDA content (nmol / mg prot) = 32.258 × ΔA ÷ Cpr.

[0125] Example 10: Detection of GSH content in wound tissue

[0126] 1. Grind the frozen wound tissue into a homogenate and centrifuge to obtain the supernatant. Use a kit (model) to detect the GSH content in the supernatant and measure the absorbance of each sample at 412 nm;

[0127] 2. Calculate the GSH content using the formula (GSH = Total Glutathione - GSSG × 2).

[0128] The experimental group data of the above embodiment are expressed as mean ± standard deviation and analyzed using SPSS 25.0 statistical software. Variance analysis was used to compare the groups and analyze the main effects of aging, CMF and HA, and to test the interaction effects of two factors. P < 0.05 indicates that the difference is statistically significant.

[0129] Experimental Results

[0130] 1. Figure 1 As a result of D-gal-induced senescence, Figure 1 It can be seen that the protein levels of aging-related genes GLB1, P53, and P16 in the elderly group were significantly increased, indicating that the aging model of the present invention was successfully constructed.

[0131] 2. The results of CMF+HA accelerating wound healing in aging rats. Figure 2 It can be seen that compared with the young control group, the wound healing rate of the aged control group rats on days 3, 7, and 14 was significantly decreased, and the wound bed diameter was significantly increased (P < 0.05). However, after applying CMF, HA, and CMF+HA, the wound healing rate on days 3, 7, and 14 was significantly increased, and the wound bed diameter was significantly decreased (P < 0.05), and the combination of drugs was significantly better than the single drug in terms of wound healing.

[0132] Compared with the aged control group, the wound healing rates of the aged CMF group, the aged HA group, and the aged CMF+HA group increased significantly and the wound bed diameter decreased significantly on the 3rd, 7th, and 14th days (P<0.05). Compared with the aged CMF group, the wound healing rates of the aged CMF+HA group increased significantly and the wound bed diameter decreased significantly on the 3rd, 7th, and 14th days (P<0.05). Compared with the aged HA group, the wound bed diameter decreased significantly on the 3rd, 7th, and 14th days (P<0.05), and the wound healing rates of the aged CMF+HA group increased significantly on the 7th and 14th days (P<0.05). There was no statistically significant difference in the wound healing rates and wound bed diameters on the 3rd, 7th, and 14th days between the aged CMF rats and the aged HA rats.

[0133] Age and HA have a synergistic effect in regulating wound healing rate on D14. CMF and HA have a synergistic effect in regulating wound healing rate on D7.

[0134] 3. Figure 3 This is the result of CMF+HA synergistically promoting extracellular matrix arrangement and collagen synthesis in aging wounds. Figure 3It can be seen that at D3 and D7, the wound tissue under CMF+HA treatment showed coarser wavy collagen fibers and more extensive collagen deposition, and was neatly arranged. Collagen I and collagen III are the main components of the dermal ECM, and they play an indispensable role in wound repair. Therefore, Western Blot was used to detect the content of collagen I and collagen III in wound tissue. The results showed that compared with the young control group, the expression levels of collagen I and collagen III in the aged control group were significantly decreased (P < 0.05); compared with the aged control group, the expression levels of collagen I and collagen III in the aged CMF group, aged HA group, and aged CMF+HA group were significantly increased (P < 0.05); compared with the aged CMF group, the expression levels of collagen I and collagen III in the aged CMF+HA group were significantly increased (P < 0.05); compared with the aged HA group, the expression levels of collagen I and collagen III in the aged CMF+HA group were significantly increased (P < 0.05), indicating that the combination of the two drugs can produce a synergistic promoting effect on collagen I and collagen III in the wound tissue of aged individuals.

[0135] 4. Figure 4 The result is that CMF+HA synergistically alleviates inflammation in aging wound tissue. Figure 5 It can be seen that compared with the young control group, the IL-10 level in the aged control group was significantly decreased, and the levels of TNF-α and MCP-1 were significantly increased (P < 0.05); compared with the aged control group, the IL-10 level in the aged CMF group, the aged HA group, and the aged CMF+HA group was significantly decreased, and the levels of TNF-α and MCP-1 were significantly increased (P < 0.05); compared with the aged CMF group and the aged HA group, the IL-10 level in the aged CMF+HA group was further decreased, and the levels of TNF-α and MCP-1 were further increased (P < 0.05); there was no statistically significant difference in the levels of IL-10, TNF-α and MCP-1 between the aged CMF group and the aged HA group, indicating that the combined use of CMF and HA can synergistically alleviate inflammation in aging wound tissue.

[0136] 5. Figure 5 The result is that CMF+HA synergistically enhances the proliferation and angiogenesis of aging wound cells. Figure 4 It can be seen that there is almost no angiogenesis in aging wounds, but after combined use, a larger number of new blood vessels can be seen, indicating that the combined use of CMF and HA can synergistically promote angiogenesis.

[0137] Immunohistochemistry results showed that a small number of Ki67-positive cells were observed in the tissues of the aging control group on the 7th day of intervention. Compared with the aging control group, Ki67 was highly expressed in the aging CMF group, aging HA group and aging CMF+HA group, among which the cells in the proliferation stage were the most abundant in the aging CMF+HA group, indicating that the combined use of CMF and HA can synergistically promote the proliferation of wound cells.

[0138] 6. Figure 6 The result is that CMF+HA synergistically reduces ferroptosis in aging wound tissue. Figure 6 It can be seen that the wound tissue of the aging control group on the 7th day showed more iron deposition, higher ptgs2 mRNA expression level and MDA content, and lower GSH content. After intervention with CMF, HA, and CMF+HA, iron deposition, ptgs2 mRNA expression level, and MDA content in aging wounds were significantly reduced, and GSH content was significantly increased. The effect of CMF+HA intervention was significantly better than that of single medication, indicating that the combination of CMF and HA has a synergistic inhibitory effect on ferroptosis.

[0139] Western Blot results showed that the expression of Fpn, SLC7A11, and Gpx4 proteins in the wound tissue of the aging control group was reduced, while the treatment of CMF, HA, and CMF+HA groups reversed this phenomenon, and the reversal effect of combined medication was the most significant. The expression level of Tfr1 and the Tfr1 / Fpn ratio in the aging control group were significantly higher than those in the young control group. Compared with the aging control group, the expression level of Tfr1 in the aging HA group and the aging CMF+HA group decreased, and the Tfr1 / Fpn ratio in the aging CMF group, the aging HA group, and the aging CMF+HA group decreased, and the difference was statistically significant. In addition, the Tfr1 / Fpn ratio in the aging CMF+HA group was significantly lower than that in the aging CMF group.

[0140] The above results indicate that in wound injuries of aging individuals, the combination of CMF and HA has a synergistic inhibitory effect on ferroptosis and synergistically regulates ferroptosis-related proteins.

Claims

1. A combination agent for promoting wound healing in aging individuals, characterized in that: The combined medicament is composed of humic acid and a traditional Chinese medicine compound preparation, and the traditional Chinese medicine compound preparation is composed of four medicines: dragon's blood, lithospermum officinale, borneol, and puffball; The humic acid in the combined agent is sodium humate, and the concentration of the sodium humate is 5 g / L; In the combined medicament, the preparation method of each 500ml of the Chinese medicine compound preparation is as follows: (1) Soak 100g of Lithospermum officinale in 500ml of sesame oil for 24h, filter and boil; (2) 6 g of Sanguisorba officinalis, 0.75 g of borneol, and 5 g of puffball powder are ground into powder, added into the soaking solution and mixed evenly to obtain the traditional Chinese medicine compound preparation; The method of using the combined drug is to firstly follow 0.5 ml / cm 2 Apply sodium humate externally, and then apply the Chinese herbal compound preparation.

2. Use of a combined pharmaceutical composition as claimed in claim 1 in the preparation of a drug for promoting wound healing in aging individuals, characterized in that: The combined medicament is composed of sodium humate and a traditional Chinese medicine compound preparation, and the traditional Chinese medicine compound preparation is composed of four medicines: dragon's blood, lithospermum officinale, borneol, and puffball; The concentration of sodium humate in the combined agent is 5 g / L; In the combined medicament, the preparation method of each 500ml of the Chinese medicine compound preparation is as follows: (1) Soak 100g of Lithospermum officinale in 500ml of sesame oil for 24h, filter and boil; (2) 6 g of Sanguisorba officinalis, 0.75 g of borneol, and 5 g of puffball powder are ground into powder, added into the soaking solution and mixed evenly to obtain the Chinese medicine compound preparation.

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