CN112143806A

CN112143806A - Medicine and method for treating lung diseases by taking LDL-LDLR (low-density lipoprotein-LDLR) metabolic axis as target point

Info

Publication number: CN112143806A
Application number: CN201910576362.8A
Authority: CN
Inventors: 王久存; 石祥广; 梅雪倩
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2019-06-28
Filing date: 2019-06-28
Publication date: 2020-12-29
Anticipated expiration: 2039-06-28
Also published as: CN112143806B

Abstract

The invention relates to a medicine and a method for treating lung diseases by taking an LDL-LDLR metabolic axis as a target point. In particular, the invention relates to the use of an agent targeting the LDL-LDLR metabolic axis that promotes LDLR expression and/or increases LDLR protein activity in the manufacture of a medicament for the treatment or prevention of a disease that benefits from inhibition of pulmonary endothelial and/or epithelial apoptosis, and/or inhibition of pulmonary endothelial mesenchymal transition. The invention also provides a pharmaceutical composition comprising an LDL-targeting lipid lowering agent and a PCSK 9-targeting lipid lowering agent.

Description

Medicine and method for treating lung diseases by taking LDL-LDLR (low-density lipoprotein-LDLR) metabolic axis as target point

Technical Field

The invention relates to a medicine and a method for treating lung diseases by taking an LDL-LDLR metabolic axis as a target point.

Background

The development of scleroderma pulmonary fibrosis is a complex process involving several processes including immune dysfunction, pulmonary inflammatory response, pulmonary capillary injury, the transformation of endothelial cell morphology into stroma, loss of type II alveolar epithelial cell (ATII) surface active components leading to the destruction of the first barrier of the lung, epithelial-stromal transformation, and fibroblast activation, ultimately leading to massive collagen secretion. The complicated pathogenesis of the scleroderma, namely the pulmonary fibrosis is a difficult problem to overcome in the medical field, and the WHO classifies the scleroderma, namely the pulmonary fibrosis as one of the intractable diseases. In 10 months 2014, pirfenidone (pirfenidone) and nintedanib (nintedanib) are approved by the FDA to be used for treating idiopathic pulmonary fibrosis, however, both of the drugs have strong toxic and side effects, wherein nintedanib increases the toxic and side effects of the liver, and pirfenidone has strong toxic and side effects on the liver and the kidney, and the two drugs are expensive and too costly to treat. Worse still, these two drugs are used for treating idiopathic pulmonary fibrosis, and no specific drug for scleroderma pulmonary fibrosis exists clinically, and although cyclophosphamide is once used for treating scleroderma pulmonary fibrosis, sufficient evidence still exists, and no high-quality clinical test proves that the condition of patients taking cyclophosphamide can be obviously improved. Therefore, in order to deeply and carefully conduct comprehensive research on the scleroderma pulmonary fibrosis, a wider research means must be adopted to excavate potential pathogenic mechanisms, intervention methods, drug targets and the like.

Low Density Lipoproteins (LDL) are composed of free cholesterol, cholesterol esters, phospholipids, triglycerides and apolipoproteins, mainly produced by the liver and catabolism of Very Low Density Lipoproteins (VLDL). Abnormally elevated LDL can induce a number of cardiovascular diseases including hypercholesterolemia, atherosclerosis, myocardial infarction, and the like. This is associated with the ability of LDL to cause vascular endothelial cytotoxicity, increased superoxide radical production, and the like.

In addition to inducing endothelial cell dysfunction in cardiovascular diseases, LDL may also induce epithelial cell dysfunction, such as apoptosis; but also can induce the expression of TGF-beta and Fibronection of glomerular epithelial cells. More importantly, studies have shown that LDL plays an important role in surfactant metabolism in damaged alveolar epithelial cells. TGF-beta induced by the strain is a crucial regulator in the pathogenesis of acute lung injury and other respiratory diseases. Existing studies also mention more or less the relevance of LDL to scleroderma (SSc). However, these studies indicate that LDL is either elevated, lowered, or unchanged in patients with SSc, and thus remain highly controversial. Moreover, importantly, there has not been any report of changes in LDL levels in SSc-PF.

The level of LDL is almost determined by its receptor Low Density Lipoprotein (LDLR) in the metabolic mechanism. LDLR is a transmembrane receptor protein present on the cell surface, which is internalized when bound to LDL, and is taken up into the cell in endocytosed form by various cofactors such as ARH/DAB 2. Under intracellular acidic pH conditions, the complex is degraded by association with lysosomes, where LDL is broken down into free fatty acids and amino acids; the LDLR returns to the cell membrane to be continuously utilized or degraded by PCSK 9-based protein.

Patients with high levels of LDL often have different forms of LDLR dysfunction, including lower expression, genetic mutants or SNPs. Although increased LDL has been found to be associated with dysfunctional LDLR in many cardiovascular diseases, the role of LDL and LDLR in SSc-associated pulmonary fibrosis remains unclear.

Disclosure of Invention

The invention provides application of an agent which takes an LDL-LDLR metabolic axis as a target and promotes LDLR expression and/or improves LDLR protein activity in preparing a medicament for treating or preventing diseases benefiting from the inhibition of lung endothelial and/or epithelial cell apoptosis and/or the inhibition of lung endothelial intercellular transformation.

In one or more embodiments, the disease is selected from: acute respiratory distress syndrome, pulmonary fibrosis, lung injury, pulmonary hypertension, pulmonary edema, and lung cancer.

In one or more embodiments, the pulmonary fibrosis is idiopathic interstitial pneumonia or scleroderma pulmonary fibrosis.

In one or more embodiments, the agent that promotes LDLR expression is selected from the group consisting of:

(1) an LDLR expression vector;

(2) an expression vector of a nucleic acid molecule capable of promoting the expression of the LDLR gene carried by the host cell; and

(3) a lipid lowering agent selected from statin lipid lowering agents and bile acid sequestrants.

In one or more embodiments, the agent that increases LDLR protein activity is a PCSK9 inhibitor selected from the group consisting of:

(1) agents that inhibit PCSK9 expression and/or its activity, such as sirnas, antisense RNAs, ribozymes, and gene editing vectors, such as CRISPR-CAS9 gene editing vectors or TALEN gene editing vectors;

(2) anti-PCSK 9 antibodies;

(3) a small molecule inhibitor;

(4) a PCSK9 vaccine; and

(5) a homologous recombination vector comprising a nucleotide sequence encoding a mutated inactive or reduced activity PCSK 9.

In one or more embodiments, the statin lipid lowering drug is selected from: lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin and pharmaceutically acceptable salts thereof.

The invention also provides the use of a LDL-targeting lipid lowering agent and a PCSK 9-targeting lipid lowering agent in the manufacture of a medicament for the treatment or prevention of a disease benefiting from the inhibition of apoptosis of pulmonary endothelial and/or epithelial cells, and/or the inhibition of interstitial transformation of pulmonary endothelial cells.

The invention also provides the application of the statin lipid-lowering drugs and the lipid-lowering drugs targeting PCSK9 in the preparation of drugs for treating or preventing pulmonary fibrosis; preferably, the statin lipid lowering drug is selected from: lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin and pharmaceutically acceptable salts thereof; preferably, the PCSK 9-targeting lipid lowering agent is an anti-PCSK 9 antibody.

The invention also provides a pharmaceutical composition comprising an LDL-targeting lipid lowering agent and a PCSK 9-targeting lipid lowering agent.

In one or more embodiments, the pharmaceutical composition contains a statin lipid lowering drug and an anti-PCSK 9 antibody.

Drawings

FIG. 1: biochemical blood index of normal people, scleroderma patients and scleroderma pulmonary fibrosis patients. *: p < 0.05; **: p < 0.01; ***: p < 0.001; means. + -. SEM.

FIG. 2: LDLR expression in normal, scleroderma patients, and scleroderma-pulmonary fibrosis patients. *: p < 0.05; **: p < 0.01; ***: p < 0.001; means. + -. SEM.

FIGS. 3A-3B: lung histopathological examination of mice in different treatment groups. FIG. 3A, HE section analysis of pathological change results and Masson collagen staining results; figure 3B, Ashcroft scoring the degree of lung tissue damage in mice; WT-Saline: wild type saline group; ldlr-/-salt: an Ldlr gene knock-out mouse normal saline group; WT-BLM: a wild-type bleomycin group; Ldlr-/-BLM: knocking out the bleomycin group by the Ldlr gene; n is more than or equal to 6; *: p < 0.05; **: p < 0.01; ***: p < 0.001; means. + -. SEM.

FIGS. 3C-3D: and (3) detecting the collagen expression of lung tissues of mice in different treatment groups. FIG. 3C, the Sircol assay method, analyzes extracellular matrix levels in lung tissue of mice of different treatment groups; FIG. 3D, qPCR shows the detection of collagen-related gene expression in lung tissue from different treatment groups; WT-Saline: wild type saline group; ldlr-/-salt: an Ldlr gene knock-out mouse normal saline group; WT-BLM: a wild-type bleomycin group; Ldlr-/-BLM: knocking out the bleomycin group by the Ldlr gene; n is more than or equal to 6; *: p < 0.05; **: p < 0.01; ***: p < 0.001; means. + -. SEM.

FIG. 3E: and analyzing blood lipid components in the blood plasma of mice of different treatment groups. WT-Saline: wild type saline group; ldlr-/-salt: an Ldlr gene knock-out mouse normal saline group; WT-BLM: a wild-type bleomycin group; Ldlr-/-BLM: knocking out the bleomycin group by the Ldlr gene; n is more than or equal to 6; *: p < 0.05; **: p < 0.01; ***: p < 0.001; means. + -. SEM.

FIG. 3F: the proportion of each cell in the alveolar lavage fluid of mice of different treatment groups is changed. WT-Saline: wild type saline group; ldlr-/-salt: an Ldlr gene knock-out mouse normal saline group; WT-BLM: a wild-type bleomycin group; Ldlr-/-BLM: knocking out the bleomycin group by the Ldlr gene; n is more than or equal to 6; *: p < 0.05; **: p < 0.01; ***: p < 0.001; means. + -. SEM.

FIGS. 4A-4B: increased endothelial and epithelial apoptosis in Ldlr-/-mouse lung tissue. Fig. 4A, TUNEL staining of lung tissue shows: TUNEL positive cells were significantly increased in Ldlr-/-mice compared to wild-type mice, and this difference was further exaggerated under BLM stimulation; FIG. 4B, by TUNEL staining co-localization experiments with either endothelial marker molecule CD31 or epithelial marker molecule SP-C, shows that apoptotic cells are in fact endothelial cells and type II alveolar epithelial cells.

FIG. 4C: TUNEL results of pulmonary apoptotic cell proliferation in Ldlr-/-mice showed that the number of apoptotic cells in the lung tissue of Ldlr-/-mice was significantly higher than that of wild-type mice on day 7 or day 21, and that the number of apoptotic cells in the lung tissue of Ldlr-/-mice was dramatically increased after stimulation with BLM, much higher than that of WT-BLM group. N is more than or equal to 6; *: p < 0.05; **: p < 0.01; ***: p < 0.001; means. + -. SEM.

FIGS. 4D-4E: caspase-3 enzyme activity was expressed in mouse lung tissue. Western blot results showed that, on day 7 or day 21, total caspase-3 levels in lung tissues of Ldlr-/-mice were significantly higher than those of wild-type mice, but the active form cleaved caspase-3 only rose slightly; cleaved caspase-3 was dramatically elevated in Ldlr-/-mouse lung tissue when stimulated by BLM, much higher than in the WT-BLM group. N is 6; *: p < 0.05; **: p < 0.01; ***: p < 0.001; means. + -. SEM.

FIG. 5A: the lung EndoMT fluorescence co-localization experiment induced by Ldlr deletion shows that the lung tissues of Ldlr-/-mice have a significantly larger number of alpha-SMA and CD31 double-positive cells than wild-type mice, and the trend is further enlarged under the stimulation of BLM.

FIG. 5B: deletion of Ldlr induced pulmonary EMT. Double positive cells for α -SMA and SP-C were significantly more abundant in lung tissue of Ldlr-/-mice than in wild-type mice, and this trend was further exaggerated also under BLM stimulation.

FIG. 5C: deletion of Ldlr induced changes in pulmonary EndoMT and EMT molecular markers. The qRT-PCR results show that the endothelial cell marker molecules CD31 and Cdh5 are significantly down-regulated, while the fibroblast marker molecules S100a4 and Fn1 are significantly up-regulated in the lung tissues of Ldlr-/-mice no matter on day 7 or day 21. N is more than or equal to 6, P <0.05, P <0.01, P < 0.001.

FIG. 6: LDL redundancy induces endothelial and epithelial apoptosis, while LDLR deletion induces EndoMT and EMT phenomena. Detecting the number of endothelial cells and epithelial cell flow cytometry apoptotic cells after LDL treatment (A, E), detecting the activity of apoptotic protein caspase-3 (B, F), detecting endothelial tight junction genes (C) and cell permeability (D), detecting epithelial cell surfactant active protein genes (G) and detecting epithelial cell surfactant active lipid genes (H); endothelial cells after LDLR knockdown treatment, morphology detection of epithelial cells (I, L), detection of molecular surface markers (J, M) and immune double fluorescence detection of EndoMT and EMT cell numbers (K, N); p <0.05, P <0.01, P < 0.001.

FIG. 7: an imbalance in LDL-LDLR metabolism induces a fibroblast fibrotic response. (A-B) carrying out immune dual-fluorescence detection on the influence of the endothelial cell culture medium treated by LDL and si-LDLR on the expression of fibroblast alpha-SMA; (C-F) carrying out Western detection on the influence of the endothelial cell culture medium treated by LDL and si-LDLR on the expression of fibroblast collagen, alpha-SMA and p-SMAD 2/3; (G-H) detecting the expression change of the fibroblast alpha-SMA after LDL and si-LDLR treatment by using immune dual fluorescence; (I-L) Western detection of the changes in fibroblast collagen, alpha-SMA and p-SMAD2/3 expression after treatment with LDL and si-LDLR.

FIG. 8: an imbalance in LDL-LDLR metabolism promotes secretion of cellular fibrotic factors. A. TGF-beta 1 factor expression in supernatants of LDL-treated endothelial cells EA.hy926, epithelial cells MLE 12 and fibroblasts HFL 1; B. the expression of ET-1 factor in supernatant of endothelial cells EA.hy926, epithelial cells MLE 12 and fibroblasts HFL1 after LDLR knockdown treatment; p <0.05, P <0.01, P < 0.001.

FIG. 9: the combination treatment effectively relieves pulmonary fibrosis, inhibits apoptosis of lung cells and interstitial transformation. (A-B) the result of histopathological examination shows that the combined use of the atorvastatin and the PCSK9 antibody effectively improves the histopathological structure of the lung and inhibits the proliferation of fibrous tissues of the lung; (C) detecting the extracellular matrix components of the lung tissue after the combined treatment by using Sircol assay; (D) detecting the expression of the collagen related gene of the lung tissue; (E) inflammatory cell counts in alveolar lavage fluid; (F) detecting the expression of inflammatory genes; (G) detecting the blood lipid level; (H) detecting the LDLR recovery condition after the drug treatment; n is more than or equal to 6, P <0.05, P <0.01, P < 0.001; (I) western blot assay lung tissue apoptosis following combined use of atorvastatin and PCSK9 antibodies (J) immuno-bifluorescence assay lung EndMT following combined use of atorvastatin and PCSK9 antibodies; (K) pulmonary EMT status following a combination of atorvastatin and PCSK9 antibodies in an immuno-dual fluorescence assay; (L) detecting the expression of EndoMT/EMT related genes; n is more than or equal to 6, P <0.05, P <0.01, P < 0.001; # P <0.05, # P <0.01, # P < 0.00.

Detailed Description

It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., the embodiments) may be combined with each other to constitute a preferred embodiment.

The invention discovers that LDL redundantly induces the apoptosis of endothelial cells and epithelial cells, while LDLR deletion induces the phenomena of EndoMT and EMT; furthermore, LDLR deletions contribute to LDL redundancy. Accordingly, the present invention targets the LDL-LDLR metabolic axis to treat or prevent diseases that benefit from inhibition of apoptosis of the pulmonary endothelium and/or epithelial cells (especially type II alveolar epithelial cells, AT II), and/or inhibition of pulmonary endothelial interstitial transformation, by promoting LDLR expression and/or increasing LDLR protein activity. Such disorders include, but are not limited to, acute respiratory distress syndrome (COPD), pulmonary fibrosis, lung injury, pulmonary hypertension, pulmonary edema, and lung cancer, among others.

Herein, pulmonary fibrosis is the terminal change in a large group of lung diseases characterized by fibroblast proliferation and massive extracellular matrix aggregation with inflammatory injury, destruction of tissue structure; including pulmonary fibrosis of unknown etiology (idiopathic), also known as Idiopathic Interstitial Pneumonia (IIP). The most common type of disease with pulmonary fibrosis as the main manifestation in Idiopathic Interstitial Pneumonia (IIP) is Idiopathic Pulmonary Fibrosis (IPF). In some embodiments herein, the pulmonary fibrosis is scleroderma pulmonary fibrosis.

Herein, the expression level of LDLR in cells can be increased by administering an LDLR expression vector. The expression vector is used to express a polypeptide of interest, such as LDLR, in a host cell. Herein, unless otherwise specified, LDLR refers to the LDL receptor well known in the art, preferably the human LDL receptor, whose amino acid sequence and nucleotide sequence are available in NCBI and the like-related databases. It is understood that different species, even different individuals of the same species, may differ in the amino acid or nucleotide sequence of the LDLR, but such LDLR species may be used in the present invention as long as the biological effects of the LDLR species are the same. Any plasmid or vector that is capable of replication and stability in a host may be used in the present invention, including bacterial plasmids, bacteriophages, yeast plasmids, and viruses (e.g., adenovirus, retrovirus, adeno-associated virus, herpes virus, and lentivirus), among others. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, and translation control elements. The coding sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs from retrovirus, and other known promoters that control gene expression in eukaryotic cells. The translation control element includes a ribosome binding site for translation initiation, a transcription terminator, and the like. When LDLR is expressed in higher eukaryotic cells, transcription will be enhanced if enhancer sequences are inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene. Examples include the SV40 enhancer at the late side of the replication origin at 100 to 270 bp, the polyoma enhancer at the late side of the replication origin, and adenovirus enhancers. Thus, in some embodiments, an enhancer is also included in the expression vectors of the invention. In addition, the expression vector may optionally comprise one or more selectable marker genes to provide a phenotypic trait, such as a fluorescent protein, for selection of transformed host cells.

Methods for constructing expression vectors containing LDLR coding sequences are well known in the art. For example, the coding sequence for LDLR can be obtained by chemical synthesis. Alternatively, the coding sequence of LDLR can be obtained by PCR amplification of DNA/RNA. The coding sequence is then cloned into an appropriate expression vector. Methods well known in the art can be used to construct expression vectors containing the coding sequences described herein and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

In the present invention, the expression vector also includes an expression vector containing a nucleic acid molecule capable of promoting the expression of the LDLR gene carried by the host cell itself.

Alternatively, the administration of lipid-lowering drugs can inhibit the rate-limiting enzyme in the early stage of intracellular cholesterol synthesis, which results in the reduction of intracellular free cholesterol and feedback up-regulation of the expression of LDLR on the cell surface, thereby increasing the number and activity of LDLR. Suitable lipid lowering agents include, but are not limited to, statin lipid lowering agents and bile acid sequestrants. Statins lipid lowering agents and bile acid sequestrants well known in the art may be used. Exemplary statin lipid lowering agents include, but are not limited to, lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin, and pharmaceutically acceptable salts thereof and the like.

Herein, the method of increasing the activity of LDLR protein comprises administering suitable lipid lowering agents, especially statin lipid lowering agents, as described above, which increase the number and activity of LDLR. In some embodiments, the method of increasing LDLR protein activity comprises overexpressing an LDLR mutant in a over-host cell having enhanced biological activity compared to wild-type LDLR. The expression vector was constructed as described above.

Since LDLR is degraded by PCSK9 (protein convertase subtilisin kexin 9) -based proteins, methods of increasing LDLR protein activity further comprise administering an inhibitor of PCSK 9. Inhibitors of PCSK9 include agents that inhibit PCSK9 expression and/or activity thereof, including but not limited to proteins, nucleic acids, and small molecule compounds. For example, the protein may be an antibody to PCSK 9. The antibody is preferably a monoclonal antibody. A variety of anti-PCSK 9 antibodies, such as alirocumab and evolocumab, have been disclosed in the art and may be used in the practice of the present invention. The nucleic acid can be an siRNA, an antisense RNA, a ribozyme, and a gene editing vector, such as a CRISPR-CAS9 gene editing vector or a TALEN gene editing vector. Small molecule compounds include those known in the art to inhibit PCSK9 enzymatic activity, such as the benzylalkital class of compounds disclosed in CN107158002A, which is incorporated herein by reference in its entirety. In some embodiments, the PCSK9 inhibitor is a PCSK9 vaccine. In some embodiments, the agent that inhibits expression of PCSK9 is a homologous recombinant vector comprising a nucleotide sequence encoding a mutant inactive or reduced-activity PCSK9 that knocks down the wild-type PCSK9 gene in a host cell to express the mutant inactive or reduced-activity PCSK 9. For example, it is known in the art that there are two nonsense mutations (Y142X and C679X) in the gene encoding PCSK9 that cause loss or reduction of PCSK9 activity. Therefore, inhibition of protein activity can be achieved by expressing such mutated PCSK9 in a cell of a subject by homologous recombination techniques.

In a particularly preferred embodiment of the invention, a lipid lowering agent targeting LDL is administered concurrently with a lipid lowering agent targeting PCSK9, thereby more effectively inhibiting apoptosis of the pulmonary endothelium and/or epithelial cells (especially type II alveolar epithelial cells, AT II), and/or interstitial transformation of the pulmonary endothelial cells. In a particularly advantageous embodiment, the present invention simultaneously administers a lipid lowering agent targeting LDL and a lipid lowering agent targeting PCSK9 for the prevention and treatment of pulmonary fibrosis.

Accordingly, the present invention provides the use of an agent targeting the LDL-LDLR metabolic axis that promotes LDLR expression and/or increases LDLR protein activity in the manufacture of a medicament for the treatment or prevention of a disease benefiting from the inhibition of pulmonary endothelial and/or epithelial apoptosis, and/or the inhibition of pulmonary endothelial mesenchymal transition, including but not limited to acute respiratory distress syndrome, pulmonary fibrosis, lung injury, pulmonary hypertension, pulmonary edema and lung cancer, particularly idiopathic interstitial pneumonia or scleroderma pulmonary fibrosis, as hereinbefore described.

The invention also provides application of the reagent which takes the LDL-LDLR metabolic axis as a target and promotes LDLR expression and/or improves LDLR protein activity in preparing a medicament for inhibiting apoptosis of lung endothelium and/or epithelial cells and/or inhibiting interstitial transformation of lung endothelium cells.

The invention also provides a method of treatment or prophylaxis of a disease which benefits from inhibition of apoptosis in pulmonary endothelial and/or epithelial cells, and/or inhibition of interstitial conversion in pulmonary endothelial cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of an agent which promotes LDLR expression and/or increases LDLR protein activity, targeting the LDL-LDLR metabolic axis.

Agents targeting the LDL-LDLR metabolic axis that promote LDLR expression and/or increase LDLR protein activity suitable for use and methods described herein include the various formulations described previously. For example, agents that promote LDLR expression include, but are not limited to, LDLR expression vectors, expression vectors that promote expression of the LDLR gene carried by the host cell itself, and lipid lowering agents. Preferred lipid lowering agents are selected from statin lipid lowering agents and bile acid sequestrants. More preferably, the statin lipid lowering drug is selected from: lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, and rosuvastatin. Agents that increase LDLR protein activity are PCSK9 inhibitors including but not limited to agents that inhibit PCSK9 expression and/or activity thereof, such as siRNA, antisense RNA, ribozymes, and gene editing vectors, such as CRISPR-CAS9 gene editing vectors or TALEN gene editing vectors; anti-PCSK 9 antibodies; a small molecule inhibitor; a PCSK9 vaccine; and a homologous recombination vector comprising a nucleotide sequence encoding mutated inactive or reduced activity PCSK 9.

In particularly preferred embodiments of the above uses and methods, the agents comprise an LDL-targeting lipid lowering agent and a PCSK 9-targeting lipid lowering agent, more preferably an LDL-targeting statin lipid lowering agent and an anti-PCSK 9 antibody. In some embodiments, the agent is atorvastatin in combination with an anti-PCSK 9 antibody such as alirocumab and evolocumab.

In a particularly preferred embodiment, the present invention provides the use of a statin, a lipid lowering drug and a PCSK 9-targeted lipid lowering drug in the manufacture of a medicament for the treatment or prevention of pulmonary fibrosis; preferably, the statin lipid lowering drug is selected from: lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, and rosuvastatin; preferably, the PCSK 9-targeting lipid lowering agent is an anti-PCSK 9 antibody.

The invention also provides a pharmaceutical composition comprising an LDL-targeting lipid lowering agent and a PCSK 9-targeting lipid lowering agent. Preferably, the pharmaceutical composition contains a statin lipid lowering drug and an anti-PCSK 9 antibody. More preferably, the statin lipid lowering drug is selected from: lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin and pharmaceutically acceptable salts thereof; the anti-PCSK 9 antibody is alirocumab and/or evolocumab. In the medicament or the pharmaceutical composition, the LDL-targeting lipid-lowering drug and the PCSK 9-targeting lipid-lowering drug can be independently packaged. The agents or pharmaceutical compositions of the present invention may also be provided in admixture, provided that the two lipid lowering agents do not interfere with each other in their respective biological activities and stability when contacted. In some embodiments, the pharmaceutical compositions of the present invention are provided in the form of a kit, which may contain one or more doses of a statin, a lipid-lowering drug, and one or more doses of an anti-PCSK 9 antibody, as desired.

Besides the active ingredients, the medicine or the pharmaceutical composition also contains various pharmaceutically acceptable carriers or excipients which are commonly used in the field. As used herein, "pharmaceutically acceptable carriers" refer to those carriers and diluents which do not significantly irritate the organism and which do not otherwise impair the biological activity and performance of the agents in the pharmaceutical compositions being administered. "pharmaceutically acceptable excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the agent. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

The agent is present in the pharmaceutical composition in a therapeutically effective amount or a prophylactically effective amount. An effective amount is an amount administered sufficient to ameliorate or in some way reduce the symptoms associated with the disease. The amount administered is an amount effective to ameliorate or eliminate one or more symptoms and can be determined by one of ordinary skill in the art based on the age, sex, physical condition, etc. of the subject. The amount administered may be sufficient to cure the disease, but is generally administered to ameliorate the symptoms of the disease. Repeated administration is generally required to achieve the desired improvement in symptoms.

The pharmaceutical composition of the present invention may be formulated into any suitable dosage form for administration orally, intravenously, topically, or the like.

It is to be understood that herein, when referring to lipid lowering agents, it is generally meant herein the active ingredient that acts to lower lipid; however, in some embodiments, the lipid lowering agent may be a lipid lowering formulation, i.e., a pharmaceutically acceptable carrier or excipient in addition to the active ingredient for its lipid lowering effect. Thus, in some embodiments, the medicaments or pharmaceutical compositions of the invention contain two or more agents, such as a lipid lowering agent comprising a LDL targeting agent and a lipid lowering agent targeting PCSK 9.

Herein, the subject is a mammal, especially a human.

The present invention will be illustrated below by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.

Materials and methods

The following materials and experimental methods were used for the experiments of the present invention. Other materials and methods not mentioned are all conventional experimental materials and methods in the art, and are available from commercial sources, or can be performed by reference to well-known experimental methods.

2. Experimental methods

(1) Model establishment and administration mode

A pulmonary fibrosis mouse model is established by adopting a trachea perfusion method. Calculating the volume of the bleomycin solution according to the weight, then supplementing 50 mul with physiological saline, and injecting the bleomycin into the trachea by the model group; the normal control group was given a 50. mu.l physiological saline perfusion to the trachea. Before molding, 0.1ml of 10% chloral hydrate solution is injected into abdominal cavity for anesthesia, and a micro-injector is inserted into the trachea at 15 degrees for tracheal perfusion. Mice were sacrificed one week and three weeks after molding and corresponding samples were collected.

The dose of BLM of an Ldlr-/-knockout mouse batch experimental mouse is 2.0mg/kg, and single tracheal perfusion is carried out; treatment group batch experimental mouse BLM dosage is 4.0mg/kg, single tracheal perfusion. The atorvastatin is used at a concentration of 10 mg/kg/day; performing intragastric administration; the time from 3 days before molding to the time before killing the mice. The PCSK9 antibody was used at a concentration of 3 mg/kg/week; subcutaneous injection; the time from 7 days before molding to the time before killing the mice. Mice were fasted overnight without water deprivation prior to sacrifice. And (4) detecting blood biochemistry after the EDTA anticoagulation tube centrifugally collects blood plasma.

(2) Cell processing method

In vitro cell experiments, when the effects of LDL on endothelial cells, epithelial cells and fibroblasts were studied, the control group was supplemented with a non-fat serum medium (basal medium + non-fat serum LPDS), the experimental group used a LDL medium (basal medium + LDL), and neither group used FBS. The activity of the cell supernatant protein factor ELISA experiment and the intracellular signal path is detected after the cell is treated for 12 hours, and is not suitable to exceed 24 hours.

When the effect of LDLR on endothelial cells, epithelial cells and fibroblasts is researched, the cell morphology, the expression of the supernatant factor and the activity of intracellular channels are detected 48 to 72 hours after the LDLR is knocked down. The medium used was a complete medium normally containing 10% FBS.

When the cell supernatant was collected, the supernatant was centrifuged at 1000g for 5min to remove dead cells. The collected supernatant is immediately detected or stored at-80 ℃ to avoid repeated freeze thawing.

(3) Primer and method for producing the same

Second, result in

1. LDL is gradually increased in normal people, scleroderma patients and scleroderma pulmonary fibrosis patients

Endothelial injury is thought to be the initiating factor in the initiation of SSc, and plays an important role in the development of SSc. To verify whether SSc and later secondary SSc-PF are accompanied by abnormalities in LDL levels, we included 1642 normal volunteers, 52 SSc patients, 185 SSc-PF patients, and tested their LDL levels strictly according to clinical criteria. At the same time we also measured indicators related to vascular dysfunction. Including HDL (high density lipoprotein), TC (total cholesterol), TG (triglyceride) and GLU (glucose).

Experimental results show a significant increase in LDL levels in SSc compared to normal humans (1.61 ± 0.52vs.2.06 ± 0.67, P < 0.001). More importantly, patients with SSc-PF showed higher LDL levels than patients with SSc (2.06 ± 0.67vs.2.39 ± 0.85, P <0.001) (fig. 1, a). In addition, the HDL levels in patients with SSc were significantly reduced compared to normal (1.61. + -. 0.36vs. 1.17. + -. 0.33, P <0.001), but were not further down-regulated in SSc-PF (1.17. + -. 0.33vs. 1.19. + -. 0.37), with no statistical difference (FIG. 1, B). Another independent index for assessing the risk index of vascular disease, the LDL/HDL ratio, increased from normal to SSc and then to SSc-PF (1.05. + -. 0.43vs. 1.81. + -. 0.58vs. 2.1. + -. 0.72, P <0.001, P <0.01), with similar trends in change as LDL (FIG. 1, C).

Furthermore, we found by parallel examination that TC was reduced in SSc compared to normal (4.51 ± 0.93vs.4.02 ± 0.86, P <0.001), but almost as high as normal in SSc-PF patients (4.02 ± 0.86vs.4.54 ± 1.86, P <0.01) (fig. 1, D). Furthermore, levels of TG (1.86. + -. 1.43vs. 1.75. + -. 0.88vs. 1.65. + -. 0.95, NS) and GLU (5.04. + -. 1.00vs. 4.71. + -. 0.86, p <0.05, 4.71. + -. 0.86vs. 4.66. + -. 1.43, NS) were nearly comparable in patients with HDs, SSc and SSc-PF (FIGS. 1, E and F). These results show that, unlike other indices, LDL levels and LDL/HDL ratios exhibit a tendency to increase progressively in SSc and SSc-PF patients, suggesting that LDL may play an important role in the pathogenesis of SSc and in the progression of SSc to SSc-PF.

2. LDLR is gradually reduced in normal persons, scleroderma patients and scleroderma-pulmonary fibrosis patients

Given the important function of LDLR in LDL clearance, we speculate that altered LDLR transcript levels may be a significant cause of increased LDL in patients with SSc and SSc-PF. To elucidate this, we first analyzed the differentially expressed gene profile in pulmonary fibrosis tissues using public databases and found that LDLR expression was significantly low expressed in pulmonary fibrosis lung tissues. We then isolated total leukocyte RNA from normal humans, SSc and SSc-PF patients, and detected LDLR mRNA levels using RT-qPCR (quantitative reverse transcription-PCR) method. The results show that LDLR mRNA levels in SSc patients are reduced compared to normal humans (P <0.001), and LDLR mRNA levels in SSc-PF patients are reduced compared to SSc (P < 0.05). Our results are consistent with the chip results of Eileen Hsu, with LDLR mRNA lower than SSc for patients with SSc-PF. We also examined LDLR levels in lung biopsies of pulmonary fibrosis and found that LDLR protein levels were also significantly reduced in IPF compared to normal persons (P <0.001) (see figure 2).

3.1 Ldlr knockout aggravates Bleomycin (BLM) -induced changes in lung pathology

To investigate the role of LDL and LDLR in the development of scleroderma-induced pulmonary fibrosis, we first compared the histopathological differences between LDLR knockout mice and wild-type (WT) mice 7 and 21 days after molding with saline or BLM (2 mg/kg). HE staining and Masson staining showed that BLM was able to significantly induce damage and fibrosis of lung tissue in Ldlr-/-mice at day 7, while WT mice only increased slightly. BLM-induced pulmonary fibrosis in Ldlr-/-mice remained without any remission on day 21, and WT mice also developed significant fibrosis (fig. 3, a). Furthermore, semi-quantitative evaluation by using Ashcroft score also showed that BLM-induced pulmonary fibrosis was more severe in the Ldlr-/-than in the WT lungs, especially at 7 days (fig. 3, B). Moreover, these histopathological changes were associated with low survival of Ldlr-/-mice after BLM perfusion (the survival of BLM-treated Ldlr-/-mice in the 7-day group was 6/10, the survival of 21-day group was 7/10; the survival of BLM-treated WT mice in the 7-day group was 8/10, and the survival of 21-day group was 10/10).

3.2 Ldlr knockout aggravates BLM-induced collagen deposition in Lung tissue

We then determined the levels of collagen in lung tissue by the sircol method and showed that the soluble collagen content in lung tissue of Ldlr-/-mice under BLM stimulation was significantly higher than in WT-BLM treated group, especially on day 7 (fig. 3, C). Consistent with these results, fibrosis-associated genes such as Col1a1, Col1a2, Col3a1, α -Sma and Ctgf were significantly elevated in the lung tissue of Ldlr-/-mice 7 days after BLM treatment, whereas these collagen genes were only slightly increased in WT mice and were not statistically significant. Fibrosis-associated genes were significantly increased in both WT and Ldlr-/-lung tissues 21 days after BLM treatment. However, it is noteworthy that α -Sma and Col3a1 expression in the lungs of Ldlr-/-mice was still higher than in WT mice, indicating that the absence of the Ldlr gene makes the mice more susceptible to BLM-induced pulmonary fibrosis (fig. 3, D).

3.3 Ldlr knockout results in a dramatic increase in LDL

To study the mechanism of BLM-induced pulmonary fibrosis in scleroderma aggravated by defects in the Ldlr gene, we first needed to determine whether Ldlr-/-could alter LDL levels in plasma. To this end, we used an automated biochemical analyzer to measure blood lipid levels in the plasma of WT and Ldlr-/-mice. As shown in fig. 3(G, H), the LDL concentration and LDL/HDL ratio in plasma of Ldlr-/-mice were significantly increased on either day 7 or day 21, indicating that Ldlr plays a critical role in LDL metabolism. These results are consistent with the lipid changes reported by Matsuzaka et al in Ldlr-/-mice under normal dietary conditions. Thus, an increase in LDL levels in Ldlr-/-mice could be attributed to impaired LDL uptake resulting from downregulation of Ldlr expression. Furthermore, the magnitude of the rise in LDL was further increased in the BLM-treated Ldlr-/-mice compared to the WT mice, again demonstrating that Ldlr-/-mice are more sensitive to BLM than WT mice. Taken together, these results indicate that deletion of the Ldlr gene induces pathological changes or fibrosis in the lung and that Ldlr knockout mice rapidly show very severe lesions once BLM stimulation is given (fig. 3, E).

3.4 Ldlr knockout of increased proportion of leukocytes and lymphocytes in mouse alveolar lavage fluid

The percentage of leukocytes and lymphocytes in the lf (alveolar lavage fluid) of Ldlr-/-mice was found to be significantly higher than in the WT group by routine blood tests, while the percentage of neutrophils and monocytes tended to decrease (fig. 3, F). This suggests that Ldlr deficiency mediated pulmonary fibrosis in scleroderma is to some extent associated with immune cells.

4. Ldlr-/-aggravation of BLM Induction of apoptosis in pulmonary endothelial and epithelial cells in mice

We compared the fluorescent TUNEL staining of WT or Ldlr-/-lungs after saline or BLM tracheal perfusion with caspase-3 activity to assess apoptosis. As shown in the immunofluorescence image of fig. 4(a), TUNEL positive cells were significantly increased in lung tissue of mice after BLM perfusion at day 7, but apoptotic cells were significantly more in lung tissue of Ldlr-/-mice, and also significantly more apoptotic cells than wild-type in saline-treated Ldlr-/-mice. By observing the apoptotic cell localization we found that the apoptotic cells of the lung tissue of Ldlr-/-mice were not only increased in number, but also both endothelial and epithelial cells were apoptotic. As a result of TUNEL staining of lung in mice of day 21, TUNEL positive cells were found to be decreased in lung tissue of WT mice after BLM perfusion, indicating that the fibrotic progression of the body may be restored, whereas apoptotic cells in lung tissue of Ldlr-/-mice were not significantly attenuated, indicating that the lesion was still persistent.

To confirm that apoptotic cells were derived from endothelium and type II alveolar epithelium, we performed double staining of lung tissue with TUNEL and CD31 (endothelial marker molecule) or SP-C (type II alveolar epithelial marker molecule) using immuno-co-fluorescence, the results are shown in fig. 4 (B). The results show that these apoptotic cells are indeed endothelial and type II alveolar epithelial cells.

Furthermore, it was also shown by TUNEL positive cell counting that the number of TUNEL positive cells was significantly increased in Ldlr-/-mice compared to WT mice 7 and 21 days after BLM treatment (fig. 4, C). To further elucidate apoptotic cell proliferation in the lung tissue of Ldlr-/-mice from the molecular level, western results after lung tissue homogenization showed that BLM was able to significantly induce caspase-3 enzyme activity in both Ldlr-/-and WT mice on both days 7 and 21 (FIG. 4, D). Levels of apoptotic active caspase-3 were greatly increased in 7-day and 21-day Ldlr-/-mice when exposed to BLM compared to WT mice (FIG. 4, E, left panel). It was encouraging that the Ldlr-/-mice had significantly higher total caspase-3 expression in themselves than the WT mice, but only slightly increased caspase-3 enzyme activity was exhibited by the saline (FIG. 4, E, right panel). This revealed why Ldlr-/-mice were highly sensitive to BLM-induced pulmonary fibrosis in scleroderma.

5. Ldlr-/-promoting mouse lung endothelial cell matrix transformation

Since Ldlr-/-mice exhibited an earlier, more severe fibrotic lesion, we speculated that Ldlr-/-exacerbates the EndoMT and EMT phenomena. Therefore, the EndoMT condition is identified by adopting an in vivo mouse lung tissue fluorescence co-localization experiment and co-staining endothelial cell marker molecule CD31 and fibroblast marker molecule alpha-SMA. As a result, it was found that EndoMT in lung tissue of Ldlr-/-mice was significantly higher than that of wild-type mice (FIG. 5, A).

EMT was identified by co-staining with type II alveolar epithelial cell marker SP-C and fibroblast marker alpha-SMA. As a result, the number of EMT cells in the lung tissue of Ldlr-/-mice was found to be significantly higher than that of the lung tissue of wild-type mice, and this phenomenon was particularly aggravated by BLM stimulation (fig. 5, B).

In contrast to immunofluorescence, gene expression detection showed that all of the endothelial marker molecules CD31, Cdh5 (fig. 5, C) and type II alveolar epithelial marker molecule Sftps in the lung tissue of Ldlr-/-mice were significantly down-regulated, while the fibroblast marker molecules α -SMA, S100a4 and Fn1 were significantly up-regulated. The experiments show that Ldlr-/-mice not only cause massive endothelial and epithelial apoptosis, but also promote the transformation of the two cells into fibroblasts. This further explains why Ldlr-/-mice exhibit earlier, more severe fibrotic lesions under BLM stimulation.

6. LDL promotes endothelial and epithelial cell apoptosis, LDLR deletion induces EndoMT/EMT

To further elucidate the apoptotic and EndoMT/EMT phenomena in the lung tissue of Ldlr-/-mice, we investigated the role of abnormal LDL and Ldlr levels in inducing fibrosis by in vitro experiments, respectively. Flow cytometry technology, western, qPCR and other multiple experimental results show that LDL significantly induces endothelial cell apoptosis and apoptosis-related protein cspase-3 activation, and destroys the original biological functions of cells. Including increased endothelial permeability, loss of tight junction molecules, decreased expression of epithelial cell surface active proteins (FIG. 6, A-H). Interestingly, after the expression of LDLR is knocked down, endothelial cell EndoMT and epithelial cell EMT phenomena are respectively induced, and our conclusion is derived from the technologies of cell morphology observation, epithelial/endothelial-mesenchymal protein expression detection, mesenchymal transformed cell staining and the like (FIG. 6, I-N). Our in vitro experiments showed that apoptosis and EndoMT/EMT phenomena in the lung tissue of Ldlr-/-mice were induced by LDL redundancy and Ldlr depletion, respectively. The imbalance between the two contributes to a variety of pathological phenomena in the lung tissue of Ldlr-/-mice.

7. LDL-LDLR metabolic imbalance induces fibroblast fibrotic response

To test whether LDL or LDLR-induced endothelial, epithelial cell dysfunction contributes to the development of fibrosis, we cultured LDL or si-LDLR (siRNA of LDLR) pretreated ea.hy926 cell conditioned medium in human lung fibroblasts HFL 1. Western blot results showed that both LDL-ea.hy926 and si-LDLR-ea.hy926 conditioned media were able to significantly promote the fibrotic response of fibroblasts, as evidenced by increased α -SMA and collagen expression (fig. 7, a-F). Similarly, LDL or si-LDLR pretreated MLE 12 cell conditioned medium also significantly promoted the fibrotic response of mouse fibroblasts, NIH3T 3. Furthermore, we treated LDL or si-LDLR directly into fibroblasts, and as a result, found that significant fibrotic responses were also occurring in the fibroblasts (fig. 7, G-L). These results indicate that LDL-induced endothelial, epithelial apoptosis and si-LDLR-induced endothelial, epithelial mesenchymal transition both promote fibroblast fibrotic response; may be associated with apoptosis, release of certain pro-fibrotic factors by transformed endothelial and epithelial cells, and formation of a pro-fibrotic microenvironment. These fibrotic factors after LDL or si-LDLR treatment can also be directly secreted by fibroblasts.

8. LDL-LDLR metabolic imbalance induces profibrotic factor secretion

To further illustrate key factors inducing fibroblast fibrosis response after LDL or si-LDLR treatment, we used ELISA technology to detect the expression levels of key fibrosis factors ET-1 and TGF-beta 1 in endothelial cells, epithelial cells and fibroblasts after LDL or si-LDLR treatment. The results show that LDL mainly promotes the secretion of TGF-beta 1 in the three types of cells, and the secretion of ET-1 in the three types of cells is mainly caused by LDLR deletion. Our results indicate that LDL redundancy is different from the cytopathological phenotype induced by LDLR depletion, and that the secreted profibrotic factors are also different between the two. The imbalance in LDL-LDLR metabolism leads collectively to fibrotic lesions in the lung through different molecular pathways.

9. Combination of atorvastatin and PCSK9 antibodies for inhibition of BLM-induced pulmonary fibrosis

To investigate whether restoring LDLR expression could protect against lung injury and fibrogenesis, we pre-treated mice 3 days prior to tracheal perfusion of BLM with atorvastatin or PCSK9 antibody (alirocumab) alone or in combination with both drugs. Results from histological staining, ashcroft scoring and soluble collagen content analysis experiments showed that lung tissue fibrotic regions were reduced in mice treated with a combination of atorvastatin and alirocumab on day 21 post-BLM perfusion, while BLM model mice treated with atorvastatin alone or the alirocumab group alone were only slightly remitting (fig. 9, a-C). Also, fibrosis genes, such as α -Sma, Ctgf, Col1a1, Col1a2, and Col3a1, were significantly inhibited in the mice of the combined treatment group (fig. 9, D). The improvement in histopathology and marker indices correlated with lower mortality and weight gain (with only 3 mice from the combination treatment group dying, weighing 17.0 + -2.1 grams, and 9 mice from the BLM model group 15 dying, weighing 22.6 + -1.5 grams). In addition, lymphocytes in BALF of the combination treatment group reduced the expression of inflammatory genes such as IL-6, MCP-1, etc. in lung tissue (FIG. 9, E-F). These results indicate that atorvastatin in combination with alirocumab effectively reduces PF by protecting lung injury and fibrogenesis. We then measured the lipid composition in plasma. These results further support the notion that the combination-mediated PF-relief effect correlates with LDL lowering, compared to atorvastatin or alirocumab alone, the combination treatment significantly blocked BLM-induced LDL elevation and LDL/HDL ratio, returning nearly to normal physiological levels (fig. 9, G). Next, we examined the protein levels of pulmonary LDLR and found that combination therapy significantly promoted LDLR expression (50% increase in saline and 80% increase in BLM), while atorvastatin or alirocumab alone increased only slightly (FIG. 9, H). Furthermore, we also examined the protein levels of PCSK9 in the plasma of SSc-PF patients and PF mice, and found that PCSK9 expression levels were about 2-fold higher than controls (not shown). We therefore used a combination of alirocumab and atorvastatin to treat pulmonary fibrosis. Overall, these results support the view that: i.e., the benefits of combination therapy are primarily LDL-LDLR metabolic axis dependence.

10. Combination of atorvastatin and PCSK9 antibodies inhibits BLM-induced apoptosis and mesenchymal transition of endothelial and epithelial cells

We have previously demonstrated that Ldlr-/-mice exhibit higher apoptotic and mesenchymal transition in endothelial/epithelial cells compared to WT mice. Therefore, we tested lung apoptotic activity, EndoMT and EMT with atorvastatin in combination with alirocumab or with both drugs alone. As shown in FIG. 9(I), lung-activated caspase-3 protein was significantly inhibited in the combination-treated mice. Furthermore, the tight junctions and surfactant gene loss induced by BLM-induced apoptosis were restored to nearly physiological levels by combination therapy, including Ocln, Cldn-5, JAMs, ZOs endothelial marker molecules and Sftps, lpcaps ATII cell marker molecules (not shown). Given that LDLR expression loss can induce the phenomenon of EndoMT/EMT in cells in addition to causing cells, we followed the examination of pulmonary EndoMT/EMT in mice after combination therapy. The results showed that the combination treatment group was CD31⁺α-SMA⁺Double positive cells and SP-C⁺α-SMA⁺Both double positive cells were significantly reduced (FIG. 9, J-K). Furthermore, the transcriptional levels of the endothelial markers CD31, Cdh5 and ATII marker Sftps were enhanced, while the mesenchymal markers S100a4, Fn1 (fig. 9, L) were reduced. These show that the combination treatment group not only reduced BLM-induced apoptosis but also inhibited EndoMT/EMT behavior, while further illustrating that the effectiveness of the combination treatment group was based on the LDL-LDLR metabolic axis.

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Claims

1. Use of an agent targeting the LDL-LDLR metabolic axis that promotes LDLR expression and/or increases LDLR protein activity in the manufacture of a medicament for the treatment or prevention of a disease that benefits from inhibition of pulmonary endothelial and/or epithelial apoptosis, and/or inhibition of pulmonary endothelial mesenchymal transition.

2. The use according to claim 1, wherein the disease is selected from the group consisting of: acute respiratory distress syndrome, pulmonary fibrosis, lung injury, pulmonary hypertension, pulmonary edema, and lung cancer.

3. The use of claim 2, wherein the pulmonary fibrosis is idiopathic interstitial pneumonia or scleroderma pulmonary fibrosis.

4. The use of any one of claims 1 to 3, wherein the agent that promotes LDLR expression is selected from the group consisting of:

(1) an LDLR expression vector;

5. The use of any one of claims 1 to 3, wherein the agent that increases LDLR protein activity is a PCSK9 inhibitor selected from the group consisting of:

(2) anti-PCSK 9 antibodies;

(3) a small molecule inhibitor;

(4) a PCSK9 vaccine;

6. The use of claim 4, wherein the statin, lipid-lowering drug is selected from the group consisting of: lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin and pharmaceutically acceptable salts thereof.

7. Use of a LDL-targeting lipid lowering agent and a PCSK 9-targeting lipid lowering agent in the manufacture of a medicament for the treatment or prevention of a disease benefiting from the inhibition of apoptosis of pulmonary endothelial and/or epithelial cells, and/or the inhibition of pulmonary endothelial mesenchymal transition.

8. The use of a statin lipid lowering drug and a PCSK 9-targeted lipid lowering drug in the preparation of a medicament for the treatment or prevention of pulmonary fibrosis; preferably, the statin lipid lowering drug is selected from: lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin and pharmaceutically acceptable salts thereof; preferably, the PCSK 9-targeting lipid lowering agent is an anti-PCSK 9 antibody.

9. A pharmaceutical composition comprising an LDL-targeting lipid lowering agent and a PCSK 9-targeting lipid lowering agent.

10. The pharmaceutical composition of claim 9, comprising a statin lipid-lowering drug and an anti-PCSK 9 antibody.