Application of amino acid substituted polypeptide R13A-MOTS-c in preparation of medicine for treating radiation-induced lung injury

Abstract

The invention discloses application of amino acid replacement polypeptide R13A-MOTS-c in preparation of a medicine for treating radiation-induced lung injury, and belongs to the field of biological medicine. According to the invention, the polypeptide R13A-MOTS-c with improved cell membrane penetrating power is prepared, and the polypeptide R13A-MOTS-c is applied to treatment of radiation-induced lung injury individuals. A cell model and an animal model verify that the polypeptide R13A-MOTS-c prepared by the invention can significantly relieve oxidative stress, inflammatory response, mitochondrial function damage and apoptosis of radiation-induced lung injury, and provides a new means and idea for prevention and treatment of radiation-induced lung injury.

Description

Technical Field

[0001] The present invention relates to the application of amino acid substituted polypeptide R13A-MOTS-c in preparing a drug for treating radiation-induced lung injury, and belongs to the field of biomedicine. Background Art

[0002] Radiation-induced lung injury (RILI) is common in patients undergoing radiotherapy for thoracic tumors (such as lung and breast cancer), which not only reduces patients' quality of life but also inhibits the therapeutic efficacy of radiotherapy. Currently, clinical treatments for RILI primarily include antioxidants, glucocorticoids, immunomodulators, and steroids. These drugs primarily focus on symptom relief, but fail to fundamentally address the underlying lung injury and are associated with significant side effects. Alveolar epithelial cells are the primary target cells for RILI and are key regulators of repair and pathological progression. Protecting their function or promoting their repair capacity is crucial for the prevention and treatment of RILI. The mechanisms of cellular radiation damage can be categorized as direct and indirect. Although the actual duration of radiation exposure in both mechanisms is extremely brief, the biological effects they trigger are central to the development and progression of RILI. Within this complex process, mitochondria, lacking the protection of histones and nucleosomes, deficient in their own DNA repair mechanisms, and as the primary site of reactive oxygen species (ROS) production, become prime targets of radiation damage. Therefore, alleviating mitochondrial dysfunction in alveolar epithelial cells is the key to the prevention and treatment of RILI.

[0003] Mitochondrial-derived peptide (Mitochondrial open reading frame of the 12S rRNA type-c, MOTS-c) is a 16-amino acid polypeptide encoded by mitochondrial DNA (mtDNA). MOTS-c exerts its protective effect by enhancing the activity of transcription factors. Under hydrogen peroxide or high sugar conditions, MOTS-c polypeptide can be translocated from the mitochondria to the nucleus within 30 minutes, and interact with the nuclear transcription factor Nrf2, initiating mitochondrial-nucleus "dialogue" and promoting the expression of various key genes necessary for mitochondrial development. However, the poor cell membrane permeability of polypeptide substances limits the exertion of their biological activity. Therefore, finding membrane proteins that can efficiently achieve transmembrane delivery of polypeptide substances will provide a very promising solution for breaking through the bottleneck of clinical application of MOTS-c therapeutic polypeptides. Summary of the Invention

[0004] The purpose of the present invention is to provide an amino acid substituted polypeptide R13A-MOTS-c for use in a drug for preventing and / or treating radiation-induced lung injury, thereby providing a new idea and method for the prevention and treatment of radiation-induced lung injury.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The first object of the present invention is to provide the use of R13A-MOTS-c peptide, R13A-MOTS-c peptide derivatives and / or R13A-MOTS-c peptide chimeras in the preparation of drugs for preventing and / or treating radiation-induced lung injury.

[0007] In one embodiment, the amino acid sequence of the R13A-MOTS-c peptide is MRWQEMGYIFYPAKLR (shown in SEQ ID NO. 1).

[0008] In one embodiment, the radiation lung injury includes early radiation pneumonitis and late radiation pulmonary fibrosis.

[0009] In one embodiment, the drug contains one or more of the active ingredients R13A-MOTS-c peptide, R13A-MOTS-c peptide derivatives, and R13A-MOTS-c peptide chimeras, and pharmaceutically acceptable excipients.

[0010] In one embodiment, the excipients include any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

[0011] In one embodiment, the combination of at least two agents, such as a combination of a diluent and an excipient, a combination of a binder and a wetting agent, a combination of an emulsifier and a solubility aid, and any other combinations can be selected and will not be described in detail here.

[0012] In one embodiment, the vector is a virus, a liposome, or a nanoparticle.

[0013] In one embodiment, the excipient is one or more of mannitol, lactose, fatty acid, and polyethylene glycol.

[0014] In one embodiment, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops.

[0015] In one embodiment, the administration route of the drug includes oral administration, sublingual administration, rectal administration, mucocutaneous administration, inhalation administration or injection administration.

[0016] In one embodiment, the prevention and / or treatment of radiation-induced lung injury includes at least one of the following aspects:

[0017] (1) Activate Nrf2 protein;

[0018] (2) Alleviate oxidative stress and inflammatory response in lung epithelial cells;

[0019] (3) inhibiting oxidative stress and inflammatory responses in lung tissue;

[0020] (4) Alleviate mitochondrial damage in lung epithelial cells;

[0021] (5) Inhibit apoptosis of lung epithelial cells;

[0022] (6) Inhibit apoptosis and mitochondrial damage in lung epithelial cells and lung tissue.

[0023] The present invention also provides a method for improving the cell membrane penetration ability of the polypeptide MOTS-c, which comprises replacing the arginine at position 13 of the MOTS-c amino acid sequence (MRWQEMGYIFYPRKLR) with alanine.

[0024] Beneficial effects:

[0025] (1) The polypeptide MOTS-c mutant R13A-MOTS-c provided by the present invention can effectively improve the oxidative stress, inflammatory response, mitochondrial function damage and apoptosis of lung tissue induced by X-ray irradiation.

[0026] (2) The polypeptide MOTS-c mutant R13A-MOTS-c provided by the present invention has the advantages of high efficiency, low toxicity, and high selectivity. It can inhibit X-ray-induced oxidative stress, inflammatory response, mitochondrial dysfunction, and apoptosis in alveolar epithelial cells, and has a protective effect against RILI. The present invention provides a new target for the preparation of drugs to protect against radiation-induced lung injury and provides a new approach for the development of RILI therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The membrane permeability verification results of R13A-MOTS-c; wherein, A is the conservation analysis result of amino acid residues of MOTS-c polypeptides from 14 different species; B is the 6 types of MOTS-c synthesized by the present invention; C is the average hydrophilicity coefficient of the three types of polypeptides; D and E are the immunofluorescence detection results of the three types of polypeptides entering the cell nucleus (scale: 2μm).

[0028] Figure 2The results show that R13A-MOTS-c can activate Nrf2. A, B, and C show the immunofluorescence analysis of cells stained with Nrf2 (green) and DAPI (blue) (scale bar: 20 μm). E shows the expression level of Nfe2l2 detected by RT-qPCR. F and G show the protein content of Nrf2 in MLE-12 cells detected by Western Blot.

[0029] Figure 3 The effect of R13A-MOTS-c on alleviating radiation-induced oxidative stress and inflammatory response in MLE-12 cells; A and B are CCK8 assays to detect the effects of MOTS-c and R13A-MOTS-c on MLE-12 cells; CF are the levels of LDH, GSH, SOD, and MDA in MLE-12 cells; G and H are DCFH-DA fluorescent probes to detect intracellular ROS content (scale bar: 100 μm); I is RT-qPCR detection of the mRNA levels of Il-6 and Tnf-α.

[0030] Figure 4 Figure 3. Effect of R13A-MOTS-c on alleviating oxidative stress and inflammatory response in RILI mice; A and B are HE staining of lung tissue sections (scale bar: 50 μm) and the corresponding lung injury scores; C is the protein concentration in BALF; DG are the levels of LDH, GSH, MPO, and MDA in mouse serum; H is the mRNA levels of Il-6 and Tnf-α in lung tissue detected by RT-qPCR.

[0031] Figure 5 The results show that R13A-MOTS-c inhibits radiation-induced mitochondrial damage in MLE-12 cells; A and B are JC-1 staining kits (scale bar: 20 μm) used to detect mitochondrial membrane potential in MLE-12 cells; C and D are MitoSOX red fluorescent probes used to detect mtROS levels in MLE-12 cells (scale bar: 20 μm); E is the ATP content in MLE-12 cells; F is RT-qPCR detection of the mRNA expression levels of CoxI, Cox IV and Opa1; G and H are Western Blot detection of the protein levels of COX I, COX IV and OPA1 in MLE-12 cells.

[0032] Figure 6The results show that R13A-MOTS-c can alleviate the radiation-induced apoptosis of MLE-12 cells. A and B show the apoptosis level of MLE-12 cells detected by Tunel staining (scale bar: 20 μm); C shows the mRNA expression levels of Bcl2, Bax, Caspase9 and Cyt-c in MLE-12 cells detected by RT-qPCR; D and E show the protein levels of Bcl2, Bax, Caspase9 and Cyt-c in MLE-12 cells detected by Western Blot.

[0033] Figure 7 The results show that R13A-MOTS-c can inhibit apoptosis and mitochondrial damage in lung tissue of RILI mice; A is the RT-qPCR detection of the mRNA expression levels of Bcl2, Bax, Caspase9 and Cyt-c in lung tissue; B, C are the Western blot detection of the protein levels of Bcl2, Bax, Caspase9 and Cyt-c in lung tissue; D is the RT-qPCR detection of the mRNA levels of Opa1, Cox I and Cox IV in lung tissue; E, F are the Western blot detection of the protein levels of OPA1, COX I and COX IV; G, H are the immunofluorescence analysis of lung tissue stained with OPA1 (green) and DAPI (blue) (Scale bar: 20 μm). DETAILED DESCRIPTION

[0034] The technical solutions provided by the present invention will be described in detail below with reference to specific embodiments. However, it should be understood that the following specific embodiments are only used to illustrate the present invention and do not limit the present invention in any form.

[0035] The synthesis of MOTS-c, R13A-MOTS-c, R16A-MOTS-c, FITC-MOTS-c, FITC-R13A-MOTS-c and FITC-R16A-MOTS-c was entrusted to Wuxi Maimtop Biotechnology Co., Ltd., with a purity greater than 95%; mouse alveolar epithelial cells MLE-12 were purchased from ATCC, USA.

[0036] Example 1: R13A-MOTS-c has stronger membrane permeability

[0037] WebLogo 3 was used to align the sequences of MOTS-c peptides from 14 different species, including human, chimpanzee, bonobo, orangutan, mouse, rat, naked mole rat, dog, cow, zebrafish, lion, bear, horse, and dolphin. The grand average of hydropathicity (GRAVY) of the three peptides was predicted using ProtParam (https: / / web.expasy.org / protparam). MLE-12 cells were pretreated with 5 μM FITC-MOTS-c, FITC-R13A-MOTS-c, and FITC-R16A-MOTS-c for 30 minutes, and cell membrane penetration was observed using confocal microscopy.

[0038] (2) Experimental results

[0039] The results are as follows Figure 1 As shown, the sequence of the wild-type MOTS-c polypeptide is Met-Arg-Trp-Gln-Glu-Met-Gly-Tyr-Ile-Phe-Tyr-Pro-Arg-Lys-Leu-Arg, of which the first 11 amino acid residues are highly conserved in cross-species comparisons ( Figure 1 A). It is worth noting that among these 11 core residues, only glycine (Gly), leucine (Leu) and proline (Pro) are non-polar amino acids, and the number is only 3; the rest are polar amino acids. Particularly prominent is the arginine residues at positions 13 and 16, whose hydropathic index is as high as -4.50, showing extremely strong hydrophilicity. Peptides in which the arginine at positions 13 and 16 were mutated to alanine were constructed and named R13A-MOTS-c and R16A-MOTS-c, respectively. The average hydrophilicity coefficient was calculated, and the results showed that the average hydrophilicity coefficient of the mutated polypeptides increased to -0.544. This change indicates that their cell membrane penetration performance has been greatly improved ( Figure 1 B, 1C). Cell immunofluorescence experiments further confirmed that the nuclear-cytoplasmic ratio of FTIC fluorescence intensity of R13A-MOTS-c increased by 158% compared with wild-type MOTS-c, while the nuclear-cytoplasmic ratio of FTIC fluorescence intensity of R16A-MOTS-c increased by 10%, with no statistical difference ( Figure 1 D, 1E), indicating that the cell membrane permeability of R13A-MOTS-c is enhanced.

[0040] Example 2: R13A-MOTS-c has stronger Nrf2 activation ability

[0041] (1) Experimental methods

[0042] A control group (Normal), a MOTS-c-treated group (MOTS-c), and a R13A-MOTS-c-treated group (R13A-MOTS-c) were set up. MLE-12 cells were treated with MOTS-c and R13A-MOTS-c at a final concentration of 5 μM, respectively. After 24 hours of treatment with the two peptides, Nrf2 mRNA and protein levels were measured by RT-qPCR, immunofluorescence, and Western blotting.

[0043] (2) Experimental results

[0044] like Figure 2 As shown, the results of cell immunofluorescence showed that R13A-MOTS-c effectively increased the Nrf2 protein content compared with the MOTS-c group ( Figure 2 A to 2D), indicating that our synthesized R13A-MOTS-c has good physiological activity. In addition, Western Blot and RT-qPCR results further confirmed that R13A-MOTS-c can effectively increase the protein content and mRNA level of Nrf2, which increased by 54% and 74% respectively compared with MOTS-c ( Figure 2 E~2G). This suggests that R13A-MOTS-c has a stronger ability to activate Nrf2.

[0045] Example 3: R13A-MOTS-c alleviates radiation-induced oxidative stress and inflammatory response in lung epithelial cells MLE-12

[0046] (1) Experimental methods

[0047] Methods: A control group (Normal), a model group (IR), a MOTS-c intervention group (IR+MOTS-c), and an R13A-MOTS-c intervention group (IR+R13A-MOTS-c) were established. MLE-12 alveolar epithelial cells in the model, MOTS-c, and R13A-MOTS-c intervention groups were irradiated with a single dose of 8 Gy using X-rays. MOTS-c and R13A-MOTS-c were added to the cells before X-ray irradiation (final concentrations of 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 μM, respectively). Cells were analyzed for CCK8 24 hours after X-ray irradiation. In addition, 24 hours after X-ray irradiation, cells in the 5μM MOTS-c and R13A-MOTS-c intervention groups were collected for the detection of oxidative stress indicators (LDH, GSH, SOD, MDA and ROS content); on the other hand, RT-qPCR was used to detect the mRNA levels of inflammatory factors Tnf-α and Il-6.

[0048] (2) Experimental results

[0049] like Figure 3 As shown in the CCK8 results, 5 μM R13A-MOTS-c can reduce radiation-induced MLE-12 cell damage by 49%, while 5 μM wild-type MOTS-c can reduce radiation-induced cell damage by 3%, indicating that R13A-MOTS-c has a better cell protection effect ( Figure 3 A, 3B). LDH assay results showed that R13A-MOTS-c intervention reduced the LDH level in the supernatant of radiation-induced MLE-12 cells by 74% ( Figure 3 C). In addition, we examined oxidative stress-related indicators and found that R13A-MOTS-c treatment increased radiation-induced GSH and SOD levels by 20% and 99%, respectively, and decreased MDA content by 46% ( Figure 3 Meanwhile, R13A-MOTS-c intervention reduced the radiation-induced ROS level by 35% ( Figure 3 G, H). In terms of inflammatory response, R13A-MOTS-c intervention reduced the mRNA expression levels of Tnf-α and Il-6 in MLE-12 cells by 39% and 36%, respectively ( Figure 3 I). The above results suggest that R13A-MOTS-c has the ability to alleviate radiation-induced oxidative stress and inflammatory response.

[0050] Example 4: R13A-MOTS-c alleviates radiation-induced oxidative stress and inflammatory response in MLE-12 cells

[0051] (1) Experimental methods

[0052] Six- to eight-week-old C57BL / 6 mice were randomly divided into a control group (Normal), a model group (IR), a MOTS-c intervention group (IR+MOTS-c), and an R13A-MOTS-c intervention group (IR+R13A-MOTS-c), with five mice in each group (Table 1). Mice in the model group, MOTS-c group, and R13A-MOTS-c intervention groups were anesthetized with sodium pentobarbital (40 mg / kg) and their limbs were fixed with tape in an X-RAD SMART small animal radiotherapy simulator. The mice were placed in the prone position with their upper limbs facing forward and their lower limbs facing backward, with their limbs balanced and straight. Under CT guidance, the lung irradiation range was outlined, minimizing the risk of heart and spine irradiation. A single dose of 20 Gy was administered at a dose rate of 5 Gy / min. After X-ray irradiation, mice in the MOTS-c and R13A-MOTS-c intervention groups received daily intraperitoneal injections of 5 mg / kg MOTS-c and R13A-MOTS-c peptides dissolved in ddH2O for 14 days. Lung tissue, serum, and bronchoalveolar lavage fluid were collected on day 14. A portion of lung tissue was fixed with 4% paraformaldehyde and subjected to H&E staining for lung tissue pathology. Other lung tissues were analyzed by RT-qPCR for mRNA levels of the inflammatory factors Tnf-α and Il-6. Mouse serum was used to measure oxidative stress markers (LDH, GSH, MPO, SOD, and MDA). Total cell counts in bronchoalveolar lavage fluid were performed using flow cytometry.

[0053] Table 1 Experimental groups

[0054]

[0055] (2) Experimental results

[0056] like Figure 4 As shown in Figure 2, HE experiments showed that the mice that received only radiation showed morphological damage characteristics, including edema, rupture of alveolar structures, and extensive infiltration of inflammatory cells into the lung parenchyma. In contrast, the extent of these injuries was significantly reduced in the mice that received R13A-MOTS-c treatment (see Figure 2). Figure 4 A, 4B). BALF results showed that the protein concentration in BALF of R13A-MOTS-c treatment group was reduced by 43% compared with that of IR group ( Figure 4 C), while serum LDH levels decreased by 58% ( Figure 4 D), both of which reflect the reduction of lung tissue damage. To more comprehensively assess the oxidative stress status, we tested multiple biochemical indicators in mouse serum, including GSH, MPO, and MDA. The results showed that compared with the IR group, the MPO and MDA in serum of R13A-MOTS-c treatment group decreased by 58% and 74%, respectively, while the GSH content increased by 38% ( Figure 4 E~4G), these changes together indicate an improvement in oxidative stress. In addition, we also detected the mRNA expression levels of inflammatory cytokines Il-6 and Tnf-α in lung tissue. The results showed that after R13A-MOTS-c treatment, the mRNA expression levels of these two inflammatory factors decreased by 62% and 63%, respectively, compared with the IR group ( Figure 4 H). These results suggest that R13A-MOTS-c can effectively alleviate oxidative damage and inflammatory responses in RP mice.

[0057] Example 5: R13A-MOTS-c inhibits radiation-induced mitochondrial damage in MLE-12 cells

[0058] (1) Experimental methods

[0059] Methods: A control group (Normal), a model group (IR), a MOTS-c intervention group (IR+MOTS-c), and an R13A-MOTS-c intervention group (IR+R13A-MOTS-c) were established. MLE-12 alveolar epithelial cells in the model, MOTS-c, and R13A-MOTS-c intervention groups were irradiated with X-rays at a single dose of 8 Gy. MOTS-c and R13A-MOTS-c were added to the cells at a final concentration of 5 μM before X-ray irradiation in the MOTS-c and R13A-MOTS-c intervention groups, respectively. Twenty-four hours after X-ray irradiation, mitochondrial function was assessed using a JC-1 mitochondrial membrane potential staining kit, a mitochondrial reactive oxygen species staining kit, and an ATP assay. RT-qPCR and Western blotting were used to measure the mRNA and protein levels of COX I, COX IV, and OPA1.

[0060] (2) Experimental results

[0061] The results are as follows Figure 5 As shown in the JC-1 staining experiment, R13A-MOTS-c effectively alleviated the IR-induced transformation of the JC-1 fluorescent probe from polymers (red) to monomers (green), maintaining the stability of the mitochondrial membrane potential ( Figure 5 A, 5B). Cell immunofluorescence results showed that the MitoSOX red fluorescence signal in MLE-12 cells was reduced by 53% after R13A-MOTS-c supplementation compared with the IR group ( Figure 5 C, 5D), indicating that R13A-MOTS-c can reduce the generation of mtROS. Furthermore, we found that the ATP content of MLE-12 cells in the IR group decreased, while the intervention of R13A-MOTS-c increased the ATP content by 16% ( Figure 5E), demonstrating that R13A-MOTS-c can improve mitochondrial energy metabolism. In addition, R13A-MOTS-c increased the mRNA expression levels of Cox I, Cox IV, and Opa1 in radiation-induced MLE-12 cells by 136%, 172%, and 168%, respectively ( Figure 5 F). Western Blot results were consistent with RT-qPCR results. R13A-MOTS-c increased the protein expression levels of COX I, COX IV, and OPA1 in radiation-induced MLE-12 cells by 133%, 118%, and 175%, respectively ( Figure 5 G, 5H). These results suggest that R13A-MOTS-c effectively alleviates radiation-induced mitochondrial damage in MLE-12 cells by eliminating excess ROS and protecting mitochondrial function.

[0062] Example 6: R13A-MOTS-c alleviates radiation-induced apoptosis in MLE-12 cells

[0063] (1) Experimental methods

[0064] Methods: A control group (Normal), a model group (IR), a MOTS-c intervention group (IR+MOTS-c), and an R13A-MOTS-c intervention group (IR+R13A-MOTS-c) were set up. MLE-12 alveolar epithelial cells in the model, MOTS-c, and R13A-MOTS-c intervention groups were irradiated with X-rays at a single dose of 8 Gy. MOTS-c and R13A-MOTS-c were added to the cells in the MOTS-c and R13A-MOTS-c intervention groups, respectively, at a final concentration of 5 μM before X-ray irradiation. Twenty-four hours after X-ray irradiation, cell apoptosis was assessed using a Tunel staining kit. The mRNA and protein levels of Bcl2, Bax, Caspase9, and Cyt-c were measured by RT-qPCR and Western blotting.

[0065] (2) Experimental results

[0066] The results are as follows Figure 6 As shown in Figure 2, Tunel staining results showed that R13A-MOTS-c reduced the level of radiation-induced cell apoptosis by 25% ( Figure 6 A, 6B). Further studies showed that R13A-MOTS-c intervention in the IR group reduced the mRNA expression levels of pro-apoptotic genes Bax, Caspase9, and Cyt-c by 31%, 32%, and 41%, respectively, while increasing the expression level of the anti-apoptotic gene Bcl2 by 76% ( Figure 6C). In addition, at the protein level, R13A-MOTS-c intervention reduced the protein levels of Bax, Caspase9, and Cyt-c by 43%, 46%, and 45%, respectively, compared with the IR group, while increasing the protein level of Bcl2 by 270% ( Figure 6 These results suggest that R13A-MOTS-c inhibits radiation-induced cell apoptosis by regulating the expression of apoptosis-related genes.

[0067] Example 7: R13A-MOTS-c inhibits lung tissue apoptosis and mitochondrial damage in RILI mice

[0068] (1) Experimental methods

[0069] Six- to eight-week-old C57BL / 6 mice were randomly divided into a control group (Normal), a model group (IR), a MOTS-c intervention group (IR+MOTS-c), and an R13A-MOTS-c intervention group (IR+R13A-MOTS-c), with five mice in each group. Mice in the model group, MOTS-c group, and R13A-MOTS-c intervention groups were anesthetized with sodium pentobarbital (40 mg / kg) and their limbs were secured with tape in an X-RADSMART small animal radiotherapy simulator. The mice were placed in the prone position with their upper limbs facing forward and their lower limbs facing backward, with their limbs balanced and straight. CT localization was used to outline the lung irradiation range, minimizing the risk of heart and spine irradiation. A single dose of 20 Gy was administered at a dose rate of 5 Gy / min. After X-ray irradiation, mice in the MOTS-c and R13A-MOTS-c intervention groups received daily intraperitoneal injections of 5 mg / kg MOTS-c and R13A-MOTS-c peptides dissolved in ddH2O for 14 days. Lung tissues were collected on the 14th day. A portion of the lung tissue was analyzed for mRNA and protein levels of apoptosis-related and mitochondrial genes using RT-qPCR and Western blotting. Other lung tissues were fixed with 4% paraformaldehyde and immunofluorescence stained for changes in OPA1 protein.

[0070] (2) Experimental results

[0071] The results are as follows Figure 7 As shown in the results of RT-qPCR experiments, compared with the IR group, R13A-MOTS-c reduced the mRNA expression levels of pro-apoptotic genes Bax, Caspase9, and Cyt-c by 37%, 34%, and 42%, respectively, and increased the mRNA level of anti-apoptotic gene Bcl2 by 115% ( Figure 7A). Apoptosis-related protein results showed that compared with the IR group, R13A-MOTS-c reduced the protein levels of pro-apoptotic genes Bax, Caspase9, and Cyt-c by 39%, 36%, and 43%, respectively, while increasing the protein level of anti-apoptotic gene Bcl2 by 127% ( Figure 7 In addition, the mRNA expression levels of mitochondrial function-related genes Cox I, Cox IV, and Opa1 in lung tissues of the R13A-MOTS-c treatment group increased by 98%, 119%, and 151%, respectively, compared with the IR group ( Figure 7 D). The results of mitochondrial-related proteins showed that the protein levels of mitochondrial function-related genes COX I, COX IV, and OPA1 in lung tissues of the R13A-MOTS-c treatment group increased by 160%, 135%, and 121%, respectively, compared with the IR group ( Figure 7 E, 7F). Immunofluorescence staining of lung tissue further showed that the OPA1 protein content in the R13A-MOTS-c group increased by 253% compared with the IR group ( Figure 7 G, 7H). Taken together, these results suggest that R13A-MOTS-c exerts its protective effect against RILI by inhibiting cell apoptosis and improving mitochondrial function.

[0072] These results indicate that R13A-MOTS-c has an ameliorative effect on X-ray-induced radiation-induced lung injury, inhibiting radiation-induced oxidative stress, inflammatory response, mitochondrial damage, and apoptosis. This invention provides a new target for the preparation of drugs to protect against radiation-induced lung injury.

[0073] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of R13A-MOTS-c peptide, R13A-MOTS-c peptide derivatives and / or R13A-MOTS-c peptide chimeras in the preparation of a drug for preventing and / or treating radiation-induced lung injury, characterized in that: The amino acid sequence of the R13A-MOTS-c peptide is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The radiation-induced lung injury includes early radiation pneumonitis and / or late radiation-induced pulmonary fibrosis.

3. The use according to claim 1 or 2, characterized in that The drug contains one or more of R13A-MOTS-c peptide, R13A-MOTS-c peptide derivative, and R13A-MOTS-c peptide chimera, and contains pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that The excipients include one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

5. The use according to claim 5, characterized in that The carriers include viruses, liposomes or nanoparticles.

6. The use according to claim 5, characterized in that The excipients include one or more of mannitol, lactose, fatty acid, and polyethylene glycol.

7. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the drug includes suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops.

8. The use according to claim 7, characterized in that The administration routes of the drug include oral administration, sublingual administration, rectal administration, skin and mucosal administration, inhalation administration or injection administration.

9. The use according to any one of claims 1 to 7, characterized in that: The prevention and / or treatment of radiation-induced lung injury includes at least one of the following aspects: (1) Activate Nrf2 protein; (2) Alleviate oxidative stress and inflammatory response in lung epithelial cells; (3) inhibiting oxidative stress and inflammatory responses in lung tissue; (4) Alleviate mitochondrial damage in lung epithelial cells; (5) Inhibit apoptosis of lung epithelial cells; (6) Inhibit apoptosis and mitochondrial damage in lung epithelial cells and lung tissue.

10. A method for improving the cell membrane penetration ability of polypeptide MOTS-c, characterized in that: The arginine at position 13 of the MOTS-c amino acid sequence was replaced with alanine.

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