Application of peptide SRD in the preparation of drugs for treating acute lung injury

Abstract

This invention discloses the application of a peptide SRD in the preparation of drugs for treating acute lung injury, belonging to the field of biomedical technology. In vivo experiments verified the protective effect of peptide SRD on acute lung injury (ALI), particularly its effect on improving AT2 mitochondrial function. In animal models of ALI, tail vein injection of peptide SRD significantly reduced the protein concentration and total cell count in bronchoalveolar lavage fluid, significantly alleviated the severity of lung injury, lowered histological scores, significantly decreased SOD2 acetylation levels while significantly increasing total SOD2 protein levels, and significantly increased SPC levels (an indicator of AT2 survival). ELISA analysis showed a significant decrease in the levels of IL-6, IL-1β, and TNF-α, indicators of pneumonia in mice. These experimental results confirm that the antioxidant peptide SRD can improve oxidative stress damage in ALI, effectively reduce AT2 cell damage under ALI conditions, promote AT2 regeneration and repair, effectively improve lung function, and reduce the inflammatory response of damaged lungs, thus exhibiting a good therapeutic effect on ALI.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a polypeptide SRD in the preparation of drugs for treating acute lung injury. Background Technology

[0002] Acute lung injury (ALI) and its progressive stage, acute respiratory distress syndrome (ARDS), are acute disruptions of the alveolar-capillary barrier caused by various direct (pneumonia, aspiration) or indirect (sepsis, trauma, pancreatitis) factors. Pathologically, it is characterized by diffuse alveolar damage, manifested as pulmonary congestion, interstitial and alveolar edema, and infiltration of numerous inflammatory cells (neutrophils, macrophages). In severe cases, hyaline membranes form within the alveolar spaces (due to protein exudation). ALI / ARDS is a relatively common respiratory critical illness in clinical practice, mainly presenting as uncontrollable hypoxemia and respiratory distress.

[0003] Despite significant progress in the pathogenesis of ALI / ARDS in recent years, leading to the development of various new prevention and treatment measures, the clinical efficacy of treatment has not improved significantly, and the mortality rate remains high. Current treatments for ALI / ARDS mainly fall into two categories: supportive physical therapy (mechanical ventilation, prone positioning, extracorporeal membrane oxygenation, and conservative fluid therapy) and drug therapy (glucocorticoids, nitric oxide (NO), anticoagulation, and anti-cytokine monoclonal antibodies, etc.). Mechanical ventilation is one of the main treatments for ARDS, but it can lead to ventilator-induced lung injury (VILI) and oxygen toxicity, thereby exacerbating pulmonary and systemic inflammatory responses, accelerating multiple organ failure and death. In terms of drug therapy, corticosteroids have become an important treatment for ARDS; moderate doses of corticosteroids can improve the duration of mechanical ventilation, length of hospital stay, or blood oxygen saturation. However, prolonged use of steroids inevitably leads to secondary infections due to weakened immunity. Phosphodiesterase (PDE) inhibitors have broad effects, including anti-inflammatory, antioxidant, and anti-edema effects; however, clinical studies on the use of PDE inhibitors are scarce, and their efficacy is uncertain. Similarly, many other drugs with good preclinical therapeutic effects, such as statins, beta-agonists, nonsteroidal anti-inflammatory drugs (NSAIDs), antioxidants, exogenous surfactants, neutrophil elastase inhibitors, anticoagulants, and anti-TNF biologics, despite strong pathophysiological principles and preclinical data confirming their effectiveness, have failed in clinical trials. Therefore, new treatment methods are urgently needed to improve the clinical outcomes of ALI / ARDS.

[0004] When pathogenic factors induce ALI, lung macrophages are activated, recruiting neutrophils and circulating macrophages into the lesion, releasing a large number of inflammatory factors and mediators. These inflammatory factors or mediators can induce apoptosis and necrosis of lung structural cells, especially alveolar epithelial cells, with the most critical being the apoptosis and necrosis of type II alveolar epithelial cells (AT2). Multiple studies have shown that inhibiting excessive inflammatory responses in the lungs and promoting the regeneration and repair of AT2 cells are key to the treatment of ALI / ARDS. In addition to the direct damage of inflammatory factors to alveolar epithelial cells, inflammation can also indirectly induce oxidative stress damage in alveolar epithelial cells, leading to mitochondrial oxidative stress and dysfunction, further accelerating their necrosis and apoptosis. The reason for the failure of traditional drugs is that they cannot simultaneously block multiple inflammatory signals and only target the inflammatory response, neglecting to combat oxidative damage. More importantly, these drugs do not target improving mitochondrial function to alleviate and prevent lung cell damage. Given the current in-depth research on the pathogenesis of ALI / ARDS, oxidative stress leads to damage to the structure and function of lung parenchymal cells, such as lung epithelial cells and endothelial cells, resulting in mitochondria, impaired ATP production, and insufficient cellular energy supply, ultimately leading to apoptosis and necrosis—the core pathological mechanism of ALI lung injury. Therefore, targeting mitochondria for antioxidant stress treatment, which can fundamentally improve mitochondrial dysfunction and alleviate lung cell damage, may be a novel strategy for treating ALI / ARDS. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, the main objective of this invention is to provide the application of a peptide SRD in the preparation of a drug for treating acute lung injury, wherein the peptide SRD serves as a novel clinical candidate drug for ALI / ARDS, bringing hope to ALI / ARDS patients.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] Application of peptide SRD in the preparation of drugs for treating acute lung injury.

[0008] The sequence of the peptide SRD is KPPSLSYRCPCRFFESHGGSGGGRGDVY.

[0009] In some specific embodiments, the acute lung injury is lipopolysaccharide-induced acute lung injury.

[0010] Description of the drug:

[0011] Preferably, the drug further includes a pharmaceutically acceptable carrier, which includes (but is not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, binders, sprays, transdermal absorbents, humectants, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, or adsorbents.

[0012] The drug of the present invention can be prepared into various dosage forms as needed, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

[0013] The administration route of the drug of the present invention is not limited, as long as it can achieve the desired therapeutic or preventive effect, including but not limited to oral, intravenous, intramuscular, subcutaneous, sublingual, rectal, nasal spray, oral spray, local or systemic transdermal administration.

[0014] The drug of this invention can also be used in combination with other drugs for treating acute lung injury, and the combined use of multiple drugs can greatly improve the success rate of treatment.

[0015] Compared with the prior art, the present invention has at least the following advantages:

[0016] 1) The application provided by this invention verifies the protective effect of peptide SRD in acute lung injury (ALI) through in vivo experiments, especially its effect on improving AT2 mitochondrial function, thereby reducing its damage (decreasing apoptosis rate). AT2 is an important cell in the damaged lung and a key cell in the progression of ALI. The application of peptide SRD can reduce the oxidative stress damage of AT2 mitochondria in the oxidative stress microenvironment of ALI. Secondly, as an antioxidant peptide, the therapeutic application of peptide SRD can improve the high oxidative stress state of ALI lung tissue and reduce oxidative damage to lung cells other than AT2 cells, such as lung endothelial cells. This peptide can improve and enhance the enzyme activity of mitochondrial antioxidant enzyme SOD2 (reducing its acetylation level and conversely increasing the total protein and deacetylation level of SOD2), that is, reducing the level of reactive oxygen species in mitochondria, protecting mitochondrial structure and function, and reducing apoptosis in the mitochondrial pathway.

[0017] 2) The application provided by this invention verifies the protective effect of peptide SRD in acute lung injury (ALI) through in vivo experiments. In an ALI animal model induced by LPS (5 mg / mL) nebulized inhalation, it was found that the protein concentration and total cell count in the bronchoalveolar lavage fluid of mice with acute lung injury injected with 100 μM peptide SRD via tail vein were significantly reduced. Pathological observation showed that the degree of lung injury in mice was significantly reduced, the histological score was lower, the SOD2 acetylation level (an indicator of lung injury severity) was significantly decreased while the total SOD2 protein level was significantly increased, and the SPC level (an indicator of AT2 survival) was significantly increased. ELISA detection showed a significant decrease in the levels of IL-6, IL-1β, and TNF-α (indicators of pneumonia response in mice). These experimental results confirm that the antioxidant peptide SRD can improve oxidative stress damage in ALI, effectively reduce AT2 cell damage under ALI conditions, and promote AT2 regeneration and repair.

[0018] 3) The polypeptide SRD in this invention is a short peptide drug with high biological activity and good safety; it is easy to convert into a drug, its indications have strong expandability, and its application prospects are bright. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0020] Figure 1 The effect of different concentrations of peptide SRD on cell viability and proliferation of alveolar type 2 epithelial cells (MLE-12 cells) after 24 hours of pretreatment;

[0021] Figure 2 The effect of 50 μM peptide SRD pretreatment of MLE-12 on the antioxidant and anti-apoptotic activities of cells; among which Figure 2 a is a cell state diagram. Figure 2 b is a statistical graph of apoptosis measured by flow cytometry. Figure 2 c is a flow cytometry graph of apoptosis.

[0022] Figure 3 The effect of SRD pretreatment of MLE-12 with peptides on ATP, a mitochondrial energy metabolism indicator in the cellular oxidative stress microenvironment.

[0023] Figure 4 This is a diagram showing the inflammatory cell counting experiment of the peptide SRD in the bronchoalveolar lavage fluid of lung tissue after ALI modeling in mice.

[0024] Figure 5 The figure shows the effect of peptide SRD on the inflammatory cell count (left) and protein concentration (right) of bronchoalveolar lavage fluid in lung tissue after mouse ALI modeling.

[0025] Figure 6The effect of peptide SRD on the expression level of total SOD2 protein in lung tissue after mouse ALI modeling;

[0026] Figure 7 The effect of peptide SRD on the expression of total SOD2 protein acetylation level in lung tissue after mouse ALI modeling;

[0027] Figure 8 The effect of peptide SRD on the expression of SPC protein, an AT2 survival marker in lung tissue, after mouse ALI modeling;

[0028] Figure 9 This is a gross image of lung tissue after ALI modeling of the peptide SRD in mice.

[0029] Figure 10 The image shows the HE staining and tissue damage scoring results of the peptide SRD on lung tissue after mouse ALI modeling.

[0030] Figure 11 The effect of peptide SRD on IL6, an inflammatory marker in lung tissue, after mouse ALI modeling;

[0031] Figure 12 The effect of peptide SRD on lung tissue inflammatory markers IL-1β and TNFα after mouse ALI modeling. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the scope of protection of this invention. Any formal equivalent modifications made based on the concept of this invention should be considered within the scope of this invention.

[0033] The abbreviations used in this invention have the following meanings:

[0034] SOD2, superoxide dismutase 2, LPS, lipopolysaccharide

[0035] BALF (Bronchoalveolar Lavage Fluid), SPC (Sterile Protein C), and pulmonary surfactant protein C

[0036] IL-6, Interleukin-6, IL-1β, Interleukin-1β

[0037] TNFα, tumor necrosis factor-α, PMN, neutrophils

[0038] Among them, peptide SRD is a fusion peptide that can enhance cell targeting / anti-apoptosis.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] In the following examples, the peptide SRD solution used was prepared by the following method:

[0041] 10.0 mg of peptide SRD powder (the sequence of the peptide SRD is shown in SEQ ID NO.1, specifically KPPSLSYRCPCRFFESHGGSGGGRGDVY(SED), which was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.) was weighed and added to 2.64 mL of physiological saline (stock concentration 1500 μmol), thoroughly shaken and mixed, and then filtered through a 0.22 μm microporous membrane to obtain a sterile peptide SRD solution. Finally, 200 μL (blood drug concentration 100 μmol) was injected into each mouse via the tail vein for in vivo experiments.

[0042] Example 1: Effects of peptide SRD on cell-related functions of alveolar type II epithelial cell line (MLE-12)

[0043] This embodiment assesses the effects of pretreatment of MLE-12 with peptide SRD on cell proliferation, oxidative stress, and mitochondrial function. The specific testing methods are as follows:

[0044] 1) Effects of different concentrations of peptide SRD on the proliferation of alveolar type II epithelial cell line (MLE-12)

[0045] MLE-12 cells were seeded in 96-well plates at a density of 1 × 10⁶ cells / well. 4 Cells / well were stimulated with different concentrations of peptide SRD (0, 25, 50, 100 μM) for 24 hours. After 24 hours, cell proliferation was detected using a CCK-8 assay kit. The results are as follows: Figure 1 As shown, after pretreating MLE-12 cells with different concentrations of peptide SRD for 24 hours, there was no significant decrease in MLE-12 cell viability and cell proliferation (P>0.05), indicating that peptide SRD does not affect the viability and proliferation capacity of MLE-12 cells.

[0046] 2) Enhancement of cellular antioxidant and anti-apoptotic activity by pretreatment with 50 μM peptide SRD on MLE-12.

[0047] MLE-12 cells were seeded in 6-well plates in groups (NC group, SRD group, 500μM H2O2 group, and SRD+H2O2 group) at a seeding density of 4×10⁻⁶ cells / well. 5Cells / well. After 24 hours of cell culture, the SRD+H2O2 group was pretreated with 50 μM peptide SRD for 12 hours. Then, each group was further stimulated with the corresponding concentrations of peptide (50 μM) and H2O2 (500 μM) for another 24 hours. Microscopic observation at 200× revealed no abnormal cell morphology in the NC and SRD groups. The H2O2-only stimulation group showed significant vacuolation, irregular cell edges, and an increased number of suspended cell debris. In contrast, the SRD+H2O2 group, after SRD pretreatment, showed a significant reduction in vacuolation, more regular cell edges, and maintained a certain "paving stone" normal cell state. Figure 2 a).

[0048] Cells were harvested, and apoptosis was detected using an apoptosis kit. The results are as follows: Figure 2 As shown in (2b and 2c) (where ***P<0.001, **P<0.01): the apoptosis rate of cells in the NC group and the SRD group was low, and there was no significant difference between the two groups. In contrast, the H2O2 group showed a large number of apoptosis, with an apoptosis rate of 99%. The results of the SRD+H2O2 group showed that the apoptosis rate was significantly reduced, indicating that SRD pretreatment of MLE-12 can significantly enhance the cells' ability to resist oxidative stress and apoptosis.

[0049] 3) The protective effect of peptide SRD pretreatment of MLE-12 on mitochondrial energy metabolism marker ATP in the cellular oxidative stress microenvironment.

[0050] The experimental grouping, plating density, and cell culture were the same as in point 2) of Example 1, with the addition of a 400 μM H2O2 group and an SRD+400 μM H2O2 group. Changes in cellular ATP expression were detected using an ATP assay kit after cell harvesting. Results are as follows: Figure 3 As shown (where **P < 0.01, *P < 0.05): there was no significant difference in ATP expression between the NC group and the SRD group, indicating that SRD pretreatment alone did not significantly inhibit mitochondrial ATP levels in MLE-12 cells. Stimulation with 400 μM and 500 μM H2O2 significantly decreased cellular ATP and inhibited mitochondrial function. However, pretreatment with SRD for 12 hours significantly upregulated ATP in both groups of oxidative stress-damaged cells, and effectively restored mitochondrial function. This suggests that SRD pretreatment can significantly enhance the antioxidant capacity of MLE-12 cells, restore ATP synthesis in the oxidative stress microenvironment, and protect mitochondrial function.

[0051] In summary, the peptide SRD pretreatment of this application can promote ATP production and reduce apoptosis rate in MLE-12 cells under oxidative stress. This application is based on a mitochondrial protection strategy to fundamentally reduce lung injury and secondary inflammatory response in the lungs. By reducing mitochondrial damage in lung cells, it can reduce lung AT2 cell damage and enhance their repair function. At the same time, reducing mitochondrial damage can reduce the release of DAMP molecules (mitochondrial DNA, RNA, mitochondrial antiviral signaling proteins, cardiolipin, N-formyl peptide, and other mitochondrial components) from mitochondria.

[0052] Example 2: Effects of peptide SRD on lung tissue-related parameters after mouse ALI modeling

[0053] This embodiment evaluates the efficacy of peptide SRD in treating LPS nebulized inhalation-induced ALI in mice. The specific testing method is as follows:

[0054] Six- to eight-week-old male C57BL / 6 mice, weighing approximately 20g, were selected. Mice were randomly divided into three groups of five mice each: a control group (NC / Control group), an LPS-induced injury group (LPS group), and an LPS-induced injury followed by peptide therapy (SRD treatment group). In the LPS and SRD treatment groups, mice received a single ultrasonic nebulization inhalation of 5 mg / mL LPS (O55:B5, sigma) for 30 minutes. Twenty-four hours after LPS injury, mice in the SRD treatment group received a tail vein injection of 200 μL of SRD solution (100 μM blood concentration). The control group received only a tail vein injection of the same volume of saline as the SRD treatment group.

[0055] Tissue samples were collected 2 and 5 days after LPS-induced injury. The right lung was used for lavage, and the protein concentration and inflammatory cell (PMN) count in BALF were measured. The left lung underwent routine paraffin embedding, sectioning (for subsequent HE staining), and lung tissue protein extraction to detect the expression of SPC, total SOD2 protein, SOD2 acetylation level, and inflammatory cytokines IL-6, IL-1β, and TNF-α.

[0056] The specific test results are as follows:

[0057] 1) Changes in BALF protein concentration and inflammatory cell count after LPS nebulized inhalation injury-induced ALI treated with peptide SRD

[0058] Bronchoalveolar lavage was performed 2 and 5 days after LPS-induced injury. The specific method was as follows: lavage was performed three times with 1 mL of normal saline via endotracheal intubation. The collected bronchoalveolar lavage fluid was centrifuged at 3000 rpm / min and 4℃ for 10 minutes. The sediment was collected, resuspended in 200 μL of PBS, and 20 μL of the cell suspension was imaged on a fixed-area slide, stained with Wright's Giemsa stain, and the total number of nucleated cells was counted in 10 randomly selected fields under a 200× microscope. Figure 4 and Figure 5 As shown in a; the protein concentration of the supernatant was determined using the BCA method ( Figure 5 (b) As shown in the figure (where ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05): On day 2 after LPS-induced injury, the BALF protein concentration and cell count in the LPS group were significantly increased compared with the control group. At the same time point, in the SRD-treated group, the protein concentration and inflammatory cell count were significantly decreased compared with the LPS group, but still significantly higher than the control group. On day 5 after LPS injury, the protein concentration and inflammatory cell count in both the LPS and SRD-treated groups returned to normal levels. This suggests that in the early stages of ALI, SRD can significantly reduce the inflammatory response in damaged lungs and has a significant therapeutic effect on ALI.

[0059] 2) Effects of peptide SRD on the expression of total SOD2 protein and acetylation levels in lung tissue after ALI treatment

[0060] Two and five days after LPS-induced injury, right lung tissue was harvested from mice. The mice were divided into a control group, an LPS group, and an SRD treatment group, with five replicates in each group. Each sample was added to 500 μL of lysis buffer (100×RIPA + 1×PMSF), ground at -20°C for 90 seconds, and then lysed on ice for another 30 minutes. Total protein was extracted from each group, and protein concentration was determined using a BCA kit. 30 µg of total protein from each group was loaded onto a PVDF membrane, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred wet-on-wet. The membrane was then blocked with 5% skim milk-PBS for 1 hour. The following antibodies were added: SOD2, SOD2-Ace (1:1000 dilution), and internal control β-actin (1:5000 dilution), and incubated overnight on a shaker at 4°C. The next day, the membrane was washed, and secondary antibody (goat anti-rabbit, 1:5000 dilution) was added and incubated at room temperature for 1 hour. Horseradish peroxidase-labeled enhanced chemiluminescence (ECL reagent) was used for color development. The PVDF membrane was immersed in the developing solution, and images were acquired using a gel imaging system. ImageJ software was used to analyze the grayscale values ​​of the target protein and the internal control β-actin bands, and the ratio of the two bands represented the relative expression level of the target protein.

[0061] The results are as follows Figure 6 and Figure 7As shown (where ***P < 0.001, **P < 0.01): Compared with the control group, the total SOD2 protein level in the LPS group was significantly lower on day 2 after LPS injury (where ***P < 0.001, **P < 0.01). Figure 6 ), while SOD2 acetylation levels (SOD2-Ace) were significantly upregulated ( Figure 7 Compared with the LPS group, at the same number of days after LPS injury, the SRD treatment group showed significantly higher total SOD2 protein levels and significantly lower SOD2-Ace levels compared to the LPS group. These results suggest that LPS-induced injury in ALI mice leads to impaired mitochondrial degradation or synthesis, decreased lung enzyme activity, and hindered superoxide anion clearance. Treatment with peptide SRD significantly reduced mitochondrial damage indicators, increased lung enzyme activity, promoted superoxide anion clearance, and reduced oxidative stress damage in lung tissue. This indicates that peptide SRD can protect lung tissue, resist oxidative stress damage, and reduce mitochondrial damage in ALI, potentially improving the prognosis of ALI patients.

[0062] 3) Effect of peptide SRD on the expression of SPC protein, an AT2 survival marker in lung tissue, after ALI treatment

[0063] Two and five days after LPS-induced injury, samples were taken from the right lung of mice. After sample processing according to point 2) of Example 2, the following antibodies were added: SPC (1:800 dilution) and internal control β-Actin (1:5000 dilution). The samples were incubated overnight at 4°C on a shaker. The next day, the membrane was washed, and secondary antibody (goat anti-rabbit, 1:5000 dilution) was added. The membrane was incubated at room temperature for 1 hour, washed again, and then developed using a spectrophotometer. Results are as follows: Figure 8 As shown (where ***P<0.001, **P<0.01, *P<0.05): Compared with the control group, the SPC protein expression in the LPS group was significantly decreased on day 2 after LPS-induced injury, and returned to normal levels on day 5; the SRD treatment group showed significantly higher SPC protein expression on day 2 after LPS-induced injury than both the LPS group and the control group, and continued to show an upregulation trend on day 5 after injury. This indicates that peptide SRD can not only effectively resist ALI-induced AT2 damage, but also effectively promote the proliferation, regeneration, and repair of AT2 after ALI, which is key to ALI treatment.

[0064] 4) Changes in lung histology after ALI treated with peptide SRD

[0065] Processing of paraffin-embedded tissue specimens: During the experiment, lung tissue was removed from experimental mice and cut in half along its long axis. The half of the lung tissue closest to the hilum was placed in 4% paraformaldehyde solution and fixed at 4°C for 24 hours. The next day, the fixed lung tissue underwent dehydration treatment, specifically as follows: the tissue was sequentially immersed in ethanol solutions of different concentrations: 75% ethanol for 60 minutes, 85% ethanol for 60 minutes, 90% ethanol for 60 minutes, 95% ethanol for 60 minutes, then in another bottle of 95% ethanol for 60 minutes, 100% ethanol for 60 minutes, and then in another bottle of 100% ethanol for 60 minutes. Subsequently, the tissue was cleared by immersing it in xylene for 120 minutes, and finally, it was sequentially immersed in two bottles of paraffin solution at 62°C for 60 minutes each, completing the pre-embedding treatment. The treated tissue was placed in a paraffin-containing mold, and its position was adjusted to ensure proper placement (see the general diagram of the lung tissue as shown). Figure 9 (As shown). The tissue was then placed on the cooling platform of a paraffin embedding machine for cooling. After cooling, the tissue was cut into 4μm thick slices, which were then placed in a slide bleaching machine to fully unfold. Next, the unfolded slices were gently lifted with tweezers and placed on glass slides pre-coated with poly-L-lysine. The slides were numbered with a marker for subsequent identification and manipulation. The slides were then placed in a 70°C oven for drying for 1 hour to ensure close adhesion between the tissue slices and the slides.

[0066] Following this, the slides were placed in a 60°C oven and baked for another hour. After completing the above steps, the slides were removed from the oven and kept for subsequent experimental use. For HE staining of mouse lung tissue paraffin sections: the slides were placed in a 60°C oven and baked for 30 minutes. Immediately after removing the slides from the oven, they were immersed in xylene I for 15 minutes → xylene II for 15 minutes → anhydrous ethanol for 5 minutes → 95% ethanol for 5 minutes → 75% ethanol for 5 minutes → distilled water for 5 minutes. Staining: Hematoxylin staining for 45 seconds → rinse with tap water for 5 minutes (observe under a microscope to determine rinsing time and whether to counterstain) → rinse with distilled water → differentiate with hydrochloric acid ethanol for 1-2 seconds → rinse with tap water for 5 minutes (observe under a microscope to determine rinsing time and whether to counterstain) → rinse with distilled water → eosin staining for 2-3 seconds → rinse with tap water for 5 minutes (depending on the intensity of eosin staining, decide whether to place in anhydrous ethanol) → dry the slide with a hairdryer → xylene I for 2-3 minutes → xylene II for 5 minutes. Mount the slide with neutral resin and cover with a coverslip. Observe the lung histological changes on days 2 and 5 after peptide SRD treatment for ALI under a microscope (magnification: HE×400). Results are as follows: Figure 10As shown on the left (where ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05): HE staining of pathological sections from mice in the control group showed intact alveolar structure, no abnormal widening or edema of the alveolar septa, and no obvious exudation in the alveolar cavities, which is the lung tissue manifestation under normal physiological conditions. After LPS-induced injury, the lung tissue of ALI mice showed typical pathological changes of acute lung injury: alveolar structure destruction, significant widening and edema of the alveolar septa, and a large number of inflammatory cell infiltrations and protein exudates in the alveolar cavities, especially on the second day after LPS induction, the pathological manifestations were the most severe. After treatment with peptide SRD, the pathological damage of mouse lung tissue was significantly reduced, showing that the alveolar structure was more intact, the degree of alveolar septal widening and edema was reduced, and the inflammatory cell infiltration and exudate in the alveolar cavities were significantly reduced. On the fifth day after LPS-induced injury, the lung histological structure was close to the normal level. Lung tissue injury in mice was scored according to the following four scoring criteria: (1) Alveolar wall thickening: 0 points: none; 1 point: focal; 2 points: multifocal; 3 points: diffuse mild; 4 points: diffuse severe. (2) Alveolar hemorrhage / exudation: 0 points: none; 1 point: small amount; 2 points: moderate; 3 points: extensive; 4 points: diffuse with structural destruction. (3) Inflammatory cell infiltration: 0 points: none; 1 point: small amount; 2 points: moderate; 3 points: dense focal; 4 points: diffuse infiltration. (4) Alveolar structural destruction: 0 points: none; 1 point: <10%; 2 points: 10-25%; 3 points: 25-50%; 4 points: >50%. The sum of the scores for the four scoring items is the final score for lung tissue injury. The results are as follows: Figure 10 As shown in the right figure, the peptide SRD treatment for ALI can significantly reduce the damage manifestations of LPS-induced lung tissue, such as alveolar exudation and septal widening, thereby effectively reducing the pathological damage of ALI.

[0067] 5) Changes in inflammatory cytokines in lung tissue after ALI treatment with peptide SRD

[0068] Lung tissue samples were collected from mice 2 and 5 days after LPS-induced injury. The mice were divided into a control group, an LPS group, and an SRD treatment group, with five replicates in each group. Each sample was weighed, and lysis buffer (100×PBS + 1×PMSF) was added at 10 times its body weight. The samples were then homogenized at -20°C for 90 seconds and continued lysis on ice for 30 minutes. The levels of lung inflammatory cytokines IL-6, IL-1β, and TNFα were detected using an ELISA kit. Results are as follows: Figure 11 and Figure 12 As shown (where ***P<0.001, **P<0.01, *P<0.05): On day 2 after LPS-induced injury, the level of the inflammatory cytokine IL-6 in the lung tissue of the LPS group was significantly lower than that of the lung tissue of the LPS group. Figure 11 ), TNFα ( Figure 12a) and IL-1β Figure 12 b) The expression levels of these substances were significantly higher than those in the control group; after treatment with peptide SRD, the levels of IL-6, IL-1β, and TNFα in lung tissue were significantly reduced. This suggests that peptide SRD treatment can effectively reduce the degree of inflammatory response in lung tissue.

[0069] In summary, compared with the ALI group, the SRD peptide treatment group showed a significant reduction in gross pulmonary congestion area, pathological observation revealed alveolar wall thickening, increased hyaline membrane area, and decreased related pathological scores, as well as a significant decrease in the number and protein concentration of inflammatory cells in BALF. Simultaneously, the treatment group showed a significant increase in total SPC and SOD2 protein levels in lung tissue and a decrease in SOD2 acetylation levels, suggesting that SRD peptide can enhance AT2 regeneration and repair and promote the recovery of antioxidant enzyme activity (SOD2 deacetylation) in lung cell mitochondria.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. The application of polypeptide SRD in the preparation of drugs for treating acute lung injury, characterized in that, The sequence of the peptide SRD is KPPSLSYRCPCRFFESHGGSGGGRGDVY.

2. The application according to claim 1, characterized in that, The acute lung injury mentioned is lipopolysaccharide-induced acute lung injury.

3. The application according to claim 1 or 2, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

4. The application according to claim 3, characterized in that, The drug includes one of the following: tablets, solutions, granules, ointments, or capsules.

5. The application according to claim 4, characterized in that, The drug can be administered via intravenous injection.

Images