A rap polypeptide analogue and medical uses thereof

Abstract

The application discloses a RAP polypeptide analogue and medical use thereof, and belongs to the polypeptide medical field, and the amino acid sequence of the RAP polypeptide analogue is Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17, and the N terminal of the RAP polypeptide analogue is acetylated, and the C terminal amino acid is amidated. Compared with the natural RAP polypeptide, the analogue has higher stability, bioavailability and drug efficacy, meanwhile, the dosage is low, the toxic and side effects are not obvious, the production cost is low, and the analogue has good popularization and clinical application prospects. The analogue is suitable for preventing and treating acute and chronic nephritis, chronic nephropathy and renal fibrosis caused by the acute and chronic nephritis, pulmonary inflammation and pulmonary fibrosis caused by the pulmonary inflammation, chronic liver disease and liver fibrosis caused by the chronic liver disease and the like.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide medicine, specifically relating to a RAP polypeptide analog and its pharmaceutical uses. Background Technology

[0002] Chronic kidney disease (CKD) is a progressive disease characterized by excessive extracorporeal membrane (ECM) deposition, manifesting as persistent abnormalities in kidney structure, function, or both. Structural abnormalities include cysts, tumors, malformations, and atrophy, while functional abnormalities include hypertension, edema, changes in urine, and growth retardation in children. CKD is a significant public health problem worldwide. Statistics show that in 2023, there were 788 million adults with CKD globally, a significant increase from 697.5 million in 2017, and the incidence rate is rising annually, making it the ninth leading cause of death globally. As CKD is more common in the elderly, its burden will increase with the aging of the global population. The U.S. Centers for Disease Control and Prevention predicts that 7% of elderly CKD patients will eventually develop end-stage renal disease, requiring dialysis or kidney transplantation for survival.

[0003] Renal fibrosis is the common final outcome of almost all chronic and progressive kidney diseases. Its pathological features include glomerular sclerosis and interstitial fibrosis, involving almost all cell types in the kidney and ultimately leading to kidney failure. Renal fibrosis is difficult to detect, and there are currently no effective drugs for treatment. Existing treatment options are mostly limited to dialysis and kidney transplantation, which seriously affects patients' quality of life and places a huge burden on society and families.

[0004] Pulmonary fibrosis is the end-stage pathological change in the lungs of various interstitial lung diseases (ILDs). ILDs refer to a group of diffuse parenchymal lung diseases with different clinical, imaging, and pathological manifestations, encompassing more than 200 parenchymal lung diseases. ILDs that can eventually progress to pulmonary fibrosis include idiopathic pulmonary fibrosis (IPF), connective tissue disease-related ILDs (such as rheumatoid arthritis-related ILD, systemic sclerosis-related ILD, etc.), vasculitis-related ILDs (such as hypersensitivity pneumonitis), and some unclassifiable ILDs. Pulmonary fibrosis is mainly characterized by excessive deposition of ECM proteins in the lung interstitium after lung parenchymal damage, leading to alveolar structure destruction, decreased compliance and gas exchange, and impaired lung function. Clinically, patients mainly present with dry cough and progressive dyspnea, eventually dying from respiratory failure. IPF is the most common type of ILD, accounting for approximately 17%-37% of ILD diagnoses, with a global incidence of 5-15 cases per 100,000 people per year, and this is still increasing. In the United States, the median survival for IPF in patients over 65 years of age is only 3.8 years. The nonspecific symptoms in the early stages of pulmonary fibrosis make diagnosis difficult, and it is often discovered in its end stage, making timely intervention impossible. Pulmonary fibrosis has become a significant public health problem.

[0005] Liver fibrosis is a complex pathological process involving various pathogenic factors that lead to abnormal proliferation of connective tissue in the liver. It involves multiple cells, protein factors, and signal transduction pathways. The main pathological manifestation is an imbalance between ECM synthesis and degradation, resulting in ECM deposition in the liver, including collagen fibers (types I, III, and IV collagen), forming fibrous scars that cause structural changes in the liver and affect liver function. Pathologically, this disease can be classified as a chronic fibrotic inflammatory disease. According to 2017 statistics, 1.5 billion people worldwide suffer from chronic liver disease (CLD), the most common being non-alcoholic fatty liver disease (NAFLD) accounting for 60%, hepatitis B virus (HBV) for 29%, hepatitis C virus (HCV) for 9%, and alcoholic liver disease (ALD) for 2%. Approximately 2 million people die from chronic liver disease worldwide each year, making it a major global public health problem. Liver fibrosis, as an important pathological feature of chronic liver disease, is often asymptomatic and difficult to detect. If left untreated, it can lead to permanent scarring and organ dysfunction. Prolonged illness can progress to cirrhosis, which may then develop into liver cancer and ultimately death. However, there are currently no effective treatments available clinically.

[0006] Existing research, focusing on the commonalities in the pathological manifestations of fibrosis across different organs, has primarily concentrated on targeting molecules and signaling pathways related to ECM generation in the treatment of acute and chronic kidney inflammation and its induced CKD and renal fibrosis, lung inflammation and its induced pulmonary fibrosis, and chronic liver disease and its induced liver fibrosis. Numerous preclinical and clinical trial results have also indicated that novel therapeutic strategies such as modulating inflammatory responses, inhibiting pro-fibrotic growth factors, and targeting epigenetic alterations can alleviate the progression of fibrosis. Furthermore, some treatments initially thought to primarily regulate molecular pathways directly related to fibrosis can also modulate other processes, such as inflammatory responses, making it difficult to determine which factors and pathways dominate the treatment of renal fibrosis, pulmonary fibrosis, and liver fibrosis.

[0007] Experimental studies have shown that the receptor for advanced glycation end products (RAGE) is significantly upregulated in the kidney, lung, and liver tissues of patients with fibrosis. RAGE is primarily highly expressed in lung tissue, but can also be expressed in hepatic sinusoidal endothelial cells, hepatic stellate cells, and proliferating bile duct epithelial cells. It is also widely expressed in the kidneys, particularly in fibroblasts, glomerular podocytes, epithelial cells, endothelial cells, and mesangial cells. Numerous studies have reported its close association with various kidney, lung, and liver diseases at different levels. However, there are currently no reports on targeting RAGE receptors for the treatment of organ fibrosis. RAP is a RAGE antagonist peptide obtained by comparing the amino acid sequences of RAGE ligands HMGB-1 and S100P. In vivo and in vitro, it competitively blocks S100P, S100A4, and HMGB-1-mediated RAGE activation, inhibiting tumor growth and metastasis (Clin Cancer Res. 2012 Aug 15;18(16):4356-64.). Summary of the Invention

[0008] Objective of the Invention: Addressing the problems existing in the prior art, the objective of this invention is to provide a class of RAP peptide analogs or pharmaceutically acceptable salts thereof. Using RAP as the parent peptide, structural modification and sequence optimization yield a series of peptide analogs with good biological activity. These analogs, based on the parent peptide RAP, exhibit enhanced activity, improved stability, and lower in vitro and in vivo toxicity. Subsequent mechanistic studies revealed that they can target RAGE, thereby inhibiting a series of subsequent biological effects, ultimately alleviating the development of renal fibrosis, pulmonary fibrosis, and hepatic fibrosis in vitro and in vivo.

[0009] The present invention also provides pharmaceutical compositions of the aforementioned RAP polypeptide analogues and their pharmaceutical uses.

[0010] Technical solution: The RAP polypeptide analog or a pharmaceutically acceptable salt thereof of the present invention, wherein the amino acid sequence of the RAP polypeptide analog is as follows:

[0011] Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17, and the N-terminus of the RAP peptide analog is acetylated, while the C-terminal amino acid is amidated; among them...

[0012] Xaa1 is selected from Glu, Ala, Leu, D-Glu, Ser, Thr, Asp, Dap, or NMe-Glu;

[0013] Xaa2 is selected from Leu, Ala, D-Leu, Glu, or NMe-Leu;

[0014] Xaa3 is selected from Lys, Ala, D-Lys, Dap, Sar, Aib, D-Ala, Gly, Abu, or NMe-Lys;

[0015] Xaa4 is selected from Val, Ala, D-Val, Glu, Gly, Sar, Aib, Abu, Nva, OctGly, or NMe-Val;

[0016] Xaa5 is selected from Leu, Ala, D-Leu, Met, Asp, Glu, Ser, Lys, Dap, Sar, Aib, Me-Lys, Orn, P-Ser, Asn, Cit, or NMe-Lys;

[0017] Xaa6 is selected from Met, Ala, D-Met, Leu, Phe, Val, Glu, α-(4-Pentenyl)-Ala, D-Ala, Sar, Dap, Aib, Abu, ALGly, OcAla, Nle, or NMe-Met;

[0018] Xaa7 is selected from Glu, Ala, D-Glu, Val, D-Ala, Sar, Dap, Aib, Abu, Pra, D-Pra, or NMe-Glu;

[0019] Xaa8 is selected from Lys, Ala, D-Lys, or does not exist;

[0020] Xaa9 is selected from Glu, Ala, D-Glu, or may not exist;

[0021] Xaa10 is selected from Leu, Ala, D-Leu, Glu, or is not present;

[0022] Xaa11 is selected from Pro or does not exist;

[0023] Xaa12 is selected from Gly or does not exist;

[0024] Xaa13 is selected from Phe or does not exist;

[0025] Xaa14 is selected from Leu or does not exist;

[0026] Xaa15 is selected from Gln or does not exist;

[0027] Xaa16 is selected from Ser or does not exist;

[0028] Xaa17 is selected from Gly or does not exist;

[0029] If the 8th amino acid (Xaa8) is missing, then all C-terminal residues following that site in the peptide chain will be absent.

[0030] Furthermore, the amino acid sequence of the RAP polypeptide analog is selected from any of the following:

[0031]

[0032]

[0033]

[0034]

[0035]

[0036] This RAP peptide analog is a novel peptide compound that can improve organ fibrosis and fibrotic symptoms associated with organ diseases. Extensive experimental studies have demonstrated that this peptide compound has no adverse reactions and can be used to treat or improve organ fibrosis and fibrotic symptoms associated with organ diseases, particularly suitable for treating or improving renal fibrosis, pulmonary fibrosis, and hepatic fibrosis and fibrotic symptoms associated with organ diseases.

[0037] The analogues of this invention exhibit significantly enhanced efficacy in the treatment of renal fibrosis: through structural optimization and sequence modification of the parent peptide RAP through multiple generations of analogues, the in vivo therapeutic dose of the final analogue RSM25 was reduced from 5 mg / kg of the parent peptide RAP to 0.5 mg / kg, and the in vitro effective concentration was reduced from 100 μM to 1 μM (10 times in vivo, 100 times in vitro); the in vivo therapeutic dose of DRSM25 was reduced from 5 mg / kg of the parent peptide RAP to 0.1 mg / kg, and the in vitro effective concentration was reduced from 100 μM to 1 μM. The significant reduction in dose (50 times in vivo, 100 times in vitro) confirms that the optimized analogues have stronger anti-fibrotic activity and better therapeutic potential.

[0038] The use of the RAP polypeptide analogue or a pharmaceutically acceptable salt thereof described in this invention in the preparation of medicaments for the prevention and / or treatment of organ fibrosis.

[0039] Furthermore, the organ fibrosis disease is selected from renal fibrosis, pulmonary fibrosis, liver fibrosis, or a combination thereof.

[0040] Preferably, the renal fibrosis includes obstructive nephropathy, diabetic nephropathy, hypertensive nephropathy, nephritis, renal tumors, adverse drug reactions, or pathogenic microbial infections causing renal fibrosis; the pulmonary fibrosis includes pulmonary fibrosis caused by multiple risk factors such as smoking, viral infections, environmental pollution, genetic susceptibility, and medications; and the liver fibrosis includes liver fibrosis caused by non-alcoholic fatty liver disease, hepatitis B, hepatitis C, and alcoholic liver disease.

[0041] Furthermore, the RAP peptide analogue or its pharmaceutically acceptable salt can alleviate and / or inhibit the abnormal expression of fibrin and genes.

[0042] The use of the RAP polypeptide analogue or a pharmaceutically acceptable salt thereof described in this invention in the preparation of medicaments for the prevention and / or treatment of chronic organ parenchymal diseases.

[0043] Furthermore, the chronic organ parenchymal disease is selected from chronic kidney disease, pneumonia, chronic liver disease, or a combination thereof.

[0044] The present invention provides a pharmaceutical composition comprising the above-mentioned RAP polypeptide analog or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0045] Furthermore, the pharmaceutically acceptable carrier is a pharmaceutically acceptable excipient, diluent, or functional excipient.

[0046] Furthermore, the use of the pharmaceutical composition in the prevention and / or treatment of organ fibrosis diseases.

[0047] Furthermore, the formulation may be in the form of tablets, capsules, granules, oral liquids, syrups, pills, ointments and patches for skin application, aerosols, nasal sprays, suppositories, microsphere formulations, injection formulations, or lyophilized powder injections.

[0048] Furthermore, the pharmaceutical composition can be used in any suitable manner for patients in need of treatment.

[0049] Furthermore, the suitable methods include, but are not limited to, oral, rectal, nasal, local (including oral and sublingual), subcutaneous, or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and intrathecal) administration.

[0050] Mechanism of action: The function of the drugs against renal fibrosis, pulmonary fibrosis and liver fibrosis is to inhibit the binding of abnormally activated ligands to receptors on the cell surface, thereby inhibiting downstream pro-fibrotic signaling pathways; the cell surface receptors mainly include the advanced glycation end product receptor RAGE.

[0051] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0052] (1) This invention provides a series of RAP peptide analogs and studies the efficacy of RAP and its analogs, including in vitro activity and cytotoxicity, and in vivo activity assessment. The results show that, compared with the parent peptide RAP, several analogs 5 have better anti-fibrotic activity, thus proving that RAP peptide analogs can be used to prepare drugs for the prevention and / or treatment of renal fibrosis, pulmonary fibrosis and liver fibrosis.

[0053] (2) By modifying the structure and optimizing the sequence of the parent peptide RAP, the present invention has screened multiple analogues with improved activity, enhanced stability and high synthesis yield compared with the parent peptide, which are easy to scale up and greatly reduce production costs.

[0054] (3) Compared with small molecule compounds, analogues are non-toxic and have a large safety window; compared with large molecule proteins, they are more economical to prepare and have greater scalability. Attached Figure Description

[0055] Figure 1 The cytotoxicity of peptide RAP to NIH-3T3 and NRK-52E cells: A: MTT assay of RAP cytotoxicity to NIH-3T3 cells; B: MTT assay of RAP cytotoxicity to NRK-52E cells.

[0056] Figure 2 To assess the anti-renal fibrosis activity of RAP in TGF-β1-induced NIH-3T3 and NRK-52E cell fibrosis models: A: Expression levels of fibrosis marker proteins in TGF-β1-induced NIH-3T3 cells; BD: Relative expression levels of fibrosis marker proteins in NIH-3T3 cells; E: Expression levels of fibrosis marker proteins in TGF-β1-induced NRK-52E cells; FH: Relative expression levels of fibrosis marker proteins in NRK-52E cells; I: Expression levels of fibrosis marker proteins in TGF-β1-induced NRK-52E cells at RAP doses of 50 and 100 μM; FH: Relative expression levels of fibrosis marker proteins in NRK-52E cells at RAP doses of 50 and 100 μM.

[0057] Figure 3 Schematic diagram of the effect of RAP on renal function indicators in mice with UUO-induced renal fibrosis: A: BUN content; B: Scr content;

[0058] Figure 4 Schematic diagram of the expression of renal fibrosis marker proteins induced by RAP and UUO: A: Expression level of renal fibrosis-related proteins; BF: Relative expression levels of renal fibrosis-related proteins;

[0059] Figure 5 This is a schematic diagram illustrating the expression of fibrosis marker genes in kidney tissue induced by RAP and UUO.

[0060] Figure 6 To assess the anti-renal fibrosis activity of RAP analogs in a TGF-β1-induced NRK-52E cell fibrosis model: A, E, I, L: expression levels of marker proteins; B, F, J, M: relative expression levels of the marker Fibronectin; C, G, K, N: relative expression levels of the marker Collagen I; D, H: relative expression levels of the marker α-SMA.

[0061] Figure 7 A schematic diagram showing the effects of RAP and R7p on the fibrosis genes Fn1 and Vim in the NRK-52E cell fibrosis model.

[0062] Figure 8 Schematic diagram of the effect of R7p on renal function indicators in mice with UUO-induced renal fibrosis: A: BUN content; B: Scr content;

[0063] Figure 9 Schematic diagram of R7p-induced renal fibrosis marker protein expression in UUO: A: Expression level of renal fibrosis-related proteins; BG: Relative expression level of renal fibrosis-related proteins;

[0064] Figure 10 This is a schematic diagram illustrating the expression of fibrosis marker genes in kidney tissue induced by R7p and UUO.

[0065] Figure 11 To assess the anti-renal fibrosis activity of R7p analogs in a TGF-β1-induced NRK-52E cell fibrosis model: A, D, G, J, M, P: expression levels of marker proteins; B, E, H, K, N, Q: relative expression levels of the marker Fibronectin; C, F, I, L, O, R: relative expression levels of the marker Collagen I.

[0066] Figure 12 Schematic diagram showing the effects of R7p, R7p-5S, and R7p-5K on the expression of fibrosis marker proteins in the NRK-52E cell fibrosis model: A: Expression level of marker proteins; B: Relative expression level of marker Fibronectin; C: Relative expression level of marker Collagen I.

[0067] Figure 13 R7p, R7p-5S, and R7p-5K are used to investigate the effects of fibrosis genes in the NRK-52E cell fibrosis model. Fn1, Col1a1, VimSchematic diagram of the impact;

[0068] Figure 14 A schematic diagram showing the effects of R7p-5S and R7p-5K on renal function indicators BUN (A, C) and Scr (B, D) in UUO-induced renal fibrosis mice;

[0069] Figure 15 Schematic diagram of the expression of fibrosis marker proteins in renal tissue induced by R7p-5S and R7p-5K: A, G: expression level of fibrosis marker proteins; BF, HL: relative expression level of fibrosis marker proteins;

[0070] Figure 16 A schematic diagram showing the expression of fibrosis marker genes in kidney tissue induced by R7p-5S and R7p-5K in UUO-induced renal tissue.

[0071] Figure 17 To detect the anti-renal fibrosis activity of R7p and R7p-5K analogs in a TGF-β1-induced NRK-52E cell fibrosis model (RDM1-RDM30): A, D, G, J: expression levels of marker proteins; B, E, H, K: relative expression levels of the marker Fibronectin; C, F, I, L: relative expression levels of the marker Collagen I.

[0072] Figure 18 To detect the anti-renal fibrosis activity of R7p and R7p-5K analogs (RSM1-R5K7M) in a TGF-β1-induced NRK-52E cell fibrosis model: A, D, G, J: expression levels of marker proteins; B, E, H, K: relative expression levels of the marker Fibronectin; C, F, I, L: relative expression levels of the marker Collagen I.

[0073] Figure 19 To detect the anti-renal fibrosis activity of R7p and R7p-5K analogs in a TGF-β1-induced NRK-52E cell fibrosis model: A, D, G, J: expression levels of marker proteins; B, E, H, K: relative expression levels of marker Fibronectin; C, F, I, L: relative expression levels of marker Collagen I.

[0074] Figure 20 RSM25 and R5K5M are used to identify fibrosis genes in the NRK-52E cell fibrosis model. Fn1, Col1a1, Vim Schematic diagram of the impact;

[0075] Figure 21 A schematic diagram showing the effect of R5K5M on renal function indicators BUN (A) and Scr (B) in UUO-induced renal fibrosis mice;

[0076] Figure 22 Schematic diagram of R5K5M-induced renal fibrosis marker protein expression in UUO: A: Expression level of fibrosis marker protein; BF: Relative expression level of fibrosis marker protein;

[0077] Figure 23 This is a schematic diagram illustrating the expression of fibrosis marker genes in kidney tissue induced by R5K5M and UUO.

[0078] Figure 24 The effect of RSM25 on renal function indicators BUN (A) and Scr (B) in UUO-induced renal fibrosis mice, and the expression of renal injury marker genes Clu, Havcr1, and Lcn2 are shown in the diagram.

[0079] Figure 25 RSM25 serves as an inflammatory marker for UUO-induced renal fibrosis in mice. Tnf-α, IL-1β and IL-6 Schematic diagram of the impact;

[0080] Figure 26 Schematic diagram of RSM25-induced renal fibrosis marker protein expression: A, B: Expression levels of fibrosis and EMT marker proteins; CG: Relative expression levels of fibrosis marker proteins;

[0081] Figure 27 This is a schematic diagram illustrating the expression of fibrosis marker genes in kidney tissue induced by RSM25 and UUO.

[0082] Figure 28 The diagram shows the effects of RSM25 on renal function indicators BUN (A) and Scr (B) in FA-induced renal fibrosis mice, and the expression of renal injury marker genes Havcr1 and Lcn2.

[0083] Figure 29 RSM25 is an inflammatory marker for FA-induced renal fibrosis in mice. Tnf-α, IL-1β and IL-6 Schematic diagram of the impact;

[0084] Figure 30 Schematic diagram of RSM25-induced renal fibrosis marker protein expression: A, B: expression levels of fibrosis and EMT marker proteins; CG: relative expression levels of fibrosis marker proteins;

[0085] Figure 31 A schematic diagram showing the expression of fibrosis marker genes in kidney tissue induced by RSM25 and FA.

[0086] Figure 32The effect of RSM25 on organ coefficient (lung coefficient) in BLM-induced pulmonary fibrosis mice;

[0087] Figure 33 Schematic diagram of RSM25's effect on the expression of BLM-induced lung fibrosis marker proteins: A, B: Expression levels of fibrosis and EMT marker proteins; CH: Relative expression levels of fibrosis marker proteins;

[0088] Figure 34 This is a schematic diagram showing the effect of RSM25 on the inflammatory markers Tnf-α and IL-1β in BLM-induced pulmonary fibrosis mice;

[0089] Figure 35 This is a schematic diagram showing the expression of fibrosis marker genes in BLM-induced pulmonary fibrosis tissue by RSM25.

[0090] Figure 36 A schematic diagram showing the effect of RSM25 on liver function indicators AST (A) and ALT (B) in CCl4-induced liver fibrosis mice;

[0091] Figure 37 Schematic diagram of RSM25-induced expression of CCl4-induced liver fibrosis marker proteins: A, B: Expression levels of fibrosis and EMT marker proteins; CG: Relative expression levels of fibrosis marker proteins;

[0092] Figure 38 This is a schematic diagram illustrating the effect of RSM25 on the inflammatory markers Tnf-α and IL-6 in CCl4-induced liver fibrosis mice.

[0093] Figure 39 This is a schematic diagram showing the expression of fibrosis marker genes in liver fibrosis tissue induced by RSM25 and CCl4.

[0094] Figure 40 The diagram shows the effects of DRSM25 on renal function indicators BUN (A) and Scr (B) in UUO-induced renal fibrosis mice, and the expression of renal injury marker genes Clu, Havcr1, and Lcn2.

[0095] Figure 41 DRSM25 as an inflammatory marker in mice with UUO-induced renal fibrosis Tnf-α, IL-1β and IL-6 Schematic diagram of the impact;

[0096] Figure 42 Schematic diagram of DRSM25-induced renal fibrosis marker protein expression in UUO-induced renal tissue: A, B: Expression levels of fibrosis and EMT marker proteins; CG: Relative expression levels of fibrosis marker proteins;

[0097] Figure 43 This is a schematic diagram illustrating the expression of fibrosis marker genes in kidney tissue induced by DRSM25 and UUO.

[0098] Figure 44 The effect of DRSM25 on organ coefficient (lung coefficient) in BLM-induced pulmonary fibrosis mice;

[0099] Figure 45 Schematic diagram of DRSM25-induced expression of BLM-induced lung fibrosis marker proteins: A, B: Expression levels of fibrosis and EMT marker proteins; CG: Relative expression levels of fibrosis marker proteins;

[0100] Figure 46 This is a schematic diagram showing the expression of fibrosis marker genes in BLM-induced pulmonary fibrosis tissue by DRSM25.

[0101] Figure 47 A schematic diagram showing the effect of DRSM25 on liver function indicators AST (A) and ALT (B) in CCl4-induced liver fibrosis mice;

[0102] Figure 48 Schematic diagram of DRSM25-induced expression of CCl4-induced liver fibrosis marker proteins: A, B: Expression levels of fibrosis and EMT marker proteins; CG: Relative expression levels of fibrosis marker proteins;

[0103] Figure 49 DRSM25 serves as an inflammatory marker for CCl4-induced liver fibrosis in mice. Tnf-α, IL-6 Schematic diagram of the impact;

[0104] Figure 50 This is a schematic diagram showing the expression of fibrosis marker genes in CCl4-induced liver fibrosis tissue by DRSM25. Detailed Implementation

[0105] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0106] The present invention will be further described below with reference to specific embodiments.

[0107] This invention modifies existing RAP precursor peptides to obtain novel RAP polypeptide analogs. The polypeptide compounds SEQ ID NO:1-123 in the embodiments of this invention can all be directly synthesized artificially using a polypeptide solid-phase method.

[0108] For specific synthesis methods, please refer to the literature: Feng et al. reported the Fmoc-solid phase method, "The CaMKIIInhibitory Peptide AIP Alleviates Renal Fibrosis via TGF-β / Smad and RAF / ERK Pathways," J. Pharmacol. Exp. Ther. 2023, 123, 001621.1; and "Novel Peptide PEP-Z-2 Treats Renal Fibrosis In Vivo and In Vitro by Modulating TGF-β / Smad / AKT / MAPK Signaling," Eur. J. Pharmacol. 2024, 176942. Different designs and preparations can be made according to existing methods.

[0109] Example 1 Synthesis of polypeptide compounds

[0110] For ease of explanation, this embodiment uses the synthesis of the precursor peptide RAP as an example. The polypeptide analog was synthesized using the Fmoc solid-phase synthesis method, purified by reversed-phase HPLC, and characterized by mass spectrometry. The specific synthesis steps are as follows:

[0111] (1) Add dichloromethane to 4-methyldiphenylmethane resin (MBHA), and after the resin is fully swollen, dry it under vacuum. Add a deprotecting agent, shake at low speed, dry under vacuum, and wash. Add a mixture of amino acids, N-hydroxybenzotriazole (HOBt) and O-benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU) and N,N-diisopropylethylamine, shake, dry under vacuum, and wash.

[0112] (2) Couple the amino acids one by one to the MBHA resin according to step (1);

[0113] (3) Acidification of the amino terminus: The last amino acid at the N-terminus of the completed peptide chain is acetylated and the reaction is completed under reduced pressure, then tested for indole and washed.

[0114] (4) Add DCM to the dried resin to swell, add anhydrous methanol and shake at low speed; dry, cut, wash, concentrate, add ice-cold ether to precipitate the peptide, add water to dissolve the peptide completely in water, filter, purify, and obtain pure peptide.

[0115] The polypeptide compounds 1-123 synthesized based on the above steps are shown in Table 1 below:

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] Example 2: Detection of in vitro cytotoxicity of precursor peptide RAP (SEQ ID NO. 123) by MTT assay

[0122] Cells: mouse embryonic fibroblasts (NIH-3T3) and rat renal tubular epithelial cells (NRK-52E).

[0123] Culture medium: NIH-3T3 cells were cultured in high-glucose DMEM medium containing 10% FBS, and NRK-52E cells were cultured in high-glucose DMEM medium containing 5% FBS.

[0124] Culture conditions: 37℃, 5% CO2 incubator.

[0125] 7×10 3 Cell suspensions were prepared and mixed, then added to 96-well plates at 100 μL / well. After culturing for 24 h, the precursor peptide RAP (SEQ ID NO. 123) from Example 1 was added at concentration gradients of 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM. After culturing for 24 h, MTT (5 mg / mL, Solarbio, 715F0528) was added, and the plates were incubated at 37°C for 3 h. The absorbance at 570 nm was measured using a microplate reader.

[0126] Experimental results are as follows Figure 1 As shown, within the concentration range of 0-200 μM, the precursor peptide RAP has no toxic side effects on either type of cell.

[0127] Example 3: In vitro activity screening of the precursor peptide RAP (SEQ ID NO. 123) in improving and treating renal fibrosis.

[0128] Cells: mouse embryonic fibroblasts (NIH-3T3) and rat proximal renal tubular epithelial cells (NRK-52E).

[0129] Culture medium: NIH-3T3 cells were cultured in high-glucose DMEM medium containing 10% FBS, and NRK-52E cells were cultured in high-glucose DMEM medium containing 5% FBS.

[0130] Culture conditions: 37℃, 5% CO2 incubator.

[0131] 1. Cell Culture: When the cell confluence reaches 80-90%, discard the upper culture medium. Digest the cells with trypsin until they are round. Then, add high-glucose DMEM medium containing 10% FBS (NIH-3T3) or high-glucose DMEM medium containing 5% FBS (NRK-52E) to stop the digestion. Repeatedly pipette the cells to detach them from the cell wall and transfer them to centrifuge tubes. Centrifuge at 800 rpm for 5 min, discard the supernatant, and resuspend the cells in high-glucose DMEM medium containing 10% FBS (NIH-3T3) or high-glucose DMEM medium containing 5% FBS (NRK-52E) to form a single-cell suspension. Add an appropriate amount of cells to a culture dish for cell passage.

[0132] 2. Western blot analysis of the effect of precursor peptide RAP on the expression of TGF-β1-induced fibrosis markers in NIH-3T3 and NRK-52E cells.

[0133] The fibrosis markers detected in this experiment included α-smooth muscle actin (α-SMA), fibronectin, and type I collagen (Collagen I).

[0134] 2.1 Drug administration: The cells cultured in step 1 were administered at a rate of 3 × 10⁻⁶. 5 Cell suspensions were prepared at 2 mL / well and added to 6-well plates. After culturing for 24 h, the cells were starved for 10 h using serum-free high-glucose DMEM medium. Two concentrations of the precursor peptide RAP (water and DMSO mixture, adjusted for solubility) prepared in Example 1 were selected: 50 μM and 100 μM. A cell fibrosis model was induced using 5 ng / ml TGF-β1. The experimental groups were: control group, TGF-β1 group, 50 μM precursor peptide RAP group (TGF-β1 + 50 μM RAP), and 100 μM precursor peptide RAP group (TGF-β1 + 100 μM RAP).

[0135] 2.2 Total protein extraction from cells: After incubating cells with RAP for 24 h, the supernatant of the culture medium was discarded, the cells were washed 3 times with PBS, dried, and then RIPA lysis buffer containing 1% PMSF was added. The protein lysate was scraped off with a scraper and collected into a 1.5 mL centrifuge tube. The cells were lysed on ice for 30 min, centrifuged at 12000 rpm for 30 min, and the supernatant was collected.

[0136] 2.3 Western Blotting: Protein concentration was detected using a BCA kit. The protein loading volume was adjusted to 20 μg. The protein was denatured by heating in a 100℃ metal bath. Proteins of different molecular weights were separated using 10% SDS-PAGE gel electrophoresis. The stacking gel was run at 80 V for 30 min, and the separating gel at 120 V for 1.5 h. After electrophoresis, the target protein was transferred to a PVDF membrane and analyzed using GenScript eBlot. TM The membrane was transferred using a wet transfer apparatus for 17 minutes. After transfer, the membrane was blocked with 5% skim milk powder for 1.5 hours, followed by TBST washing for 10 minutes × 3 times. The membrane was then incubated with primary antibody overnight at 4°C. The next day, the PVDF membrane was removed, washed with TBST for 10 minutes × 3 times, and then incubated with secondary antibody (HRP-labeled goat anti-rabbit IgG, Beyotime, A0208) at room temperature for 1 hour, followed by TBST washing for 10 minutes × 3 times. Finally, a chemiluminescence reagent kit was added for color development.

[0137] First, in vitro cell fibrosis models were established by inducing NIH-3T3 cells and NRK-52E cells with TGF-β1 (5 ng / mL). RAP at doses of 50 μM and 100 μM was administered, and after 24 h of treatment, total cell protein was extracted. Western blotting was used to detect the expression of Fibronectin, Collagen I, and α-SMA. Results are as follows: Figure 2 As shown, after TGF-β1 induction, the expression of markers α-SMA, Fibronectin, and Collagen I in NIH-3T3 and NRK-52E cells was significantly upregulated. When a 100 μM dose of the precursor peptide RAP was administered, the expression of the above markers was significantly inhibited, indicating that the precursor peptide RAP has in vitro antifibrotic activity.

[0138] Example 4: In vivo activity assay of the precursor peptide RAP (SEQ ID NO. 123) in improving and treating renal fibrosis

[0139] Experimental animals: Male C57BL / 6 mice, 6-8 weeks old, purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The temperature was maintained at 25℃, with alternating lighting for 12 hours, free access to food and water, and bedding changed twice a week.

[0140] 1. Experimental grouping, modeling, and drug administration:

[0141] Experimental groups: sham operation group (Sham), surgical group (UUO, unilateral ureteral ligation), low-dose group (UUO + 2.5 mg / kg RAP), and high-dose group (UUO + 5.0 mg / kg RAP), with 8 mice in each group. Establishment of a mouse model of unilateral ureteral obstruction: After anesthesia, the skin on the left back of the mouse was incised, the muscle layer was torn open, and the left ureter was freed. Ligation was performed at the renal pelvis and the upper 1 / 3 of the ureter. The ureter was then cut between the ligation lines, and the muscle layer and skin were sutured. Drug administration: Drug administration began on the second day after surgery, via subcutaneous injection, once daily for 2 weeks.

[0142] 2. Renal function index detection: Blood was collected from the orbital cavity of mice after anesthesia and placed in 1.5 mL EP tubes. The tubes were centrifuged twice at 5000 rpm for 15 min, and the supernatant was collected. Serum Scr and BUN levels were measured according to the instructions of the creatinine (Scr) assay kit (Nanjing Jiancheng Bioengineering Institute, C011-2-1) and the blood urea nitrogen (BUN) assay kit (Nanjing Jiancheng Bioengineering Institute, C013-2-1), respectively. The experimental results are as follows: Figure 3 As shown, RAP significantly inhibited the abnormal increase in BUN and Scr levels caused by renal fibrosis.

[0143] 3. Detection of the effect of the mother peptide RAP (SEQ ID NO.123) on the expression of fibrosis markers at the animal level

[0144] Western blotting was used to detect the expression of biomarkers Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin; qRT-PCR was used to detect fibrosis genes. Fn1, Col1a1, Acta2, Mmp2, Vim The expression of .

[0145] 3.1 Detection of tissue fibrosis marker expression by Western blotting

[0146] Tissue protein extraction: Weigh 15 mg of mouse kidney tissue from each group, add 100 μL of RIPA lysis buffer containing 1% PMSF, add one grinding steel ball of each size, grind at 60 Hz for 120 s, remove the grinding steel ball, allow to stand at low temperature to lyse the tissue protein for 30 min, centrifuge at 12000 rpm for 30 min, and repeat twice. Collect the supernatant to obtain the total tissue protein.

[0147] Western blotting of proteins: The procedure is the same as in cell experiments 2.3.

[0148] Experimental results are as follows Figure 4As shown in the figure, the downregulation of the expression of fibrosis-related proteins Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin by the precursor peptide RAP under the influence of UUO indicates that RAP has in vivo anti-renal fibrosis activity at the protein level.

[0149] 3.2 qRT-PCR was used to detect the expression of fibrosis genes.

[0150] RNA extraction from tissues: Weigh 15 mg of mouse kidney tissue from each group, add 1000 μL of RNA extraction buffer, add one grinding steel ball of each size, grind at 60 Hz for 120 s, let stand at room temperature for 10 min, transfer the lysis buffer to a new enzyme-free EP tube, add 200 μL of chloroform, shake vigorously, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 15 min, aspirate the colorless aqueous phase, add 500 μL of isopropanol, mix well, let stand for 10 min, centrifuge at 12000 rpm for 10 min, discard the supernatant, a white gel-like precipitate forms on the side and bottom of the tube, add 1000 μL of 75% ethanol, gently shake to detach the precipitate from the tube wall, centrifuge at 7500 rpm for 5 min, aspirate the supernatant, air dry the precipitate at room temperature, and dissolve the precipitate in enzyme-free sterile water.

[0151] Reverse transcription: RNA concentration was detected using a micro UV-Vis spectrophotometer. Samples with an RNA A260 / A280 value between 1.8 and 2.2 met the requirements for subsequent experimental procedures. The reverse transcription system was prepared according to the proportions in Table 2, mixed thoroughly, and added to the PCR instrument. The reverse transcription conditions were set as follows: 37℃ for 2 min, 55℃ for 15 min, and 85℃ for 5 min. After reverse transcription, the synthesized cDNA was stored at -20℃.

[0152] Table 2 Reverse Transcription System Configuration Table

[0153]

[0154]

[0155] Table 3 Amplification System Configuration Table

[0156]

[0157] Primer sequences are shown in Table 4:

[0158] Table 4

[0159]

[0160] Amplification program: Pre-denaturation: 95℃, 5 min. Amplification 40 cycles: 95℃, 10 s; 60℃, 30 s. Melting: 95℃, 1 min; 55℃, 30 s; 95℃, 30 s. Calculate relative expression based on gene Ct values. .

[0161] Experimental results are as follows Figure 5 As shown, RAP significantly inhibited the fibrosis gene induced by UUO. Fn1, Col1a1, Acta2 Mmp2, Vim The abnormal expression of RAP indicates that RAP also inhibits renal fibrosis induced by UUO at the gene level.

[0162] Example 5: In vitro activity screening of RAP peptide analogs (SEQ ID NO.1-SEQ ID NO.23) for improving and treating renal fibrosis.

[0163] This embodiment is the same as Example 3, except that the drug treatment experimental group is different, as detailed below:

[0164] 1. Western blot analysis of the effects of RAP and its analogues on the expression of TGF-β1-induced fibrosis markers in NRK-52E cells.

[0165] 2. Administration: 3×10 5 Cell suspension was prepared at 2 mL / well and added to 6-well plates. After culturing for 24 h, the cells were starved for 10 h using serum-free high-glucose DMEM medium. In the screening experiment for the antifibrotic activity of the RAP peptide analogs (SEQ ID NO.1-SEQ ID NO.23) prepared in Example 1, the dosage of the peptide analogs was 50 μM. The experimental groups were: control group, TGF-β1 group, TGF-β1 + RAP group, and TGF-β1 + RAP analog group. The remaining procedures were the same as in Example 3.

[0166] Using a TGF-β1-induced NRK-52E cell fibrosis model, the pharmacophore of the parent peptide RAP was determined by alanine scanning, D-peptide, and reverse peptide modification. The optimal active fragment of the parent peptide RAP was screened by sequence extension and reduction. RAP peptide analogs (SEQ ID NO.1-SEQ ID NO.23) were administered at a dose of 50 μM. After 24 h of treatment, total cell protein was extracted, and the expression of Collagen I and Fibronectin was detected by Western blotting.

[0167] The results are as follows Figure 6As shown, after TGF-β1 induction, the expression of the markers Collagen I and Fibronectin was significantly upregulated. Among the 23 modified analogs in this batch, the shortened peptide R7p (SEQ ID NO.14 prepared in Example 1) significantly inhibited the expression of the above markers. Thus, it is determined that the RAP peptide analog R7p (SEQ ID NO.14 prepared in Example 1) can maintain the in vitro antifibrotic activity that is basically consistent with the parent peptide RAP based on sequence shortening, and is even superior to the parent peptide.

[0168] 3. qRT-PCR detection of the effect of R7p (SEQ ID NO.14) on TGF-β1-induced fibrosis marker genes in NRK-52E cells.

[0169] 3.1 Total RNA Extraction from Cells: Add 500 μL of RNA lysis buffer to each well of a 6-well plate, repeatedly pipet and transfer to an enzyme-free 1.5 mL EP tube. Add 100 μL of chloroform, vortex vigorously, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 15 min, aspirate the colorless aqueous phase, add 250 μL of isopropanol, mix well, let stand for 10 min, centrifuge at 12000 rpm for 10 min, discard the supernatant, a white gel-like precipitate will form on the sides and bottom of the tube. Add 500 μL of 75% ethanol, gently shake to detach the precipitate from the tube wall, centrifuge at 7500 rpm for 5 min, aspirate the supernatant, air-dry the precipitate at room temperature, and dissolve the precipitate in enzyme-free sterile water. Subsequent operations are the same as in 3.2 of Example 4.

[0170] Experimental results Figure 7 The effect of R7p on the expression of fibrosis-related genes Fn1 and Vim in TGF-β1-induced NRK-52E cells was investigated. As shown in the figure, TGF-β1 induction significantly upregulated the expression of the marker genes Fn1 and Vim. At a dose of 50 μM, R7p significantly inhibited the abnormal expression of these genes induced by TGF-β1, indicating that R7p significantly inhibits TGF-β1-induced renal fibrosis in NRK-52E cells at the gene level.

[0171] Example 6: In vivo activity assay of peptide R7p (SEQ ID NO.14) in improving and treating renal fibrosis

[0172] The source of experimental animals and the operation method in this embodiment are the same as those in Example 4. The difference is that the RAP used is the RAP analog R7p (SEQ ID NO.14) prepared in Example 1.

[0173] Depend on Figure 8 It can be seen that R7p significantly reduced the abnormal overexpression of Scr and BUN induced by UUO, indicating that R7p can alleviate kidney damage caused by UUO.

[0174] Based on this, the in vivo anti-renal fibrosis activity of R7p was evaluated, and the expression of fibrosis markers was detected at both the protein and gene levels. Figure 9 The results showed that after UUO induction, the expression of fibrosis markers Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin was significantly upregulated. When R7p was administered, the overexpression of the above markers was significantly inhibited. Moreover, a low dose of R7p (1.25 mg / kg) could inhibit the expression of all the above markers, thus indicating that the in vivo anti-renal fibrosis activity of R7p was better than that of the parent peptide RAP.

[0175] Fibrosis marker genes Col1a1, Fn1, Acta2, Mmp2, Vim The detection results of the expression situation are as follows Figure 10 As shown in the figure, R7p significantly downregulated the overexpression of the above-mentioned genes, indicating that R7p also inhibits renal fibrosis induced by UUO at the gene level.

[0176] Example 7: In vitro activity screening of peptide compound R7p (SEQ ID NO.14) for improving and treating renal fibrosis.

[0177] This embodiment is the same as Example 5, except that the drug treatment experimental group is different, as detailed below:

[0178] 1. Western blot analysis of the effects of R7p and its analogues on the expression of TGF-β1-induced fibrosis markers in NRK-52E cells.

[0179] 2. Administration: 3×10 5 Cell suspension was prepared at a concentration of 2 mL / well in 6-well plates. After incubation for 24 h, the cells were starved for 10 h using serum-free medium. In the R7p analog antifibrotic activity screening experiment, the dosage of the peptide analog was 25 μM. The experimental groups were: control group, TGF-β1 group, TGF-β1 + R7p group, and TGF-β1 + R7p analog group. The remaining procedures were the same as in Example 5.

[0180] A TGF-β1-induced fibrosis model of NRK-52E cells was established. The sequence of the precursor peptide R7p was modified and its structure optimized using alanine scanning, D-amino acid scanning, and single point mutation. The activity of R7p peptide analogs was assessed using selection doses of 25 μM, 12.5 μM, and 6 μM. After 24 h of treatment, total cell protein was extracted, and Western blotting was used to detect the expression of Collagen I and Fibronectin. Results are as follows: Figure 11 , Figure 12As shown, after TGF-β1 induction, the expression of the markers Collagen I and Fibronectin was significantly upregulated. Among the 36 modified analogs (SEQ ID NO.24-SEQ ID NO.59) in this batch, the analogs R7p-5S and R7p-5K significantly inhibited the expression of the above markers. Therefore, it was determined that the peptides R7p-5S (SEQ ID NO.51) and R7p-5K (SEQ ID NO.52) have better in vitro antifibrotic activity than the parent peptide RAP analog R7p (SEQ ID NO.14).

[0181] 3. qRT-PCR detection of the effects of R7p-5S and R7p-5K on TGF-β1-induced fibrosis marker genes in NRK-52E cells.

[0182] 3.1 Total RNA Extraction from Cells: Add 500 μL of RNA lysis buffer to each well of a 6-well plate, repeatedly pipette and transfer to an enzyme-free 1.5 mL EP tube. Add 100 μL of chloroform, vortex vigorously, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 15 min, aspirate the colorless aqueous phase, add 250 μL of isopropanol, mix well, let stand for 10 min, centrifuge at 12000 rpm for 10 min, discard the supernatant, a white gel-like precipitate will form on the sides and bottom of the tube. Add 500 μL of 75% ethanol, gently shake to detach the precipitate from the tube wall, centrifuge at 7500 rpm for 5 min, aspirate the supernatant, air-dry the precipitate at room temperature, and dissolve the precipitate in enzyme-free sterile water. Subsequent operations are the same as in 3.2 of Example 4.

[0183] Experimental results Figure 13 R7p-5S and R7p-5K are genes related to fibrosis in TGF-β1-induced NRK-52E cells. Fn1, Col1a1, Vim The effects of these genes on the expression status were investigated. As shown in the figure, when the drug dose was 6 μM, R7p-5S and R7p-5K significantly inhibited the abnormal expression of the above-mentioned genes induced by TGF-β1, indicating that R7p-5S and R7p-5K significantly inhibited TGF-β1-induced renal fibrosis in NRK-52E cells at the gene level.

[0184] Example 8: In vivo activity assays of peptide compounds R7p-5S (SEQ ID NO.51) and R7p-5K (SEQ ID NO.52) in improving and treating renal fibrosis.

[0185] This embodiment is the same as Example 4, except that the RAP used is the RAP analog R7p-5S (SEQ ID NO.51) and R7p-5K (SEQ ID NO.52) prepared in Example 1.

[0186] Depend on Figure 14 It can be seen that R7p-5S and R7p-5K significantly reduced the abnormal overexpression of kidney function indicators Scr and BUN induced by UUO, indicating that both R7p-5S and R7p-5K can alleviate kidney damage caused by UUO.

[0187] Based on this, the in vivo anti-renal fibrosis activity of R7p-5S and R7p-5K was evaluated, and the expression of fibrosis markers was detected at both the protein and gene levels. Figure 15 The results showed that after UUO induction, the expression of fibrosis markers Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin was significantly upregulated. When R7p-5S and R7p-5K were administered, the overexpression of the above markers was significantly inhibited. Furthermore, low doses of R7p-5S and R7p-5K (0.5 mg / kg) were sufficient to inhibit the expression of all the above markers, indicating that the in vivo anti-renal fibrosis activity of R7p-5S and R7p-5K was better than that of the parent peptide R7p.

[0188] Fibrosis marker genes Col1a1, Fn1, Acta2, Mmp2, Vim The detection results of the expression situation are as follows Figure 16 As shown in the figure, R7p-5S and R7p-5K significantly downregulated the overexpression of the above genes, indicating that R7p-5S and R7p-5K also inhibit UUO-induced renal fibrosis at the gene level.

[0189] Example 9: In vitro activity screening of peptide compounds R7p-5S (SEQ ID NO.51) and R7p-5K (SEQ ID NO.52) for improving and treating renal fibrosis.

[0190] The cells and culture conditions used in this embodiment are the same as in Example 3.

[0191] 1. Western blot analysis of the effects of R7p, R7p-5K and their analogues on the expression of TGF-β1-induced fibrosis markers in NRK-52E cells.

[0192] 2. Administration: 3×10 5 Cell suspensions were prepared at a concentration of 2 mL / well and added to 6-well plates. After culturing for 24 h, the cells were starved for 10 h using serum-free medium. In the R7p analog antifibrotic activity screening experiment, the dosage of the peptide analog was 6 μM. The experimental groups were: control group, TGF-β1 group, TGF-β1 + R7p group / TGF-β1 + R7p-5K group, and TGF-β1 + R7p analog group / TGF-β1 + R7p-5K analog group. The remaining procedures were the same as in Example 3.

[0193] A TGF-β1-induced NRK-52E cell fibrosis model was established. Further sequence modification and structural optimization of the precursor peptides R7p and R7p-5K were performed using single-site mutation, double-site mutation, and N-methylation scanning. The activity of R7p and R7p-5K peptide analogs was assessed sequentially at screening doses of 6 μM, 3 μM, and 1 μM. After 24 h of treatment, total cell protein was extracted, and Western blotting was used to detect the expression of Collagen I and Fibronectin. Results are as follows: Figure 17 , Figure 18 , Figure 19 As shown, after TGF-β1 induction, the expression of the markers Collagen I and Fibronectin was significantly upregulated. Among the 63 modified analogs (SEQ ID NO.60-SEQ ID NO.122) in this batch, the analogs RSM25 and R5K5M significantly inhibited the expression of the above markers. Therefore, it was determined that the peptides RSM25 and R5K5M have better in vitro antifibrotic activity than the parent peptides R7p and R7p-5K.

[0194] 3. qRT-PCR detection of the effects of RSM25 (SEQ ID NO.114) and R5K5M (SEQ ID NO.119) on TGF-β1-induced fibrosis marker genes in NRK-52E cells.

[0195] 3.1 Total RNA Extraction from Cells: Add 500 μL of RNA lysis buffer to each well of a 6-well plate, repeatedly pipette and transfer to an enzyme-free 1.5 mL EP tube. Add 100 μL of chloroform, vortex vigorously, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 15 min, aspirate the colorless aqueous phase, add 250 μL of isopropanol, mix well, let stand for 10 min, centrifuge at 12000 rpm for 10 min, discard the supernatant, a white gel-like precipitate will form on the sides and bottom of the tube. Add 500 μL of 75% ethanol, gently shake to detach the precipitate from the tube wall, centrifuge at 7500 rpm for 5 min, aspirate the supernatant, air-dry the precipitate at room temperature, and dissolve the precipitate in enzyme-free sterile water. Subsequent operations are the same as in 3.2 of Example 4.

[0196] Experimental results Figure 20 RSM25 and R5K5M are TGF-β1-induced fibrosis-related genes in NRK-52E cells. Fn1, Col1a1, Vim The effects of these genes on their expression were investigated. As shown in the figure, when the drug dose was 1 μM, RSM25 and R5K5M significantly inhibited the abnormal expression of the above-mentioned genes induced by TGF-β1, indicating that RSM25 and R5K5M significantly inhibited TGF-β1-induced renal fibrosis in NRK-52E cells at the gene level.

[0197] Example 10: In vivo activity assay of peptide compound R5K5M (SEQ ID NO. 119) in improving and treating renal fibrosis.

[0198] This embodiment is the same as that of embodiment 4, except that the RAP used is the RAP analog R5K5M (SEQ ID NO.119) prepared in embodiment 1.

[0199] Depend on Figure 21 It can be seen that after UUO modeling, the renal function indicators Scr and BUN were abnormally elevated. R5K5M significantly reduced the abnormal overexpression of renal function indicators Scr and BUN induced by UUO, indicating that R5K5M can alleviate the renal damage caused by UUO.

[0200] Based on this, the in vivo anti-renal fibrosis activity of R5K5M was evaluated, and the expression of fibrosis markers was detected at both the protein and gene levels. Figure 22 The results showed that after UUO induction, the expression of fibrosis markers Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin was significantly upregulated. When R5K5M was administered, the overexpression of the above markers was significantly inhibited. Moreover, a low dose of R5K5M (0.1 mg / kg) could inhibit the expression of all the above markers, thus indicating that the in vivo anti-renal fibrosis activity of R5K5M was better than that of the parent peptide R7p-5K.

[0201] Fibrosis marker genes Col1a1, Fn1, Acta2, Mmp2, Vim, Tgfb1 The detection results of the expression situation are as follows Figure 23 As shown in the figure, after UUO induction, the fibrosis marker genes... Col1a1, Fn1, Acta2, Mmp2, Vim, Tgfb1 R5K5M significantly upregulated the expression of the above genes, and R5K5M significantly downregulated the overexpression of the above genes, indicating that R5K5M also inhibits renal fibrosis induced by UUO at the gene level.

[0202] Example 11: In vivo activity assay of peptide compound RSM25 (SEQ ID NO. 114) in improving and treating renal fibrosis

[0203] This embodiment is the same as Example 4, except that the RAP used is the RAP analog prepared in Example 1, which is RSM25 (SEQ ID NO.115).

[0204] 1. Experimental grouping, modeling, and drug administration in a unilateral ureteral obstruction (UUO) model.

[0205] The experimental animals and procedures used in this part are the same as those in Example 4.

[0206] First, the effect of RSM25 (SEQ ID NO. 114) on UUO-induced kidney injury was evaluated, and the results are as follows: Figure 24 As shown, RSM25 (SEQ ID NO. 114) significantly reduced the abnormal overexpression of kidney function markers Scr and BUN induced by UUO; and reduced the expression of kidney injury-related biomarkers. Clu, Havcr1, Lcn2 The test results, as shown in the figure, indicate that RSM25 (SEQ ID NO. 114) significantly reduced the abnormal expression of the aforementioned genes after UUO induction. These results suggest that RSM25 (SEQ ID NO. 114) can alleviate kidney damage induced by UUO.

[0207] By analyzing inflammation-related genes TnF-α, IL-1b, IL6 The test revealed that after UUO induction, inflammatory factors were found in the kidney tissue. TnF-α, IL-1b, IL6 The expression of these markers was significantly upregulated, while RSM25 (SEQ ID NO. 114) was able to inhibit the abnormal expression of these markers, indicating that RSM25 can alleviate the inflammatory response in kidney tissue caused by UUO. The results are as follows. Figure 25 As shown.

[0208] Based on this, the in vivo anti-renal fibrosis activity of RSM25 (SEQ ID NO.114) was evaluated, and the expression of fibrosis markers was detected at both the protein and gene levels. Figure 26 The results showed that after UUO induction, the expression of fibrosis markers Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin was significantly upregulated. When RSM25 (SEQ ID NO.114) was administered, the overexpression of the above markers was significantly inhibited. Moreover, a low dose of RSM25 (SEQ ID NO.114) (0.1 mg / kg) could inhibit the expression of all the above markers, thus indicating that the in vivo anti-renal fibrosis activity of RSM25 (SEQ ID NO.114) was better than that of the parent peptide R7p-5K.

[0209] Fibrosis marker genes Col1a1, Fn1, Acta2, Vim, Tgfb1 The detection results of the expression situation are as follows Figure 27 As shown in the figure, RSM25 (SEQ ID NO.114) significantly downregulated the overexpression of the above-mentioned genes, indicating that RSM25 (SEQ ID NO.114) also inhibits renal fibrosis induced by UUO at the gene level.

[0210] 2. Folic acid-induced renal fibrosis model (FA) experimental grouping, modeling, and drug administration:

[0211] Experimental groups: Normal group, model group (FA), 0.5 mg / kg RSM25 (SEQ ID NO.114) group, and 1.25 mg / kg RSM25 (SEQ ID NO.114) group, with 8 mice in each group. Establishment of a mouse model of folic acid-induced renal fibrosis: Prepare the required volume of 0.3 mol / L NaHCO3 solution according to the number of mice. Weigh folic acid powder at a dose of 250 mg / kg and dissolve it in the NaHCO3 solution to achieve a final folic acid concentration of 25 mg / mL. Intraperitoneal injection was then administered according to the mice's body weight to establish the model. Starting from the day after surgery, mice in the treatment group received daily subcutaneous injections of 0.5 mg / kg or 1.25 mg / kg RSM25 (SEQ ID NO.114), while the normal group and FA model group received the same volume of PBS solution. After 7 days of continuous treatment, the mice were euthanized, and bilateral kidney tissue was removed for subsequent experiments.

[0212] Administration: Administer the medication starting the day after surgery, via subcutaneous injection, once daily for one week.

[0213] The remaining operations are the same as those for the UUO model.

[0214] Similarly, the effect of RSM25 (SEQ ID NO. 114) on FA-induced kidney injury was first evaluated, and the results are as follows: Figure 28 As shown in the figure, RSM25 (SEQ ID NO. 114) significantly reduced the abnormal overexpression of FA-induced renal function markers Scr and BUN. The detection results of renal injury-related biomarkers Havcr1 and Lcn2 also show that RSM25 (SEQ ID NO. 114) significantly reduced the abnormal expression of these genes after FA induction. These results indicate that RSM25 (SEQ ID NO. 114) can alleviate renal injury induced by UUO.

[0215] By analyzing inflammation-related genes TnF-α, IL-1b, IL6 The test results showed that RSM25 (SEQ ID NO. 114) could alleviate the inflammatory response in kidney tissue caused by FA, ​​as shown in the following results. Figure 29 As shown.

[0216] Based on this, the in vivo anti-renal fibrosis activity of RSM25 (SEQ ID NO.114) was evaluated, and the expression of fibrosis markers was detected at both the protein and gene levels. Figure 30The results showed that after FA induction, the expression of fibrosis markers Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin was significantly upregulated. When RSM25 (SEQ ID NO.114) was administered, the overexpression of the above markers was significantly inhibited, indicating that RSM25 (SEQ ID NO.114) also has good anti-renal fibrosis activity in the FA-induced renal fibrosis model.

[0217] Fibrosis marker genes Col1a1, Fn1, Acta2, Mmp2, Vim, Tgfb1 The detection results of the expression situation are as follows Figure 31 As shown in the figure, RSM25 (SEQ ID NO.114) significantly downregulated the overexpression of the above-mentioned genes, indicating that RSM25 (SEQ ID NO.114) also inhibits FA-induced renal fibrosis at the gene level.

[0218] Example 12 In vivo activity assay of peptide compound RSM25 (SEQ ID NO. 114) in improving and treating pulmonary fibrosis

[0219] Experimental animals: Female C57BL / 6 mice, 6-8 weeks old, purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The temperature was maintained at 25℃, with alternating lighting for 12 hours, free access to food and water, and bedding changed twice a week.

[0220] 1. Experimental grouping, modeling, and drug administration:

[0221] Experimental groups: Normal group, surgical group (BLM injection), low-dose group (BLM + 0.5 mg / kg RSM25), and high-dose group (BLM + 1.25 mg / kg RSM25), with 10 mice in each group. Establishment of a BLM-induced mouse pulmonary fibrosis model: C57BL / 6J mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (50 mg / kg). The neck skin of the mice was disinfected with 75% alcohol. A 1 cm incision was made in the skin using sterile surgical instruments, and the trachea was exposed by blunt dissection of the muscles. 50 μL of bleomycin solution (4 mg / kg) was injected intratracheally to induce pulmonary fibrosis, resulting in mice with pulmonary fibrosis. The BLM-induced pulmonary fibrosis model was characterized by: alveolar structure destruction, alveolar cavity deformation, widening of alveolar septa, inflammatory cell infiltration, and fibroblast proliferation within the alveolar septa. Normal group mice were injected intratracheally with an equal volume of PBS using the same method.

[0222] Administration: Administer the medication starting the day after surgery, via subcutaneous injection, once daily for 21 days.

[0223] 2. Sample Collection and Organ Coefficient Calculation: Blood and lung tissue samples were collected after 21 days of continuous drug administration. Mouse body weight and lung tissue weight were measured to calculate the organ coefficient. Lung tissue proteins were extracted to detect the expression of pulmonary fibrosis-related proteins and genes. The lung coefficient is an important factor in evaluating the pathological state of lung tissue, such as… Figure 32 As shown, RSM25 (SEQ ID NO.114) significantly inhibited the increase in lung tissue weight induced by BLM, as evidenced by the decrease in lung coefficient after RSM25 (SEQ ID NO.114) treatment.

[0224] 3. Animal-level detection of the effect of RSM25 (SEQ ID NO.114) on the expression of fibrosis markers

[0225] Western blotting was used to detect the expression of biomarkers Fibronectin, Collagen I, MMP2, α-SMA, E-Cadherin, and Vimentin; qRT-PCR was used to detect fibrosis genes. Fn1, Col1a1, Acta2, Mmp2, Tgfb1, Vim, Tnf- α, IL-1b The expression of .

[0226] 3.1 Detection of tissue fibrosis marker expression by Western blotting

[0227] Tissue protein extraction: Weigh 15 mg of lung tissue from each of the above groups of mice, add 100 μL of RIPA lysis buffer containing 1% PMSF, add one grinding steel ball of each size, grind at 60 Hz for 120 s, remove the grinding steel ball, allow to stand at low temperature to lyse the tissue protein for 30 min, centrifuge at 12000 rpm for 30 min, and centrifuge twice. Collect the supernatant to obtain the total tissue protein.

[0228] Western blot: The procedure is the same as in Example 2, Cell Experiment 2.3.

[0229] Experimental results are as follows Figure 33 As shown in the figure, RSM25 downregulates the abnormal expression of fibrosis-related proteins Fibronectin, Collagen I, MMP2, α-SMA, E-Cadherin, and Vimentin induced by BLM, indicating that RSM25 has in vivo anti-fibrotic activity at the protein level.

[0230] 3.2 qRT-PCR was used to detect the expression of fibrosis genes.

[0231] The experimental procedure is the same as 3.2 in Example 4.

[0232] like Figure 34 As shown, by targeting inflammation-related genes Tnf-α, IL-1bThe tests revealed that RSM25 can alleviate the inflammatory response in lung tissue caused by BLM.

[0233] Fibrosis marker genes Col1a1, Fn1, Acta2, Mmp2, Vim, Tgfb1 The detection results of the expression situation are as follows Figure 35 As shown in the figure, RSM25 significantly downregulated the overexpression of the above-mentioned genes in BLM-induced pulmonary fibrosis tissue, indicating that RSM25 also inhibits BLM-induced pulmonary fibrosis at the gene level.

[0234] Example 13 In vivo activity assay of peptide compound RSM25 (SEQ ID NO. 114) in improving and treating liver fibrosis

[0235] Experimental animals: Male C57BL / 6 mice, 6-8 weeks old, purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The temperature was maintained at 25℃, with alternating lighting for 12 hours, free access to food and water, and bedding changed twice a week.

[0236] 1. Experimental grouping, modeling, and drug administration:

[0237] Experimental groups: control group (Normal), surgical group (CCl4 injection), low-dose group (CCl4 + 0.5 mg / kg RSM25), and high-dose group (CCl4 + 1.25 mg / kg RSM25), with 8 mice in each group. Establishment of a CCl4-induced mouse liver fibrosis model: After 3 weeks of intraperitoneal injection of corn oil or CCl4, mice were administered either low- or high-dose RSM25 or saline. Three weeks after administration, tissue samples were collected from the treated mice. Serological markers were detected by blood collection from the eyeballs, and liver tissue was collected for subsequent analysis.

[0238] 2. Liver function index detection: Blood was collected from the orbital cavity of mice after anesthesia and placed in 1.5 mL EP tubes. The tubes were centrifuged twice at 5000 rpm for 15 min, and the supernatant was collected. Serum AST and ALT levels were measured according to the instructions of the Aspartate Aminotransferase (AST) assay kit (Nanjing Jiancheng Bioengineering Institute, C010-2-1) and the Alanine Aminotransferase (ALT) assay kit (Nanjing Jiancheng Bioengineering Institute, C009-2-1), respectively. The experimental results are as follows: Figure 36 As shown, RSM25 significantly inhibited the abnormal increase in AST and ALT levels caused by liver fibrosis.

[0239] 3. Animal-level detection of the effect of RSM25 (SEQ ID NO.114) on the expression of fibrosis markers

[0240] Western blotting was used to detect the expression of biomarkers Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin; qRT-PCR was used to detect fibrosis genes. Col1a1, Acta2, Mmp2, Tgfb1, Vim, Tnf-α, IL-6 The expression of .

[0241] 3.1 Detection of tissue fibrosis marker expression by Western blotting

[0242] Tissue protein extraction: Weigh 15 mg of mouse liver tissue from each group, add 100 μL of RIPA lysis buffer containing 1% PMSF, add one grinding steel ball of each size, grind at 60 Hz for 120 s, remove the grinding steel ball, allow to stand at low temperature to lyse the tissue protein for 30 min, centrifuge at 12000 rpm for 30 min, and repeat twice. Collect the supernatant to obtain the total tissue protein.

[0243] Western blotting of proteins: The procedure is the same as in cell experiments 2.3.

[0244] Experimental results are as follows Figure 37 As shown in the figure, RSM25 downregulates the abnormal expression of CCl4-induced liver fibrosis-related proteins Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin, indicating that RSM25 has in vivo anti-liver fibrosis activity at the protein level.

[0245] 3.2 qRT-PCR was used to detect the expression of fibrosis genes.

[0246] The experimental procedure is the same as 3.2 in Example 4.

[0247] like Figure 38 As shown, by detecting the inflammation-related genes Tnf-α and IL-6, it was found that RSM25 can alleviate the inflammatory response in liver tissue caused by CCl4.

[0248] Fibrosis marker genes Col1a1, Acta2, Mmp2, Vim, Tgfb1 The detection results of the expression situation are as follows Figure 39 As shown in the figure, RSM25 significantly downregulated the overexpression of the above-mentioned genes in CCl4-induced liver fibrosis tissue, indicating that RSM25 also inhibits CCl4-induced liver fibrosis at the gene level.

[0249] Example 14 In vivo activity assay of peptide compound DRSM25 (SEQ ID NO.122) in improving and treating renal fibrosis

[0250] The experimental animals and procedures were the same as those described in Example 4.

[0251] First, the effect of DRSM25 on UUO-induced kidney injury was assessed, and the results were as follows: Figure 40 As shown in the figure, DRSM25 significantly reduced the abnormal overexpression of kidney function indicators Scr and BUN induced by UUO. The results of detection of kidney injury-related biomarkers Clu, Havcr1, and Lcn2 also show that DRSM25 significantly reduced the abnormal expression of these genes after UUO induction. These results indicate that DRSM25 can alleviate kidney injury induced by UUO.

[0252] By analyzing inflammation-related genes TnF-α, IL-1b, IL6 The tests revealed that DRSM25 could alleviate the inflammatory response in kidney tissue caused by UUO, as shown in the results. Figure 41 As shown.

[0253] Based on this, the in vivo anti-renal fibrosis activity of DRSM25 was evaluated, and the expression of fibrosis markers was detected at both the protein and gene levels. Figure 42 The results showed that after UUO induction, the expression of fibrosis markers Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin was significantly upregulated. When DRSM25 was administered, the overexpression of the above markers was significantly inhibited, indicating that DRSM25 has good in vivo anti-renal fibrosis activity.

[0254] Fibrosis marker genes Col1a1, Fn1, Acta2, Mmp2, Tgfb1, Vim The detection results of the expression situation are as follows Figure 43 As shown in the figure, DRSM25 significantly downregulated the overexpression of the above-mentioned genes, indicating that DRSM25 also inhibits renal fibrosis induced by UUO at the gene level.

[0255] Example 15 In vivo activity assay of peptide compound DRSM25 (SEQ ID NO.122) in improving and treating pulmonary fibrosis

[0256] The experimental animals and procedures were the same as those described in Example 12.

[0257] 1. Sample Collection and Organ Coefficient Calculation: Blood and lung tissue samples were collected after 21 days of continuous drug administration. Mouse body weight and lung tissue weight were measured to calculate the organ coefficient. Lung tissue proteins were extracted to detect the expression of pulmonary fibrosis-related proteins and genes. The lung coefficient is an important factor in evaluating the pathological state of lung tissue, such as… Figure 44 As shown, DRSM25 significantly inhibited the increase in lung tissue weight induced by BLM, as evidenced by the decrease in lung coefficient after DRSM25 treatment.

[0258] 2. Animal-level detection of the effect of DRSM25 (SEQ ID NO.122) on the expression of fibrosis markers

[0259] Western blotting was used to detect the expression of biomarkers Fibronectin, Collagen I, MMP2, α-SMA, and E-Cadherin; qRT-PCR was used to detect fibrosis genes. Fn1, Col1a1, Mmp2, Vim The expression of .

[0260] 2.1 Detection of tissue fibrosis markers by Western blotting

[0261] Experimental results are as follows Figure 45 As shown in the figure, DRSM25 downregulates the abnormal expression of fibrosis-related proteins Fibronectin, Collagen I, MMP2, α-SMA, and E-Cadherin induced by BLM, indicating that DRSM25 has in vivo anti-fibrotic activity at the protein level.

[0262] 2.2 qRT-PCR was used to detect the expression of fibrosis genes.

[0263] The experimental procedure is the same as 3.2 in Example 4.

[0264] Fibrosis marker genes Fn1, Col1a1, Mmp2, Vim The detection results of the expression situation are as follows Figure 46 As shown in the figure, DRSM25 significantly downregulated the overexpression of the above-mentioned genes in BLM-induced pulmonary fibrosis tissue, indicating that DRSM25 also inhibits BLM-induced pulmonary fibrosis at the gene level.

[0265] Example 16 In vivo activity assay of peptide compound DRSM25 (SEQ ID NO.122) in improving and treating liver fibrosis

[0266] The experimental animals and procedures were the same as those described in Example 13.

[0267] 1. Liver function index testing: Experimental results are as follows Figure 47 As shown, DRSM25 significantly inhibited the abnormal increase in AST and ALT levels caused by liver fibrosis.

[0268] 2. Animal-level detection of the effect of DRSM25 on the expression of fibrosis markers

[0269] Western blotting was used to detect the expression of biomarkers Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin; qRT-PCR was used to detect fibrosis genes. Fn1, Col1a1, Acta2, Mmp2, Tgfb1, Tnf-α, IL-6 The expression of .

[0270] 2.1 Detection of tissue fibrosis markers by Western blotting

[0271] Experimental results are as follows Figure 48 As shown in the figure, DRSM25 downregulates the abnormal expression of CCl4-induced liver fibrosis-related proteins Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin, indicating that DRSM25 has in vivo anti-liver fibrosis activity at the protein level.

[0272] 2.2 qRT-PCR was used to detect the expression of fibrosis genes.

[0273] The experimental procedure is the same as 3.2 in Example 4.

[0274] like Figure 49 As shown, by targeting inflammation-related genes Tnf-α, IL-6 The test results showed that DRSM25 can alleviate the inflammatory response in liver tissue caused by CCl4.

[0275] Fibrosis marker genes Fn1, Col1a1, Acta2, Mmp2, Tgfb1 The detection results of the expression situation are as follows Figure 50 As shown in the figure, DRSM25 significantly downregulated the overexpression of the above-mentioned genes in CCl4-induced liver fibrosis tissue, indicating that DRSM25 also inhibits CCl4-induced liver fibrosis at the gene level.

Claims

1. A RAP polypeptide analogue or a pharmaceutically acceptable salt thereof, characterized in that, The amino acid sequence of the RAP polypeptide analog is as follows: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17, and the N-terminus of the RAP peptide analog is acetylated, while the C-terminal amino acid is amidated; among them... Xaa1 is selected from Glu, Ala, Leu, D-Glu, Ser, Thr, Asp, Dap, or NMe-Glu; Xaa2 is selected from Leu, Ala, D-Leu, Glu, or NMe-Leu; Xaa3 is selected from Lys, Ala, D-Lys, Dap, Sar, Aib, D-Ala, Gly, Abu, or NMe-Lys; Xaa4 is selected from Val, Ala, D-Val, Glu, Gly, Sar, Aib, Abu, Nva, OctGly, or NMe-Val; Xaa5 is selected from Leu, Ala, D-Leu, Met, Asp, Glu, Ser, Lys, Dap, Sar, Aib, Me-Lys, Orn, P-Ser, Asn, Cit, or NMe-Lys; Xaa6 is selected from Met, Ala, D-Met, Leu, Phe, Val, Glu, α-(4-Pentenyl)-Ala, D-Ala, Sar, Dap, Aib, Abu, ALGly, OcAla, Nle, or NMe-Met; Xaa7 is selected from Glu, Ala, D-Glu, Val, D-Ala, Sar, Dap, Aib, Abu, Pra, D-Pra, or NMe-Glu; Xaa8 is selected from Lys, Ala, D-Lys, or does not exist; Xaa9 is selected from Glu, Ala, D-Glu, or may not exist; Xaa10 is selected from Leu, Ala, D-Leu, Glu, or is not present; Xaa11 is selected from Pro or does not exist; Xaa12 is selected from Gly or does not exist; Xaa13 is selected from Phe or does not exist; Xaa14 is selected from Leu or does not exist; Xaa15 is selected from Gln or does not exist; Xaa16 is selected from Ser or does not exist; Xaa17 is selected from Gly or does not exist; If the 8th amino acid (Xaa8) is missing, then all C-terminal residues following that site in the peptide chain will be absent.

2. The RAP polypeptide analogue or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The amino acid sequence of the RAP polypeptide analog is selected from any of the following: 。 3. Use of a RAP polypeptide analog of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of organ fibrosis.

4. The use according to claim 3, characterized in that, The organ fibrosis disease is selected from renal fibrosis, pulmonary fibrosis, liver fibrosis, or a combination thereof.

5. The use according to claim 4, characterized in that... The renal fibrosis includes obstructive nephropathy, diabetic nephropathy, hypertensive nephropathy, nephritis, renal tumors, adverse drug reactions, or pathogenic microbial infections. The pulmonary fibrosis includes pulmonary fibrosis caused by various risk factors such as smoking, viral infections, environmental pollution, genetic susceptibility, and medications. The liver fibrosis includes liver fibrosis caused by non-alcoholic fatty liver disease, hepatitis B, hepatitis C, and alcoholic liver disease.

6. The use of a RAP polypeptide analog of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of chronic kidney disease, pneumonia, and chronic liver disease.

7. A pharmaceutical composition comprising the RAP polypeptide analogue of any one of claims 1 or 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutically acceptable carrier is a pharmaceutically acceptable excipient, diluent, or functional excipient.

9. Use of the pharmaceutical composition of claim 7 in a medicament for the prevention and / or treatment of organ fibrosis.

10. The pharmaceutical composition according to claim 7, characterized in that, The formulations include tablets, capsules, granules, oral liquids, syrups, pills, ointments and patches for skin application, aerosols, nasal sprays, suppositories, microsphere formulations, injection formulations, or lyophilized powder injections.

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