The role of a novel peptide UPCP in the treatment of organ fibrosis

Abstract

The application discloses a novel peptide UPCP and an application of the peptide in treatment of organ fibrosis. The application provides an application of the UPCP polypeptide in treatment or improvement of organ fibrosis, reduction of cardiovascular or muscle tissue fibrosis degree of an animal and the like. Experiments prove that the UPCP can treat organ fibrosis; application of the UPCP peptide strategy to reverse cell aging in organ tissues can possibly fundamentally reverse tissue fibrosis, and has important significance for radical treatment of organ fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the role of a novel peptide, UPCP, in the treatment of organ fibrosis. Background Technology

[0002] Population aging is a social problem facing the world. It is predicted that my country's elderly population will reach 487 million around 2050, accounting for 34.9% of the total population, far exceeding the standard for population aging. Organ fibrosis is a progressive disease characterized by an increase in fibrous connective tissue and a decrease in parenchymal cells within organs, ultimately leading to organ deformation and functional failure. Statistics show that nearly 45% of deaths from various diseases can be attributed to tissue fibrosis. Research indicates that the progression of fibrosis is highly correlated with age; its incidence and mortality rates both increase with age, and the accumulation of senescent cells due to aging is key to the development of fibrosis.

[0003] Senescent cells are essentially in a state of growth arrest, no longer proliferating, but still maintaining metabolic activity. Senescent cells are primarily characterized by the senescence-associated secretory phenotype (SASP), which involves the secretion of inflammatory cytokines, growth factors, and proteases. The progression of organ fibrosis, including cardiac fibrosis, muscle fibrosis, liver fibrosis, kidney fibrosis, and idiopathic pulmonary fibrosis, is largely influenced by SASP. Currently, the main treatments for organ fibrosis include organ transplantation and drug therapy. However, organ transplantation is difficult to meet the needs of most patients with fibrosis due to the shortage of donor organs and the unavoidable risk of rejection. The most important targets for anti-fibrotic drugs currently include extracellular matrix-producing cells, fibrotic cytokines, and the extracellular matrix itself. Commonly used anti-fibrotic drugs targeting these targets include pentoxifylline, statins, calcium channel blockers, and antioxidants. Currently, there is no cure for fibrosis, indicating that more effective drugs and interventional treatment strategies need to be developed.

[0004] Peptide drugs have advantages such as small molecular weight, simple structure, and high cell membrane permeability.

[0005] Therefore, there is a need to find new strategies to cure organ fibrosis. Summary of the Invention

[0006] The purpose of this invention is to provide the uses of UPCP peptides.

[0007] On the one hand, the present invention provides the use of UPCP peptides in the preparation of products having any of the following functions.

[0008] 1) To treat or improve organ fibrosis;

[0009] 2) Reduces the degree of fibrosis in the cardiovascular or muscle tissue of animals;

[0010] 3) Improves the body weight of animals with idiopathic pulmonary fibrosis;

[0011] 4) Improves the motor function of animals with idiopathic pulmonary fibrosis;

[0012] 5) Improves lung tissue fibrosis in animals with idiopathic pulmonary fibrosis;

[0013] 6) Reduce the expression of aging-related markers in the lung tissue of animals with idiopathic pulmonary fibrosis or the liver tissue of animals with liver fibrosis;

[0014] 7) Alleviates cell aging;

[0015] 8) Inhibits the secretion of SASP factor in senescent cells;

[0016] 9) Treatment of liver function damage in animals caused by liver fibrosis;

[0017] 10) Reduce fibrosis in animals with liver fibrosis.

[0018] In the above text, the treatment or improvement of organ fibrosis refers to the treatment or improvement of cardiac fibrosis, the treatment or improvement of muscle fibrosis, the treatment or improvement of liver fibrosis, or the treatment or improvement of idiopathic pulmonary fibrosis.

[0019] On the other hand, the present invention also provides a product comprising the above-mentioned polypeptide or the polypeptide with a labeled group, wherein the active ingredient of the product is a polypeptide, and other auxiliary ingredients may be added.

[0020] The above products have any of the following functions:

[0021] 1) Treatment of organ fibrosis;

[0022] 2) Reduces the degree of fibrosis in the cardiovascular or muscle tissue of animals;

[0023] 3) Improves the body weight of animals with idiopathic pulmonary fibrosis;

[0024] 4) Improves the motor function of animals with idiopathic pulmonary fibrosis;

[0025] 5) Improves lung tissue fibrosis in animals with idiopathic pulmonary fibrosis;

[0026] 6) Reduce the expression of aging-related markers in the lung tissue of animals with idiopathic pulmonary fibrosis or the liver tissue of animals with liver fibrosis;

[0027] 7) Alleviates cell aging;

[0028] 8) Inhibits the secretion of SASP factor in senescent cells;

[0029] 9) Treatment of liver function damage in animals caused by liver fibrosis;

[0030] 10) Reduce fibrosis in animals with liver fibrosis.

[0031] In the above text, reducing the degree of fibrosis in animal cardiovascular or muscle tissue is reflected in: alleviating the degree of fibrosis in animal vascular adventitia, improving (weakening) the degree of fibrosis in animal heart tissue, or reducing the degree of fibrosis in animal muscle tissue.

[0032] Specifically, in the embodiments of the present invention, the animal mentioned above is a premature aging animal, specifically Lmna. G609G / G609G Premature aging mice.

[0033] In the above description, reducing aging-related markers in the lung tissue of animals with idiopathic pulmonary fibrosis or the liver tissue of animals with liver fibrosis is specifically defined in the embodiments of the present invention as p21 protein or p16 protein.

[0034] In the above, the mitigation of cell senescence in the embodiments of the present invention specifically takes BLM-induced cell senescence as an example;

[0035] In the above text, the SASP factors that inhibit the secretion of SASP factors in senescent cells are specifically IL6, IL8, and MMP9 in the embodiments of the present invention.

[0036] In the above text, the animal mentioned above is a mouse, which is used as an example in this invention.

[0037] Compared to the elimination of senescent cells, this invention innovatively proposes that "reversing cellular senescence" may serve as a novel strategy for the radical treatment of age-related organ fibrosis. Experiments of this invention demonstrate that UPCP can treat organ fibrosis; applying the UPCP peptide strategy to reverse cellular senescence in organ tissues may fundamentally reverse tissue fibrosis, which is of great significance for the radical treatment of organ fibrosis. Attached Figure Description

[0038] Figure 1 UPCP improves cardiovascular and muscle fibrosis in premature aging mice; A: Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Representative Masson staining image of the aorta of mice 13 weeks after birth, scale bars = 100 μm. B: Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G The width of the adventitia of the mouse aorta was statistically analyzed, n=3, *, P<0.05, **, P<0.01. C:Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609GRepresentative images of Masson staining of mouse heart tissue after 13 weeks, scale bars = 100 μm. D: for Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G The degree of cardiac fibrosis in mice was statistically analyzed (n=3, **, P<0.01). E: Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Representative images of Masson staining of mouse muscle tissue after 13 weeks, scale bars = 150 μm. F: Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G The degree of fibrosis in mouse muscle tissue was statistically analyzed (n=3, *, P<0.05, **, P<0.01).

[0039] Figure 2 Construction and validation of a mouse model of idiopathic pulmonary fibrosis; A: Schematic diagram of constructing a mouse model of idiopathic pulmonary fibrosis by endotracheal injection of bleomycin. B: Masson assay to detect fibrosis in mouse lung tissue; (B) is a representative image of Masson staining in mouse lung tissue, scale bars = 100 μm. C: Immunohistochemical assay to detect the expression level of α-SMA in mouse lung tissue, scale bars = 100 μm.

[0040] Figure 3 To illustrate the diffusion of UPCP in mice, mice were injected intraperitoneally with 20 mg / kg of FITC-UPCP. Six hours after injection, mouse lungs were harvested, and FITC fluorescence signals were collected using a mouse in vivo imaging system.

[0041] Figure 4 To improve body weight and motor dysfunction in mice with idiopathic pulmonary fibrosis using UPCP; A: Changes in body weight in mice in the control group, model group, and UPCP treatment group. B: Visualized images of mouse open field test trajectories and statistics of total movement distance, *, P<0.05.

[0042] Figure 5 To reduce α-SMA expression and collagen deposition in the lung tissue of mice with idiopathic pulmonary fibrosis using UPCP; A: Immunohistochemical assay to detect α-SMA expression in the lung tissue of mice in the control group, model group, and UPCP treatment group. (A) is a representative image, scale bars = 100 μm. B: Masson staining to detect fibrosis in the lung tissue of mice in the control group, model group, and UPCP treatment group. (B) is a representative image, scale bars = 100 μm.

[0043] Figure 6 To reduce p21 expression in the lung tissue of mice with idiopathic pulmonary fibrosis treated with UPCP; A: Western blot analysis was used to detect p21 expression in the lung tissue of mice in the control group, model group, and UPCP treatment group. B: Real-time PCR analysis was used to detect p21 expression in the lung tissue of mice in the control group, model group, and UPCP treatment group. *, P<0.05.

[0044] Figure 7 For UPCP cell safety analysis: A: In A549 cells, Western blot was used to detect the effect of UPCP on the expression of the cell proliferation marker Cyclin A2. B, C: In A549 cells, immunofluorescence assay was used to detect and statistically analyze the expression of Ki67. Scale bars = 100 μm, ns. No significant difference was observed (***, P < 0.001). D: A549 cells were seeded in 96-well plates and cultured for 24 h. Then, different concentrations of UPCP (0 μM, 10 μM, 20 μM, 30 μM) were added. OD values ​​were measured at 0 h, 24 h, 48 h, and 72 h of culture (ns). No significant difference was observed (***, P < 0.001). E: A549 cells were seeded in 96-well plates, and different concentrations of UPCP (0 μM, 10 μM, 20 μM, 30 μM) were added on the second day. After 48 h of culture, the OD value was detected. Cell viability = OD value of UPCP treatment group / OD value of control group, ns. There was no significant difference, ***, P<0.001.

[0045] Figure 8 To induce senescence in A549 cells using BLM: A: Western blot analysis was used to detect the expression level of p21 in A549 cells after treatment with different concentrations of bleomycin, with β-actin as an internal control. B, C: SA-β-gal staining assays were used to detect and statistically analyze the positive proportion of senescent cells in A549 cells after treatment with different concentrations of bleomycin. **, P < 0.01, ***, P < 0.001, Scale bars = 100 μm. D, E: Immunofluorescence assays were used to detect and statistically analyze the expression of Ki67 in A549 cells after treatment with different concentrations of bleomycin. Scale bars = 100 μm, ***, P < 0.001.

[0046] Figure 9To alleviate BLM-induced senescence in A549 cells, UPCP was added to BLM-induced senescent A549 cells. Western blot was used to detect p21 expression levels, with β-actin as an internal control. B and C: UPCP was added to BLM-induced senescent A549 cells. SA-β-gal staining was used to detect the effect of UPCP on BLM-induced senescent A549 cells. ***, P < 0.001, Scale bars = 100 μm. D and E: UPCP was added to BLM-induced senescent A549 cells. Immunofluorescence assay was used to detect and statistically analyze the effect of UPCP on Ki67 expression in BLM-induced senescent A549 cells. ***, P < 0.001, *, P < 0.05, Scale bars = 100 μm.

[0047] Figure 10 UPCP was used to inhibit the secretion of SASP-related factors and improve fibrosis. AC: UPCP was added to BLM-induced senescent A549 cells, and the mRNA expression levels of IL6, IL8, and MMP9 were detected by RT-qPCR. *, P<0.05, **, P<0.01. D: UPCP was added to BLM-induced senescent A549 cells, and the expression levels of p21, IL6, and IL8 were detected by Western blot, with β-actin as an internal control.

[0048] Figure 11 Construction and validation of a mouse model of carbon tetrachloride-induced liver fibrosis; A: Schematic diagram of the construction of a mouse model of carbon tetrachloride-induced liver fibrosis. B: Sirius red staining experiment to detect fibrosis in mouse liver tissue. (B) is a representative image of Sirius red staining in mouse liver tissue, scale bars = 200 μm. C: Immunohistochemical experiment to detect the expression of α-SMA in mouse liver tissue, scale bars = 200 μm.

[0049] Figure 12 To demonstrate that UPCP can improve liver fibrosis in mice with liver fibrosis: A: Sirius red staining assay to detect fibrosis in mouse liver tissue. (A) is a representative image of Sirius red staining in mouse liver tissue, scale bars = 200 μm. B: Immunohistochemical assay to detect the expression of α-SMA in mouse liver tissue. C: RT-PCR to detect the expression of TGF-β and α-SMA in mouse liver tissue. *, P < 0.05, **, P < 0.01. D, E: Kits to detect the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice in the control group, model group, and UPCP treatment group. *, P < 0.05, *, P < 0.05, ***, P < 0.001.

[0050] Figure 13 UPCP was used to reduce the expression of p21 and p16 in the liver tissue of mice with liver fibrosis; AB: RT-PCR was used to detect the expression of p21 and p16 in mouse liver tissue, *, P<0.05, **, P<0.01. Detailed Implementation

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0053] The relevant information regarding the antibodies, kits, and other reagents used in the following examples is shown in Table 1 below:

[0054] Table 1 is a summary table of antibodies and reagents.

[0055]

[0056] The cells used in the following examples are the human non-small cell lung cancer cell line A549, purchased from the American Type Culture Collection (ATCC).

[0057] The mice used in the experiment were all fed according to a 12-hour diurnal rhythm, and the experimental procedures complied with the animal use guidelines of Northeast Normal University.

[0058] The following examples used live animals, namely wild-type mice of the ICR strain and Lmna strain. G609G / G609G Premature aging mice and C57BL / 6 male wild-type mice. Among them, ICR wild-type mice and C57BL / 6 male wild-type mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0059] In addition, Lmna G609G / G609G The premature aging mouse model was prepared according to the following method:

[0060] Lmna + / G609G Male and female mice were crossbred, and mice with the genotype G609G / G609G were selected as Lmna mice. G609G / G609G A premature aging mouse model was established. Genotyping of the mice was performed using a Mouse Direct PCR Kit (For Genotyping) (Bimake 820049). The primer sequences were: mLMNA_fwd:CGAAGGCTTCCTGGCTATTT; mLMNA_rev:TGCTGTAGGGCAGAGATGA. The target fragments were then recovered and sequenced by Bimake to identify the mouse genotypes.

[0061] The above Lmna + / G609G The mice were obtained by replacing the Lmna gene (Gene ID: 169052022-2-6) in the genome of ICR wild-type mice with an allele carrying the c.1827C>T; p.G609G mutation (the C at position 1827 of the sequence shown in genbank Gene ID: 16905, 2022-2-6 is mutated to T). In these mice, one chromosome contains the Lmna gene carrying the c.1827C>T; p.G609G mutation, and the other chromosome contains the Lmna gene (Gene ID: 16905 2022-2-6).

[0062] The Realtime PCR primer sequences used in the following examples are shown in Table 2 below:

[0063] Table 2 shows the primer sequences.

[0064]

[0065] The experimental methods in the following examples are as follows:

[0066] I. RNA extraction, reverse transcription, and RT-qPCR

[0067] Conventional method.

[0068] II. Western Blot

[0069] Conventional method.

[0070] III. Age-related β-galactosidase (SA-β-gal) staining assay

[0071] Conventional method.

[0072] IV. Immunohistochemical assay (IHC)

[0073] Standard methods were used. Tissue samples were obtained from mouse models (control group, model group, and UPCP treatment group) bred in our laboratory. After ex vivo fixation with 4% paraformaldehyde for 72 hours, tissue blocks were prepared and sections were performed by pathologists at the First Bethune Hospital of Jilin University. Subsequent experiments were conducted using the Zhongshan Jinqiao IHC kit (a universal two-step kit and a DAB chromogenic kit).

[0074] V. Masson staining

[0075] In this experiment, Absin's Masson trichrome staining solution (abs9347) was used to stain the tissue.

[0076] VI. Sky Blue and Scarlet Staining

[0077] In this experiment, Absin Sirius Staining Kit (absin TOP0294) was used to stain the tissue.

[0078] VII. Behavioral Analysis of Mice

[0079] This experiment will use the open field test to assess the motor abilities of mice. The specific steps are as follows:

[0080] 1. Place the mice in the experimental environment one day in advance to allow them to become familiar with the environment (the experiment is conducted in a quiet environment).

[0081] 2. Place the mouse in the center of the bottom of the box, and the camera will capture images and stop recording after a certain time (clean the inside wall and bottom of the box promptly after each mouse finishes the experiment to avoid affecting the next mouse).

[0082] 3. Different parameters are detected using computer software, and the corresponding data is obtained and analyzed.

[0083] The novel peptide UPCP described in the following examples is composed of 47 L-type amino acids, including the transmembrane peptide HIV-TAT, and has the molecular formula C. 211 H 360 N 80 O 66 S4.

[0084] The amino acid sequence of UPCP is as follows: YGRKKRRQRRRYNLRSRTVLCGTCGQPADKASASGSGAQSPQNCSIM (Sequence 1).

[0085] The aforementioned UPCP can be prepared through artificial synthesis or prokaryotic expression.

[0086] In the examples below, PBS is a 1X buffer solution with a pH of 7.4.

[0087] Example 1: UPCP improves Lmna G609G / G609G Cardiovascular and muscle fibrosis in premature aging mice

[0088] Childhood progeria-growth syndrome (HGPS) is a rare but serious disease of premature aging. The main clinical manifestation of HGPS is cardiovascular abnormalities, such as adventitia. These cardiovascular abnormalities contribute to the disease progression and mortality of HGPS.

[0089] (1) Administration of UPCP

[0090] male Lmna + / G609G Mice and female Lmna + / G609GMice were hybridized, and the offspring mice were genotyped one week after birth. The genotypes of the mice were identified using a Mouse Direct PCR Kit (For Genotyping) (Bimake820049) for PCR fragment amplification. The primer sequences were: mLMNA_fwd:CGAAGGCTTCCTGGCTATTT; mLMNA_rev:TGCTGTAGGGCAGAGATGA. The target fragments were then recovered and sequenced by Kumei Company to identify the mouse genotypes.

[0091] Selecting Lmna at 14 days old G609G / G609G Premature aging mice were administered UPCP via intraperitoneal injection at a dose of 20 mg / kg every other day. The control group showed Lmna... G609G / G609G Premature aging mice were injected with the same amount of PBS using the same method. The mice were continuously fed, their condition was observed, and the therapeutic effect of UPCP on premature aging mice was evaluated using Masson staining (Absin, abs9347, following the prescribed staining kit). Details are as follows:

[0092] UPCP processing group (UPCP+ or Lmna) G609G / G609G +UPCP): In Lmna G609G / G609G Fourteen days after birth, mice were injected intraperitoneally with 20 mg / kg UPCP into Lmna via every other day. G609G / G609G In premature aging mice;

[0093] PBS-treated groups (UPCP- or Lmna) G609G / G609G ): Premature aging mice in the control group (Lmna) G609G / G609G Inject an equal volume of PBS.

[0094] Lmna + / + Group: ICR wild-type mice were injected with an equal volume of PBS.

[0095] (2) Masson staining to assess the effect of UPCP on improving cardiovascular and muscle fibrosis in premature aging mice.

[0096] The effects of UPCP on Lmna were assessed by measuring the degree of lateral fibrosis in the aorta after treating mice in the UPCP-treated and PBS-treated groups. G609G / G609G Effects on the cardiovascular system of premature aging mice.

[0097] Masson staining of the aorta of mice treated with UPCP and PBS was performed 13 weeks after treatment (Absin, abs9347, according to the staining kit).

[0098] Experimental results showed that, similar to wild-type Lmna+ / + Compared to the aorta in mice, Lmna G609G / G609G In premature aging mice, the adventitia of the aorta showed fibrosis and thickening, while UPCP treatment could alleviate Lmna to some extent. G609G / G609G The degree of fibrosis in the vascular adventitia of premature aging mice and a certain degree of reduction in adventitia thickness. Figure 1 AB).

[0099] Consistent with this, UPCP treatment can significantly improve Lmna G609G / G609G Cardiac pathological characteristics in premature aging mice, such as reducing the degree of cardiac tissue fibrosis: Masson staining of heart tissue from mice in the UPCP-treated group and the PBS-treated group 13 weeks after treatment (heart tissue was removed after perfusion and stained). The results showed that UPCP significantly improved (reduced) Lmna G609G / G609G The degree of cardiac tissue fibrosis in premature aging mice ( Figure 1 CD).

[0100] Meanwhile, Masson staining of muscle tissue from mice in the UPCP-treated group and the PBS-treated group 13 weeks after treatment (muscle tissue from the mouse leg was removed after perfusion and stained) showed that UPCP could also reduce Lmna G609G / G609G The degree of fibrosis in the muscle tissue of prematurely aged mice ( Figure 1 EF).

[0101] The above experimental results demonstrate that UPCP treatment can significantly improve Lmna G609G / G609G The degree of fibrosis in the cardiovascular and muscle tissues of prematurely aged mice.

[0102] Example 2: UPCP improves bleomycin-induced idiopathic pulmonary fibrosis in mice.

[0103] 1. Construction of a mouse model of idiopathic pulmonary fibrosis

[0104] Bleomycin group mice: Nine-week-old male wild-type mice of the C57BL / 6 strain were injected intratracheally with bleomycin (5 mg / Kg, diluted with physiological saline) one week after being fed.

[0105] Control group mice: Nine-week-old male wild-type mice of strain C57BL / 6 were injected intratracheally with an equal volume of physiological saline after one week of rearing. Figure 2 A).

[0106] One week after modeling, mice were euthanized by cervical dislocation, and lung tissue was dissected for histopathological analysis. Collagen fiber deposition in the lungs is a major clinicopathological feature of idiopathic pulmonary fibrosis, and Masson staining is an effective method for detecting collagen fiber expression in tissues.

[0107] Masson staining revealed that collagen deposition in the lungs of mice injected intratracheally with bleomycin was significantly higher than that in the mice injected with saline. Figure 2 B).

[0108] Extracellular matrix deposition occurs in pulmonary fibrosis, mainly due to the fibrotic effect of myofibroblasts. α-SMA (α-smooth muscle actin) is a major marker of myofibroblast activation. The expression of α-SMA in the cytoplasm enables myofibroblasts to contract, and this contractile property is crucial to the fibrotic process.

[0109] The expression of α-SMA in the lung tissue of mice was analyzed by immunohistochemistry.

[0110] Immunohistochemical results showed that the expression level of α-SMA in the lungs of mice injected intratracheally with bleomycin was significantly higher than that in the control group. Figure 2 C).

[0111] Based on the above experimental results, a bleomycin-induced mouse model of idiopathic pulmonary fibrosis was successfully constructed.

[0112] 2. UPCP can diffuse from the peritoneal cavity to the lungs of mice.

[0113] To detect the uptake of UPCP in the lung tissue of mice, FITC-UPCP (with a FITC group labeled at the N-terminus of UPCP) was injected into mice via intraperitoneal injection.

[0114] The results showed that FITC-UPCP diffused into mouse lung tissue 6 hours after injection, and a strong FITC signal was clearly detected in the mouse lung tissue. Figure 3 This indicates that FITC-UPCP has strong penetrability and can diffuse from the abdominal cavity to the lung tissue in a relatively short time.

[0115] 3. UPCP can delay the decrease in body weight and improve motor dysfunction in mice with idiopathic pulmonary fibrosis.

[0116] 1) UPCP administration

[0117] To investigate the therapeutic effect of UPCP, male wild-type mice of strain C57BL / 6 were divided into a control group, a model group, and a UPCP treatment group. Ten-week-old mice were anesthetized with pentobarbital, and their tracheas were surgically exposed. The mice were then treated as follows:

[0118] Control group: 0.9% saline was injected into the airway, and the wound was then sutured. The mice were then fed normally after they recovered.

[0119] Model group: 5 mg / kg of bleomycin (diluted with physiological saline) was injected into the airway, and then the wound was sutured. After the mice recovered, they were fed normally, and physiological saline was injected into the peritoneum daily starting from the second day.

[0120] UPCP treatment group: 5 mg / kg of bleomycin (diluted with physiological saline) was injected into the airway, and then the wound was sutured. After the mice recovered, they were fed normally, and UPCP (20 mg / kg) was injected into the peritoneum daily starting from the second day.

[0121] 2) Mouse weight monitoring

[0122] To evaluate the therapeutic effect of UPCP, the weight of mice was monitored throughout the treatment process.

[0123] The results are as follows Figure 4 As shown in Figure A, compared with the control group, the body weight of mice in the model group (treated with bleomycin) showed a significant decreasing trend. The decreasing trend of body weight of mice after UPCP treatment was significantly improved compared with the untreated group (model group), but the body weight of the mice did not recover to the initial body weight.

[0124] This indicates that UPCP can improve the body weight of mice with idiopathic pulmonary fibrosis to some extent.

[0125] 3) Mouse motor ability test

[0126] Patients with idiopathic pulmonary fibrosis have reduced lung capacity and vital capacity, and lower blood oxygen levels, which significantly affects their behavioral abilities.

[0127] To further investigate the therapeutic effect of UPCP on behavioral impairment caused by idiopathic pulmonary fibrosis, the therapeutic effect of UPCP was evaluated by an open field test on day 7 after intraperitoneal injection.

[0128] Regarding motor function, compared with the control group, the model group mice showed significantly weakened motor function, while the mice treated with UPCP showed significantly enhanced motor function. Figure 4 B).

[0129] The experimental results show that UPCP can improve the motor function of mice with idiopathic fibrosis.

[0130] 4. UPCP can improve pulmonary fibrosis in mice with idiopathic pulmonary fibrosis.

[0131] Abnormal activation of fibroblasts is a key marker in the development of idiopathic pulmonary fibrosis, and numerous studies have shown that α-SMA is an important marker of activated myofibroblasts. Furthermore, the expression of α-SMA in cells endows myofibroblasts with contractile properties, which are closely related to pulmonary fibrosis.

[0132] On day 10 after intraperitoneal injection of UPCP, the expression of α-SMA in the lung tissue of mice in each group of 3(1) above was detected by immunohistochemistry.

[0133] Compared with the control group, the α-SMA content in the model group was significantly upregulated and diffuse. After UPCP treatment, the α-SMA content decreased significantly. Figure 5 A).

[0134] Therefore, UPCP treatment significantly reduced the content of α-SMA in the lung tissue of mice with bleomycin-induced idiopathic pulmonary fibrosis, suggesting that UPCP treatment of idiopathic pulmonary fibrosis may be achieved by reducing the expression of α-SMA.

[0135] Furthermore, the main clinicopathological feature of idiopathic pulmonary fibrosis is the deposition of collagen fibers in the lungs, affecting alveolar structure and function. Masson staining is an effective method for detecting collagen fiber expression in tissues.

[0136] Therefore, Masson staining was used to evaluate the therapeutic effect of UPCP on idiopathic pulmonary fibrosis, as detailed below:

[0137] (1) Mice were anesthetized with 3% sodium pentobarbital and fixed to the sampling board with pins;

[0138] (2) Use scissors to trim the hair on the mouse's chest and disinfect it with an alcohol swab;

[0139] (3) Cut open the mouse's thoracic cavity to expose the lung tissue;

[0140] (4) The lungs of mice were dissected, fixed in paraformaldehyde for 72 hours, made into wax blocks and sectioned;

[0141] (5) Mouse lung tissue was stained using the Masson staining kit (Absin, abs9347).

[0142] The experimental results showed that the model group had significant collagen deposition in the lungs compared to the control group; however, after UPCP treatment, the collagen deposition in the lungs was significantly reduced. Figure 5 B).

[0143] Experimental results show that UPCP can reduce collagen deposition in the lungs of model mice.

[0144] Therefore, UPCP can improve lung tissue fibrosis in mice with idiopathic pulmonary fibrosis.

[0145] 5. UPCP reduces p21 expression in lung tissue of mice with idiopathic pulmonary fibrosis.

[0146] Idiopathic pulmonary fibrosis (IPF), an age-related disease, has an increasing incidence with age. Most IPF patients are diagnosed at age 65 or older, and studies have shown the presence of senescent cell accumulation in the lungs of IPF patients. Therefore, attention has focused on the age-related biomarker p21 (Gene ID: 12575, updated 2022-08-11).

[0147] Proteins were extracted from the lung tissues of mice in each group of 3(1) above and detected by immunoblotting.

[0148] The results showed that, compared with the control group, the p21 protein level in mouse lung tissue was significantly upregulated in the model group, indicating that pulmonary fibrosis is closely related to aging, and that aging-related biomarkers change during pulmonary fibrosis. In the UPCP treatment group, the p21 level in mouse lung tissue showed a significant downregulation. Figure 6 A).

[0149] Total RNA was extracted from mouse lung tissue, and consistent results were obtained by RT-PCR analysis. Figure 6 B).

[0150] The above experiments suggest that UPCP can downregulate the expression of the aging-related marker p21, implying that UPCP may play a role in improving pulmonary fibrosis through aging-related pathways.

[0151] Example 3: Preliminary exploration of the mechanism by which UPCP improves idiopathic pulmonary fibrosis

[0152] 1. UPCP security testing

[0153] To further investigate whether UPCP has cytotoxicity, A549 cells (ATCC, CCL-185) were cultured for 48 h in medium containing 6 μM UPCP (DMEM complete medium + 6 μM UPCP). Cells cultured in medium without UPCP served as a control.

[0154] Western blot analysis was used to detect the expression of the proliferation marker Cyclin A2 in A549 cells after 48 hours of culture. The results are as follows: Figure 7 As shown in Figure A, UPCP was found to have no significant effect on the expression of Cyclin A2, a proliferation marker in A549 cells. Figure 7 A).

[0155] Immunofluorescence assays were used to detect Ki67 expression in A549 cells after 48 hours of culture. Results are as follows: Figure 7 As shown in B and 7C, UPCP had no effect on Ki67 expression in A549 cells. Figure 7 B and Figure 7 C).

[0156] Next, the Cell Counting Kit-8 (CCK-8) (APExBIO k1018) was used to detect the effect of UPCP on the viability of A549 cells after culturing in medium containing different concentrations of UPCP for 72 h.

[0157] CCK-8 test results are as follows Figure 7 As shown in AC, 6 μM UPCP did not affect cell proliferation compared to the control group. Figure 7 Furthermore, low concentrations of UPCP (10 μM, 20 μM) had no effect on A549 cell growth, while high concentrations of UPCP (30 μM) inhibited A549 cell growth with increasing culture time. Figure 7 D).

[0158] A549 cells were cultured for 48 hours in medium containing different concentrations of UPCP. The effect of different concentrations of UPCP on cell viability was statistically analyzed, and the results are as follows: Figure 7 As shown in Figure E, low concentrations of UPCP (10 μM, 20 μM) did not affect A549 cell survival. However, high concentrations of UPCP (30 μM) inhibited A549 cell survival to some extent. Figure 7 E).

[0159] The above results indicate that low concentrations of UPCP have no significant toxic effect on cells.

[0160] 2. UPCP alleviates BLM-induced senescence in A549 cells.

[0161] (1) BLM induces senescence in A549 cells

[0162] UPCP has been shown to improve pulmonary fibrosis in mice at the individual level. In this process, it was found that after UPCP was injected into mice, the level of p21, a marker of aging of lung tissue proteins, was significantly downregulated compared with the model group. This suggests that UPCP may alleviate pulmonary fibrosis by slowing down cellular senescence.

[0163] To investigate the role and specific mechanism of UPCP in alleviating idiopathic pulmonary fibrosis, a BLM-induced A549 cell senescence model was constructed.

[0164] Healthy A549 cells were cultured, and when the cell confluence reached 70%-80%, bleomycin (BLM) at concentrations of 0 μM, 50 μM, 100 μM, and 200 μM was added for 16 h. A control group was used without BLM.

[0165] Western blot results showed that BLM induced an increase in p21 expression in A549 cells. Furthermore, the upregulation of p21 protein levels was more significant at 100 μM bleomycin, while at 200 μM bleomycin, p21 protein levels showed a downregulation trend compared to 100 μM. Figure 8 A).

[0166] SA-β-gal staining experiments showed that the proportion of positive staining in BLM-treated A549 cells was significantly higher than that in the control group, and this increase was concentration-dependent. The highest number of positive cells was observed at a bleomycin concentration of 100 μM, while apoptosis was observed at a bleomycin concentration of 200 μM. Figure 8 (B and C).

[0167] Immunofluorescence assays showed that the number of Ki67-positive A549 cells treated with BLM was significantly lower than that in the control group, indicating that BLM could reduce the proliferation capacity of A549 cells in a dose-dependent manner. It was found that the proportion of Ki67-positive cells was low at a bleomycin concentration of 100 μM; however, at a bleomycin concentration of 200 μM, cells entered the apoptosis program. Figure 8 (D and E).

[0168] Based on the above experimental results, a BLM concentration of 100 μM can significantly induce senescence in A549 cells. Therefore, a Bleomycin concentration of 100 μM was selected for subsequent experiments.

[0169] (2) UPCP alleviates BLM-induced senescence in A549 cells

[0170] Experimental group: A549 cells in good condition were cultured. When the cell confluence was 70%-80%, bleomycin (BLM) at a concentration of 100 μM was added and the cells were treated for 16 h. The medium was then replaced with new DMEM + 10% FBS medium, and UPCP at a concentration of 6 μM was added and the cells were cultured for 48 h.

[0171] Control group: Unlike the experimental group, the control group did not include BLM.

[0172] Proteins were extracted from cells after 48 hours of UPCP culture and detected by Western blot.

[0173] Experimental results showed that p21 expression was significantly upregulated in BLM-treated A549 cells compared to the control group, indicating that BLM induces A549 cell senescence. When UPCP was added to senescent A549 cells, p21 expression was downregulated to some extent. Figure 9 A).

[0174] After 48 hours of UPCP culture, cells from each group were stained with SA-β-gal. The experiment showed that the proportion of senescent A549 cells positive for staining was significantly increased in BLM-treated A549 cells compared to the control group, while the proportion of SA-β-gal positive cells in senescent A549 cells was significantly decreased in UPCP-treated cells. Figure 9 (B and 9C).

[0175] After 48 hours of UPCP culture, immunofluorescence was performed on cells from each group. The results showed that the number of Ki67-positive cells in A549 cells treated with BLM was significantly lower than the proportion of Ki67-positive cells in the control group. However, UPCP treatment of senescent A549 cells significantly increased the proportion of Ki67-positive cells. Figure 9 (D and 9E).

[0176] The above experimental results indicate that UPCP can alleviate BLM-induced senescence in A549 cells.

[0177] (3) UPCP inhibits the secretion of SASP-related factors and improves fibrosis.

[0178] A key characteristic of cellular senescence is the development of the senescence-associated secretory phenotype (SASP), which includes pro-inflammatory cytokines, growth factors, chemokines, and matrix remodeling enzymes (such as metalloproteinases). Since UPCP can delay cellular senescence, does it also have an effect on the secretion of SASP-related factors? The following investigation examined the effect of UPCP on the expression of SASP-related factors.

[0179] Realtime-PCR was performed on cells treated with BLM for 16 h and cultured with UPCP for 48 h in the experimental and control groups in (2) above.

[0180] Experiments showed that after 16 h of BLM treatment, the levels of SASP-related components IL6, IL8, and MMP9 in A549 cells were significantly upregulated compared to the control group. This indicates that cellular senescence can induce the secretion of SASP-related factors IL6, IL8, and MMP9. Furthermore, after 48 h of UPCP treatment in BLM-induced senescent A549 cells, the levels of IL6, IL8, and MMP9 showed a uniform downward trend compared to senescent A549 cells. Figure 10 A, 10B, and 10C).

[0181] Western blot analysis was performed on cells treated with BLM for 16 h and cultured with UPCP for 48 h in the experimental and control groups mentioned in (2) above. The experiment showed that after BLM treatment of A549 cells, the level of p21 in the cells was significantly upregulated compared to the control group, while the level of p21 recovered somewhat after the addition of UPCP. Simultaneously, the levels of SASP-related factors IL6 and IL8 were significantly upregulated compared to the control group, and the levels of IL6 and IL8 also recovered somewhat after UPCP treatment. Figure 10 D).

[0182] The above results indicate that UPCP can delay BLM-induced senescence of A549 cells while inhibiting the secretion of SASP factor in senescent cells.

[0183] Example 4: UPCP improves carbon tetrachloride-induced liver fibrosis in mice

[0184] (1) Construction of a mouse model of liver fibrosis

[0185] Male wild-type C57BL / 6 mice aged 6-8 weeks were intraperitoneally injected with 100 μL of carbon tetrachloride solution (CCl4, 1 mL / Kg (20%), diluted with corn oil (Selleck, S6701, dilution ratio: 8 mL carbon tetrachloride + 32 mL corn oil)) after one week of feeding. Control mice were intraperitoneally injected with the same volume of corn oil. The administration frequency was twice a week. Figure 11 A). Six weeks after modeling, liver tissue from mice was dissected for histopathological analysis.

[0186] Sirius red is a strongly acidic anionic dye that readily reacts with the basic groups in collagen fibers, making it a specific collagen dye. Hepatic collagen fiber deposition is a major clinicopathological feature of liver fibrosis patients. Sirius red staining was used to stain mouse liver tissue.

[0187] The results showed that, compared with the control group, mice injected intraperitoneally with carbon tetrachloride for 6 weeks had significant collagen deposition in their liver tissue. Figure 11 B).

[0188] Furthermore, extracellular matrix deposition and synthesis occur during liver fibrosis, primarily due to the fibrotic effect of myofibroblasts. α-SMA (α-smooth muscle actin) is a major marker of myofibroblast activation, and its expression in the cytoplasm endows myofibroblasts with contractility, a property crucial for the fibrotic process. Immunohistochemical experiments were used to detect α-SMA expression in mouse liver tissue. The results showed that the expression level of α-SMA in the liver tissue of mice injected intraperitoneally with carbon tetrachloride was significantly higher than that in the control group. Figure 11 C).

[0189] Based on the above experimental results, a carbon tetrachloride-induced mouse liver fibrosis model was successfully constructed.

[0190] (2) UPCP can improve liver tissue fibrosis in mice with liver fibrosis.

[0191] 1) UPCP administration

[0192] To investigate the therapeutic effect of UPCP, male wild-type mice of strain C57BL / 6 were divided into UPCP treatment group, model group and control group.

[0193] Control group: 8-week-old mice were injected intraperitoneally with 100 μL of corn oil twice a week for 6 weeks. After 6 weeks, they were injected intraperitoneally with 100 μL of physiological saline every other day for 4 weeks.

[0194] Model group: 8-week-old mice were intraperitoneally injected with 100 μL of carbon tetrachloride solution (20% diluted with corn oil) twice a week for 6 weeks. After 6 weeks, the carbon tetrachloride solution was still injected twice a week, and 100 μL of physiological saline was injected intraperitoneally every other day for 4 weeks.

[0195] UPCP treatment group: 8-week-old mice were intraperitoneally injected with 100 μL of carbon tetrachloride solution (20% diluted with corn oil) twice a week for 6 weeks. After 6 weeks, the mice continued to be injected with carbon tetrachloride solution twice a week and were also treated with intraperitoneal injection of 100 μL of UPCP (20 mg / Kg) every other day for 4 weeks.

[0196] 2) UPCP can improve liver fibrosis in mice.

[0197] Liver fibrosis patients are mainly characterized by an increase in myofibroblasts and collagen fiber deposition in the liver, affecting liver structure and function. Sirius red staining was used to evaluate the therapeutic effect of UPCP on liver fibrosis.

[0198] Liver samples were taken from different groups of mice in 1) above, and liver tissue was obtained. Sirius red staining was performed. The staining results showed that, compared with the control group mice, the model group mice had significant collagen deposition in their liver tissue. After UPCP treatment, collagen deposition in the liver tissue of the liver fibrotic mice was significantly reduced. Figure 12 A).

[0199] Experimental results showed that UPCP could reduce fibrosis in mice with liver fibrosis.

[0200] In addition, abnormal activation of fibroblasts is an important marker in the development of liver fibrosis. Studies have shown that α-SMA and TGF-β are important markers of liver fibrosis.

[0201] Liver samples were taken from different groups of mice in 1) above to obtain mouse liver tissue, and the expression of α-SMA in the mouse liver tissue was detected by immunohistochemistry. Compared with the control group mice, the expression level of α-SMA in the liver tissue of the model group mice was significantly upregulated.

[0202] RNA was extracted from liver tissues of different groups of mice in 1). RT-PCR experiments revealed that, compared with the control group, the levels of α-SMA and TGF-β in the liver tissue of the model group mice were significantly upregulated, while the levels of α-SMA and TGF-β in the liver tissue of mice with liver fibrosis treated with UPCP were significantly decreased. Figure 12 (B and C).

[0203] The above experiments show that UPCP treatment can significantly reduce the expression of α-SMA and TGF-β in liver tissue of mice with carbon tetrachloride-induced liver fibrosis, suggesting that UPCP treatment of liver fibrosis in mice may be achieved by reducing the expression of α-SMA and TGF-β.

[0204] 3) UPCP improves liver function in mice with liver fibrosis.

[0205] Liver fibrosis disrupts the liver's microstructure and leads to abnormal liver function. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important indicators for monitoring liver function. Numerous studies have shown that ALT and AST levels are low in normal serum, but abnormal changes occur when liver function is impaired.

[0206] Blood samples were collected from mice in different groups as described in section 1) via orbital blood sampling. The therapeutic effect of UPCP on liver function was evaluated using corresponding AST (Nanjing Jiancheng GOT kit C010-2-1) and ALT (Nanjing Jiancheng GPT kit C009-2-1) assay kits. It was found that compared with the control group, the serum ALT and AST levels in the model group mice were significantly increased, while the serum ALT and AST levels in mice with liver fibrosis treated with UPCP were significantly decreased. Figure 12 (D and E).

[0207] The above experimental results show that UPCP treatment can significantly reduce the levels of ALT and AST in the serum of mice with liver fibrosis, suggesting that UPCP has a certain therapeutic effect on liver function damage caused by liver fibrosis.

[0208] 4) UPCP reduces the expression of p21 and p16 in the liver tissue of mice with liver fibrosis.

[0209] Studies have found that the aging of various liver cells plays an important role in chronic liver diseases such as liver fibrosis. Therefore, the focus has been on aging-related biomarkers p21 and p16.

[0210] RNA was extracted from liver tissues of different groups of mice in group 1). RT-PCR experiments revealed that, compared with the control group, the levels of p21 and p16 in the liver tissues of the model group mice were significantly upregulated, while the levels of p21 and p16 in the liver tissues of fibrotic mice in the UPCP treatment group were significantly downregulated. Figure 13 (A and B).

[0211] The above results indicate that liver fibrosis is closely related to aging, and that there are changes in aging-related biomarkers during the process of liver fibrosis. UPCP can downregulate the expression of aging-related markers p21 and p16, suggesting that UPCP may play a role in improving liver fibrosis through aging-related pathways.

Claims

1. Application of UPCP peptides in the preparation of products that treat or improve cardiac fibrosis in premature aging mice; The amino acid sequence of the UPCP is sequence 1.

2. Application of UPCP peptides in the preparation of products that treat or improve muscle fibrosis in prematurely aging mice; The amino acid sequence of the UPCP is sequence 1.

3. Application of UPCP peptides in the preparation of products that treat or improve liver fibrosis in mice; The amino acid sequence of the UPCP is sequence 1.

4. Application of UPCP peptides in the preparation of products that treat or improve idiopathic pulmonary fibrosis in mice; The amino acid sequence of the UPCP is sequence 1.

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