Application of recombinant human sDR5-Fc fusion protein in preparation of medicine for preventing and / or treating radioactive skin injury

Abstract

The invention discloses an application of a recombinant human sDR5-Fc fusion protein in preparation of a medicine for preventing and / or treating radioactive skin injury. The recombinant human sDR5-Fc fusion protein has a very good protection effect on ionizing radiation skin injury, and the expression of TRAIL and TRAIL-R receptors in skin tissues after gamma ray irradiation is up-regulated; after the ionizing radiation, the expression of the secreted TRAIL on the surface of the human keratinocyte HaCat and the receptors TRAIL-R1 and TRAIL-R2 of the human keratinocyte HaCat is increased. Ionizing radiation induces HaCat cell apoptosis and inhibits proliferation thereof in a time and dose dependent manner, which can be blocked by the TRAIL blocking protein sDR5-Fc portion. In addition, the sDR5-Fc can relieve radioactive skin injury of mice. Therefore, the sDR5-Fc fusion protein has a good application prospect in prevention and / or treatment of radioactive skin injury caused by ionizing radiation.

Description

Technical Field

[0001] The present invention relates to the field of radiation protection protein drugs and medical technology, and in particular to the use of a recombinant human sDR5-Fc fusion protein in the preparation of a drug for preventing and / or treating radiation skin damage. Background Art

[0002] The skin is the outermost organ that protects the body from harmful effects such as mechanical damage and ionizing radiation. Ionizing radiation-induced skin damage, leading to radiation dermatitis and fibrosis, is widely recognized in the medical, military, and industrial fields as a significant challenge. At the molecular level, exposure to ionizing radiation upregulates cell death, cytokines, and fibro-inflammatory pathways, which can persist for a long time, causing skin damage. The main radiation-induced skin damage is caused by X-rays, beta rays, and gamma rays. X-rays and gamma rays have strong penetrating power, damaging not only the skin but also subcutaneous tissue and even bone, sometimes causing ulcers that do not heal for a long time. Ionizing radiation-induced skin injury can be acute or chronic. Acute skin injury is the result of a combination of reduced functional stem cells, changes in skin endothelial cells, inflammation, and skin cell necrosis. Chronic skin injury can persist for years, is difficult to heal, and often develops into fibrosis in the late stages.

[0003] Currently, drug treatments for radiation-induced skin damage mainly focus on antioxidants, antibacterial and anti-infection, growth factor supplementation, and provision of a moist healing environment.

[0004] (1) Antioxidants: These drugs include superoxide dismutase liquid dressing and superoxide dismutase. They are relatively rarely used in clinical practice and are mostly used to prevent radiation-induced skin damage.

[0005] (2) Supplementation of growth factors: Supplementation of growth factors can promote the synthesis of DNA, RNA and hydroxyproline in the process of skin and mucosal wound tissue repair, induce epithelial cell proliferation and migration, and promote angiogenesis and wound healing.

[0006] (3) Antibacterial and anti-infection: Microbial infection is an important factor affecting wound healing, and antibacterial treatment is a means to promote wound healing. Broad-spectrum antibiotics such as compound sulfamethoxazole tablets (Baiyanjing), non-antibiotic bactericidal / antibacterial agents such as Baikerui disinfectant spray, and physical sterilization drugs such as silver ion dressings have all achieved certain therapeutic effects.

[0007] (4) Drugs that promote healing by creating a moist environment: Representative examples include dressings and gel-type drugs. The drugs themselves do not have therapeutic effects. They mainly promote wound healing by creating a moist environment on the wound surface, forming a protective film, and isolating pollution sources.

[0008] These drugs have certain therapeutic effects on radiation-induced skin damage, but there are fewer types of drugs, most of them are expensive, the efficacy is relatively limited, and there is less attention paid to the patient's local and systemic symptoms.

[0009] The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) system, which consists of the ligand TRAIL and five different membrane receptors in the human body, has the ability to kill tumor cells. TRAIL is a key mediator of apoptosis and inflammation. Binding to its receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) induces caspase activation and ultimately apoptosis. In addition to inducing cell death, binding to its receptors TRAIL-R1, TRAIL-R2, and TRAIL-R4 also induces an inflammatory response. The TRAIL system plays a crucial role in viral, drug-induced, autoimmune, metabolic, and other liver diseases. Soluble DR5 (sDR5) exists naturally in the human body but lacks transmembrane and intracellular domains and is not expressed on the cell membrane. sDR5 exerts its effects by inhibiting TRAIL; however, its small size, instability, and short half-life limit its use as a drug. To enhance the activity, stability, and half-life of sDR5, an sDR5-Fc fusion protein was constructed. Recent studies have demonstrated that sDR5-Fc has multiple pharmacological effects, such as effective treatment of autoimmune diseases, drug-induced hepatitis, liver failure, improvement of myocardial ischemia, acute ulcerative colitis, etc. However, the prior art has not reported that it can be directly applied to the skin and is associated with skin damage caused by ionizing radiation. Summary of the Invention

[0010] To address the issues raised in the aforementioned background art, the present invention aims to provide a recombinant human sDR5-Fc fusion protein for use in the preparation of a medicament for preventing and / or treating radiation-induced skin damage. This recombinant human sDR5-Fc fusion protein can prevent and / or treat radiation-induced skin damage by inhibiting ionizing radiation-induced cell apoptosis and inflammatory responses. The present invention creatively applies the recombinant human sDR5-Fc fusion protein to prevent and treat radiation-induced skin damage, specifically alleviating radiation-induced skin damage, reducing skin damage scores, thinning the epidermis, increasing hair follicles, and reducing collagen fibers.

[0011] In order to achieve the above objectives, the technical solutions adopted by the present invention are as follows: On the one hand, the present invention provides a use of a recombinant human sDR5-Fc fusion protein in the preparation of a drug for preventing and / or treating radiation-induced skin damage.

[0012] Furthermore, the ionizing radiation is beta rays, gamma rays, X rays and alpha rays.

[0013] Furthermore, radiation skin damage is skin atrophy, skin ulcers, skin collagen deposition, epidermal hyperplasia, increased hair follicles, decreased collagen fibers, dermal fibrosis and dermal lymphocyte and neutrophil infiltration caused by β rays, γ rays, X rays or α rays.

[0014] Furthermore, the prevention and / or treatment of ionizing radiation-induced inhibition or reduction of radiation-induced skin damage is the secretion of inflammatory factors and inflammatory cell infiltration in keratinocytes caused by beta rays, gamma rays, X rays or alpha rays.

[0015] Furthermore, the ionizing radiation induces high expression of TRAIL and TRAIL-R2 in skin tissue and HaCat cells, thereby inducing epidermal cell apoptosis.

[0016] Furthermore, the radiation skin damage induced by the ionizing radiation is irradiation dose-dependent damage.

[0017] Furthermore, the recombinant human sDR5-Fc fusion protein blocks or closes the TRAIL signaling pathway to block cell apoptosis induced by ionizing radiation and prevents radiation-induced skin damage.

[0018] Furthermore, the recombinant human sDR5-Fc fusion protein blocks TRAIL-induced epidermal cell apoptosis by inhibiting the TRAIL signaling pathway.

[0019] Furthermore, the recombinant human sDR5-Fc fusion protein blocks TRAIL-induced epidermal cell proliferation arrest by inhibiting the TRAIL signaling pathway.

[0020] Furthermore, the recombinant human sDR5-Fc fusion protein reduces the secretion of inflammatory factors by reducing epidermal cell proliferation and inflammatory cell infiltration, thereby alleviating radiation-induced skin atrophy, skin ulcers, epidermal hyperplasia and dermal fibrosis.

[0021] Furthermore, the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0022] Furthermore, the drug is a transdermal preparation, and furthermore, a transdermal microneedle, patch, cataplasm, ointment, gel, cream, latex or spray.

[0023] In another aspect, the present invention provides a method for preventing and / or treating radiation-induced skin damage, comprising the step of applying a recombinant human sDR5-Fc fusion protein to the skin.

[0024] Furthermore, the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0025] Furthermore, the ionizing radiation is beta rays, gamma rays, X rays and alpha rays.

[0026] Furthermore, radiation skin damage refers to skin atrophy, skin ulcers, skin collagen deposition, epidermal hyperplasia, dermal fibrosis and dermal lymphocyte and neutrophil infiltration caused by β rays, γ rays, X rays or α rays.

[0027] In another aspect, the present invention provides a use of a recombinant human sDR5-Fc fusion protein in the preparation of a drug for blocking apoptosis of skin tissue cells induced by ionizing radiation.

[0028] Furthermore, the skin tissue cells are dermal cells.

[0029] Furthermore, the recombinant human sDR5-Fc fusion protein blocks epidermal cell apoptosis and inflammatory response induced by ionizing radiation by blocking or closing the TRAIL signaling pathway.

[0030] Furthermore, the recombinant human sDR5-Fc fusion protein blocks TRAIL-induced epidermal cell apoptosis and inflammatory response by inhibiting the TRAIL signaling pathway.

[0031] Furthermore, the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0032] In another aspect, the present invention provides a use of a recombinant human sDR5-Fc fusion protein in the preparation of a medicament for inhibiting an inflammatory response in skin tissue or cells induced by ionizing radiation or inhibiting the expression of inflammatory cytokines in skin tissue or cells induced by ionizing radiation.

[0033] Furthermore, the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0034] Furthermore, the ionizing radiation is beta rays, gamma rays, X rays and alpha rays.

[0035] Furthermore, the inflammatory cytokine is selected from IL-6. In another aspect, the present invention provides a pharmaceutical composition for preventing or treating skin damage caused by ionizing radiation, wherein the pharmaceutical composition comprises a recombinant human sDR5-Fc fusion protein.

[0036] Furthermore, the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0037] Furthermore, the recombinant human sDR5-Fc fusion protein is the only active ingredient.

[0038] Furthermore, the pharmaceutical composition also contains a Caspase inhibitor and an anti-TRAIL neutralizing antibody.

[0039] Furthermore, the caspase inhibitor is zVAD-FMK.

[0040] The beneficial effects of the present invention are:

[0041] This invention is the first to discover that ionizing radiation leads to an upregulation of TRAIL-DR5 protein expression in normal epidermal tissue cells and demonstrates that the TRAIL-DR5-mediated apoptosis signaling pathway plays a key role in ionizing radiation-induced apoptosis in normal tissue cells. This invention also proposes the use of a recombinant human sDR5-Fc fusion protein in the preparation of a drug for the prevention and / or treatment of radiation-induced skin damage. The recombinant human sDR5-Fc fusion protein targets the TRAIL-DR5 system and inhibits the TRAIL-DR5 apoptosis signaling pathway, thereby suppressing the excessive apoptosis and inflammatory response of normal tissue cells caused by ionizing radiation. Therefore, it is effective for the prevention and / or treatment of radiation-induced skin damage.

[0042] Recombinant human sDR5-Fc fusion protein has a strong protective effect against radiation-induced skin damage. This is demonstrated by upregulation of TRAIL and its TRAIL-R receptor expression in mouse skin tissue following 30 Gy of gamma irradiation. Furthermore, irradiation significantly increases the expression of secreted TRAIL (sTRAIL) and its receptors TRAIL-R1 and TRAIL-R2 on the surface of human keratinocytes (HaCat cells). TRAIL induces apoptosis and inhibits proliferation of HaCat cells in a dose-dependent manner in vitro. This effect can be partially blocked by the TRAIL-blocking protein sDR5-Fc. Furthermore, sDR5-Fc can alleviate radiation-induced skin damage in mice, significantly reducing skin lesion scores, thinning the epidermis, increasing hair follicles, and reducing collagen fibers. Furthermore, because sDR5 is a human protein, it has the advantages of low toxicity and non-immunogenicity. Therefore, the sDR5-Fc fusion protein holds great promise for the prevention and / or treatment of radiation-induced skin damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The following table shows the expression of TRAIL in human HaCat cells before and after ionizing radiation in Example 1 of the present invention. A: Surface expression of TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 in human HaCat cells; B: Secreted TRAIL (sTRAIL) levels in the culture supernatant of human HaCat cells (pg / mL); C: Total and surface expression levels of TRAIL in human HaCat cells.

[0044] Figure 2 This is Example 2 of the present invention, showing TRAIL-induced apoptosis and proliferation arrest in HaCat cells. A and C: Analysis of HaCat cell apoptosis following treatment with TRAIL, zVAD-FMK, sDR5-Fc, and anti-TRAIL Ab, either alone or in combination. B: TRAIL-induced apoptosis in HaCat cells. D: MTS analysis of the cytotoxic activity of TRAIL in HaCat cells. E: Effect of sDR5-Fc on TRAIL-induced cytotoxic activity.

[0045] Figure 3 In Example 3 of the present invention, ionizing radiation promotes apoptosis and inhibits cell proliferation in HaCat cells. A: Ionizing radiation inhibits HaCat cell proliferation. B and C: Increasing the ionizing radiation dose induces apoptosis in HaCat cells through direct and bypass effects. D: Effect of 15 Gy irradiation on the expression of Caspase-3 and Caspase-8 proteins in HaCat cells.

[0046] Figure 4The effects of sDR5-Fc on ionizing radiation-induced HaCat cell damage in Example 3 of the present invention are shown. A: sDR5-Fc and zVAD-fmk reduce ionizing radiation-induced HaCat cell apoptosis through direct and bypass effects. B: Effect of sDR5-Fc on caspase-3 expression in ionizing radiation-induced HaCat cells within 48 hours. C: sDR5-Fc improves HaCat cell viability after ionizing radiation treatment. D: sDR5-Fc increases total cellular protein after ionizing radiation exposure. E: sDR5-Fc reduces IL-6 levels in cell cultures after ionizing radiation exposure.

[0047] Figure 5 This is an illustration of the improvement of radiation-induced skin damage by sDR5-Fc in Example 4 of the present invention. A: Representative skin images of each group on days 7, 14, and 21 after irradiation. B: Representative hematoxylin-eosin (H&E) and Masson staining of mouse skin tissue. C: Radiation-induced skin damage scores of each group on days 1, 3, 7, 14, and 21 after ionizing radiation. D: Quantification of average epidermal height, dermal height, and hair follicle density of each group on days 7 and 21.

[0048] Figure 6 This is Example 4 of the present invention, demonstrating that sDR5-Fc alleviates ionizing radiation-induced skin cell apoptosis on days 3 and 7. A: Cellular immunoassay of apoptosis (TUNEL+, green) in skin tissue from each group. B: Number of TUNEL+ cells in skin tissue from each group on day 3. C: Number of TUNEL+ cells in skin tissue from each group on day 7. Five cases per group. Three fields of view were randomly selected for each scan. DETAILED DESCRIPTION

[0049] The term "TRAIL (TNF-related apoptosis-inducing ligand)" as used herein refers to the tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10), a member of the tumor necrosis factor superfamily. It is expressed on activated T cells and other cells. Five receptors have been identified in humans: TRAIL-R1 (DR4), TRAIL-R2 (DR5), TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), and OPG. TRAIL binding to DR4 / DR5 (containing a death domain in the cytoplasmic region) induces apoptosis. However, TRAIL binding to DcR1 / DcR2 (lacking a cytoplasmic region or an incomplete death domain) fails to transmit apoptotic signals and inhibits TRAIL-induced apoptosis in DR4 / DR5-positive cells. The term "sDR5 (soluble DR5)" as used herein refers to the soluble form of DR5 that lacks the transmembrane region. Due to this lack of a transmembrane region, it is not expressed on the cell membrane and is secreted extracellularly. sDR5 is expressed at low levels in normal human peripheral blood. While sDR5 retains its ability to bind to TRAIL ligands, it cannot transmit apoptotic signals into cells, thereby blocking TRAIL-DR5-mediated apoptotic responses. The term "recombinant human sDR5-Fc fusion protein" herein refers to a recombinant fusion protein derived from human sDR5 and the Fc region of IgG1 through genetic recombination.

[0050] The present invention is described in detail below with reference to specific embodiments and accompanying drawings.

[0051] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and description are to be considered as illustrative in nature and not restrictive.

[0052] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0053] The methods for obtaining the various biological materials described in the examples merely provide experimental methods for achieving the disclosed objectives and should not be construed as limiting the sources of the biological materials used in the present invention. In fact, the sources of the biological materials used are diverse, and any legally and ethically accessible biological material may be substituted for and used as indicated in the examples.

[0054] The experimental animals, reagents, and instruments used in the following examples and their sources:

[0055] 1) Experimental animals

[0056] C578BL / 6N male mice, 6–8 weeks old, weighing 18–22 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (animal quality certificate number: SCXK-(Jing)2016-00001). Five mice were housed in a constant temperature of 25 ± 1°C with alternating cycles of 12 h light and 12 h dark for 1 week.

[0057] 2) Reagents and instruments

[0058] sDR5-Fc fusion protein: SEQ ID NO: 4, test code AS1501, 25 mg / vial, from Shenzhen Zhongke Aishen Pharmaceutical Co., Ltd.;

[0059] Rabbit polyclonal antibodies to Caspase-3 and Caspase-8 were purchased from Abcam;

[0060] Mouse monoclonal antibodies GAPDH and β-Actin were purchased from Beijing Pulilai Gene Technology Co., Ltd.;

[0061] Recombinant human TRAIL protein was purchased from Abcam;

[0062] Annexin V-FITC / PI Apoptosis Kit, purchased from BD Biosciences, USA;

[0063] Actinomycin D was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0064] TRAIL-R1-PE / CY7, TRAIL-R2-APC, TRAIL-R3-PE, TRAIL-4-PE, TRAIL-PE, Fas-L-PE /

[0065] CY7 and Fas-Percp / cy5.5 antibodies were purchased from Biolegend;

[0066] HaCat cells: donated by Professor Gao Yue's laboratory at the Academy of Military Medical Sciences;

[0067] Microplate reader: Purchased from Molecular Devices, USA.

[0068] Fusion protein:

[0069] SEQ ID NO.1:

[0070] ITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEGSSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0071] SEQ ID NO.2:

[0072] ITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0073] SEQ ID NO.3:

[0074] AAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0075] SEQ ID NO.4:

[0076] SSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0077] Example 1: Effects of ionizing radiation on TRAIL expression in human HaCat cells

[0078] In this example, HaCat cells were seeded into 6-well or 96-well plates in DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin and cultured in a 5% CO2, 37°C incubator. When the cells reached a confluence of 70%-80%, they were exposed to 60Co gamma irradiation at three different intensities: 5, 10, and 15 Gy, with a dose rate of 55.06 cCy / min and irradiation durations of 9, 18, and 27 minutes, respectively. The unirradiated group served as the normal control.

[0079] In this embodiment, the results are as follows Figure 1 A. Figure 1 B and Figure 1 As shown in Figure C, 48 hours after irradiation with 5, 10, and 15 Gy of gamma rays, the expression of TRAIL-R1 and TRAIL-R2 in HaCat cells was significantly upregulated. TRAIL-R2 expression increased with increasing irradiation dose, but TRAIL-R1 expression did not differ significantly between different irradiation doses. TRAIL-R3 and TRAIL-R4 were not expressed on the surface of HaCat cells, and ionizing radiation had no effect on their expression. Furthermore, we found that endogenous TRAIL protein was consistently expressed at high levels in HaCat cells and was unaffected by ionizing radiation. Expression of cell membrane-bound TRAIL protein was minimal but increased in a dose-dependent manner. Furthermore, the level of secreted TRAIL (sTRAIL) in the culture supernatant was found to be 1.5-fold higher than that in the control group 48 hours after irradiation. These data indicate that ionizing radiation significantly upregulates the expression of TRAIL, TRAIL-R1, and TRAIL-R2 in HaCat cells, with the greatest effect on TRAIL-R2 expression.

[0080] Example 2: TRAIL induces apoptosis and inhibits proliferation of HaCat cells

[0081] In this example, HaCat cells were trypsinized and diluted to 1×10 5 / mL, inoculated in 96-well plates, 100μL per well. Cells were treated with exogenous recombinant TRAIL (15-60ng / mL) alone or in combination with cycloheximide (CHX), where CHX is a cell apoptosis inducer and serves as a positive control in this experiment. TRAIL alone induced apoptosis in HaCat cells in a dose-dependent manner; while the combination of TRAIL and CHX significantly accelerated apoptosis. TRAIL alone (30ng / mL) induced apoptosis in HaCat cells at a rate of 46.75% + 1.36% ( Figure 2 B) 30 ng / mL TRAIL was selected for subsequent experiments. In the TRAIL-induced HaCat cell apoptosis assay, the pan-caspase inhibitor zVAD-FMK, the TRAIL blocker sDR5-Fc, and the anti-TRAIL neutralizing antibody anti-TRAIL were added, respectively. The results showed that TRAIL-induced HaCat cell apoptosis was effectively blocked. Figure 2 As shown in A and 2C.

[0082] In addition, TRAIL effectively inhibits the proliferation of HaCat. In this example, TRAIL diluted with 0.5 μg / mL actinomycin D (an inhibitor of RNA transcription and replication) was added to the above 96-well plate, 200 μL per well, and each concentration was repeated three times. The cells were placed in a cell culture incubator for 20 hours, and then 20 μL of MTS solution was added to each well. After incubation for 3 hours under standard cell culture conditions, the absorbance was immediately measured at 490 nm using a microplate reader. The results showed a dose-dependent blocking effect on HaCat cell proliferation ( Figure 2 D), with EC50 and EC90 values of 0.62 ng / mL and 6.26 ng / mL, respectively. In the sDR5-Fc neutralization assay, HaCat cells were seeded into 96-well plates and incubated as described above. sDR5-Fc was prepared by diluting 2-fold, and each diluted sample was mixed with an equal volume of recombinant human TRAIL protein and 0.5 μ / mL actinomycin D. After 20 hours of incubation, MTS was added, incubated for 3 hours, and the absorbance was measured as described above. The results showed that sDR5-Fc treatment had a dose-dependent inhibitory effect on TRAIL-induced proliferation inhibition ( Figure 2 E), and the EC50 value was 0.85 ng / mL.

[0083] Example 3: sDR5-Fc and zVAD-fmk can prevent ionizing radiation-induced HaCat damage

[0084] In this example, HaCat cells were exposed to ionizing radiation at doses of 5, 10, and 15 Gy. HaCat cell apoptosis was assessed using the Annexin V-FITC / PI Cell Apoptosis Detection Kit 24, 48, and 72 hours after ionizing radiation. Cells were trypsinized at 1000 rpm for 5 minutes and collected into EP tubes. The cells were resuspended in 1× Annexin V Binding Buffer, and 5 μL of Annexin V-FITC and 5 μL of PI reagent were added at room temperature. The tubes were then incubated in the dark for 15 minutes, and 400 μL of diluted 1× Annexin V Binding Buffer was added to each tube. Cell apoptosis was assessed by flow cytometry within 1 hour.

[0085] Figure 3 A shows that the activity of HaCat cells is gradually inhibited as the irradiation dose increases; Figure 3 B and C show that the apoptosis rate of HaCat cells gradually increased after escalating treatment with γ-irradiation (5–15 Gy) for 24–72 h; Figure 3 D shows that the expression of cleaved caspase-3 (17 kDa) increased after 15 Gy irradiation, and increased by 2.44 ± 0.15 times compared with the control group at 72 h.

[0086] Next, sDR5-Fc (50 μg / mL), anti-TRAIL antibody (neutralizing antibody, 1 μg / mL), and zVAD-fmk were added to the culture medium 1 hour before exposure to 15 Gy irradiation. Figure 4 Results (A and C) showed increased cell proliferation and partial inhibition of radiation-induced apoptosis. Figure 4 B shows that sDR5-Fc prevents radiation-induced caspase-3 cleavage.

[0087] In order to determine whether the molecules secreted in the cell supernatant after irradiation have a pro-apoptotic effect on normal HaCat cells, the supernatant of HaCat cells directly irradiated for 24 hours was added to normal HaCat cells and cultured for 48 hours, and the apoptosis rate of each group of cells was measured. Figure 3 As shown in B and 3C, the apoptosis rate was relatively low, but in the 15Gy high-dose transfer group, apoptosis increased, which was 1.35 times higher than that in the non-irradiated medium transfer group. This suggests that ionizing radiation can induce apoptosis in HaCat cells through bystander effects. In another set of experimental results, Figure 4 A shows that the apoptosis rate of normal HaCat cells treated with the supernatant of the 15Gy+sDR5-Fc and 15Gy+zVAD-fmk groups was lower than that of normal HaCat cells treated with the supernatant of 15Gy. This indicates that both sDR5-Fc and zVAD-fmk can inhibit apoptosis of HaCat cells after irradiation, and that sDR5-Fc or zVAD-fmk in the culture supernatant partially blocks sTRAIL-mediated extrinsic apoptosis.

[0088] After different time periods of ionizing radiation, the cell supernatants of each HaCat cell group were collected and centrifuged. The ELISA kit was used to quantitatively determine the content of sTRAIL and interleukin-6 (IL-6). The absorbance of each well was measured at OD450 nm using an enzyme-linked immunosorbent assay. The concentrations of sTRAIL and IL-6 were calculated from the standard curve and expressed in pg / mL. The results are shown in Table 1. Figure 4 As shown in Figure E, ionizing radiation promoted IL-6 expression in HaCat cells, while the sDR5-Fc group inhibited IL-6 expression 24 and 72 hours after irradiation. In addition to IL-6, the expression of TNF-α and IL-1β, both of which are associated with radiation-induced skin damage, was observed in the cell supernatants of each group. The concentrations of both factors were below the detection limit. This data demonstrates that sDR5-Fc reduced ionizing radiation-induced IL-6 expression in HaCat cells, suggesting that blocking the TRAIL signaling pathway can ameliorate the inflammatory response to ionizing radiation.

[0089] Example 4: sDR5-Fc can alleviate ionizing radiation-induced skin damage in mice

[0090] In this example, mice were locally irradiated with 60Coγ rays to observe the degree of damage to the skin of mice in each group caused by ionizing radiation.

[0091] After acclimation, C578BL / 6N mice were randomly divided into a blank control group, a saline control group, and an sDR5-Fc treatment group. In the saline control group, mice were anesthetized and fixed to a glass frame with a clamp. The buttocks were shaved and covered with a lead plate. The buttocks were subjected to a single local irradiation. After irradiation, a cotton swab dipped in saline solution was applied to the perforated skin area. In the sDR5-Fc treatment group, mice were anesthetized and fixed to a glass frame with a clamp. The buttocks were shaved and covered with a lead plate. The buttocks were subjected to a single local irradiation. After irradiation, a cotton swab dipped in 10 mg / kg sDR5-Fc solution was applied to the perforated skin area. In the blank control group, mice were anesthetized and fixed to a glass frame with a clamp. The buttocks were shaved and covered with a lead plate. The buttocks were subjected to a single local irradiation. After irradiation, a cotton swab dipped in 10 mg / kg sDR5-Fc solution was applied to the perforated skin area. In the blank control group, mice were anesthetized and fixed to a glass frame with a clamp. The buttocks were shaved, but no irradiation was performed. The mice then resumed their normal diet.

[0092] Irradiation dose: Both the mice in the irradiation group and the sDR5-Fc administration irradiation group received a single local irradiation of the buttocks with 60Coγ rays, with an irradiation dose of 30Gy, an irradiation dose rate of 125.72cGy / min, and an irradiation time of 23min.

[0093] Sample Collection: 2, 48, and 96 hours after irradiation, the buttocks skin of mice in the irradiated and sDR5-Fc-treated groups was perforated with a 0.75 mm disposable medical skin roller. A cotton swab dipped in saline solution (saline control group) or a 10 mg / kg sDR5-Fc solution (sDR5-Fc-treated group) was applied to the perforated skin area. Skin lesion scores were recorded 1, 3, 5, 7, 14, and 21 days after irradiation. Five animals per group were sacrificed on days 3, 7, and 21 after irradiation, and skin tissue from the irradiated area was collected for histopathological examination.

[0094] Injury score: The skin injury score adopts a semi-quantitative scoring system, with 1 point (no injury), 1.5 points (mild erythema, mild dryness), 2.0 points (moderate erythema, dryness), 2.5 points (obvious erythema, dry desquamation), 3 points (dry desquamation, mild dry scab), 3.5 points (dry desquamation, dry scab, mild epidermal spot scar), 4.0 points (patchy wet desquamation, moderate scar), 4.5 points (large confluent wet desquamation, ulcer, large), 5 points (open wound, full-thickness skin loss), and 5.5 points (necrosis).

[0095] Immunocytochemistry, Histology, TUNEL, and Image Analysis: Skin biopsies were collected from the irradiated area on days 3, 7, and 21 after irradiation. The tissues were fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin, and sectioned for hematoxylin and eosin (H&E), Masson's staining, and TUNEL staining. Immunohistochemistry was performed using antibodies against TRAIL and TRAIL-R2. Pathological scoring was performed based on H&E staining results and indicators such as epidermal and dermal thickness and hair follicle number. Masson's staining, TUNEL staining, and immunohistochemical analysis were used to quantify collagen deposition, apoptosis rate, and mean fluorescence intensity of TRAIL and TRAIL-R2 using Image J software.

[0096] Statistical analysis: Data are expressed as mean ± standard deviation (SD) and analyzed using SPSS 20.0 statistical software. Multiple comparisons were performed using one-way analysis of variance followed by the Student-Newman-Keuls test.

[0097] In this example, C57BL / 6N mice were irradiated with a single dose of 30 Gy on their buttocks and then injected with sDR5-Fc. The changes in radiation skin damage scores of each group of mice were recorded 1, 3, 7, 14, and 21 days after irradiation. Figure 5 As shown in A, on the 14th day after irradiation, the skin damage in the irradiation control group was the most serious. Figure 5 As shown in C, the sDR5-Fc administration group effectively alleviated the skin damage caused by ionizing radiation on days 7, 14, and 21. Figure 5 As shown in B, compared with the normal control group, the irradiated control group showed moderate epidermal thickening (black arrows), lymphocyte and neutrophil infiltration (green arrows); while the sDR5-Fc treatment group significantly reduced epidermal hyperplasia and inflammatory cell infiltration. In addition, Masson staining results showed that Figure 5 As shown in D, the sDR5-Fc administration group can also alleviate radiation-induced skin atrophy. On the 21st day after irradiation, the dermal tissue fibrosis of the irradiation control group was obvious, while the sDR5-Fc microneedle administration group reduced skin collagen deposition and fibrosis. TUNEL staining results are shown in Figure 6 Figures A, 6B, and 6C show that the sDR5-Fc-treated group reduced skin cell apoptosis compared to the irradiated control group on days 3 and 7 after irradiation. Seven days after irradiation, the number of hair follicles in the skin decreased, and sDR5-Fc may exert a protective effect by partially inhibiting the TRAIL signaling pathway. These results demonstrate that sDR5-Fc, as a TRAIL blocker, effectively alleviates skin ulceration, epidermal hyperplasia, and dermal fibrosis on the buttocks of mice following local irradiation.

[0098] From the results of the above examples, it can be seen that the TRAIL signaling pathway is involved in the pathogenesis of radiation-induced skin damage, and the TRAIL blocker sDR5-Fc can reduce the incidence of radiation-induced skin damage in vivo and in vitro. Based on the results that the sDR5-Fc fusion protein can effectively reduce the cell apoptosis rate, it is proved that the TRAIL-DR5-mediated cell apoptosis signaling pathway plays an important role in ionizing radiation-induced cell apoptosis. Based on this, the inventors creatively used the sDR5-Fc fusion protein for the prevention and / or treatment of radiation-induced skin damage caused by ionizing radiation. By utilizing the principle that the sDR5-Fc fusion protein can block the TRAIL-DR5-mediated cell apoptosis signaling pathway, it effectively protects normal tissue cells from the effects of radiation during ionizing radiation and promotes the recovery of cells in normal tissues after ionizing radiation damage. Specifically, it can significantly reduce skin ulceration, epidermal hyperplasia and dermal fibrosis after ionizing radiation damage, and promote its rapid recovery to normal state. Therefore, sDR5-Fc fusion protein has good application prospects in the prevention and / or treatment of radiation-induced skin damage caused by ionizing radiation, and provides a new method for the treatment of radiation-induced skin damage.

[0099] The sDR5-Fc fusion proteins with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 have similar effects to the sDR5-Fc fusion protein with amino acid sequence as shown in SEQ ID NO: 4.

[0100] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Use of a recombinant human sDR5-Fc fusion protein in the preparation of a medicament for preventing and / or treating radiation-induced skin damage; Preferably, the ionizing radiation is beta rays, gamma rays, X rays and alpha rays.

2. The use according to claim 1, wherein radiation skin damage is skin atrophy, skin ulcers, skin collagen deposition, epidermal hyperplasia, increased hair follicles, decreased collagen fibers, dermal fibrosis, and dermal lymphocyte and neutrophil infiltration caused by β-rays, γ-rays, X-rays, or α-rays; More preferably, the prevention and / or treatment of ionizing radiation-induced inhibition or reduction of radiation-induced skin damage is the secretion of inflammatory factors and inflammatory cell infiltration in keratinocytes caused by β rays, γ rays, X rays or α rays.

3. The use according to claim 1, wherein the recombinant human sDR5-Fc fusion protein blocks ionizing radiation-induced cell apoptosis by blocking or occluding the TRAIL signaling pathway to prevent ionizing radiation-induced skin damage; Preferably, the recombinant human sDR5-Fc fusion protein blocks TRAIL-induced epidermal cell apoptosis by inhibiting the TRAIL signaling pathway; Preferably, the recombinant human sDR5-Fc fusion protein blocks TRAIL-induced epidermal cell proliferation arrest by inhibiting the TRAIL signaling pathway; Preferably, the recombinant human sDR5-Fc fusion protein reduces the secretion of inflammatory factors by reducing epidermal cell proliferation and inflammatory cell infiltration, thereby alleviating radiation-induced skin atrophy, skin ulcers, epidermal hyperplasia and dermal fibrosis.

4. The use according to claim 1, wherein the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

5. The use according to claim 1, wherein the drug is a transdermal preparation; Preferably, the transdermal administration preparation is a transdermal administration microneedle, patch, cataplasm, ointment, gel, cream, latex or spray.

6. Use of a recombinant human sDR5-Fc fusion protein in the preparation of a drug for blocking apoptosis of skin tissue cells induced by ionizing radiation; Preferably, the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

7. The use according to claim 6, wherein the skin tissue cells are dermal cells; Preferably, the recombinant human sDR5-Fc fusion protein blocks epidermal cell apoptosis and inflammatory response induced by ionizing radiation by blocking or closing the TRAIL signaling pathway; Preferably, the recombinant human sDR5-Fc fusion protein blocks TRAIL-induced epidermal cell apoptosis and inflammatory response by inhibiting the TRAIL signaling pathway.

8. Use of a recombinant human sDR5-Fc fusion protein in the preparation of a medicament for inhibiting an inflammatory response in skin tissue or cells induced by ionizing radiation or inhibiting the expression of inflammatory cytokines in skin tissue or cells induced by ionizing radiation; Preferably, the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; Preferably, the ionizing radiation is beta rays, gamma rays, X rays and alpha rays; Preferably, the inflammatory cytokine is selected from IL-6.

9. A pharmaceutical composition for preventing or treating skin damage caused by ionizing radiation, comprising a recombinant human sDR5-Fc fusion protein; the pharmaceutical composition is a transdermal formulation, preferably a transdermal microneedle, patch, cataplasm, ointment, gel, cream, latex, or spray; Preferably, the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; Preferably, the recombinant human sDR5-Fc fusion protein is the only active ingredient; Preferably, the pharmaceutical composition further comprises a Caspase inhibitor and an anti-TRAIL neutralizing antibody; More preferably, the caspase inhibitor is zVAD-FMK.

10. A method for preventing and / or treating radiation-induced skin damage, comprising the step of applying a recombinant human sDR5-Fc fusion protein to the skin; Preferably, the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; Preferably, the ionizing radiation is beta rays, gamma rays, X rays and alpha rays; Preferably, the radiation skin damage is skin atrophy, skin ulcer, skin collagen deposition, epidermal hyperplasia, dermal fibrosis and dermal lymphocyte and neutrophil infiltration caused by β rays, γ rays, X rays or α rays.

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