CN117338931A - PHLPP1 and application of related substances thereof in treating pulmonary fibrosis - Google Patents
PHLPP1 and application of related substances thereof in treating pulmonary fibrosis Download PDFInfo
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- CN117338931A CN117338931A CN202311051558.8A CN202311051558A CN117338931A CN 117338931 A CN117338931 A CN 117338931A CN 202311051558 A CN202311051558 A CN 202311051558A CN 117338931 A CN117338931 A CN 117338931A
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- pulmonary fibrosis
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Abstract
The invention relates to the technical field of biomedical engineering, and provides an application of PHLPP1 and related substances thereof in treating pulmonary fibrosis. In particular to PHLPP1 protein, nucleic acid molecule encoding the same, promoter or recombinant expression vector thereof, and the use thereof in preparing products for treating pulmonary fibrosis and related symptoms or other complications, and corresponding recombinant vector or pharmaceutical composition thereof. Experimental results show that the over-expression of PHLPP1 can effectively inhibit the level of the pro-fibrosis factor, and has effective application value for relieving and treating the pathology and other symptoms or other complications of pulmonary fibrosis. Therefore, the invention provides a new target point for treating pulmonary fibrosis and has wide clinical application prospect.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to application of leucine-rich repeat protein phosphatase 1 (PHLPP 1) with homologous sequence of type 1 protein phosphatase plackstrin as a target in preparation of a medicine for treating pulmonary fibrosis, especially idiopathic pulmonary fibrosis, and also relates to a carrier and a pharmaceutical composition containing the same.
Background
Fibrotic diseases are important causes of death, whether toxin, genetic, tumor, infectious and autoimmune related, or idiopathic, and affect the function of any organ. Pulmonary fibrosis (pulmonary fibrosis, PF), a chronic progressive pathological change driven by repeated lung injury and inflammation, has a median survival of only 3 years. Although a small fraction of anti-fibrotic therapeutic drugs are now able to slow the progression of PF, there is currently a lack of intervention to effectively reverse pulmonary fibrosis due to insufficient knowledge of its etiology. Therefore, elucidation of the occurrence and development of pulmonary fibrosis after lung injury and key regulatory mechanisms are of great clinical significance for the development of pharmaceutical means to prevent pulmonary fibrosis and pulmonary failure after lung injury.
Lung fibroblasts are the most important effector cells for pulmonary fibrosis, producing a large amount of extracellular matrix in the alveoli and interstitium (extracellular matrix, ECM). Differentiation and activation of lung fibroblasts is triggered and maintained by various cytokines and growth factors released by alveolar epithelial cells and immune cells. In immune cells, alveolar macrophages (Alveolar macrophages, AMs) interact with activated myofibroblasts, which can trigger chronic inflammation and PF. AMs is located in the airways and develops by local proliferation of macrophages differentiated from monocytes (Mo-AMs) or resident from tissue (TR-AMs), where myeloid derived macrophages are important cell types that affect chronic inflammation of tissues and pulmonary fibrosis following lung injury. When lung tissue is damaged, macrophages migrate to the damaged site and differentiate into macrophages of different phenotypes and functions according to the stimulation signals of the microenvironment, i.e., the balance of the M1 with the pro-inflammatory phenotype and the M2 with the pro-fibrotic phenotype, the M1/M2 phenotypes determines the fate of the lung tissue after inflammation or damage. In early stages of lung tissue injury inflammation AMs is recruited to the injury site and prone to M1 polarization, and secretion of inflammatory factors is greatly increased, such as TNF- α, IL-1β, NO, ROS, etc., induces excessive inflammatory responses in tissues, resulting in exacerbation of lung tissue injury. As the injury progresses, the proportion of M2 type macrophages increases significantly and important fibroproliferative mediators are secreted to participate in tissue repair and healing; however, when the chronic inflammatory phase is progressed, excessive infiltration of M2 type macrophages into lung tissues causes accumulation of ECM components such as vascular growth factors and matrix metalloproteinases, and causes abnormal repair and vascular remodeling of lung tissues, triggering fibrosis. In summary, AMs is involved in mediating inflammatory injury to lung tissue via M1 polarization and in fibrotic scar tissue formation via M2 polarization. Therefore, accurate regulation of macrophage polarization after lung injury directly affects pathological homing after lung injury, and elucidation of key regulation mechanism of macrophage polarization is important.
PHLPP1 (pleckstrin homology domain leucine-rich repeat protein phosphatase 1), also known as SCOP or PPM3A or PLEKHE1, belongs to a family member of protein serine/threonine phosphatases, and PHLPP1 has been demonstrated to directly dephosphorylate corresponding conserved sites of proteins such as AKT, protein Kinase C (PKC), and S6 kinase (S6K), thereby regulating a variety of biological functions of cells. Current studies on PHLPP1 focus on its target molecule AKT. PHLPP1 dephosphorylates AKT to reduce the activation of regulatory T cell receptor signaling, affect T cell development, or dephosphorylate STAT1, regulate phagocytosis of macrophages, and thus participate in inflammatory immune responses. Furthermore, PHLPP1 deficiency can alleviate ischemic injury of the heart and brain by upregulating Akt activation, however, the role of PHLPP1 in pulmonary fibrosis is not yet clear.
In view of the foregoing, there is a great need in the art to develop new strategies for alleviating and treating pulmonary fibrosis, inhibiting immune cell function alterations and pathogenic effects in the local microenvironment of pulmonary fibers following lung injury, and controlling fibrosis and its associated symptoms and/or other complications resulting therefrom.
Disclosure of Invention
The invention is carried out aiming at the problems, firstly, the relation between the abnormal expression of PHLPP1 and pulmonary fibrosis is confirmed, then, the influence of the change of PHLPP1 expression on pulmonary fibrosis pathology and the polarization of M2 macrophages is observed based on the target point, and further, the effect of PHLPP1 in the progress of pulmonary fibrosis is determined.
The research process of the invention is as follows:
the inventor performs mining analysis on the data of lung tissues and alveolar lavage fluid macrophages of patients with idiopathic pulmonary fibrosis in a public database and performs differential gene analysis on the data and healthy people, and finds that the expression of PHLPP1 is remarkably reduced in the patients with pulmonary fibrosis and is inversely related to the severity of the pulmonary fibrosis, so that the PHLPP1 is possibly related to the occurrence of the pulmonary fibrosis.
And then, knocking out PHLPP1 in the mouse marrow line cells by using a Loxp-cre system, wherein the lung tissue fibrosis reaction is obviously aggravated and collagen in lung tissues is deposited in a lung fibrosis model of the PHLPP1 marrow line cell knockout mouse. PHLPP1 deficiency promotes the conversion of alveolar macrophages to M2 type, and its culture supernatant can promote the expression of fibrotic genes in lung fibroblasts in co-culture systems. By adopting specific small interfering RNA to target and silence PHLPP1 expression, M2 type macrophage marker gene expression can be up-regulated in bone marrow-derived macrophages; and the PHLPP1 is over-expressed in bone marrow-derived macrophages, so that the marker gene expression of M2 type macrophages can be inhibited. Therefore, we conclude that the significantly reduced expression of PHLPP1 in alveolar macrophages promotes their conversion to M2 type macrophages, and up-regulates the expression of pro-fibrotic genes, promoting the activation and fibrotic effects of lung fibroblasts, playing an important role in the progression of pulmonary fibrotic disease.
Based on the above research, the specific technical scheme of the invention is as follows:
in a first aspect of the invention, there is provided the use of a PHLPP1 promoter in the manufacture of a medicament for the treatment of pulmonary fibrosis and its associated symptoms and/or other complications.
Among them, pulmonary fibrosis, including idiopathic pulmonary fibrosis, is characterized by lung inflammation leading to persistent injury of alveoli and repeated destruction, repair, reconstruction and excessive deposition of extracellular matrix, whereas pulmonary fibroblast activation produces a large amount of extracellular matrix in alveoli and interstitium and thickens tissues around and between pulmonary air sacs (alveoli), which makes oxygen more difficult to enter the blood, leading to irreversible lung function decline, even respiratory failure.
Preferably, the PHLPP1 promoter is selected from any one or more of the following: exogenous PHLPP1 protein or nucleic acid encoding the same, a substance that promotes expression or active function of PHLPP1 protein, a substance that promotes overexpression of a nucleic acid molecule encoding PHLPP1 protein, a liposome or nanomaterial that encapsulates PHLPP1 encoding nucleic acid, a precursor protein capable of being converted to PHLPP1 in vivo, or a conjugate or complex thereof.
Further preferred, the PHLPP1 protein is selected from any one of the following:
(a) A polypeptide having the amino acid sequence shown in SEQ ID NO. 2;
(b) Proteins or polypeptides having the amino acid sequence homology shown in SEQ ID No.2 or having sequence identity (e.g., more than 80% homology or more than 80% sequence identity, such as 80%, 85%, 90%, 95%, 98%, 99%), and having the ability to inhibit fibrosis and treat pulmonary fibrosis and symptoms and/or other complications associated therewith;
(c) A protein or polypeptide having one or more amino acids substituted, deleted or added in the amino acid sequence of (a) or (b) and having the functions of inhibiting a fibrosis reaction and treating pulmonary fibrosis, or a protein or polypeptide derived from (a) or (b), wherein the nucleic acid molecule encoding PHLPP1 protein is selected from any one of the following
(i) A nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 1;
(ii) A molecule which hybridizes under stringent conditions to a nucleotide sequence defined in (i);
(iii) A nucleic acid molecule homologous or having sequence identity to the nucleotide sequence set forth in SEQ ID No.1 and encoding a protein or polypeptide having the ability to inhibit a fibrotic response and treat pulmonary fibrosis and symptoms associated therewith;
(iv) A nucleic acid molecule which is substituted, deleted or added with one or more nucleotides from the nucleotide sequence of (i), (ii) or (iii) and which encodes a protein or polypeptide having the ability to inhibit the fibrotic response and to treat pulmonary fibrosis and its associated symptoms or other complications.
In a second aspect of the present invention, there is provided a PHLPP1 recombinant vector comprising an expression vector and a nucleic acid molecule encoding a PHLPP1 protein inserted into the expression vector, the nucleic acid molecule encoding a PHLPP1 protein being as described above.
In a third aspect, the invention provides the use of a PHLPP1 promoter recombinant vector in the manufacture of a medicament for the treatment of pulmonary fibrosis and symptoms associated therewith and/or other complications.
The vectors include viral vectors and non-viral vectors.
The "viral vectors" include adenoviruses, adeno-associated viruses, lentiviruses, coxsackie viruses, herpes simplex viruses, measles viruses, newcastle disease viruses, parvoviruses, polioviruses, reoviruses, vaccinia viruses, vesicular stomatitis viruses, and the like. Suitable viral vectors are well known to those of ordinary skill in the art.
The non-viral vector comprises liposome or lipid complex, cationic polymer, chitosan polymer, nanoparticle vector and the like. Suitable non-viral vectors are well known to those of ordinary skill in the art.
In a fourth aspect of the invention, there is provided a pharmaceutical composition for the treatment of pulmonary fibrosis and its associated symptoms and/or other complications, comprising an active ingredient together with a pharmaceutically acceptable excipient, carrier or diluent. Wherein the active component is the PHLPP1 promoter or the PHLPP1 recombinant vector.
The recombinant virus and pharmaceutically acceptable auxiliary materials together form a pharmaceutical composition for treating pulmonary fibrosis and related symptoms and/or other complications, so that the curative effect is exerted more stably.
In a pharmaceutical form aspect, a medicament or pharmaceutical composition suitable for administration by a means selected from the group consisting of: oral administration, injection (e.g., direct naked DNA or protein injection, liposome coated DNA or mRNA injection), gold coated gene gun bombardment, plasmid DNA carrying for propagation defective bacteria, DNA carrying for replication defective adenovirus or protein encoded by the target gene, electroporation, nasal administration, pulmonary administration, oral administration. Aerosol inhalation administration.
In general, liquid formulations can be stored at 2 ℃ to 8 ℃ for at least one year, and lyophilized formulations remain stable at 30 ℃ for at least six months. The preparation can be suspension, water injection, freeze-drying preparation and the like commonly used in the pharmaceutical field.
When the composition of the present invention is administered to animals including humans, the administration amount varies depending on the age and weight of the patient, the nature and severity of the disease, and the administration route, and the results and various conditions of animal experiments can be referred to, and the total administration amount cannot exceed a certain range.
Further, the pharmaceutical compositions of the present invention may be used in combination with other pharmaceutical compositions for the treatment of pulmonary fibrosis and its associated symptoms and/or other complications.
In a fifth aspect, the invention provides the use of a reagent for detecting PHLPP1 expression level in the preparation of a pulmonary fibrosis detection kit or a drug screening kit.
The experimental results show that PHLPP1 expression is significantly reduced in pulmonary fibrosis patients and is inversely related to collagen fiber gene expression. PHLPP1 knockout promoted the occurrence and development of mouse pulmonary fibrosis, suggesting that PHLPP1 is a protective factor for pulmonary fibrosis and has the effect of inhibiting fibrosis reaction. From this, PHLPP1 can be inferred to be a diagnostic marker or drug screening marker for pulmonary fibrosis.
The method for drug screening with PHLPP1 as a marker is as follows:
(A) Treating cells, tissues or animals in a pulmonary fibrosis or simulated pulmonary fibrosis response or other fibrotic environment with a candidate substance;
(B) Detecting the level of PHLPP1 or a nucleic acid molecule encoding the protein in the cell, tissue or animal; and
(C) If the level of PHLPP1 or a nucleic acid molecule encoding the protein is higher than the level prior to treatment of the candidate substance or higher than in a normal control, it is indicated that the candidate substance has the effect of treating pulmonary fibrosis by promoting PHLPP1.
In a sixth aspect of the present invention, there is provided a pulmonary fibrosis diagnostic kit or a drug screening kit comprising reagents for detecting the expression level of PHLPP1 in a biological sample, consisting of a reverse transcription system, a primer system and an amplification system. Diagnosis or drug screening is accomplished by detecting the nucleic acid expression level of PHLPP1.
The invention has the beneficial effects that:
the invention provides application of PHLPP1 promoter in preparing medicines for treating pulmonary fibrosis and related symptoms and/or other complications, experiments prove that PHLPP1 knockout promotes occurrence and development of mouse pulmonary fibrosis, enhances M2 type conversion and infiltration of pro-fibrotic macrophages in a lung microenvironment, and prompts PHLPP1 to be a protective factor of pulmonary fibrosis and to have an inhibiting effect of fibrosis reaction. PHLPP1 overexpression can inhibit activation of pro-fibrotic macrophages, which is shown as inhibiting production of Arg1, retnla and TGF-beta in macrophages, thereby realizing alleviation and treatment of pulmonary fibrosis. Therefore, the invention provides a new target point for treating pulmonary fibrosis, especially idiopathic pulmonary fibrosis, aiming at the overexpression or functional promotion of PHLPP1, and has wide clinical application prospect.
Drawings
FIG. 1 shows that PHLPP1 expression is significantly reduced in pulmonary fibrosis patients and is inversely related to the expression of pulmonary fibrosis collagen fiber genes; wherein A, PHLPP1 expression levels in alveolar lavage fluid macrophages of healthy and Idiopathic Pulmonary Fibrosis (IPF) patients; b, PHLPP1 expression levels in lung tissue of healthy humans and patients with idiopathic pulmonary fibrosis; c, analysis of the expression correlation of PHLPP1 expression with Collagen fiber genes Collagen I (Col 1) and Collagen III (Col 3) in lung tissue of patients with idiopathic pulmonary fibrosis.
FIG. 2 shows that PHLPP1 knockout mice (cKO) have increased pulmonary fibrosis in a model of pulmonary fibrosis induced with Bleomycin (BLM); eosin & hematoxylin and masson staining of lung tissue of mice with a, WT and cKO; fibrosis scores for B, WT and cKO mice; c, WT and cKO mice lung tissue collagen fiber expression assay.
FIG. 3 shows that PHLPP1 knockout promotes alveolar macrophage to M2 phenotype transition; wherein the expression level of the M2 phenotype gene and the pro-fibrotic gene in lung tissue in a bleomycin-induced pulmonary fibrosis model in mice a, WT and cKO; immunofluorescent staining of M2 type macrophages in lung tissue of B, WT and cKO mice.
FIG. 4 shows that PHLPP1 knockdown macrophages are able to inhibit the expression of fibrotic genes in lung fibroblasts. Bone marrow-derived macrophages of WT and cKO mice were co-cultured with lung fibroblasts after induction or non-induction with IL-4, and examined for collagen expression in the lung fibroblasts.
FIG. 5 shows that interfering with PHLPP1 expression in bone marrow-derived macrophages promotes expression of M2-type marker genes and pro-fibrotic genes. Wherein A, the protein expression level of PHLPP1 after transfection of PHLPP1 targeting siRNA and control siRNA in bone marrow derived macrophages; b, mRNA expression level of PHLPP1 after transfection of bone marrow-derived macrophages with siRNA targeting PHLPP1 and control siRNA; c, transfection of PHLPP 1-targeting siRNA and control siRNA into bone marrow-derived macrophages, and stimulation or non-stimulation with IL-4 are performed to obtain the expression level of M2 phenotype gene and pro-fibrosis gene in cells.
FIG. 6 shows that over-expression of PHLPP1 in bone marrow-derived macrophages using lentivirus can inhibit the expression of M2-type marker genes and pro-fibrotic genes. Wherein A, the protein expression level of PHLPP1 after infection of the bone marrow-derived macrophages with lentivirus that overexpresses PHLPP1 and a control lentivirus; b, the bone marrow-derived macrophage utilizes lentivirus to over-express PHLPP1 or control virus, and then uses IL-4 to stimulate the expression level of M2 phenotype gene and pro-fibrosis gene in the cell.
Detailed Description
The following examples and experimental examples are provided to further illustrate the present invention and should not be construed as limiting the present invention. Examples do not include detailed descriptions of conventional methods, such as PCR methods, those used to construct vectors and texturesMethods of cloning, inserting a gene encoding a protein into such vectors and plasmids, or introducing a plasmid into a host cell. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, including Sambrook, j., fritsch, e.f. and maniis, t. (1989) Molecular Cloning: a Laboratory Manual,2 nd edition，Cold spring Harbor Laboratory Press。
Percentages and parts are by volume unless otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, and the preferred embodiments described herein are for illustration only.
Example 1: PHLPP1 expression was significantly reduced in patients with pulmonary fibrosis and was inversely related to the severity of pulmonary fibrosis
The data of lung tissues and alveolar lavage fluid macrophages of patients with idiopathic pulmonary fibrosis in the public database are mined and analyzed, and the index of the severity of the response pulmonary fibrosis is subjected to correlation analysis with the expression of PHLPP1.
The results show that: compared to healthy humans, macrophages in lung tissue and alveolar lavage fluid of patients with idiopathic pulmonary fibrosis expressed lower levels of PHLPP1 (fig. 1A and 1B), and PHLPP1 expression levels were inversely correlated with the expression of collagen fiber genes Col1 and Col3, respectively (fig. 1C).
The results show that: PHLPP1 plays a protective role in human pulmonary fibrosis disease.
Example 2: PHLPP1 knockout exacerbates the fibrotic pathology following lung injury in mice
PHLPP1 myeloid cell conditional knockout mice were constructed. Wherein PHLPP1 fl/fl The mice are constructed by the Sai industry biology and are hybridized with Lyz-Cre mice to obtain PHLPP1 fl/fl Lyz2-Cre +/– Mice, called cKO mice, achieve specific knockout of PHLPP1 gene in myeloid cells, while also obtaining PHLPP11 as a control mouse fl/fl Lyz2-Cre –/– (referred to as WT mice). These mice were kept in a clean class pathogen free (SPF) environment.
WT and cKO mice were given single Bleomycin (BLM), a model of BLM-induced pulmonary fibrosis was constructed, their lung tissues were fixed with paraformaldehyde, and then eosin & hematoxylin stained for observation of pathological levels of mouse lung tissues, and detection indicators included lung tissue fibrosis degree score and alveolar structural integrity of mice. The no fibrosis is counted for 1 minute, and the light, medium and heavy lung fibers are counted for 2,3 and 4 minutes respectively. Mild fibrosis mainly affects the pleural and subpleural pulmonary interstitium, alveolar structural disturbances, with an affected area of less than 20%; the involved area of moderate fibrosis accounts for 20% -50% of the whole lung; the severe fibrosis affected area accounts for more than 50% of the total lung, and the lung parenchyma is disordered and has fusion, and the air cavities of different sizes are visible.
The results show that: compared to control WT mice, cKO mice had more severe alveolar structure destruction, more cellular infiltration (fig. 2A), higher fibrosis scores (fig. 2B), and significantly increased expression of both Collagen fiber Collagen I (Col 1) and myofibroblast activation marker α -SMA in lung tissue (fig. 2C).
The results show that: PHLPP1 knockout promoted the pathological severity of mouse pulmonary fibrosis, suggesting that PHLPP1 plays a protective role in pulmonary fibrosis disease.
Example 3: PHLPP1 knockout promotes alveolar macrophage to M2 phenotype transition
In view of the close correlation between the regression of inflammation and the formation of fibrosis following lung injury and the polarization of M2-type macrophages, we next model BLM in cKO mice and control WT mice, taking their lung tissue extracted RNA for quantitative analysis and fluorescent staining.
The results show that: the expression of the marker genes Arg1, retnla and the pro-fibrosis factor Tgfb1 of M2-type macrophages in lung tissue of PHLPP1 knockout mice was significantly increased (FIG. 3A), while the number of alveolar macrophages positive for Arg1 expression was also significantly higher than that of WT mice (FIG. 3B).
The results show that: PHLPP1 knockdown promotes alveolar macrophage to M2 phenotype switching in pulmonary fibrosis mice, exacerbating the pulmonary fibrosis response following lung injury.
Example 4: PHLPP1 knocked-out macrophages promote expression of fibrotic genes in lung fibroblasts
Extracting cKO mice and control WT mice bone marrow-derived macrophages, culturing in vitro, stimulating with IL-4 (20 ng/ml), co-culturing with mouse lung primary fibroblasts, extracting fibroblast RNA, performing reverse transcription PCR, and detecting the expression of fibrotic genes in the fibroblasts by real-time fluorescence quantitative PCR.
The results show that: lung fibroblasts co-cultured with PHLPP1 knockout mouse bone marrow-derived macrophages expressed higher levels of Collagen I (Col 1) and α -SMA (fig. 4).
The results show that: PHLPP1 reduces the secretion of mediators that activate lung fibroblasts by macrophages, inhibits activation and differentiation of fibroblasts, and thus plays a protective role in pulmonary fibrosis.
Example 5: interfering with PHLPP1 enhancing macrophage to pro-fibrotic M2 phenotype conversion
Bone marrow-derived macrophages of wild-type mice were obtained, and siRNA sequences (sequences see SEQ ID NO.3-6, synthesized by Dharmacon) and control sequences (both from Dharmacon) were transfected to target PHLPP1 in the mice. Bone marrow-derived macrophages (as in example 3) were stimulated with IL-4 (20 ng/ml) to mimic the activation state of macrophages in pulmonary fibrosis 48 hours after transfection, total RNA from macrophages was harvested, and the activation of macrophages was detected by reverse transcription PCR followed by fluorescent real-time quantitative PCR.
The specific sequences of siRNA (PHLPP 1-siRNA) that specifically interfered with PHLPP1 expression are as follows:
siRNAD-058853-01：GUAAAUAACUUCCGUGACA(SEQ ID NO.3)；
siRNAD-058853-02：UAAAGGCACUCUAUGCUUC(SEQ ID NO.4)；
siRNAD-058853-03：GGAGGAUCCUUUACCCUGA(SEQ ID NO.5)；
siRNAD-058853-04：GAAGUCAAGUGUGUAGA(SEQ ID NO.6)。
the results show that: compared with the control group (lanes 1,2 and 3 in FIG. 5A), the interference PHLPP1 (lanes 4,5 and 6 in FIG. 5A) has significantly reduced PHLPP1 protein expression and significantly reduced mRNA level (FIG. 5B), indicating that the siRNAs can effectively interfere with PHLPP1 expression in macrophages; macrophages after interference with PHLPP1 expressed higher levels of the marker genes Arg1, retnla and the pro-fibrotic factor Tgfb1 of M2 type macrophages (fig. 5C).
The results show that: the siRNA can enhance the M2 transformation of macrophages to pro-fibrosis by interfering with the expression of PHLPP1, thereby promoting the activation of lung fibroblasts and the fibrosis effect.
Example 6: overexpression of PHLPP1 inhibits macrophage to pro-fibrotic M2 phenotype conversion
The gene company Ji Kai in Shanghai was entrusted to clone and amplify the DNA fragment of PHLPP1 gene and insert it into a lentiviral vector named GV643, so that a lentivirus in which PHLPP1 was overexpressed was successfully constructed and named as Lv-PHLPP1. The Lv-PHLPP1 and the control empty virus Vector are respectively infected with bone marrow-derived macrophages, and the infection efficiency of the lentivirus is detected by immunoblotting to confirm the over-expression effect. Then, the above lentivirus was infected with bone marrow-derived macrophages (same as in example 3), activated by IL-4 (20 ng/ml) to simulate the activation state of macrophages in pulmonary fibrosis, total RNA from the macrophages was collected, and the Arg1, retnla and Tgfb1 levels were detected by reverse transcription PCR followed by fluorescent real-time quantitative PCR.
The results show that: lv-PHLPP1 (FIG. 6A lane 2) showed significantly increased expression of PHLPP1 compared to Vector (FIG. 6A lane 1); the expression of marker genes Arg1, retnla and pro-fibrotic factor Tgfb1 was significantly reduced in M2 type macrophages in bone marrow-derived macrophages infected with Lv-PHLPP1 compared to Vector-infected macrophages (FIG. 6B).
The results show that: overexpression of PHLPP1 inhibits macrophage transformation to pro-fibrotic M2, thereby limiting the activation of lung fibroblasts and the fibrotic effects.
The nucleotide sequence of the PHLPP1 protein is shown in SEQ ID NO. 1:
cataatcccctggaacggccgcgcacaacgccattggcttctcccttctccgcgcgccgccgccgtctcccacctccgcctcatcgcctccctctccgcccgct
gcctccggagctgggggggaaacgcgaagccccactgcaatggagcccgccgccgcggccacggtacagcgactccccgagctcggcagggaggacc
gagcttcggctccggcggccgccgctgcggcagcagcagcagcagcggcggccgcggcggctctggcggcggcggccgggggcggccggagtccg
gagcccgcgctgaccccggcggccccgagcggcgggaacggcagcggcagcggggcgcgggaagaggccccaggcgaggcgccgccggggccg
ctgccgggcagagcggggggtgccgggcgcaggaggcggcgcggggcgccccagcccattgccggcggggctgcccccgtacccggggccggcgg
cggcgccaactccctcctgctgaggagagggcggctgaagaggaatctgtccgcggccgccgcggccgcctcctcgtcgtcgtcgtcctcggccgctgctg
cctcgcactcccccggcgctgccggcctccccgcctcctgctcggcctcggcgtcgctgtgcacccggagcctggacaggaagacgctgcttctgaagcac
cggcagacgctgcagctgcagccgtcggaccgggactgggtgaggcaccagctccagcgcggctgcgtgcacgtcttcgaccgccacatggcctcgacct
acctgcgcccggtgctctgcacactggacaccacggccggcgaggtggccgcccgcctgctgcagctgggccacaaaggcggcggcgtggtgaaggtg
ctgggccaggggcccggagccgccgccgcccgggagcccgctgaaccgccccccgaggccggcccccggctggcgcccccggagccgcgggactc
ggaggtaccgcccgcgaggagcgcgccgggtgccttcggggggcctccgcgcgcgccccccgccgacctacccctgcccgtcggcggcccgggcggg
tggtcgcgccgcgccagcccagcgccctcggactccagccccggcgagccgttcgttgggggccctgtctcttcgccccgcgccccacggcctgtggtctc
cgacaccgagagcttcagtctgagtcccagcgccgagagcgtgtctgaccggttggacccctacagcagcggcggcggctcctcgtcgtcgtcggaagag
ctcgaggccgacgcagcctcggccccgacgggggtcccgggccagccccgccgtcccggccaccccgcgcagcccctcccgcttccccagacggcttc
ctcgcctcagccgcagcagaaagccccgagggccattgacagcccgggcggggccgtccgcgaggggtcgtgcgaggagaaggcagcggcagccgtg
gccccgggaggcctccagtctacccccgggaggagcggggtgaccgcggagaaggcgcctccgccgcccccgccgcccaccctgtacgtgcagctcca
cggagagaccacccggcgcttggaggcggaggagaagccattgcagatccaaaatgactacctcttccaactgggatttggggagctgtggagggtgcag
gaggaaggcatggactcggagattggctgcctcatccgcttctatgcaggaaaacctcacagcacgggtagctctgaacggattcagctctcaggaatgtata
atgtccgtaaaggcaagatgcagttgccagtgaaccgatggacaagacgccaagtcatcctatgtgggacctgcctgatagtatcatctgtgaaagacagcttg
accggaaagatgcatgttctgccactaattggtggaaaagtagaagaagtgaaaaagcaccaacactgtttagcatttagctcctctggaccccaaagccagac
ttactacatttgctttgatactttcacagaatacttaaggtggctgcgacaagtctccaaggttgcatcccagcgcattagctcagtggacctctcgtgttgtagcct
ggaacatctgcctgccaacctcttctacagccaagacctcactcatctcaatttaaaacaaaacttcctaaggcagaaccctagccttccagctgccagggggct
taatgaactgcaaaggttcaccaagttgaagagtcttaacctttccaataatcatttaggggacttcccactggcagtctgcagtattccaaccctggcagagctg
aacgtgtcctgcaatgccctgcgatcagtcccggcagccgttggagtgatgcacaacttacagacatttttgttggatggaaactttctccaatcccttcctgctga
gttggagaacatgaagcagcttagttatctgggtctttctttcaatgaatttactgacattcccgaagtattggagaaattgactgctgtggataaactttgtatgtctg
gaaactgtgtggagacccttaggctacaggctttaagaaaaatgcctcacattaaacatgtggatctaaggttgaacgtaattaggaagctgatagcagatgaag
tggactttctacagcatgttactcagcttgacctacgagacaataagcttggtgatctagatgctatgattttcaacaacattgaagttttacactgtgaaaggaatca
actggtcacattagacatctgtggctatttcctaaaagcgctctatgcctcttctaatgaacttgttcaacttgatgtttacccagttccaaattatctgtcctacatgga
tgtttcaaggaaccgcttagaaaatgtgcctgagtgggtatgtgaaagccgaaagctagaagttttggatattggccataatcaaatatgtgaacttcctgcccgc
ttattttgtaatagcagtctccggaaactactggcaggacacaaccagttggcaaggctgcctgaaaggctagaaagaacctcggtggaggtcttggatgtgca
acacaaccagctccttgagctcccacctaaccttctgatgaaggctgacagcctgagattcctgaacgcctctgcgaacaaactggaaagccttcctccagcca
cgctttccgaagagacaaacagtatcttacaagagttgtatttgacaaataacagcctcacagacaaatgtgtgcccttgttaacgggacacccccatttgaagat
ccttcacatggcctataaccgacttcagagttttccagcaagtaaaatggcgaaactggaggaacttgaagaaattgatctcagtgggaataagctgaaagccat
cccaacaacgatcatgaattgcaggcgcatgcacaccgtgattgctcactccaactgcatcgaggtctttcccgaagttatgcagctcccagagatcaagtgtgt
ggacctgagctgtaatgagctaagtgaagtcacattaccagaaaacctgcctcccaaactgcaggagctagacctgactggaaacccgcgccttgtccttgat
cacaaaaccctggaactactgaataatatccgctgtttcaagattgatcagccttctacaggagacgcttccggagccccagctgtatggagtcatggttacact
gaagcttcgggggtaaaaaacaagttgtgtgtcgcagccctgtcggtgaataacttctgtgacaaccgcgaagccctgtatggtgtgtttgacggagaccgga
atgtggaggtgccctaccttctccagtgcactatgagtgacattttggctgaagagctgcaaaaaacaaaaaacgaagaagaatacatggtcaatacattcattg
tcatgcaaaggaaacttggaactgctgggcagaagcttggtggtgccgctgtcctttgtcatatcaagcatgaccctgtggatccaggaggatccttcaccttga
cctctgctaatgtgggcaagtgccaaacagttctctgtcgaaatggaaagccgctgcctctgtccagatcttacatcatgagctgtgaagaagagctgaagagg
attaaacagcacaaggccattatcactgaggatggcaaggtgaacggagtgactgagtccacgcgcatcctgggctacaccttcctccatcccagtgtggtgc
ctcgcccccacgtgcagtccgtgctcctgactccccaggatgagttcttcatcctaggcagtaaggggttgtgggacagcctgtccgtcgaggaggccgtgga
agccgtgcgcaacgtgcccgatgccctggctgctgccaagaagctgtgtaccctggcccagagctacggctgccacgacagcatcagcgctgtggtggtgc
agctcagtgtcactgaggacagcttctgctgctgcgagctcagcgccggtggggctgtgccaccacccagtcctggcatctttcctccctcagtgaacatggtg
atcaaggatcggccctcagatgggctgggcgtgccgtcctccagcagcggcatggcttccgagattagcagtgagctctccacttctgagatgagcagcgag
gtggggtcaacagcctccgatgagcccccgcccggagccctaagcgagaacagccctgcctaccccagtgagcag c gctgcatgctccaccccatctgtct
gtccaactccttccag c gccagctatccagcgccacgttctctagcgccttctccgacaacggccttgacagtgacgatgaggagcccatcgagggcgtcttc
accaacggcagccgggtggaggtggaggtggacatccactgcagccgggccaaggagaaggagaaacagcagcacctgcttcaggtgccagcagagg
ccagtgatgagggcattgtcatcagcgccaacgaggatgagccaggtctgcccaggaaggcagacttctctgccgttgggaccattgggcgccggagggc
caatggctctgttgcgccccaggaaaggagccacaatgtgatagaggtggctacagacgcacctcttcgaaagcctggaggctattttgctgccccggctcag
ccggatcctgatgatcagtttatcatacccccggagctggaagaggaggtcaaagaaatcatgaagcatcaccaggagcaacagcagcagcagcagccgc
caccaccccctcagctccagccgcagctgccgcggcactaccagctggaccagctgccagattattacgacacgccactatgacccagccgagctgtttaac
aaataaactaaccacaaaagactgagttgcaagagtctcccaggctcacattaaaccaggggttttactccacatccttcccccagacactgttcccaacctgtc
atcgcagctaatctgtaggttctctttctttgggttatttttttaagtaatcaccactttcttctagtgatgctttaccaatatgatttacatttgttaacttctccccctaacat
atcagatatgtaaagacaaagaacaaaaggtttaatatattacagagaaacagttaatgataatgtaatattttttaaaatggctttttgttgtttgtttggaaggcagg
gcaggctgccgttgctaaatgatttaataatattgtaattctgtatttctttggggggaaaaggcttttgttttgttttgttttgttttgttttgtttttgtcttgaaaataataga
catttgtagaatatggagactaactcctaggagttgctttactctgtcaggtgacttaagtcactgggattcactaattttctctgagagaacagctgattgagaattt
ccattgtaaatagctcagtgttgtatagtgaggcttacgatgttttgtagtcttggcgtaaggacacagcccaagtaactgacgtttcccctccccctcccctctgag
gagcctgcctgcctcacaactcaccctcacttcactgaatgaggaggctgagcagctgcagtgtttctgtccggaggaaatggatcttaggccactggacaag
aacctgcacccaagggccctgaacccattttcctcccctgtcccagccttcccactttgacagacacttttaactgtgttccttactgctgccacaatcagcatggtt
gtatagtgccccattaggccatttacatacccagagttatactcaagcagaatgcacaaatggacatgtcataatttttgttacaataaatatgaaatttacaagta
the amino acid sequence of PHLPP1 protein in the invention is shown in SEQ ID NO. 2:
MEPAAAATVQRLPELGREDRASAPAAAAAAAAAAAAAAAALAAAAGGGRSPEPALTPAAPSGGNG
SGSGAREEAPGEAPPGPLPGRAGGAGRRRRRGAPQPIAGGAAPVPGAGGGANSLLLRRGRLKRNLSA
AAAAASSSSSSSAAAASHSPGAAGLPASCSASASLCTRSLDRKTLLLKHRQTLQLQPSDRDWVRHQL
QRGCVHVFDRHMASTYLRPVLCTLDTTAGEVAARLLQLGHKGGGVVKVLGQGPGAAAAREPAEPP
PEAGPRLAPPEPRDSEVPPARSAPGAFGGPPRAPPADLPLPVGGPGGWSRRASPAPSDSSPGEPFVGGP
VSSPRAPRPVVSDTESFSLSPSAESVSDRLDPYSSGGGSSSSSEELEADAASAPTGVPGQPRRPGHPAQP
LPLPQTASSPQPQQKAPRAIDSPGGAVREGSCEEKAAAAVAPGGLQSTPGRSGVTAEKAPPPPPPPTLY
VQLHGETTRRLEAEEKPLQIQNDYLFQLGFGELWRVQEEGMDSEIGCLIRFYAGKPHSTGSSERIQLSG
MYNVRKGKMQLPVNRWTRRQVILCGTCLIVSSVKDSLTGKMHVLPLIGGKVEEVKKHQHCLAFSSS
GPQSQTYYICFDTFTEYLRWLRQVSKVASQRISSVDLSCCSLEHLPANLFYSQDLTHLNLKQNFLRQN
PSLPAARGLNELQRFTKLKSLNLSNNHLGDFPLAVCSIPTLAELNVSCNALRSVPAAVGVMHNLQTFL
LDGNFLQSLPAELENMKQLSYLGLSFNEFTDIPEVLEKLTAVDKLCMSGNCVETLRLQALRKMPHIKH
VDLRLNVIRKLIADEVDFLQHVTQLDLRDNKLGDLDAMIFNNIEVLHCERNQLVTLDICGYFLKALYA
SSNELVQLDVYPVPNYLSYMDVSRNRLENVPEWVCESRKLEVLDIGHNQICELPARLFCNSSLRKLLA
GHNQLARLPERLERTSVEVLDVQHNQLLELPPNLLMKADSLRFLNASANKLESLPPATLSEETNSILQE
LYLTNNSLTDKCVPLLTGHPHLKILHMAYNRLQSFPASKMAKLEELEEIDLSGNKLKAIPTTIMNCRR
MHTVIAHSNCIEVFPEVMQLPEIKCVDLSCNELSEVTLPENLPPKLQELDLTGNPRLVLDHKTLELLNN
IRCFKIDQPSTGDASGAPAVWSHGYTEASGVKNKLCVAALSVNNFCDNREALYGVFDGDRNVEVPY
LLQCTMSDILAEELQKTKNEEEYMVNTFIVMQRKLGTAGQKLGGAAVLCHIKHDPVDPGGSFTLTSA
NVGKCQTVLCRNGKPLPLSRSYIMSCEEELKRIKQHKAIITEDGKVNGVTESTRILGYTFLHPSVVPRP
HVQSVLLTPQDEFFILGSKGLWDSLSVEEAVEAVRNVPDALAAAKKLCTLAQSYGCHDSISAVVVQL
SVTEDSFCCCELSAGGAVPPPSPGIFPPSVNMVIKDRPSDGLGVPSSSSGMASEISSELSTSEMSSEVGST
ASDEPPPGALSENSPAYPSEQRCMLHPICLSNSFQRQLSSATFSSAFSDNGLDSDDEEPIEGVFTNGSRV
EVEVDIHCSRAKEKEKQQHLLQVPAEASDEGIVISANEDEPGLPRKADFSAVGTIGRRRANGSVAPQE
RSHNVIEVATDAPLRKPGGYFAAPAQPDPDDQFIIPPELEEEVKEIMKHHQEQQQQQQPPPPPQLQPQL
PRHYQLDQLPDYYDTPL
while the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.
Claims (10)
- Use of phlpp1 promoter in the preparation of a medicament for treating pulmonary fibrosis or complications thereof.
- 2. Use according to claim 1, characterized in that: the lung fibrosis is pulmonary fibrosis induced by alveolar macrophages and lung fibroblast pathogens, including idiopathic pulmonary fibrosis.
- 3. Use according to claim 1, characterized in that: the PHLPP1 promoter is selected from any one or more of the following conditions: exogenous PHLPP1 protein or nucleic acid encoding the same, a substance that promotes expression or active function of PHLPP1 protein, a substance that promotes overexpression of a nucleic acid molecule encoding PHLPP1 protein, a liposome or nanomaterial that encapsulates nucleic acid encoding PHLPP1 protein, a precursor protein capable of being converted to PHLPP1 in vivo, or a conjugate or complex thereof.
- 4. Use according to claim 3, characterized in that:wherein, PHLPP1 protein is selected from any one of the following cases:(a) A polypeptide having the amino acid sequence shown in SEQ ID NO. 2;(b) A protein or polypeptide having homology or sequence identity to the amino acid sequence shown in SEQ ID NO.2 and having the ability to inhibit the fibrotic response and treat pulmonary fibrosis and its associated symptoms and/or other complications;(c) A protein or polypeptide having one or more amino acids substituted, deleted or added in the amino acid sequence of (a) or (b) and having a function of inhibiting a fibrosis reaction and treating pulmonary fibrosis, or a protein or polypeptide derived from (a) or (b),the nucleic acid molecule encoding PHLPP1 protein is selected from any one of the following:(i) A nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 1;(ii) A molecule which hybridizes under stringent conditions to a nucleotide sequence defined in (i);(iii) A nucleic acid molecule homologous or having sequence identity to the nucleotide sequence set forth in SEQ ID No.1 and encoding a protein or polypeptide having the ability to inhibit a fibrotic response and treat pulmonary fibrosis and symptoms associated therewith;(iv) A nucleic acid molecule which is substituted, deleted or added with one or more nucleotides from the nucleotide sequence of (i), (ii) or (iii) and which encodes a protein or polypeptide having the ability to inhibit the fibrotic response and to treat pulmonary fibrosis and its associated symptoms.
- 5. A PHLPP1 recombinant vector, comprising an expression vector and a nucleic acid molecule encoding a PHLPP1 protein inserted into the expression vector, wherein the nucleic acid molecule encoding the PHLPP1 protein is as defined in claim 4.
- 6. The PHLPP1 recombinant vector of claim 5, wherein:wherein the expression vector is a plasmid vector, a cosmid vector, a phage vector or a viral vector, and the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, coxsackie virus, herpes simplex virus, measles virus, newcastle disease virus, parvovirus, poliovirus, reovirus, vaccinia virus and vesicular stomatitis virus.
- 7. Use of the PHLPP1 recombinant vector of claim 5 in the preparation of a medicament for treating pulmonary fibrosis.
- 8. A pharmaceutical composition for treating pulmonary fibrosis, characterized in that the pharmaceutical composition comprises an active ingredient and a pharmaceutically acceptable excipient, carrier or diluent,wherein the active component is the PHLPP1 promoter of any one of claims 1 to 4 or the PHLPP1 recombinant vector of any one of claims 5 to 6.
- 9. The application of the reagent for detecting PHLPP1 expression level in preparing a pulmonary fibrosis detection kit or a drug screening kit.
- 10. A lung fibrosis diagnostic kit or drug screening kit comprising reagents for detecting PHLPP1 expression levels in a biological sample.
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