WO2015158177A1 - Uses of recombinant ganoderma lucidum immunoregulatory protein in preparing medicines for treating skin tissue aging and wounds - Google Patents

Abstract

The present invention provides uses of recombinant ganoderma lucidum immunoregulatory protein (rLZ-8) in preparing medicines for treating skin tissue aging caused by endogenous and exogenous stimuli and skin wounds caused by external force, sports, postoperation and the like. In the present invention, a rat skin aging model and a skin wound model are separately created; after rLZ-8 drug therapy, a result indicates that the rLZ-8 improves a physiological index of the skin aging model, stimulates secretion of skin tissue growth factors and increases wound healing of rat skin tissue.

Description

Application of recombinant Ganoderma lucidum immunomodulatory protein in the preparation of drugs for treating skin tissue aging and trauma Technical field

The invention relates to the field of biopharmaceuticals, and relates to the application of recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) in preparing skin tissue aging and trauma drugs, in particular to the establishment of a rat model of skin aging and a model of skin trauma; a therapeutic procedure Statistical analysis of design and treatment outcomes, focusing on the application of recombinant Ganoderma lucidum immunomodulatory proteins in the treatment of skin aging rat models and skin wound rat models.

Background technique

Skin aging is mainly caused by internal causes and external causes. Among them, internal factors: 1) natural decline in the function of skin accessory organs such as sweat glands of the skin, decreased function of the skin glands, reduction of secretions, lack of moisture in the skin membrane and stratum corneum of the skin. Dry, causing dry lines and peeling. 2) As the skin's metabolism is slowed down, the moisturizing factor in the dermis is reduced, which causes the function of the elastic fibers and collagen fibers in the dermis to decrease, causing skin tension and weakening of elasticity, making the skin prone to wrinkles. 3) The skin of the face is thinner than the skin of other parts of the body. Due to the nutritional disorders of the skin, the storage of subcutaneous fat is gradually reduced, and the cells and fibrous tissues are malnourished and the performance is degraded. 4) The enzymes in the living body are gradually reduced, and the skills of the body are reduced in various aspects, so that a large amount of free radicals damage the human cells and cause the cells to die. External factors: 1) improper maintenance, lack of skin care, or incorrect skin Nursing procedures. 2) The cold and dry climate makes the skin's various functions diminish and the skin lacks moisture. 3) Excessive exposure to sunlight causes skin aging. 4) The main cause of skin aging is that the pores are usually blocked by dead cells, which affects metabolism.

Skin trauma, the mildest trauma is limited to the epidermal layer of the skin, which can be healed by epithelial regeneration. Slightly severe cases have skin and subcutaneous tissue rupture and wounds; severe trauma can have muscles, muscle bonds, nerve breaks and fractures. If the skin appendages (hair follicles, sweat glands, and sebaceous glands) are completely destroyed, they cannot be completely regenerated and scar repair occurs. After the tendon rupture, the scar is repaired at the beginning, but with the continuous improvement of functional exercise, the collagen fibers can be arranged in the direction of the original tendon fibers to achieve complete regeneration. Skin wound healing process:

1. Early changes in wounds There are varying degrees of tissue necrosis and vascular rupture in the wound. Inflammation occurs within a few hours, manifesting as hyperemia, serous exudation and leukocyte migration, so local redness. The fibrinogen in the blood and exudate of the wound quickly solidifies to form a clot, and some of the clot surface is dried to form a suede, and the clot and suede act to protect the wound.

2. After 2 to 3 days of wound contraction, the entire layer of skin and subcutaneous tissue on the edge move toward the center, so the wound shrinks rapidly and stops until about 14 days. The significance of wound contraction is to reduce the wound surface.

3. Granulation tissue hyperplasia and scar formation began from the third day from the bottom of the wound and the edge of the granulation tissue to fill the wound. On the 5th to 6th day, the fibroblasts produced collagen fibers, and the collagen fibers formed very active one week later, and gradually slowed down later. As more and more collagen fibers appear, scar formation occurs, and the scar is completely formed about one month after the injury.

4. Epidermal and other tissue regeneration rupture trauma within 24 hours, basal cells at the edge of the wound That is, hyperplasia begins and migrates beneath the clot to the center of the wound, forming a single layer of epithelium covering the surface of the granulation tissue. When these cells meet each other, migration ceases and proliferates and differentiates into squamous epithelium. Healthy granulation tissue is important for epidermal regeneration because it provides the nutrients and growth factors needed for epithelial regeneration. If the granulation tissue does not fill the wound for a long time and forms a scar, the epithelial regeneration will be delayed; in another case, the excessive growth of the granulation tissue (Exuberant granulation) due to foreign body and infection, etc., is higher than the skin surface. Epidermal regeneration is also prevented, so it is often necessary to remove it. If the wound is too large (generally considered to be more than 20 cm in diameter), it is difficult for the regenerated epidermis to completely cover the wound, often requiring skin grafting.

The Fungal Immunomodulatory Protein of Ganoderma lucidium (LZ-8) was the first fungal immunoregulatory protein isolated from Ganoderma lucidum mycelium extract in 1989. Previous studies have shown that LZ-8 has a systemic allergic reaction that inhibits mouse peripheral blood lymphocyte proliferation and immunological activity of cytokines such as IL-2, TNF-α and IFN-γ. The recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) was obtained by recombinant expression of rLZ-8 and the corresponding pilot purification process using the constructed Pichia pastoris GS115 strain. As a recombinant protein immunomodulatory preparation, rLZ-8 has a variety of immune functions, which can avoid a series of toxic and side effects caused by chemotherapy in the anti-cancer and anti-cancer effects.

Summary of the invention

The object of the present invention is to provide a focus on the application of recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) in the preparation of skin repairing drugs, focusing on the effects of skin aging rat model and skin wound rat model on the treatment effect, and establishing skin aging Rat model and skin trauma rat model, adopted The mathematical statistics software tests the data and concludes that rLZ-8 has a significant effect in the treatment of the skin aging rat model and the skin trauma rat model. The specific invention is as follows:

Skin repair model was established: 1) Skin aging rat model: Healthy female Wistar rats were selected, and D-galactose was injected subcutaneously into the neck and back for several weeks. Microscopic observation of the model establishment. 2) Rat model of skin trauma: Healthy female SD rats were used, weighing between 240-290 g, rats were depilated with 5% Na ₂ S back, and a full thickness skin defect wound was created on the back, with a diameter of 1.5 cm. Deep into the muscle layer.

Experimental grouping and administration methods: 1) Skin aging rats: 78 female Wistar rats were randomly divided into 6 groups (n=13): normal control group, aging model group, positive control group, LZ-8 low dose group. LZ-8 medium dose group, LZ-8 high dose group; normal control group and aging model group were injected with normal saline (0.1ml), positive control group was injected with vitamin E (80mg/kg/d), rLZ-8 low dose group (8ug/kg/d), rLZ-8 medium dose group (16ug/kg/d), rLZ-8 high dose group (32ug/kg/d). Continuous injection for 14 days. 2) Rat model of skin trauma: The wound control group was treated with the same amount of normal saline, and the wounds of the treatment group were directly treated (the drug was directly applied to the wound wound of rats, and the concentration of rLZ-8 was 125ug/kg. 250 ug/kg, 500 ug/kg), 4 weeks of peripheral drug administration (administered at 4 weeks outside the rat wound, rLZ-8 at a concentration of 125 ug/kg, 250 ug/kg, 500 ug/kg) and irrigation For gastric administration, rLZ-8 was administered at a concentration of 125 ug/kg, 250 ug/kg, and 500 ug/kg. Each group was administered in an independent feeding mode and administered once a day for 2 weeks.

Test items and methods: 1) Skin aging model: The main test items are skin moisture content, hydroxyproline content in skin, hyaluronic acid content in skin, SOD content in body, MDA in vivo The content, the content of lipofuscin (LF) in the body and the pathological changes observed under HE microscope. Among them, superoxide dismutase (SOD) and ELISA test kit (R&D) were used to measure the SOD content in rat plasma and skin tissues, and the operation was carried out according to the SOD kit instructions; the ELISA kit (R&D) was used to measure the large amount. The content of HA in the plasma and skin tissues of rats was operated according to the kit instructions; the skin protein content was determined using the Coomassie brilliantin assay kit, and the method was followed according to the kit instructions; using the hydroxyproline (Hyp) ELISA kit (R&D) measures the content of Hyp in rat plasma and skin tissues; MDA content in rat plasma and skin tissues was determined by malondialdehyde (MDA) ELISA kit (R&D). 2) Skin wound model: The main test items are rat epidermal growth factor (EGF), rat vascular endothelial growth factor (VEGF), rat fibroblast growth factor (FGF) growth factor and hydroxypeptidic content of granulation tissue. Wait. Among them, FGF, EGF, VEGF and other items were determined by the corresponding enzyme-linked immunosorbent assay, and the hydroxyproline content of granulation tissue was determined by hydroxyproline (Hyp) ELISA kit (R&D).

Experimental results statistics: The present invention mainly through skin moisture content, hydroxyproline content in the skin, hyaluronic acid content in the skin, SOD content in the body, MDA content in the body, lipofuscin (LF) content in the body, and HE staining under skin tissue. Observing pathological changes and other indicators to investigate the effect of rLZ-8 rat skin aging model on the treatment effect, through rat epidermal growth factor (EGF), rat vascular endothelial growth factor (VEGF), rat fibroblast growth factor (FGF) Growth factor and hydroxypeptidate content of granulation tissue The therapeutic effect of rLZ-8 on rat skin wound model was examined. Based on the experimental data of the above indicators, SPSS statistical software was used for data analysis. According to the analysis of each treatment group, the statistical results were used to evaluate the application of rLZ-8 in skin repair drugs.

Skin aging: rLZ-8 can increase the content of hyaluronic acid in skin tissue and inhibit the decrease of skin tissue moisture content caused by aging; rLZ-8 can increase the important extracellular matrix component of skin tissue---collagen The content of the skin, thereby improving the elasticity of the skin, reducing the level of lipofuscin in the skin tissue, slowing down aging; rLZ-8 can enhance the level of superoxide dismutase (SOD) in rats and reduce the malformaldehyde of the lipid metabolism product malondialdehyde The level of (MDA) indicates that rLZ-8 can effectively antagonize the senescence of skin cells induced by D-galactose.

Skin wounds: All three routes of administration can achieve consistent therapeutic effects at the same time point. The experimental results show that rLZ-8 can significantly regulate rat epidermal growth factor (EGF) and rats within 7 days after trauma. The expression levels of vascular endothelial growth factor (VEGF) and rat fibroblast growth factor (FGF) growth factor promote wound repair, while steadily increasing the hydroxyproline content of granulation tissue, and indirectly reacting by measuring the content of hydroxyproline. The content of collagen is used to understand the collagen metabolism during wound healing. As the synthesis of collagen continues to increase, the defect tissue is gradually filled, the wound is shrunk, and then healed.

The objectives, features, and advantages of the invention are disclosed in the Detailed Description of the Detailed Description of the Drawings.

DRAWINGS

Fig.1 Comparison of HE staining photos of skin tissue of rats in each group at 40x magnification

Fig.2 Comparison of HE staining images of skin tissue of rats in each group at 200x magnification

detailed description

Example 1 Therapeutic effect of rLZ-8 on skin aging in rats

1.1 Experimental methods

1.1.1 Experimental reagents and materials:

rLZ-8, vitamin E, physiological saline, D-galactose, distilled water, 1 mL disposable syringe, superoxide dismutase (SOD) ELISA test kit, malondialdehyde (MDA) ELISA kit, Coomassie brilliant blue protein assay Kit, Hydroxyproline (Hyp) ELISA Kit, HA ELISA Kit (R&D)

1.1.2 Instrument and equipment:

Analytical balance, vertical pressure steam sterilizer, centrifuge, double clean bench, inverted microscope, 4 ° C refrigerator, low temperature refrigerator (-80 ° C), common microplate reader.

1.1.3 Experimental animals:

Healthy adult Wistar rats, female, weighing 240-260 grams, SPF grade. Raised under the SPF level conditions of Jilin University. During the experiment, the room temperature was controlled at (20 ± 2) °C for 12 hours, and the mice were given free access to water and food.

1.1.4 Experimental grouping and drug administration procedures:

78 female Wistar rats were randomly divided into 6 groups (n=13): normal control group, aging model group, vitamin E positive control group, rLZ-8 low dose group, rLZ-8 medium dose group, rLZ-8 high. The dose group; the normal control group was injected subcutaneously with normal saline 0.lml/10g BW every day, and the other groups were injected subcutaneously with D-galactose 1000mg/kg per day (equivalent to the injection volume). 0.1 ml/l0g BW) modeling for 7 weeks. A subacute aging model of rats was established. The drug-administered group was intragastrically administered with vitamin E or intraperitoneal injection of rLZ-8, and the normal control group and the aging model group were given an equal volume of physiological saline. The drug dosage is shown in the table below:

Table 1 Skin aging rat model treatment grouping and drug administration procedures

1.2 Experimental results:

The invention mainly adopts the skin moisture content, the hydroxyproline content in the skin, the hyaluronic acid content in the skin, the SOD content in the body, the MDA content in the body, the lipofuscin (LF) content in the body, and the pathological changes observed under the tissue microscope of HE staining. The indicators were used to investigate the effects of the treatment of skin aging model in rLZ-8 rats. The specific detection methods and results are as follows:

1.2.1 Effect of rLZ-8 on water content of rat skin aging model

The skin of the back of the mouse was taken 2 cm × 2 cm, and the wet weight was accurately weighed. It was baked in a drying oven at 80 ° C for 12 hours and weighed to dry weight. Calculate the moisture content.

The moisture content of the skin = (skin wet weight - dry skin weight) / wet weight of the skin x 100%.

The skin moisture content showed that the water content of the rat skin was significantly lower than that of the control group after D-galactose modeling (p<0.05), and the other groups were given V _E or high, medium and low doses of rLZ- 8, can significantly increase the moisture content in the skin and return to normal levels (Table 2).

Table 2. Skin moisture content of each group of rat models

#: Compared with the normal control group, p<0.05; ※: compared with the model group, p<0.05.

1.2.2 Effect of rLZ-8 on the content of hydroxyproline in skin of rat skin aging model

The present invention measures the content of Hyp in rat plasma and skin tissue using a hydroxyproline (Hyp) ELISA kit (R&D Co., Ltd.), and operates according to the method of the kit. The results showed that except for the VE positive control group, the content of hydroxyproline (Hyp) in the skin of the other experimental groups was significantly lower than that of the normal control group (p<0.05), but by administering high dose and low dose. rLZ-8 significantly increased the content of hydroxyproline in rat skin. The hydroxyproline content in the skin of these two groups was significantly higher than that in the model group (Table 3). It is suggested that rLZ-8 can promote the synthesis of skin collagen and make the skin tend to change young.

Table 3. Hydroxyproline content in skin of each group of rats (ng/g)

a: p<0.05 compared with the normal control group; b: compared with the model group, p<0.05.

1.2.3 Effect of rLZ-8 on hyaluronic acid content in skin of rat skin aging model

The present invention measures the HA content in rat plasma and skin tissue using an ELISA kit (R&D Co., Ltd.), and operates according to the method of the kit. The results show that the aging model group There was no significant difference in the amount of hyaluronic acid in the skin compared with the normal control group. However, the content of hyaluronic acid in the skin of high concentration and low concentration group was significantly higher than that of the model group and the normal control group (p<0.05). The specific results are shown in Table 4.

Table 4 Hyaluronic acid content (ng/g) in the skin of each group of rats

a: p<0.05 compared with the normal control group; b: compared with the model group, p<0.05.

1.2.4 Effects of rLZ-8 on SOD content in plasma and skin tissues of rat skin aging model

The present invention measures the SOD content in plasma and skin tissues of a rat model using a superoxide dismutase (SOD) ELISA test kit (R&D Co., Ltd.), and operates according to the method of the SOD kit. The results showed that compared with the normal control group, the content of SOD in plasma and skin tissue of the aging model group decreased, but the difference was not significant; however, the content of SOD in plasma and tissue of rLZ-8 group was significant. Higher than the model group and the normal control group (p<0.05), the VE group was higher than the model group (Table 5). It shows that rLZ-8 can enhance the skin's protection against free radicals.

Table 5. SOD content in each group of rats

a: p<0.05 compared with the normal control group; b: compared with the model group, p<0.05.

1.2.5 Effect of rLZ-8 on malondialdehyde (MDA) content in rat skin aging model

The present invention employs a malondialdehyde (MDA) ELISA kit (R&D Co., Ltd.) to measure the MDA content in rat plasma and skin tissues, and operates according to the kit instructions. The results showed that the content of MDA in the aging model group was significantly higher than that in the normal control group (p<0.05). After the administration of rLZ-8, the MDA content in plasma and skin tissues was significantly decreased, among the medium and low concentration groups. Even lower than the normal control group (p < 0.05) (Table 6).

It is suggested that rLZ-8 can reduce the accumulation of lipid peroxidation products.

Table 6. MDA content in rats of each group

a: p<0.05 compared with the normal control group; b: compared with the model group, p<0.05.

1.2.6 Effect of rLZ-8 on the content of lipofuscin (LF) in rat skin aging model

In the present invention, the LF content in rat plasma and skin tissues is measured by a lipofuscin (LF) ELISA kit (R&D Co., Ltd.), and the method is carried out according to the method of the kit. The experimental results showed that the lipofuscin level in the model group was significantly higher than that in the normal control group. After three doses of rLZ-8, the level of lipofuscin in the skin tissue was significantly decreased, and the LF in the skin of the high and low dose groups was observed. The content was comparable to that of the normal control group (Table 7).

Table 7 LF content in each group of rats

a: p<0.05 compared with the normal control group; b: compared with the model group, p<0.05.

1.2.7 Histopathological changes in skin of rats in each group

The invention adopts the HE staining method to observe the tissue cells of the rat model and observes the specific method: the skin of the back of the mouse is 0.5cm×0.5cm in the middle of the back, and is fixed in 4% paraformaldehyde for 12-24h, and is conventionally dehydrated and transparent. , dipping wax, embedding, paraffin sectioning, thickness 5um, patch to slide. Xylene dewaxing, stepwise ethanol rehydration to water washing, hematoxylin staining, tap water washing, hydrochloric acid ethanol differentiation, tap water soaking, HE staining, conventional dehydration, transparent neutral gum seal, observation under light microscope, photomicrography. The results showed that the skin thickness of the normal control group was normal, the epidermal tissue structure was intact, the cell layer was clear, the dermis layer was thick, and the wavy collagen fibers were densely arranged. Compared with the normal control group, the aging model group rats skin dermis The layer was significantly thinner, the collagen fibers were reduced, and the arrangement was loose. Compared with the aging model group, the rat dermis layer was thicker, the collagen fibers were more dense, and the arrangement was denser; the rLZ-8 group was larger than the aging model group. The dermis layer of the mouse was significantly thickened, the collagen fibers were significantly increased, and the arrangement was dense, which was close to the normal control group. It can be seen that rLZ-8 can significantly improve the degree of aging of skin tissue (Figure 1, 2).

Example 2 Application of rLZ-8 in the treatment of wound healing in rats

2.1 Experimental methods

2.1.1 Experimental reagents and materials:

rLZ-8, physiological saline, distilled water, 1mL disposable syringe, sodium bicarbonate, sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, formaldehyde, absolute ethanol, EGF growth factor enzyme immunoassay kit , hydroxyproline assay kit, VEGF growth factor enzyme-linked immunosorbent kit, FGF growth factor enzyme-linked immunoassay kit, protein content determination kit

2.1.2 Equipment:

Constant temperature magnetic stirrer, high pressure steam sterilizer, low speed centrifuge, plate balance, digital thermostat water bath, tissue homogenizer, electronic balance, plastic centrifuge tube, EP tube, gun head, tissue embedding machine, tissue section Machine, electric heating constant temperature water tank, electric constant temperature drying oven, electric blast drying oven, microplate reader, ultraviolet spectrophotometer, low temperature high speed centrifuge, analytical balance, vertical pressure steam sterilizer, double super clean bench, Optical inverted microscope, 4 ° C refrigerator, low temperature refrigerator (-80 ° C), common microplate reader.

2.1.3 Experimental animals:

Healthy adult SD rats, female, weighing 240-260 grams, SPF grade. Raised under the SPF level conditions of Jilin University. During the experiment, the room temperature was controlled at (20±2) °C, and the lighting was alternated for 12 hours, free to drink and feed.

2.1.4 Experimental grouping and drug administration procedures:

There were 100 male SD rats, totaling 200. Randomly divided into 10 groups, model group (M group) 20, administered into rLZ-8 low dose group (group A), rLZ-8 medium dose group (group B), rLZ-8 high dose group (C Group); 20 in each group, a total of 9 groups of 3 modes of administration. Weighing Remember. The specific administration procedures are shown in Table 8.

Table 8 Rat skin wound model experimental grouping and administration procedures (n=20)

2.1.5 Experimental testing items and methods:

On the 3rd, 7th, and 14th day after model establishment in rats, anesthetized with ether, 6 rats in each group were sacrificed for blood collection from the fundus venous plexus, granulation tissue of the wound surface of the rat, and skin around the wound. Detection of growth factors and some functional indicators. Serum, skin, and granulation tissue samples were stored in a -80 degree refrigerator and stored. The detection method is provided by various commercially available growth factor detection kits.

2.2 Experimental results:

The invention mainly relates to wound healing rate, hydroxyproline content in wound granulation tissue, rat epidermal growth factor (EGF) content, rat fibroblast growth factor (FGF) content, and rat vascular endothelial growth factor (VEGF) content. The indicators were used to investigate the effect of rLZ-8 on the repair and treatment of rat skin wound model. The specific detection methods and results are as follows:

2.2.1 Effect of rLZ-8 on healing rate of rat skin wound model

The invention adopts the wound healing standard: the granulation completely covers the wound surface as the curing standard, and the wound healing standard time is the wound healing time; the time required for the wound healing after the modeling of each group is recorded.

Wound healing rate: wound healing rate = (original wound area - residual wound area) / original Wound area × 100%. On the 3rd, 7th and 14th postoperative day, the morphology of each group of granulation flaps was accurately imaged with sulfuric acid paper. A scale was placed next to the wound surface. The images were taken into the computer and analyzed by image analysis software Photoshop. Data were obtained and data were obtained. The wound healing rate was calculated and the wound healing rate was 100% for wound healing. The specific experimental results are as follows (Table 9):

Table 9 Effect of rLZ-8 on healing rate of rat skin wound model (%n=6)

^▲ There was a significant difference between the experimental group and the model group (p<0.05).

The experimental results showed that the direct administration of the drug was compared with other routes of administration. On the third day after the model was established, the low-dose group of rLZ-8 had the fastest healing rate, which was significantly different from the model group. (p<0.05). On the 7th day after the model administration, the low-dose group of rLZ-8 was prominent in the four-point administration route, and the healing rate reached 59.2±10.5%, which was significantly different from the model group (p<0.05). On the 14th day after the model was administered, we found that the healing rate of the group was still the highest, reaching a mean value of 95.0% or more. At the same time, we found that the intragastric administration route was not outstanding compared with the above two administration routes. There was also a significant difference in the model group comparison, which was not statistically significant (p>0.05), indicating direct administration of the therapeutic route of administration in the rat skin wound model. Four-point administration is more suitable. At the same time, we can also see that rLZ-8 can rapidly promote the healing of wounds and improve the wound healing rate during the treatment of rat skin wound model.

2.2.2 Effect of rLZ-8 on hydroxyproline content in granulation tissue of rat skin wounds

The invention adopts a commercially available hydroxyproline (Hyp) ELISA kit (R&D company) to determine the content of hydroxyproline in the granulation tissue of rat skin wound, and operates according to the method of hydroxyproline (Hyp) ELISA kit. The specific test results are shown in Table 10.

Table 10 Effect of rLZ-8 on hydroxyproline content in granulation tissue of rat skin wounds (μg/mg wet weight) (x±s, n=6)

^▲ There was a significant difference between the experimental group and the model group (p<0.05).

The experimental results showed that the levels of hydroxyproline in each group increased gradually after modeling, and reached the highest on the 14th day. On the third day after administration, the expression of hydroxyproline in the low dose group of rLZ-8 in the direct administration route was significantly increased, which was significantly different from that in the model group. On the 7th day after model establishment, rLZ- The hydroxyproline content in the granulation tissue of each model group was significantly increased (p<0.05). On the 14th day after model establishment, it can be seen that only the low dose of rLZ-8 in the direct administration route. Group and The model group showed a significant increase in hydroxyproline content in granulation tissue, which was statistically significant (p<0.05). It is indicated that rLZ-8 has a significant effect on promoting the expression of hydroxyproline in granulation tissue under direct administration, and it has a positive effect on the repair of skin wounds, and also indicates the mode of administration of direct administration in comparison. More suitable for skin wound repair treatment.

2.2.3 Effect of rLZ-8 on the content of epidermal growth factor (EGF) in rats

The invention adopts a commercially available rat epidermal growth factor (EGF) enzyme-linked immunosorbent kit to measure the change of the growth factor content in the epidermis of rats, and operates according to the method of epidermal growth factor (EGF) enzyme-linked immunoassay kit, and specifically detects The results are shown in Table 11.

Table 11 Effect of rLZ-8 on the content of epidermal growth factor (EGF) in rats (unit: ug/L) (x±s, n=6)

^▲ There was a significant difference between the experimental group and the model group (p<0.05).

The results of the experiment showed that the expression of EGF in the early stage of rats decreased after the model administration, and the expression of EGF in the skin of the rats after trauma was a slower change. On the third day, the results showed that the expression of EGF was slower. High, the lowest on the 7th day, then gradually rises, the first At 14 days, it was higher than that at 7 days, but it still reached the expression level at 3 days. From the drug dose, the post-traumatic EGF content of rLZ-8 low-dose group was better than medium and high doses. Four-point administration and intragastric administration showed outstanding performance after 14 days of modeling, and there was significant difference compared with the model group, which was statistically significant (p<0.05).

2.2.4 Effect of rLZ-8 on the content of fibroblast growth factor (FGF) in rats

The invention adopts a commercially available rat fibroblast growth factor (FGF) enzyme-linked immunosorbent assay kit to determine the change of rat fibroblast growth factor (FGF) content according to fibroblast growth factor (FGF) enzyme-linked immunosorbent reagent. The box instruction method is operated, and the specific test results are shown in Table 12.

Table 12 Effect of rLZ-8 on rat fibroblast growth factor (FGF) content (unit: ug/L) (x±s, n=6)

The results showed that after trauma, the expression of FGF in the skin of rats after trauma was different from other factors, which was a gradual increase. The FGF level was the lowest on the third day and began to increase on the 7th day. On the 14th day, the FGF level no longer rises and is basically stable; From the dose of drug, the FGF level was the highest in the low dose group in the different time periods, and the FGF level was the lowest in the middle dose group and the high dose group, but the FGF levels in the different dose groups were higher than the model group, the difference was not significant; In terms of the route of administration, the expression level of FGF in the skin of the rats after trauma and the administration route were not significantly affected, and there was no significant difference between the groups and the model group.

2.2.5 Effect of rLZ-8 on the content of vascular endothelial growth factor (VEGF) in rats

The invention adopts a commercially available rat vascular endothelial growth factor (VEGF) enzyme-linked immunosorbent kit to measure the change of rat vascular endothelial growth factor (VEGF) content according to the rat vascular endothelial growth factor (VEGF) enzyme-linked immunosorbent assay kit. The method of the specification is operated, and the specific test results are shown in Table 13.

Table 13 Effect of rLZ-8 on vascular endothelial growth factor (VEGF) content in rats (unit: ug/L) (x±s, n=6)

The results showed that the expression of VEGF in the skin of post-traumatic rats was a slower change from time to time. It was higher on the third day, the lowest on the 7th day, and then gradually increased. On the 14th day, the expression level was basically reached at the 3rd day; At the dose, the post-traumatic VEGF concentration The rLZ-8 low-dose group was higher than the middle and high doses. The VEGF level and model ratio of each dose group were lower than the model group, but the difference was not significant, and there was no statistical significance, indicating that rLZ-8 on rat vascular endothelial growth factor ( The effect of VEGF) content is small. From the comparison of the route of administration, there was no significant difference in the concentration of VEGF between the administration groups at each time point after the model establishment, and there was no statistical significance.

2.2.6 Effect of rLZ-8 on albumin content in rat skin

The invention adopts a commercially available albumin detection kit to measure the change of albumin content in the skin tissue of rats, and operates according to the method of the albumin content detection kit, and the specific detection results are shown in Table 14.

Table 14 Effect of rLZ-8 on albumin content in rat skin (unit: ug/L) (x±s, n=6)

The experimental results show that, from the time of wound healing, the albumin content of each group of skin tissue decreased with the increase of healing time. After the literature reported that the albumin content in serum showed a slight increase in early stage and a significant decrease in the later stage. Trauma caused by trauma and related proteins Compensatory changes. The experimental results are consistent in the relevant literature reports. From the drug dose, the protein content of each dose group was lower than that of the model and the positive drug group, but there was no statistical difference (p>0.05). From different routes of administration, there was no significant change in protein content between the experimental groups of different administration routes and the model and positive drug groups, and there was no statistical difference (p>0.05).

Through the above embodiments, the object of the present invention has been fully achieved. Those skilled in the art will appreciate that the present invention includes, but is not limited to, the drawings and the details described in the Detailed Description. Any modifications that do not depart from the functional and structural principles of the invention are intended to be included within the scope of the appended claims.

Claims (5)

1. Recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) is used in the preparation of skin tissue aging and trauma drugs.
2. Skin aging according to claim 1 wherein the symptoms include skin aging caused by endogenous and exogenous stimuli.
3. The skin wound according to claim 1, wherein the symptom comprises skin trauma caused by external force, exercise, postoperative or the like.
4. The medicament according to claim 1, characterized in that the core component of the pharmaceutical preparation consists of the fusion protein of claim 1 and optionally a pharmaceutically acceptable adjuvant.
5. The pharmaceutical preparation according to claim 1 is administered orally and parenterally, wherein oral administration includes oral liquids, tablets, pills and capsules; parenteral administration includes external preparations and injections.

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