CN115068590B - New application of tumor necrosis factor related apoptosis inducing ligand mutant - Google Patents

Abstract

The present invention provides a new use of a tumor necrosis factor-related apoptosis-inducing ligand mutant TRAIL-Mu3. TRAIL-Mu3 has small toxic and side effects, can reduce lung wet weight, reduce lung collagen deposition, lung tissue inflammatory lesions, and lung tissue erosion; by promoting the expression of lung tissue DR5, it can induce the apoptosis of radiation-activated lung fibroblasts, effectively improve radiation-induced pulmonary fibrosis, and has excellent clinical application value.

Description

New application of a tumor necrosis factor-related apoptosis-inducing ligand mutant

Technical Field

The invention belongs to the field of biomedicine, and specifically relates to a new use of a tumor necrosis factor-related apoptosis-inducing ligand mutant.

Background Art

Thoracic malignancies are the most serious malignant tumors that threaten human life and affect the quality of life worldwide. Radiotherapy is one of the main treatments for thoracic tumors, but about 5% to 20% of patients will develop radiation-induced lung injury (RILI) during and after chest radiotherapy, and radiation-induced lung fibrosis (RILF) may occur 4-6 months after the end of radiotherapy. At present, there is still a lack of effective prevention and treatment for RILF. Clinically, adrenal glucocorticoids and fibrosis inhibitors (pirfenidone and nintedanib) are recommended to control the progression of RILF and relieve patient symptoms. However, the fundamental problem cannot be solved, and the long-term use of large amounts of glucocorticoids and fibrosis inhibitors will bring many side effects. Therefore, exploring safer and more effective treatment methods is of great significance for the prevention and treatment of RILF.

Current studies believe that RILF is closely related to immune response and inflammatory cytokines. Since lung tissue is relatively sensitive to radiation, after receiving a certain dose of radiation, bronchial alveolar epithelial and endothelial cells will be damaged, accompanied by the release of inflammatory factors. In addition, active immune inflammatory cells, alveolar epithelial cells and fibroblasts lead to high expression of transforming factor-β, promote the activation of fibroblasts into fibrocytes, lead to collagen deposition and then induce lung tissue fibrosis. Therefore, the key to preventing and treating RILF is to reduce lung damage caused by cytokine release and inhibit the fibrotic reaction caused by myofibroblast aggregation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily, also known as human apoptosis factor 2 ligand. Studies have found that it can induce apoptosis in a variety of tumor cells, while having little toxicity to normal cells. It has been recognized as a potential anti-tumor drug with great development and application prospects.

TRAIL-Mu3 is a TRAIL mutant, which has a mutated N-terminus to eight consecutive arginines (amino acids 114 to 122) based on the wild-type TRAIL soluble fragment. Compared with TRAIL, the yield of the target protein in fermentation is increased, and it is mainly expressed in the form of soluble protein, which improves the purification efficiency and is conducive to improving the quality of the finished drug. However, the effect of TRAIL-Mu3 on fibrotic diseases is still unclear, and its use in improving and treating radiation-induced pulmonary fibrosis has not been reported. Clarifying the role and mechanism of TRAIL-Mu3 in RILF lesions will be of great significance for the clinical treatment of RILF.

Summary of the invention

The purpose of the present invention is to provide a new use of a tumor necrosis factor-related apoptosis-inducing ligand mutant TRAIL-Mu3.

The present invention provides use of a tumor necrosis factor-related apoptosis-inducing ligand mutant TRAIL-Mu3 in preparing a medicine for treating radiation-induced pulmonary fibrosis.

Furthermore, the sequence of the above TRAIL-Mu3 is shown in SEQ ID NO:1.

Furthermore, the above-mentioned drug is a drug for reducing lung wet weight.

Furthermore, the above-mentioned medicine is a medicine for reducing lung tissue erosion.

Furthermore, the above-mentioned drug is a drug for reducing inflammatory lesions of lung tissue, preferably a drug for downregulating the expression of TNF-α, IL-6, and IL-13.

Furthermore, the above-mentioned drug is a drug for reducing lung collagen deposition.

Furthermore, the above-mentioned drug is a drug that inhibits collagen secretion of lung fibroblasts in lung tissue, and is preferably a drug that downregulates the expression of α-SMA.

Furthermore, the above-mentioned drug is a drug that induces apoptosis of lung fibroblasts, and is preferably a drug that upregulates DR5 expression.

The present invention also provides a medicine for treating radiation-induced pulmonary fibrosis, which is a medicine prepared by taking tumor necrosis factor-related apoptosis-inducing ligand mutant TRAIL-Mu3 as an active ingredient and adding pharmaceutically acceptable excipients.

Furthermore, the sequence of the above-mentioned tumor necrosis factor-related apoptosis-inducing ligand mutant TRAIL-Mu3 is shown in SEQ ID NO:1.

The experimental results show that TRAIL-Mu3 has small toxic and side effects, can reduce lung wet weight, reduce lung collagen deposition, lung tissue inflammatory lesions, and lung tissue erosions; by promoting the expression of DR5 in lung tissue, it can induce apoptosis of radiation-activated lung fibroblasts, effectively improve radiation-induced pulmonary fibrosis, and has excellent clinical application value.

The tumor necrosis factor-related apoptosis-inducing ligand mutant TRAIL-Mu3 described in the present invention is a truncated mutant obtained by mutating the amino acid sequence (VRERGPQR) of positions 114 to 121 of the wild-type tumor necrosis factor-related apoptosis-inducing ligand TRAIL to 8 consecutive arginines (RRRRRRRR), and has the sequence of SEQ ID.NO:1.

Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the pathological results of the radiation-induced pulmonary fibrosis model in C57BL/6 mice established by single radiotherapy of both lungs with 15 Gy. Scale bar = 100 μm.

Figure 2 shows the typical appearance and lung wet weight of each group of mice in the radiation-induced pulmonary fibrosis model

Figure 3 shows the micro-CT images of lung tissues of mice with radiation-induced pulmonary fibrosis at different time periods.

Figure 4 shows the lung tissue pathological results of radiation-induced pulmonary fibrosis mice in each group, scale bar = 100 μm

Figure 5 shows the lung tissue pathological scores of each group of mice with radiation-induced pulmonary fibrosis

Figure 6 shows the results of Masson staining and scoring of lung tissues in each group of mice with radiation-induced pulmonary fibrosis. Scale bar = 50 μm

Figure 7 shows the CVF values of lung tissues of mice with radiation-induced pulmonary fibrosis in each group stained with Masson staining

Figure 8 shows the results of α-SMA immunohistochemical staining and expression levels in lung tissues of mice with radiation-induced pulmonary fibrosis in each group, scale bar = 50 μm

Figure 9 shows the results of DR5 immunohistochemical staining and expression levels in lung tissues of mice with radiation-induced pulmonary fibrosis in each group, scale bar = 50 μm

Figure 10 shows the DR5 mRNA levels in lung tissues of mice with radiation-induced pulmonary fibrosis in each group

Figure 11 shows the DR5 and Caspase 3 protein levels in lung tissues of mice with radiation-induced pulmonary fibrosis in each group

Figure 12 shows the levels of serum IL-6, TNF-α, TGF-β1, and IL-13 in each group of mice with radiation-induced pulmonary fibrosis Figure 13 shows the changes in hair and body weight in each group of mice with radiation-induced pulmonary fibrosis

FIG. 14 shows the H&E staining results of the main organs of each group of radiation-induced pulmonary fibrosis mice, scale bar = 50 μm

DETAILED DESCRIPTION

The TRAIL-Mu3 of the present invention is prepared by referring to the method disclosed in Chinese Patent CN105555799B. Unless otherwise specified, other raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

Example 1: Drugs for treating radiation-induced pulmonary fibrosis according to the present invention

The preparation is prepared by taking tumor necrosis factor-related apoptosis-inducing ligand mutant TRAIL-Mu3 as the active ingredient and adding pharmaceutically acceptable excipients.

The beneficial effects of TRAIL-Mu3 of the present invention are demonstrated by experimental examples below.

Experimental Example 1: Establishment of RILF model

1. Experimental methods

Before modeling, prepare 1% sodium pentobarbital, sterile saline and a 1 mL sterile syringe.

(1) After C57BL/6 mice are transferred to the modeling environment and adapted, an appropriate amount of 1% sodium pentobarbital is injected intraperitoneally for anesthesia. The injection dose is 50 mg/kg. After the abdominal skin is disinfected, the needle is inserted into the right lower abdominal cavity at about 45 degrees and the depth of the needle is about 1 cm. A breakthrough feeling indicates that the syringe has entered the abdominal cavity. According to the weight fluctuation of the mouse, 90 to 110 μL is injected for each mouse of 18 to 20 g. After anesthesia, observe the state of the mouse to ensure that there is no overdose of anesthesia. Fix the mouse after it is completely anesthetized.

(2) The mouse was fixed in a supine position on a cardboard mold. The head, neck, abdomen, and limbs were blocked by lead blocks, leaving only the chest exposed. A 1.5-2 cm tissue compensation membrane was spread on the front chest of the mouse. The whole lung was irradiated with a single anterior field and a single shot of linear accelerator X-ray. The source-skin distance was 100 cm, the energy was 6 MV, the dose rate was 1.37 Gy/min, the irradiation field was 20×3 cm, and the prescription dose was 15 Gy/1f.

(3) After irradiation, carefully place the mice back into their cages and wait for them to wake up naturally. After waking up, transfer them back to the animal room for observation and subsequent experiments.

2. Experimental results

After the whole lung of C57BL/6 mice was irradiated with 15Gy radiation for 16 weeks, three mice were randomly selected and their lung tissues were taken for pathological section H&E staining to observe and evaluate the modeling effect. The results are shown in Figure 1. In the lung tissues of the three mice, there were many inflammatory cell infiltrations around blood vessels (yellow arrows); the alveolar wall epithelial cells decreased, and fibrinoid exudates and foam cells were seen in the extensive alveolar cavities (black arrows); the alveolar cavity structure was blurred and solidified, and some alveolar cavities were narrowed; strong eosinophilic agglutinates were seen locally (blue arrows). Among them, a large amount of fibrous exudates in the lungs were accompanied by the infiltration of multiple inflammatory cells and changes in the lung structure, especially the solidification of the alveoli, which all indicated that after 16 weeks of irradiation of the lungs of C57BL/6 mice with 15Gy X-rays, their lungs could form obvious pathological changes of pulmonary fibrosis.

The above results indicate that the present invention successfully established a mouse model of radiation-induced pulmonary fibrosis.

Experimental Example 2: Therapeutic effect of TRAIL-Mu3 on RILF in mice

1. Experimental methods

1.1 TRAIL-Mu3 treatment plan and experimental methods for mouse RILF

(1) Sixteen weeks after the whole lung of C57BL/6 mice was irradiated with a single 15Gy X-ray, mice with equivalent fibrosis grades were randomly divided into three groups by micro-CT scanning, with 10 mice in each group. Group 1 was the negative control group, which was given saline and was recorded as Con; Group 2 was the positive control group, which was given dexamethasone sodium phosphate injection 2mg/kg/mouse and was recorded as Dex; Group 3 was the treatment group, which was given TRAIL-Mu3 45mg/kg/mouse and was recorded as TRAIL-Mu3. The volume of the drug in the above three groups was 100μL/mouse, and the drug was given every other day for a total of 10 times for a total of 3 weeks, and micro-CT images were collected once a week. After the last dose, micro-CT images were collected the next day, and the mice were killed for anatomical observation, lung wet weight was weighed, and samples were collected and fixed for subsequent analysis.

1.2 Histopathological analysis

1.2.1 Tissue embedding, sectioning and hydration

(1) Soak fresh mouse lung tissue in 4% paraformaldehyde solution, fix for 48 hours, place in an embedding box, and rinse under running tap water overnight. Soak the tissue in 75% alcohol for 30 minutes, 85% alcohol for 30 minutes, 95% alcohol for 30 minutes twice, 100% alcohol for 20 minutes three times, xylene for 30 minutes twice, and finally soak in paraffin twice for a total of 1 hour. After soaking, place the embedding box in new wax for embedding.

(2) After embedding, place the cooled wax block on ice to cool completely before slicing on a microtome. The surface of the wax block can be trimmed before slicing. The slice thickness is about 3 to 5 μm. Then, bleach the slices and control the temperature at 42°C. After the tissue is fully unfolded, use a glass slide to spread the slices on a slicing machine at a temperature of 65°C. After the slicing is completed, dry the paraffin in a slicing machine and take out the slices for hydration.

(3) After baking the slices in a dryer for 2 h, take them out and soak them in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% alcohol for 5 min. Finally, rinse the slices in distilled water.

1.2.2 H&E staining

(1) Hematoxylin staining: The sections were stained in hematoxylin stain for 3 to 5 min, rinsed with tap water, differentiated with differentiation solution, rinsed with tap water, bluing solution, and rinsed with running water.

(2) Eosin staining: The sections were placed in 85% and 95% alcohol for dehydration for 5 min, and then placed in eosin staining solution for staining for 5 min.

(3) Dehydration and sealing: Soak the sections in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min, and finally seal the sections with neutral gum.

(3) Observation and photography: Use a microscope to perform microscopic examination and use an image acquisition system to collect and analyze images.

(4) Scoring of inflammatory response under microscopy after H&E staining of lung tissue: Two researchers randomly collected 5 visual fields under an Olympus CX41RF light microscope. Referring to previous literature reports, the inflammatory score needs to comprehensively consider the level of inflammatory cell infiltration in the alveolar cavity, alveolar wall thickening, and the proportion of interstitial edema in the lung tissue to score the degree of inflammatory lesions in the lung tissue of each group of mice. ≤10% was scored as 0-1, with 0 points; 11%-30% was scored as 2-4, with 1 point; 31%-50% was scored as 5-7, with 2 points; 51%-70% was scored as 8-9, with 3 points; >70% was scored as 10, with 4 points. The average score of the 5 visual fields was taken as the final score and recorded in order.

1.2.3 Masson staining

(1) After the sections are dehydrated, they are immersed in Masson's compound neutral staining solution for 5 minutes, then rinsed in 0.2% acetic acid aqueous solution for 2 minutes, 8% phosphotungstic acid for 10 minutes, 0.2% acetic acid aqueous solution for 2 minutes, 0.2% aniline blue solution for 5 minutes, 0.2% acetic acid aqueous solution for 2 minutes twice, 85% alcohol for 2 minutes, 95% alcohol for 2 minutes, 100% alcohol for 2 minutes, xylene for 5 minutes, and finally sealed with neutral gum. Then observe under a microscope and collect images for analysis.

(2) Scoring of lung tissue fibrosis under the microscope: Two researchers randomly collected 5 fields of view under an Olympus CX41RF light microscope and graded them according to the cumulative area of collagen fiber deposition and the degree of damage to the lung tissue structure, which was divided into 0 to 8 grades. Grade 0 represents normal lung tissue, which is scored as 0 points; Grade 1 to 2: Mild thickening of alveolar septa or peribronchiolar fibers, which is scored as 1 point; Grade 3 to 4: Moderate thickening of alveolar septa or peribronchiolar fibers, which is scored as 2 points; Grade 5 to 6: Fiber bands and local fibrous foci are formed, accompanied by a certain degree of damage to the lung tissue structure, which is scored as 3 points; Grade 7: Large fibrous foci are formed with severe damage to the lung tissue structure, which is scored as 4 points; Grade 8: Extensive fibrosis in the whole lung, which is scored as 5 points. The scoring results of each group are the average of all mice in that group.

1.2.4 Immunohistochemical staining

(1) Antigen repair: Place the tissue sections in a citric acid antigen repair buffer and then place them in a microwave oven for antigen repair. Start with medium heat for 8 minutes until boiling, stop the heat for 8 minutes to keep warm, and then turn to medium to low heat for 7 minutes. During the repair process, prevent the buffer from evaporating excessively and causing the sections to dry out. After natural cooling, place the sections in PBS and wash them on a decolorizing shaker 3 times, 5 minutes each time.

(2) Blocking endogenous peroxidase: Place the slides in 3% hydrogen peroxide and incubate at room temperature in the dark for 25 minutes. Then place the slides in PBS and wash them three times on a decolorizing shaker for 5 minutes each time.

(3) Serum blocking: Add 3% BSA solution or rabbit serum in the histochemical circle to evenly cover the tissue and block at room temperature for 30 minutes.

(4) Add primary antibody: Carefully shake off the blocking solution, place the slices flat in a humidified chamber, add the primary antibody prepared in PBS solution, and incubate at 4°C overnight.

(5) Adding secondary antibody: Place the slices in PBS and wash on a decolorizing shaker three times, 5 minutes each time. After carefully drying the slices, add HRP-labeled secondary antibody of the same species as the primary antibody to cover the tissue and incubate at room temperature for 50 minutes.

(6) DAB color development: Place the slices in PBS and wash them three times on a decolorizing shaker, 5 minutes each time. After carefully drying the slices, add DAB color development solution to the circle and control the color development time under a microscope. The positive color is brown-yellow. After satisfactory color development, rinse the slices under tap water to terminate the color development.

(7) Re-staining of cell nuclei: Re-stain with hematoxylin for 3 min, rinse with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, re-blue with hematoxylin bluing solution, and rinse with running water.

(8) Dehydration and sealing: Place the slices in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min to dehydrate and make them transparent. Then take out the slices and air dry them, and seal them with neutral gum.

(9) Observation and photography: Observe under a microscope and collect and analyze images through an image acquisition system.

2. Experimental results

Effect of TRAIL-Mu3 on lung wet weight in RILF mice

After the RILF model of C57BL/6 mice was successfully established, mice with equivalent fibrosis grades were randomly divided into three groups by micro-CT scanning. After the treatment cycle, the mice were killed, dissected, and the whole lungs of the mice were photographed and weighed. The results are shown in Figure 2. From the appearance of the mouse lungs, the lung tissue of the Con group mice was dark blood red, while the lung tissue of the Dex group and the TRAIL-Mu3 group mice was pink. In terms of color, the Con group was significantly darker than the Dex group and the TRAIL-Mu3 group. In addition, extensive erosive tissue structures were visible in the lung tissue of the Con group, while the corresponding areas of the Dex group and the TRAIL-Mu3 group were significantly less than those of the Con group. When the wet weight of the lungs of each group was weighed and compared, the results showed that the wet weight of the lungs of the Dex group and the TRAIL-Mu3 group mice was lower than that of the Con group (P<0.001), both about 0.3g, while the wet weight of the lungs of the Con group mice was as high as 0.5±0.1g. There was no significant difference in the wet weight of the lungs of the TRAIL-Mu3 group and the Dex group mice.

Effects of TRAIL-Mu3 on micro-CT images of lungs in RILF mice

During the anti-fibrosis treatment of RILF mice, the progress of RILF in each group of mice was regularly tracked and observed using micro-CT technology. The first day of drug administration was Day 1, and each group of mice was scanned with micro-CT four times on Day 0, Day 7, Day 14, and Day 21, and the images were compared and analyzed. As shown in Figure 3, during the treatment, the degree of fibrosis in the Con group mice gradually worsened over time; while the ground-glass shadows in the lungs of the Dex group and TRAIL-Mu3 group mice gradually subsided and the fibrous cord shadows gradually decreased.

2.3 Pathological effects of TRAIL-Mu3 on lung tissue of RILF mice

The appearance of lung tissue can only judge its lesions to a certain extent, while pathological sections can more accurately compare and analyze the degree of lesions. Therefore, we took out the lung tissues of mice in each group and performed pathological analysis by H&E staining, as shown in Figure 4. After the treatment cycle, the lung tissues of mice in the Con group showed more inflammatory cell infiltration around blood vessels and airways (yellow arrows); the number of alveolar wall epithelial cells decreased, and cellulose exudates and foam cells were seen in a wide range of alveolar cavities (black arrows); the alveolar cavity structure was blurred and solidified in some parts; some alveolar cavities were narrowed (blue arrows); and strong eosinophilic coagulation was seen in some alveolar cavities. Under the microscope, a small amount of inflammatory cell infiltration around a small number of blood vessels was observed in the lung tissues of mice in the Dex group (yellow arrows); the number of alveolar wall epithelial cells decreased, and foam cells were seen in many alveolar cavities (black arrows); and some alveolar cavities were narrowed (blue arrows). In the lung tissues of mice in the TRAIL-Mu3 group, more alveolar cavities were narrowed, the number of epithelial cells decreased, and a small number of foam cells were seen (black arrows); the alveolar cavities were solidified in some parts, and the normal structure disappeared (blue arrows).

The degree of inflammatory lesions in the lung tissues of mice in each group was scored, and the results are shown in Figure 5. Among them, the degree of inflammatory lesions in the lung tissues of mice in the Dex group and TRAIL-Mu3 group were lower than that in the Con group (P<0.05, P<0.01), and the degree of lung lesions in the TRAIL-Mu3 group was the lowest among the three groups, but there was no significant difference between it and the Dex group.

Effect of TRAIL-Mu3 on collagen deposition in lung tissue of RILF mice

Mice undergo fibrotic pathological changes after high-dose radiation exposure, and activated fibroblasts excrete a large amount of collagen, resulting in a significant increase in collagen deposition in the extracellular matrix. Therefore, the RILF process caused by RILI is accompanied by an increase in the amount of collagen deposition in the lungs, and the degree of collagen deposition in the lungs also reflects the severity of pulmonary fibrosis. To study the effect of TRAIL-Mu3 on RILF in mice, we detected the collagen proliferation in the lungs of mice by masson staining. As shown in Figure 6, the blue collagen staining area visible in the lung tissue of mice in the Con group was the largest, followed by the Dex group, and the staining area in the TRAIL-Mu3 group was the smallest. In the Con group, more diffuse blue collagen staining was seen, and it was widely distributed in both the trachea and alveoli; while the blue collagen staining in the lung tissue of mice in the Dex group was mostly distributed around the trachea, and a small amount of collagen staining was seen in the alveolar structure; the collagen staining area in the TRAIL-Mu3 group was smaller and also mostly concentrated around the trachea. The results of Masson staining showed that the fibrosis score in the TRAIL-Mu3 group was lower than that in the Con group (P<0.05), and there was no significant difference between the Dex group and the Con group.

Image J was used to analyze the results of masson staining of lung tissues of mice in each group, and the ratio of the collagen-positive blue area to the total tissue area was calculated to obtain the collagen volume fraction (CVF) to compare and analyze the degree of collagen deposition. The results are shown in Figure 7. The CVF values in the lung tissues of mice in the Dex group and TRAIL-Mu3 group were lower than those in the Con group (P<0.01, P<0.001). Among them, the CVF value of the lung tissue of mice in the TRAIL-Mu3 group was the lowest, not only lower than that of the Con group, but also significantly different from that of the Dex group (P<0.01).

Effect of TRAIL-Mu3 on α-SMA levels in lung tissue of RILF mice

After high-dose chest irradiation, the lung fibroblasts in the lung tissue of mice may be activated and begin to secrete collagen and other extracellular matrices in large quantities, leading to fibrosis. These activated abnormally proliferating lung fibroblasts highly express α-SMA, so the expression results of α-SMA can also explain the effect of TRAIL-Mu3 on lung fibroblasts in mouse lung tissue. As shown in Figure 8, after different treatments, the expression of α-SMA in the lung tissue of each group of mice also changed differently. The positive staining degree of the TRAIL-Mu3 group and the Dex group was lower than that of the Con group, among which the α-SMA positive staining area in the lung tissue of the TRAIL-Mu3 group of mice was the smallest, significantly lower than that of the Con group. By imageJ, the α-SMA level of the lung tissue of each group was statistically analyzed, and the α-SMA expression level of the TRAIL-Mu3 group was the lowest among the three groups, with significant differences from the Con group (P<0.01). In addition, the Dex group also had significant differences from the Con group (P<0.01), but there was no significant difference between the Dex group and the TRAIL-Mu3 group.

The above results indicate that TRAIL-Mu3 is an effective treatment for radiation-induced pulmonary fibrosis, can reduce lung wet weight, reduce lung tissue erosion and inflammatory lesions, and reduce α-SMA expression in lung fibroblasts, and the effect is comparable to that of the positive control dexamethasone sodium phosphate; TRAIL-Mu3's reduction in lung collagen deposition is even significantly higher than that of the positive control dexamethasone sodium phosphate, reflecting an excellent effect in the treatment of radiation-induced pulmonary fibrosis.

Experimental Example 3: Effect of TRAIL-Mu3 on cytokine and DR expression in RILF treatment of mice

3.1 Immunohistochemical detection of DR5 in lung tissue of RILF mice

TRAIL-Mu3 is a recombinant protein modified based on TRAIL-Mu3 protein. Its target is highly specific, namely death receptor (DR). In mice, only DR5 can effectively induce apoptosis, and DR4 is not expressed or expressed at very low levels in mice. Therefore, in order to explore the reason why TRAIL-Mu3 alleviates pulmonary fibrosis in RILF model mice, we detected the level of its specific target DR5 in mice.

After the observation of the fibrosis stage, the mice were killed and their lung tissues were taken for DR5 immunohistochemical analysis. The results are shown in Figure 9. Compared with the Con group, the positive staining area and depth of DR5 in the lung tissue of the TRAIL-Mu3 group mice were significantly increased, and the DR5 expression in the Dex group was also increased relative to the Con group. To further analyze the results, we used imageJ to perform statistical analysis on the DR5 expression levels of each group. The results showed that the DR5 expression level in the lung tissue of the TRAIL-Mu3 group mice was the highest among the three groups, and the DR5 expression in the Dex group was also higher than that in the Con group (P<0.01), but there were significant differences between the TRAIL-Mu3 group and the Con group and the Dex group (P<0.001, P<0.05).

3.2 qRT-PCR and Western Blot Detection of DR5 in Lung Tissue of RILF Mice

To further confirm that TRAIL-Mu3 induces effective apoptosis by upregulating the level of DR5, we analyzed the mRNA level of DR5 in the lung tissues of mice in each group and the protein level of DR5 and Caspase 3. As shown in Figure 10, in the lung tissues of mice in the TRAIL-Mu3 group, the mRNA level of DR5 was higher than that in the Con group and the Dex group (P<0.001). At the protein level, as shown in Figure 11, the expression of DR5 in the lung tissues of mice treated with TRAIL-Mu3 was significantly upregulated (P<0.001), and the uncut Caspase 3 in the TRAIL-Mu3 group was significantly reduced (P<0.001).

The above results indicate that TRAIL-Mu3 upregulates the expression level of DR5 in mouse lung tissue, induces the Caspase cascade reaction, promotes the cleavage of the apoptotic effector Caspase 3, thereby inducing apoptosis of activated lung fibroblasts and thereby treating pulmonary fibrosis.

Effects of TRAIL-Mu3 on cytokine levels in the blood of RILF mice

At the end of the fibrosis observation, blood was collected from the mouse eyeballs to obtain serum, and the levels of IL-6, TNF-α, TGF-β1, and IL-13 factors were detected using liquid chip technology. The results are shown in Figure 12. The levels of IL-6, TNF-α, and IL-13 in the serum of mice in the TRAIL-Mu3 group and the Dex group were significantly reduced, and there were significant differences compared with the Con group (P<0.05). The TNF-α and IL-13 levels in the TRAIL-Mu3 group were significantly lower than those in the Dex group (P<0.05). In addition, compared with the Con group, the TGF-β1 level in the Dex group was significantly reduced. However, in terms of TGF-β1 level, the TRAIL-Mu3 group was significantly higher than the Con group (P<0.05) and the Dex group (P<0.01).

After TRAIL-Mu3 intervention, serum cytokines TNF-α, IL-6, and IL-13 in RILF mice were all downregulated, indicating that TRAIL-Mu3 can reduce inflammatory responses and thus alleviate the occurrence of fibrosis. The upregulation of TGF-β1 may be related to a variety of factors. According to previous literature reports, TGF-β1 can increase the expression of DR5, while IL-13 downregulation can relieve DR5 inhibition. Therefore, although there are differences in the effects of TRAIL-Mu3 and Dex groups on the cytokine levels of RILF mice, according to the changes in cytokines after TRAIL-Mu3 intervention, it can be proved that TRAIL-Mu3 can effectively reduce inflammatory responses and promote DR5 expression, thereby alleviating the occurrence of pulmonary fibrosis and treating pulmonary fibrosis.

Experimental Example 4: Toxic and side effects of TRAIL-Mu3 on RILF mice

During the experiment, no obvious abnormalities in the diet, excretion and behavior of the mice were observed. However, the chests of the mice in each group faded and turned white after receiving large doses of X-ray radiation, and there was varying degrees of hair loss. As shown in Figure 13, the degree of hair loss was the lowest in the TRAIL-Mu3 group. During the experiment, the weight of the mice was recorded every two days. Except for a slight decrease in the weight of the mice in the Con group, the weight of the mice in the Dex group and the TRAIL-Mu3 group remained basically stable.

After the observation, the hearts, livers, spleens and kidneys of mice in each group were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced for H&E staining. The results are shown in Figure 14. No obvious abnormalities were found in the heart, liver, spleen and kidneys.

The above results indicate that TRAIL-Mu3 has low toxicity and side effects, which is conducive to clinical application.

In summary, the present invention provides a new use of a tumor necrosis factor-related apoptosis-inducing ligand mutant TRAIL-Mu3. TRAIL-Mu3 has small toxic and side effects, can reduce lung wet weight, reduce lung collagen deposition, lung tissue inflammatory lesions, and lung tissue erosion; by promoting the expression of lung tissue DR5, it induces apoptosis of radiation-activated lung fibroblasts, effectively improves radiation-induced lung fibrosis, and has excellent clinical application value.

SEQUENCE LISTING

<110> West China Hospital, Sichuan University

<120> A new use of a tumor necrosis factor-related apoptosis-inducing ligand mutant

<130> GYKH1670-2021P0112705CC

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 169

<212> PRT

<213> TRAIL-Mu3 sequence

<400> 1

Met Arg Arg Arg Arg Arg Arg Arg Arg Val Ala Ala His Ile Thr Gly

1 5 10 15

Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu

20 25 30

Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly

35 40 45

His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile

50 55 60

His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe

65 70 75 80

Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln

85 90 95

Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys

100 105 110

Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr

115 120 125

Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile

130 135 140

Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala

145 150 155 160

Ser Phe Phe Gly Ala Phe Leu Val Gly

165

Claims (1)

1. The application of TRAIL-Mu3 in preparing medicine for treating radiation pulmonary fibrosis is disclosed, wherein the TRAIL-Mu3 sequence is shown in SEQ ID NO. 1.