WO2018236194A1 - Composition for prevention or treatment of fibrosis, comprising gas6 protein or receptor activator thereof - Google Patents

Abstract

The present invention relates to a composition for prevention or treatment of fibrosis, comprising Gas6 protein or a receptor activator thereof. Gas6 protein can induce the secretion and production of PGE2, PGD2, and HGF through a receptor thereof in epithelial cells and inhibit an EMT phenomenon in an auto-secretion manner through a downstream signaling pathway thereof, thereby preventing and treating fibrosis. Thus, Gas6 protein and a receptor activator thereof has an excellent effect for use in prevention or treatment of fibrosis.

Description

Composition for preventing or treating fibrosis comprising a Gas6 protein or a receptor activator thereof

The present invention relates to a composition for preventing or treating fibrosis comprising Gas6 protein or a receptor activator thereof.

Fibrosis is a disease in which abnormal formation, accumulation, and deposition of extracellular matrix by fibroblasts occurs and is caused by fibrosis of an organ or tissue. Fibrosis is a very fatal disease that causes organ damage. For example, IPF (idiopathic pulmonary fibrosis) is a consequence of recurrent alveolar epithelial cell injury associated with fibroblast accumulation and fibroblast differentiation, and is associated with the extracellular matrix (ECM) of the lung parenchyma ), Which is a chronic, progressive, and lethal disease. However, there is no effective treatment for fibrosis until now (Fibrogenesis Tissue Repair, 2012, 5 (1): 11; N Engl J Med, 2001, 345 (7): 517) Development continues to be required.

As a result of intensive efforts to develop a composition for the prevention or treatment of fibrosis, the present inventors have found that the new role of Gas6 protein and the mechanism of action of Gas6 protein on epithelial to mesenchymal transition (EMT), migration and invasion of alveolar type II epithelial cells And confirmed the preventive and therapeutic effects of Gas6 in a bleomycin-induced pulmonary fibrosis mouse model, thereby completing the present invention.

One object of the present invention is to provide a composition for preventing or treating fibrosis, which comprises a receptor activator of Gas6 protein or Gas6 protein.

It is another object of the present invention to provide a method for preventing or treating fibrosis, which comprises administering to a subject a receptor activator of Gas6 protein or Gas6 protein.

Gas6 proteins induce secretion production of PGE _2, PGD ₂ and HGF with its receptor in the epithelial cells and through those of the sub-transmission path self-inhibiting EMT developed with a secretion system, and carries the fibrosis prevention and can be treated in accordance with Therefore, the Gas6 protein and its receptor activator have excellent effects for the prevention or treatment of fibrosis.

FIG. 1 shows the results of confirming the shape of LA-4 cells by TGF-β and Gas6 treatment using a phase contrast microscope. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-? Was treated for 48 hours or 72 hours.

FIG. 2 shows the results of confirming the expression level of EMT marker protein in LA-4 cells following treatment with TGF-β and Gas6. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-? Was treated for 48 hours or 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 3 shows the results of confirming the expression level of EMT marker mRNA in LA-4 cells according to TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-? Was treated for 48 hours or 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 4 shows the results of confirming the expression level of EMT marker protein in HEK-293 kidney epithelial cells following treatment with TGF-β and Gas6. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-beta was treated for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 5 shows the results of confirming the expression levels of Snai1 / 2, Zeb1 / 2 and Twist1 mRNA in LA-4 cells according to TGF-beta and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-? Was treated for 48 hours or 72 hours.

FIG. 6 shows the results of confirming the expression levels of Snai1 / 2, Zeb1 / 2, and Twist1 mRNA in HEK-293 cells following treatment with TGF-β and Gas6. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-beta was treated for 72 hours.

FIG. 7 shows the results of confirming phosphorylation of Smad2 or Smad3 protein in LA-4 cells following treatment with TGF-beta and Gas6. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-beta was treated for 30 minutes or 1 hour. * P <0.05 compared to control.

FIG. 8 shows the results of confirming phosphorylation of ERK1 / 2 protein in LA-4 cells following treatment with TGF-beta and Gas6. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 5 minutes, 30 minutes or 1 hour. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 9 shows the results of confirming phosphorylation of AKT protein in LA-4 cells by TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-beta was treated for 1 hour, 3 hours or 8 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 10 shows the results of confirming phosphorylation of P38 protein in LA-4 cells following treatment with TGF-β and Gas6. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 15 minutes, 1 hour, or 6 hours. * P <0.05 compared to control.

Fig. 11 shows the result of confirming the level of COX-2 or COX-1 mRNA in LA-4 cells according to Gas6 treatment. 400 ng / ml Gas6 were each treated with the indicated times. * P <0.05 compared to control.

Fig. 12 shows the result of confirming the level of COX-2 or COX-1 protein in LA-4 cells according to Gas6 treatment. 400 ng / ml Gas6 were each treated with the indicated times. * P <0.05 compared to control.

FIG. 13 shows the results of confirming the level of PGE ₂ or PGD ₂ protein in the LA-4 cell culture medium according to Gas6 treatment. 400 ng / ml Gas6 was treated for 8 hours or 20 hours. * P <0.05 compared to control.

FIG. 14 shows the results of confirming the levels of COX-2 protein in LA-4 cells following treatment with COX-2 specific siRNA. LA-4 cells were transfected with COX-2 specific siRNA or control siRNA for 6 hours. * P <0.05 compared to control.

FIG. 15 shows the results of confirming the level of PGE ₂ or PGD ₂ protein in LA-4 cell culture medium according to treatment with COX-2 specific siRNA. LA-4 cells were transfected with COX-2 specific siRNA or control siRNA for 6 hours and treated with 400 ng / ml Gas6 for 20 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values

Fig. 16 shows the results of confirming morphological changes of LA-4 cells according to NS-398 treatment. LA-4 cells were treated with 10 μM NS-398 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 72 hours.

FIG. 17 shows the results of confirming mRNA levels of EMT markers in LA-4 cells according to NS-398 treatment. LA-4 cells were treated with 10 μM NS-398 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Fig. 18 shows the results of confirming the protein level of EMT markers in LA-4 cells according to NS-398 treatment. LA-4 cells were treated with 10 μM NS-398 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 19 shows the results of confirming mRNA levels of EMT markers in LA-4 cells according to COX-2-specific siRNA treatment. LA-4 cells were transfected with COX-2 siRNA or control siRNA for 6 hours, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 20 shows the results of confirming the protein level of EMT markers in LA-4 cells according to COX-2-specific siRNA treatment. LA-4 cells were transfected with COX-2 siRNA or control siRNA for 6 hours, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 21 shows the results of confirming mRNA levels of Snai1, Zeb1 and Twist1 in LA-4 cells according to NS-398 treatment. LA-4 cells were treated with 10 μM NS-398 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 22 shows the results of confirming mRNA levels of Snai1, Zeb1 and Twist1 in LA-4 cells following treatment with COX-2 siRNA. LA-4 cells were treated with COX-2 siRNA or control siRNA for 6 hours, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 23 shows the results of confirming the phosphorylation level of ERK1 / 2 protein in LA-4 cells according to COX-2 siRNA treatment. LA-4 cells were treated with COX-2 siRNA or control siRNA for 6 hours, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 5 minutes. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

24 shows the results of confirming the phosphorylation level of AKT protein in LA-4 cells according to COX-2 siRNA treatment. LA-4 cells were treated with COX-2 siRNA or control siRNA for 6 hours, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 8 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

25 shows the result of confirming morphological changes of LA-4 cells according to receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and 10 mM of each receptor antagonist EP2 (AH-6809), EP4 (AH-23848), DP1 (BW-A868C) or DP2 (BAY-u3405) The morphological changes of TGF-β were observed at 48 hours or 72 hours after administration or no administration.

26 shows the results of confirming mRNA levels of EMT markers in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-β for 48 hours in the absence or administration of 10 mM of each receptor antagonist EP2 (AH-6809) or EP4 (AH-23848) Respectively. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 27 shows the results of confirming mRNA levels of EMT markers in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-beta for 48 hours in the presence or absence of 10 mM DP2 antagonist (BAY-u3405). * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

28 shows the results of confirming the level of mRNA of EMT markers in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-beta for 72 hours in the presence or absence of 10 mM DP1 antagonist (BW-A868C). * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 29 shows the results of confirming the protein level of EMT marker in LA-4 cells according to receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-β for 48 hours in the absence or administration of 10 mM of each receptor antagonist EP2 (AH-6809) or EP4 (AH-23848) Respectively. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 30 shows the results of confirming the protein level of EMT markers in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-β1 for 48 hours in the presence or absence of 10 mM DP2 antagonist (BAY-u3405). * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

31 shows the results of confirming the protein level of EMT markers in LA-4 cells according to receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-beta for 72 hours in the presence or absence of 10 mM DP1 antagonist (BW-A868C). * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 32 shows the results of confirming the levels of Snai1, Zeb1 and Twist1 mRNA in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with 10 mM of each receptor antagonist EP2 (AH-6809), EP4 (AH-23848) or DP2 (BAY-u3405) TGF-beta was treated for 48 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 33 shows the results of confirming the levels of Snai1, Zeb1 and Twist1 mRNA in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-beta for 72 hours in the presence or absence of 10 mM DP1 antagonist (BW-A868C). * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 34 shows the results of confirming the levels of HGF mRNA in LA-4 cells following Gas6 treatment. LA-4 cells were treated with 400 ng / ml Gas6 for the indicated time. * P <0.05 compared to control.

FIG. 35 shows the result of confirming the HGF protein level in the LA-4 cell culture medium according to the Gas6 treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 8 or 20 hours. * P <0.05 compared to control.

Fig. 36 shows the result of confirming the level of HGF protein in LA-4 cells according to Gas6 treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 8 or 20 hours. * P <0.05 compared to control.

FIG. 37 shows the results of confirming the level of RhoA protein in LA-4 cells according to RhoA-specific siRNA treatment. LA-4 cells were treated with RhoA-specific siRNA or control siRNA for 24 hours. * P <0.05 compared to control.

FIG. 38 shows the results of confirming the level of EMT marker mRNA in LA-4 cells according to RhoA-specific siRNA treatment. LA-4 cells were treated with RhoA-specific siRNA or control siRNA for 24 hours, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 48 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 39 shows the results of confirming the level of EMT marker protein in LA-4 cells according to RhoA-specific siRNA treatment. LA-4 cells were treated with RhoA-specific siRNA or control siRNA for 24 hours, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 48 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

40 shows the results of confirming morphological changes of LA-4 cells according to Y-27632 treatment. LA-4 cells were treated with 10 mM Y-27632 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 48 hours. The following morphological changes were observed.

41 shows the results of confirming the level of EMT marker mRNA in LA-4 cells according to Y-27632 treatment. LA-4 cells were treated with 10 mM Y-27632 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 48 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 42 shows the results of confirming the level of EMT marker protein in LA-4 cells according to Y-27632 treatment. LA-4 cells were treated with 10 mM Y-27632 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 48 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 43 shows the results of confirming the levels of Snai1, Zeb1 and Twist1 mRNA in LA-4 cells according to RhoA-specific siRNA treatment. LA-4 cells were transfected with RhoA siRNA or control siRNA for 24 hours, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 44 shows the results of confirming the levels of Snai1, Zeb1 and Twist1 mRNA in LA-4 cells according to Y-27632 treatment. LA-4 cells were treated with 10 mM Y-27632 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 48 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 45 shows the results of confirming the phosphorylation level of ERK1 / 2 protein in LA-4 cells according to RhoA-specific siRNA treatment. LA-4 cells were transfected with RhoA siRNA or control siRNA for 24 hours, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 5 minutes. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 46 shows the results of confirming the phosphorylation level of AKT protein in LA-4 cells according to RhoA-specific siRNA treatment. LA-4 cells were transfected with RhoA siRNA or control siRNA for 24 hours, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 8 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 47 shows the results of confirming the morphology of LA-4 cells according to c-Met antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-β for 72 hours in the presence or absence of 250 nM of c-Met antagonist PHA-665752.

48 shows the result of confirming the mRNA level of EMT marker in LA-4 cells according to c-Met antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-β for 72 hours in the presence or absence of 250 nM of c-Met antagonist PHA-665752. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

49 shows the results of confirming the protein level of the EMT marker in LA-4 cells according to the c-Met antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-β for 72 hours in the presence or absence of 250 nM of c-Met antagonist PHA-665752. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 50 shows the results of confirming mRNA levels of Snai1, Zeb1 and Twist1 in LA-4 cells following c-Met antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-β for 72 hours in the presence or absence of 250 nM of c-Met antagonist PHA-665752. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 51 shows the results of confirming the protein levels of EMT markers in LA-4 cells following PGE ₂ , PGD ₂ or HGF treatment. LA-4 cells were treated with PGE ₂ (35 or 118 pg / ml), PGD ₂ (6 or 28 pg / ml) or HGF (169 or 194 pg / ml) with 10 ng / ml TGF- . * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 52 shows the results of confirming the protein level of EMT markers in LA-4 cells following HGF treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 h and then replaced with fresh medium. HGF (169 or 194 pg / ml) was treated with 10 ng / ml TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 53 shows the results of confirming c-Met, EP2, EP4, DP1 or DP2 receptor protein levels in LA-4 cells following Gas6 treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 12 or 20 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 54 shows the results of confirming the level of Axl or Mer expression in LA-4 cells following Gas6 treatment. 400 ng / ml Gas6 was treated for 0, 5, 15, 30, 60 and 120 minutes. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 55 shows the results of confirming Axl or Mer protein expression levels in LA-4 cells following Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours and treated with 400 ng / ml Gas6. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 56 shows the results of confirming the levels of COX-2 mRNA, PGE ₂ and PGD ₂ expression in LA-4 cells following Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours and treated with 400 ng / ml Gas6. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 57 shows the results of confirming RhoA activity, HGF mRNA and protein expression level in LA-4 cells according to Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours and treated with 400 ng / ml Gas6. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 58 shows the results of confirming the mRNA level of EMT marker in LA-4 cells according to Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours, treated with 400 ng / ml Gas6 for 20 hours, and treated with TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 59 shows the results of confirming the level of protein expression of EMT markers in LA-4 cells following Axl or Mer-specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours, treated with 400 ng / ml Gas6 for 20 hours, and treated with TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 60 shows mRNA levels of Snai1, Zeb1 and Twist1 in LA-4 cells following Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours, treated with 400 ng / ml Gas6 for 20 hours, and treated with TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 61 shows the results of confirming the phosphorylation of ERK1 / 2 protein in LA-4 cells following Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours, treated with 400 ng / ml Gas6 for 20 hours, and treated with TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Figure 62 shows the results of confirming phosphorylation of AKT protein in LA-4 cells following treatment with AxI or Mer specific siRNA. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours, treated with 400 ng / ml Gas6 for 20 hours, and treated with TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

63 shows the results of confirming the expression level of EMT marker mRNA in the mouse AT II cell according to Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, TGF-beta was treated for 48 or 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

Fig. 64 shows the result of confirming the expression level of EMT marker protein in the mouse AT II cell according to Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, TGF-beta was treated for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 65 shows the results of confirming the expression levels of Snai1 / 2, Zeb1 / 2 and Twist1 mRNA in the mouse AT II cells according to TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-? Was treated for 48 hours or 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 66 shows the results of confirming the levels of COX-2 mRNA in premature mouse AT II cells following treatment with Gas6. 400 ng / ml Gas6 were each treated with the indicated times. * P <0.05 compared to control.

67 shows the results of confirming the expression levels of EMT marker mRNA in primary mouse AT II cells following treatment with NS-398 and Gas6. 10 μM NS-398 was treated for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and treated with 10 ng / ml of TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

68 shows the results of confirming the expression levels of Snai1 / 2, Zeb1 / 2 and Twist1 mRNA in the mouse AT II cells according to NS-398 and Gas6 treatment. 10 μM NS-398 was treated for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and treated with 10 ng / ml of TGF-β for 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

69 shows the results of confirming the expression level of EMT marker mRNA in the early mouse AT II cells according to the treatment with AH6, BAY and Gas6. After treatment with 400 ng / ml Gas6 for 20 hours, treatment with TGF-β alone, treatment with 10 μM of EP2 (AH-6809) or treatment with 10 μM of DP2 (BAY-u3405) were performed for 48 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 70 shows the results of confirming the expression levels of Snai1 / 2, Zeb1 / 2 and Twist1 mRNA in the mouse AT II cells according to treatment with AH6, BAY and Gas6. After treatment with 400 ng / ml Gas6 for 20 hours, treatment with TGF-β alone, treatment with 10 μM of EP2 (AH-6809) or treatment with 10 μM of DP2 (BAY-u3405) were performed for 48 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

71 shows the result of confirming the level of HGF mRNA in the mouse AT II cell according to Gas6 treatment. LA-4 cells were treated with 400 ng / ml Gas6 for the indicated time. * P <0.05 compared to control.

FIG. 72 shows the results of confirming the level of mature AT II cell EMT marker mRNA in the wild-type mouse according to Y-27632 treatment. LA-4 cells were treated with 10 mM Y-27632 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 48 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

73 shows the results of confirming the levels of Snai1, Zeb1, and Twist1 mRNA of the premature mouse AT II cells according to Y-27632 treatment. LA-4 cells were treated with 10 mM Y-27632 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF-β for 48 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

74 shows the results of confirming mRNA levels of EMT markers in primary mouse AT II cells following c-Met antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-β for 72 hours in the presence or absence of 250 nM of c-Met antagonist PHA-665752. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 75 shows the results of confirming the levels of Snai1, Zeb1 and Twist1 mRNA in the mouse AT II cells according to the c-Met antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF-β for 72 hours in the presence or absence of 250 nM of c-Met antagonist PHA-665752. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

76 is a graph showing the effect of TGF-beta and Gas6 on human lung adenocarcinoma cells (549 cells) And the expression level of the EMT marker mRNA was confirmed. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-? Was treated for 48 hours or 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 77 shows the results of confirming the level of EMT marker protein expression in human lung adenocarcinoma cells (549 cells) following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-? Was treated for 48 hours or 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

78 shows the results of confirming the levels of Snai1 / 2, Zeb1 / 2 and Twist1 mRNA in human lung adenocarcinoma cells (549 cells) following TGF-beta and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-? Was treated for 48 hours or 72 hours. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 79 shows the results of in vivo blomycin and Gas6 treatment, in which the shape of primary mouse AT II cells was confirmed by a phase contrast microscope.

80 shows the results of confirming the expression level of EMT marker mRNA in the first mouse AT II cells in vivo by treatment with bleomycin and Gas6. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

81 shows the results of confirming the expression levels of Snai1, Zeb1, and Twist1 mRNA of invasive mouse AT II cells in vivo by treatment with bleomycin and Gas6. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 82 shows the results of confirming COX-2 mRNA levels in the mouse AT II cells according to in vivo bleomycin and Gas6 treatment. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

83 shows the results of confirming the level of PGE ₂ or PGD ₂ protein in primary mouse AT II cells in vivo by treatment with bleomycin and Gas6. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

84 shows the results of confirming the level of HGF mRNA in primary mouse AT II cells in vivo by treatment with bleomycin and Gas6. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

85 shows the results of confirming levels of HGF and TGF-beta protein in primary mouse AT II cells in vivo by treatment with bleomycin and Gas6. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

86 shows the results of confirming the expression levels of EMT markers and extracellular matrix proteins in the lung tissue at day 14 following treatment with in vivo bleomycin and Gas6. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

FIG. 87 shows the results of confirming the expression levels of EMT markers and extracellular matrix proteins in the lung tissue at day 21 following in vivo treatment with bromomycin and Gas6. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

88 shows the results of confirming the level of hydroxyproline in the lung tissue at day 21 following treatment with in vivo bleomycin and Gas6. * P < 0.05, compared to control group; + P < 0.05, comparison of displayed values.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.

One aspect of the present invention for achieving the above object is a pharmaceutical composition for preventing or treating fibrosis, which comprises Gas6 (Growth arrest-specific 6) protein or Gas6 protein receptor activator as an active ingredient.

The term " fibrosis " in the present invention means that excessive fibrous connective tissue is formed in an organ or tissue. This can be distinguished from fibrous tissue as a normal component in the organ or tissue. Because of the excessive accumulation of extracellular metrix such as fibronectin and collagen by fibroblast, fibrosis can be understood as a fatal disease that eventually leads to organ damage.

The term " epithelial to mesenchymal transition " (EMT) in the present invention refers to a phenomenon in which epithelial cells are transformed into mesenchymal cells, and is associated with embryonic development, organ development, wound healing and stem cell behavior, (J Clin Invest, 2009, 119 (6): 1420). Fibroblasts are classified into (1) proliferation and differentiation of resident lung fibroblasts, (2) EMTs converted from (ave) epithelial cells to (my) fibroblasts, and (3) (Curr Rheumatol Rep, 2006, 8: 145). The results reported so far suggest that EMT is a major event in the development of fibrosis such as IPF (Am J Pathol, 2009, 175: 3; J Exp Med, 2011,208: 1339). It is therefore obvious to those skilled in the art that EMT inhibition can have preventive and therapeutic effects on fibrosis.

In the present invention, the fibrotic syndrome may be selected from the group consisting of lung, kidney, liver, heart, brain, blood vessels, joints, bowel, skin, soft tissues, bone marrow, penis, peritoneum, lance, muscle, spine, testis, ovary, breast, thyroid, Gallbladder, bladder, or prostate. &Lt; / RTI &gt; Specifically, in the present invention, fibrosis is a disease caused by fibrosis occurring in each tissue of the body, such as abnormal wound healing, alcohol-induced hepatic injury induced fibrosis, connective fibrosis, Crohn's disease (intestinal fibrosis), pancreatic and lung cystic Fibrosis, fibrosis caused by Graft-Versus-Host Disease (GVHD), fibrosis of the spleen, fibrosis of the spleen and retinal fibrosis, which may occur as complications of intermuscular injection, especially in children, endocardial myocardial fibrosis or cardiac fibrosis Fibrosis complications of surgery or injection fibrosis, glomerulonephritis, epileptic fibrosis, keloids and hypertrophic scarring (skin fibrosis), macular degeneration, mediastinal fibrosis (soft tissue fibrosis of the mediastinum), morphea, Fibrosis, bone marrow fibrosis, renal systemic fibrosis (skin fibrosis), nodular epidermal fibrosis (e. G., Benign fibrous tissue &lt; Proliferative fibrosis, pipestem fibrosis, postfiber fibrosis, progressive bundle fibrosis (pulmonary fibrosis type, complications of coal worker's pneumoconiosis), pleural fibrosis, fibrosis as a result of surgery (for example, (Fibrosis of the retroperitoneal soft tissue), post-surgical scarring, post-operative fibrosis, fibromyalgia, fibromyalgia, chronic fibrosis, chronic myelogenous leukemia, Scleroderma / systemic sclerosis (skin fibrosis), epithelial cells, uterine fibrosis, or viral hepatitis induced fibrosis.

In the present invention, pulmonary fibrosis is classified as idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, acute interstitial pneumonia, cryptogenic organizing pneumonia, respiratory bronchitis-related epilepsy But not limited to, Respiratory Bronchiolitisassociated Interstitial Lung, Desquamative Interstitial Pneumonia, Lymphoid Interstitial Pneumonia, Interstitial Pulmonary Fibrosis, and Diffuse Pulmonary Fibrosis. It is not.

As used herein, the term " treatment " means any action that improves or alleviates the symptoms of fibrosis with the administration of the pharmaceutical composition, and " prevention " means inhibiting the onset of fibrosis by administration of the pharmaceutical composition Or delaying any action that

In the present invention, it has been confirmed for the first time that the activator of Gas6 protein or Gas6 protein receptor can inhibit the EMT phenomenon, thereby confirming that the activator of Gas6 protein or Gas6 protein receptor can be used for prevention and treatment of fibrosis.

The term " Growth arrest-specific protein 6 (Gas6) " in the present invention refers to a secretable vitamin K-dependent protein, also called AXSF or AXLLG. In the present invention, the Gas6 protein may have, for example, an amino acid sequence of the NCBI accession number (NP_062394.2) (SEQ ID NO: 35), but it is not limited thereto, and may have homology with known Gas6 protein, Deletion, addition or substitution of a part of the sequences as long as they have the same activity or Gas6 proteins derived from human or other animals can all be included in the scope of the present invention. In the present invention, the sequence homology may be 70% or more, 80% or more, 90% or more, 95% or more, 98% or more or 99% or more of the sequence of the wild type Gas6 protein. In addition, fragments obtained by cleaving some domains for activity in the wild-type Gas6 protein, and fragments having the same activity as the wild-type Gas6 protein in the form of a fusion protein in which known peptides are bound to increase the expression, isolation, purification, May be included within the Gas6 protein category of the present invention. In the present invention, the Gas6 protein can be produced by a method known in the art, for example, through peptide synthesis or production of a protein using transformed cells, but is not limited thereto. In an exemplary embodiment of the present invention, the murine Gas6 protein of SEQ ID NO: 35 was synthesized and used (R & D Systems; Minneapolis, MN, USA).

Gas6 contains a large C-terminal region that is homologous to the N-terminal gamma-carboxyglutamic acid (Gla) domain, four epidermal growth factor (EGF) -like domains, and sex hormone binding globulin (SHBG) Biol, 1993, 13: 4976; Blood Cells Mol Dis, 2006, 36: 373). Gas6 is expressed in the lung, heart, kidney, intestine, fibroblasts, endothelial cells, bone marrow cells, vascular smooth muscle, white blood cells and neurons and is known as a common ligand of the TAM (Tyro3 / Axl / Mer) 1995, 82: 355; Nature Reviews Immunology, 2008, 8: 327). Thus, the Gas6 protein receptor in the present invention includes, but is not limited to, AXL receptor tyrosine kinase, Mer tyrosine kinase, or TYRO3. These receptors share considerable domain similarity, including both extracellular N-terminal immunoglobulin-like domains and two fibronectin-III-like domains and the tyrosine kinase domain located at the following C-terminal cytoplasmic end of the receptor . TAM activity by Gas6 has been reported to induce signals that mediate cell survival, proliferation, predation, differentiation, platelet function and thrombolytic stabilization. Recent studies have also shown that Gas6 signaling in macrophages and dendritic cells regulates the immune response by regulating TLR-induced cytokine secretion. Furthermore, recently, we have found that macrophages are reprogrammed by Gas6 to play a role in the paracrine of HGF induced by Mer / RhoA / Akt / MAP kinase signaling pathway in macrophages, leading to epithelial cell proliferation and wound repair . However, the direct role of Gas6 on epithelial cell homeostasis remains unknown.

Activators of Gas6 protein receptor in the present invention is to enable the Gas6 protein receptor means a substance capable of activating the sub-transmission path, particularly in the purposes of PEG2, PGD ₂ or HGF of the present invention to produce, secrete Quot; refers to the active material for the signal transduction pathway. For example, the activator of the Gas6 protein receptor may be, but is not limited to, Protein S (Pros1), Tubby and tubby-like protein 1, or Galectin3.

In a specific example of the present invention, Gas6-inhibiting effect of EMT induced by TGF-beta in alveolar and renal epithelial cells was directly confirmed through changes in cell morphology and EMT marker mRNA and protein levels 4). In another embodiment this anti-EMT activity of Gas6 was shown to be blocked by blocking Smad-independent TGF-ss signals including ERK and Akt pathways (Figures 5 to 10). In addition, it was confirmed that PGE ₂ , PGD ₂ and HGF secretion were induced by treatment with Gas6, and showed an anti-EMT effect mainly through EP2, DP2 and c-Met receptors (FIGS. 11 to 53). Gas6 acts on Axl or Mer receptor tyrosine kinase distributed in the cell membrane to block the TGF-β signal through COX-2-derived PGE ₂ , PGD ₂ secretion and RhoA-dependent HGF secretion, thus confirming anti-EMT effect (Figs. 5 to 62). In particular, mouse invitation in alveolar epithelial cells was confirmed the effect of the anti--EMT natdeut receive from mouse alveolar epithelial cells, PGE _2, PGD ₂ and HGF-dependent signal Gas6 (Fig. 63 to Fig. 75). Furthermore, the anti-EMT effect was similarly observed when human Gas6 recombinant protein was administered to human lung carcinoma cells (A549 cells) (Figs. 76 to 78). In another embodiment, lung fibrosis was improved by administration of Gas6 in an animal model in which pulmonary fibrosis was induced (Figs. 79 to 88).

Thus compositions of the invention produce PEG2, PGD ₂ or HGF in the cell, and secretion can be suppressed EMT (epithelial to mesenchymal transition) by autocrine manner, because of the excellent effects in the prevention and treatment of fibrosis, the fibrosis Can be very useful for prevention or treatment.

In addition, the pharmaceutical composition of the present invention may further comprise an appropriate carrier, excipient or diluent conventionally used in the production of a pharmaceutical composition. Compositions comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration may include tablet 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 may include sterile 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 ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may also be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, nonaqueous solutions, suspensions, emulsions, A pharmaceutical preparation and a suppository.

The composition may be administered in a dose of 100 占 퐂 / kg to 500 占 퐂 / kg, but is not limited thereto. The composition of the present invention can be administered at a different time or at the same time as the already known fibrosis treatment agent or can be applied together with known fibrosis treatment methods already known. The composition may also be administered singly or multiply. 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 side effects, which can be easily determined by a person skilled in the art.

The term " administration " as used herein refers to the introduction of a pharmaceutical composition of the present invention to a subject by any suitable method, and the administration route can be administered through various routes of oral or parenteral administration as long as it can reach the target tissue .

The pharmaceutical composition may be appropriately administered to a subject according to the purpose or necessity, depending on the conventional method, route of administration and dosage used in the art. Examples of routes of administration include oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal routes, and parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. The appropriate dosage and the frequency of administration may be selected according to methods known in the art, and the amount and the frequency of administration of the pharmaceutical composition of the present invention to be actually administered depends on the type of symptom to be treated, route of administration, sex, , Diet, age and weight of the individual, and the severity of the disease.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to inhibit or alleviate an increase in vascular permeability at a reasonable benefit / risk ratio applicable to medical use, and effective dosage levels will vary depending on the species and severity, Sex, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts.

In the present invention, the term " individual " means all animals including humans, which have the fibrosis disease or the disease of the present invention. By administering the pharmaceutical composition of the present invention to an individual, fibrosis can be prevented or treated.

Another aspect of the present invention is a method of preventing or treating fibrosis, comprising administering to a subject a receptor activator of Gas6 protein or Gas6 protein.

The use of Gas6 protein, its receptor activator, for the prevention or treatment of fibrosis has been described above.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example 1: Cell line and culture

LA-4 and HEK-293 cells were purchased from ATCC. LA-4 cells were cultured in F12K medium (Lonza, Switzerland) containing 15% FBS (fetal bovine serum) inactivated by heat treatment at 37 ° C and 5% CO ₂ . HEK-293 cells 37 ℃ 5% 10% in CO ₂ conditions, FBS, 2 mM L- glutamine, 100 U / ml penicillin and 100 mg / ml streptomycin, DMEM containing (Dulbecco's modified Eagle's medium; US Media Tech, Inc.) Lt; / RTI &gt;

Example 2: Isolation and culture of early cells

The primary mouse AT II (alveolar type II) epithelial cells were isolated and purified from BALB / c mice.

Pulmonary artery was infused with 0.9% saline to remove pulmonary blood. After washing the lungs with 1 ml of saline, 100 units of dispase were injected into the mouse lungs and then incubated at room temperature for 45 minutes. Then, the lungs were separated from the large bronchus by mechanical means, and the separated lung tissues were cultured in DMEM medium containing 0.01% of DNase I at 37 ° C for 10 minutes. The cells were filtered, centrifuged and resuspended in a cell culture Petri dish coated with mouse IgG 0.75 mg / ml for 1 hour at 37 ° C to remove macrophages and fibroblasts, respectively, by sequential plating. The final cell isolates _{15 mM HEPES, 0.8 mM CaCl 2} , 0.25% BSA, 5 mg / ml insulin, 5 mg / ml transferrin, Ham 's F12 medium containing 5 ng / ml sodium selenite and 2% mouse serum Lt; RTI ID = 0.0 &gt; 35-mm &lt; / RTI &gt;

The purity of AT II cells evaluated by pro-SP-C immunofluorescence staining was over 90%.

Example 3: Incubation of epithelial cells

LA-4 and HEK-293 cells were inoculated (2 x 10 ⁵ cells / well) in 6-well culture dishes and incubated overnight in 200 μl of RPMI 1640 or DMEM containing 10% FBS. The primary AT II cells were inoculated into type 1 collagen-coated culture dishes (1 x 10 ⁶ cells / well) and cultured for 48 hours. Cells were treated with 400 ng / ml Gas6 for 20 hours in the presence or absence of 10 ng / ml TGF-beta (R & D Systems Inc). In some experiments, 10 μM NS-398 was used for COX-2 inhibition and 30 mM Y-27632 was used for Rho kinase inhibition. Each inhibitor was added 1 hour before the treatment of Gas6. AH-6809, AH-23848, BW-A868C, BAY-u3405 10 mM or PHA-665752 250 nM were used as antagonists for EP2, EP4, DP1, DP2 or c-Met, respectively. Each receptor antagonist was added 1 hour before TGF-beta treatment.

Example 4: Transient transfection &lt; RTI ID = 0.0 &gt;

1 μg / ml of COX-2 or RhoA-specific siRNA or control siRNA was transiently transfected into LA-4 cells using 5 μl siRNA transfection reagent (Genlantis). 75 nM of Axl or Mer specific siRNA was used. The siRNA sequences used are shown in Table 1.

[Table 1]

Before the experiment, cells were cultured in serum-free medium containing COX-2 siRNA for 6 hours, in serum-free medium containing RhoA siRNA for 24 hours, or in serum-free medium containing Axl or Mer siRNA for 48 hours. No significant effect on cell survival was observed for all siRNA used.

Example 5: Immunoblot analysis

To confirm epithelial marker and hepatic lobe marker expression, the cells were lysed in lysis buffer containing 0.5% Triton X-100 and loaded onto 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The membrane was blocked with TBS (Tris-buffered saline) containing 3% BSA or 5% skim milk at room temperature and then incubated with each anti-mouse primary antibody at room temperature to attach anti-mouse HRP-conjugated secondary antibody . Protein bands were identified using enhanced chemiluminescence.

Example 6 Measurement of Enzyme-Linked Immunosorbent Assay (ELISA)

Cell culture supernatants were collected and subjected to ELISA. The levels of PGE ₂ PGD ₂ , HGF and TGF-β in active form were measured by ELISA kit (R & D Systems, Minneapolis, MN).

Example 7: qPCR (Quantitative real-time PCR)

Gene expression was analyzed by real-time qPCR in a StepOnePlus system (Applied Biosystems, Life Technologies, Carlsbad, Calif., USA). For each qPCR assay, a total of 50 ng of cDNA was used. Primer Express software was used to design primer sets for PCR-based amplification. The primers used in the present invention are shown in Table 2.

[Table 2]

cDNA was normalized to the amount of HPRT (hypoxanthine-guanine phosphoribosyltransferase) and expressed as fold-change in the control.

Example 8: RhoA activity assay

RhoA activity was measured in LA-4 cell lysate using ELISA-based RhoA activation assay Biochem Kit (G-LISA; Cytoskeleton). Cell lysates were added to RhoA-GTP affinity plates coated with Rhotekin binding domain of RhoA for 30 min. The active GTP-binding form of RhoA was measured using indirect immunoassay and the chromaticity response was measured at 490 nm with a microplate spectrophotometer.

Example 9: Measurement of hydroxyproline

The content of hydroxyproline was measured using a hydroxyproline assay kit (Nanjing Jiancheng Bioengineering Co., Ltd.) according to the manufacturer's instructions.

Example 10: Animal experiment

Male C57BL / 6 mice (Orient Bio, Seongnam, Korea) were used for all experiments with 20-25 g of specific pathogen. A test solution containing bleomycin (30 μl of 5 U / kg body weight) was administered by aspiration of the mouse pharynx. Saline alone or Gas6 (50 μg / kg, i.p.) was administered one day before the administration of bleomycin. The inhibitors after the first bleomycin dosing were administered once a day and the mice were euthanized on days 14 and 21 of bleomycin administration.

Example 11: Statistical analysis

Mean values were expressed as ± SEM. Analysis of variance (ANOVA) was applied for various analyzes and Tukey's post hoc test was applied. Student's t-tests were used to compare the sample mean of the two samples. A p value of 0.05 or less was considered statistically significant. All data were analyzed using JMP software (SAS Institute, Cary, NC).

Experimental Example 1: Inhibitory effect of Gas6 on TGF-beta induced EMT in lung and kidney epithelial cells

TGF-β signaling has been shown to play an important role in EMT and fibrosis development (Am J Physiol Lung Cell Mol Physiol, 2007, 293: L525). We sought to determine if Gas6 treatment modulates the TGF-beta induced EMT process in mouse alveolar type 2-like lung lung epithelial cells (LA-4).

First, it was confirmed that LA-4 cells were converted into spindle-like morphology when stimulated with TGF-β at 10 ng / ml for 48 hours or 72 hours. However, when TGF-β was stimulated with 400 ng / ml of Gas6 inhibited spindle-like morphological transformation of LA-4 cells and showed morphology of epithelial cells (Fig. 1). In addition, when stimulated with TGF-β at 10 ng / ml for 48 or 72 hours, expression of E-cadherin, an adherens junction protein, was decreased and N-cadherin and α-SMA (α- Smooth muscle actin expression was increased, but the expression of EMT markers was suppressed at the level of protein and mRNA when treated with 400 ng / ml of Gas6 together with TGF-β (FIGS. 2 and 3). This data suggests that synthetic and secreted mediators after Gas6 treatment can block TGF-β signaling in an autocrine manner in LA-4 cells. In addition, the present HEK-293 human kidney epithelial cell (FIG. 4) also confirmed the inhibition of TGF-β induced EMT marker change by Gas6 treatment at the protein level.

Experimental Example 2: Effect of Gas6 treatment on TGF-β-induced Smad-independent signal and EMT regulatory transcription factor expression in LA-4 cells

In EMT induction, the activated Smad or non-Smad signal mediates transcriptional regulation of the Snai, Zeb and Basic helix-loop-helix transcription factor families, inhibiting the expression of epithelial marker genes, (Sci Signal, 2014, 7: re8). Thus, the present inventors sought to determine whether Gas6 inhibits the expression of the transcription factor in LA-4 cells stimulated with TGF- ?.

As a result of treatment of LA-4 cells or HEK 293 cells with TGF-β after 20 hours of treatment with Gas6, it was confirmed that mRNA expression of TGF-β-induced Snai1 / 2, Zeb1 / 2 and Twist1 was all suppressed (Figures 5 and 6, respectively). Gas6 treatment in LA-4 cells did not affect the phosphorylation of Smad2 and Smad3 mediated by TGF-β (FIG. 7), but the TGF-β induced extracellular signal-regulated kinase (ERK) (Figs. 8 and 9). There was no effect on the phosphorylation of p38 MAP (mitogen-activated protein) kinase (Fig. 10). The data show that the bioactive agents synthesized or secreted following Gas6 treatment substantially block Smad-independent TGF-β signals including ERK and Akt pathways, thereby preventing the EMG process in LA-4 cells .

Experimental Example 3: Confirmation of COX-2-derived PGE ₂ and PGD ₂ secretion and their EMT inhibitory effects by Gas6 treatment in LA-4 cells

The COX-2 / PGE ₂ and PGD ₂ pathways are known to inhibit EMT in lung and kidney epithelial cells (Sci Rep, 2016, 6: 20992). Therefore, the inventors sought to determine whether COX-2-derived PGE ₂ and PGD ₂ secreted from LA-4 in response to Gas6 mediate the anti-EMT effect in LA-4 cells by autocrine secretion.

First, the present inventors confirmed mRNA and protein expression of COX-1 and COX-2 by Gas6 and changes in production of PGE ₂ and PGD ₂ in LA-4 cells. As a result, the level of COX-2 mRNA peaked at 1 hour after treatment with Gas6, returned to its original level at 20 hours, and the expression of COX-1 mRNA was not changed within 24 hours after treatment with Gas6 (FIG. COX-2 protein expression gradually increased until 24 hours after Gas6 treatment, but COX-1 expression did not change during this period (FIG. 12). In addition, PGE ₂ and PGD ₂ secretion measured by EIA in LA-4 cells increased 3.3 and 2.5-fold at 20 hours after Gas6 treatment (Fig. 13).

Next, LA-4 cells were transfected with a COX-2 specific siRNA or a negative-control siRNA in order to confirm that COX-2 induction by Gas6 stimulation enhanced PGE ₂ and PGD ₂ production in LA-4 cells And cultured for 6 hours. As a result, the negative control siRNA did not affect the amount of intracellular COX-2 protein but the amount of COX-2 protein was ~ 60% or more higher than that of wild-type LA-4 cells in the cells transfected with COX-2 specific siRNA (Fig. 14). 15 inhibited Gas6-induced PGE ₂ and PGD ₂ secretion when COX-2 siRNA was injected (FIG. 15), suggesting that increased production of Gas6-induced PGE ₂ and PGD ₂ in LA-4 cells resulted in COX-2 expression Induction &lt; / RTI &gt;

In order to further confirm the functional relevance of the COX-2 signal in the anti-EMT effect of Gas6, LA-4 cells were treated with the highly selective COX-2 inhibitor NS-398 for 1 hour before treatment with Gas6. As a result, NS-398 reversed both the cell morphology changes induced by TGF-β and E-cadherin loss at gene and protein levels and the effect of Gas6 on the synthesis of α-SMA and N-cadherin (FIGS. 18). In addition, knockdown of the COX-2 gene in LA-4 cells also reversed the effect of Gas6 (FIGS. 19 and 20). Furthermore, both NS-398 and COX-2 siRNA reversed the effect of Gas6 on reducing TGF-β-induced Snai1, Zeb1 and Twist1 mRNA expression (FIGS. 21 and 22). In addition, COX-2 siRNA reversed the effect of Gas6, which reduces phosphorylation of ERK1 / 2 and AKT induced by TGF-beta in LA-4 cells, but this result was not observed in negative control siRNA (Fig. 23 and Fig. 24 ).

Next, in order to confirm whether PGE ₂ and PGD ₂ secretion derived from COX-2 mediate the anti-EMT effect in LA-4 cells, LA-4 cells were pretreated with TGF- EP2 PGE _2, such as (E-prostanoid-2 receptor) antagonists (AH-6809), EP4 antagonists (AH-23848), DP1 antagonist (BW-A868C) or DP2 antagonists (BAY-u3405) - or PGD ₂ - specific Receptor antagonist. As a result, the anti-EMT effect of Gas6 was significantly reversed by antagonists of EP2 and DP2 (Figs. 25-27, 29, 30 and 32). However, EP4 antagonist and DP1 antagonist had little or no effect on the anti-EMT effect of Gas6 (Figs. 25, 26, 28, 29, 31 and 33). These results suggest that the anti-EMT effect in LA-4 cells is mediated primarily by COX-2-derived PGE ₂ and PGD ₂ signals through EP2 and DP2 receptors, respectively.

Experimental Example 4: Confirmation of RhoA pathway-dependent HGF secretion and its EMT inhibitory effect by Gas6 treatment in LA-4 cells

We have previously reported that Gas6 induces mRNA and protein production of HGF in macrophages via the RhoA-dependent pathway (J Pharmacol Exp Ther, 2014, 350: 563). HGF mediates EMT induced by TGF-? In LA-4 cells (Sci. Rep., 2016, 6: 20992). Therefore, the inventors sought to determine the role of RhoA-dependent HGF secretion in LA-4 cells in the anti-EMT effect induced by Gas6.

As a result of treatment of LA-4 cells with Gas6, Gas6-induced HGF mRNA expression showed the highest level at 3 hours and then decreased to half level at 6 hours (Fig. 34). HGF secretion of LA-4 cells as measured by ELISA increased somewhat due to treatment with Gas6 as compared to the basal secretion level (Fig. 35). Immunoblot analysis of the LA-4 cell lysate using the anti-HGF? Chain antibody showed that the intracellular HGF protein level was also increased by Gas6 treatment (FIG. 36).

Next, in order to suppress the RhoA / Rho kinase signal, RhoA-specific siRNA was transfected into LA-4 cells before Gas6 stimulation for 24 hours or the Rho kinase inhibitor Y-27632 was treated for 1 hour. As a result, both RhoA knockdown and Rho kinase inhibition reversed the effect of Gas6 on TGF-beta induced EMT (Figures 37-44). In addition, RhoA siRNA completely reversed the effect of Gas6 on phosphorylation of TGF-beta induced ERK1 / 2 and AKT in LA-4 cells, but negative control did not show this effect (FIGS. 45 and 46). In addition, the c-Met antagonist PHA-665752 was treated with LA-4 cells 1 hour before TGF-beta treatment to invert the anti-EMT effect of Gas6 (Figs. 47 to 50) . These data strongly suggest that RhoA / Rho kinase-dependent HGF production in LA-4 cells also mediates the anti-EMT effect of Gas6 through c-Met signaling by self-secretion.

Experimental Example 5: Enhancement of EP2, DP2 and c-Met Expression of Gas6

As confirmed in the previous examples, PGE ₂ , PGD ₂ and HGF produced by the Gas6 treatment mainly cause anti-EMT signals through EP2, DP2 and c-MET, respectively. In order to evaluate whether PGE ₂ , PGD ₂ and HGF acted in a self-secretion manner as an EMT inhibitor, the effects of the soluble mediator on LA-4 cells were evaluated at basal concentrations (35, 6 and 169 pg / ml, respectively) Stimulation concentrations (118, 28 and 194 pg / ml, respectively).

As a result, stimulation concentrations of PGE ₂ and PGD ₂ inhibited the change of EMT markers induced by TGF-β at the protein level as compared with the basal concentration (FIG. 51). The data support that EMT inhibition is significantly induced by PGE ₂ and PGD ₂ release induced in Gas-6 in LA-4 cells.

In contrast, HGF did not show an anti-EMT effect at both stimulation (194 pg / ml) and basal (169 pg / ml) concentrations (Figure 51), but after 20 hours of pretreatment with Gas6, When 194 pg / ml was added, changes in EMT markers induced by TGF-β were inhibited (FIG. 52). However, basal HGF did not show anti-EMT effect under the above experimental conditions. Furthermore, the amount of c-MET protein was increased after 20 hours of Gas6 treatment (Fig. 53). This data suggests that despite the relatively low HGF production, HGF may have anti-EMT activity because it increases c-MET expression in the experimental conditions. Gas6 treatment in LA-4 cells also increased the amount of EP2 and DP2 protein, but did not increase the amount of EP4 and DP1 protein (FIG. 53). On the other hand, when all of the mediators were added at the stimulation concentration in LA-4 cells, they did not show synergistic effect (Fig. 51), suggesting that they inhibit TGF-beta induced signal pathway through the same target molecule.

Experimental Example 6 Effect of Axl or Mer on Gas6-induced EMT Inhibition in LA-4 Cells

Next, the present inventors tried to determine whether the Gas6 / Ax1 or Mer signal was regulated by Gas6 in LA-4 cells.

In the LA-4 cells, 400 ng / ml of Gas6 was treated with time (0, 5, 15, 30, 60 and 120 minutes) and the level of phosphorylation of Axl or Mer was measured (Fig. 54). As a result, the levels of AxI or Mer phosphorylation all peaked at 30 minutes after treatment with Gas6, and gradually decreased.

Next, LA-4 cells were transfected with AxI or Mer specific siRNA or negative-control siRNA, cultured for 48 hours, and the level of AxI or Mer expression was measured (Fig. 55). As a result, the negative control siRNA did not affect the amount of AxI or Mer protein, but the amount of AxI or Mer protein decreased by 70% or more in the cells transfected with AxI or Mer specific siRNA compared with wild type LA-4 cells .

In addition, when Axl or Mer siRNA was transfected, it inhibited Gas6-induced COX-2 mRNA elevation and PGE ₂ and PGD ₂ secretion (Fig. 56), indicating that Gas6-induced PGE ₂ and PGD ₂ production is derived from induction of Axl or Mer expression.

In addition, when Axl or Mer siRNA was transfected, it inhibited Gas6-induced increase of RhoA activity and increase of HGF mRNA and protein level (Fig. 57), indicating that Gas6-induced increase of RhoA activity and HGF mRNA And that increased protein levels are derived from inducing Axl or Mer expression.

In addition, the present inventors tried to confirm the effect of Axl or Mer on EMT inhibition by Gas6 in LA-4 cells.

LA-4 cells were transfected with AxI or Mer-specific siRNA or negative-control siRNA, cultured for 48 hours, and mRNA and protein expression levels of EMT markers were measured (FIGS. 58 and 59). As a result, E-cadherin induced by Gas6 and α-SMA and N-cadherin decreased when Axl or Mer siRNA was transfected. This suggests that the increase in E-cadherin and decrease in? -SMA and N-cadherin induced in Gas-6 in LA-4 cells result from induction of Axl or Mer expression.

In addition, we sought to determine the effect of Axl or Mer on the expression of EMT regulatory transcription factors and non-Smad TGF-β1 signal by Gas6 in LA-4 cells.

LA-4 cells were transfected with AxI or Mer specific siRNA or negative-control siRNA, cultured for 48 hours, and mRNA and protein expression levels of EMT regulated transcription factors were measured (Figure 60). As a result, expression of mRNA of Snai1, Zeb1 and Twist1 inhibited by Gas6 was increased. In addition, both ERK (extracellular signal-regulated kinase) suppressed by Gas6 and phosphorylation of Akt were both increased (Figs. 61 and 62). This suggests that the EMT transcription factor and ERK and Akt phosphorylation inhibition induced in Gas-6 in LA-4 cells are derived from induction of Axl or Mer expression.

Experimental Example 7: Effect of Gas6 on EMT in primary mouse AT II cells

7-1. Effect of pretreatment of Gas6 on TGF-β1-induced EMT in invasive mouse AT II cells

We sought to determine if Gas6 treatment modulates the TGF-beta induced EMT process in invasive mouse AT II cells. AT II cells were treated with 400 ng / ml of Gas6 and treated with TGF-β at 10 ng / ml for 48 hours or 72 hours after 20 hours, and the level of EMT marker mRNA and protein expression was confirmed. mRNA expression levels were measured on the basis of Hprt mRNA (Figures 63 and 64). This data suggests that synthetic and secreted mediators after Gas6 treatment can block TGF-beta signaling in autocrine manner in AT II cells. In addition, the present inventors confirmed that TGF-β treatment of AT II cells after 20 hours of treatment with Gas6 inhibited the expression of mRNA of Snai1 / 2, Zeb1 / 2, and Twist1 induced by TGF-β 65). These data suggest that the GasE6 treatment reduces the transcriptional regulators to block the EMT process in AT II cells.

7-2. COX-2 signaling for Gas6-induced EMT inhibition in invasive mouse AT II cells

The present inventors intend to confirm the regulation of COX-2 signal by Gas6 in early mouse AT II cells.

We treated 400 ng / ml Gas6 with time (0, 1, 2, 3, and 6 hours) and measured levels of COX-2 mRNA expression in primary mouse AT II cells (Fig. 66). COX-2 mRNA expression levels were maximal after one hour of treatment with Gas6 and gradually decreased.

Next, in order to further confirm the functional relevance of the COX-2 signal in the anti-EMT effect of Gas6, we used NS-398, a highly selective COX-2 inhibitor, for 1 hour before treatment with Gas6 in primary mouse AT II cells Lt; / RTI &gt; As a result, NS-398 reversed the effects of Gas6 on E-cadherin loss, synthesis of α-SMA and N-cadherin, and reduction of Snai1, Zeb1 and Twist1 mRNA expression at the gene level induced by TGF-β 67 and 68).

Next, the present inventors examined whether or not TGF-beta-treated primary mouse AT II cells were treated with EP2 (E-prostanoid-2 receptor) antagonist (AH-6809) or DP2 antagonist (BAY- u3405) Antagonists of PGE ₂ - or PGD ₂ - specific receptors were treated. As a result, the anti-EMT effect of Gas6 was significantly reversed by antagonists of EP2 and DP2 (Fig. 69 and Fig. 70).

These results suggest that the anti-EMT effect in AT II cells is mediated primarily by COX-2-derived PGE ₂ and PGD ₂ signals through EP2 and DP2 receptors, respectively.

7-3. Identification of RhoA pathway-dependent HGF-signaling for Gas6-induced EMT inhibition in invasive mouse AT II cells

The present inventors have determined to modulate RhoA pathway-dependent HGF-signal by Gas6 in primary mouse AT II cells.

As a result of treatment of Inv6 mouse AT II cells with Gas6, expression of HGF mRNA induced by Gas6 gradually increased (FIG. 71).

Next, in order to suppress the RhoA / Rho kinase signal, RhoA-specific siRNA was transfected into the invasive mouse AT II cells before Gas6 stimulation for 24 hours or the Rho kinase inhibitor Y-27632 was treated for 1 hour. As a result, both RhoA knockdown and Rho kinase inhibition reversed the effect of Gas6 on TGF-beta induced EMT (FIGS. 72 and 73). In addition, treatment of PHA-665752, a c-Met antagonist, with AT II cells 1 hour before TGF-beta treatment reversed the anti-EMT effect of Gas6 (FIGS. 74 and 75).

The data strongly suggest that RhoA / Rho kinase-dependent HGF production in AT II cells also mediates the anti-EMT effect of Gas6 via self-secretion of the c-Met signal.

Experimental Example 8: Effect of pretreatment of Gas6 on TGF-β1-induced EMT in human lung adenocarcinoma cells (A549 cells)

We sought to determine whether administration of Gas6 in A549 cells regulates the TGF-beta induced EMT process.

A549 cells were treated with 400 ng / ml of Gas6, and after 20 hours, 10 ng / ml of TGF-beta was treated for 72 hours to confirm the level of EMT marker mRNA and protein expression. mRNA expression levels were measured on the basis of Hprt mRNA (Figures 76 and 77). This data suggests that synthetic and secreted mediators after Gas6 treatment may block TGF-β signaling in a self-secreted manner in A549 cells.

In addition, the present inventors confirmed that TGF-beta treatment of A549 cells after 20 hours of treatment with Gas6 inhibited the expression of mRNA of Snai1 / 2, Zeb1 / 2 and Twist1 induced by TGF-beta 78). These data suggest that the GasE6 treatment reduces the transcriptional regulators to prevent EMT processes in A549 cells.

Experimental Example 9: EMT inhibition effect of Gas6 in an animal model of pulmonary fibrosis

9-1. Inhibitory effect of Gas6 on bleomycin-induced EMT in an animal model of pulmonary fibrosis

The present inventors sought to determine whether administration of Gas6 regulates the EMT process in lung epithelial cells in vivo.

C57BL / 6 mice were divided into five groups by Control, Gas6 group, Bleomycin group (BLM) and Gas6 + Bleomycin group (Gas6 + BLM). Each day, 50 μg / kg of Gas6 was administered according to each taxon before administration of bleomycin. At day 14 after bleomycin administration, primary mouse AT II cells were isolated from the lungs of mice.

The morphology of isolated mouse cells was determined. As a result, it was confirmed that the cells of the mice to which bromomycin was administered were converted into spindle-like morphology, but the cells of the mice to which Gas6 was administered before the bleomycin administration showed a decrease in the spindle-like morphology 79).

In order to confirm the expression pattern of EMT markers (E-cadherin, N-cadherin and a-SMA) and EMT-activated transcription factors (Snai1, Zeb1 and Twist1), each mRNA level was measured (FIGS. 80 and 81) . E-cadherin expression and N-cadherin and α-SMA expression were increased in bleomycin-treated mice, but the level of EMT marker expression was inhibited by Gas6 treatment at the mRNA level. In addition, it was confirmed that expression of mRNA of Snai1, Zeb1, and Twist1 induced by bleomycin was all inhibited when Gas6 was treated. These results suggest that Gas6 treatment may inhibit bleomycin-induced EMT in a pulmonary fibrosis mouse model.

9-2. Effect of Gas6 Treatment on COX-2-Derived PGE ₂ , PGD ₂ and HGF Secretion in Pulmonary Fibrosis Animal Model

The present inventors sought to determine whether administration of Gas6 in vivo regulates PGE ₂ , PGD ₂ and HGF pathways.

C57BL / 6 mice were divided into five groups by Control, Gas6 group, Bleomycin group (BLM) and Gas6 + Bleomycin group (Gas6 + BLM). Each day, 50 μg / kg of Gas6 was administered according to each taxon before administration of bleomycin. At day 14 after bleomycin administration, primary mouse AT II cells were isolated from the lungs of mice. The production of mRNA, PGE ₂ , PGD ₂ , HGF and TGF-β of COX-2 and HGF was confirmed in isolated mouse cells.

As a result, the mRNA level of COX-2 was increased more than twice as much as that of the control group due to the administration of Gas6, the PGE ₂ secreted by the control group was secreted by 4000 pg / ml or more, and the PGD ₂ protein level was about 4 times (Figs. 82 and 83). These results suggest that EM6 suppression due to Gas6 in pulmonary fibrosis mouse models results from increased COX-2 expression, PGE ₂ and PGD ₂ production.

In addition, the mRNA level of HGF was increased more than 3 times as compared to the control group due to the administration of Gas6, HGF was secreted by 2500 pg / ml or more, and the level of TGF-beta protein increased by bleomycin was decreased due to administration of Gas6 And Fig. 85). These results suggest that inhibition of EM due to Gas6 in the lung fibrosis mouse model results from HGF increase and TGF-beta inhibition.

9-3. Effect of Gas6 treatment on bleomycin-induced EMT markers and collagen deposition changes in pulmonary fibrosis animal models

The present inventors sought to determine whether administration of Gas6 in vivo controls changes in EMT markers and collagen deposition.

C57BL / 6 mice were divided into five groups by Control, Gas6 group, Bleomycin group (BLM) and Gas6 + Bleomycin group (Gas6 + BLM). Each day, 50 μg / kg of Gas6 was administered according to each taxon before administration of bleomycin. Early mouse AT II cells were isolated from the lungs of mice on days 14 and 21 after administration of bleomycin. Each separated mouse cell was Western blotted using anti-E-cadherin, anti-N-cadherin, anti-α-SMA, anti-type 1 collagen 1 or anti-fibronectin antibody. Relative density of each band was determined based on Α-tubulin.

As a result, E-cadherin levels decreased by bleomycin in mouse lung tissue on days 14 and 21 were elevated due to the administration of Gas6, and the levels of N-cadherin, a-SMA, type 1 collagen and fibronectin increased by bleomycin (Fig. 86 and Fig. 87).

In addition, the collagen accumulation of the entire lung was measured at the 21th day of hydroxyproline level (Fig. 88). As a result, the level of hydroxyproline increased by bromomycin was reduced to a level similar to that of the control group due to administration of Gas6.

These results suggest that fibromyxia induced by in vivo bleomycin is improved by administration of Gas6.

Taken together, all of the above results, Gas6 has induced the secretory production of PGE _2, PGD ₂ and HGF in epithelial cells and, through a signal transmission path through their receptors party by acting as a secretion system, induced by TGF-β EMT-inhibiting effect of EMT.

In addition, Gas6 in LA-4 cells secretes COX-2 signal pathway and RhoA pathway dependent HGF based on Axl or Mer signal pathway and has the same effect on LA-4 cell in primary mouse AT II cell and human lung adenocarcinoma cell .

In addition, it was confirmed through animal experiments that administration of Gas6 suppressed EMT induced by bromomycin in vivo, and thus had an excellent prophylactic and therapeutic effect on fibrosis.

Therefore, Gas6 can be very usefully used for the prevention or treatment of fibrosis.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (10)

1. A pharmaceutical composition for preventing or treating fibrosis, comprising as an active ingredient an activator of Gas6 (Growth arrest-specific 6) protein or Gas6 protein receptor.
2. The pharmaceutical composition according to claim 1, wherein the Gas6 protein receptor is any one or more selected from the group consisting of AXL receptor tyrosine kinase, Mer tyrosine kinase, and TYRO3.
3. The pharmaceutical composition according to claim 1, wherein the activator of the Gas6 protein receptor is at least one selected from the group consisting of Protein S (Pros1), Tubby and tubby-like protein 1, or Galectin3.
4. The method of claim 1, wherein the fibrosis is selected from the group consisting of lung, kidney, liver, heart, brain, blood vessels, joints, bowel, skin, soft tissue, bone marrow, penis, peritoneum, lens, muscle, vertebra, testes, , Pancreas, gall bladder, bladder or prostate.
5. The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.
6. 2. The pharmaceutical composition according to claim 1, wherein the composition is administered in a dose of 100 [mu] g / kg to 500 [mu] g / kg.
7. The pharmaceutical composition according to claim 1, wherein the composition is administered orally or parenterally.
8. The pharmaceutical composition according to claim 1, wherein the composition inhibits the epithelial to mesenchymal transition (EMT) of the cell.
9. A method for preventing or treating fibrosis, comprising administering a receptor activator of Gas6 protein or Gas6 protein to a subject having fibrosis or at risk of developing fibrosis.
10. Use of a Gas6 protein or a receptor activator thereof for the prophylaxis or treatment of fibrosis.

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