Application of Thonningianin A in the preparation of drugs that promote the polarization of M1 macrophages into M2 macrophages, drugs and preparation methods

Abstract

This invention relates to novel uses of compounds, disclosing the application of Thonningianin A (TA) in the preparation of a drug that promotes the polarization of M1 macrophages into M2 macrophages, the drug itself, and its preparation method. This invention, through the preparation of a wound dressing system that stably releases TA to the wound surface, observes the role of TA in the healing of diabetic wounds in in vivo experiments, and further explores the effect of TA on the polarization of M1 macrophages into M2 macrophages in in vitro experiments. It clarifies that TA promotes the healing of diabetic wounds by facilitating the polarization of M1 macrophages into M2 macrophages, providing a new direction for the treatment of chronic diabetic wounds, and possessing significant theoretical and clinical value.

Description

Technical Field

[0001] This invention relates to novel uses of compounds, specifically to the application of Thonningianin A in the preparation of a drug that promotes the polarization of M1 macrophages into M2 macrophages, and the drug and its preparation method. Background Technology

[0002] Delayed wound healing is a serious complication of diabetes, and excessive inflammatory infiltration is a major contributing factor to the poor healing of diabetic wounds. Macrophages play a crucial role in the inflammatory process: during the inflammatory phase of a wound, M1 macrophages produce pro-inflammatory factors that promote the release of inflammatory mediators and combat pathogens, but their excessive accumulation often exacerbates tissue damage; M2 macrophages mainly inhibit the inflammatory response by releasing various anti-inflammatory factors and promote cell proliferation, granulation tissue formation, angiogenesis, and wound healing. Promoting the differentiation of M1 macrophages into M2 macrophages during the inflammatory phase of a wound, thus transitioning the inflammatory phase to the proliferative phase, is an important target for treating refractory diabetic wounds.

[0003] PCP (Polygonum cuspidatum) is a traditional Chinese herbal medicine. Several active extracts of PCP have been identified, among which Thonningianin A (TA) is a novel monomeric compound extracted from PCP. The structural formula of Thonningianin A, an extract of PCP, is disclosed in patent document CN113143943A. Currently, research on TA is limited, and its potential for further expansion remains to be studied. Summary of the Invention

[0004] This invention has verified that TA can promote the polarization of M1 macrophages into M2 macrophages, and therefore provides the following technical solution:

[0005] The first aspect of this invention provides the use of Thonningianin A in the preparation of a drug that promotes the polarization of M1 macrophages into M2 macrophages.

[0006] In some embodiments, the drug is used to prevent and / or treat delayed wound healing caused by or associated with diabetes.

[0007] In some embodiments, the delayed wound healing includes diabetic ulcers.

[0008] In some implementations, the delayed wound healing occurs in the foot.

[0009] A second aspect of the present invention provides a medicament for treating diabetic complications, wherein the active ingredient of the medicament includes Thonningianin A.

[0010] In some embodiments, the drug further includes a carrier of the active ingredient, wherein the carrier is chitosan.

[0011] In some embodiments, the mass ratio of the carrier to the active ingredient is 1:0.2-0.8.

[0012] In some embodiments, the drug further includes a dressing for adhering to the wound, the dressing being an aqueous solution of a polyoxyethylene polyoxypropylene ether triblock copolymer.

[0013] In some embodiments, the dressing is a 15-25% (w / v) aqueous solution of polyoxyethylene polyoxypropylene ether triblock copolymer, wherein the volume-to-mass ratio of the aqueous solution of polyoxyethylene polyoxypropylene ether triblock copolymer to the active ingredient is 100 μL: 0.02-0.08 mg.

[0014] In some embodiments, the drug is a topical medication.

[0015] In some embodiments, the drug is in an aqueous solution state when the temperature is <20°C, and transforms into a gel state when the temperature is ≥20°C.

[0016] A third aspect of the present invention provides a method for preparing a medicament for the treatment of diabetic complications, wherein the method includes encapsulating the active ingredient Thonningianin A using a carrier, and then dispersing the carrier encapsulating the active ingredient Thonningianin A in an aqueous solution of a polyoxyethylene polyoxypropylene ether triblock copolymer.

[0017] In some embodiments, the process of using a carrier to encapsulate the active ingredient Thonningianin A includes: dissolving 1 mg of chitosan in a first solution, and then adjusting the pH to 5-6 to obtain a chitosan solution;

[0018] 0.2-0.8 mg of Thonningianin A is dissolved in a second solution to form a Thonningianin A solution, and then the Thonningianin A solution is mixed with the chitosan solution to obtain a third solution;

[0019] A fourth solution is added dropwise to the third solution to completely precipitate the chitosan containing Thonningianin A, and the chitosan nanoparticles containing Thonningianin A are obtained by centrifugation.

[0020] In some embodiments, the process of dispersing the carrier encapsulating the active ingredient Thonningianin A in an aqueous solution of a polyoxyethylene polyoxypropylene ether triblock copolymer includes: dissolving the polyoxyethylene polyoxypropylene ether triblock copolymer in distilled water to obtain 1000 μL of a 15-25% (w / v) aqueous solution of the polyoxyethylene polyoxypropylene ether triblock copolymer, and then dispersing all of the chitosan nanoparticles encapsulating Thonningianin A into the aqueous solution of the polyoxyethylene polyoxypropylene ether triblock copolymer.

[0021] There are no special requirements for the amount of the first and second solutions used in the preparation method, as long as they can completely dissolve the corresponding components. Specifically, the mass ratio of chitosan to the volume of the first solution can be 3 mg: 1 mL, and the concentration of Thonningianin A after dissolving in the second solution can be 0.5-2 mg / mL.

[0022] The amount of the fourth solution added is generally determined by the point at which the milky white suspension no longer increases, indicating that the chitosan containing Thonningianin A has been completely precipitated. The mass ratio of chitosan to the volume of the fourth solution can be 3 mg: 1 mL.

[0023] In some embodiments, the first solution is a 1-3 wt% acetic acid solution.

[0024] In some embodiments, the second solution is a mixture of PEG400, physiological saline, and anhydrous ethanol, wherein the volume ratio of PEG400, physiological saline, and anhydrous ethanol is 5-7:2-4:1.

[0025] In some embodiments, the fourth solution is a 0.1-0.2% (w / v) sodium tripolyphosphate solution.

[0026] In the treatment of diabetic wounds, maintaining a constant drug concentration at the site of application is crucial for the healing process. Chitosan nanoparticles (CNPS) make it possible to maintain a constant drug concentration. Diabetic wounds are complex and irregular in shape, making it difficult for conventional dressings to adhere evenly. Polyoxyethylene polyoxypropylene ether triblock copolymers possess unique thermal reversibility; they are liquid at low temperatures and gradually transform into a semi-solid gel form as the temperature rises. When applied to the wound at low temperatures, the dressing transforms into a gel state upon contact with body temperature, thus adapting to the complex and irregular spaces of diabetic wounds.

[0027] This invention prepares a wound dressing system that stably releases TA to the wound surface, observes the role of TA in the healing of diabetic wounds in in vivo experiments, and further explores the effect of TA on the polarization of M1 macrophages to M2 macrophages in in vitro experiments. It clarifies that TA promotes the healing of diabetic wounds by promoting the polarization of M1 macrophages to M2 macrophages, providing a new direction for the treatment of chronic diabetic wounds, and has important theoretical value and clinical significance.

[0028] In the drug provided by this invention, TA can be the sole active ingredient. Attached Figure Description

[0029] Figure 1 This is the preparation process of TA-CNPS-PF;

[0030] Figure 2 This describes the changes in the rats' wounds during the drug concentration screening experiment;

[0031] Figure 3 This is a statistical graph of blood glucose changes in rats during an experiment analyzing the effect of TA on diabetic wound healing.

[0032] Figure 4 This is a statistical graph showing the changes in body weight of rats in an experiment analyzing the effect of TA on the healing of diabetic wounds.

[0033] Figure 5 This study analyzed the changes in the wounds of rats during an experiment analyzing the effects of TA on the healing of diabetic wounds.

[0034] Figure 6 This is a statistical analysis of the wound healing rate in an experiment analyzing the effect of TA on diabetic wound healing;

[0035] Figure 7 These are the results of a cytotoxicity test;

[0036] Figure 8 These are the results of an immunocytochemical experiment;

[0037] Figure 9 These are the results of a flow cytometry experiment. Detailed Implementation

[0038] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0039] The preparation process of TA-CNPS-PF is as follows: Figure 1As shown in the figure, the pink spheres in the beaker represent chitosan (CNPS) and the blue spheres represent TA. After chitosan encapsulates TA, it precipitates in the presence of sodium tripolyphosphate solution (TPP solution). After centrifugation, TA-CNPS nanoparticles are obtained. The prepared drug is obtained by dispersing the TA-CNPS nanoparticles in polyoxyethylene polyoxypropylene ether triblock copolymer (PF-127, the blue mesh-like substance in the figure), and is denoted as TA-CNPS-PF.

[0040] Unless otherwise specified, all reagents used in the following preparation examples are commercially available. Among them, polyoxyethylene polyoxypropylene ether triblock copolymer (PF-127) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number Sigma-P2443.

[0041] Preparation Example 1

[0042] 1. Preparation of TA-CNPS nanoparticles: Dissolve 45 mg of chitosan in 15 ml of acetic acid solution (1 wt%). To ensure that the chitosan is fully and uniformly mixed and dissolved with the acetic acid solution, add it slowly at a rate of 22.5 mg / min while stirring at 600 rpm until dissolved. Then adjust the pH of the obtained solution to 5.5 ± 0.5 with 2 mol / L NaOH to obtain the chitosan solution.

[0043] Prepare a mixed solution of PEG400, physiological saline, and anhydrous ethanol, wherein the volume ratio of PEG400, physiological saline, and anhydrous ethanol is 6:3:1 to obtain a second solution.

[0044] Prepare a 0.1% (w / v) sodium tripolyphosphate solution, i.e., the fourth solution, using distilled water;

[0045] Take 9 mg of TA and dissolve it in 15 ml of the second solution to form a TA solution. Then, mix the obtained TA solution with the chitosan solution to obtain a third solution. Add the fourth solution dropwise to the third solution at a rate of 40 drops / min. A milky white suspension begins to appear in the mixture. Add 15 ml of sodium tripolyphosphate solution. Then, centrifuge the obtained liquid at 1000 rpm for 30 minutes. Wash the obtained solid particles three times with deionized water and then freeze-dry them at -80℃ for 72 hours to obtain chitosan nanoparticles embedded with TA, which are denoted as TA-CNPS nanoparticles and stored in a dark environment at -20℃ for later use.

[0046] 2. Preparation of TA-CNPS-PF hydrogel: Dissolve PF-127 in distilled water at 4℃ and stir magnetically to form a 20% (w / v) PF-127 clear solution (45000 μL). Then, at 4℃, disperse all the TA-CNPS nanoparticles obtained in step 1 in the PF-127 clear solution to obtain TA-CNPS-PF hydrogel, which is stored at 4℃ for later use. In the TA-CNPS-PF hydrogel prepared in this example, the mass ratio of chitosan to TA is 1:0.2, and the volume-to-mass ratio of the polyoxyethylene polyoxypropylene ether triblock copolymer aqueous solution to TA is 100 μL:0.02 mg.

[0047] The obtained TA-CNPS-PF hydrogel is a flowing aqueous solution at temperatures below 20℃ and becomes a non-flowing gel at temperatures above 20℃.

[0048] Preparation Example 2

[0049] This preparation example uses the same method as Preparation Example 1, except that 18 mg of TA is dissolved in 15 ml of a second solution to form a TA solution. In the TA-CNPS-PF hydrogel prepared in this example, the mass ratio of chitosan to TA is 1:0.4, and the volume-to-mass ratio of the polyoxyethylene polyoxypropylene ether triblock copolymer aqueous solution to TA is 100 μL:0.04 mg.

[0050] Preparation Example 3

[0051] This preparation example uses the same method as Preparation Example 1, except that 27 mg of TA is dissolved in 15 ml of a second solution to form a TA solution. In the TA-CNPS-PF hydrogel prepared in this example, the mass ratio of chitosan to TA is 1:0.6, and the volume-to-mass ratio of the polyoxyethylene polyoxypropylene ether triblock copolymer aqueous solution to TA is 100 μL:0.06 mg.

[0052] Preparation Example 4

[0053] This preparation example uses the same method as Preparation Example 1, except that 36 mg of TA is dissolved in 15 ml of a second solution to form a TA solution. In the TA-CNPS-PF hydrogel prepared in this example, the mass ratio of chitosan to TA is 1:0.8, and the volume-to-mass ratio of the polyoxyethylene polyoxypropylene ether triblock copolymer aqueous solution to TA is 100 μL:0.08 mg.

[0054] Preparation Example 5

[0055] This preparation example uses the same method as Preparation Example 1, except that no TA is dissolved in the second solution.

[0056] The TA-CNPS nanoparticles prepared by the above method have an embedding rate of TA within chitosan of over 99%.

[0057] I. Drug Concentration Screening

[0058] 1. Experimental materials and instruments

[0059] (1) Experimental animals: SD rats, each weighing about 200g, purchased from the Animal Experiment Center of Southwest Medical University.

[0060] (2) 60% high-fat feed: purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., product code: D12492;

[0061] (3) SPF-grade rat and mouse maintenance feed was purchased from Huanyu Biotechnology Co., Ltd.;

[0062] (4) Streptozotocin (STZ): Purchased from Sichuan Philbos Technology Co., Ltd., product number: S8050-100mg;

[0063] (5) Aike test strips, including instruments: purchased from Benyuansheng Experimental Equipment Store, Jiangyang District, Luzhou City, item number: EZ3-200.

[0064] 2. Establish a diabetic mouse model

[0065] Six rats were selected, one of which was a normal group and fed an SPF-grade rat maintenance diet. The other five rats were a model group. The model group rats were fed a 60% high-fat diet for 4 weeks and then fasted (but allowed water) for 12 hours. Each rat was then given 30 mg / kg of 1% streptozotocin solution (STZ was dissolved in 0.1 mol / L sodium citrate buffer, freshly prepared to a concentration of 10 mg / mL, and sterilized by filtering with a 0.22 μm filter. The solution was prepared fresh and protected from light) via intraperitoneal injection for 4 consecutive days. Then, the rats were continued to be fed a 60% high-fat diet for 2 weeks.

[0066] 3. Establish a diabetic wound model

[0067] Make a 2cm diameter circular full-thickness skin incision at the junction of the neck and back of the rat, extending to the fascia layer.

[0068] 4. Drug concentration screening: Five model rats from the first group were used, one of which served as a diabetic control group with no wound treatment. The wounds of the other four rats were uniformly coated with 200 μL of the TA-CNPS-PF hydrogel prepared in Examples 1-4. After coating, the TA-CNPS-PF hydrogel in the aqueous solution state quickly transformed into a non-flowing gel state and adhered firmly to the wound surface. Based on the average weight of the rats of 200g, the drug dosages applied to the rats in Examples 1-4 reached 0.2 mg / Kg, 0.4 mg / Kg, 0.6 mg / Kg, and 0.8 mg / Kg, respectively.

[0069] Change the dressing every two days and take photos at 0D, 3D, 7D, and 14D.

[0070] 5. Results Analysis: Random blood glucose, body weight, and wound healing status of rats were monitored. Blood glucose levels were measured using tail vein blood collection. The results are as follows.

[0071] The results of random blood glucose measurements in rats are shown in Table 1.

[0072] Table 1 Blood glucose monitoring values

[0073]

[0074] As shown in Table 1, the random blood glucose levels of the model group rats were ≥16.7 mmol / L 3 days after STZ injection.

[0075] The results of rat weight monitoring before and after modeling are shown in Table 2.

[0076] Table 2 Weight Monitoring

[0077]

[0078] As shown in Table 2, the normal diet group gained weight after STZ injection and continued feeding for 14 days, while the control group and the diabetes model group both experienced weight loss after STZ injection and continued feeding for 14 days.

[0079] The criteria for successful establishment of a type 2 diabetes model are: a random blood glucose level ≥16.7 mmol / L 3 days after STZ injection, and the rats exhibiting polydipsia, polyphagia, polyuria, dull yellow coat, reduced activity, and weight loss. Therefore, the rats fed using this modeling method were successfully modeled as having diabetes.

[0080] Changes in rat wounds as follows Figure 2 As shown.

[0081] from Figure 2The first column, from top to bottom, shows the wounds of rats in the normal diet group (denoted as Ctrl) on days 0, 3, 7, and 14. The second column, from top to bottom, shows the wounds of rats in the diabetic control group (denoted as DM) on days 0, 3, 7, and 14. The third column, from top to bottom, shows the wounds of rats in the diabetic model group (denoted as 0.2 mg / kg TA-CNPS-PF) administered at a TA dosage of 0.2 mg / kg on days 0, 3, 7, and 14. The fourth column, from top to bottom, shows the wounds of rats in the diabetic model group (denoted as 0.4 mg / kg TA-CNPS-PF) administered at a TA dosage of 0.4 mg / kg on days 0. The images show wound photographs of TA-CNPS-PF rats on days 0, 3, 7, and 14. The fifth column, from top to bottom, shows wound photographs of diabetic model rats (denoted as 0.6 mg / Kg TA-CNPS-PF) administered at a dose of 0.6 mg / Kg, on days 0, 3, 7, and 14. The sixth column, from top to bottom, shows wound photographs of diabetic model rats (denoted as 0.8 mg / Kg TA-CNPS-PF) administered at a dose of 0.8 mg / Kg, on days 0, 3, 7, and 14.

[0082] from Figure 2 As can be seen, wound healing was not significant on days 3 and 7. On day 14, the wound healing rate was lowest in the diabetic control group compared to the normal diet group. The wound healing rate was highest in the model group with a TA dosage of 0.6 mg / Kg. The wound healing rates of the three model groups with TA dosages of 0.2 mg / Kg, 0.4 mg / Kg, and 0.8 mg / Kg were higher than those of the normal diet group and the diabetic control group. Therefore, 0.6 mg / Kg of TA is the most suitable concentration for further experiments.

[0083] Additionally from Figure 2 The comparison also revealed that, in diabetic rats treated with TA, wound healing was better than in the untreated group on a normal diet, indicating that TA can significantly promote wound healing in diabetic patients. TA could also be used as the sole active ingredient in drugs for treating diabetic ulcers.

[0084] II. Analysis of the impact of TA on diabetic wound healing

[0085] 1. Experimental Materials and Methods

[0086] The experimental materials, methods for establishing the diabetic mouse model, the method for establishing the diabetic wound model, and the blood glucose measurement methods used in this part of the experiment are the same as those in Part 1—Drug Concentration Screening.

[0087] Twenty SD rats were selected and randomly divided into a normal diet group (n=5, denoted as Ctrl) and a model group (n=15) using a random number table. The normal diet group was fed SPF-grade mouse maintenance diet. After the model group rats successfully developed diabetes, diabetic lesions were created at the neck-dorsal junction of the normal diet group and the model group rats.

[0088] 2. Administration

[0089] The wounds of the normal diet group were not treated in any way. The model group was divided into three groups. One group was treated without wound treatment and was designated as the diabetes control group (DM). One group was coated with the gel prepared in Example 5 and was designated as the drug blank control group (CNPS-PF). The other group was coated with TA-CNPS-PF hydrogel prepared in Example 3 at a ratio of 0.6 mg / Kg and was designated as the treatment group (TA-CNPS-PF). The amount of gel coated was 200 μL / time / mouse.

[0090] 3. Data Recording

[0091] ① Blood glucose measurement: To avoid death from hyperosmolar hyperglycemia in rats due to excessively high blood glucose levels, random blood glucose levels were measured at 0D, 3D, 7D, and 14D.

[0092] ② Body weight measurement: The body weight of the rats was measured on day 0 and day 14.

[0093] ③ Wound monitoring: Dressings were changed every two days. Wound photos were taken and measured at 0D, 3D, 7D, and 14D, with the long axis of the wound as a (cm) and the short axis as b (cm). The wound healing rate on day T postoperatively was assessed using the following formula:

[0094] Wound area healing % = (wound area on day 0 - wound area on day T) ÷ wound area on day 0 × 100%, where wound area S = (a × b)π / 4.

[0095] 4. Results Analysis

[0096] ① Blood glucose monitoring results as follows Figure 3 As shown in the figure, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001. From Figure 3 It can be seen that the random blood glucose level of all model group rats was ≥16.7mmol / L 3D after STZ injection, and since the number of rats was 5, the increase in blood glucose was statistically significant.

[0097] ② Weight monitoring results as follows Figure 4As shown in the figure, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001. After STZ injection, on day 14, compared to day 0, the model group rats showed a decrease in body weight, while the normal diet group showed an increase in body weight. Furthermore, because the number of rats was 5, the increase in blood glucose was statistically significant.

[0098] Combination Figure 3 and Figure 4 It can be seen that the rat model of diabetes was successfully established.

[0099] ③ Wound monitoring results:

[0100] like Figure 5 The images show the wound changes in rats. In the first column, the four images from top to bottom are wound photos of rats in the normal diet group (Ctrl) on days 0, 3, 7, and 14. In the second column, the four images from top to bottom are wound photos of rats in the diabetic control group (DM) on days 0, 3, 7, and 14. In the third column, the four images from top to bottom are wound photos of rats in the drug blank control group (CNPS-PF) on days 0, 3, 7, and 14. In the fourth column, the four images from top to bottom are wound photos of rats in the treatment group (TA-CNPS-PF) on days 0, 3, 7, and 14.

[0101] Rat wound healing rate statistics are as follows Figure 6 As shown in the figure, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001. Specific blood glucose test values ​​are shown in Table 1.

[0102]

[0103] Combination Figure 5 , Figure 6 As shown in Table 1, at 3D, the wound healing rate of the diabetes control group (DM) was lower than that of other groups. The wound healing rates of the normal diet group (Ctrl) and the drug blank control group (CNPS-PF) were similar, with no statistically significant difference. The wound healing rate of the treatment group (TA-CNPS-PF) was the highest.

[0104] At day 7, the overall wound healing rate was higher than that at day 3. The wound healing rate in the diabetic control group (DM) was the lowest, but there was no statistically significant difference compared with the normal diet group (Ctrl). The wound healing rate in the drug blank control group (CNPS-PF) was higher than that in the normal diet group (Ctrl), and the wound healing rate in the treatment group (TA-CNPS-PF) was the highest.

[0105] At the 14th day, the wound healing rate of the diabetic control group rats (DM) was lower than that of the normal diet group rats (Ctrl). The wound healing rate of the treatment group (TA-CNPS-PF) was the highest. The wound healing rate of the drug blank control group rats (CNPS-PF) was similar to that of the normal diet group rats (Ctrl), and the difference was not statistically significant.

[0106] III. In vitro experiments

[0107] 1. Experimental materials and instruments

[0108] 特级胎牛血清:购自四川信标生物科技有限公司,货号:AC03L055; Special fetal bovine serum: Purchased from Sichuan Xinbiao Biotechnology Co., Ltd., product number: AC03L055;

[0109] 青霉素-链霉素-两性霉素B:购自喀斯玛商城泸州配送中心(Beyotime / 碧云天),货号:C0224-100ml; Penicillin-streptomycin-amphotericin B: Purchased from the Luzhou Distribution Center of Kasmart Mall (Beyotime), product number: C0224-100ml;

[0110] DMEM培养基:购自喀斯玛商城泸州配送中心(Gibco代服务),货号:C11885500BT; DMEM medium: Purchased from the Luzhou Distribution Center of Kasmart Mall (Gibco proxy service), product number: C11885500BT;

[0111] RAW264.7巨噬细胞:购自喀斯玛商城泸州配送中心(Procell / 普诺赛代服务),货号:CL-0190; RAW264.7 macrophages: Purchased from the Luzhou Distribution Center of Kasmart Mall (Procell proxy service), product number: CL-0190;

[0112] CCK8试剂盒:泸州市龙马潭区万丰实验器材经营部(DOJINDO / 同仁化学),货号:CK04; CCK8 kit: Purchased from Wanfeng Experimental Equipment Business Department in Longmatan District, Luzhou City (DOJINDO), product number: CK04;

[0113] 4%多聚甲醛:购自喀斯玛商城泸州配送中心(Solarbio / 索莱宝),货号:P1110-500ml; 4% paraformaldehyde: Purchased from the Luzhou Distribution Center of Kasmart Mall (Solarbio), product number: P1110-500ml;

[0114] Triton X-100:购自西格玛奥德里奇(上海)贸易有限公司,货号:T8787; Triton X-100: Purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number: T8787;

[0115] 牛血清白蛋白(BSA):购自西格玛奥德里奇(上海)贸易有限公司,货号:A1933-1G; Bovine serum albumin (BSA): Purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number: A1933-1G;

[0116] 一抗CD86 Antibody,购自深圳市益百顺科技有限公司; Primary antibody CD86 Antibody, purchased from Shenzhen Yibaisun Technology Co., Ltd.;

[0117] 一抗Anti-Mouse CD206 / MMR Antibody,购自喀斯玛商城泸州配送中心(abcam / 艾博抗),货号:ab64693; Primary antibody Anti-Mouse CD206 / MMR Antibody, purchased from the Luzhou Distribution Center of Kasmart Mall (abcam), product number: ab64693;

[0118] Donkey Anti-Rabbit Fluorescent Secondary Antibody, Alexa Fluor 555: Purchased from Guangzhou Guihai Biotechnology Co., Ltd. (Invitrogen), Catalog No.: A31572;

[0119] Donkey Anti-Mouse Fluorescent Secondary Antibody, Alexa Fluor 488: Purchased from Guangzhou Guihai Biotechnology Co., Ltd. (Invitrogen), Catalog No.: A21202;

[0120] APC Anti-Mouse CD206 / MMR Antibody was purchased from Sichuan Aichi Biotechnology Co., Ltd. (Elabscience), Catalog No.: E-AB-F1135E;

[0121] Anti-fluorescence quenching mounting medium (containing DAPI): Shanghai Beyotime Biotechnology Co., Ltd., Catalog No.: P0131-25ml;

[0122] Inverted fluorescence microscope: OLYMPUS;

[0123] Flow cytometer: ACEA NovoCyte TM 2070;

[0124] Cell incubator: Thermo Fisher Scientific cell incubator i160.

[0125] 2. Cell culture

[0126] Normal culture medium: DMEM medium containing 10% fetal bovine serum (FBS), 100 U / mL -1 penicillin-streptomycin-amphotericin B;

[0127] High-glucose culture medium: DMEM medium containing 10% fetal bovine serum (FBS), 100 U / mL -1 penicillin-streptomycin-amphotericin B, 40 mmol / L glucose concentration;

[0128] TA + high-glucose culture medium: DMEM medium containing 10% fetal bovine serum (FBS), 100 U / mL -1 penicillin-streptomycin-amphotericin B, 40 mmol / L glucose concentration, 2 μM TA;

[0129] TA culture medium: DMEM medium containing 10% fetal bovine serum (FBS), 100 U / mL -1 penicillin-streptomycin-amphotericin B, 2 μM TA.

[0130] RAW264.7 macrophages were cultured in the corresponding culture medium according to the grouping, and the culture conditions were all 37 °C, 5% CO2, so as to obtain the corresponding cultured cells.

[0131] 3. Cytotoxicity test

[0132] RAW264.7 macrophages cultured in normal culture medium were used to prepare a RAW264.7 cell suspension for cell counting at 3×10⁻⁶ cells / mL. 4 Approximately 100 μL of cell suspension was seeded into each well of a 96-well plate, with 6 replicates per group. Macrophages were treated with 0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM TA, respectively. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h, followed by 10 μL of CCK8 and incubation for another 2 h. Absorbance was measured at 450 nm, and the IC50 was calculated. 50 Determine the TA dosage concentration.

[0133] 4. Immune cell experiments

[0134] RAW264.7 macrophages cultured in normal culture medium were seeded onto slides and used as a control group (Ctrl).

[0135] RAW264.7 macrophages cultured in TA medium were seeded onto slides and used as the TA drug group (TA).

[0136] AW264.7 macrophages cultured in high-glucose medium were seeded onto slides and used as the diabetes group (Highglucose).

[0137] RAW264.7 macrophages cultured in TA+high glucose medium were seeded and used as the diabetes + TA drug group.

[0138] Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 5% BSA, incubated with primary antibody at 4℃ for 12 h, incubated with secondary antibody at 25℃ for 1 h, stained with DAPI, and then blocked again. Immunofluorescence microscopy was used to photograph M1 and M2 macrophages and observe morphological changes. Primary antibodies CD86 and CD206 were used to observe M1 and M2 macrophages and their morphological changes.

[0139] 5. Flow cytometry experiment

[0140] The same methods as those used in immune cell experiments were used to obtain the Ctrl group, TA group, High glucose group, and Highglucose+TA group, respectively.

[0141] After cell processing and counting, centrifuge at 1000 rpm for 5 min, discard the supernatant, wash cells with PBS cell washing buffer containing 2% BSA, fix cells with 4% paraformaldehyde at room temperature, resuspend cells with a cell permeabilizing agent, and add to 1.5 mL EP tubes, 1×10⁶ cells / min. 6 / tube, volume 100μL / tube. A blank control (containing RAW264.7 macrophages, without antibody, used for calibrating cell autofluorescence) and an isotype control (without RAW264.7 macrophages, containing antibody, used for calibrating flow cytometry baseline) were set up. After incubation with APC Anti-Mouse CD206 / MMRAntibody at 25℃ for 1 h, the cells were washed three times before flow cytometry analysis.

[0142] 6. Experimental Results

[0143] (1) Cytotoxicity test

[0144] like Figure 7 As shown, where Figure 7 (a) is a line graph of cell viability calculated from the OD value measured at 450 nm using an ELISA reader. It can be seen that cell viability decreases as TA concentration increases. Figure 7 (b) is to calculate IC using Prism 10.1.2 software for 7(a). 50 The method of calculating the obtained graph, the calculated IC 50 The concentration was 4.798 μM, therefore the safe concentration of TA for macrophages was ultimately chosen to be 2 μM.

[0145] (2) Immune cell experiments

[0146] The results are as follows Figure 8 As shown, the first row of five photos in the image sequentially represents the macrophage state under a light microscope in the control group, DAPI-stained cell nuclei, green fluorescent staining of CD86 (M1 macrophage marker), red fluorescent staining of CD206 (M2 macrophage marker), and a combination of the second, third, and fourth images; the second row of five photos sequentially represents the macrophage state under a light microscope in the diabetes group (High glucose), DAPI-stained cell nuclei, green fluorescent staining of CD86 (M1 macrophage marker), red fluorescent staining of CD206 (M2 macrophage marker), and a combination of the second, third, and fourth images; the third row of five photos sequentially represents the macrophage state under a light microscope in the diabetes + TA drug group (High glucose + 2μM TA), DAPI-stained cell nuclei, green fluorescent staining of CD86 (M1 macrophage marker), red fluorescent staining of CD206 (M2 macrophage marker), and a combination of the second, third, and fourth images; the fourth row of five photos sequentially represents the macrophage state under a light microscope in the TA drug group (2μM TA), DAPI-stained cell nuclei, green fluorescent staining of CD86 (M1 macrophage marker), red fluorescent staining of CD206 (M2 macrophage marker), and a combination of the second, third, and fourth images; The image is a combination of the following images: macrophage state under a light microscope, cell nuclei stained with DAPI, CD86 (marker of M1 macrophages) stained with green fluorescence, CD206 (marker of M2 macrophages) stained with red fluorescence, and the second, third, and fourth images.

[0147] Under a light microscope, the macrophage morphology in the diabetes + TA drug group (High glucose + 2μM TA) changed from round to spindle-shaped (M2 type macrophage morphology) (first photo in the third row). A small number of spindle-shaped macrophages were also observed in the TA drug group (first photo in the fourth row). Cells in the control group (Ctrl) and the diabetes group (High glucose) were round (first and second photos in the first and second rows, respectively). Immunocell experiments showed enhanced green fluorescence in the diabetes group (High glucose), indicating that 40 mmol / L high glucose can induce M1 type macrophages (third photo in the second row), and TA drug can induce M1 type macrophages to polarize into M2 type macrophages (third and fourth photos in the third row).

[0148] (3) Flow cytometry experiment

[0149] The results are as follows Figure 9 As shown in the figure, the first row of three photos represents, in order, the total number of cells detected by the P1 gate in the control group, the total number of cells after adhesion removal detected by the P2 gate, and the number of M2 macrophages detected by APC fluorescence; the second row of three photos represents, in order, the total number of cells detected by the P1 gate in the diabetes group, the total number of cells after adhesion removal detected by the P2 gate, and the number of M2 macrophages detected by APC fluorescence; the third row of three photos represents, in order, the total number of cells detected by the P1 gate in the diabetes + TA drug group (High glucose + TA), the total number of cells after adhesion removal detected by the P2 gate, and the number of M2 macrophages detected by APC fluorescence; the fourth row of three photos represents, in order, the total number of cells detected by the P1 gate in the TA drug group, the total number of cells after adhesion removal detected by the P2 gate, and the number of M2 macrophages detected by APC fluorescence.

[0150] The first column of the graphs shows All Events, representing 20,000 cells examined. The P1 gate selects the target cell population from these 20,000 cells, reducing noise and interference. The second column shows further removal of adherent cells after the P1 gate. The third column shows the result after removing adhesion signals; APC is a fluorescent dye labeled CD206, and the P4 gate represents the number of M2 macrophages. It can be seen that M2 macrophages accounted for 1.44% in the control group (Ctrl), 2.58% in the diabetes group (High glucose), 14.61% in the diabetes + TA drug group (High glucose + TA), and 10.55% in the TA drug group. This indicates that TA can induce M2 macrophage polarization.

[0151] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention. Those skilled in the art, under the guidance of the present invention, can make various similar representations without departing from the spirit and claims of the present invention, and such modifications all fall within the protection scope of the present invention.

Claims

1. Application of Thonningianin A in the preparation of drugs that promote the polarization of M1 macrophages into M2 macrophages; in, The drug is a topical medication used to treat delayed healing of wounds caused by or associated with diabetes, specifically diabetic ulcers occurring on the foot. The active ingredient of the drug includes Thonningianin A, and the drug also includes a carrier of the active ingredient, wherein the carrier is chitosan.

2. The application according to claim 1, wherein, The mass ratio of the carrier to the active ingredient is 1:0.2-0.

8.

3. The application according to claim 1 or 2, wherein, The medication also includes a dressing for adhering to the wound, the dressing being an aqueous solution of a polyoxyethylene polyoxypropylene ether triblock copolymer.

4. The application according to claim 3, wherein, The dressing is a 15-25% (w / v) aqueous solution of polyoxyethylene polyoxypropylene ether triblock copolymer, wherein the volume-to-mass ratio of the aqueous solution of polyoxyethylene polyoxypropylene ether triblock copolymer to the active ingredient is 100 μL: 0.02-0.08 mg.

5. The application according to claim 4, wherein, When the temperature is <20℃, the drug is in an aqueous solution state; when the temperature is ≥20℃, the drug transforms into a gel state.

6. The application according to claim 1, wherein the method for preparing the drug comprises: The active ingredient Thonningianin A was encapsulated using a carrier, and then the carrier encapsulated with Thonningianin A was dispersed in an aqueous solution of polyoxyethylene polyoxypropylene ether triblock copolymer.

7. The application according to claim 6, wherein, The process of using a carrier to encapsulate the active ingredient Thonningianin A includes: dissolving 1 mg of chitosan in a first solution, and then adjusting the pH value to 5-6 to obtain a chitosan solution; 0.2-0.8 mg of Thonningianin A is dissolved in a second solution to form a Thonningianin A solution, and then the Thonningianin A solution is mixed with the chitosan solution to obtain a third solution; A fourth solution is added dropwise to the third solution to completely precipitate the chitosan containing Thonningianin A, and the chitosan nanoparticles containing Thonningianin A are obtained by centrifugation.

8. The application according to claim 7, wherein, The process of dispersing the carrier encapsulating the active ingredient Thonningianin A in an aqueous solution of polyoxyethylene polyoxypropylene ether triblock copolymer includes: dissolving the polyoxyethylene polyoxypropylene ether triblock copolymer in distilled water to obtain 1000 μL of a 15-25% (w / v) polyoxyethylene polyoxypropylene ether triblock copolymer aqueous solution, and then dispersing all the chitosan nanoparticles encapsulating Thonningianin A into the polyoxyethylene polyoxypropylene ether triblock copolymer aqueous solution.

9. The application according to claim 8, wherein, The first solution is a 1-3 wt% acetic acid solution; And / or, the second solution is a mixed solution of PEG400, physiological saline and anhydrous ethanol, wherein the volume ratio of PEG400, physiological saline and anhydrous ethanol is 5-7:2-4:1; And / or, the fourth solution is a 0.1-0.2% (w / v) sodium tripolyphosphate solution.

Images