Application of PARVA as target spot in treatment of pulmonary fibrosis

Abstract

The invention belongs to the technical field of biological medicine, discloses application of PARVA as a target spot in treatment of pulmonary fibrosis, and particularly relates to application of a PARVA down-regulating agent in preparation of medicine for treating pulmonary fibrosis. Aiming at the problem that the existing pulmonary fibrosis treatment medicine is difficult to target the pain point of a core pathological link, the medicine disclosed by the invention takes a PARVA gene in lung fibroblasts or an encoding protein thereof as a target spot, and comprises a knockdown medicine (such as shRNA, siRNA and miRNA), a knockout medicine (such as a Cre / LoxP system) or a targeted blocking medicine (such as a small molecule compound and polypeptide), preferably an injection, an atomizing agent and other dosage forms. The down-regulating agent realizes inhibition of activation and proliferation of lung fibroblasts and extracellular matrix deposition by down-regulating PARVA expression or inhibiting PARVA activity, so as to block pulmonary fibrosis progress. The invention has the advantages of strong target specificity, clear action mechanism, various drug types, obvious treatment effect and the like, and provides a new effective strategy for pulmonary fibrosis treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of PARVA as a target in treatment of pulmonary fibrosis, and particularly relates to a pharmaceutical composition containing a PARVA down-regulator and application of the pharmaceutical composition in preparation of a drug for treating pulmonary fibrosis. BACKGROUND

[0002] Pulmonary fibrosis is a chronic progressive disease characterized by diffuse fibrosis of lung tissue and destruction of normal lung structure. The pathogenesis of pulmonary fibrosis is complex and has not been fully elucidated.

[0003] Abnormal activation and proliferation of lung fibroblasts and excessive deposition of extracellular matrix are the core pathological links in the occurrence and development of pulmonary fibrosis. Existing treatment methods are mainly to relieve symptoms or delay disease progression, and there is a lack of specific drugs that can fundamentally reverse or block the pathological process. There is an urgent need for such specific drugs in clinical practice.

[0004] PARVA (Parvin alpha) belongs to the Parvins family and is an actin-binding protein that mainly participates in key biological processes such as assembly of cytoskeleton, cell adhesion, migration and signal transduction. PARVA is widely expressed, especially in high expression in heart, skeletal muscle, kidney and liver.

[0005] As a cell adhesion-related protein, PARVA participates in the regulation of cell proliferation, differentiation, migration and other biological processes. The specific role of PARVA in pulmonary fibrosis and its application as a therapeutic target have not been reported. Therefore, exploring the regulatory mechanism of PARVA in pulmonary fibrosis and developing related therapeutic drugs may have important clinical significance.

[0006] In addition, on the one hand, there are differences in understanding of the skilled person in the art, and on the other hand, the applicant has studied a large number of literatures and patents when making the present application, but due to the limited space, all the details and contents have not been listed in detail, which does not mean that the present application does not have these characteristics of the prior art. On the contrary, the present application already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art in the background art. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application aims to solve at least one or more technical problems in the prior art.

[0008] The application aims to provide an application of a PARVA down-regulator (i.e. an agent for reducing the expression of PARVA or an agent for inhibiting the activity of a PARVA protein, specifically, an agent for inhibiting the expression of PARVA and increasing the degradation of PARVA, which are different mechanisms but lead to the same result, i.e. the reduction of the level of PARVA) in the preparation of a drug for treating pulmonary fibrosis, so as to solve the problems of limited effect of the existing drugs for treating pulmonary fibrosis and difficulty in targeted intervention in the core pathological link, and provide a new effective strategy for the treatment of pulmonary fibrosis.

[0009] In order to achieve the above-mentioned application purposes, the application provides, in a first aspect, an application of a PARVA down-regulator in the preparation of a drug for treating pulmonary fibrosis.

[0010] Preferably, the drug targets the PARVA gene or the encoded protein thereof in lung fibroblasts, and plays a role in treating pulmonary fibrosis by down-regulating the expression of PARVA or inhibiting the activity thereof.

[0011] Preferably, the drug comprises or is at least one of a knockdown drug for the PARVA gene, a knockout drug or a targeted blocking drug for the PARVA protein.

[0012] Preferably, the knockdown drug for the PARVA gene comprises or is any one of an shRNA, an siRNA, an miRNA or a CRISPR / Cas-mediated knockdown drug targeting the PARVA gene.

[0013] Preferably, the knockout drug for the PARVA gene comprises or is a drug containing a Cre / LoxP system, which can specifically knockout the PARVA gene in lung fibroblasts.

[0014] Preferably, the targeted blocking drug for the PARVA protein comprises or is any one of a small molecule compound, a polypeptide or an antibody.

[0015] The mechanism of action of the drug is that by targeting and down-regulating PARVA in lung fibroblasts, the activation and proliferation of lung fibroblasts are inhibited, and the deposition of extracellular matrix is reduced, so as to block the pathological progression of pulmonary fibrosis and achieve the therapeutic effect.

[0016] In some embodiments of the application, the dosage form of the drug comprises or is any one of a needle injection, a powder, a tablet, a capsule, an aerosol or a granule.

[0017] In some preferred embodiments, the dosage form of the drug is a needle injection or an aerosol. The needle injection can be administered by injection, and the aerosol can be administered by inhalation through the respiratory tract, both of which can further improve the targeted accumulation efficiency of the drug in the lungs.

[0018] In some embodiments of the present invention, the bioactive component of the injection preferably includes at least one of the following: a knockdown or knockout reagent targeting the PARVA gene in lung fibroblasts, or a targeted blocking reagent targeting the PARVA protein in lung fibroblasts. Preferably, the knockdown reagent includes or is shRNA or siRNA targeting the PARVA gene and a vector. The vector may include or be any one of adeno-associated virus vectors, adenovirus vectors, lentiviral vectors, retroviruses, Sendai virus vectors, or liposomes. The knockout reagent includes or is a Cre / LoxP system or CRISPR / Cas system reagent targeting the PARVA gene. The targeted blocking reagent includes or is a small molecule compound, peptide, or antibody targeting the PARVA protein. Preferably, using the known PARVA structure or its functional site, high-throughput screening of small molecule libraries and protein-small molecule interaction detection (such as SPR, ITC) are used to screen candidate compounds. Preferably, monoclonal antibodies are prepared by immunizing animals with recombinant PARVA protein, and then neutralizing antibodies capable of blocking PARVA and its interaction partners (such as integrin complex, FAK, etc.) are obtained through screening.

[0019] In some embodiments of the present invention, the injection may further comprise one or more of human serum albumin, glycerol, mannitol, and water. Preferably, the human serum albumin in the injection comprises 0.01% to 0.1% by mass, for example, 0.01%, 0.02%, 0.05%, or 0.1% by mass. Preferably, the glycerol in the injection comprises 1% to 20% by mass, for example, 1%, 2%, 5%, 10%, 15%, or 20% by mass. Preferably, the mannitol in the injection comprises 0.1% to 10% by mass, for example, 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, or 10% by mass.

[0020] In a second aspect, the present invention provides a medicament for treating pulmonary fibrosis, the medicament comprising a PARVA downregulator and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

[0021] Preferably, the PARVA downregulator is a reagent that downregulates PARVA expression or a reagent that inhibits PARVA protein activity.

[0022] Preferably, the reagent that inhibits PARVA gene expression can be a knockdown reagent targeting the PARVA gene, such as any one of shRNA, siRNA, miRNA or CRISPR / Cas targeting the PARVA gene.

[0023] Preferably, the agent for inhibiting PARVA gene expression can also be a knockout agent targeting the PARVA gene, such as a drug of the Cre / LoxP system for specifically knocking out the PARVA gene in the lung fibroblasts.

[0024] The reagent that inhibits the activity of PARVA protein can be a blocking agent targeting PARVA protein, such as a blocking agent in the form of a small molecule compound, peptide, or antibody targeting the PARVA protein.

[0025] Preferably, the pharmaceutically acceptable vector can be any one of adeno-associated virus vector, adenovirus vector, lentivirus vector, retrovirus, Sendai virus vector, and liposome.

[0026] In this invention, there are no special requirements for the pharmaceutically acceptable diluents or excipients. Conventional pharmaceutically acceptable diluents or excipients can be used, as long as they are conducive to administration, conducive to the preparation of the target dosage form, and do not bring unacceptable adverse effects.

[0027] Preferably, the dosage form of the pharmaceutical composition can be any one of injection, powder, tablet, capsule, nebulizer, and granule. More preferably, the dosage form of the pharmaceutical composition is an injection or nebulizer. Injections can be administered by injection; nebulizers can be administered by inhalation through the respiratory tract. Both can further improve the targeted accumulation efficiency of the drug in the lungs.

[0028] When the dosage form is an injection, the injection may further contain one or more selected from human serum albumin, glycerol, mannitol, and water. Preferably, the human serum albumin in the injection contains 0.01% to 0.1% by mass, for example, 0.01%, 0.02%, 0.05%, or 0.1% by mass. Preferably, the glycerol in the injection contains 1% to 20% by mass, for example, 1%, 2%, 5%, 10%, 15%, or 20% by mass. Preferably, the mannitol in the injection contains 0.1% to 10% by mass, for example, 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, or 10% by mass.

[0029] Compared with the prior art, the present invention has the following outstanding advantages: (1) High target specificity: The drug of the present invention directly targets the PARVA gene or protein in lung fibroblasts and can precisely intervene in the core pathological process of pulmonary fibrosis through the PARVA downregulator contained therein, thereby reducing the impact on normal cells; (2) The mechanism of action is clear: it regulates the activation and proliferation of lung fibroblasts and the deposition of extracellular matrix by downregulating PARVA, and the treatment logic is clear; (3) Diverse drug types: covering multiple mechanisms of action such as gene knockdown, knockout and protein blocking, different types of drugs can be developed according to actual needs, with wide applicability; (4) Significant therapeutic effect: It can fundamentally delay or block the progression of pulmonary fibrosis, providing a new and effective means for clinical treatment. Attached Figure Description

[0030] Figure 1 The experimental results from human pulmonary fibrosis patients in Example 1 are shown: PARVA, α-SMA (α-smooth muscle actin), and Collagen I (type I collagen, Col1A1) staining showed that the expression levels of all three were significantly higher in the lung tissue of fibrotic patients (Lung Fibrosis) than in normal lung tissue (Control). Immunohistochemical staining analysis of lung tissue sections was performed using specific antibodies against PARVA, α-SMA, or Collagen I, with irrelevant rabbit IgG as a negative control. Samples were obtained from pulmonary fibrosis patients and non-fibrotic controls (n=5), with a scale bar of 100 micrometers.

[0031] Figure 2 This illustration shows Western blot analysis of PARVA or α-SMA expression levels in fibroblasts derived from human fibrotic lung tissue and control non-fibrotic lung tissue, as described in Example 1. GAPDH in the figure represents glyceraldehyde-3-phosphate dehydrogenase. The protein expression levels of PARVA or α-SMA in fibroblasts from fibrotic lung tissue were quantified using densitometric assay and compared with the expression levels in the control group (normalized to 1; n=3). The results showed that the expression levels of PARVA and α-SMA were significantly increased in cells from fibrotic lung tissue.

[0032] Figure 3 The experimental results of Example 2 are shown: Immunofluorescence co-localization staining was performed on human fibrotic lung tissue and non-fibrotic control lung tissue using anti-α-SMA murine monoclonal antibody and anti-PARVA rabbit polyclonal antibody; simultaneously, the mean fluorescence intensity of PARVA in α-SMA positive cells in fibrotic lung tissue was quantitatively analyzed and compared with the non-fibrotic control group (n=5 patients in each group). Scale bar: 50 μm.

[0033] Figure 4The results of knockdown of PARVA in human lung fibroblasts in Example 3 are shown. The two fragments used for knockdown were labeled sh-PA-1 and sh-PA-2. Western blot results of PARVA showed that sh-PA-1 and sh-PA-2 significantly reduced the protein level of PARVA.

[0034] Figure 5 The results of PARVA knockdown in human lung fibroblasts in Example 3 are shown. The two fragments used for knockdown were labeled sh-PA-1 and sh-PA-2. α-SMA fluorescence intensity detection showed that the cell viability of the PARVA knockdown groups (sh-PA-1 and sh-PA-2) was significantly lower than that of the untreated group (HLF) and the negative control group (Sh-NC). This result suggests that the expression level of PARVA is closely related to the activity of human lung fibroblasts, and that reducing PARVA expression affects the activation state of lung fibroblasts.

[0035] Figure 6 The experimental results of the effect of PARVA knockdown on the contractile function of human lung fibroblasts in Example 3 are shown. Both fragments used for knockdown were labeled sh-PA-1 and sh-PA-2. α-SMA fluorescence intensity detection showed that the contractile function of the PARVA knockdown groups (sh-PA-1 and sh-PA-2) was significantly lower than that of the untreated group (HLF) and the negative control group (Sh-NC), further validating the regulatory role of PARVA on the contractile function of human lung fibroblasts.

[0036] Figure 7 This demonstrates that knocking out PARVA in a mouse pulmonary fibrosis model in Example 4 prevented the progression of pulmonary fibrosis. A is a flowchart of the experimental design; B is a gross morphological image of the lung after knocking out PARVA in the mouse pulmonary fibrosis model, showing that knocking out PARVA specifically for lung fibroblasts reduced lung parenchyma volume and lightened staining; C shows the results of PARVA staining, α-SMA staining, and Collagen I staining in cKO mice, indicating that knocking out PARVA specifically for lung fibroblasts reduced the degree of pulmonary fibrosis, and that collagen deposition in the lung tissue of cKO mice was significantly reduced compared to WT (wild-type) mice. Scale bar: 100 micrometers. Detailed Implementation

[0037] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the examples are all conventional molecular biology and cell biology methods; the materials and reagents used are all commercially available unless otherwise specified.

[0039] Example 1: PARVA is highly expressed in pulmonary fibrosis tissue. 1. Sample Collection: Lung tissue samples were collected from 5 patients with surgically removed lung tissue diagnosed by pathologists as having pulmonary fibrosis, and 5 samples of normal lung tissue removed surgically due to benign lung lesions. All sample collection was approved by the hospital's ethics committee, and patients signed informed consent forms.

[0040] 2. Immunohistochemical detection of PARVA protein expression: Paraffin-embedded sections of tissue samples were dewaxed and antigen-retrievaled. Anti-PARVA monoclonal antibody (from ProteinTech Wuhan Sanying, catalog number 55268-1-AP, 1:500 dilution) was added and incubated overnight at 4°C. After incubation with HRP-labeled secondary antibody, DAB staining was performed. Results showed that the positive expression intensity of PARVA protein in the lung tissue of patients with pulmonary fibrosis was significantly higher than that in normal lung tissue. Figure 1 (P<0.001).

[0041] 3. Western blotting detection of PARVA protein expression: The protein levels of PARVA in human idiopathic pulmonary fibrosis cell lines and normal lung fibroblast cell lines were detected by Western blotting. The results showed that the expression level of PARVA in idiopathic pulmonary fibrosis fibroblasts was significantly higher than that in normal lung fibroblasts. Figure 2 ).

[0042] Example 2: PARVA localization in α-SMA-positive fibroblasts Immunofluorescence detection of PARVA protein cellular localization: Paraffin-embedded sections of pulmonary fibrosis tissue samples from Example 1 were dewaxed and antigen-retrieved. Anti-PARVA monoclonal antibody (1:500 dilution) and α-SMA (a marker of fibroblast activation) were added for co-staining, and the sections were incubated overnight at 4°C. After incubation with fluorescently labeled secondary antibody, the sections were observed under a fluorescence microscope. Results showed that PARVA was co-expressed in α-SMA-positive cells (…). Figure 3 ).

[0043] Example 3: PARVA reduces the effects of lung fibroblast activation and contractile function. 1. Cell Culture and Transfection: Human fibroblast cell lines were selected and routinely cultured in DMEM medium containing 10% FBS. Lentiviral viruses targeting PARVA (shPA-1, shPA-2) and a negative control lentivirus (ShNC) were constructed. Subsequent experiments were performed 48 hours after infecting human fibroblasts with the constructed viruses. The DNA sequences used to express the shRNAs are shown in Table 1 below, which can be used by those skilled in the art to obtain the corresponding shRNAs.

[0044] Table 1

[0045] 2. Verification of PARVA knockdown: Proteins were extracted from human fibroblast ShNC and shPARVA cells, and PARVA protein expression levels were detected by Western blot. Results showed that PARVA protein expression levels were significantly decreased in shPARVA cells. Figure 4 ).

[0046] 3. α-SMA immunostaining experiment: Human fibroblasts in logarithmic growth phase (wild-type, ShNC, shPA-1, shPA-2 groups) were immunostained with 2×10⁻⁶ cells / mL. 4 The cells were seeded at a density equal to the pre-placed coverslips in 24-well plates and cultured for 24 hours to allow cell adhesion. The culture medium was discarded, and the cells were washed three times with PBS for 5 minutes each time. 4% paraformaldehyde solution was added and fixed at room temperature for 20 minutes, followed by three washes with PBS. 0.1% Triton X-100 was added and permeabilized at room temperature for 15 minutes, followed by three washes with PBS. 5% BSA blocking buffer was added, and the cells were blocked at 37°C for 1 hour. The blocking buffer was discarded, and primary antibody (rabbit anti-human α-SMA antibody, 1:200 dilution) was added and incubated overnight at 4°C. The next day, the cells were washed three times with PBS, followed by addition of fluorescently labeled secondary antibody (goat anti-rabbit Alexa Fluor 488, 1:500 dilution) and incubated at 37°C in the dark for 1 hour. The cells were washed three times with PBS, and DAPI staining solution was added and stained at room temperature in the dark for 10 minutes, followed by three washes with PBS. The coverslips were inverted onto a slide (containing an anti-fluorescence quencher), and the cells were observed and photographed under a fluorescence microscope. The results showed that knocking down the PARVA gene significantly reduced the fluorescence intensity of α-SMA in human fibroblasts, demonstrating that myofibroblast differentiation was inhibited. Figure 5 ).

[0047] 4. Fibroblast Contraction Assay: The cell contraction ability was detected using a collagen gel contraction assay. Type I rat tail collagen was diluted to 3 mg / mL with PBS to obtain a collagen gel solution. Human fibroblasts in logarithmic growth phase (including wild-type, shNC, shPA-1, and shPA-2 groups) were digested and counted using 0.25% trypsin. Based on the counting results, 50,000 cells from each group were placed in 1.5 ml centrifuge tubes, and the supernatant was removed after centrifugation. Each tube was resuspended in 400 μL of MEM basal medium, and then 200 μL of collagen gel and 2 μL of 1M NaOH were added and mixed thoroughly. The mixture was immediately added to 24-well plates (600 μL per well) and incubated at 37°C, 5% CO2 for 15-30 minutes to allow the collagen to solidify. After solidification, 1 mL of complete culture medium was added to each well, and the cells were cultured further. The shrinkage of collagen gels was observed at 6, 12, 24, 48, and 72 hours of culture (medium changed every 24 hours), and images were taken. The gel area was measured using ImageJ software. The relative shrinkage rate of gels in each group at the same time point was calculated, with the wild-type gel area at a given time point defined as 100%. The results showed that knocking down the PARVA gene significantly weakened the collagen gel shrinkage ability of human fibroblasts, with significant differences in shrinkage. Figure 6 ).

[0048] Example 4: Effects of PARVA on a mouse model of pulmonary fibrosis 1. Construction of lung fibroblast-specific PARVA knockout mice: The construction method is as follows: Using Cre-LoxP targeted editing technology, Col1a1-Cre / ERT2 tool mice were crossed with PARVA Flox mice to obtain Col1a1Cre;PARVAFlox / Flox mice (cKO mice). After tamoxifen induction, the PARVA protein in lung fibroblasts of these mice was specifically knocked out; PARVAFlox / Flox mice were used as wild-type controls (WT mice).

[0049] 2. Establishment of a mouse model of pulmonary fibrosis: Eight-week-old WT mice and cKO mice were randomly divided into two groups of five each. Pulmonary fibrosis was established by intratracheal injection of bleomycin (2.5 mg / kg), while the control group was injected with an equal volume of physiological saline.

[0050] 3. Sample Collection and Detection: Mice were sacrificed on day 14 after bleomycin injection, and lung tissue was collected. One portion of the lung tissue was used for HE staining to observe pathological changes and collagen deposition; the other portion of the lung tissue was used for IHC to detect the protein expression levels of α-SMA (a marker of fibroblast activation) and Collagen I (type I collagen, a major component of the extracellular matrix).

[0051] The results showed that compared with WT mice, cKO mice exhibited significantly reduced inflammatory infiltration in lung tissue and a significantly decreased collagen deposition area; the protein expression levels of α-SMA and Collagen I were significantly reduced, indicating that knocking out PARVA in lung fibroblasts could inhibit the progression of pulmonary fibrosis. Figure 7 ).

[0052] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; terms such as "preferredly," "according to a preferred embodiment," or "optionally" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept.

Claims

1. Use of a PARVA down-regulator in the manufacture of a medicament for treating pulmonary fibrosis.

2. Use according to claim 1, characterized in that, The down-regulator is a drug targeting the PARVA gene or its encoded protein in lung fibroblasts.

3. Use according to claim 2, characterized in that, The medicament comprises at least one of a knock-down drug against the PARVA gene, a knock-out drug against the PARVA gene, or a targeted blocking drug against the PARVA protein.

4. Use according to claim 3, characterized in that, The knock-down drug against the PARVA gene comprises any one of an shRNA drug, an siRNA drug, an miRNA drug, or a CRISPR / Cas drug targeting the PARVA gene.

5. Use according to claim 3, characterized in that, The knock-out drug against the PARVA gene comprises a drug for a Cre / LoxP system or a CRISPR / Cas system for specifically knocking out the PARVA gene in the lung fibroblasts.

6. Use according to claim 3, characterized in that, The targeted blocking drug against the PARVA protein comprises a small molecule compound, a polypeptide, or an antibody.

7. Use according to claim 1, characterized in that, The medicament comprises a drug for inhibiting the activated proliferation of lung fibroblasts by PARVA.

8. The use according to claim 1, characterized in that, The medicament comprises a drug for reducing extracellular matrix deposition by PARVA.

9. The use according to claim 2, characterized in that, The dosage form of the medicament comprises any one of a needle injection, a powder, a tablet, a capsule, an aerosol, and a granule.

10. Use according to claim 9, characterized in that, The biologically active ingredient of the needle injection comprises at least one of a knock-down reagent against the PARVA gene in the lung fibroblasts, a knock-out reagent against the PARVA gene in the lung fibroblasts, or a targeted blocking reagent against the PARVA protein in the lung fibroblasts.

11. Use according to claim 10, characterized in that, In the knock-down reagent or the knock-out reagent against the PARVA gene in the lung fibroblasts, the knock-down reagent comprises an shRNA targeting the PARVA gene or an siRNA targeting the PARVA gene, and a pharmaceutically acceptable carrier; and the knock-out reagent comprises a reagent targeting a Cre / LoxP system or a CRISPR / Cas system of the PARVA gene.

12. Use according to claim 11, characterized in that, The carrier comprises any one of an adeno-associated virus carrier, an adenovirus carrier, a lentivirus carrier, a retrovirus, a Sendai virus carrier, and a liposome.

13. The use according to claim 10, characterized in that, The targeted blocking reagent comprises a small molecule compound, a polypeptide, or an antibody targeting the PARVA protein.

14. Use according to any one of claims 10 to 13, characterized in that, The needle injection further comprises any one or more of human blood albumin, glycerol, mannitol, and water.

15. A pharmaceutical composition for treating pulmonary fibrosis, characterized by, The pharmaceutical composition comprises a PARVA down-regulator.

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