Application of silybum marianum alcohol extract in preparation of deep II-degree burn and scald medicine

Abstract

The invention belongs to the technical field of traditional Chinese medicine and application thereof, and particularly relates to application of silybum marianum alcohol extract in preparation of medicine for treating deep II-degree burns and scalds. When the silybum marianum alcohol extract is applied to preparation of the medicine for treating deep II-degree burns and scalds, oxidative stress injury is improved by activating an Nrf2 / HO-1 pathway, and fibrosis is inhibited by regulating a TGF-beta / Smad pathway so as to promote repair of the deep II-degree burns and scalds. According to the method, the yield of the silybum marianum alcohol extract is improved by optimizing the extraction conditions of the silybum marianum; it is clear that the silybum marianum alcohol extract has multiple mechanisms of resisting oxidation, inhibiting fibrosis and resisting inflammation, the silybum marianum alcohol extract has a remarkable promoting effect on treatment and repair of deep II-degree burns and scalds, and theoretical basis and experimental support are provided for silybum marianum serving as a potential treatment medicine for scar-free healing after deep II-degree scalds.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine and its application, and particularly relates to application of a silymarin ethanol extract in preparation of a medicament for treating deep Ⅱ degree burn and scald. BACKGROUND

[0002] Burn and scald are caused by long-term contact of skin with low-heat objects higher than body temperature. The depth of scald is classified into shallow Ⅱ degree, deep Ⅱ degree, Ⅲ / Ⅳ degree, etc. according to the degree of thermodynamic damage, wherein the deep Ⅱ degree is damage to the reticular layer of the dermis, and the clinical manifestations are: lack of blisters or local necrosis (pale base), dull touch, and 50%-70% of skin appendages are damaged in tissue biopsy. The wound caused by scald is deep, and even deep tissue necrosis occurs in severe cases, and then ulceration occurs, which cannot heal for a long time, threatening the health of the human body. At present, the treatment methods for deep Ⅱ degree burn and scald mainly include non-surgical treatment and surgical treatment. Surgical intervention includes debridement, skin transplantation and use of biological materials; non-surgical treatment usually uses cold compress, drug application and appropriate dressing to promote healing and relieve pain. Since the surgical intervention method has the problems of surgical risk, postoperative recovery and high surgical cost, non-surgical treatment has become the focus of attention.

[0003] At present, non-surgical treatment of deep Ⅱ degree scald is mainly achieved by drugs. Common drugs include western medicine and traditional Chinese medicine. Western medicine mainly includes analgesics, topical antibacterial drugs, antibiotics, growth factors and enzyme preparations, etc. Western medicine only implements "symptomatic treatment" for the symptoms such as pain and infection of patients after scald, in order to alleviate the pain of patients, prevent infection and promote the healing of wounds. However, this treatment method does not actively intervene in the core mechanism of wound repair, scar formation, local microenvironment balance and systemic complications, resulting in long healing period, many sequelae of scars, and insufficient control of systemic risks. At the same time, since the western medicine scald medicine is mainly a steroid hormone, or a corticosteroid is added to the antibacterial drug, the side effects on the human body are high; the treatment method of traditional Chinese medicine is mainly external preparation, and the treatment principles mainly include clearing heat and resolving toxins, promoting blood circulation to remove blood stasis, relieving swelling and pain, and promoting healing; therefore, more and more researches on traditional Chinese medicine are carried out.

[0004] Traditional Chinese medicine has accumulated profound history and rich clinical experience in the treatment of deep Ⅱ degree burns, and shows unique treatment advantages. It has been pointed out in the literature that many traditional Chinese medicines such as honeysuckle, forsythia, radix scrophulariae, safflower, angelica, radix lithospermi, borneol, chuanxiong and radix astragali are frequently used in the treatment of burns and become common ingredients for preparing burn treatment drugs. However, traditional Chinese medicine believes that burns are not only surface damage to the skin, but also damage to qi and blood and imbalance of yin and yang. According to the dialectical treatment of traditional Chinese medicine, the treatment of burns needs to be classified according to different pathological states, such as 'hot and toxic' and 'deficiency of both qi and blood'. For 'hot and toxic' burns, traditional Chinese medicine such as honeysuckle and forsythia is often used to help inflammation and promote healing; and for 'deficiency of both qi and blood' burns, traditional Chinese medicine such as ginseng and radix astragali is often used to tonify qi and nourish blood. It can be seen that traditional Chinese medicine needs to consider the pathology of deep Ⅱ degree burns and the mechanism of traditional Chinese medicine when treating burns.

[0005] Silybum marianum L. belongs to the family of Asteraceae, with bitter taste, cool nature, and liver and gallbladder meridians, with the effects of clearing heat and resolving toxicity, soothing liver and promoting bile. Silymarin is a safe and well-tolerated flavonolignan compound extracted from the seed coat of Silybum marianum L.; Silymarin includes Silybinin (SB) with the highest content, the strongest activity and the greatest effect, as well as isosilybin, silydianin (SCH) and silychristin (SD), etc. Silybinin is a mixture of two enantiomers, silybinin A and silybinin B, with equal content. Due to its anti-inflammatory specificity, low toxicity and outstanding effect in liver protection and anti-inflammatory, it has been widely used in the field of liver disease treatment for many years. Although Silymarin has antioxidant and anti-inflammatory effects and can promote blood vessel regeneration, there are few reports on the active substances extracted from Silybum marianum L. for skin repair, especially deep Ⅱ degree burns. Meanwhile, the mechanism of Silybum marianum L. in the treatment of burns is not clear, which limits its application in the treatment of deep Ⅱ degree burns. SUMMARY

[0006] In view of the technical problem that there are few reports on the active substances extracted from Silybum marianum L. for skin repair, especially deep Ⅱ degree burns, and the mechanism of Silybum marianum L. in the treatment of burns is not clear, which limits its application in the treatment of deep Ⅱ degree burns, the application of Silybum marianum L. ethanol extract in the preparation of a drug for treating deep Ⅱ degree burns is provided.

[0007] The present application improves the yield of the ethanol extract of S. marianum by optimizing the extraction conditions of S. marianum; and through the efficacy and treatment mechanism of the ethanol extract of S. marianum, it is clear that the ethanol extract of S. marianum has multiple mechanisms of antioxidant, inhibition of fibrosis and anti-inflammatory, and has a significant promoting effect on the treatment and repair of deep Ⅱ degree burns, which provides a theoretical basis and experimental support for S. marianum as a potential therapeutic drug for scarless healing after deep Ⅱ degree burns.

[0008] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0009] The application of the ethanol extract of S. marianum in the preparation of a drug for treating deep Ⅱ degree burns.

[0010] Further limitation, when the ethanol extract of S. marianum is applied in the preparation of a drug for treating deep Ⅱ degree burns, the repair of deep Ⅱ degree burns is promoted by activating the Nrf2 / HO-1 pathway to improve oxidative stress injury.

[0011] Further limitation, when the ethanol extract of S. marianum is applied in the preparation of a drug for treating deep Ⅱ degree burns, the repair of deep Ⅱ degree burns is promoted by regulating the TGF-β / Smad pathway to inhibit fibrosis.

[0012] Further limitation, when the ethanol extract of S. marianum is applied in the preparation of a drug for treating deep Ⅱ degree burns, the expression of Nrf2, HO-1 and NQO-1 can be up-regulated; and the expression of TGF-β1 and p-Smad2 / 3 can be down-regulated.

[0013] Further limitation, when the ethanol extract of S. marianum is applied in the preparation of a drug for treating deep Ⅱ degree burns, the release of inflammatory factors TNF-α and IL-1 can be reduced.

[0014] Further limitation, when the ethanol extract of S. marianum is applied in the preparation of a drug for treating deep Ⅱ degree burns, the ethanol extract of S. marianum improves oxidative stress injury through the Nrf2 / TGF-β1 pathway, and promotes the benign proliferation of HaCaT cells.

[0015] Further limitation, the extraction method of the ethanol extract of S. marianum is: taking S. marianum powder, extracting twice by ethanol reflux method, and combining the filtrates of the two extractions; evaporating and concentrating, and freeze-drying to obtain the ethanol extract of S. marianum in the form of extract.

[0016] Further limitation, during each extraction, 8mL of ethanol with a volume fraction of 85% is added for each g of S. marianum powder; and the extraction time is 1.5h.

[0017] Further limitation, the extraction method of the ethanol extract of S. marianum further comprises: adding ethanol and Tween 80 to the ethanol extract of S. marianum, swelling for 20min, and then grinding and stirring until completely dissolved; then adding distilled water and stirring uniformly, high-temperature sterilization, and filtering to obtain the S. marianum spray.

[0018] Further limit, add 6mL ethanol, 10mL Tween 80 and 184mL distilled water to every 1.538g S. miltiorrhiza alcohol extract; the volume fraction of ethanol is 85%.

[0019] Compared with the prior art, the present application has the beneficial effects that:

[0020] 1、The present application obtains S. miltiorrhiza alcohol extract by optimizing the extraction process of S. miltiorrhiza, and evaluates the efficacy of S. miltiorrhiza alcohol extract by observing the wound healing rate, tissue moisture content and pathological section analysis. The results show that S. miltiorrhiza alcohol extract can effectively promote wound healing, reduce inflammatory response, and promote the regeneration of granulation tissue and skin appendages, and is superior to the positive control MEBO in terms of collagen proliferation.

[0021] 2、After treatment with S. miltiorrhiza alcohol extract, the healing rate of the wound is significantly improved, and HE staining results show that it has obvious promoting effect on epithelialization. In addition, collagen Masson staining shows that S. miltiorrhiza spray can effectively increase the collagen content in the granulation tissue of rats; therefore, S. miltiorrhiza has significant effect in promoting wound healing.

[0022] 3、The present application studies the influence of S. miltiorrhiza alcohol extract on Nrf2 / HO-1 and TGF-β / Smad signaling pathways by Western blotting and immunohistochemical staining. The results show that S. miltiorrhiza alcohol extract can up-regulate the expression of Nrf2, HO-1 and NQO-1, enhance antioxidant capacity, and down-regulate the expression of TGF-β1 and p-Smad2 / 3, regulate fibrosis process. This finding reveals the dual mechanism of S. miltiorrhiza in burn repair, that is, promoting healing through antioxidant and fibrosis regulation.

[0023] 4、The present application research shows that S. miltiorrhiza alcohol extract has significant promoting effect in burn repair, which is specifically manifested in: improving oxidative stress damage by activating Nrf2 / HO-1 pathway, and regulating TGF-β / Smad pathway to achieve inhibition of fibrosis, and also having anti-inflammatory effect. It is clear that S. miltiorrhiza alcohol extract has multiple mechanisms of antioxidant, fibrosis inhibition and anti-inflammatory, and has significant promoting effect on the treatment and repair of deep Ⅱ degree burn, which provides theoretical basis and experimental support for S. miltiorrhiza as a potential therapeutic drug for scarless healing after burn, and expands the new use of S. miltiorrhiza in deep Ⅱ degree burn. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The influence of S. miltiorrhiza alcohol extract on the levels of SOD, GSH-Px and MDA in HaCaT cells in each group; (x±s, n=5); (Note: compared with the Control group ## P<0.01 or #P<0.05; compared with the Model group* P<0.01 or * P<0.05);

[0025] Figure 2 The effect of SFE on Nrf2 and TGF-β1 in each group of cells;

[0026] Figure 3 The quantitative results of the effect of SFE on Nrf2 in each group of cells;

[0027] Figure 4 The effect of SFE on Nrf2 / HO-1 and TGF-β1 / Smad signaling pathways in each group of cells; (a) Western blotting detects the effect of SFE on Nrf2 / HO-1 and TGF-β1 / Smad signaling pathways in cells; (b) Western blotting detects the quantitative analysis of the effect of SFE on Nrf2 / HO-1 and TGF-β1 / Smad signaling pathways;

[0028] Figure 5 The effect of SFE on Nrf2 / HO-1 and TGF-β1 / Smad signaling pathways in each group of cells after adding Nrf2 inhibitor; (a) Western blotting detects the effect of SFE on Nrf2 / HO-1 and TGF-β1 / Smad signaling pathways after adding Nrf2 inhibitor; (b) Western blotting detects the quantitative analysis of the effect of SFE on Nrf2 / HO-1 and TGF-β1 / Smad signaling pathways after adding Nrf2 inhibitor (x±s, n=3);

[0029] Figure 6 HE staining of skin burn wounds of rats with different degrees of scald; A: normal tissue; B: skin Ⅰ degree scald; C: skin shallow Ⅱ degree scald; D: skin deep Ⅱ degree scald;

[0030] Figure 7 The effect of SFE on the liver and kidney indices of rats with deep Ⅱ degree scald; (a) liver indices of rats in each group; (b) kidney indices of rats in each group;

[0031] Figure 8 The wound healing process chart of SD rats (n=6); M: model group; NC: negative control group; SM-L: low-dose SFE spray group; SM-M: medium-dose SFE spray group; SM-H: high-dose SFE spray group; PC: MEBO group (MEBO);

[0032] Figure 9Effect of S. mooreana on the healing rate of deep Ⅱ degree scald in rats (Note: compared with Model group * P<0.01 or * P<0.05);

[0033] Figure 10 Effect of S. mooreana on the water content of wound tissue at different time points in rats with scald (Note: compared with Model group * P<0.01 or * P<0.05);

[0034] Figure 11 Pathological histological changes of wound in rats (HE×100, n=6);

[0035] Figure 12 Pathological histological changes of wound in rats (MASSON×100, n=6);

[0036] Figure 13 Effect of S. mooreana spray on SOD, GSH-Px and MDA in serum of deep Ⅱ degree scalded rats (x±s, n=6) (Note: compared with Control group ## P<0.01 or # P<0.05; compared with Model group * P<0.01 or * P<0.05);

[0037] Figure 14 Effect of S. mooreana spray on SOD, GSH-Px and MDA in skin tissue of deep Ⅱ degree scalded rats (x±s, n=6) (Note: compared with Control group ## P<0.01 or # P<0.05; compared with Model group * P<0.01 or * P<0.05);

[0038] Figure 15 Effect of S. mooreana spray on IL-1 level in serum and skin tissue of deep Ⅱ degree scalded rats (x±s, n=6) (a) serum; (b) skin tissue (Note: compared with Control group ## P<0.01 or # P<0.05; compared with Model group * P<0.01 or * P<0.05);

[0039] Figure 16Effects of milk thistle spray on serum and skin tissue levels in rats with deep second-degree burns (x±s, n=6) (a) Serum; (b) Skin tissue (Note: compared with the control group) ## P < 0.01 or # P < 0.05; compared with the Model group* * P < 0.01 or * P < 0.05);

[0040] Figure 17 The effect of milk thistle spray on Nrf2 in the skin tissue of rats in each group;

[0041] Figure 18 The quantitative results show the effect of milk thistle spray on Nrf2 in the skin tissue of rats in each group;

[0042] Figure 19 The effects of milk thistle spray on the Nrf2 / HO-1 pathway in the skin tissue of rats in each group were investigated. (a) The effects of Western blotting on Nrf2 / HO-1 and TGF-β1 / Smad pathway proteins in rat skin tissue after 7 days were investigated. (b) Quantitative analysis of the effects of Western blotting on Nrf2 / HO-1 and TGF-β1 / Smad pathway proteins in rat skin tissue after 7 days were investigated (x±s, n=3).

[0043] Figure 20 (c) Effects of milk thistle spray on the TGF-β1 / Smad pathway in the skin tissue of rats in each group; (d) Quantitative analysis of the effects of Western blotting on Nrf2 / HO-1 and TGF-β1 / Smad pathway proteins in the skin tissue of rats after 21 days (x±s, n=3). Detailed Implementation

[0044] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but the embodiments of the present invention are not limited thereto. Other methods for preparing the compounds of the present invention, with some conventional modifications to the reaction conditions according to the present invention, are considered to be within the scope of the present invention.

[0045] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0046] The technical solution of the present invention will be described in detail below with reference to several embodiments.

[0047] It should be noted that, in the following examples, unless otherwise specified, the chemicals and reagents used are commercially available products that are conventional in the art.

[0048] It should be noted that, in the following examples, unless otherwise specified, the operations used are conventional operations; for example, unless otherwise specified, the operation temperature is room temperature operation. Unless otherwise specified, the test method is the standard test method available in the art.

[0049] I. Preparation method and condition optimization of Silymarin ethanol extract

[0050] The purpose of the present application is to obtain Silymarin ethanol extract (denoted as SM) from Silybum marianum by alcohol extraction method, and to optimize the conditions.

[0051] The preparation method of Silymarin ethanol extract includes the following steps: grinding dried Silybum marianum into powder, and extracting with ethanol to obtain an extract; then evaporating and concentrating the sample under vacuum at 40℃ using a rotary evaporator, and finally freeze-drying the obtained concentrated extract in a freeze-drying machine for 48 hours to obtain Silymarin ethanol extract (brownish-brown extract 13g, extract yield 13%, concentration 7.69g / g extract containing crude drug)

[0052] The present application uses orthogonal experiment to screen the optimal extraction process of Silybum marianum, and uses L9(3 4 ) orthogonal experiment table for design. The present application selects four factors to investigate solvent multiple (A), extraction time (B), ethanol content (C), and extraction times (D) as the main factors affecting the extraction process, and the factor levels are shown in Table 1.

[0053] Table 1 Factor level table for extraction method investigation

[0054]

[0055] Taking the content of silybin in Silybum marianum as the investigation index, the peak area corresponding to silybin is obtained by high performance liquid chromatography (see Table 2), so as to determine the optimal process.

[0056] Table 2 Intuitive analysis table

[0057]

[0058]

[0059] As can be seen from Table 2, the orthogonal experiment results are analyzed by range analysis, and the results show that the influence degree of the four factors on the experiment is C>A>D>B in turn, so the optimal extraction method combination is determined as A1B2C2D2, that is, 8 times of 85% ethanol is added, extracted for 1.5h, and extracted for 2 times.

[0060] The present application extracts by using the above preferred conditions. The specific process is: dry Silybum marianum (100g) is made into powder, and extracted with 800ml of 85% ethanol, 2 times, 1.5h each time. The two filtrates are combined, and then the sample is evaporated and concentrated under vacuum at 40℃ using a rotary evaporator, and finally the obtained concentrated extract is placed in a freeze dryer for 48 hours to obtain Silybum marianum ethanol extract (Silybum marianum ethanol extract is a brownish brown extract, a total of 13g, the extract yield is 13%, and the concentration is 7.69g / g extract containing crude drug).

[0061] The present application also uses the above Silybum marianum ethanol extract in the form of extract to prepare Silybum marianum spray.

[0062] The preparation process of the Silybum marianum spray of the present application is as follows:

[0063] Take 1.538g of Silybum marianum ethanol extract, add about 6ml of 85% ethanol, and then add about 10ml of Tween 80, swell for 20 minutes, grind and stir to make the solution completely dissolved, then slowly add about 184ml of distilled water and stir uniformly, sterilize at high temperature, and then filter to obtain Silybum marianum spray.

[0064] The Silybum marianum spray is a light brown clear liquid, equivalent to 60mg of raw medicine per milliliter, and the Silybum marianum spray is stored in a cool place in a sealed manner.

[0065] II. In vitro study on the mechanism of Silybum marianum in treating deep Ⅱ degree scald

[0066] The present application verifies the mechanism of the above prepared Silybum marianum ethanol extract (SM) in treating deep Ⅱ degree scald through in vitro experiments.

[0067] 2.1, experimental method

[0068] 2.1.1, kit determination of the influence of Silybum marianum ethanol extract on SOD, GSH-Px and MDA in each group of cells

[0069] After HaCaT cell culture, model construction and drug intervention, the experiment is terminated when SM is treated for 24h, the cell supernatant is discarded, the cells are gently washed with pre-cooled sterile PBS for 3 times to remove residual culture medium components, and then 2ml of protein lysis buffer system containing protease and phosphatase inhibitors is added to each well, the monolayer cells are mechanically peeled off by using a cell scraper, the obtained cell suspension is centrifuged at 10000r·min -1The speed of the centrifuge was 10,000 rpm, and the centrifugation time was 10 min. The supernatant was collected in a new centrifuge tube, and the precipitate was collected. The washing and centrifugation steps were repeated to remove soluble interferents. Finally, the cells were broken by a low-temperature cell homogenizer to prepare a homogenized lysate. The quantitative analysis of each oxidative stress-related index was completed according to the operation specification of the superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) detection kits.

[0070] 2.1.2, The effect of Silymarin ethanol extract on the expression of Nrf2 protein in each group of HaCaT cells was determined by cell fluorescence.

[0071] After standard subculture, disease model induction, and drug intervention treatment, the HaCaT cells were subjected to immunofluorescence staining experiments on sterile 24-well plates. Before the experiment, 10 mm round coverslips coated with polylysine were sterilized by high-temperature sterilization and placed at the bottom of the well plate. Then, cells in the logarithmic growth phase were inoculated at 5 × 10 3 cells / well, and cultured under conventional culture conditions for 24 h. Then, 1 mL / well of drug-containing medium was added for further incubation for 24 h. During the staining process, the culture solution was gently removed, 1-2 mL / well of PBS buffer was added to balance the cell microenvironment for 3 min, and the rinsing was repeated twice to remove metabolic residues. Then, 1 mL / well of 4% paraformaldehyde solution was added to fix the cells at room temperature for 15-30 min to maintain the structural integrity. Subsequently, 0.5% Triton X-100 (1 mL / well) was used for permeabilization at room temperature for 10-20 min, and then 1 mL / well of 5% BSA blocking solution was used to block non-specific binding sites for 50-60 min. Further, 1:400 diluted primary antibody working solution was used for incubation at 4°C overnight, after which the antibody solution was recovered, and 1 mL / well of PBS buffer was used for washing 3 times (5 min each time) to remove unbound antibodies. Then, 1:400 diluted Alexa Fluor-labeled secondary antibody was prepared under light shielding conditions, and 500 μL / well was used for light shielding incubation for 1 h, followed by thorough washing with PBS. Finally, DAPI staining solution was used for incubation under light shielding conditions for 10 min to label the cell nucleus, and the free dye was washed again. Finally, the samples were fixed with an anti-fluorescence quenching mounting agent, and the fluorescence images were systematically collected under a laser confocal microscope. Each group of experiments was independently repeated three times to ensure data reliability.

[0072] 2.1.3, The effect of Silymarin ethanol extract on the Nrf2 / HO-1 and TGFβ1 / Smad signaling pathways in each group of cells was determined by Western blotting.

[0073] 2.1.3.1, Cell grouping

[0074] The culture, modeling and administration of cells were performed, and the administration was performed after dissolving the extract of S. mooreana to DMSO. The cells were divided into a normal administration group and an Nrf2 inhibitor group, and the normal administration group was divided into groups as follows:

[0075] (1) Control group (Control): H2O2: 0 nm, SM: 0 mg / mL;

[0076] (2) Cell model group (Model): H2O2: 25 mM, SM: 0 mg / mL;

[0077] (3) SM low-dose group (SM-L): H2O2: 25 mM, SM: 7.812 mg / mL;

[0078] (4) SM medium-dose group (SM-M): H2O2: 25 mM, SM: 15.625 mg / mL;

[0079] (5) SM high-dose group (SM-H): H2O2: 25 mM, SM: 31.25 mg / mL;

[0080] From the experimental data of the normal group, it can be seen that when the concentration of SM is 31.25 mg / mL, the activation effect on the Nrf2 / HO-1 and TGFβ1 / Smad signaling pathways is the most significant, and therefore the concentration of SM used in the Nrf2 inhibitor group is 31.25 mg / mL, and the Nrf2 protein inhibitor group is divided into groups as follows:

[0081] (1) Control group (Control): H2O2: 25 mM, SM: 0 mg / mL;

[0082] (2) Cell model group (Model): H2O2: 25 mM, SM: 0 mg / mL;

[0083] (3) H2O2+ML385: H2O2: 25 mM, ML385: 5 mM;

[0084] (4) H2O2+SM: H2O2: 25 mM, SM: 31.25 mg / mL;

[0085] (5) H2O2+SM+ML385: H2O2: 25 mM, SM: 31.25 mg / mL, ML385: 5 mM;

[0086] 2.1.3.2, HaCaT cell total protein extraction

[0087] (1) After the cells are cultured to the predetermined time, the culture vessel is removed from the constant temperature incubator, the culture medium in the hole is gently aspirated, and then the cells are slowly rinsed three times along the hole wall, and the buffer used is pre-cooled sterile phosphate buffer (PBS);

[0088] (2) Using a micropipette, 100 μL of pre-prepared cell lysate (RIPA buffer containing 1% protease inhibitor and 1 mM PMSF) was added vertically along the wall of each group of culture medium

[0089] (3) The cell sample was placed on a pre-cooled ice bath platform for lysis treatment, and the whole process was strictly maintained at 0-4°C environment to fully protect the protein activity;

[0090] (4) The liquid in the culture medium was thoroughly blown and mixed using a pipette gun, and then the cells were broken using a cell ultrasonic crusher to ensure that the cell proteins could be completely extracted;

[0091] (5) After transferring the liquid in the culture dish to the centrifuge tube, the centrifuge speed was set to 14000 rpm, and centrifugation was performed for 10 minutes. After centrifugation, the supernatant was collected;

[0092] (6) The collected cell supernatant was transferred to a new centrifuge tube, and the BCA method was used for protein quantification.

[0093] (7) According to the volume of the supernatant, 5x protein loading buffer was added and mixed thoroughly, then heated in a 95°C metal bath for 5 to 10 minutes. After the sample cooled to room temperature, it was aliquoted and stored in a -80°C refrigerator for later use.

[0094] 2.1.3.3. BCA protein quantification

[0095] According to the BCA protein quantification kit operation specification, first refer to the pre-optimized concentration gradient (0.5-2.0 mg / mL) to prepare bovine serum albumin (BSA) standard solution, and according to the kit instructions to prepare BCA working solution, vortex mixing after mixing, and store in the dark. When quantitatively detecting, use a precision pipette to dispense 20 μL of standard, sample and blank (double distilled water) into the 96-well plate according to the pre-set hole position, and set double duplicate holes for each group to control experimental error. Then add 200 μL BCA working solution to each well, incubate at 37°C for 30 min, and then measure the absorbance value of each well at 562 nm wavelength using a microplate reader. Wear powder-free gloves throughout the operation to avoid fingerprint contamination of the optical detection surface. Establish a standard curve (R 2 ≥0.99), and calculate the sample protein concentration and group concentration balance to ensure equal sample loading for subsequent experiments.

[0096] 2.1.3.4. Western blot analysis

[0097] (1) Preparation of separating gel and stacking gel: Wash the thin plate and the thick plate, prepare the stacking gel according to the proportions in Table 3, gently press it flat with pure water, and add the stacking gel after the separating gel and pure water have a boundary line. After the stacking gel solidifies, it is ready for use.

[0098] (2) Protein sample loading: Load samples sequentially according to the order, and the loading amount is calculated based on the BCA protein quantification.

[0099] (3) Electrophoresis: Electrophoresis constant current 30mA, bromophenol blue at the separating gel, electrophoresis constant current 40mA, when bromophenol blue runs to the bottom of the glass plate groove, stop electrophoresis and prepare for membrane transfer;

[0100] (4) Transfer: Soak the pre-cut PVDF membrane in methanol for 1 min, and transfer the membrane according to the "sandwich" method. The transfer is carried out under a constant current of 200 mA. The transfer time is determined by the molecular weight of the protein.

[0101] (5) Sealing: Prepare 5% skim milk with 1×TBST and seal at room temperature for 1 hour;

[0102] (6) Incubation of primary antibody: Prepare antibody according to Table 4, incubate primary antibody overnight at 4°C;

[0103] (7) Incubation with secondary antibody: On the second day, wash the membrane, recover the primary antibody, and incubate with secondary antibody (antibody dilution ratio is shown in Table 4 below) at room temperature for 1 hour. After incubation, wash the membrane.

[0104] (8) Prepare the luminescent solution according to the instructions, and use it immediately. Drop the luminescent solution evenly onto the PVDF membrane. Expose the membrane using a gel imaging system, and analyze the target band using ImageJ software with GAPDH as an internal reference.

[0105] Table 3. Proportions of stacking gel and separating gel

[0106] 5% concentrated gel 8% resolving gel 10% resolving gel Reagents Volume (mL) Reagents Volume (mL) Volume (mL) H2O 4.51 H2O 6.95 5.95 30% AB 1.33 30% AB 4.00 5.00 Tris-HCL 2.00 Tris-HCL 3.75 3.75 10% SDS 0.08 10% SDS 0.15 0.15 10% AP 0.08 10% AP 0.15 0.15 TEMED 0.008 TEMED 0.012 0.012 Total volume 8 Total volume 15 15

[0107] Table 4 Antibody Dilution Ratio

[0108]

[0109] 2.2 Results

[0110] 2.2.1 Effects of milk thistle ethanol extract on SOD, GSH-Px and MDA levels in HaCaT cells of different groups

[0111] There is a set of endogenous antioxidant system in cells, which regulates ROS level by generating antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-PT) and so on, so as to offset the harmful effects of lipid peroxidation, protein oxidation and DNA damage on cells. Therefore, in the study of cell damage caused by oxidative stress, antioxidant enzymes such as SOD, GSH-Px and lipid peroxidation products such as MDA are often used as potential biomarkers. The experimental results are shown in Figure 1 As shown in Table 2, after H2O2-induced oxidative stress, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the model group HaCaT cells were significantly lower than those in the normal control group (P<0.01), and their relative activities were only 40.10% and 19.79% of the control group, respectively. After pretreatment with different concentrations of SM, the activities of the above antioxidant enzymes showed a dose-dependent recovery. Compared with the Control group, the SOD and GSH-Px contents in the Model group HaCaT cells were significantly decreased (P<0.01 or P<0.05). After administration, each dose of SM significantly increased the SOD content (P<0.01), and SM-M and SM-H significantly increased the GSH-Px level (P<0.01), while SM-L had no significant effect on the GSH-Px level. Compared with the Control group, the MDA level in the Model group HaCaT was significantly increased (P<0.01), and after administration, each dose of SM significantly decreased the MDA level (P<0.01), i.e. up-regulated the activities of endogenous antioxidant enzymes (SOD, GSH-Px) and synergistically inhibited the lipid peroxidation chain reaction, effectively reduced the oxidative damage induced by H2O2, thereby maintaining the redox homeostasis of HaCaT cells.

[0112] 2.2.2, Effect of Silymarin Ethanol Extract on Nrf2 Protein Expression in HaCaT Cells in Each Group

[0113] The results of cell immunofluorescence are shown in Figure 2 Compared with the Control group, the expression of Nrf2 in the Model group was significantly decreased (P<0.01), and each dose of SM could increase the expression of Nrf2 to different degrees (P<0.01 or P<0.05), as shown in Figure 3

[0114] 2.2.3, Effect of Silymarin Ethanol Extract on Nrf2 / HO-1 and TGFβ1 / Smad Pathways in HaCaT Cells in Each Group

[0115] ​Nrf2 as an important nuclear transcription factor in oxidative stress response and its downstream HO-1 and NQO-1 are all related to the key role of anti-oxidative stress and prevention of cell damage. In burn repair, NRF2 and TGF-β pathway may form a dynamic balance: NRF2 inhibits excessive TGF-β activity in the early stage through antioxidant effect to reduce fibrosis; while TGF-β promotes tissue remodeling in the later stage, but needs the balance of NRF2 to avoid excessive scar formation. In addition to applying SM to verify Nrf2 / TGF-β1 in the experiment, Nrf2 protein signal inhibitor ML385 was further applied to verify the upstream and downstream pathway relationship, and the results are as follows Figure 4 As shown in Fig. 5a, compared with the Control group, the expression of Nrf2, HO-1 and NQO-1 proteins in the Model group decreased significantly (P<0.01); after drug administration, each dose of SM significantly increased the expression of Nrf2 (P<0.01); SM-L and SM-H significantly increased the expression of HO-1 protein (P<0.05); each dose of SM significantly increased the expression of NQO-1 protein (P<0.01); compared with the Control group, the expression of TGF-β1, Smad2 / 3 and Keap-1 proteins in the Model group increased significantly (P<0.05 or P<0.01); after drug administration, each dose of SM significantly reduced the expression of TGF-β1 and Smad2 / 3, Keap-1 proteins (P<0.01), and the statistical results are shown in Fig. 5b. Figure 4

[0116] After completing the modeling of SM intervention HaCaT cells and drug treatment, Nrf2 / HO-1 and TGF-β1 / Smad signal pathway were verified, and Nrf2 signal inhibitor ML385 was used for intervention to further verify the correlation of the signal pathway. The experimental results are shown in Fig. 6a. Figure 5 As shown in Fig. 6a, compared with the Control group, the expression of Nrf2, HO-1 and NQO-1 in the Model group decreased significantly (P<0.01); TGF-β1, Smad2 / 3 and Keap-1 increased significantly (P<0.01). After applying ML385, compared with the Model group, the expression of Nrf2, HO-1, NQO-1 and Smad2 / 3 in the H2O2+ML385 group further decreased significantly (P<0.01), and TGF-β1, Smad2 / 3 and Keap-1 further increased significantly (P<0.01). After applying SM and ML385, compared with the normal drug administration group H2O2+SM, the expression of Nrf2, HO-1 and NQO-1 in the H2O2+SM+ML385 group decreased significantly (P<0.01 or P<0.05), and the expression of TGF-β1, Smad2 / 3 and Keap-1 increased significantly (P<0.01 or P<0.05), and the statistical results are shown in Fig. 6b. Figure 5 ​As shown in Fig. 5B. The above results suggest that the SOD activity of SOD in S. miltiorrhiza ethanol extract may inhibit the TGF-β1 / Smad pathway by activating the Nrf2 / HO-1 pathway, and the activation of NRF2 may simultaneously reduce oxidative stress and inhibit TGF-β, thereby promoting cell proliferation.

[0117] The experiment used hydrogen peroxide to simulate oxidative stress environment to some extent, and explored the possible mechanism of S. japonica in treating scald. First, the expression levels of oxidative stress markers SOD, MDA and GSH-Px were discussed. The results showed that the dynamic balance of the three was broken after the keratinocytes were damaged by oxidation, and the S. japonica alcohol extract could reverse this situation. The outbreak of ROS in HaCaT cells after scalding activated the TGF-β / Smad2 / 3 pathway, leading to lipid peroxidation and MDA damage, and driving the TGF-β / Smad2 / 3 pathway overactivation, forming a vicious cycle of "oxidative stress-fibrosis". Based on this, the relationship between Nrf2 / HO-1 pathway and TGFβ / Smad pathway was explored. In the early stage of scald, the endogenous antioxidant defense was enhanced by activating the Nrf2 pathway, Keap-1 perceived oxidative damage and released Nrf2, and initiated the endogenous antioxidant program; HO-1 generated antioxidant molecules by decomposing hemoglobin, synergistically inhibited inflammation and promoted angiogenesis; NQO-1 blocked the ROS generation chain reaction, protecting cells from secondary oxidative damage. They together provided a molecular hub for cross-pathway regulation of oxidative stress-inflammation-repair; therefore, targeting the activation of this pathway can achieve antioxidant, anti-inflammatory and pro-repair at the same time, breaking through the limitations of traditional single therapy; TGF-β1 / Smad2 / 3 pathway plays a dual regulatory role in wound repair and pathological process. When scald occurs, TGF-β1 / Smad2 / 3 pathway plays a key role in repair by promoting inflammation subsidence, initiating extracellular matrix (ECM) synthesis and enhancing cell migration, and its expression characteristics are characterized by rapid secretion of TGF-β1, enhanced phosphorylation and nuclear translocation of Smad2 / 3. However, the overactivation or continuous activation of this pathway may lay the foundation for pathological fibrosis. Western blotting results showed that after keratinocytes were damaged by oxidation, S. japonica alcohol extract could up-regulate Nrf2, HO-1 and NQO-1 proteins to achieve antioxidant effect. After H2O2-treated HaCaT cells, TGF-β increased, but oxidative stress inhibited proliferation. After giving S. japonica freeze-dried powder, TGF-β1 and Smad2 / 3 expression decreased, oxidative stress expression decreased and cell proliferation increased, so TGF-β may be regulated to an appropriate level, leading to a decrease in TGF-β expression. The specific up-regulation of Nrf2 / HO-1 pathway also increased the expression of antioxidant enzymes (SOD and GSH-Px activity), not only antagonized ROS accumulation, but also reprogrammed the TGF-β signal from the pro-fibrotic phenotype to the pro-repair phenotype by competitively inhibiting Smad2 / 3 expression. This finding breaks through the traditional "antioxidant-anti-fibrosis" single target treatment idea and proposes a new paradigm of "oxidative repair synergistic regulation": through the dynamic balance of keratinocyte Nrf2-TGF-β axis, both epidermal reepithelialization and inhibition of dermal fibroblast abnormal activation can be accelerated, providing a theoretical basis for scarless healing after scald.

[0118] In summary, the Silymarin ethanol extract can improve oxidative stress injury and promote benign proliferation of HaCaT cells in keratinocytes through the Nrf2 / TGF-β1 pathway, thereby providing a theoretical basis for scarless healing after scalding.

[0119] III. Pharmacodynamic evaluation of Silybum marianum in the treatment of deep Ⅱ degree scald in rats

[0120] The Silybum marianum spray prepared in the first part (denoted as SM) is used to evaluate the pharmacodynamics of Silybum marianum in the treatment of deep Ⅱ degree scald in rats.

[0121] 3.1, Experimental animals

[0122] 126 SPF level SD male rats (animal source: Chengdu Dashuo Experimental Animal Company, license number: SCXK (Chuan) 2020-0030), 8 weeks old, weighing 180±20g. The animals were raised in the Traditional Chinese Medicine Pharmacology Laboratory of Shaanxi University of Chinese Medicine, with a temperature of 16-25℃, and free access to water and food. The experiment was conducted after 7 days of adaptive feeding. The experiment was approved by the Animal Ethics Committee of Shaanxi University of Chinese Medicine.

[0123] 3.2, Experimental method

[0124] 3.2.1, Preparation method of drugs and reagents

[0125] 3.2.1.1, Dose conversion and concentration grouping of Silybum marianum spray for rats

[0126] Reference the crude drug dosage of the positive drug control group Mebo Moist Exposed Burn Cream, the dosage of Mebo Moist Exposed Burn Cream is 1g of ointment ≈0.21g of crude drug, and 0.5g of ointment is applied each time, consuming 0.1g of crude drug. Unit area crude drug dosage: known application area 4cm 2 Apply 0.1g of crude drug to the wound, then the unit area dosage is 0.1g÷4cm 2 =0.025g / cm 2 Therefore, the unit area crude drug dosage ≈25mg / cm 2 , the dosage of Silybum marianum spray (SM) is close to the unit area crude drug dosage of Mebo Moist Exposed Burn Cream, so the absorption of crude drug per unit area of skin after spraying should not be less than 25mg / cm 2 . The dose calculation formula of the spray is: crude drug amount (mg / cm 2 )=spray volume (mL / cm 2 )×spray concentration (mg / mL), adjust the low dose spray concentration to make the unit area crude drug dosage equivalent to 25mg / cm 2 of Mebo Moist Exposed Burn Cream, and each scald wound is given 3 times (about 0.3mL) when given, then the spray concentration is designed, and the final animal efficacy experiment dose is:

[0127] (1) SM-L: Silybum marianum spray low dose (10% w / v): 30 mg / cm 2 (consistent with MEBO Burn Cream)

[0128] (2) SM-M: Silybum marianum spray medium dose (20% w / v): 60 mg / cm 2 (2 times)

[0129] (3) SM-H: Silybum marianum spray high dose (40% w / v): 120 mg / cm 2 (4 times)

[0130] (4) PC: Positive control group MEBO Burn Cream: 25 mg / kg

[0131] 3.2.2. Establishment of rat deep Ⅱ degree burn model and grouping and administration

[0132] 3.2.2.1. Establishment of rat deep Ⅱ degree burn model

[0133] Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution (30 mg / kg). Eighteen 8-week-old male SD rats were selected for each experiment, and modeling was carried out after 1 week of adaptive feeding. YLS-5Q type electric heating temperature control burn instrument was used, which realizes precise control of temperature, pressure and action time through electric heating conduction principle. The experimental process includes: ① Pretreatment stage: 4 cm 2 specification high temperature flat head, and the heat conducting paste was applied for preheating; ② Anesthesia and hair removal: after anesthesia, the back hair was removed by electric shaver and evenly coated with depilatory cream. After standing for 6-8 minutes, the residual hair was gently scraped off, and the experimental area was marked after physiological saline cleaning; ③ Gradient burn: 80°C, 90°C, 100°C burn temperature was used in turn, each maintained for 8 seconds of action time, and vertical pressure was applied to the predetermined sites on both sides of the rat spine; one hour after the burn, 5 mL of lactated Ringer's solution was given to the rat intraperitoneally to prevent shock. ④ Sample treatment: the animals were anesthetized again 24 hours after the operation, and the burn skin tissue was obtained after blood sampling from the abdominal aorta and killing the animals, and was fixed with formalin and stored at -80°C; ⑤ Pathological analysis: dehydration, embedding and section preparation were completed after 3 days of fixation, and the histopathological changes were evaluated by HE staining. The contact time and rubbing force of the depilatory cream were strictly controlled during the whole modeling process to ensure the integrity of the epidermis and the repeatability of the experiment.

[0134] 3.2.2.2. Animal grouping and administration

[0135] After 7 days of adaptive feeding, 126 SD male rats were randomly divided into 7 groups, namely, blank control (Control), model group (Model), low-dose Shui Fei Ji spray group (SM-L), medium-dose Shui Fei Ji spray group (SM-M), high-dose Shui Fei Ji spray group (SM-H), matrix negative control group (NC, pure water and appropriate amount of Tween 80), and positive drug control group (PC, Meibao Moist Burn Cream). After modeling, the positive control group was immediately given Meibao Moist Burn Cream to the scalded site, with a thickness of about 0.5 mm, once a day. The negative control group was given the matrix solution, and the experimental high, medium and low dose groups were given the corresponding drug solution, 3 sprays, about 0.3 mL. After drug administration, the wound surface was kept open, and the rats in each group were single-caged and normally fed and watered (the experimental cage was disinfected and cleaned every two days, and the experimental room was disinfected once a day to avoid external contamination of the wound surface). All rats were given drugs twice a day in the morning and evening in the first week, and once a day from the second week, for 14 consecutive days. The model group was not treated, and the normal group was regularly shaved for 21 consecutive days. The rats were sacrificed at 24 hours, 7 days, 14 days and 21 days after scalding, and the full-thickness skin of the scalded site was taken. Part of the tissue was fixed in 10% neutral formalin solution and prepared into paraffin sections for immunohistochemical study; another part was stored at -80℃ for related biochemical index determination and protein extraction.

[0136] 3.2.3, Determination of the effect of Shui Fei Ji on the organ coefficient of rats in each group

[0137] After drug administration, the rats were anesthetized according to their body weight, blood was collected from the abdominal aorta and centrifuged to obtain the upper serum, the rat's left and right kidneys were taken, the rat's complete liver was taken, the physiological saline was used to remove the blood and then the filter paper was used to absorb the moisture, and then the weight was taken. Each group of rats was weighed before anesthesia, and the following calculation formula of liver and kidney coefficient was used:

[0138] Liver coefficient = wet weight of liver (g) / body weight of rat (g)

[0139] Kidney coefficient = wet weight of kidney (g) / body weight of rat (g)

[0140] 3.2.4, Determination of wound index of scalded rats

[0141] 3.2.4.1, Wound healing rate of scalded tissue of rats

[0142] On the 7th, 14th, and 21st day after scalding, 6 rats were taken from each group, and pictures of the wound surface were collected by a camera. When taking the pictures, a ruler was placed at the edge of the wound surface, the camera position was fixed, the back of the rat was placed in the same position for taking pictures, and the corresponding wound surface area was measured using ImageJ image analysis software. The scalded area on the 1st day after scalding was taken as the original scalded area, and at 1 hour and 7th, 14th, 21st, and 28th days after the model was successfully established, the mirror surface should be kept relatively parallel to the wound surface when taking pictures, the distance between the mirror surface and the wound surface should be kept about 15 cm, and a ruler with clear scales was placed at the edge of the wound surface as a scale. After taking pictures, the pictures were stored in the computer in time, and the date of taking pictures and grouping were accurately recorded, as well as other useful phenomena. The imageJ image processing and analysis system was used to measure the area of the wound surface in terms of the proportion of pixels.

[0143]

[0144] 3.2.4.2. Water content of rat scalded tissue

[0145] At 7th, 14th, and 21st days after scalding, 3 rats were taken from each group, about 200 mg of full-thickness skin of the wound surface was taken, the blood was absorbed with filter paper, the wet weight was weighed on an electronic balance, and the dry weight was weighed after being placed in an 80℃ oven for 24 hours. The water content of the tissue was calculated by dry and wet methods.

[0146] Tissue water content (%) = (tissue wet weight - tissue dry weight) / tissue wet weight x 100%.

[0147] 3.2.4.3. H&E staining of skin tissue of rats in each group

[0148] The scalded skin samples obtained in the experiment of 2.2.2 were taken for the experiment, and were cut into 4 μm thick sections. After being dewaxed with xylene, anhydrous ethanol, and 75% alcohol, the sections were subjected to hematoxylin staining, differentiation, counterstaining, dehydration, and then stained with eosin staining solution, and finally mounted.

[0149] 3.2.4.4. Effect of Masson staining on skin tissue of rats in each group

[0150] The skin samples obtained in the experiment of 3.2.2.2 were taken for the experiment. After dehydration of the paraffin-embedded block, the tissue sample was immersed in dimethylbenzene for 10 to 15 minutes, and the process was repeated twice. Subsequently, the sample was immersed in anhydrous ethanol and 75% concentration ethanol respectively, and each time the immersion time was 10 minutes. After water washing, the tissue was soaked with potassium dichromate overnight. The next day, staining was performed using hematoxylin staining solution, followed by differentiation treatment in hydrochloric acid alcohol. Then, staining was performed by adding carmine staining solution, and then the sample was immersed in phosphomolybdic acid solution for 1 to 2 minutes. After removing the liquid in the section, staining was performed using aniline blue. Finally, differentiation treatment was performed using glacial acetic acid, and after removing the liquid and air-drying, observation was performed.

[0151] 3.3, Experimental results

[0152] 3.3.1, Establishment of deep Ⅱ degree burn model

[0153] The experimental animals were subjected to conventional HE staining after sampling. The results showed that the normal group of rats had complete skin in all layers, the stratum corneum was complete, the hair follicle and skin appendage structure was complete, the structure of the prickle cell layer was clear, the skin muscle layer located in the deep layer of the dermis was arranged in order, and the muscle cross striation was obvious Figure 6 A); when the model was established at a temperature of 80°C for 8 seconds, only the stratum corneum and hair follicle were damaged, and the rest of the structure was intact, indicating that the rat was injured by Ⅰ degree skin burn Figure 6 B); and in the model establishment process at 90°C for 8 seconds, the epidermis layer showed necrosis, confirming that the rat was injured by shallow Ⅱ degree skin burn Figure 6 C); finally, in the model establishment process at 100°C for 8 seconds, HE staining showed that part of the dermal papilla layer was shed, the collagen fibers in the dermis were damaged, arranged in disorder, and part of the collagen fibers had disappeared. In addition, a "leukocyte infiltration zone" was visible between the necrotic tissue and the surviving tissue; in the deep layer of the dermis, part of the skin appendage structure remained, and leukocyte infiltration was significant, confirming that the rat was injured by deep Ⅱ degree skin burn Figure 6 D). In summary, the HE staining results showed that under the condition of 100°C for 8 seconds, the skin injury was confirmed as deep Ⅱ degree burn. It was shown that the rat model of deep Ⅱ degree burn was successfully established, which provided an important basis for the subsequent application research of silymarin extract.

[0154] 3.3.2, Effect of silymarin on organ coefficients and body weight of rats in each group

[0155] The experimental results are shown in Figure 7 a and b indicate that each group has no significant effect on liver index and kidney index, indicating that the drug is safe and non-toxic; each group has no significant effect on the body weight of rats (Table 5).

[0156] Table 5 Effects of S. marianum on the body weight index of rats with deep Ⅱ degree scald

[0157]

[0158]

[0159] (Note: control group compared with ## P<0.01 or # P<0.05; compared with Model group * P<0.01 or * P<0.05)

[0160] 3.3.3, Determination of wound index of scalded rats

[0161] 3.3.3.1 Comparison of S. marianum on the wound healing rate of scalded rats

[0162] Deep Ⅱ degree scald affects the deep layer of the dermis, with only a small amount of deep skin appendages remaining. The recovery process depends on two aspects: (1) normal cells around the wound migrate to the center of the wound by proliferation to cover the wound; (2) fibroblasts at the bottom proliferate to fill the wound, and epidermal stem cells at the bottom also need to proliferate and differentiate to form new epidermis, so as to achieve the healing of the wound. On the first day after scalding, the wound healing rate of scalded tissues in each group showed no significant difference. Compared with the model group, the wound healing rate of rats in each drug group showed an upward trend over time after scalding. In particular, on the 14th day after scalding, the wound healing rate of the SM-M, SM-H dose groups and the PC group was significantly improved; and on the 14th and 21st days after scalding, the wound healing rate of the SM groups and the PC group was also significantly increased. Compared with the model group, these differences were statistically significant (P<0.05, P<0.01). These results show that SM can significantly promote wound healing, and its effect is related to the dose and time. The specific results are shown in Figure 8 and Figure 9 From the photos of the dynamic process of scalded wound healing, it can be seen that SM significantly promotes wound healing, and the efficacy is dose-dependent, but the medium dose group is slightly better than the MEBO group.

[0163] 3.3.3.2 Effects of S. marianum on the water content of scalded wound tissue of rats

[0164] The water content of the scalded tissue of each group showed no significant difference on the first day after scalding. Compared with the model group, the water content of the tissue of each group of rats showed a gradual downward trend after scalding over time. On the 7th day, the tissue water content of the SM-M, SM-H and PC groups had significantly decreased. After 14 days and 21 days of scalding, the tissue water content of the SM-L group further decreased, and the difference was statistically significant compared with the model group (P<0.05, P<0.01); see Table 3 for details Figure 10 .

[0165] 3.3.4. Influence of S. mooreana on the histopathology of scalded wounds in rats

[0166] 3.3.4.1. Influence of S. mooreana on H&E staining of each group of rats

[0167] Through dynamic pathological evaluation of the scalded skin of rats, it was found that each experimental group showed a differential repair process on the 7th, 14th and 21st day after treatment Figure 11 ). The normal control group maintained the integrity of the full-thickness skin structure, with clear keratin layer-epidermis-dermis layering and regular distribution of hair follicles and appendages. The model group showed progressive pathological changes at different stages after scalding: on the 7th day after treatment, the epidermal structure disintegrated, there were many necrotic hair follicle structures and inflammatory cell infiltration in the dermis, and a large amount of necrotic tissue was deposited; by the 14th day, necrotic tissue lysis still existed, the wound was covered with weak granulation tissue, epithelial regeneration was delayed, and collagen fibers were arranged in disorder; on the 21st day after treatment, there was still incomplete epidermalization, the healing area lacked hair follicle structure and formed pathological scar. The SM-M, SM-H and PC groups showed mild inflammatory reaction with active granulation proliferation on the 7th day after treatment; by the 14th day, the scab had fallen off and was covered with dense granulation matrix, the inflammatory cell infiltration was significantly reduced, and the epidermal cells began to crawl; by the 21st day, complete epithelial coverage was achieved, and collagen fibers were deposited in an orderly manner; new hair follicles and regular collagen networks were observed in the dermis. In contrast, the SM-H group had a large number of hair follicles on the 14th day, which basically filled the tissue defect, and the epidermal cells began to crawl, and the necrotic scab fell off, while the MEBO group still had residual necrotic tissue. Pathological evaluation showed that SM could significantly accelerate the process of epidermal regeneration and promote the reconstruction of skin appendages, and its repair efficiency was superior to that of traditional MEBO therapy.

[0168] 3.3.4.2. Influence of S. mooreana on Masson staining of each group of rats

[0169] The Masson staining results showed that Figure 12), the collagen fibers in the dermis of normal rat skin were arranged in a tight blue bundle structure, and the boundary with the epidermis layer was clear. The collagen fibers in the skin tissue were stained blue. In the Model group, the collagen fibers in the rat were less, and no new hair follicles were observed. The collagen fibers were small and loose. After the administration of S. mooreana and the positive drug, the degree of collagen deposition in the dermis of the rat was significantly increased compared with the Model group. The collagen at the epidermis-dermis junction was arranged vertically, and the collagen fibers were dense.

[0170] A deep Ⅱ degree burn model was established on the back of rats by using a super temperature control burn instrument, and the depth of the burn was verified by HE staining of pathological sections. The results show that under the condition of 100 DEG C, when the burn time is 8 seconds, the epidermis and skin appendages such as hair follicles in the burn area of the rat appear necrosis, and the subcutaneous tissue has serious edema, and a large number of vacuoles are formed in the dermis. This burn injury involves the deep layer of the dermis and belongs to deep Ⅱ degree burn. Under this condition, the model is constructed, which is simple to operate, can accurately control the burn area and depth, and has high repeatability, which provides an important basis for subsequent research on the treatment effect of drugs on experimental burns and its related mechanism.

[0171] The present application analyzes by observing the healing rate of the wound surface and the change of the water content of the wound surface tissue. The water content of the wound surface tissue can not only quantify the degree of edema, and then evaluate the intensity of acute inflammatory reaction and the change of blood vessel permeability, but also reflect the hypoxic injury and oxidative stress level caused by microcirculation disorder. Therefore, this index can be used as an important marker for dynamically evaluating the treatment effect of anti-inflammatory and antioxidant therapy. In addition, higher water content can predict the risk of scar hyperplasia and susceptibility to infection, and provide key experimental basis for interpreting the activity of NRF2 / TGF-β signal pathway and optimizing wound repair strategy. Through routine HE staining and MASSON staining, the influence of drugs on the histopathology in the healing process of deep Ⅱ degree burn on the back of experimental animals is observed. The results show that S. mooreana can effectively reduce the infiltration of inflammatory cells in the wound surface, accelerate the removal of necrotic tissue, promote the formation of granulation tissue and skin appendages such as hair follicles, and promote the proliferation of collagen fibers, showing good effect of promoting the healing of burn wound. The efficacy is equivalent to that of the positive drug. MEBO, as a positive control, can also promote the repair of the wound, but its main effect is to promote the proliferation of collagen, and in the aspects of re-epithelialization of the wound surface and regeneration and repair of skin appendages such as hair follicles, the effect is not as good as that of S. mooreana.

[0172] IV. Mechanism research on the pharmacodynamic evaluation of S. mooreana in the treatment of deep Ⅱ degree burn in rats

[0173] Based on the pathological process of deep Ⅱ degree burn, the S. mooreana spray prepared in the first part is used to evaluate the pharmacodynamic mechanism of S. mooreana in the treatment of deep Ⅱ degree burn in rats.

[0174] 4.1 Experimental method

[0175] 4.1.1 Detection of antioxidant enzyme activity and lipid peroxidation products

[0176] The treated burn skin tissue was placed in a pre-labeled centrifuge tube and pre-cooled normal saline was added. Then, the skin tissue was cut into pieces using scissors and homogenized with a tissue grinder. Next, a refrigerated centrifuge was used to centrifuge at 3000 rpm for 10 minutes. After separation, the supernatant was transferred to a new centrifuge tube, its concentration was determined, and the supernatant was ensured to be 10% of the tissue homogenate for standby use. In addition, the rat upper serum stored at -80°C was taken out and placed on ice for standby use. According to the instructions for SOD, GSH-PX and MDA, the effects of SOD, GSH-PX and MDA on the antioxidant enzyme activity and lipid peroxidation products in the skin tissue and serum of rats in each group were evaluated.

[0177] 4.1.2 ELISA determination of TNFα and IL-1 content in serum and tissue of rats in each group

[0178] First, the kit was warmed and the reagents were diluted according to the requirements of the instructions. Then, the standard was pre-added according to the concentration gradient, and a duplicate hole was set for each standard hole. Next, the sample was diluted and added, and then enzyme-labeled reagent was added to the sample hole. After mixing the liquid, it was incubated in a 37°C constant temperature environment. After incubation, washing was performed, and after 3-5 times of washing, color developing agent was added and incubated in a 37°C incubator for 15 minutes. After incubation, 50 μL of stop solution was added to each test hole to terminate the reaction. Finally, the absorbance (OD value) of each hole was measured at 450 nm, and the regression equation was calculated according to the standard, and then the concentration of the sample was calculated according to the OD value.

[0179] 4.1.3 Immunohistochemical staining to determine the effect of S. japonica on Nrf2 expression in rats in each group

[0180] The prepared rat skin tissue paraffin sections were deparaffinized, repaired, placed in a 3% H2O2 solution (avoid light, incubate), washed the sections, blocked with 3% BSA, removed the blocking solution, incubated the first antibody NLRP3 (dilution ratio 1:500, 4°C overnight), washed the sections, incubated the second antibody (dilution ratio 1:200), added DAB staining solution, washed the sections, stained with hematoxylin staining solution, dehydrated, transparent, mounted, and photographed for observation. The positive expression area was brown, and 6 random fields were selected from each section, and the positive expression was semi-quantitatively analyzed in Image J software.

[0181] 4.1.4 Western blotting method for detecting the expression of related pathway proteins

[0182] 4.1.4.1, Total protein extraction of rat skin

[0183] (1) Put the grinding block and the labeled centrifuge tube in the refrigerator in advance, open the low-temperature high-speed centrifuge for precooling, take out the skin tissue stored at -80, and place it on ice;

[0184] (2) Weigh 100 mg of rat skin tissue on an analytical balance and place it in a 2 mL labeled centrifuge tube. Add 100 μL of prepared lysis buffer to each 10 mg of tissue;

[0185] (3) Use scissors to cut the tissue in the centrifuge tube as much as possible, then use a tissue grinder to homogenize the tissue fragments, 65 Hz, 60 s, rest for 15 s, a total of 9 times. After grinding, take out the centrifuge tube to check if the grinding is sufficient. If the grinding is not sufficient, grind again;

[0186] (4) After 10 min of rest on ice, centrifuge at 14000 rpm for 10 min. After centrifugation, collect the supernatant;

[0187] (5) Transfer the collected scalded full-thickness skin tissue supernatant to a new centrifuge tube and use the BCA method for protein quantification;

[0188] (6) Add 5x protein loading buffer to the supernatant according to its volume, mix evenly, heat in a 95°C metal bath for 5-10 minutes, cool to room temperature, aliquot, and store in a -80°C freezer.

[0189] 4.1.4.2, BCA protein quantification

[0190] The operation method is the same as that in Section 2.1.3.3.

[0191] 4.1.4.3, Western blot analysis

[0192] The operation method is the same as that in Section 2.1.3.4

[0193] 4.2, Results

[0194] 4.2.1, Effect of S. japonica on oxidative stress level in deep Ⅱ degree scalded rats

[0195] 4.2.1.1, Effect of S. japonica on SOD, MDA, and GSH-Px levels in serum of deep Ⅱ degree scalded rats

[0196] The antioxidation of S. miltiorrhiza is reflected in its ability to promote the expression of antioxidant enzymes. In oxidative stress, SOD, as part of the antioxidant enzyme system, can scavenge free radicals, and significantly protect the inflammatory response and structural damage of tissue cells. GSH-Px, as a peroxide decomposing enzyme, can scavenge ROS and lipid oxidation, and cooperates with SOD to scavenge oxidants. MDA, as the product of lipid peroxidation, reflects the changes in the body's antioxidant capacity to some extent. The increase of serum antioxidant enzyme activity indicates that the body initiates systemic antioxidant defense mechanism to neutralize excess free radicals in the circulatory system and prevent oxidative damage to multiple organs. The experimental results of serum detection are shown in Table 2. Figure 13 As shown in Table 2, compared with the Control group, the SOD and GSH-Px contents in the Model group and serum were significantly decreased (P<0.01 or P<0.05), and the MDA content was sharply increased (P<0.01 or P<0.05); after administration, each dose of SM significantly increased the SOD content in serum (P<0.01 or P<0.05), SM-M, SM-H and PC significantly increased the GSH-Px (P<0.01 or P<0.05), and the MDA level was significantly decreased in each dose of SM and PC group (P<0.01 or P<0.05). Therefore, when the rat scald occurs, SM can significantly up-regulate the expression of these enzymes, thereby enhancing the antioxidant capacity of cells.

[0197] 4.2.1.2, Effect of S. miltiorrhiza on the levels of SOD, MDA and GSH-Px in the skin tissue of deep Ⅱ degree scald rats

[0198] The antioxidant enzymes in the tissue more directly reflect the local damage and repair, especially the oxidative stress state around the scald wound and the self-repairing ability of cells. The experimental results in the tissue are shown in Table 3. Figure 14 As shown in Table 3, compared with the Control group, the SOD and GSH-Px of the Model and NC groups were significantly decreased (P<0.01 or P<0.05), and the MDA was significantly increased (P<0.01); after administration, each dose of SM and the positive drug could significantly increase the SOD content in the skin tissue of deep Ⅱ degree scald rats (P<0.01 or P<0.05), SM-M, SM-H and the positive drug could significantly increase the GSH-Px content, and each dose of SM and the positive drug could significantly decrease the MDA content (P<0.01).

[0199] 4.2.2, Effect of S. miltiorrhiza on the inflammation level of deep Ⅱ degree scald rats

[0200] 4.2.2.1, Effect on the IL-1 level in the serum and skin tissue of rats

[0201] As shown in Table 4, compared with the Control group, the IL-1 level in the serum and skin tissue of the Model group was significantly increased (P<0.01 or P<0.05); after administration, each dose of SM and the positive drug could significantly decrease the IL-1 level in the serum and skin tissue of deep Ⅱ degree scald rats (P<0.01 or P<0.05). Figure 15As shown, compared with the Control group, the expression of IL-1 in the Model and NC groups was significantly reduced in rat serum and tissues (P<0.01 or P<0.05); after administration, all doses of SM and the positive control drug significantly reduced the IL-1 content in the skin tissue and serum of rats with deep second-degree burns (P<0.01 or P<0.05).

[0202] 4.2.2.2 Effects on TNF-α levels in rat serum and skin tissue

[0203] like Figure 16 As shown, compared with the Control group, the expression of TNF-α in the Model and NC groups was significantly reduced in rat serum and tissues (P<0.01 or P<0.05); after administration, all doses of SM and the positive control drug significantly reduced the content of TNF-α in the skin tissue and serum of rats with deep second-degree burns (P<0.01 or P<0.05).

[0204] 4.2.3 Effects of milk thistle on Nrf2 expression in rats of different groups

[0205] To verify the effect of milk thistle on Nrf2 protein expression in rats with deep second-degree burns, immunohistochemistry was used to detect the localization and expression of Nrf2 in skin tissue, with dark brown tissue sections indicating positive expression. The experimental results are as follows: Figure 17 As shown, compared with the Control group, the expression of Nrf2 protein in the prostate tissue of rats in the Model group was significantly reduced (P<0.01). After drug administration, the SM-M and SM-H groups significantly increased the expression of Nrf2 in the tissue (P<0.01), and the PC group also significantly increased the expression of Nrf2 (P<0.01). The statistical results are as follows. Figure 18 As shown.

[0206] 4.2.4 Effects of milk thistle on the Nrf2 / HO-1 and TGFβ1 / Smad signaling pathways in rats of different groups

[0207] like Figure 19 As shown, at 7 days of wound healing, compared with the Control group, the expression of Nrf2, HO-1, and NQO-1 in the Model group rats was significantly decreased (P < 0.01); compared with the Model group, administration of SM at various doses and the PC group significantly increased the expression of Nrf2, HO-1, and NQO-1 proteins (P < 0.01 or P < 0.05), and the SM group showed higher expression of HO-1 and NQO-1 proteins than the PC group. Compared with the Control group, the expression of TGF-β1 and p-smad proteins in the Model group rats was significantly decreased, while the expression of SM at various doses and the PC group could reverse this condition, and the SM-M group significantly increased TGF-β1 and p-smad2 / 3 (P < 0.01). At 21 days of wound healing, as Figure 20As shown, SM each dose and PC group can continuously make Nrf2, HO-1, NQO-1 protein expression significantly increased (P<0.01 or P<0.05) after administration, SM-M group is still higher than PC group in HO-1, NQO-1 protein expression, which shows that it can continue to resist oxidation in the healing process of the wound, at this stage, it can be found that the expression of TGF-β1 and p-smad2 / 3 protein in Model group rats is significantly increased (P<0.01 or P<0.05), while SM each dose and PC group can significantly down-regulate the expression of TGF-β1 and p-smad (P<0.01 or P<0.05), these results suggest that SM each dose group up-regulates TGF-β1 and activates Smad2 / 3 in the proliferation phase, induces the transdifferentiation of epidermal stem cells to myofibroblasts, and promotes wound healing, and continuously up-regulates HO-1, NQO-1 protein and activates Nrf2 in the maturation phase, continuously reduces inflammation and the risk of possible scar, SM can activate the Nrf2 / HO-1 pathway related to antioxidant stress and then inhibit the TGF-β1 / Smad pathway, reduce oxidative stress and regulate TGF-β to an appropriate level, which can achieve a dynamic balance of inflammation, fibrosis and tissue repair, thereby significantly improving the healing quality and reducing scar formation.

[0208] The results of the present application show that the expression of TNF-α and IL-1 in the Model rats is increased, and the release of inflammatory factors TNF-α and IL-1 can be reduced after SM administration, and the double anti-inflammatory effect forms a synergistic mechanism with the antioxidant effect, which promotes the wound repair process together. Immunohistochemistry shows that Nrf2 is sharply increased in each dose group of SM, which also means that Nrf2 plays a crucial role in the SM-mediated oxidation stress antagonism after scalding. Western blotting detects the expression of Nrf2 and TGF-β1 / Smad signaling pathway and related proteins in rat skin tissue on the 7th day and the 14th day. On the 7th day, the protein expression of Keap-1 in the Model group is increased, and the protein expression of TGF-β1 and smad2 / 3 is decreased. After administration, each dose group of SM and the PC group can increase the protein of antioxidant Nrf2, HO-1 and NQO-1, and also increase the protein expression of TGF-β1 and smad2 / 3, which shows that the activation of Nrf2 pathway by SM has a concentration-dependent effect, that is, the effect is more significant with the increase of the dose, which shows that the mechanism of SM is directly or indirectly related to the regulation of Nrf2. In this stage, the increase of TGF-β1 and smad2 / 3 protein shows that the water fly can promote the healing of the wound. On the 21st day of wound healing, the protein of TGF-β1 and smad2 / 3 in the Model group begins to increase, and TGF-β1 is over-activated, which may bring about sustained inflammatory response of the wound, leading to poor healing and causing scar formation. The protein of TGF-β1 and smad2 / 3 in each dose group of SM begins to decrease gradually, and the anti-oxidative stress effect still exists, which shows that SM can treat scalding through multiple pathways and targets, and become an ideal external treatment agent for wounds, which has significant development potential. The above WB and immunohistochemistry results show that the treatment mechanism promotes wound healing through Nrf2 / HO-1 and TGF-β / Smad signaling pathways.

[0209] The present application first optimizes the extraction process of water fly; then studies the influence of water fly alcohol extract on Nrf2 / HO-1 and TGF-β / Smad signaling pathway. The results show that the water fly alcohol extract can up-regulate the expression of Nrf2, HO-1 and NQO-1, enhance the antioxidant capacity, and down-regulate the expression of TGF-β1 and p-Smad2 / 3, regulate the fibrosis process. This finding reveals the double mechanism of water fly in scald repair, that is, promoting healing through antioxidant and regulating fibrosis.

[0210] In summary, the water fly alcohol extract has a significant promoting effect in scald repair, improves oxidative stress damage by activating Nrf2 / HO-1 pathway, and regulates TGF-β / Smad pathway to achieve inhibition of fibrosis, which provides a theoretical basis and experimental support for water fly as a potential therapeutic drug for non-scarring healing after scalding.

[0211] The above are several more preferred embodiments of the preparation method of the present application, but cannot be taken as a limitation on the technical solutions protected by the present application. Any alternative solution based on the technical idea of the present application and obtained by a person of ordinary skill in the art without making any creative effort shall fall within the protection scope of the present application.

Claims

1. Application of a Silybum marianum ethanol extract in the preparation of a medicament for treating deep Ⅱ degree burns and scalds.

2. The use of the Silybum marianum ethanol extract according to claim 1 for the preparation of a medicament for treating deep Ⅱ degree burn and scald, characterized in that, When the Silybum marianum ethanol extract is applied in the preparation of a medicament for treating deep Ⅱ degree burns and scalds, the Nrf2 / HO-1 pathway is activated to improve oxidative stress damage and promote the repair of deep Ⅱ degree burns and scalds.

3. The use of the Silybum marianum ethanol extract according to claim 1 in the preparation of a medicament for treating deep Ⅱ degree burns and scalds, characterized in that, When the Silybum marianum ethanol extract is applied in the preparation of a medicament for treating deep Ⅱ degree burns and scalds, the TGF-β / Smad pathway is regulated to inhibit fibrosis and promote the repair of deep Ⅱ degree burns and scalds.

4. The use of the Silybum marianum ethanol extract according to claim 1 in the preparation of a medicament for treating deep Ⅱ degree burns and scalds, characterized in that, When the Silybum marianum ethanol extract is applied in the preparation of a medicament for treating deep Ⅱ degree burns and scalds, the expression of Nrf2, HO-1 and NQO-1 is up-regulated, and the expression of TGF-β1 and p-Smad2 / 3 is down-regulated.

5. The use of the Silybum marianum ethanol extract according to claim 1 for the preparation of a medicament for treating deep Ⅱ degree burn and scald, characterized in that, When the Silybum marianum ethanol extract is applied in the preparation of a medicament for treating deep Ⅱ degree burns and scalds, the release of inflammatory factors TNF-α and IL-1 is reduced.

6. The use of the Silybum marianum oil extract according to claim 1 for the preparation of a medicament for the treatment of deep 2nd degree burns. When the Silybum marianum ethanol extract is applied in the preparation of a medicament for treating deep Ⅱ degree burns and scalds, the Silybum marianum ethanol extract improves oxidative stress damage through the Nrf2 / TGF-β1 pathway and promotes the benign proliferation of HaCaT cells.

7. The use of the Silybum marianum oil extract according to claim 1 for the preparation of a medicament for the treatment of deep 2nd degree burns. The extraction method of the Silybum marianum ethanol extract is as follows: Silybum marianum powder is extracted twice by ethanol reflux, and the filtrates of the two extractions are combined; the filtrates are evaporated and concentrated, and then freeze-dried to obtain the Silybum marianum ethanol extract in the form of extract.

8. The use of the Silybum marianum oil extract according to claim 7 for the preparation of a medicament for the treatment of deep 2nd degree burns, characterized in that, During each extraction, 8 mL of ethanol with a volume fraction of 85% is added per g of Silybum marianum powder; and each extraction lasts for 1.5 h.

9. The use of the Silybum marianum oil extract according to claim 7 for the preparation of a medicament for the treatment of deep 2nd degree burns, characterized in that, The extraction method of the Silybum marianum ethanol extract further includes: adding ethanol and Tween 80 to the Silybum marianum ethanol extract, swelling for 20 min, and then grinding and stirring until completely dissolved; then adding distilled water and stirring uniformly, high-temperature sterilization, and filtration to obtain a Silybum marianum spray.

10. The use of the Silybum marianum oil extract according to claim 9 for the preparation of a medicament for the treatment of deep 2nd degree burns, characterized in that, 6 mL of ethanol, 10 mL of Tween 80 and 184 mL of distilled water are added per 1.538 g of Silybum marianum ethanol extract; and the volume fraction of ethanol is 85%.

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