Use of phlpp1 and related substances in the treatment of pulmonary fibrosis

Abstract

The application relates to the technical field of biological medicine engineering, and provides application of PHLPP1 and related substances in treatment of pulmonary fibrosis. Specifically, the application relates to the role of PHLPP1 protein, a nucleic acid molecule for coding the protein, a promoter or a recombinant expression vector in preparation of products for treating pulmonary fibrosis and related symptoms or other complications, and also relates to corresponding recombinant vectors or pharmaceutical compositions. Experimental results show that overexpression of PHLPP1 can effectively inhibit the level of a fibrosis promoting factor, and has effective application value in alleviating and treating the pathology of pulmonary fibrosis and other symptoms or other complications. Therefore, the application provides a new target for treatment of pulmonary fibrosis, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of type 1 protein phosphatase Pleckstrin homologous sequence leucine-rich repeat fragment protein phosphatase 1 (PHLPP1) as a target in the preparation of drugs for treating pulmonary fibrosis, especially idiopathic pulmonary fibrosis, and also to carriers and pharmaceutical compositions containing it. Background Technology

[0002] Fibrotic diseases are a major cause of death, and whether they are toxin-related, genetic, tumor-related, infectious, autoimmune-related, or idiopathic, they can affect the function of any organ. Pulmonary fibrosis (PF) is a chronic, progressive pathological change driven by repeated lung injury and inflammation, with a median survival of only 3 years. Although some anti-fibrotic drugs can slow the progression of PF, due to insufficient understanding of its etiology, there are currently no effective interventions to reverse pulmonary fibrosis. Therefore, elucidating the occurrence, development, and key regulatory mechanisms of pulmonary fibrosis after lung injury is of significant clinical importance for developing drug interventions to prevent post-injury pulmonary fibrosis and lung failure.

[0003] Lung fibroblasts are the most important effector cells in pulmonary fibrosis, producing large amounts of extracellular matrix (ECM) in the alveoli and interstitium. The differentiation and activation of lung fibroblasts are triggered and maintained by various cytokines and growth factors released by alveolar epithelial cells and immune cells. Among immune cells, alveolar macrophages (AMs) interact with activated myofibroblasts, inducing chronic inflammation and pulmonary fibrosis (PF). AMs are located within the airways and develop from macrophages that differentiate into monocytes (Mo-AMs) or tissue-resident (TR-AMs) through local proliferation. Myeloid-derived macrophages are a crucial cell type influencing chronic inflammation and pulmonary fibrosis after lung injury. During lung tissue injury, macrophages migrate to the site of injury and differentiate into macrophages with different phenotypes and functions based on different microenvironmental stimuli: M1 with a pro-inflammatory phenotype and M2 with a pro-fibrotic phenotype. The balance of the M1 / M2 phenotype determines the fate of lung tissue after inflammation or injury. In the early inflammatory stage of lung tissue injury, macrophages (AMs) are recruited to the injury site and tend to polarize in the M1 region. The secretion of inflammatory factors, such as TNF-α, IL-1β, NO, and ROS, increases significantly, inducing an excessive inflammatory response and exacerbating lung tissue damage. As the injury progresses, the proportion of M2 macrophages increases significantly, and they secrete important fibrosis regulators to participate in tissue repair and healing. However, in the chronic inflammatory phase, excessive infiltration of M2 macrophages in lung tissue leads to a large accumulation of ECM components such as angiogenesis factor and matrix metalloproteinases, causing abnormal lung tissue repair, vascular remodeling, and triggering fibrosis. In summary, AMs can mediate inflammatory damage in lung tissue through M1 polarization and fibrotic scar tissue formation through M2 polarization. Therefore, precise regulation of macrophage polarization after lung injury directly affects the pathological outcome of lung injury, making the elucidation of the key regulatory mechanisms of macrophage polarization particularly important.

[0004] PHLPP1 (pleckstrin homology domain leucine-rich repeat protein phosphatase 1), also known as SCOP, PPM3A, or PLEKHE1, belongs to the protein serine / threonine phosphatase family. PHLPP1 has been shown to directly dephosphorylate conserved sites of proteins such as AKT, protein kinase C (PKC), and S6 kinase (S6K), thereby regulating various cellular biological functions. Current research on PHLPP1 largely focuses on its target molecule, AKT. PHLPP1 dephosphorylation of AKT reduces regulatory T cell receptor signaling activation, affecting T cell development, or dephosphorylates STAT1, regulating macrophage phagocytosis and thus participating in inflammatory immune responses. Furthermore, PHLPP1 deficiency can alleviate ischemic damage to the heart and brain by upregulating Akt activation; however, the role of PHLPP1 in pulmonary fibrosis remains unclear.

[0005] In summary, there is an urgent need in this field to develop new strategies for alleviating and treating pulmonary fibrosis, inhibiting the changes in immune cell function and pathogenic effects in the local microenvironment of pulmonary fibrosis after lung injury, and controlling the resulting fibrosis and its related symptoms and / or other complications. Summary of the Invention

[0006] This invention addresses the aforementioned problems by first confirming the relationship between abnormal PHLPP1 expression and pulmonary fibrosis, and then observing the effects of changes in PHLPP1 expression on pulmonary fibrosis pathology and M2 macrophage polarization based on this target, thereby determining the role of PHLPP1 in the progression of pulmonary fibrosis.

[0007] The research process of this invention is as follows:

[0008] The inventors mined and analyzed data from lung tissue and bronchoalveolar lavage fluid macrophages of patients with idiopathic pulmonary fibrosis in a public database, and performed differential gene analysis on them with healthy individuals. They found that the expression of PHLPP1 was significantly reduced in patients with pulmonary fibrosis and was negatively correlated with the severity of pulmonary fibrosis, suggesting that PHLPP1 may be related to the occurrence of pulmonary fibrosis.

[0009] Subsequently, PHLPP1 was knocked out in mouse myeloid cells using the Loxp-cre system. The results showed that in a PHLPP1 knockout mouse model of pulmonary fibrosis, pulmonary fibrosis was significantly aggravated, with substantial collagen deposition in the lung tissue. PHLPP1 deficiency promoted the transformation of alveolar macrophages into M2-type macrophages, and its culture supernatant promoted the expression of fibrosis genes in lung fibroblasts in a co-culture system. By using specific small interfering RNA to target and silence PHLPP1 expression, the expression of M2-type macrophage marker genes was upregulated in bone marrow-derived macrophages; conversely, overexpression of PHLPP1 in bone marrow-derived macrophages inhibited the expression of M2-type macrophage marker genes. Therefore, we conclude that significantly reduced PHLPP1 expression in alveolar macrophages promotes their transformation into M2-type macrophages, upregulates the expression of pro-fibrosis genes, and promotes the activation and fibrotic effects of lung fibroblasts, playing a crucial role in the progression of pulmonary fibrosis.

[0010] Based on the above research, the specific technical solution of the present invention is as follows:

[0011] In a first aspect, the invention provides the use of a PHLPP1 promoter in the preparation of a medicament for treating pulmonary fibrosis and its related symptoms and / or other complications.

[0012] Pulmonary fibrosis, including idiopathic pulmonary fibrosis, is characterized by persistent damage to alveoli and repeated destruction, repair, reconstruction and excessive deposition of extracellular matrix caused by lung inflammation. Activation of pulmonary fibroblasts produces a large amount of extracellular matrix in the alveoli and interstitium, and thickens the tissue around and between the lung air sacs (alveoli). This makes it more difficult for oxygen to enter the blood, leading to irreversible decline in lung function and even respiratory failure.

[0013] Preferably, the PHLPP1 promoter is selected from any one or more of the following: exogenous PHLPP1 protein or nucleic acid encoding it, substances that promote the expression or activity of PHLPP1 protein, substances that promote the overexpression of nucleic acid molecules encoding PHLPP1 protein, liposomes or nanomaterials encapsulating PHLPP1-encoding nucleic acid, precursor proteins or their conjugates or complexes that can be converted into PHLPP1 in vivo.

[0014] Further preferred, the PHLPP1 protein is selected from any of the following:

[0015] (a) A polypeptide having the amino acid sequence shown in SEQ ID NO.2;

[0016] (b) Proteins or polypeptides that are homologous to or have sequence similarity (e.g., more than 80% homology or more than 80% sequence similarity, such as 80%, 85%, 90%, 95%, 98%, 99%) of the amino acid sequence shown in SEQ ID NO.2, and that inhibit fibrotic reactions and treat pulmonary fibrosis and its related symptoms and / or other complications.

[0017] The nucleic acid molecule encoding the PHLPP1 protein is selected from any of the following categories: (c) a protein or polypeptide whose amino acid sequence of (a) or (b) has been substituted, deleted, or added with one or more amino acids, and which has the function of inhibiting fibrosis and treating pulmonary fibrosis; or a protein or polypeptide derived from (a) or (b).

[0018] (i) A nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO.1;

[0019] (ii) Molecules that hybridize with the nucleotide sequence defined in (i) under strict conditions;

[0020] (iii) A nucleic acid molecule that is homologous to or has the same sequence as the nucleotide sequence shown in SEQ ID NO.1 and encodes a protein or polypeptide that inhibits fibrotic response and treats pulmonary fibrosis and its related symptoms;

[0021] (iv) A nucleic acid molecule that has one or more nucleotides substituted, deleted or added from the nucleotide sequence in (i), (ii) or (iii) and encodes a protein or polypeptide that inhibits the fibrotic response and treats pulmonary fibrosis and its related symptoms or other complications.

[0022] In a second aspect, the present invention provides a PHLPP1 recombinant vector, the recombinant vector comprising an expression vector and a nucleic acid molecule encoding a PHLPP1 protein inserted thereon, the nucleic acid molecule encoding the PHLPP1 protein as described above.

[0023] In a third aspect, the present invention provides the use of a PHLPP1 promoter recombinant carrier in the preparation of a medicament for treating pulmonary fibrosis and its related symptoms and / or other complications.

[0024] The vectors mentioned include viral vectors and non-viral vectors.

[0025] The term "viral vector" includes adenovirus, adeno-associated virus, lentivirus, Coxsackievirus, herpes simplex virus, measles virus, Newcastle disease virus, parvovirus, poliovirus, reovirus, vaccinia virus, and vesicular stomatitis virus, etc. Suitable viral vectors are well known to those skilled in the art.

[0026] The term "non-viral carrier" includes liposomes or lipid complexes, cationic polymers, chitosan polymers, and nanoparticle carriers. Suitable non-viral carriers are well known to those skilled in the art.

[0027] A fourth aspect of the present invention provides a pharmaceutical composition for treating pulmonary fibrosis and its related symptoms and / or other complications, comprising an active ingredient and a medically acceptable excipient, carrier, or diluent. The active ingredient is the aforementioned PHLPP1 promoter or PHLPP1 recombinant carrier.

[0028] The recombinant virus of the present invention, together with pharmaceutically acceptable excipients, forms a pharmaceutical composition for treating pulmonary fibrosis and its related symptoms and / or other complications, thereby achieving a more stable therapeutic effect.

[0029] Regarding drug form, drugs or drug combinations suitable for administration via a selection from the following methods are acceptable: oral administration, injection (e.g., direct injection of naked DNA or protein, liposome-encapsulated DNA or mRNA injection), gold-coated gene gun bombardment, plasmid DNA carried by replication-defective bacteria, target DNA carried by replication-defective adenovirus, or protein encoded by the target gene, electroporation, nasal administration, pulmonary administration, oral administration, and nebulized inhalation.

[0030] Under normal circumstances, liquid formulations can be stored stably at 2℃-8℃ for at least one year, while lyophilized formulations remain stable at 30℃ for at least six months. The formulations can be commonly used in the pharmaceutical industry, such as suspensions, injections, and lyophilized formulations.

[0031] When the composition of the present invention is administered to animals, including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The dosage can be determined with reference to the results of animal experiments and various other factors, but the total dosage should not exceed a certain range.

[0032] Furthermore, the pharmaceutical compositions of the present invention can be used in combination with other pharmaceutical compositions for treating pulmonary fibrosis and its related symptoms and / or other complications.

[0033] In a fifth aspect, the invention provides the use of a reagent for detecting PHLPP1 expression levels in the preparation of a pulmonary fibrosis detection kit or a drug screening kit.

[0034] Experimental results showed that PHLPP1 expression was significantly decreased in patients with pulmonary fibrosis and was negatively correlated with collagen fiber gene expression. PHLPP1 knockout promoted the development and progression of pulmonary fibrosis in mice, suggesting that PHLPP1 is a protective factor against pulmonary fibrosis and has an inhibitory effect on the fibrotic response. Therefore, it can be inferred that PHLPP1 could serve as a diagnostic biomarker or drug screening biomarker for pulmonary fibrosis.

[0035] The method for drug screening using PHLPP1 as a biomarker is as follows:

[0036] (A) Treat cells, tissues, or animals with pulmonary fibrosis or simulated pulmonary fibrosis response or other fibrotic environments with candidate substances;

[0037] (B) Detecting the level of PHLPP1 or the nucleic acid molecule encoding this protein in the cells, tissues, or animals; and

[0038] (C) If the level of PHLPP1 or the nucleic acid molecule encoding the protein is higher than the level before the candidate substance treatment or higher than the level in the normal control, it indicates that the candidate substance has the effect of treating pulmonary fibrosis by promoting PHLPP1.

[0039] In a sixth aspect, the present invention provides a diagnostic kit or drug screening kit for pulmonary fibrosis, comprising reagents for detecting the expression level of PHLPP1 in biological samples, and consisting of a reverse transcription system, a primer system, and an amplification system. Diagnosis or drug screening is achieved by detecting the nucleic acid expression level of PHLPP1.

[0040] The beneficial protections and effects of this invention are as follows:

[0041] This invention provides the use of PHLPP1 promoters in the preparation of drugs for treating pulmonary fibrosis and its related symptoms and / or other complications. Experiments have demonstrated that PHLPP1 knockout promotes the development and progression of pulmonary fibrosis in mice, enhancing the M2 transformation and infiltration of pro-fibrotic macrophages in the lung microenvironment, suggesting that PHLPP1 is a protective factor against pulmonary fibrosis and has an inhibitory effect on the fibrotic response. PHLPP1 overexpression can inhibit the activation of pro-fibrotic macrophages, manifested by inhibiting the production of Arg1, RetnIa, and TGF-β in macrophages, thereby achieving the relief and treatment of pulmonary fibrosis. Therefore, this invention provides a new target for the treatment of pulmonary fibrosis, especially idiopathic pulmonary fibrosis, by targeting the overexpression or functional promotion of PHLPP1, and has broad clinical application prospects. Attached Figure Description

[0042] Figure 1 The study showed that PHLPP1 expression was significantly decreased in patients with pulmonary fibrosis and was negatively correlated with the expression of collagen fiber genes in pulmonary fibrosis. Specifically, A represents the PHLPP1 expression level in bronchoalveolar lavage fluid macrophages of healthy individuals and patients with idiopathic pulmonary fibrosis (IPF); B represents the PHLPP1 expression level in lung tissue of healthy individuals and patients with IPF; and C represents the correlation analysis between PHLPP1 expression in lung tissue of patients with IPF and the expression of collagen fiber genes Collagen I (Col1) and Collagen III (Col3).

[0043] Figure 2 The images show that pulmonary fibrosis is aggravated in PHLPP1 knockout mice (cKO) in a bleomycin (BLM)-induced pulmonary fibrosis model. A shows the eosin, hematoxylin, and masson staining results of lung tissues from WT and cKO mice; B shows the fibrosis scores of WT and cKO mice; and C shows the expression of collagen fibers in lung tissues from WT and cKO mice.

[0044] Figure 3 The results show that PHLPP1 knockout promotes the conversion of alveolar macrophages to the M2 phenotype; the expression levels of M2 phenotype genes and pro-fibrosis genes in lung tissue in bleomycin-induced pulmonary fibrosis models of A, WT and cKO mice; and the immunofluorescence staining results of M2 macrophages in lung tissue of B, WT and cKO mice.

[0045] Figure 4 PHLPP1 knockout macrophages showed that they could suppress the expression of fibrosis genes in lung fibroblasts. Bone marrow-derived macrophages from WT and cKO mice were co-cultured with lung fibroblasts after IL-4 induction or non-induction, and the expression of collagen in lung fibroblasts was detected.

[0046] Figure 5 The study showed that interfering with the expression of PHLPP1 in bone marrow-derived macrophages promoted the expression of M2 marker genes and profibrotic genes. A represents the protein expression level of PHLPP1 in bone marrow-derived macrophages after transfection with PHLPP1-targeting siRNA and control siRNA; B represents the mRNA expression level of PHLPP1 in bone marrow-derived macrophages after transfection with PHLPP1-targeting siRNA and control siRNA; and C represents the expression levels of M2 phenotype genes and profibrotic genes in bone marrow-derived macrophages after transfection with PHLPP1-targeting siRNA and control siRNA, followed by IL-4 stimulation or non-stimulation.

[0047] Figure 6 The results show that lentiviral overexpression of PHLPP1 in bone marrow-derived macrophages can inhibit the expression of M2 phenotype marker genes and profibrosis genes. A represents the protein expression level of PHLPP1 in bone marrow-derived macrophages after infection with lentivir overexpressing PHLPP1 and control lentivir; B represents the expression levels of M2 phenotype genes and profibrosis genes in bone marrow-derived macrophages after stimulation with IL-4 following lentiviral overexpression of PHLPP1 or control virus. Detailed Implementation

[0048] The following examples and experimental cases further illustrate the present invention and should not be construed as limiting the invention. The examples do not include detailed descriptions of conventional methods, such as PCR methods, methods used to construct vectors and plasmids, methods for inserting protein-encoding genes into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold spring Harbor Laboratory Press.

[0049] Unless otherwise stated, percentages and parts are by volume. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to this invention, and the preferred methods and materials described herein are for illustrative purposes only.

[0050] Example 1: PHLPP1 expression was significantly decreased in patients with pulmonary fibrosis, and was negatively correlated with the severity of pulmonary fibrosis.

[0051] We conducted data mining and analysis on macrophage data from lung tissue and bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis in public databases, and performed correlation analysis between indicators reflecting the severity of pulmonary fibrosis and the expression of PHLPP1.

[0052] The results showed that, compared with healthy individuals, macrophages in the lung tissue and bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis expressed lower levels of PHLPP1 (…). Figure 1 (A and 1B), and the expression level of PHLPP1 was negatively correlated with the expression of collagen fiber genes Col1 and Col3, respectively. Figure 1 C).

[0053] These results indicate that PHLPP1 plays a protective role in human pulmonary fibrosis.

[0054] Example 2: PHLPP1 knockout exacerbates fibrosis pathology after lung injury in mice

[0055] PHLPP1 myeloid cell conditional knockout mice were constructed. Among them, PHLPP1... fl / fl The mouse was constructed by Cyagen Biosciences and then crossed with Lyz2-Cre mice to obtain PHLPP1. fl / fl Lyz2-Cre + / –Mice, called cKO mice, were used to specifically knock out the PHLPP1 gene in myeloid cells, while also obtaining control mice with PHLPP11. fl / fl Lyz2-Cre – / – (These are called WT mice). These mice are raised in a specific pathogen-free (SPF) environment.

[0056] WT and cKO mice were given a single dose of bleomycin (BLM) to establish a BLM-induced pulmonary fibrosis model. Lung tissue was fixed with paraformaldehyde and stained with eosin and hematoxylin to observe the pathological level of the lung tissue. The indicators included the degree of pulmonary fibrosis score and the integrity of alveolar structure. No fibrosis was scored as 1 point, while mild, moderate, and severe pulmonary fibrosis were scored as 2, 3, and 4 points, respectively. Mild fibrosis mainly affected the pleura and subpleural interstitium, with disordered alveolar structure and an affected area of ​​less than 20%. Moderate fibrosis affected 20%–50% of the entire lung. Severe fibrosis affected more than 50% of the entire lung, with disordered lung parenchyma and fusion, and visible air cysts of varying sizes.

[0057] The results showed that, compared with the control group WT mice, cKO mice had more severe alveolar structural damage and more cell infiltration. Figure 2 A), higher fibrosis score ( Figure 2 B), the expression of collagen I (Col1) and the myofibroblast activation marker α-SMA in lung tissue were both significantly increased. Figure 2 C).

[0058] These results indicate that PHLPP1 knockout promotes the pathological severity of pulmonary fibrosis in mice, suggesting that PHLPP1 plays a protective role in pulmonary fibrosis.

[0059] Example 3: PHLPP1 knockout promotes the conversion of alveolar macrophages to the M2 phenotype.

[0060] Given that the resolution of inflammation and the formation of fibrosis after lung injury are closely related to the polarization of M2 macrophages, we will next establish BLM model in cKO mice and control WT mice, and extract RNA from their lung tissue for quantitative analysis and fluorescent staining.

[0061] The results showed that the expression of Arg1, Retnla, and the pro-fibrotic factor Tgfb1, marker genes of M2 macrophages, was significantly increased in the lung tissue of PHLPP1 knockout mice. Figure 3 A), and the number of Arg1-positive alveolar macrophages was also significantly higher than that in WT mice ( Figure 3 B).

[0062] The results indicate that PHLPP1 knockout promotes the conversion of alveolar macrophages to the M2 phenotype in pulmonary fibrosis mice, exacerbating the pulmonary fibrosis response after lung injury.

[0063] Example 4: PHLPP1 knockout macrophages promote the expression of fibrosis genes in lung fibroblasts.

[0064] Macrophages derived from the bone marrow of cKO mice and control WT mice were cultured in vitro and stimulated with IL-4 (20 ng / ml). They were then co-cultured with primary lung fibroblasts of mice. RNA was extracted from the fibroblasts and reverse transcription PCR was performed, followed by real-time quantitative PCR to detect the expression of fibrosis genes in the fibroblasts.

[0065] The results showed that lung fibroblasts co-cultured with bone marrow-derived macrophages from PHLPP1 knockout mice expressed higher levels of Collagen I (Col1) and α-SMA (Col1). Figure 4 ).

[0066] The results indicate that PHLPP1 reduces the secretion of macrophage mediators that activate lung fibroblasts, inhibits the activation and differentiation of fibroblasts, and thus plays a protective role in pulmonary fibrosis.

[0067] Example 5: Interference with PHLPP1 enhances macrophage transformation to the pro-fibrotic M2 phenotype.

[0068] Wild-type mouse bone marrow-derived macrophages were obtained and transfected with a siRNA sequence targeting mouse PHLPP1 (sequences shown in SEQ ID NO. 3-6, synthesized by Dharmacon) and a control sequence (both purchased from Dharmacon). Forty-eight hours after transfection, the bone marrow-derived macrophages (same as in Example 3) were stimulated with IL-4 (20 ng / ml) to simulate the activation state of macrophages in pulmonary fibrosis. Total RNA from the macrophages was collected, and the activation status was detected by reverse transcription PCR followed by real-time quantitative PCR.

[0069] The specific sequence of the siRNA that specifically interferes with PHLPP1 expression (PHLPP1-siRNA) is as follows:

[0070] siRNAD-058853-01: GUAAUAACUUCCGUGACA (SEQ ID NO. 3);

[0071] siRNAD-058853-02:UAAAGGCACUCUAUGCUUC(SEQ ID NO.4);

[0072] siRNAD-058853-03:GGAGGAUCCUUUACCCUGA (SEQ ID NO.5);

[0073] siRNAD-058853-04: GAAGUCAAGUGUGUAGA (SEQ ID NO. 6).

[0074] The results showed that interference with PHLPP1 ( Figure 5 A (lanes 4, 5, 6) and control group ( Figure 5 Compared to A1, A2, and A3, PHLPP1 protein expression was significantly reduced, and its mRNA level was also significantly decreased. Figure 5 B) indicates that these siRNAs can effectively interfere with the expression of PHLPP1 in macrophages; macrophages after PHLPP1 interference express higher levels of the marker genes Arg1, Retnla, and the profibrotic factor Tgfb1 of M2 macrophages. Figure 5 C).

[0075] The results indicate that siRNA interference with PHLPP1 expression can enhance the transformation of macrophages into the pro-fibrotic M2 type, thereby promoting the activation and fibrotic effects of lung fibroblasts.

[0076] Example 6: Overexpression of PHLPP1 inhibits the transformation of macrophages to the pro-fibrotic M2 phenotype.

[0077] The DNA fragment of the PHLPP1 gene was cloned and amplified by Shanghai Jikai Gene Co., Ltd., and inserted into a lentiviral vector named GV643, successfully constructing a lentivirus overexpressing PHLPP1, which was named Lv-PHLPP1. Lv-PHLPP1 and a control empty viral vector were used to infect bone marrow-derived macrophages, and the infection efficiency of the lentivirus was detected by Western blotting to confirm its overexpression effect. Subsequently, bone marrow-derived macrophages (as in Example 3) were infected with the above lentivirus, and IL-4 (20 ng / ml) was used to stimulate them to simulate the activated state of macrophages in pulmonary fibrosis. Total RNA from macrophages was collected, and the levels of Arg1, RetnIa, and Tgfb1 were detected by reverse transcription PCR followed by real-time quantitative PCR.

[0078] The results showed that: Lv-PHLPP1 ( Figure 6 A-path 2) compared to Vector Figure 6 In Lv-PHLPP1-infected bone marrow-derived macrophages, the expression of PHLPP1 was significantly increased; compared with Vector-infected macrophages, the expression of Arg1, Retnla, and the profibrotic factor Tgfb1, marker genes of M2 macrophages, was significantly decreased. Figure 6 B).

[0079] The results showed that overexpression of PHLPP1 inhibited the transformation of macrophages into the pro-fibrotic M2 type, thereby limiting the activation and fibrotic effects of lung fibroblasts.

[0080] The nucleotide sequence encoding the PHLPP1 protein in this invention is shown in SEQ ID NO.1:

[0081] cataatcccctggaacggccgcgcacaacgccattggcttctcccttctccgcgcgccgccgccgtctcccacctccgcctcatcgcctccctctccgcccgct

[0082] gcctccggagctgggggggaaacgcgaagccccactgcaatggagcccgccgccgcggccacggtacagcgactccccgagctcggcagggaggacc

[0083] gagcttcggctccggcggccgccgctgcggcagcagcagcagcagcggcggccgcggcggctctggcggcggcggccgggggcggccggagtccg

[0084] gagcccgcgctgaccccggcggccccgagcggcgggaacggcagcggcagcggggcgcgggaagaggccccaggcgaggcgccgccggggccg

[0085] ctgccgggcagagcggggggtgccgggcgcaggaggcggcgcggggcgccccagcccattgccggcggggctgcccccgtacccggggccggcgg

[0086] cggcgccaactccctcctgctgaggagagggcggctgaagaggaatctgtccgcggccgccgcggccgcctcctcgtcgtcgtcgtcctcggccgctgctg

[0087] cctcgcactcccccggcgctgccggcctccccgcctcctgctcggcctcggcgtcgctgtgcacccggagcctggacaggaagacgctgcttctgaagcac

[0088] cggcagacgctgcagctgcagccgtcggaccgggactgggtgaggcaccagctccagcgcggctgcgtgcacgtcttcgaccgccacatggcctcgacct

[0089] acctgcgcccggtgctctgcacactggacaccacggccggcgaggtggccgcccgcctgctgcagctgggccacaaaggcggcggcgtggtgaaggtg

[0090] ctgggccaggggcccggagccgccgccgcccgggagcccgctgaaccgccccccgaggccggcccccggctggcgcccccggagccgcgggactc

[0091] ggaggtaccgcccgcgaggagcgcgccgggtgccttcggggggcctccgcgcgcgccccccgccgacctacccctgcccgtcggcggcccgggcggg

[0092] tggtcgcgccgcgccagcccagcgccctcggactccagccccggcgagccgttcgttgggggccctgtctcttcgccccgcgccccacggcctgtggtctc

[0093] cgacaccgagagcttcagtctgagtcccagcgccgagagcgtgtctgaccggttggacccctacagcagcggcggcggctcctcgtcgtcgtcggaagag

[0094] ctcgaggccgacgcagcctcggccccgacgggggtcccgggccagccccgccgtcccggccaccccgcgcagcccctcccgcttccccagacggcttc

[0095] ctcgcctcagccgcagcagaaagccccgagggccattgacagcccgggcggggccgtccgcgaggggtcgtgcgaggagaaggcagcggcagccgtg

[0096] gccccgggaggcctccagtctacccccgggaggagcggggtgaccgcggagaaggcgcctccgccgcccccgccgcccaccctgtacgtgcagctcca

[0097] cggagagaccacccggcgcttggaggcggaggagaagccattgcagatccaaaatgactacctcttccaactgggatttggggagctgtggagggtgcag

[0098] gaggaaggcatggactcggagattggctgcctcatccgcttctatgcaggaaaacctcacagcacgggtagctctgaacggattcagctctcaggaatgtata

[0099] atgtccgtaaaggcaagatgcagttgccagtgaaccgatggacaagacgccaagtcatcctatgtgggacctgcctgatagtatcatctgtgaaagacagcttg

[0100] accggaaagatgcatgttctgccactaattggtggaaaagtagaagaagtgaaaaagcaccaacactgtttagcatttagctcctctggaccccaaagccagac

[0101] ttactacatttgctttgatactttcacagaatacttaaggtggctgcgacaagtctccaaggttgcatcccagcgcattagctcagtggacctctcgtgttgtagcct

[0102] ggaacatctgcctgccaacctcttctacagccaagacctcactcatctcaatttaaaacaaaacttcctaaggcagaaccctagccttccagctgccagggggct

[0103] taatgaactgcaaaggttcaccaagttgaagagtcttaacctttccaataatcatttaggggacttcccactggcagtctgcagtattccaaccctggcagagctg

[0104] aacgtgtcctgcaatgccctgcgatcagtcccggcagccgttggagtgatgcacaacttacagacatttttgttggatggaaactttctccaatcccttcctgctga

[0105] gttggagaacatgaagcagcttagttatctgggtcttctttcaatgaatttactgacattcccgaagtattggagaaattgactgctgtggataaactttgtatgtctg

[0106] gaaactgtgtggagacccttaggctacaggctttaagaaaaatgcctcacattaaacatgtggatctaaggttgaacgtaattaggaagctgatagcagatgaag

[0107] tggactttctacagcatgttactcagcttgacctacgagacaataagcttggtgatctagatgctatgattttcaacaacattgaagttttacactgtgaaaggaatca

[0108] actggtcacattagacatctgtggctatttcctaaaagcgctctatgcctcttctaatgaacttgttcaacttgatgtttacccagttccaaattatctgtcctacatgga

[0109] tgtttcaaggaaccgcttagaaaatgtgcctgagtgggtatgtgaaagccgaaagctagaagttttggatattggccataatcaaatatgtgaacttcctgcccgc

[0110] ttattttgtaatagcagtctccggaaactactggcaggacacaaccagttggcaaggctgcctgaaaggctagaaagaacctcggtggaggtcttggatgtgca

[0111] acacaaccagctccttgagctcccacctaaccttctgatgaaggctgacagcctgagattcctgaacgcctctgcgaacaaactggaaagccttcctccagcca

[0112] cgctttccgaagagacaaacagtatcttacaagagttgtatttgacaaataacagcctcacagacaaatgtgtgcccttgttaacgggacacccccatttgaagat

[0113] ccttcacatggcctataaccgacttcagagttttccagcaagtaaaatggcgaaactggaggaacttgaagaaattgatctcagtgggaataagctgaaagccat

[0114] cccaacaacgatcatgaattgcaggcgcatgcacaccgtgattgctcactccaactgcatcgaggtctttcccgaagttatgcagctcccagagatcaagtgtgt

[0115] ggacctgagctgtaatgagctaagtgaagtcacattaccagaaaacctgcctcccaaactgcaggagctagacctgactggaaacccgcgccttgtccttgat

[0116] cacaaaaccctggaactactgaataatatccgctgtttcaagattgatcagccttctacaggagacgcttccggagccccagctgtatggagtcatggttacact

[0117] gaagcttcgggggtaaaaaacaagttgtgtgtcgcagccctgtcggtgaataacttctgtgacaaccgcgaagccctgtatggtgtgtttgacggagaccgga

[0118] atgtggaggtgccctaccttctccagtgcactatgagtgacattttggctgaagagctgcaaaaaacaaaaaacgaagaagaatacatggtcaatacattcattg

[0119] tcatgcaaaggaaacttggaactgctgggcagaagcttggtggtgccgctgtcctttgtcatatcaagcatgaccctgtggatccaggaggatccttcaccttga

[0120] cctctgctaatgtgggcaagtgccaaacagttctctgtcgaaatggaaagccgctgcctctgtccagatcttacatcatgagctgtgaagaagagctgaagagg

[0121] attaaacagcacaaggccattatcactgaggatggcaaggtgaacggagtgactgagtccacgcgcatcctgggctacaccttcctccatcccagtgtggtgc

[0122] ctcgcccccacgtgcagtccgtgctcctgactccccaggatgagttcttcatcctaggcagtaaggggttgtgggacagcctgtccgtcgaggaggccgtgga

[0123] agccgtgcgcaacgtgcccgatgccctggctgctgccaagaagctgtgtaccctggcccagagctacggctgccacgacagcatcagcgctgtggtggtgc

[0124] agctcagtgtcactgaggacagcttctgctgctgcgagctcagcgccggtggggctgtgccaccacccagtcctggcatctttcctccctcagtgaacatggtg

[0125] atcaaggatcggccctcagatgggctgggcgtgccgtcctccagcagcggcatggcttccgagattagcagtgagctctccacttctgagatgagcagcgag

[0126] gtggggtcaacagcctccgatgagcccccgcccggagccctaagcgagaacagccctgcctaccccagtgagcag c gctgcatgctccaccccatctgtct

[0127] gtccaactccttccag c gccagctatccagcgccacgttctctagcgccttctccgacaacggccttgacagtgacgatgaggagcccatcgagggcgtcttc

[0128] accaacggcagccgggtggaggtggaggtggacatccactgcagccgggccaaggagaaggagaaacagcagcacctgcttcaggtgccagcagagg

[0129] ccagtgatgagggcattgtcatcagcgccaacgaggatgagccaggtctgcccaggaaggcagacttctctgccgttgggaccattgggcgccggagggc

[0130] caatggctctgttgcgccccaggaaaggagccacaatgtgatagaggtggctacagacgcacctcttcgaaagcctggaggctattttgctgccccggctcag

[0131] ccggatcctgatgatcagtttatcatacccccggagctggaagaggaggtcaaagaaatcatgaagcatcaccaggagcaacagcagcagcagcagccgc

[0132] caccaccccctcagctccagccgcagctgccgcggcactaccagctggaccagctgccagattattacgacacgccactatgacccagccgagctgtttaac

[0133] aaataaactaaccacaaaagactgagttgcaagagtctcccaggctcacattaaaccaggggttttactccacatccttcccccagacactgttcccaacctgtc

[0134] atcgcagctaatctgtaggttctctttctttgggttatttttttaagtaatcaccactttcttctagtgatgctttaccaatatgatttacatttgttaacttctccccctaacat

[0135] atcagatatgtaaagacaaagaacaaaaggtttaatatattacagagaaacagttaatgataatgtaatattttttaaaatggctttttgttgtttgtttggaaggcagg

[0136] gcaggctgccgttgctaaatgatttaataatattgtaattctgtatttctttggggggaaaaggcttttgttttgttttgttttgttttgttttgtttttgtcttgaaaataataga

[0137] catttgtagaatatggagactaactcctaggagttgctttactctgtcaggtgacttaagtcactgggattcactaattttctctgagagaacagctgattgagaattt

[0138] ccattgtaaatagctcagtgttgtatagtgaggcttacgatgttttgtagtcttggcgtaaggacacagcccaagtaactgacgtttcccctccccctcccctctgag

[0139] gagcctgcctgcctcacaactcaccctcacttcactgaatgaggaggctgagcagctgcagtgtttctgtccggaggaaatggatcttaggccactggacaag

[0140] aacctgcacccaagggccctgaacccattttcctcccctgtcccagccttcccactttgacagacacttttaactgtgttccttactgctgccacaatcagcatggtt

[0141] gtatagtgccccattaggccatttacatacccagagttatactcaagcagaatgcacaaatggacatgtcataatttttgttacaataaatatgaaatttacaagta

[0142] In the present invention, the amino acid sequence of PHLPP1 protein is shown as SEQ ID NO. 2:

[0143] MEPAAAATVQRLPELGREDRASAPAAAAAAAAAAAAAAAALAAAAGGGRSPEPALTPAAPSGGNG

[0144] SGSGAREEAPGEAPPGPLPGRAGGAGRRRRRGAPQPIAGGAAPVPGAGGGANSLLLRRGRLKRNLSA

[0145] AAAAASSSSSSSAAAASHSPGAAGLPASCSASASLCTRSLDRKTLLLKHRQTLQLQPSDRDWVRHQL

[0146] QRGCVHVFDRHMASTYLRPVLCTLDTTAGEVAARLLQLGHKGGGVVKVLGQGPGAAAAREPAEPP

[0147] PEAGPRLAPPEPRDSEVPPARSAPGAFGGPPRAPPADLPLPVGGPGGWSRRASPAPSDSSPGEPFVGGP

[0148] VSSPRAPRPVVSDTESFSLSPSAESVSDRLDPYSSGGGSSSSSEELEADAASAPTGVPGQPRRPGHPAQP

[0149] LPLPQTASSPQPQQKAPRAIDSPGGAVREGSCEEKAAAAVAPGGLQSTPGRSGVTAEKAPPPPPPPTLY

[0150] VQLHGETTRRLEAEEKPLQIQNDYLFQLGFGELWRVQEEGMDSEIGCLIRFYAGKPHSTGSSERIQLSG

[0151] MYNVRKGKMQLPVNRWTRRQVILCGTCLIVSSVKDSLTGKMHVLPLIGGKVEEVKKHQHCLAFSSS

[0152] GPQSQTYYICFDTFTEYLRWLRQVSKVASQRISSVDLSCCSLEHLPANLFYSQDLTHLNLKQNFLRQN

[0153] PSLPAARGLNELQRFTKLKSLNLSNNHLGDFPLAVCSIPTLAELNVSCNALRSVPAAVGVMHNLQTFL

[0154] LDGNFLQSLPAELENMKQLSYLGLSFNEFTDIPEVLEKLTAVDKLCMSGNCVETLRLQALRKMPHIKH

[0155] VDLRLNVIRKLIADEVDFLQHVTQLDLRDNKLGDLDAMIFNNIEVLHCERNQLVTLDICGYFLKALYA

[0156] SSNELVQLDVYPVPNYLSYMDVSRNRLENVPEWVCESRKLEVLDIGHNQICELPARLFCNSSLRKLLA

[0157] GHNQLARLPERLERTSVEVLDVQHNQLLELPPNLLMKADSLRFLNASANKLESLPPATLSEETNSILQE

[0158] LYLTNNSLTDKCVPLLTGHPHLKILHMAYNRLQSFPASKMAKLEELEEIDLSGNKLKAIPTTIMNCRR

[0159] MHTVIAHSNCIEVFPEVMQLPEIKCVDLSCNELSEVTLPENLPPKLQELDLTGNPRLVLDHKTLELLNN

[0160] IRCFKIDQPSTGDASGAPAVWSHGYTEASGVKNKLCVAALSVNNFCDNREALYGVFDGDRNVEVPY

[0161] LLQCTMSDILAEELQKTKNEEEYMVNTFIVMQRKLGTAGQKLGGAAVLCHIKHDPVDPGGSFTLTSA

[0162] NVGKCQTVLCRNGKPLPLSRSYIMSCEEELKRIKQHKAIITEDGKVNGVTESTRILGYTFLHPSVVPRP

[0163] HVQSVLLTPQDEFFILGSKGLWDSLSVEEAVEAVRNVPDALAAAKKLCTLAQSYGCHDSISAVVVQL

[0164] SVTEDSFCCCELSAGGAVPPPSPGIFPPSVNMVIKDRPSDGLGVPSSSSGMASEISSELSTSEMSSEVGST

[0165] ASDEPPPGALSENSPAYPSEQRCMLHPICLSNSFQRQLSSATFSSAFSDNGLDSDDEEPIEGVFTNGSRV

[0166] EVEVDIHCSRAKEKQQHLLQVPAEASDEGIVISANEDEPGLPRKADFSAVGTIGRRRANGSVAPQE

[0167] RSHNVIEVATDAPLRKPGGYFAAPAQPDPDDQFIIPPELEEEVKEIMKHHQEQQQQQQPPPPPQLQPQL

[0168] PRHYQLDQLPDYYDTPL

[0169] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. The use of PHLPP1 promoter in the preparation of drugs for treating idiopathic pulmonary fibrosis, characterized in that: The PHLPP1 promoter is selected from any one or more of the following: exogenous PHLPP1 protein or nucleic acid encoding it, liposomes or nanomaterials encapsulating the nucleic acid encoding the PHLPP1 protein. Wherein, the PHLPP1 protein is a polypeptide having the amino acid sequence shown in SEQ ID NO.2; the nucleic acid molecule encoding the PHLPP1 protein is a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO.

1.

2. A PHLPP1 recombinant vector, characterized in that, The recombinant vector includes an expression vector and a nucleic acid molecule encoding the PHLPP1 protein inserted onto the expression vector, the nucleic acid molecule encoding the PHLPP1 protein as described in claim 1.

3. The PHLPP1 recombinant vector according to claim 2, characterized in that: in, The expression vector is a plasmid vector, a granular vector, a bacteriophage vector, or a viral vector, wherein the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, Coxsackievirus, herpes simplex virus, measles virus, Newcastle disease virus, parvovirus, poliovirus, reovirus, vaccinia virus, and vesicular stomatitis virus.

4. Use of the PHLPP1 recombinant vector according to claim 2 in the preparation of a drug for treating idiopathic pulmonary fibrosis.

5. A pharmaceutical composition for treating idiopathic pulmonary fibrosis, characterized in that, The pharmaceutical composition includes an active ingredient and medically acceptable excipients, carriers, or diluents. The active component is the PHLPP1 promoter according to claim 1 or the PHLPP1 recombinant vector according to any one of claims 2 to 3.

6. Application of reagents for detecting PHLPP1 expression levels in the preparation of idiopathic pulmonary fibrosis detection kits.

Images