WO2018236194A1 - Composition pour la prévention ou le traitement de la fibrose, comprenant la protéine gas6 ou un activateur du récepteur de celle-ci - Google Patents

Composition pour la prévention ou le traitement de la fibrose, comprenant la protéine gas6 ou un activateur du récepteur de celle-ci Download PDF

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WO2018236194A1
WO2018236194A1 PCT/KR2018/007128 KR2018007128W WO2018236194A1 WO 2018236194 A1 WO2018236194 A1 WO 2018236194A1 KR 2018007128 W KR2018007128 W KR 2018007128W WO 2018236194 A1 WO2018236194 A1 WO 2018236194A1
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gas6
cells
hours
treatment
protein
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이지희
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이화여자대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1732—Lectins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration

Definitions

  • 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.
  • IPF idiopathic pulmonary fibrosis
  • ECM extracellular matrix
  • 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.
  • 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- ⁇ 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- ⁇ 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- ⁇ 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- ⁇ 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- ⁇ 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- ⁇ 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- ⁇ 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- ⁇ and Gas6. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF- ⁇ 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 ⁇ M NS-398 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF- ⁇ 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 ⁇ M NS-398 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF- ⁇ for 72 hours.
  • 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 ⁇ M NS-398 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF- ⁇ for 72 hours.
  • 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- ⁇ for 72 hours.
  • 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- ⁇ for 72 hours.
  • 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 ⁇ M NS-398 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and stimulated with 10 ng / ml TGF- ⁇ for 72 hours.
  • 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- ⁇ for 72 hours.
  • 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- ⁇ for 5 minutes.
  • 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- ⁇ for 8 hours.
  • 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)
  • EP2 AH-6809
  • EP4 AH-23848
  • DP1 BW-A868C
  • DP2 BAY-u3405
  • LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF- ⁇ for 48 hours in the absence or administration of 10 mM of each receptor antagonist EP2 (AH-6809) or EP4 (AH-23848) Respectively.
  • EP2 AH-6809
  • EP4 AH-23848
  • 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.
  • 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- ⁇ for 48 hours in the absence or administration of 10 mM of each receptor antagonist EP2 (AH-6809) or EP4 (AH-23848) Respectively.
  • EP2 AH-6809
  • EP4 AH-23848
  • 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- ⁇ 1 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.
  • 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.
  • EP2 AH-6809
  • EP4 AH-23848
  • DP2 BAY-u3405
  • 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- ⁇ for 48 hours.
  • 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- ⁇ for 48 hours.
  • 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- ⁇ for 48 hours. The following morphological changes were observed.
  • 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- ⁇ for 48 hours.
  • 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- ⁇ for 48 hours.
  • 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- ⁇ for 72 hours.
  • 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- ⁇ for 48 hours.
  • 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- ⁇ for 5 minutes.
  • 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- ⁇ for 8 hours.
  • 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- ⁇ for 72 hours in the presence or absence of 250 nM of c-Met antagonist PHA-665752.
  • LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF- ⁇ 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.
  • LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF- ⁇ 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- ⁇ for 72 hours in the presence or absence of 250 nM of c-Met antagonist PHA-665752.
  • 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- .
  • PGE 2 35 or 118 pg / ml
  • PGD 2 (6 or 28 pg / ml)
  • HGF 169 or 194 pg / ml
  • 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- ⁇ 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.
  • 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.
  • 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- ⁇ for 72 hours.
  • 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- ⁇ for 72 hours.
  • 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- ⁇ for 72 hours.
  • 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- ⁇ for 72 hours.
  • 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- ⁇ for 72 hours. * P ⁇ 0.05, compared to control group; + P ⁇ 0.05, comparison of displayed values.
  • TGF-beta 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- ⁇ 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 ⁇ M NS-398 was treated for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and treated with 10 ng / ml of TGF- ⁇ for 72 hours.
  • 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 ⁇ M NS-398 was treated for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and treated with 10 ng / ml of TGF- ⁇ for 72 hours.
  • 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.
  • treatment with 400 ng / ml Gas6 for 20 hours treatment with TGF- ⁇ alone, treatment with 10 ⁇ M of EP2 (AH-6809) or treatment with 10 ⁇ M of DP2 (BAY-u3405) were performed for 48 hours.
  • 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- ⁇ for 48 hours.
  • 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- ⁇ for 48 hours.
  • LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and treated with TGF- ⁇ 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- ⁇ for 72 hours in the presence or absence of 250 nM of c-Met antagonist PHA-665752.
  • 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- ⁇ 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.
  • 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.
  • 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.
  • 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.
  • Gas6 Heating arrest-specific 6
  • Gas6 protein receptor activator as an active ingredient.
  • 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.
  • epithelial to mesenchymal transition 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 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.
  • 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), morph
  • 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.
  • surgery for example, (Fibrosis of the retroperitoneal soft tissue), post-surgical scarring, post-operative fibrosis, fibromyalgia, fibromyalgia, chronic fibrosis, chronic myelogenous leukemia, Scleroderma / systemic
  • 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.
  • treatment means any action that improves or alleviates the symptoms of fibrosis with the administration of the pharmaceutical composition
  • prevention means inhibiting the onset of fibrosis by administration of the pharmaceutical composition Or delaying any action 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.
  • Growth arrest-specific protein 6 in the present invention refers to a secretable vitamin K-dependent protein, also called AXSF or AXLLG.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • Gas6 acts on Axl or Mer receptor tyrosine kinase distributed in the cell membrane to block the TGF- ⁇ signal through COX-2-derived PGE 2 , PGD 2 secretion and RhoA-dependent HGF secretion, thus confirming anti-EMT effect (Figs. 5 to 62).
  • 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).
  • 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).
  • lung fibrosis was improved by administration of Gas6 in an animal model in which pulmonary fibrosis was induced (Figs. 79 to 88).
  • 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.
  • 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.
  • Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like.
  • 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.
  • non-aqueous solvent and the suspending agent examples include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
  • injectable ester such as ethyl oleate.
  • the suppository base examples 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 ⁇ ⁇ / kg to 500 ⁇ ⁇ / 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.
  • administration 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.
  • routes of administration include oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal routes
  • 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.
  • pharmaceutically effective amount 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.
  • the term "individual" means all animals including humans, which have the fibrosis disease or the disease of the present invention.
  • 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.
  • Gas6 protein its receptor activator
  • 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 .
  • FBS fetal bovine serum
  • HEK-293 cells 37 °C 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 >
  • 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 purity of AT II cells evaluated by pro-SP-C immunofluorescence staining was over 90%.
  • LA-4 and HEK-293 cells were inoculated (2 x 10 5 cells / well) in 6-well culture dishes and incubated overnight in 200 ⁇ l 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).
  • 10 ⁇ M 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.
  • siRNA transfection reagent Genlantis
  • 75 nM of Axl or Mer specific siRNA was used.
  • the siRNA sequences used are shown in Table 1.
  • 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.
  • cDNA was normalized to the amount of HPRT (hypoxanthine-guanine phosphoribosyltransferase) and expressed as fold-change in the control.
  • 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.
  • the content of hydroxyproline was measured using a hydroxyproline assay kit (Nanjing Jiancheng Bioengineering Co., Ltd.) according to the manufacturer's instructions.
  • mice Male C57BL / 6 mice (Orient Bio, Seongnam, Korea) were used for all experiments with 20-25 g of specific pathogen.
  • 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.
  • TGF- ⁇ signaling has been shown to play an important role in EMT and fibrosis development (Am J Physiol Lung Cell Mol Physiol, 2007, 293: L525).
  • LA-4 cells were converted into spindle-like morphology when stimulated with TGF- ⁇ at 10 ng / ml for 48 hours or 72 hours.
  • TGF- ⁇ 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).
  • 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).
  • the present inventors sought to determine whether Gas6 inhibits the expression of the transcription factor in LA-4 cells stimulated with TGF- ?.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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 < / RTI >
  • LA-4 cells were treated with the highly selective COX-2 inhibitor NS-398 for 1 hour before treatment with Gas6.
  • NS-398 reversed both the cell morphology changes induced by TGF- ⁇ and E-cadherin loss at gene and protein levels and the effect of Gas6 on the synthesis of ⁇ -SMA and N-cadherin (FIGS. 18).
  • knockdown of the COX-2 gene in LA-4 cells also reversed the effect of Gas6 (FIGS. 19 and 20).
  • both NS-398 and COX-2 siRNA reversed the effect of Gas6 on reducing TGF- ⁇ -induced Snai1, Zeb1 and Twist1 mRNA expression (FIGS. 21 and 22).
  • 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 ).
  • 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.
  • 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.
  • 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.
  • RhoA knockdown and Rho kinase inhibition reversed the effect of Gas6 on TGF-beta induced EMT ( Figures 37-44).
  • 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).
  • PGE 2 , PGD 2 and HGF produced by the Gas6 treatment mainly cause anti-EMT signals through EP2, DP2 and c-MET, respectively.
  • 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).
  • 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,
  • 194 pg / ml was added, changes in EMT markers induced by TGF- ⁇ were inhibited (FIG. 52).
  • basal HGF did not show anti-EMT effect under the above experimental conditions.
  • 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.
  • 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).
  • 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 .
  • 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).
  • E-cadherin induced by Gas6 and ⁇ -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.
  • 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).
  • expression of mRNA of Snai1, Zeb1 and Twist1 inhibited by Gas6 was increased.
  • 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.
  • the present inventors intend to confirm the regulation of COX-2 signal by Gas6 in early mouse AT II cells.
  • NS-398 a highly selective COX-2 inhibitor, for 1 hour before treatment with Gas6 in primary mouse AT II cells Lt; / RTI > As a result, NS-398 reversed the effects of Gas6 on E-cadherin loss, synthesis of ⁇ -SMA and N-cadherin, and reduction of Snai1, Zeb1 and Twist1 mRNA expression at the gene level induced by TGF- ⁇ 67 and 68).
  • 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.
  • EP2 E-prostanoid-2 receptor
  • DP2 antagonist BAY- u3405
  • Antagonists of PGE 2 - or PGD 2 - specific receptors were treated.
  • the anti-EMT effect of Gas6 was significantly reversed by antagonists of EP2 and DP2 (Fig. 69 and Fig. 70).
  • the present inventors have determined to modulate RhoA pathway-dependent HGF-signal by Gas6 in primary mouse AT II cells.
  • 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.
  • RhoA knockdown and Rho kinase inhibition reversed the effect of Gas6 on TGF-beta induced EMT (FIGS. 72 and 73).
  • 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).
  • 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.
  • 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- ⁇ signaling in a self-secreted manner in A549 cells.
  • 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).
  • GasE6 treatment reduces the transcriptional regulators to prevent EMT processes in A549 cells.
  • 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 ⁇ g / 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).
  • EMT markers E-cadherin, N-cadherin and a-SMA
  • EMT-activated transcription factors Snai1, Zeb1 and Twist1
  • each mRNA level was measured (FIGS. 80 and 81) .
  • E-cadherin expression and N-cadherin and ⁇ -SMA expression were increased in bleomycin-treated mice, but the level of EMT marker expression was inhibited by Gas6 treatment at the mRNA level.
  • gas6 treatment it was confirmed that expression of mRNA of Snai1, Zeb1, and Twist1 induced by bleomycin was all inhibited when Gas6 was treated.
  • 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 ⁇ g / 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- ⁇ of COX-2 and HGF was confirmed in isolated mouse cells.
  • 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 ⁇ g / 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- ⁇ -SMA, anti-type 1 collagen 1 or anti-fibronectin antibody. Relative density of each band was determined based on ⁇ -tubulin.
  • 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- ⁇ EMT-inhibiting effect of EMT.
  • 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 .
  • Gas6 can be very usefully used for the prevention or treatment of fibrosis.

Abstract

La présente invention concerne une composition pour la prévention ou le traitement de la fibrose, comprenant la protéine Gas6 ou un activateur du récepteur de celle-ci. La protéine Gas6 peut induire la sécrétion et la production de PGE2, PGD2 et HGF dans les cellules épithéliales par l'intermédiaire d'un récepteur de celle-ci, et inhiber le phénomène de la TEM par auto-sécrétion et par l'intermédiaire d'une voie de signalisation en aval de celui-ci, ce qui permet de prévenir et de traiter la fibrose. Ainsi, la protéine Gas6 et un activateur du récepteur de celle-ci ont un excellent effet pour une utilisation dans la prévention ou le traitement de la fibrose.
PCT/KR2018/007128 2017-06-23 2018-06-22 Composition pour la prévention ou le traitement de la fibrose, comprenant la protéine gas6 ou un activateur du récepteur de celle-ci WO2018236194A1 (fr)

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CN114272382A (zh) * 2021-12-31 2022-04-05 上海中医药大学 一种Galectin-3抑制剂的用途及药物组合物
CN114592009A (zh) * 2022-04-20 2022-06-07 东南大学 一种促进心肌再生的重组腺相关病毒gas6的制备方法及运用
CN114592009B (zh) * 2022-04-20 2023-10-03 东南大学 一种促进心肌再生的重组腺相关病毒gas6的制备方法及运用

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