KR20180120341A - Pharmaceutical composition for preventing or treating pulmonary fibrosis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating pulmonary fibrosis, containing a cystatin C (CST3) protein.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating pulmonary fibrosis,

The present invention relates to a pharmaceutical composition for preventing or treating pulmonary fibrosis.

Pulmonary fibrosis is a respiratory disease that causes severe respiratory distress due to hardening of the lung tissue. The hardening of the lungs means excessive accumulation of fibrous connective tissue and this process is called fibrosis. As pulmonary fibrosis progresses, various complications arise, including systemic hypoxia caused by decreased spirometry and oxygen diffusing disorder, pulmonary hypertension due to compression of pulmonary arteries and capillaries as the fibrous tissue contracts, and blood circulation in the right ventricle due to pulmonary circulation Induced heart failure, as well as thrombosis and pneumonia. In addition, inflammation and fibrosis can increase lung cancer incidence.

Pulmonary fibrosis is caused by inhalation of toxic substances or persistent inflammation, but there are also idiopathic pulmonary fibrosis that can not reveal the specific cause.

There is no way to recover lung fibrosis due to pulmonary fibrosis. The average survival time after diagnosis is fatal, ranging from 2 to 3 years. Currently, steroids and immunosuppressants that suppress inflammation are used as therapeutic agents for pulmonary fibrosis. However, after the inflammatory reaction of the disease progresses, fibrosis progresses, and most patients visit the hospital because of dyspnea. Therefore, there is a problem that it is difficult to expect the effect of anti-inflammatory treatment.

Korean Patent No. 10-0860903

The present invention provides a pharmaceutical composition for preventing or treating pulmonary fibrosis and a method for treating pulmonary fibrosis.

1. A pharmaceutical composition for preventing or treating pulmonary fibrosis comprising cystatin C (CST3) protein.

2. The pharmaceutical composition for preventing or treating pulmonary fibrosis according to 1 above, wherein the cystatin C protein comprises the amino acid sequence of SEQ ID NO: 1.

3. The pharmaceutical composition for preventing or treating pulmonary fibrosis according to item 1 above, wherein said cystatin C protein inactivates the TGF-Smad signaling pathway.

4. The pharmaceutical composition for preventing or treating pulmonary fibrosis according to 1 above, wherein the cystatin C protein inhibits the growth or activity of fibroblasts.

5. The pharmaceutical composition for preventing or treating pulmonary fibrosis according to item 1, further comprising a GDF-15 (Growth Differentiation Factor-15) protein.

6. The pharmaceutical composition for preventing or treating pulmonary fibrosis according to claim 5, wherein the GDF-15 protein comprises the amino acid sequence of SEQ ID NO: 2.

7. A method for treating pulmonary fibrosis comprising administering to a subject other than a human the composition of any one of claims 1 to 6.

The pharmaceutical composition according to the present invention can be treated by targeting fibroblasts as a new strategy for treating pulmonary fibrosis. Pulmonary fibrosis is caused by over-activation of fibroblasts and abnormal wound recovery accompanied by dissolution of alveolar epithelium. The pharmaceutical composition of the present invention targeting fibroblasts is more effective in preventing or treating pulmonary fibrosis than conventional anti-inflammatory drugs great.

In addition, since the pharmaceutical composition according to the present invention uses a protein as an active ingredient, the side effects are less than those of conventional chemical synthetic drugs and the success rate of clinical trials is high.

In addition, the cystatin C protein and the GDF-15 protein, which inhibit the growth or activity of the fibroblast of the present invention, are more mixed than when they are used alone, and are more excellent in the effect of inhibiting the growth or activity of the fibroblast.

Figure 1 shows the growth inhibitory effect of fibroblasts on conditioned media.
Figure 2 shows the proliferation, apoptosis and degree of necrosis of fibroblasts in conditioned media.
Fig. 3 shows the results of immunoblotting of α-SMA and collagen-1α of fibroblasts in the conditioned medium.
FIG. 4 shows the growth inhibitory effect of heat-treated epithelial HaCaT-derived factors on fibroblasts and fibroblast proliferation, apoptosis and necrosis.
FIG. 5 shows the growth inhibitory effect of fibroblasts of trypsin-treated epithelial HaCaT-derived factors and the inhibitory effect of fibroblasts on the growth of fibroblasts of two fractions obtained from the medium containing the epithelial HaCaT-derived factors.
Figure 6 is a profile of the cytokines involved in conditioned media collected from epithelial HaCaT and lung cancer cell A549 cells.
Figure 7 shows the results of the profiling of the cytokines contained in conditioned media collected from epithelial HaCaT and lung cancer cell A549 cells.
Figure 8 shows cytokine candidate substances that inhibit cytokines and fibroblasts rich in epithelial HaCaT-conditioned media.
Fig. 9 shows the growth inhibitory effect of fibroblasts on conditioned media obtained from epithelial HaCaT transfected with CST3, GDF-15 or IL-lα siRNA, respectively.
Fig. 10 shows the results of immunoblotting by incubation time in conditioned medium from epithelial HaCaT transfected with CST3 or GDF-15 siRNA, respectively, and immunoblotting results on siRNAs knocking down other mRNA of CST3 or GDF-15 .
Fig. 11 (a) shows the results of immunoblotting of conditioned medium obtained from epithelial cell RPMI2650 transfected with CST3 or GDF-15 siRNA, respectively, and fibroblast growth inhibitory effect.
Figures 11 (b) and (c) show the effect of fibroblasts on conditioned media from epithelial HaCaT transfected with either CST3 or GDF-15 siRNA respectively, or with CST3 and GDF-15 siRNA Proliferation, apoptosis and necrosis.
Fig. 12 shows the effect of inhibiting fibroblast proliferation and ECM production of CST3 and GDF-15 proteins.
FIG. 13 shows the results of immunoblotting and mRNA expression levels of α-SMA and collagen-1α of fibroblasts treated with CST3, GDF-15 or CST3 + GDF-15 protein.
Figure 14 (a) shows the phosphorylation inhibitory effect of Smad2 and Smad3 of fibroblasts treated with CST3 or GDF-15 protein in the presence of FBS (Serum).
FIG. 14 (b) shows the phosphorylation inhibitory effect of TGF-β receptor, Smad2 and Smad3 of fibroblasts treated with CST3 or GDF-15 protein at the time of stimulation with TGF-β1.
Figures 15 (a) and 15 (b) show the growth of fibroblasts stimulated by TGF-? 1 and the effect of CST3 and GDF-15 protein on ECM production.
Figure 15 (c) shows the inhibitory effect of CST3 and GDF15 on the TGF-Smad signaling pathway.
Figure 16 shows the experimental design of injecting bleomycin and peptide into mice used as an animal model of pulmonary fibrosis.
FIG. 17 shows the body weight and survival rate of CST3 and GDF15 injected mice.
FIG. 18 is a result of checking the degree of pulmonary fibrosis by staining a control and a lung and tissue section treated with bleomycin.
Figure 19 shows the results of immunoblotting against CST3 and GDF-15 in the control and bleomycin treated lungs.
FIG. 20 shows the fibrosis improvement effect upon administration of CST3, GDF-15 or CST3 + GDF-15 protein to the bleomycin-treated lung.
FIG. 21 is a graph showing immunohistochemical staining results of α-SMA and collagen-1α upon administration of CST3, GDF-15 or CST3 + GDF-15 protein to a bleomycin-treated lung and a numerical value thereof.
Figure 22 shows the results of cell cycle analysis of fibroblasts in the conditioned medium.
23 shows the results of analysis of the cell proliferation cycle and the cell death cycle of the fibroblasts in the conditioned medium.
24 shows the results of analysis of cell proliferation cycle and cell death cycle of fibroblasts of heat-treated epithelial HaCaT-derived factors.
25 shows the growth inhibitory effect of fibroblasts on conditioned medium from lung cancer cell A549 transfected with siRNA of GDF-15 or IL-l [alpha], respectively.
26 shows the results of analysis of cell proliferation cycle and cell death cycle of fibroblasts in epithelial cell-conditioned medium transfected with CST3, GDF-15 and CST3 + GDF-15 siRNA, respectively.
FIG. 27 shows the inhibitory effect of CST3 and GDF-15 proteins on epithelial HaCaT and cancer cell A549.

Hereinafter, specific embodiments of the present invention will be described. However, this is merely an example and the present invention is not limited thereto.

The present invention provides a pharmaceutical composition for preventing or treating pulmonary fibrosis comprising cystatin C (CST3) protein.

Pulmonary fibrosis in the present invention means idiopathic pulmonary fibrosis (IPF). Idiopathic pulmonary fibrosis is a chronic progressive pulmonary disease associated with excessive extracellular matrix deposition and collapse of the lung parenchymal architecture leading to severe respiratory failure with a survival rate of 2 to 4 years.

The fibrous features of idiopathic pulmonary fibrosis are due to the hyperproliferation of activated fibroblasts. In most tissues, epithelial-mesenchymal homeostasis should be maintained for normal structure and function. When the epithelium is damaged, a variety of cells, including epithelial cells, fibroblasts and vascular cells, work together in harmony to restore the broken barriers, and wound healing processes must be completed to ensure proper recovery. Damaged epithelial cells during the inflammatory phase release a variety of cytokines that stimulate immune cells and fibroblasts. During the proliferation phase, resident fibroblasts proliferate and differentiate into aggressive myofibroblasts, producing collagen and other matrix proteins to rapidly restore tissue structure. The epithelium is also reconstituted to restore barrier function. Finally, in the maturation phase, all activated signals are stopped to allow restoration of homeostasis of epithelial-mesenchyme. In particular, to reduce the fibrous burden required for emergency healing, myofibroblasts undergo apoptosis and are removed from the restored tissue.

Idiopathic pulmonary fibrosis is now understood to be a disorder of homeostasis of the epithelium-mesenchyme during wound healing rather than a direct consequence of inflammation. Several hypotheses have been proposed to explain why damaged epithelium causes fibrosis progression. In response to injury, epithelial cells secrete proinflammatory cytokines and produce fibroblast-stimulating cytokines via epithelial-mesenchymal transition. After repeated wound regeneration, epithelial cells lose their potential to regenerate themselves. As a result, since the integrity of the epithelium is not restored, the wound healing process can not be stopped and fibroblast stimulation continues. In addition, myofibroblasts induce epithelial cell death and inhibit the epithelial repair process. Damaged epithelial cells and grown myofibroblasts in IPF tissue activate the positive feedback loop and cause vast fibrosis and alveolar destruction.

Despite much efforts to understand the pathogenesis of IPF, little is known about the mechanism of epithelial cell regulation of fibroblasts in the homeostasis of epithelial-mesenchyme. Identifying fibroblast-controlling cytokines can provide new peptide drugs for IPF therapy.

Accordingly, the present invention provides cystatin C protein, an epithelial cell-induced cytokine that inhibits the growth and activation of fibroblast, to prevent or treat pulmonary fibrosis.

In the present invention, the cystatin C protein comprises the amino acid sequence shown in SEQ ID NO: 1 (Table 1). Specifically, the cystatin C protein may be composed of 120 amino acids, but is not limited thereto.

SEQ ID NO: Amino acid sequence One SSPGKPPRLVGGPMDASVEEEGVRRALDFAVGEYNKASNDMYHSRALQVVRARKQIVAGVNYFLDVELGRTTCTKTQPNLDNCPFHDQPHLKRKAFCSFQIYAVPWQGTMTLSKSTCQDA

Also, in the case where at least one amino acid is added or removed from the terminal region of the above-mentioned sequence or a part of the amino acid of the above sequence is mutated so long as the protein comprising the amino acid sequence of SEQ ID NO: 1 shows the effect of preventing or treating pulmonary fibrosis, It is obvious that it is included in the scope of.

In addition, the protein comprising the amino acid sequence of SEQ ID NO: 1 is an amino acid sequence which is 90% or more, specifically 96% or more, more specifically 98% or more, and more particularly 99% Are also included in the scope of the present invention.

The protein of the present invention is chemically modified or protected with an organic group at its N-terminus and / or C-terminus in order to protect it from protein cleavage enzymes in vivo and increase stability, Lt; / RTI > In particular, in the case of a chemically synthesized protein, the N-terminal and the C-terminal are charged, so that the N-terminal is acetylated and / or the C-terminal is amidated, But is not limited thereto.

The protein of the present invention may be synthesized by a method well known in the art, for example, an automatic peptide synthesizer depending on its length, and may be produced by genetic engineering techniques. For example, a fusion gene encoding a fusion protein, including a fusion partner and a protein of the present invention, is produced by genetic engineering, and the resulting fusion gene is transformed into a host cell, expressed in the form of a fusion protein, The desired protein can be produced by cleaving and separating the protein of the present invention from the fusion protein. To this end, a DNA sequence coding for an amino acid residue which can be cleaved by, for example, a proteolytic enzyme such as Factor Xa or enterokinase, a compound such as CNBr or hydroxylamine is used as a fusion partner and a polynucleotide encoding the protein of the present invention As shown in FIG.

In one embodiment of the present invention, the cystatin C protein can inactivate the TGF-Smad signaling pathway. Also, in one embodiment of the present invention, the cystatin C protein can inhibit the growth or activity of fibroblasts.

In the present invention, the TGF-Smad signaling pathway is part of the TGF-beta signaling pathway. TGF-beta signaling pathways play a crucial role in cell growth, differentiation and developmental regulation in a wide range of biological systems. The TGF-β signaling pathway is summarized as follows. TGF-beta superfamily ligands bind to type II receptors, and type II receptors recruit and phosphorylate type I receptors. Type I receptors phosphorylate receptor-regulated SMADs (R-SMADs), which can now bind coSMAD SMAD4. The R-SMAD / coSMAD complex induces gene transcription by accessing the promoter of the target gene as a transcription factor.

The growth and activation of fibroblasts is highly dependent on the TGF-Smad signaling pathway. Specifically, cystatin C protein can inactivate TGF-beta receptor and Smad2 / 3 (Figs. 14a and 14b). The cystatin C protein may directly counteract the TGF-? Signaling pathway and interfere with fibrogenesis. The cystatin C protein can physically bind to the TGF-beta receptor and inhibit the interaction of TGF-beta with its receptor. Thus, the cystatin C protein can inactivate the TGF-Smad signaling pathway and inhibit fibroblast growth or activity through inactivation of the TGF-Smad signaling pathway.

In one embodiment of the present invention, the pharmaceutical composition of the present invention may further comprise a GDF-15 (Growth Differentiation Factor-15) protein.

In the present invention, the GDF-15 protein comprises the amino acid sequence shown in SEQ ID NO: 2 (Table 2). Specifically, the GDF-15 protein is composed of 112 amino acids, but is not limited thereto.

SEQ ID NO: Amino acid sequence 2 ≪ RTI ID = 0.0 >

Also, in the case where at least one amino acid is added or removed from the terminal region of the above-mentioned sequence or a part of the amino acid of the above sequence is mutated as long as the protein comprising the amino acid sequence of SEQ ID NO: 2 shows the effect of preventing or treating pulmonary fibrosis, It is obvious that it is included in the scope of.

In addition, the protein comprising the amino acid sequence of SEQ ID NO: 2 is an amino acid sequence which is 90% or more, specifically 96% or more, more specifically 98% or more, and more particularly 99% Are also included in the scope of the present invention.

The protein of the present invention is chemically modified or protected with an organic group at its N-terminus and / or C-terminus in order to protect it from protein cleavage enzymes in vivo and increase stability, Lt; / RTI > In particular, in the case of a chemically synthesized protein, the N-terminal and the C-terminal are charged, so that the N-terminal is acetylated and / or the C-terminal is amidated, But is not limited thereto.

The protein of the present invention may be synthesized by a method well known in the art, for example, an automatic peptide synthesizer depending on its length, and may be produced by genetic engineering techniques. For example, a fusion gene encoding a fusion protein, including a fusion partner and a protein of the present invention, is produced by genetic engineering, and the resulting fusion gene is transformed into a host cell, expressed in the form of a fusion protein, The desired protein can be produced by cleaving and separating the protein of the present invention from the fusion protein. To this end, a DNA sequence coding for an amino acid residue which can be cleaved by, for example, a proteolytic enzyme such as Factor Xa or enterokinase, a compound such as CNBr or hydroxylamine is used as a fusion partner and a polynucleotide encoding the protein of the present invention As shown in FIG.

In one embodiment of the present invention, the GDF-15 protein may deactivate the TGF-Smad signaling pathway. In one embodiment of the present invention, the GDF-15 protein may inhibit fibroblast growth or activity. The growth and activation of fibroblasts is highly dependent on the TGF-Smad signaling pathway. Specifically, the GDF-15 protein can inactivate the TGF-beta receptor and Smad2 / 3 (FIGS. 14a and 14b). The GDF-15 protein may act as a partial agonist for the TGF-beta receptor. Generally, a weak agonist can stimulate the receptor in the absence of a complete agonist, but acts to inactivate receptor signaling by competing with the receptor in the presence of a complete agonist. GDF-15 may act as an inhibitor of fibrogenesis, given the TFGF-rich environment in the IPF lung. Thus, the GDF-15 protein can inactivate the TGF-Smad signaling pathway and inhibit fibroblast growth or activity through inactivation of the TGF-Smad signaling pathway.

In one embodiment of the invention, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.

The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, it may be prepared using diluents or excipients such as fillers, weights, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esterol such as ethyl oleate.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories It can have one formulation.

The composition of the present invention may be administered in a pharmaceutically effective amount.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level will vary depending on the species and severity, age, sex, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts.

The prophylactic or therapeutic agent for pulmonary fibrosis of the present invention may be administered daily or intermittently, and the number of administration per day may be administered once or two or three times. In addition, the composition of the present invention can be used alone or in combination with other drug therapy for the prevention or treatment of pulmonary fibrosis. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

It is known that the cystatin C or GDF-15 of the present invention is a protein, and when such a protein is developed as a pharmaceutical product and is put into practical use, it acts specifically on diseases, and thus the side effects are relatively lower and the success rate of clinical trials is higher than that of conventional chemical synthetic drugs . On the other hand, gene therapy techniques have limited theoretical and experimental backgrounds, and there are many limitations in clinical clinical use. Also, injecting genes has the risk of destroying the arrangement of other genes. In this respect, the development of a protein as a therapeutic agent for a specific disease is the most commercially available in terms of stability and technical aspects.

The present invention provides a method of treating pulmonary fibrosis comprising administering a composition of the present invention to a subject other than a human.

The term " individual " means all animals, including humans, suffering from the prevention or treatment of pulmonary fibrosis, and the disease can be effectively prevented or treated by administering the pharmaceutical composition. The term means the whole mammal including dogs, cows, horses, rabbits, mice, rats, chickens or humans, but the mammal of the present invention is not limited by the above examples. Specifically, the object may be an object other than a human.

The route of administration of the pharmaceutical composition may be administered through any conventional route so long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, subcutaneously, intradermally, or orally, but not limited to, parenteral administration, as desired. The composition may also be administered by any device capable of transferring the active agent to the target cell. The number of administrations is as described above.

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to be illustrative of the invention and are not intended to limit the scope of the claims. It will be apparent to those skilled in the art that such variations and modifications are within the scope of the appended claims.

Example

Example  1. Identification of cells producing fibroblast inhibitor

To determine the cells producing the fibroblast inhibitor, a conditioned medium (Hodgkin's yeast) was cultured in various epithelial cells containing three normal cell lines (HaCaT, RPMI 2650 and hNEC) and two carcinoma cell lines (A549 and HCT116) , CM), fibroblast cell line CCD-18Lu and MEF were cultured.

Cell culture and conditioned media (CMs) were prepared as follows.

(Normal lung epithelial cells), A549 (lung cancer) and HCT116 (colon cancer) were purchased from ATCC (American Type Culture Collection, Manassas, Va.) And HaCaT Skin epithelium) and MEF (mouse embryonic fibroblast) cell line were purchased from Korean Cell Line Bank (Seoul, Korea). Cells were cultured in EMEM (Eagle's Minimum Essential Medium) medium containing 10% heat-inactivated fetal bovine serum (FBS), Dulbecco's Modified Eagles Medium medium or RPMI 1640 medium (GIBCO). To prepare conditioned media (CMs), the cells were seeded in a 100-mm dish at 80% confluency and incubated for 24 hours. The next day, cells were washed with PBS and cultured in fresh serum-free medium. The cells were centrifuged at 2,000 x g for 5 minutes to remove cell debris on the 2nd or 3rd day after the exchange of the medium, and the cells were filtered with a 0.20-μm syringe filter (Corning, Corning, NY).

The cell proliferation assay procedure is as follows. A CCD-18Lu cells and MEF cells in 24-well plates and inoculated with 2 × 10 ⁵ was placed overnight in a density of cell / well. The next day, the cells were cultured in conditioned medium for 24 hours. Cells were isolated using trypsin and EDTA and stained with trypan blue. Unstained viable cells were counted using a hemocytometer.

CCD-18Lu grew well in cancer cell-conditioned media (CMs), but none of the epithelial-CMs could grow (Fig. 1a, * compared to CCD-18Lu CM, P <0.05). Likewise, MEF stopped growth in HaCaT-CM (FIG. 1b, * is P <0.05 versus HCT116 or A549 CM). 22), the lower G1 (sub-G1) group, meaning cell death, was significantly higher in CCD-18Lu cells incubated with HaCaT-CM (Fig. 1C , * * P <0.05 compared to CCD-18Lu or A549 CM). In this experiment, CCD-18Lu-CM was used as a reference for the normal cell cycle pattern of CCD-18Lu. According to the results, normal epithelial cells are likely to release several factors that counteract fibroblast proliferation.

Example  2. Count of proliferation, apoptosis or necrotic fibroblast

2-1. Cell cycle analysis and BrdU ( bromodeoxyuridine ) Incorporation Assay  Analysis of cell proliferation using

CCD-18Lu cells were plated on a 60-mm dish at 40% confluency. The next day, cells were cultured in a mixture of fresh medium and conditioned medium (v: v, 1: 1). Cells were fixed in 70% ethanol overnight at -4 ° C and stained with 20 μg / ml propidium iodide (PI) for 30 min at room temperature. The stained cells were applied to BD FACSCanto II flow cytometry analyzer (BD Biosciences) and cell cycle steps were analyzed using CellQuest software.

To examine cell proliferation, CCD-18Lu cells cultured in a media mixture containing 50% conditioned media were treated with 10 μM BrdU for 1 hour and harvested. Cells were fixed, permeabilized, treated with DNase, and stained with FITC-conjugated anti-BrdU (BD Biosciences). Total DNA and cloned DNA were designated 7-AAD and FITC-BrdU, respectively, and analyzed by flow cytometry. The group of cells labeled BrdU in step S was considered to be a proliferating cell population.

2-2. Cell death and necrosis analysis

Cell death and necrosis were determined using annexin V-FITC / PI cell death detection kit (BD Biosciences). Cells at a density of 1 x 10 ⁵ / well were stained with 1.25 μl of annexin V-FITC and 50 μg / ml of PI for 20 min at room temperature in the dark. Samples were analyzed with BD FACSCanto II (BD Biosciences) and CellQuest software. Cells in the lower right quadrant panel and the upper left quadrant panel were considered apoptotic and necrotic, respectively.

To understand the characteristics of fibroblast growth arrest, proliferation, apoptotic or necrotic CCD-18Lu cells were counted. (FIG. 23 (a)), the proliferation of CCD-18Lu cells was stopped in HaCaT-CM (Fig. 2a, *) was compared with CCD-18Lu or A549CM in consideration of the fact that BrdU- P < 0.05). (Fig. 23 (b)), and HaCaT-CM induced apoptosis or necrosis in CCD-18Lu cells (Fig. 2b, 2c, P <0.05 compared to CCD-18Lu or A549 CM).

Next, we analyzed the cellular levels of α-SMA and α-SMA, the representative markers of activated myofibroblasts. Considering that both markers increased incubation time dependently, CCD-18Lu cells were spontaneously activated under fetal serum and CM. However, this fibroblast activation did not occur in HaCaT-CM (Fig. 3).

Example  3. Determine whether a fibroblast inhibitor is a polypeptide

The HaCaT-CM was heated to denature the polypeptide to see if the HaCaT-derived factor was composed of the polypeptide. For thermal inactivation of the protein, HaCaT-CM was incubated at 95 DEG C for 30 minutes and centrifuged at 1,000 xg for 5 minutes.

After heating, HaCaT-CM almost completely lost its ability to inhibit fibroblast growth (Fig. 4a, * vs P <0.05 versus naive CM). In contrast, there was no significant difference in fibroblast growth between naive cancer cells-CM and heat-inactivated cancer cells-CM. Furthermore, CCD-18Lu cells were more aggressively differentiated in heat-inactivated HaCaT-CM than naive CM (Fig. 4b, * vs P <0.05 compared to naive CM, Fig. 24 (a) Cell death and necrosis were significantly reduced in inactivated CM (FIG. 4c, * vs P <0.05 compared to naive CM, FIG. 24 (b)).

To further characterize the HaCaT-inducing factor, the CM was pre-incubated with trypsin to digest the polypeptide and then treated with trypsin inhibitor to exclude the effect of residual trypsin on cell growth.

For tryptic digestion of the proteins, CM was treated with 200 ㎍ / ml trypsin (dissolved in 1 mM HCl) at 37 째 C for 8 hours. To stop trypsin treatment, 400 [mu] g / ml trypsin inhibitor (Sigma-Aldrich) was added to the medium. All media was filtered through a 0.20-μm membrane to remove microorganisms.

Trypsin cleavage abolished the fibroblast-inhibiting effect of HaCaT-CM (Fig. 5a, * = P <0.05 compared to naive CM). To roughly determine the size of the HaCaT-derived factor, the CM was filtered through a 30 kDa Centricon (R) filter and the volume of the residual medium was adjusted to the volume of the original medium by adding fresh medium. The retentate of the two fractions of HaCaT-CM showed the same fibroblast-inhibiting effect as the original medium, but the filtrate was not (Fig. 5b, * is P <0.05 compared to naive CM). These results strongly indicate that the fibroblast-inhibitory factor is composed of the polypeptide.

Example  4. Identification of fibroblast-inhibiting cytokines

4-1. Cytokine candidate substance

To identify peptide-based cytokines in CM, HaCaT and A549 CM were tested with the Human XL cytokine array kit. The cytokine profiling process is as follows.

The cytokine profiling was performed using a proteome profiler Human XL cytokine array kit (# ARY022) supplied by R & D Systems. Cells were cultured in serum-free medium for 3 days, and conditioned media was prepared as described above. The medium was applied to a nitrocellulose-based array membrane and incubated overnight at 4 ° C. The array membranes were treated with detection antibody cocktail (R & D Systems) for 1 hour and treated with streptavidin-HRP solution for 30 minutes. Immune complexes were visualized using a Chemi-Reagent Mix kit and the arrays were exposed to X-ray film. Protein concentrations in each conditioned medium were normalized by cell number and reference dots of nitrocellulose-based array membranes were evidence that each conditioned medium contained similar amounts of protein.

The red boxes (1-10) show more abundant cytokines in HaCaT-CM than A549-CM (Fig. 6). The quantified cytokine levels in the pixels of the ImageJ program are displayed as a bar graph (Fig. 7, * P <0.05 compared to A549 CM). The abundant cytokines in HaCaT-CM are summarized in FIG. Three candidate substances were determined as fibroblast-inhibiting cytokines: cystatin C (CST3), growth differentiation factor 15 (GDF15), interleukin 1α (IL-1α).

4-2. Screening for cytokines that inhibit fibroblast growth in cytokine candidates

To investigate which of the candidates inhibited fibroblast growth, we prepared CM from each cytokine knocked-down HaCaT cell.

Cells with 50% confluency were transfected with siRNA using RNAi-MAX reagent (Thermo Fisher Scientific) to prepare the knockdown cells of each cytokine. After stabilization for 48 hours, cells were applied to the indicated experiment. siRNAs were synthesized by Integrated DNA Technologies (Coralville, IA) and the sequences (5 'to 3') are shown in Table 3.

SEQ ID NO: Name of sample The base sequence (5'-3 ') 3 CST3 # 1 GCACAAUGACCUUGUCGAAAUCCAC 4 CST3 # 2 CCUAUCACCUCUUAUGCACACCUCC 5 CST3 # 3 CGCCCGCAAGCAGAUCGUAGCUGGG 6 GDF15 # 1 GCUGGGAAGAUUCGAACACCGACCT 7 GDF15 # 2 AGACUCCAGAUUCCGAGAGUUGCGG 8 GDF15 # 3 CGGAUACUCACGCCAGAAGUGCGGC 9 IL-1 alpha # 1 GCACAAUAGCAGUUGAAACAAGAAG 10 IL-1 alpha # 2 AGAGGAUCAAGACUUCUUUGUGCTC

CM from CST3- or GDF15-knockdown HaCaT did not inhibit CCD-18Lu growth, but CM from IL-1 alpha -drown HaCaT effectively inhibited growth (Figure 9, * indicates heat-inactivation, P < 0.05, # for P <0.05 compared to control CM). Considering that the growth of CCD-18Lu is not increased in CM of A549 transfected with CST3 and GDF15 siRNA (Fig. 25), siRNAs remaining in CM itself do not affect the growth of CCD-18Lu.

The efficiency of siRNA was assessed by immunoblotting. The process of immunoblotting is as follows. Cells were lysed in denaturing SDS sample buffer. The protein of the cell lysate was separated from SDS / 10-15% polyacrylamide gel and transferred to Immobilon-P membrane. Membranes were preincubated with 5% skim milk for 30 min to block nonspecific reactions, incubated overnight at 4 ° C with primary antibody (1: 1000 dilution), washed three times, and HRP-conjugated secondary And incubated with antibody (1: 5000) for 1 hour at room temperature. The immune complexes were visualized using the SuperSignal West Femto kit (Thermo Scientific, Waltham, Mass.).

The cleaved caspase-3, PARP, Smad2, p-Smad2, Smad3 and p-Smad3 antibodies were purchased from Cell Signaling Tech. (Danvers, MA); Caspase-9 and TGFβR1 antibodies were purchased from Santa Cruz Biotech (Dallas, TX); α-SMA, collagen-1α and p-TGFβR1 antibodies were purchased from Abcam (Cambridge, MA); anti-CST3 was purchased from R & D Systems (Minneapolis, MN); Anti-GDF15 was purchased from Fisher Scientific (Pittsburgh, Pa.).

In the immunoblot analysis, CST3 and GDF15 accumulated over time in HaCaT-CM but could be neglected in A549-CM (Fig. 10A). Fibroblast inhibitory effects of CST3 and GDF15 siRNA targeting different regions of each mRNA were assessed and confirmed (Fig. 10b, *, heat-inactivation, P <0.05 versus control CM, P <0.05 compared to CM).

We have already tested whether RPMI 2650 cells regulate fibroblast growth through these cytokines, as RPMI 2650, another epithelial cell line, has shown to inhibit the growth of CCD-18Lu as effectively as HaCaT (Fig. 1a). Both CST3 and GDF15 were detected in RPMI 2650-CM and each knockdown cytokine rescued the CCD-18Lu growth inhibited by CM (Figure 11a, * indicates heat-inactivation, P <0.05 compared to control CM, P < 0.05 compared to control CM). When both cytokines were all knocked down, both cell proliferation and survival were additionally improved ((Fig. 11b, 11c, *, heat-inactivation, P <0.05 versus control CM, # vs. P <0.05 compared to control CM) 26a, 26b). These results suggest that CST3 and GDF15 are secreted from normal epithelial cells and are involved in the inhibition of fibroblast growth.

Example  5. Recombination CST3  And GDF15's  Fibroblast Growth and Inhibition of Activity ( in vitro )

CCD-18Lu was treated with recombinant peptides of human CST3 (rCST3) and human GDF15 (rGDF15) to determine the effect of CST3 and GDF15 on fibroblast growth, and after 24 hours the number of cells was counted.

Both peptides decreased the number of CCD-18Lu cells in a concentration-dependent manner (Fig. 12A). Based on the IC ₅₀ (half-maximal inhibitory concentration) value, rGDF15 and rCST3 appear to have similar potency. In contrast, the peptides did not inhibit cell growth in HaCaT and A549 (Fig. 27), supporting the fibroblast specific action of these peptides. To test the synergistic effect of rCST3 and rGDF15, we compared the inhibitory effect of 1 ng / mL of each peptide on fibroblasts and the half concentration of both peptides (0.5 + 0.5 ng / mL). The combination showed a greater (> 2-fold) effect than the single treatment of each peptide (Figure 12b). This strongly indicates the synergistic action of the two peptides, since the total amount of peptide administered is the same in all cases.

Except for growth inhibition, both peptides down-regulated collagen-1 alpha and alpha-SMA protein and mRNA levels (Figures 13a, 13b, * were compared with untreated control (Ctrl) P <0.05, # treated only with CST3 or GDF15 P <0.05 compared to the army). These results have encouraged us to investigate whether peptides are applicable to prevent pulmonary fibrosis.

Total RNA was extracted from CCD-18Lu cells using TRIzol kit (Thermo Fisher Scientific). CDNA was synthesized from 2 ug of RNA using M-MLV reverse transcriptase (Promega, San Luis Obispo, Calif.). The cDNA was amplified with SYBR Green (Enzymes, Daejeon, Korea) and quantitated using a 7900HT real-time PCR detection system (Bio-Rad, Hercules, Calif.). In each PCR, mRNA levels were normalized to the amount of 18S ribosomal RNA. Each sample was measured three times and at least three independent samples were analyzed for each experimental condition. The base sequences of the PCR primers are shown in Table 4.

SEQ ID NO: Amplification target The base sequence (5'-3 ') 11 α - SMA Forward cccttgagaagagttacgag 12 Reverse tgtaggtggtttcatggatg 13 Collagen 1α Forward tacaaaaccaccaagacctc 14 Reverse agtttacaggaagagagacag 15 18S rRNA Forward gttaattccgataacgaacg 16 Reverse cacagacctgttattgctca

Example  6. Recombination CST3  And GDF15's TGF  Evaluation of inhibition of signal transduction pathway

Considering that fibroblast growth and activation is highly dependent on the TGF-Smad pathway, we first ascertained whether CST3 and GDF15 inhibit the signaling pathway.

The process of the Luciferase reporter assay is as follows. Cells were transfected with SBE-luciferase and CMV- [beta] -galactosidase plasmids using Lipofectamine 3000 reagent (Invitrogen). The total concentration of DNA (1 μg / 100 mm dish) was adjusted with pcDNA. The SBE-Luc reporter construct contains nine repeated Smad binding elements (SBEs) from Kim SG (Seoul National University, Seoul). After stabilization overnight, the transfected cells were inoculated into 12-well plates and further incubated for 24 hours. Cells were incubated with recombinant peptides for 2 hours, treated with TGF- [beta] l for 24 hours, and lysed to determine luciferase activity. To normalize the transfection efficiency, the? -Galactosidase activity was measured using o-nitrophenyl-β-D-galactopyranoside. Assays were repeated four times and each experiment was repeated three times.

As expected, Smad2 / 3 of CCD-18Lu was phosphorylated (or activated) in the presence of FBS. Interestingly, phosphorylation was attenuated by recombinant CST3 and / or GDF15 (Fig. 14A). When the TGF signaling pathway was stimulated by TGF-β1, the TGF-β receptor and Smad2 / 3 were inactivated by CST3 and / or GDF15 (FIG. 14b).

CCD-18Lu growth (FIG. 15A) and ECM production (FIG. 15B) were stimulated by TGF-β1, which was inhibited by recombinant peptides. We assessed TGF / Smad dependent gene expression using SBE-luciferase reporter and confirmed the inhibitory effect of CST3 and GDF15 on the TGF-Smad signaling pathway (Fig. 15c, * shows P <0.05 compared to control, P < 0.05 compared to the group treated with TGF- beta 1 only).

Example  7. Recombination CST3  And GDF15's  Evaluation of lung fibrosis improvement

To induce pulmonary fibrosis, C57 / B6 mice underwent a single challenge of bleomycin via bronchial instillation. Six days later, mice were systemically injected twice weekly with PBS, rCST3 (100 ug / kg), rGDF15 (100 ug / kg) or two peptides (50 ug / kg). At 21 days after bleomycin treatment, lung tissue was excised and histological analysis was performed. The experimental protocol is shown in Fig.

Bleomycin-induced pulmonary fibrosis is common in animal models for testing potential IPF therapies. Bleomycin mimics pulmonary inflammation and leads to chronic progression of pulmonary fibrosis. As a molecular feature of fibrosis, proinflammatory cytokines such as IL-1, IL-6 and TNF-α are increased in the early stages of bleomycin-treated lung tissue. Later, pro-fibrotic markers such as fibronectin, procollagen-1 and TGF-beta will gradually increase in tissue and reach peak levels after about two weeks after treatment with bleomycin. The transition between inflammation and fibrosis occurs around 9 days after bleomycin treatment. For prophylactic treatment, anti-inflammatory agents have begun to be administered at an early stage, but anti-fibrotic compounds have been suggested to be effective when administered at the "fibrotic" stage, irrespective of disease progression. Treatment of acute inflammation may not be practical in a hospital because it can actually diagnose pulmonary fibrosis after radiological evidence of respiratory symptoms. Thus, treatment may begin at the fibrosis stage. Taking into consideration the actual treatment period, the recombinant peptide was started to be injected after 6 days of bleomycin treatment. Although pulmonary fibrosis has already begun, peptides have been effective enough to stop disease progression (Figures 17-21). Therefore, cytokine therapy is expected to be effective in patients already diagnosed with pulmonary fibrosis.

The detailed experimental procedure is as follows. C57BL / 6J mice (male, 11 weeks) were purchased from Central Laboratory Animal Inc. (Seoul, Korea) and stored in a clean room. Mice were anesthetized with a mixture of tiletamine / zolazepam (30 mg / kg) and xylazine (10 mg / kg), and saline or bleomycin sulfate (2 mg / kg, MBcell, CA, USA) were injected intracorally in a total volume of 50 μl. CST3 / GDF15 peptides (50 [mu] g / kg each) or PBS were intraperitoneally injected into mice at 6, 9, 12, 15 and 18 days after the administration of the bleomycin. Recombinant peptides of mature CST3 (aa. 27-146, NM_000099) and mature GDF15 (aa. 195-308, NM_004864) were purchased from Abcam and Sino Biological Inc. (Beijing, China), respectively. On day 21, lung tissue was removed from the mice and divided into two lobes. The left lobe was fixed with 4% paraformaldehyde and the right lobe was stored in liquid nitrogen. All procedures were approved by SNU-SNU-141006-3.

The mice were continuously monitored for body weight to monitor their apparent health status. Mouse weight decreased progressively over 21 days, but was significantly restored by the combined peptides (Fig. 17a, * vs P <0.05 for control, # for P <0.05 versus Bleo-PBS group). Mouse survival rate was also improved by combination (Fig. 17B).

To evaluate the extent of pulmonary fibrosis, lung samples were removed from mice with Masson's trichrome and hematoxylin / eosin on day 21 (Fig. 18). Paraffin sections (4 μm) of the lungs were deparaffinized and rehydrated and stained with Weigert's iron hematoxylin and biebrich scarlet-acid fuchsin for 10 minutes. Finally, the sections were stained with 2.5% aniline blue for 10 minutes and decolored with 1% glacial acetic acid for 3 minutes. Microscopic photographs were taken at random in four sections of tissue sections.

As expected, bleomycin induced thickening of the alveolar sacs with inter-alveolar septa, leading to excessive collagen accumulation in the lung epilepsy.

Moreover, both CST3 and GDF15 were significantly down-regulated in the bleomycin-treated lungs compared to the control lungs (Fig. 19, * P <0.05 compared to the control). Inhibition of these cytokines by bleomycin provides the rationale for restoring cytokines for the treatment of pulmonary fibrosis.

Administration of CST3 or GDF15 peptides reduced airway space and fibrosis in the blomycin-treated lungs, and a half dose of these peptide combinations was more effective than any single administration (Fig. 20A). To quantify fibrous changes, fibronectin sites and Ashcroft scores were measured for Masson's trichrome staining samples. The Ashcroft score was assessed on the basis of the Ashcroft fibrosis scoring system for the severity (0 to 8) of interstitial fibrosis.

To quantify fibrotic changes, the hydroxyproline levels in lung tissue were biochemically analyzed (Figure 20b, 20c, 20d, * for P <0.05 versus control, # for P <0.05 versus Bleo-PBS group).

The hydroxyproline assay process is as follows. The frozen right lung was homogenized in distilled water (100 [mu] l / 10 mg tissue). The homogenate was incubated at 95 ° C. for 3 hours and centrifuged at 16,000 × g for 10 minutes. The supernatant collagen content was measured using a hydroxyproline colorimetric assay kit provided by BioVision (Milpitas, Calif.). The level of hydroxyproline was measured with a spectrophotometer based on absorbance at 560 nm.

The results of these fibrosis parameters supported the hypothesis that the combination of rCST3 and rGDF15 attenuated pulmonary fibrosis by bleomycin. Next, we evaluated the degree of fibroblast activation by analyzing α-SMA and collagen-1α levels.

Paraffin sections (4 μm) of lung tissue were deparaffinized, rehydrated, and sterilized in 100 mM citrate buffer (pH 6.0) at 121 ° C. for 10 minutes to recover the antigen. After treatment with 3% hydrogen peroxide for 10 minutes, the sections were incubated in 10% bovine serum for 1 hour at room temperature to block non-specific interactions. (1: 250 dilution; Abcam) or α-SMA (1: 500; Abcam) overnight with the biotin secondary antibody (1: 500) provided by Vector Laboratories (Burlingame, CA) Lt; / RTI &gt; Immunoconjugates were visualized using the VECTASTAIN ABC Kit (Vector laboratories). To analyze the staining area, four high power fields were randomly selected in each section using the ImageJ program (NIH, Bethesda, Md.).

Both markers were stained heavily in the bleomycin treated lungs but less evident in the peptides treated lungs (Figure 21, * P <0.05 compared to control, P = 0.05 compared to Bleo-PBS group).

Wound healing is a dynamic and complex process involving harmonious interactions between epithelial cells and fibroblasts. Fibroblasts grown after tissue regeneration are usually inactivated and reduced. However, if this process does not occur, the tissue undergoes pathologic fibrosis. The mechanism causing pulmonary fibrosis is not well understood, and it is difficult to prevent or cure diseases. However, the present invention provides a pharmaceutical composition comprising epithelial-derived cytokine capable of alleviating pulmonary fibrosis.

A variety of anti-inflammatory drugs have been tested as IPF treatments on the assumption that inflammation leads to fibrosis, but anti-inflammatory drugs have not lowered the mortality of IPF in clinical trials and caused side effects. IPF is believed to be due to overactive activation of interstitial fibroblasts and abnormal wound healing involving disruption of the alveolar epithelium. Based on this concept, a new strategy to treat IPF has focused on fibroblast-target therapy and has established an anti-fibrotic strategy to enhance fibroblast-suppressive signaling pathways. According to the homeostasis theory of the epithelium-mesenchyme, the lung epithelium has intrinsic ability to inhibit active fibroblasts after completion of wound repair. Thus, the inventors have discovered two fibroblast-inhibiting cytokines, cystatin C (CST3) and GDF-15 in normal epithelial cells, and demonstrated therapeutic efficacy in a bleomycin-induced pulmonary fibrosis mouse model.

CST3 is expressed in most mammalian cells and its cleaved form is ubiquitously detected in blood and body fluids. In the process of liver fibrosis, cathepsin, a cysteine protease, stimulates stellate cells to promote fiber formation and inhibit collagen deposition by inhibiting cathepsin B. In the pulmonary fibrosis process, cathepsin B stimulates TGF-β-induced differentiation of pulmonary fibroblasts to increase extracellular matrix deposition, and thus the action of cathepsin B is inhibited by CST3. Independently of its effect on cathepsin activity, it directly antagonizes the TGF-β pathway and interferes with fiber formation by CST3 itself. CST3 physically binds to the TGF-beta receptor and inhibits the interaction of TGF-beta with its receptor. The result that CST3 weakens the TGF signal in CCD-18Lu supports this mechanism.

GDF-15 is a member of the TGF-β family that is induced immediately after adverse stress. GDF-15 is thought to be involved in cellular responses to microenvironmental changes, but its biological function has not been clearly elucidated. Since GDF-15 overexpression induces apoptosis in colorectal, prostate, and lung cancer cells, GDF-15 has been shown to promote apoptosis in cancer cells. Since the GDF-15 gene has a p53 response element in the promoter region, it is reasonable to assume the role of GDF-15 as a pro-apoptotic gene. GDF-15 is expected to induce fibroblast death in adverse conditions such as hypoxia and inflammation. However, GDF-15 was unexpectedly found to inhibit fibroblast growth and activation through inhibition of the TGF-Smad signaling pathway (Figures 14-15). GDF-15 may act as a partial agonist for the TGF-beta receptor. Generally, a weak agonist can stimulate the receptor in the absence of a complete agonist, but acts to inactivate receptor signaling by competing with the receptor in the presence of a complete agonist. Considering the TGF-β rich environment in the IPF lung, GDF-15 may act as an inhibitor of fibrogenesis.

In conclusion, cytokine cystatin C (CST3) and GDF-15 are secreted from normal epithelial cells and inhibit the growth and activation of lung fibroblasts. In addition, these cytokines alleviate interstitial lung fibrosis in rats with intramuscular drops of bleomycin. CST3 and GDF15 appear to be genuine regulators that prevent excessive proliferation and activation of fibroblasts in damaged lung.

Statistical analysis of embodiments of the present invention was performed using SPSS (Windows version 15.0.0) software package. Comparisons between the two groups were analyzed by Student's t-test or Mann-Whitney U test, and the significance level was defined as P <0.05.

<110> SEOUL NATIONAL UNIVERSITY R & D FOUNDATION <120> Pharmaceutical composition comprising cystatine C for preventing          or treating pulmonary fibrosis <130> 17P04019 <160> 16 <170> KoPatentin 3.0 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> cystatin C (CST3) <400> 1 Ser Ser Pro Gly Lys Pro Pro Arg Leu Val Gly Gly Pro Met Asp Ala   1 5 10 15 Ser Val Glu Glu Glu Gly Val Arg Arg Ala Leu Asp Phe Ala Val Gly              20 25 30 Glu Tyr Asn Lys Ala Ser Asn Asp Met Tyr His Ser Arg Ala Leu Gln          35 40 45 Val Val Arg Ala Arg Lys Gln Ile Val Ala Gly Val Asn Tyr Phe Leu      50 55 60 Asp Val Glu Leu Gly Arg Thr Thr Cys Thr Lys Thr Gln Pro Asn Leu  65 70 75 80 Asp Cys Pro Phe His Asp Gln Pro His Leu Lys Arg Lys Ala Phe                  85 90 95 Cys Ser Phe Gln Ile Tyr Ala Val Pro Trp Gln Gly Thr Met Thr Leu             100 105 110 Ser Lys Ser Thr Cys Gln Asp Ala         115 120 <210> 2 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> GDF-15 <400> 2 Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg   1 5 10 15 Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp              20 25 30 Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys          35 40 45 Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser      50 55 60 Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro  65 70 75 80 Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val                  85 90 95 Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile             100 105 110 <210> 3 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA CST3 # 1 <400> 3 gcacaaugac cuugucgaaa uccac 25 <210> 4 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA CST3 # 2 <400> 4 ccuaucaccu cuuaugcaca ccucc 25 <210> 5 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA CST3 # 3 <400> 5 cgcccgcaag cagaucguag cuggg 25 <210> 6 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA GDF15 # 1 <400> 6 gcugggaaga uucgaacacc gacct 25 <210> 7 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA GDF15 # 2 <400> 7 agacuccaga uuccgagagu ugcgg 25 <210> 8 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA GDF15 # 3 <400> 8 cggauacuca cgccagaagu gcggc 25 <210> 9 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA IL-1alpha # 1 <400> 9 gcacaauagc aguugaaaca agaag 25 <210> 10 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA IL-1alpha # 2 <400> 10 agaggaucaa gacuucuuug ugctc 25 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> alpha-SMA forward primer <400> 11 cccttgagaa gagttacgag 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> alpha-SMA reverse primer <400> 12 tgtaggtggt ttcatggatg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> collage 1alpha forward primer <400> 13 tacaaaacca ccaagacctc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> collage 1alpha reverse primer <400> 14 agtttacagg aagcagacag 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s rRNA forward primer <400> 15 gttaattccg ataacgaacg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s rRNA reverse primer <400> 16 cacagacctg ttattgctca 20

Claims (7)

A pharmaceutical composition for preventing or treating pulmonary fibrosis comprising cystatin C (CST3) protein.
[Claim 2] The pharmaceutical composition according to claim 1, wherein the cystatin C protein comprises the amino acid sequence of SEQ ID NO: 1.
The pharmaceutical composition according to claim 1, wherein the cystatin C protein inactivates the TGF-Smad signaling pathway.
The pharmaceutical composition for preventing or treating pulmonary fibrosis according to claim 1, wherein the cystatin C protein inhibits fibroblast growth or activity.
The pharmaceutical composition according to claim 1, further comprising a GDF-15 (Growth Differentiation Factor-15) protein.
[Claim 5] The pharmaceutical composition according to claim 5, wherein the GDF-15 protein comprises the amino acid sequence of SEQ ID NO: 2.
A method of treating pulmonary fibrosis comprising administering the composition of claims 1 to 6 to a subject other than a human.

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