WO2020088575A1 - 一种pde10a抑制剂在制备成纤维细胞活性抑制药物中的应用 - Google Patents

一种pde10a抑制剂在制备成纤维细胞活性抑制药物中的应用 Download PDF

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WO2020088575A1
WO2020088575A1 PCT/CN2019/114644 CN2019114644W WO2020088575A1 WO 2020088575 A1 WO2020088575 A1 WO 2020088575A1 CN 2019114644 W CN2019114644 W CN 2019114644W WO 2020088575 A1 WO2020088575 A1 WO 2020088575A1
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papaverine
tgf
cells
pde10a
group
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French (fr)
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汤慧芳
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浙江大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the invention relates to the application of a PDE10A inhibitor in the preparation of fibroblast activity inhibitory drugs.
  • IPF Specific pulmonary fibrosis
  • pulmonary fibrosis is a group of progressive, destructive, and irreversible lung interstitial diseases with an average survival rate of 2 to 3 years since diagnosis.
  • the pathological mechanism of IPF is that the body initiates a protective repair process in response to injury stimuli, but abnormal excessive repair leads to disorder of the internal environment of the tissue, excessive deposition of extracellular matrix, and destruction of tissue structure, resulting in Fibrosis occurs.
  • pirfenidone and nidanib have been approved for use by the FDA in 2014, and are conditionally recommended for use.
  • the pathogenesis of pulmonary fibrosis is intricate and requires in-depth research. Therefore, it has important theoretical and practical significance for finding effective treatment methods for pulmonary fibrosis.
  • Pulmonary fibrosis is characterized by progressive extracellular matrix deposition, the destruction of lung tissue structure, and ultimately leads to loss of lung function.
  • the typical pathological feature of pulmonary fibrosis is the formation of fibroblast / myofibroblast foci "Fibroblast", which is the main manifestation of active fibrosis.
  • Myofibroblasts as the main component of fibrous foci, are considered to be the core factors of pulmonary fibrosis.
  • Myofibroblasts highly express ⁇ -smooth muscle actin (SMA), which has the characteristics of migration and contractility. It can divide a large amount of extracellular matrix and lead to the formation of collagen fiber network. Therefore, as the core cell of pulmonary fibrosis, interfering with the activation of myofibroblasts is an important entry point to find the therapeutic target of pulmonary fibrosis.
  • SMA smooth muscle actin
  • TGF- ⁇ 1 is an important profibrotic cytokine, and TGF- ⁇ 1 is a major participant in fibrosis.
  • TGF- ⁇ 1 can promote the proliferation and chemotaxis of fibroblasts, promote epithelial-mesenchymal transition, promote the conversion of fibroblasts into myofibroblasts, and inhibit the apoptosis of myofibroblasts.
  • TGF- ⁇ 1 can mediate the effect of promoting fibrosis through its classical Smad pathway and non-Smad pathway.
  • PDE inhibitors ie phosphodiesterase inhibitors
  • cAMP and cGMP play an important role in tissue fibrosis.
  • the content of PDE10A in the model of bleomycin-induced pulmonary fibrosis in rats significantly increased. Therefore, in this study, we will explore the inhibitors of papaverine and PF- through PDE10A. 2545920 to inhibit the activity of PDE10A can treat experimental pulmonary fibrosis.
  • PDE10A inhibitor papaverine has a relaxing effect on cardiovascular, bronchial, gastrointestinal tract, bile duct and other smooth muscles. By relaxing vascular smooth muscle, coronary artery dilation, peripheral resistance and cerebrovascular resistance are reduced. It is mainly used for clinical relief of accompanying arteries Spastic brain and peripheral vascular disease, treatment of cerebral thrombosis, pulmonary embolism, arterial spasm and arterial embolism pain, etc .; can also be used to treat intestinal, ureteral and biliary tract spasm pain and dysmenorrhea, and as a compound bronchodilator spray One part; can also be used for hypertension, angina pectoris, cardiac ischemia complicated with arrhythmia, etc.
  • PDE10A inhibitors are currently undergoing international central nervous system clinical research, such as Huntington's disease, Alzheimer's disease, encephalitis, phobia, epilepsy, aphasia, Bell's palsy, cerebral palsy, sleep Disorders, pain, Tourette's syndrome, schizophrenia, delusions, drug-induced psychosis, and psychosis of panic disorder and obsessive-compulsive disorder, anxiety disorder, movement disorders and / or neurological disorders.
  • aphasia aphasia
  • Bell's palsy cerebral palsy
  • sleep Disorders pain
  • Tourette's syndrome schizophrenia, delusions, drug-induced psychosis, and psychosis of panic disorder and obsessive-compulsive disorder, anxiety disorder, movement disorders and / or neurological disorders.
  • schizophrenia, delusions, drug-induced psychosis, and psychosis of panic disorder and obsessive-compulsive disorder, anxiety disorder, movement disorders and / or neurological disorders are currently no successfully marketed
  • the purpose of the present invention is to provide a PDE10A inhibitor for inhibiting the activity of lung fibroblasts, which has important theoretical and practical significance for finding an effective treatment method for pulmonary fibrosis.
  • the invention provides the application of a PDE10A inhibitor in a drug for inhibiting the activity of lung fibroblasts.
  • the fibroblast is human embryo lung fibroblast HFL-1, human embryo lung fibroblast MRC-5 or embryo fibroblast MEF.
  • the PDE10A inhibitor is papaverine or PF-2545920.
  • the concentration of the PDE10A inhibitor is 10-100 ⁇ M.
  • the drug for inhibiting the activity of lung fibroblasts is a drug for preventing or treating pulmonary fibrosis, that is, the present invention also provides an application of the PDE10A inhibitor in the preparation of a drug for preventing or treating pulmonary fibrosis.
  • the medicine is an effective anti-pulmonary fibrosis medicine.
  • the beneficial effects of the present invention are mainly reflected in: the present invention provides a new application of PDE10A inhibitors in the preparation of drugs that inhibit lung fibroblast activity, and provides a new therapeutic target for pulmonary fibrosis, PDE10A, possible By inhibiting PDE10A, the effect of alleviating the development of pulmonary fibrosis is achieved, and the effect is better than the existing drug pirfenidone, and the adverse reactions are small.
  • Figure 1 The effect of papaverine and pirfenidone on the activity of human lung fibroblast HFL-1, PAP H: papaverine high dose 100 ⁇ M; PAP M: papaverine medium dose 30 ⁇ M; PAP L: papaverine low dose 10 ⁇ M; PFD H: pirfenidone high dose 0.1mg / ml; PFD M: pirfenidone medium dose 0.03mg / ml; PFD L: pirfenidone low dose 0.01mg / ml; C: blank control group; TGF- ⁇ 1 : TGF- ⁇ 1 stimulation group.
  • Figure 2 The effect of papaverine (A) and pirfenidone (B) on the viability of human lung fibroblast MRC-5.
  • PAP H papaverine high dose 100 ⁇ M
  • PAP M papaverine medium dose 30 ⁇ M
  • PAP L papaverine low dose 10 ⁇ M
  • C blank control group
  • TGF- ⁇ 1 TGF- ⁇ 1 stimulation group.
  • PFD H high dose of pirfenidone 0.1 mg / ml
  • PFD M medium dose of pirfenidone 0.03 mg / ml
  • PFD L low dose of pirfenidone 0.01 mg / ml
  • C blank control group
  • TGF- ⁇ 1 TGF- ⁇ 1 stimulation group.
  • Figure 3 Changes in mRNA levels of ⁇ -SMA, collagen I and PDE10A induced by TGF- ⁇ 1. After stimulation with TGF- ⁇ 1 for 2, 3, 4 and 7 days, HFL-1 cells were collected for PCR detection, C represents the blank control group, T represents TGF- ⁇ 1 treatment, p * ⁇ 0.05, p ** ⁇ 0.01.p ** * ⁇ 0.001.
  • FIG. 4 TGF- ⁇ 1 induced changes in PDE10A protein levels in human lung fibroblasts HLF-1.
  • A is a representative graph of WB, and B is a grayscale analysis graph.
  • FIG. 5 Effects of papaverine and pirfenidone on TGF- ⁇ 1-induced myofibrilization of human lung fibroblast HFL-1 cells.
  • A is a representative graph of immunofluorescence and B is a graph of fluorescence intensity analysis;
  • PAP papaverine;
  • PFD pirfenidone, p * ⁇ 0.05, p *** ⁇ 0.001.
  • Figure 6 Effect of papaverine and pirfenidone on TGF- ⁇ 1-induced protein markers ( ⁇ -SMA, FN, COL1) of human lung fibroblast HFL-1 cell activation.
  • a Representative image of Western blotting of ⁇ -SMA
  • b Grayscale analysis of Western blotting of ⁇ -SMA
  • c Representative image of Western blotting of FN
  • d Western analysis of Western blotting of FN
  • e Representative image of Western blotting of COL1 ; F; COL1 gray analysis of Western blotting.
  • PAP papaverine
  • PFD pirfenidone, p * ⁇ 0.05, p ** ⁇ 0.01.
  • FIG. 7 Effects of papaverine and pirfenidone on TGF- ⁇ 1-induced myofibrilization of human lung fibroblast MRC-5 cells.
  • A is a representative graph of immunofluorescence and B is a graph of fluorescence intensity analysis;
  • PAP papaverine;
  • PFD pirfenidone, p *** ⁇ 0.001.
  • Figure 8 The effect of papaverine and pirfenidone on TGF- ⁇ 1-induced protein markers of MRC-5 activation in human lung fibroblasts.
  • A is the representative image of Western blotting of ⁇ -SMA, FN, and COL1
  • B is the Western blot analysis of ⁇ -SMA
  • C is the Western blot analysis of FN
  • D is the Western blot analysis of COL1
  • PAP papaverine
  • PFD pirfenidone, p ** ⁇ 0.01, p *** ⁇ 0.001.
  • Figure 9 PF-2545920, papaverine, and pirfenidone on TGF- ⁇ 1 induced human lung fibroblast HLF-1 cell activation and its effect on signaling pathways.
  • A is a representative image of Western blotting of ⁇ -SMA, FN and ⁇ -catenin
  • B is a representative image of Western blotting of FN and GSK3- ⁇ .
  • PF PF-2545920
  • PAP papaverine
  • PFD pirfenidone.
  • PAP H papaverine 100 ⁇ M
  • PAP M papaverine 30 ⁇ M
  • PF3, 10 PF-2545920 3 ⁇ M
  • PFD H pirfenidone 0.1 mg / ml
  • PFD M pirfenidone 0.03 mg / ml
  • Control Blank control group
  • TGF- ⁇ 1 TGF- ⁇ 1 stimulation group.
  • FIG. 10 TGF- ⁇ 1-induced expression of phosphorylated smad 2/3 at different time points in human lung fibroblasts HFL-1 cells, A is a representative image of Western blot of phosphorylated smad3, B is a representative image of Western blot of phosphorylated smad2, C is the grayscale analysis of western blot of phosphorylated smad3 / 2.
  • FIG. 11 Effect of papaverine and pirfenidone on TGF- ⁇ 1 induced smad2 / 3 phosphorylation of human lung fibroblast HLF-1 cells.
  • A is a representative image of Western blot of phosphorylated smad3
  • B is a representative image of Western blot of phosphorylated smad2
  • C is a gray analysis diagram of Western blot of phosphorylated smad3
  • D is a gray analysis diagram of Western blot of phosphorylated smad2;
  • PAP Papaverine
  • PFD pirfenidone
  • Figure 12 Effects of papaverine and pirfenidone on the expression of ⁇ -catenin protein induced by TGF- ⁇ 1 in human lung fibroblast HFL-1 cells.
  • A is a representative image of Western blotting of ⁇ -catenin and PDE10A
  • B is a grayscale analysis of Western blotting of PDE10A
  • C is a grayscale analysis of Western blotting of ⁇ -catenin;
  • PAP papaverine
  • PFD pirfenidone
  • Figure 13 Effect of papaverine and pirfenidone on the expression and distribution of ⁇ -catenin in human lung fibroblast HFL-1 cells induced by TGF- ⁇ 1.
  • PAP papaverine
  • PFD pirfenidone.
  • Figure 14 Effects of papaverine and pirfenidone on ⁇ -SMA expression of mouse embryonic lung fibroblast MEF cells induced by TGF- ⁇ 1.
  • PAP papaverine
  • PFD pirfenidone.
  • Figure 15 Results of masson three-color staining of mouse lung tissue.
  • C normal saline control group; BLM: bleomycin group; DEX: dexamethasone group; PAP H: 10mg / kg high-dose group; PAP M: 3mg / kg medium-dose group; PAP L: 0.1mg / kg low Dose group.
  • PDE10A inhibitor papaverine
  • PDE10A inhibitor papaverine
  • Human lung embryonic fibroblasts HFL-1 (CCL153) and human lung embryonic fibroblasts MRC-5 (CCL-171) were purchased from ATCC. After receiving the cell lines, they were subcultured and HFL-1 cells were cultured in F12K medium Medium (containing 10% fetal bovine serum, 100 U / ml penicillin and 100 ⁇ g / ml streptomycin), MRC-5 cells were cultured in DMEM / H medium (containing 10% fetal bovine serum, 100 U / ml penicillin and 100 ⁇ g / ml streptomycin), the cells were passaged to a stable shape, and partly frozen at the third generation for later use, and partly used for the next experiment.
  • F12K medium Medium containing 10% fetal bovine serum, 100 U / ml penicillin and 100 ⁇ g / ml streptomycin
  • MRC-5 cells were cultured in DMEM / H medium (containing 10% fetal bovine serum, 100 U / ml penicillin and
  • the mouse embryonic fibroblast MEF was isolated from 14-day-old gestational mice (C57BL / 6).
  • the obtained MEF cells were cultured in DMEM / H medium (containing 10% fetal bovine serum, 100 U / ml penicillin and 100 ⁇ g / ml streptomycin), and partly frozen after being passaged to the first generation, and partly carried out for the next experiment .
  • the cells were cultured in a cell incubator at 37.0 ° C and 5% CO 2. All operations were completed in a sterile operating table.
  • mice Male 7 to 8 week old C57BL / 6 mice, weighing 19 to 21 g, were purchased from Shanghai Slake Company and raised in the SPF animal house of Zhejiang University Experimental Animal Center, with a temperature of 20 to 23 ° C and a relative humidity of 50% to 60% The photoperiod is 12h; all mice get sufficient clean water and feed, and the operation process complies with the "Administrative Regulations on Experimental Animals".
  • PDE10A inhibitor PF-2545920, Meilun Biological Company, China;
  • TGF- ⁇ 1 PeproTech, United States
  • Tris-HCL buffer (1.5M, pH8.8 or pH6.8): Wuhan Boster Bioengineering Co., Ltd., China
  • Non-toxic environmental protection hematoxylin-Eosin (HE) dye solution kit Nanjing Jiancheng Technology Co., Ltd., China
  • F12K medium Wuhan Boster Bioengineering Co., Ltd., China
  • Group 1 to Group 5 are prevention groups
  • Group 6 to Group 10 are treatment groups
  • Group 11 is a normal control group.
  • Groups 1 and 6 were both dexamethasone control group (Dex), which was injected intraperitoneally with dexamethasone 0.30 mg / kg
  • groups 2 and 7 were both low-dose papaverine groups (PAP L), and intraperitoneal injection of papaverine 1.0 mg / kg
  • Group 3 and Group 8 are middle-dose papaverine group (PAP M), intraperitoneal injection of papaverine 3.0mg / kg
  • Group 4 and Group 9 are high-dose papaverine group (PAP H), intraperitoneal injection of papaverine 10mg / kg
  • Group 5 and Group 10 are both PBS group (PBS), intraperitoneal injection of equal volume of PBS.
  • the prevention group (Group 1 to Group 5) was administered at the same time when the model was started, once a day, 0.1ml / 10g each time for 13 days; the treatment group (Group 6 to Group 10) was the 7th day after the model was started The administration was started once a day at 0.1ml / 10g each time for 13 days; Group 11 was injected with PBS at the same time at the beginning of modeling, once a day at 0.1ml / 10g each time for 13 days. After 14 days and 21 days of modeling, the mice were anesthetized with 4% chloral hydrate overdose, and the abdominal aorta was bled to death.
  • bronchoalveolar lavage fluid (BALF) is as high as 80%. Store it on ice. After mixing, take 50 ⁇ l for white blood cell count. The remaining BALF was centrifuged at 2000 rpm, 10 min, and 4 ° C. Take the cell pellet smear, dry it, and then carry out Wright staining to classify and count the cells. Retain the supernatant and store it at -80 ° C until use. Fix the liquid nitrogen in the lung lobes or perform formalin fixation for use.
  • BALF bronchoalveolar lavage fluid
  • Wax immersion Soak soft wax I for 90min and soft wax II for 90min at a temperature of 60-65 °C.
  • E. Embedding Put the tissue block after the wax immersion into the embedding frame with clean wax liquid in the same orientation. After the surface layer of the wax liquid solidifies, put it on the ice table and cool it to get the wax block containing the tissue block .
  • the tissue wax block can be stored in the refrigerator at 4 °C for future use.
  • the wax block is trimmed with a blade into a regular four-sided platform, which is slightly cooled on ice, and fixed on the wax block clamp of the rotary paraffin slicer, so that the wax block cut surface is parallel to the blade edge and screwed tightly; the slice quality is based on the wax block
  • the thickness is 4um, which contains tissue without wrinkles, knife marks, cavitation or cracks.
  • PBS 0.02M, pH7.4
  • Blocking To remove excess water, add 100 ⁇ l of 5% BSA blocking solution dropwise to each tablet. After incubating at room temperature for 10 minutes, remove excess liquid without washing.
  • E. Incubate the secondary antibody Incubate the primary antibody overnight, remove it, rinse with PBS for 3min ⁇ 3 times, shake off excess water, add 100 ⁇ l of anti-primary antibody IgG-FITC or IgG-TRIC dropwise to each tablet, place in a wet box Incubate at °C for 30min; rinse with PBS for 3min ⁇ 3 times.
  • Primary antibody Stock solution: 1ml diluted in PBS, 30 ⁇ l aliquots stored at -80 °C; Working solution: diluted 180 times with PBS before use.
  • Secondary antibody Stock solution: 1ml Reagent Diluent diluted, 30 ⁇ l aliquot -80 °C preservation; Working solution: Reagent Diluent diluted 180 times before use.
  • Standard product Stock solution: 0.5ml Reagent Diluent diluted into 10 ⁇ l aliquots -80 °C, 6 months; Working solution: 5-7 concentration gradients are diluted with Reagent Diluent etc. according to the instructions.
  • PBS 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , pH 7.2. Store at 4 ° C for 6 months, do not freeze.
  • Reagent Diluent (blocking solution): 1% BSA / PBS
  • Basal fluid Streptavidin-HRP, diluted with Reagent Diluent before use (1: 200)
  • Block block with 300 ⁇ l blocking solution, seal the plate, and incubate at 25 °C for 1h (sample preparation)
  • Secondary antibody 100 ⁇ l secondary antibody, plate sealing, 37 °C, incubate for 2h (preparation of coloring solution, avoid light)
  • Sensitization 100 ⁇ l of substrate solution, seal the plate, incubate at 25 ° C for 20 min, protected from light (preparation of substrate solution, protected from light)
  • Color development 100 ⁇ l color developing solution, seal plate, incubate at 25 °C for 20min, avoid light
  • E. Ophthalmic forceps lift the uterine horn upwards, subtract the surrounding connective tissue, place the cut uterus in a 10cm Petri dish containing PBS, and divide three consecutive 10cm Petri dishes containing PBS to wash three times and remove blood cells.
  • G Use ophthalmic scissors to remove the internal organs of the embryo (note: the internal organs are darker in color), head, limbs, and tail, retain the torso of the embryo, transfer it to a new 10cm Petri dish containing PBS, rinse with PBS three times .
  • human lung embryo fibroblasts HFL-1, human lung embryo fibroblasts MRC-5 and mouse MEF cells were used.
  • the drugs are divided into experimental drugs, control drugs and stimulating factors, all of which are formulated with PBS.
  • the dosage of experimental drugs is papaverine high dose (PAP H, 100 ⁇ M), medium dose (PAP M, 30 ⁇ M) and low dose (PAP L , 10 ⁇ M); PF-2545920 high dose (PF) 10 ⁇ M, low dose (PF3 ⁇ M);
  • the control drug doses are pirfenidone high dose (PFD H, 0.1mg / ml), medium dose (PFD H, 0.03mg / ml) and low dose (PFD H, 0.01mg / ml),
  • the stimulating factor is TGF- ⁇ 1 (10ng / ml).
  • the culture medium of HFL-1 cells is F12K of 10% FBS, and the plate is plated when it reaches 80-90% fusion, according to 3.5 ⁇ 10 ⁇ 5 (6cm dish), 1 ⁇ 10 ⁇ 5 (12-well plate) or 5 ⁇ 10 ⁇ 3 (96-well plate) was plated, when the cells reached a fusion degree of about 60%, they were treated with 0.4% FBS starvation, and the liquid was changed after 24h, and then added drugs and TGF- ⁇ 1 treatment for 48h, followed by immunoblotting, immunofluorescence Experiment or CCK-8 test; or add TGF- ⁇ 1 treatment for 48h, the detection of muscle index ⁇ -SMA and collagen I.
  • MRC-5 cells and MEF cells were cultured in DMEM high glucose medium containing 10% FBS. When starved, 0.5% FBS was used for 24 hours, and the follow-up experiment was consistent with HFL-1.
  • control well (medium containing cells, CCK-8, drug, without TGF- ⁇ 1)
  • step 5 After the cells are treated as described in step 5 (2), aspirate the cell culture medium and wash with PBS for 3 min ⁇ 3 times; add 4% formaldehyde solution and incubate at room temperature for 15 min. Wash with PBS for 3min ⁇ 3 times.
  • Blocking add 5% BSA in PBS and incubate at room temperature for 1 hour.
  • FITC-labeled goat anti-rabbit was diluted 1: 500 in PBS containing 5% BSA. After incubation at room temperature in the dark for 30 min, it was washed with PBS for 3 min ⁇ 3 times.
  • DAPI counterstaining After diluting DAPI 1: 1000 with PBS, incubate at room temperature in the dark for 10 min, and wash with PBS for 3 min ⁇ 3 times.
  • PICT Phosphatase Inhibitor Cocktail Tablets, Roche, Switzerland 1 tablet dissolved in 1ml of double distilled water to make 10 ⁇ l / ml, 150 ⁇ l aliquot preservation at -20 °C.
  • PhosSToP (Swiss Roche) 1 tablet dissolved in 1ml of double-distilled water to make 10 ⁇ l / ml, 100 ⁇ l divided and stored at -20 °C.
  • Lung tissue homogenate in a 2ml EP tube with 0.5ml lysate per whole lung at high speed, shake in an ice bath for 30min, and vortex every 5min during the period.
  • Cells 40 ⁇ l lysate in 6cm dish, transferred to 1.5EP tube ice bath, ice bath for 30min, vortex every 5 minutes during the period.
  • Electrophoresis 70V 60min concentrated into a line, 130V 45min separation.
  • PDE10A (1: 500); ⁇ -SMA (1: 600); FN (1: 1000); collagen 1 (1: 1000); P-smad2 (1: 1000); P-smad3 (1: 2000); smad2 (1: 1000); smad3 (1: 2000); beta-catenin (1: 1000); GAPDH (1: 8000);
  • Tissue homogenate 6 levels of homogenate until there is no obvious tissue block (operation on ice) shake for several minutes / shake for 30min
  • Reagent M-MLV system usage (20ul) The above template RNA / primer and other mixtures 12ul 5xPrimeScript8Buffer 4ul RNase free dd H 2 O 2ul dNTP Mixture (10mM) 1ul RNase Inhibitor (40u / ul) 0.5ul M-MLV (200u / ul) 0.5ul
  • Reagent RealTime PCR reaction system (10ul) 2 ⁇ TaqMasterMix 5ul Forword Primer (20uM) 0.25ul Reverse Primer (20uM) 0.25ul Template DNA ⁇ 0.2ug RNase-Free Water Up to 10ul
  • the present invention adopts Gaphpad Prism Version 5.01 software to analyze and process all data. All measurement data is expressed by Mean ⁇ SEM. T-test is used to test the difference between groups and between groups. P ⁇ 0.05 means significant statistical difference, P ⁇ 0.01 means extremely Significant statistical difference.
  • the dosage of papaverine and pirfenidone in lung fibroblasts is first determined by the CCK-8 experiment.
  • the cells reach a fusion degree of about 60%, 0.4% FBS starvation treatment, after changing the fluid after 24h, add drugs, we use papaverine (PAP) 100 ⁇ M, 30 ⁇ M and 10 ⁇ M are high, medium and low doses, and we also use the positive drug pirfenidone (PFD) high and medium , Low three doses, respectively 0.1mg / ml, 0.03mg / ml and 0.01mg / ml (equivalent to 540 ⁇ M, 162 ⁇ M and 54 ⁇ M).
  • PAP papaverine
  • PFD positive drug pirfenidone
  • Fig. 2 (C shows no added drugs and TGF- ⁇ ), the CCK-8 test performed on HFL-1 cells and MRC-5 cells showed that except for the high dose of PAP, the cells were reduced to a certain extent. In addition to the activity, the remaining three doses of PAP, medium and low doses and PFD have no significant effect on cell activity.
  • TGF- ⁇ 1 induced myofibroblast activation was established.
  • TGF- ⁇ 1 10ng / ml
  • TGF- ⁇ 1 10ng / ml
  • cells at the same time point were used as controls.
  • Cells were collected after 2, 3, 4, and 7 days of stimulation, respectively.
  • Trizol was used to cleave and extract RNA, and real-time quantitative PCR was used to detect fibroblast myogenic indexes ⁇ -SMA and collagen I, such as As shown in Figure 3, TGF- ⁇ 1 can significantly stimulate the expression of ⁇ -SMA and collagen I on days 2, 3, 4 and 7 and reach the highest value on the next day. Therefore, in the subsequent experiments, we used the 48h time point to harvest the cells and follow-up experiments.
  • the present invention also detected the expression of PDE10A under the stimulation of TGF- ⁇ 1, and also showed that the mRNA expression was the highest on the second day.
  • the present invention uses HFL-1 cells to investigate the effect of papaverine on TGF- ⁇ 1-induced fibroblast myogenesis.
  • papaverine and pirfenidone are treated with different doses for 30 minutes, and then 10 ng / ml TGF- ⁇ 1 is used to stimulate for 48 hours.
  • ⁇ -SMA molecule as an indicator of fibroblast muscularization.
  • Cellular immunofluorescence assay was used to detect the protein expression of ⁇ -SMA after TGF- ⁇ 1 stimulation of HFL-1 cells for 48 hours.
  • the expression of ⁇ -SMA in the stimulation group increased significantly at 48 hours, while the high level of papaverine The dose significantly reduced the expression of ⁇ -SMA.
  • both middle-dose papaverine and high-dose pirfenidone showed inhibition of HFL-1 myogenesis.
  • pirfenidone showed a tendency to activate myogenesis.
  • papaverine can inhibit fibroblast myogenesis
  • the two fibrotic protein molecules Fibronectin and collagen I generated during the process.
  • high doses of papaverine can significantly inhibit the increase of ⁇ -SMA, Fibronectin and collagen I protein levels induced by TGF- ⁇ 1 in a dose-dependent manner, in which collagen I protein levels are inhibited at the middle dose of papaverine The effect is most obvious.
  • the present invention also adopts another commonly used in vitro model cell of fibroblast myogenesis, MRC-5 cells.
  • FIG. 8 Immunoblotting assay was used to confirm the protein levels of myosin ⁇ -SMA, Fibronectin and collagen I after 48 hours of TGF- ⁇ 1 stimulation of MRC-5 cells, showing that high doses of papaverine can significantly inhibit ⁇ induced by TGF- ⁇ 1 -SMA protein level, but papaverine has no significant significance for the reduction of Fibronectin and collagen I protein levels, that is, it is also shown in Figure 8 that high doses of papaverine can significantly inhibit the increase of TGF- ⁇ 1 induced myogenesis index .
  • PF-2545920 is an effective and highly selective PDE10A inhibitor with IC50 of 0.37nM, which is more than 1000 times more selective than PDE10A.
  • Fig. 9 uses high-dose PF (10 ⁇ M) or / and low-dose PF (3 ⁇ M) to detect the protein expression of myogenic indexes ⁇ -SMA and Fibronectin after 48 hours of TGF- ⁇ 1 stimulation of HFL-1 cells. Signaling pathway protein molecules GSK-3 ⁇ and ⁇ -catenin during fibroblast activation.
  • the results show that high dose of PF-2545920 can significantly inhibit the level of ⁇ -SMA protein induced by TGF- ⁇ 1, which is more effective than papaverine at high dose, and is significantly better than pirfenidone. Therefore, the above results suggest that PDE10A inhibitors can significantly inhibit the activation of myofibroblasts induced by TGF- ⁇ 1, involving upstream regulatory pathways of ⁇ -catenin, such as GSK-3 ⁇ .
  • TGF- ⁇ 1 (10ng / ml) was added for 0.5 hours to detect the phosphorylation of smad3 and smad2, as shown in Figure 11, showing papaverine and pirfenone
  • Papaverine could not inhibit the increase of smad3 / smad2 phosphorylation, suggesting that papaverine may not affect the fibroblast muscularization process by affecting the activation of smad2 / 3.
  • TGF- ⁇ 1 may also affect fibroblast muscularization through some non-classical pathways such as wnt / ⁇ -catenin.
  • TGF- ⁇ 1 10ng / ml was added to stimulate for 48 hours, and Western immunoblotting was used to detect the protein expression of PDE10A and ⁇ -catenin.
  • papaverine can significantly reduce the increase of ⁇ -catenin content induced by TGF- ⁇ 1 after 48 hours, and it is inversely related to the dose of papaverine, which is related to the protein index of fibroblast myosin previously detected.
  • the trend is consistent, confirming that high dose papaverine can reduce the expression of PDE10A, and papaverine has a significant downward trend for ⁇ -catenin stimulated by TGF- ⁇ 1 stimulation.
  • Papaverine as an inhibitor of PDE10A can significantly inhibit the expression of PDE10A.
  • the classic wnt / ⁇ -catenin pathway activates the messy protein (Dvl) after binding to the receptor through Wnt.
  • Dvl can destroy the ⁇ -catenin degradation complex, thereby accumulating unphosphorylated ⁇ -catenin in the cytoplasm, ⁇ -Catenin enters the nucleus and regulates the expression of target genes. Therefore, in order to clarify whether papaverine has an effect on the nuclear entry of ⁇ -catenin, in HFL-1 cells, after treatment with different doses of papaverine and pirfenidone for 30 minutes, TGF- ⁇ 1 (10ng / ml) stimulation for 48 hours, we used immunofluorescence to detect the expression of ⁇ -catenin in the cell.
  • pirfenidone also showed a certain decrease in the intracellular content of ⁇ -catenin, showing that Papaverine can not inhibit the nuclear transfer of ⁇ -catenin, but can significantly reduce the content of ⁇ -catenin in the cytoplasm in a dose-dependent manner.
  • papaverine may inhibit the expression of ⁇ -catenin in the cytoplasm, thereby inhibiting the fibroblast myogenesis induced by TGF- ⁇ 1.
  • the present invention also tested the effect of papaverine in primary fibroblasts isolated from fetal rats, MEF, using fibroblasts isolated from embryonic mice (within 6 passages), adding papaverine and pirfen After 30 minutes after the treatment with ketene, exogenous TGF- ⁇ 1 (10ng / ml) was added for stimulation.
  • the cell immunofluorescence was used to detect the myochemical index ⁇ -SMA, as shown in Figure 14, we used the detection ⁇ -SMA as TGF- ⁇ 1, an indicator of fibroblast muscularization, significantly increased the muscularization of primary fibroblasts in mice, and papaverine reversed the process of fibroblast muscularization in a dose-dependent manner, showing the separation in mice
  • the addition of papaverine to the fibroblasts can obviously inhibit the increase of ⁇ -SMA caused by TGF- ⁇ 1 stimulation. Therefore, it is suggested that papaverine can inhibit the primary mouse fibroblast myocytes induced by TGF- ⁇ 1.
  • the present invention uses tracheal infusion to inject C57BL / 6 mouse bleomycin (2.5 mg / kg) to prepare a mouse model of pulmonary fibrosis, as described in the experimental method at the 21st day of modeling
  • C57BL / 6 mouse bleomycin 2.5 mg / kg
  • the papaverine in 3.0mg / kg high-dose group, 0.3mg / kg medium-dose group and 0.1mg / kg low-dose group were used for prophylactic or therapeutic treatment.
  • Masson trichromatic staining was used to detect collagen fibers in the lung tissues of 21-day mice after different treatments to observe the degree of fibrosis.
  • the high-dose and medium-dose papaverine in the prevention group significantly reduced collagen production, while in the treatment group, the high-dose papaverine also showed the effect of reducing collagen production. It is suggested that papaverine high-dose group can significantly inhibit pulmonary fibrosis induced by bleomycin in vivo.

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Abstract

一种PDE10A抑制剂在抑制肺成纤维细胞活性药物中的应用，为肺纤维化提供一个新的治疗靶点PDE10A。

Description

一种PDE10A抑制剂在制备成纤维细胞活性抑制药物中的应用 (一)技术领域
本发明涉及一种PDE10A抑制剂在制备成纤维细胞活性抑制药物中的应用。
(二)背景技术
特异性肺纤维化(IPF)是一组具有进展性、破坏性且具有不可逆转性的肺间质疾病，自诊断起平均生存率为2～3年。目前普遍认为IPF的病理机制是机体应对损伤性刺激启动了保护性的修复过程，但是异常过量的修复导致了组织内环境的紊乱，细胞外基质的过度沉积，以及组织结构的破坏，从而造成了纤维化的发生。到目前为止，只有吡非尼酮和尼达尼布在2014年被FDA批准进行使用，而且是有条件推荐使用。肺纤维化的发病机制错综复杂，需深入研究，因此对于寻找肺纤维化的有效治疗方式具有重要的理论和现实意义。
肺纤维化特征性表现为进行性的细胞外基质沉积，肺组织结构破坏，最终导致肺功能丧失。肺纤维化的典型病理特征为成纤维细胞/肌成纤维细胞聚集灶“Fibroblast foci”的形成，这是活动性纤维化的主要表现。肌成纤维细胞作为纤维灶的主要组成部分，被认为是肺纤维化发病的核心因素。肌成纤维细胞高表达α-平滑肌肌动蛋白(smooth muscle actin,SMA)，具有迁移性和收缩性的特征，可大量分细胞外基质导致胶原纤维网络的形成。因此，作为肺纤维化发生的核心细胞，干预肌成纤维细胞的活化是寻找肺纤维化治疗靶点的重要切入点。
TGF-β1是重要的促纤维化细胞因子，并且TGF-β1是纤维化的主要参与者。TGF-β1能够促进成纤维细胞的增殖和趋化，促进上皮-间质转化，促进成纤维细胞转化为肌成纤维细胞，抑制肌成纤维细胞的凋亡等。TGF-β1可通过其下游的经典的Smad通路和非Smad通路介导促纤维化效应。
之前的研究显示PDE抑制剂(即磷酸二酯酶抑制剂)能在细胞内调节cAMP和cGMP的含量，而cAMP和cGMP在组织的纤维化中扮演着重要的作用。在前期的研究中，我们发现在博来霉素诱导的小鼠肺纤维化模型中PDE10A的含量显著增加，因此，在此次的研究中，我们将探究通过PDE10A的抑制剂罂粟碱及PF-2545920来抑制PDE10A的活性是否能治疗实验性肺纤维化。
PDE10A的抑制剂罂粟碱对心血管、支气管、胃肠道、胆管等平滑肌都有松弛作用，通过松弛血管平滑肌，使冠脉扩张、外周阻力及脑血管阻力降低，临床主要用于缓解伴有动脉痉挛的大脑及外周血管疾病，治疗脑血栓、肺栓塞、肢端动脉痉挛及动脉栓塞性疼痛等；亦可用于治疗肠道、输尿管及胆道痉挛疼痛和痛经，以及作为复方支气管扩张喷雾剂的组分之一；还可用于高血压、心绞痛、并发心律失常的心脏局部缺血症等。对高血压心绞痛、幽门痉挛、胆绞痛、肠绞痛、支气管哮喘等在一般剂量下疗效不显著。而PDE10A抑制剂目前国际上主要在进行中枢神经系统的临床研究，如亨廷顿病(Huntington’s disease)、阿尔茨海默氏病、脑炎、恐惧症、癫痫、失语症、贝尔氏麻痹、脑瘫、睡眠障碍、疼痛、妥瑞氏综合症、精神分裂症、妄想症、药源性 精神病以及恐慌症和强迫症的精神病、焦虑症、运动障碍和/或神经障碍。但目前并没有成功上市的药物供临床应用。比如2016年12月16日辉瑞药企宣布了一项名为“阿玛丽利斯”的亨廷顿舞蹈症临床试验结果，该试验测试了PDE-10抑制剂PF-02545920，试验结果显示该药物不能改善亨廷顿舞蹈症症状。因此，目前并没有PDE10A抑制剂在纤维化治疗中的应用和研究。
(三)发明内容
本发明目的是提供一种PDE10A抑制剂在抑制肺成纤维细胞活性中的应用，对于寻找肺纤维化的有效治疗方式具有重要的理论和现实意义。
本发明采用的技术方案是：
本发明提供一种PDE10A抑制剂在抑制肺成纤维细胞活性药物中的应用。
进一步，所述成纤维细胞为人胚肺成纤维细胞HFL-1、人胚肺成纤维细胞MRC-5或胚胎成纤维细胞MEF。
进一步，所述PDE10A抑制剂为罂粟碱或PF-2545920。
进一步，所述PDE10A抑制剂浓度为10-100μM。
进一步，所述抑制肺成纤维细胞活性药物为预防或治疗肺纤维化的药物，即本发明还提供一种所述PDE10A抑制剂在制备预防或治疗肺纤维化药物中的应用。
进一步，所述药物为有效的抗肺纤维化药物。
本发明所述罂粟碱和PF-2545920结构式如下：
Figure PCTCN2019114644-appb-000001
与现有技术相比，本发明有益效果主要体现在：本发明提供了PDE10A抑制剂在制备抑制肺成纤维细胞活性药物中的新应用，为肺纤维化提供一个新的治疗靶点PDE10A，可能通过抑制PDE10A达到缓解肺纤维化发展的作用，而且效果优于现有的药物吡啡尼酮，不良反应小。
(四)附图说明
图1罂粟碱与吡非尼酮对人肺成纤维细胞HFL-1的活力影响，PAP H:罂粟碱高剂量100μM；PAP M:罂粟碱中剂量30μM；PAP L:罂粟碱低剂量10μM；PFD H:吡非尼酮高剂量0.1mg/ml；PFD M:吡非尼酮中剂量0.03mg/ml；PFD L:吡非尼酮低剂量0.01mg/ml；C:空白对照组；TGF-β1：TGF-β1刺激组。
图2罂粟碱(A)与吡非尼酮(B)对人肺成纤维细胞MRC-5的活力影响。PAP H:罂粟碱高剂量100μM；PAP M:罂粟碱中剂量30μM；PAP L:罂粟碱低剂量10μM；C:空白对照组；TGF-β1：TGF-β1刺激组。
PFD H:吡非尼酮高剂量0.1mg/ml；PFD M:吡非尼酮中剂量0.03mg/ml；PFD L:吡非尼酮低剂量0.01mg/ml；C:空白对照组；TGF-β1：TGF-β1刺激组。
图3 TGF-β1诱导的α-SMA，collagen I和PDE10A mRNA水平的变化。采用TGF-β1分别刺激2、3、4与7天后收集HFL-1细胞进行PCR检测，C代表空白对照组，T代表TGF-β1处理，p*<0.05,p**<0.01.p***<0.001。
图4 TGF-β1诱导的人肺成纤维细胞HLF-1中PDE10A蛋白水平的变化。A为WB代表图，B为灰度分析图。
图5罂粟碱与吡非尼酮对TGF-β1诱导的人肺成纤维细胞HFL-1细胞肌化的影响。A为免疫荧光代表图，B为荧光强度分析图；PAP：罂粟碱；PFD：吡非尼酮，p*<0.05,p***<0.001。
图6罂粟碱与吡非尼酮对TGF-β1诱导的人肺成纤维细胞HFL-1细胞活化的蛋白标志物(α-SMA，FN，COL1)的影响。a：α-SMA的蛋白印迹代表图；b：α-SMA的蛋白印迹灰度分析；c：FN的蛋白印迹代表图；d：FN的蛋白印迹灰度分析；e：COL1的蛋白印迹代表图；f；COL1的蛋白印迹灰度分析。PAP：罂粟碱；PFD：吡非尼酮，p*<0.05,p**<0.01。
图7罂粟碱与吡非尼酮对TGF-β1诱导的人肺成纤维细胞MRC-5细胞肌化的影响。A为免疫荧光代表图，B为荧光强度分析图；PAP：罂粟碱；PFD：吡非尼酮，p***<0.001。
图8罂粟碱与吡非尼酮对TGF-β1诱导的人肺成纤维细胞MRC-5活化的蛋白标志物的影响。A为α-SMA，FN，COL1的蛋白印迹代表图，B为α-SMA的蛋白印迹灰度分析图，C为FN的蛋白印迹灰度分析图，D为COL1的蛋白印迹灰度分析图；PAP：罂粟碱；PFD：吡非尼酮，p**<0.01,p***<0.001。
图9 PF-2545920与罂粟碱、吡非尼酮对TGF-β1诱导人肺成纤维细胞HLF-1细胞活化及对信号通路的影响。A为α-SMA，FN，β-catenin的蛋白印迹代表图，B为FN，GSK3-β的蛋白印迹代表图。PF：PF-2545920；PAP：罂粟碱；PFD：吡非尼酮。PAP H:罂粟碱100μM；PAP M:罂粟碱30μM；PF3，10:PF-2545920 3μM,10μM；PFD H:吡非尼酮0.1mg/ml；PFD M:吡非尼酮0.03mg/ml；Control:空白对照组；TGF-β1：TGF-β1刺激组。
图10 TGF-β1诱导的人肺成纤维细胞HFL-1细胞不同时间点磷酸化smad 2/3的表达，A为磷酸化smad3的蛋白印迹代表图，B为磷酸化smad2的蛋白印迹代表图，C为磷酸化smad3/2的蛋白印迹灰度分析图。
图11罂粟碱与吡非尼酮对TGF-β1诱导人肺成纤维细胞HLF-1细胞的smad2/3的磷酸化的影响。A为磷酸化smad3的蛋白印迹代表图，B为磷酸化smad2的蛋白印迹代表图，C为磷酸化smad3的蛋白印迹灰度分析图，D为磷酸化smad2的蛋白印迹灰度分析图；(PAP：罂粟碱；PFD：吡非尼酮)p*<0.05,p***<0.001。
图12罂粟碱与吡非尼酮对TGF-β1诱导人肺成纤维细胞HFL-1细胞β-catenin蛋白表达的影响。A为β-catenin和PDE10A的蛋白印迹代表图，B为PDE10A蛋白印迹灰度分析图，C为β-catenin蛋白印迹灰度分析图；(PAP：罂粟碱；PFD：吡非尼酮)#<0.05(#为与TGF-β1组做比较)。
图13罂粟碱与吡非尼酮对TGF-β1诱导人肺成纤维细胞HFL-1细胞β-catenin表达分布的影响。PAP：罂粟碱；PFD：吡非尼酮。
图14罂粟碱与吡非尼酮对TGF-β1诱导的小鼠胚胎肺成纤维细胞MEF细胞α-SMA表达的影响。PAP：罂粟碱；PFD：吡非尼酮。
图15小鼠肺组织masson三色染色结果。C:生理盐水对照组；BLM：博来霉素组；DEX：地塞米松组；PAP H:10mg/kg高剂量组；PAP M:3mg/kg中剂量组；PAP L:0.1mg/kg低剂量组。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1
一、实验设计
1、在细胞水平上明确PDE10A亚型的作用环节
(1)采用人胚肺成纤维细胞HFL-1以及人胚肺成纤维细胞MRC-5进行成纤维细胞的肌化实验，体外采用TGF-β1刺激，以α-smooth muscle actin(α-SMA)为转化成功标志物，通过免疫荧光进行检测。并在免疫印记中采用肌化标志蛋白α-SMA、fibronectin(FN)和collagen I(Col I)检测PDE10A抑制剂罂粟碱或PF-2545920的作用。
(2)从小鼠中分离原代胚胎成纤维细胞MEF进行培养，加入PDE10A抑制剂(罂粟碱)及TGF-β1刺激处理48h，采用免疫印记和免疫组化检测成纤维细胞肌化的指标a-SMA，明确PDE10A抑制剂(罂粟碱)的作用。
(3)针对细胞水平上发现的重要信号分子通路TGF-β1/β-catenin，通过采用免疫印记进行检测，明确PDE10A抑制剂对其信号分子的影响作用，以阐述PDE10A抑制剂在抑制PDE10A后的具体作用途径。
2、在整体动物模型上明确博来霉素(bleomycin，BLM)诱导的肺纤维化中PDE10A的表达及作用：采用PDE10A抑制剂(罂粟碱)对肺纤维化模型进行预防(造模后1-14天用药)和治疗(造模后14-21天用药)作用的研究，明确PDE10A抑制剂的效应及作用环节。
二、实验材料
1、实验细胞：
人肺胚胎成纤维细胞HFL-1(CCL153)和人肺胚胎成纤维细胞MRC-5(CCL-171)购自于ATCC，收到细胞系后进行传代培养，HFL-1细胞培养于F12K培养基中(含10％胎牛血清，100U/ml青霉素及100μg/ml的链霉素)，MRC-5细胞培养于DMEM/H培养基中(含10％胎牛血清，100U/ml青霉素及100μg/ml的链霉素)，细胞传代至形状稳定，第三代时进行部分冻存以备用，部分用于下一步实验。
小鼠胚胎成纤维细胞MEF是从14天胎龄小鼠(C57BL/6)中分离而得。获得的MEF细胞用DMEM/H培养基培养(含10％胎牛血清，100U/ml青霉素及100μg/ml的链霉素)，传代至第一代时即进行部分冻存，部分进行下一步实验。细胞培养于37.0℃，5％CO 2的细胞培养箱中，所有操作均在无菌操作台中完成。
2、实验动物
雄性7～8周C57BL/6小鼠，体重19～21g，购自上海斯莱克公司，饲养于浙江大学实验动物中心SPF级别动物房，温度为20～23℃，相对湿度为50％～60％，光照周 期为12h；所有小鼠均得到充足洁净的水与饲料，操作过程均遵守《实验动物管理条例》。
3、实验试剂
(1)罂粟碱：美仑生物公司，中国；
(2)吡非尼酮：美仑生物公司，中国；
(3)PDE10A抑制剂：PF-2545920，美仑生物公司，中国；
(4)TGF-β1:PeproTech公司，美国；
(5)十二烷基磺酸钠(SDS)：Amresco公司，美国；
(6)焦碳酸二乙酯(DEPC)：上海生工生物工程有限公司，中国
(7)TRIS盐酸：Amresco公司，美国
(8)丙烯酰胺ACRYLAMIDE：Amresco公司，美国
(9)Bis-ACRYLAMIDE:Amresco公司，美国
(10)甘氨酸Glycine:Amresco公司，美国
(11)过硫酸铵APS：Amresco公司，美国
(12)Tris-HCL缓冲液(1.5M，pH8.8或pH6.8)：武汉博士德(Boster)生物工程有限公司，中国
(13)RIPA裂解液：碧云天生物科技有限公司，中国
(14)Bi0-Rad Protein Assay Dye Reagent Concentration:Bi0-Rad公司
(15)磷酸酶抑制剂、蛋白酶抑制剂：Roche公司，瑞士
(16)SDDA-PAGE蛋白上样缓冲液(5X)：碧云天生物科技有限公司，中国
(17)抗体稀释液：武汉博士德(Boster)生物工程有限公司，中国
(18)一抗二抗去除液：武汉博士德(Boster)生物工程有限公司，中国
(19)RNAiso Plus:TaKaRa公司，日本
(20)Oligo dTPrimer(50uM)：TaKaRa公司，日本
(21)dNTP Mixture(10mM):TaKaRa公司，日本
(22)Reverse Transcriptase M-MLV(RNase H-):TaKaRa公司，日本
(23)Ribonuclease Inhibitor(RRI):TaKaRa公司，日本
(24)SYBR Green I Master Mix:Roche公司，瑞士
(25)无毒环保苏木素-伊红(HE)染液试剂盒：南京建成科技有限公司，中国
(26)pH6.0枸橼酸钠缓冲液(IHC抗原修复液，100×)：北京康为世纪生物科技有限公司，中国
(27)Masson’s三色染色试剂盒：武汉博士德(Boster)生物工程有限公司，中国
(28)CCK-8试剂：MedChem公司，美国
(29)F12K培养基：武汉博士德(Boster)生物工程有限公司，中国
(30)DMEM/H培养基：Corning公司，美国
(31)澳洲血清FBS：Corning公司，美国
(32)青链霉素双抗：科易
(33)BSA：Sigma Aldrich公司，德国
(34)NC膜：Schleicher&Schuell公司
(35)DAPI：凯基(KeyGEN)生物科技有限公司，中国
(36)FITC偶联二抗：Jackson ImmunoReserch公司，美国
(37)博来霉素(BLM)：日本化药
(38)其它：如无水乙醇、氯仿、氯化钠、磷酸盐等均为市售分析纯试剂，国药集团化学试剂有限公司
4、实验仪器
(1)低温冰箱：Thermo Scientific公司，美国
(2)冷冻离心机：SLICOGEM公司，美国
(3)磁力搅拌器、涡旋混合器、试管摇摆混合仪：其林贝尔制造有限公司，中国
(4)制冰机：Scontman公司，美国
(5)电子天平：上海天平仪器厂，中国
(6)精密天平：Mettler Toledo公司，瑞典
(7)电热恒温鼓风干燥箱(DHG-9145)：上海一恒科学仪器有限公司，中国
(8)微波炉：Galanz公司
(9)电磁炉：广东南海艾迪宝电器有限公司，中国
(10)DNA扩增仪：Eppendorf公司，德国
(11)包埋用石蜡(熔点58.0-60.0℃)：国药集团化学试剂有限公司，中国
(12)石蜡包埋机、独立冷台、轮转式石蜡切片机、切片刀、展片台、烤片台：Leica公司，德国
(13)光学显微镜：Olympus公司，日本
(14)轨道式振荡器：杭州奥盛仪器有限公司，中国
(15)二氧化碳培养箱、微量紫外分光光度计(NanoDrop 2000)：Thermo公司，美国
(16)红外激光双色图像分析系统：LI-COR公司，美国
(17)酶标仪(ELX800UX):Bio-tek instrument公司，美国
(18)蛋白电泳系统：Bio-rad公司，美国
(19)荧光定量PCR仪-480Ⅱ：Roche公司，瑞士
(20)荧光倒置显微镜(NIKON Andor/Zyla)：NIKON公司，日本
三、实验方法
1、BLM诱导肺纤维化模型制备及处理
造模方法：将C57BL/6小鼠随机分为11组，每组6只。所有小鼠在给予药物之前均用4％水合氯醛(用PBS稀释)进行麻醉。组1-组10共60只采用BLM(2.5mg/ml，PBS(pH＝7.4)配制，每只小鼠最终气道滴入量30μl，不足30μl的用PBS补足至30μl)进行气道滴入造模，组11共6只采用PBS(pH＝7.4)进行气道滴入造模。
给药方法：组1-组5为预防组，组6-组10为治疗组，组11为正常对照组。组1和组6均为地塞米松对照组(Dex)，采用腹腔注射地塞米松0.30mg/kg；组2和组7均为低剂量罂粟碱组(PAP L)，腹腔注射罂粟碱1.0mg/kg；组3和组8均为中剂量 罂粟碱组(PAP M)，腹腔注射罂粟碱3.0mg/kg；组4和组9均为高剂量罂粟碱组(PAP H)，腹腔注射罂粟碱10mg/kg；组5和组10均为PBS组(PBS)，腹腔注射等体积PBS。组11腹腔注射等体积PBS(pH＝7.4)。预防组(组1-组5)为造模开始时即同时给药，每天一次，每次0.1ml/10g，共13天；治疗组(组6-组10)为造模开始后第7天开始给药，每天一次，每次0.1ml/10g，共13天；组11为造模开始时同时注射PBS，每天一次，每次0.1ml/10g，共13天。造模14天和21天后将小鼠用4％水合氯醛过量麻醉，腹主动脉放血处死，结扎右肺后，暴露气管行气管插管，用PBS(pH7.4)0.5ml分2次进行支气管肺泡灌洗，支气管肺泡灌洗液(BALF)回收率高达80％，冰上保存，混匀后取50μl进行白细胞计数。剩余BALF离心2000rpm，10min，4℃。取细胞沉淀涂片，晾干后进行瑞氏染色，进行细胞分类计数。留取上清液，-80℃保存备用，将肺叶液氮固定或进行福尔马林固定备用。
2、肺组织病理学检测
(1)肺石蜡包埋
A.取材与固定：处死小鼠后无菌剪小鼠肺组织，置于4％的福尔马林溶液中，固定3-7天后将肺组织装入包埋盒中，用铅笔在每个盒子上做标记。
B.梯度脱水：分别为体积浓度75％乙醇水溶液1h或过夜；体积浓度85％乙醇水溶液1h；体积浓度95％乙醇水溶液Ⅰ1h；体积浓度95％乙醇水溶液Ⅱ1h；体积浓度95％乙醇水溶液Ⅲ1h；体积浓度100％乙醇水溶液Ⅰ1h；体积浓度100％乙醇水溶液Ⅱ1h；
C.透明：二甲苯Ⅰ30min；二甲苯Ⅱ30min；
D.浸蜡：在60-65℃的温度中依次浸泡软蜡Ⅰ90min；软蜡Ⅱ90min。
E.包埋：将浸蜡完全后的组织块按相同的取向放入装有干净蜡液的包埋框，待蜡液表层凝固后放于冰台上冷却，即得含组织块的蜡块。组织蜡块可存放于4℃冰箱备用。
F.切片：蜡块用刀片修整成规整的四棱台，冰上稍冷却，固定于轮转式石蜡切片机的蜡块钳上，使蜡块切面与刀刃平行，旋紧；切片质量以蜡块厚度为4um，内含组织且无皱折、刀痕、空泡或破裂为准。
G.展片：将选中的蜡片轻轻托放于水中，展片水温为46℃，展片时间以蜡片刚好舒展平整为宜，然后立即用粘附载玻片托起，甩多余水分使蜡片平整牢附于载玻片上。
H.烤片：将玻片平放于烤片台，或置于烘箱中干燥过夜；烤片温度42～46℃。次日收回切片，存放于4℃冰箱备用，待行H&E染色，Masson’s三色染色和免疫组织荧光染色。
(2)H&E染色
A.切片脱蜡：在二甲苯Ⅰ10min；二甲苯Ⅱ10min
B.切片水化：100％乙醇5min；95％乙醇5min；85％乙醇5min；75％乙醇5min；清水漂洗5min。
C.H&E染色：切片甩去多余水分，每片滴加50-100ul(视组织大小而定)，室温静置5min；清水漂洗干净后，每片滴加50ul伊红染液，约20s后再次用清水漂洗 干净；1％盐酸酒精(配法：1ml36.5％浓盐酸加入99ml75％乙醇中，混匀)分化5s左右，使染色浓淡均一、适中。
D.脱水：切片浸入100％乙醇中5min，泡两次，或适当延长时间以保证脱水彻底，取出后二甲苯透明(此步可省)，室温充分晾干。
E.封片：滴加适量(1-2滴)中性树胶于切片上，再将洁净盖玻片倾斜放下使封片剂完全覆盖组织，注意避免出现气泡。
(3)Masson’s三色染色
A.脱蜡水化(同H&E染色)
B.Masson’s三色染色：置组织切片于苏木素染液3分钟，流水冲洗，1％盐酸酒精分化3～5秒，流水冲洗，温水返蓝1分钟，流水冲洗，置于丽春红酸性品红溶液中3分钟，流水冲洗，1％磷钼酸分化1分钟，2％苯胺蓝复染1分钟。
C.脱水、透明、封片(同H&E染色)
D.显微镜下观察。
(4)免疫组织荧光染色
A.脱蜡水化(同H&E染色)
B.抗原修复：将切片浸没于500ml 0.1M枸橼酸钠缓冲液(pH6.0)，置于电磁炉上加热煮沸1min后，关闭电源静置4min，如此重复3次，共20min；取出投入冷水中冷却，PBS漂洗3min×3次。
PBS(0.02M，pH7.4)的配方如下：NaCl9g、Na 2HPO 4·12H 2O 7g、NaH 2PO 4·H 2O0.5g、去离子水加至1000ml。
C.封闭：甩去多余水分，每片滴加100μl 5％BSA封闭液，室温孵育10min后，甩去多余液体，不洗。
D.孵育一抗：按体积比1:100用抗体稀释液将一抗(α-SMA一抗、β-catenin一抗)稀释为一抗工作液；切片每片滴加100μl一抗工作液，置于湿盒中，4℃孵育过夜。
E.孵育二抗：孵育一抗过夜后取出，PBS漂洗3min×3次，甩去多余水分，每片滴加100μl抗一抗属性的IgG-FITC或者IgG-TRIC，置于湿盒中，37℃孵育30min；PBS漂洗3min×3次。
F.复染：DAPI染色5min，PBS漂洗3min×3次。
G.封片：切片晾干后，采用抗荧光淬灭剂封片，置于4℃保存。
H.显微镜下观察，图片分析。
3、ELISA测定TGF-beta
(1)试剂
一抗：储备液：1ml PBS稀释，30μl分装-80℃保存；工作液：用前用PBS稀释180倍。
二抗：储备液：1ml Reagent Diluent稀释，30μl分装-80℃保存；工作液：用前用Reagent Diluent稀释180倍。
标准品：储备液：0.5mlReagent Diluent稀释成为10μl分装-80℃，6个月；工作液：按说明书用Reagent Diluent等比稀释5-7个浓度梯度。
PBS：137mMNaCl、2.7mMKCl、8.1mMNa 2HPO 4、1.5mMKH 2PO 4，pH 7.2。4℃保存6个月，不可冻存。
Reagent Diluent(封闭液)：1％BSA/PBS
基底液：Streptavidin-HRP，使用前用Reagent Diluent稀释(1:200)
清洗液：0.05％Tween-20/PBS
显色液：0.1％H 2O 2(30％)/TMB(避光)
终止液：2N H 2SO 4
(2)步骤
1)包被：100μl一抗，封板，25℃孵育12h
2)清洗：300μl清洗液清洗3次，最后一次甩干或扣干
3)封闭：300μl封闭液封闭，封板，25℃孵育1h(样品准备)
4)清洗：300μl清洗液清洗3次，最后一次甩干或扣干
5)加样：100μl样品或标准品，封板，37℃孵育2h。
6)清洗：300μl清洗液清洗3次，最后一次甩干或扣干
7)二抗：100μl二抗，封板，37℃，孵育2h(显色液准备，避光)
8)清洗：300μl清洗液清洗3次，最后一次甩干或扣干
9)增敏：100μl基底液，封板，25℃孵育20min，避光(基底液准备，避光)
10)清洗：400μl清洗液清洗3次，最后一次甩干或扣干
11)显色：100μl显色液，封板，25℃孵育20min，避光
12)终止：50μl终止液
13)读板：在450nm波长读板。
14)绘制标准曲线
15)换算样品中细胞因子浓度
4、原代鼠胚胎成纤维细胞分离
(1)分离胚胎
A.取10个10cm培养皿，每皿加入约10ml含青链霉素双抗的PBS液备用。
B.脱颈处死14.5D的孕鼠，脱颈处死后置于75％的酒精中浸泡3min。
C.在超净台工作台中，将孕鼠仰卧于无菌的塑料薄膜上，用75％酒精消毒小鼠腹部及其周围部位。
D.用组织剪将腹部皮肤横行剪开一个小口，再用无菌镊夹住两端皮肤向上下拉开暴露腹肌，换剪刀剪开腹部肌肉及腹膜以暴露出子宫，可见双角子宫膨大，其内胎鼠整齐排列。
E.眼科镊子向上提起子宫角，减去周围结缔组织，将剪下的子宫置于1个含PBS的10cm培养皿中，连续分置3个含PBS的10cm的培养皿中清洗三遍，去除血细胞。
F.在含PBS的培养皿中用眼科剪将胚胎的包膜剪开，取出胚胎并将胚胎移至另一个含PBS的10cm皿中，用PBS清洗三遍，记录胚胎数量。
G.用眼科剪去除胚胎的内脏(注：内脏的颜色较深)、头部、四肢及尾部，保留胚胎的躯干，将其转移至新的含PBS的10cm培养皿中，用PBS冲洗三遍。
(2)胚胎组织的消化
A.取出胚胎至一个10cm皿的培养皿中(无PBS)，用眼科剪将组织剪碎(大约5-8min)。
B.在培养皿中加入约2ml的胰酶(0.05％)，继续剪2min，直至组织块更小，剪成1mm^3以下的碎块。
C.低胰酶浓度组加入约5ml的0.05％的胰酶，将培养皿置于37℃，5％的CO 2培养箱中孵育15min，期间每隔5min取出培养皿用吸管吹打0.5min，以分散组织。
D.加入与胰酶等量的DMEM高糖培养基终止消化，吹打均匀后移入15ml离心管中，用DMEM高糖培养基冲洗培养皿一遍，再将冲洗液吸入上述的15ml离心管中。
E.200g，5min离心，移弃上清后用DMEM高糖培养基重悬细胞沉淀，并收集至同一离心管中，制成细胞悬液。
(3)细胞接种
A.将细胞接种于10cm培养皿(按每个培养皿接种一个胚胎组织的细胞)，每个10cm培养皿加入10ml DMEM高糖培养基，标为N0。置于37℃，5％的CO 2培养箱中过夜孵育。
B.第二天取出培养皿镜下观察细胞形态及生长状态，并更换培养基。
(4)细胞传代与冻存
A.继续培养至细胞生长汇合至80-90％并处于指数生长期时，即可以进行传代，下一代即标为N1，待N1生长汇合至80-90％时，可以部分进行传代进行后续的实验，部分进行冻存以备用。
5、细胞培养及处理
(1)药物剂量
在体外实验中，采用人肺胚胎成纤维细胞HFL-1、人肺胚胎成纤维细胞MRC-5以及小鼠MEF细胞。药物分为实验药物、对照药物和刺激因子，均用PBS配制而成，其中实验药物剂量分别为罂粟碱高剂量(PAP H，100μM)，中剂量(PAP M，30μM)和低剂量(PAP L，10μM)；PF-2545920高剂量(PF 10μM)，低剂量(PF3μM)；对照药物剂量分别为吡非尼酮高剂量(PFD H，0.1mg/ml)、中剂量(PFD H，0.03mg/ml)和低剂量(PFD H，0.01mg/ml)，刺激因子为TGF-β1(10ng/ml)。
(2)细胞处理
HFL-1细胞的培养基为10％FBS的F12K，达80-90％融合度时进行铺板，按照3.5×10^5(6cm皿)、1×10^5(12孔板)或者5×10^3(96孔板)进行铺板，当细胞达60％左右的融合度时进行0.4％FBS饥饿处理，24h后换液，然后同时加入药物和TGF-β1处理48h，后续进行免疫印记、免疫荧光实验或者CCK-8检测；或者只加入TGF-β1处理48h，肌化指标α-SMA和collagen I的检测。
MRC-5细胞以及MEF细胞培养在含10％FBS的DMEM高糖培养基中。饥饿时采用0.5％FBS处理24h，后续实验与HFL-1一致。
6、细胞活力检测(CCK-8法检测)
(1)按步骤5中(2)所述方法处理细胞
(2)每孔加入10μl的CCK-8试剂，轻敲培养板以帮助混匀，置于37℃，5％的CO 2培养箱中孵育1-4h之后，使用酶标仪测定450nm吸光度，其中设一组空白，即 在不含细胞的培养基中加入CCK-8，测定450nm的吸光度即为空白对照组(C)。
(3)按照以下公式计算细胞存活率：
细胞存活率(％)＝[(As-Ab)/(Ac-Ab)]×100％
As：实验孔(含有细胞的培养基、CCK-8、药物、TGF-β1)
Ac：对照孔(含有细胞的培养基、CCK-8、药物、不含TGF-β1)
Ab：空白对照孔(不含有细胞和处理药物的培养基、仅含CCK-8)
7、免疫细胞荧光染色
(1)固定：当细胞按照步骤5中(2)所述方法处理后，吸去细胞培养基，用PBS洗涤3min×3次；加入4％的甲醛溶液室温下孵育15min。用PBS洗涤3min×3次。
(2)透化：加入0.5％Triton-X100室温下孵育20min，用PBS洗涤3min×3次。
(3)封闭：加入5％BSA的PBS溶液，室温下孵育1h。
(4)一抗孵育：将一抗(α-SMA、β-catenin)用一抗稀释液按体积比1:100稀释后加入12孔中，按300ul/孔(12孔板)，4℃摇床过夜后，用PBS洗涤3min×3次。
(5)二抗孵育：将FITC标记的羊抗兔按1：500稀释于含5％BSA的PBS中，室温下避光孵育30min后，用PBS洗涤3min×3次。
(6)DAPI复染：将DAPI按1：1000用PBS稀释后，室温下避光孵育10min后，用PBS洗涤3min×3次。
(7)封片：采用抗荧光淬灭剂封片，避光室温过夜后，置于4℃保存。
(8)拍片：荧光显微镜下观察，图片分析。
8、Western blot
(1)试剂：
PICT：(Phosphatase Inhibitor Cocktail Tablets、瑞士Roche公司)1片溶于1ml双蒸水制成10μl/ml，150μl分装-20℃保存。
PhosSToP：(瑞士Roche公司)1片溶于1ml双蒸水制成10μl/ml，100μl分装-20℃保存。
PMSF：(中国Boster公司)17.4mg溶于1ml异丙醇制成10μl/ml，100μl分装-20℃保存。
(2)RIPA裂解液提总蛋白(全过程冰上操作)
1)按每1ml RIPA裂解液(1×)：冰上现配现用
PICT：150μl
PhosSToP：100μl
PMSF：100μl(先室温溶解再添加)
2)匀浆机转头清洗：依次清水、75％乙醇、RIPA清洗(4mlEP管装2ml)
3)肺组织：在2mlEP管中按每全肺0.5ml裂解液高速匀浆，冰浴震摇30min，期间每5min漩涡一下。
细胞：6cm皿40μl裂解液，转入1.5EP管冰浴，冰浴30min，期间每5分钟漩涡一下。
4)4℃预冷离心：12000rpm×15min；
5)收集上清；
6)Bio-rad蛋白浓度试剂盒测定蛋白浓度。
A、96孔板每孔10μl样品
B、每孔加200μl蛋白测定1×试剂(由5×稀释而来)
C、放置5min,595nm波长测吸光率D、绘制标准曲线R 2>0.98、计算样品浓度
7)变性：
A、按蛋白提取液体积4:1比例加5X蛋白上样缓冲液
B、100℃、5min
C、12000rpm，3min离心
8)保存：Micro-EP分装，-80℃保存、备用
(3)SDS-PAGE电泳(全程双手套)
A、配10％分离胶并上覆异丙醇
表1、10％分离胶的配制
组分 体积
双蒸水 5.9ml
30％丙烯酰胺/双丙烯酰胺溶液 5.0m1
分离胶缓冲液(Tris PH8.8) 3.8ml
10％SDS溶液 150ul
饱和过硫酸铵 150ul
四甲基乙二胺 4ul
B、分离胶聚合完全后(>40min)，倾出覆盖层液体，尽可能排去凝胶上的液体
C、配5％浓缩胶：加入TEMED快速混合灌注，立即插入梳子避免混入气泡
表2、5％浓缩胶的配制
组分 体积
双蒸水 5.5ml
30％丙烯酰胺/双丙烯酰胺溶液 1.3m1
浓缩胶缓冲液(Tris PH6.8) 1.0ml
10％SDS溶液 80ul
饱和过硫酸铵 80ul
四甲基乙二胺 6ul
D、浓缩胶聚合完全后(>40min)，加电泳液后小心垂直移出梳子
E、加样：按预定顺序加样：10μl枪头或微样器加样缓慢加样，每孔加30ug总蛋白，总体积不超20μl。
F、电泳：70V 60min浓缩压成线，130V 45min分离。
(4)转膜(全程双手套)
A、剪切9张滤纸和1张NC膜，其大小都应与凝胶大小吻合。
B、如下安装转移装置：
负极(黑)-滤纸-凝胶-NC膜-滤纸-正级(白/红)
注意：a、滤纸、凝胶、NC膜需对齐
b、防止气泡产生、有气泡局部短路
C、将电泳仪器整个冰浴
D、300mA,60min或90min转膜
(5)抗体免疫反应
A、封闭0.4ml/cm 2，5％BSA-TBS中，室温摇床1h。
B、孵一抗：按抗体说明书用一抗稀释液进行稀释，4℃摇过夜
PDE10A(1:500)；α-SMA(1:600)；FN(1:1000)；collagen 1(1:1000)；P-smad2(1:1000)；P-smad3(1:2000)；smad2(1:1000)；smad3(1:2000)；beta-catenin(1:1000)；GAPDH(1:8000)；
C、洗膜：TBST 4min×6次
D、孵二抗：用二抗稀释液稀释荧光二抗，4℃摇床1-2h
兔抗：1:5000，鼠抗：1:5000
E、洗膜：TBST 4min×6次
F、红外激光双色图像分析系统处理结果
表3 抗体信息
Figure PCTCN2019114644-appb-000002
9、Realtime PCR测定mRNA表达水平
(1)总RNA提取
细胞：
1)0.5ml(6孔板每孔)Trizol吹打
2)震荡数分钟/摇30min
肺组织：
1)匀浆：50-100mg组织加1ml Trizol
2)组织匀浆：6档匀浆至无明显组织块(冰上操作)震荡数分钟/摇30min
3)萃取：室温置5min，按0.2ml/1ml Trizol加入氯仿，剧烈摇荡15s
4)离心：室温置10min，l2,000rpm 4℃离心15min
5)沉淀：小心取上层水相于新EP管，按0.5ml/1ml Trizol加入预冷异丙醇
6)离心：室温置10min，l2,000rpm 4℃离心15min
7)去杂：小心弃上清，按至少1ml/1ml Trizol加入75％乙醇/DEPC，涡旋
8)离心：室温置5min，l2,000rpm 4℃离心10min(重复步骤7、8)
9)空离：小心弃上清，9000rpm 4℃离心1-2min
10)干燥：中小枪头组合弃上清，适当室温干燥
11)溶解：加10～30μl RNase free dH 2O/DEPC水
12)NanoDrop-2000浓度测定
(2)cDNA合成(20μl体系)
1)2μg RNA加DEPC水到10μl，每份反应体系加2μl OligdT(18)
表4 反应体系
试剂 使用量
Total RNA 2ug
Oligo dT primer(50uM) 2ul
RNase free dd H 2O Up to 12ul
2)70℃保温10min，迅速在冰上急冷2-5min
3)离心数秒钟使模板RNA/引物等的混合液聚集于Microtube管底部
4)配加M-MLV逆转录反应液：
表5 逆转录反应液
试剂 M-MLV体系使用量(20ul)
上述模板RNA/引物等混合物 12ul
5xPrimeScript⑧Buffer 4ul
RNase free dd H 2O 2ul
dNTP Mixture(10mM) 1ul
RNase Inhibitor(40u/ul) 0.5ul
M-MLV(200u/ul) 0.5ul
5)42℃保温1h，70℃保温15min
6)-20℃保存备用
(3)RT-PCR(10μl体系)
按Roche试剂说明书配置反应体系和条件，使用GAPDH和β-acin作为内参基因，定量PCR仪检测基因Ct值，根据2 -ΔΔCt法分析mRNA表达水平。引物详见表7(由上海生工生物技术公司合成).
表6 RT-PCR反应体系
试剂 RealTime PCR反应体系(10ul)
2×Taq MasterMix 5ul
Forword Primer(20uM) 0.25ul
Reverse Primer(20uM) 0.25ul
Template DNA <0.2ug
RNase-Free Water Up to 10ul
表7 引物表
Figure PCTCN2019114644-appb-000003
10、数据处理和统计学分析
本发明采用Gaphpad prism Version 5.01软件分析处理所有数据，所有计量数据用Mean±S.E.M表示，采用T-test检验组内和组间差异，P<0.05即有显著统计学差异，P<0.01即具有极显著统计学差异。
四、实验结果
1、罂粟碱对TGF-β1诱导肌成纤维细胞的活化的影响
1)罂粟碱对HFL-1、MRC-5细胞的活力影响
本发明先通过CCK-8实验来确定在肺成纤维细胞(HFL-1和MRC-5细胞)中罂粟碱与吡非尼酮的使用剂量，当细胞达60％左右的融合度时进行0.4％FBS饥饿处理，24h后换液后，加入药物，我们采用罂粟碱(PAP)100μM，30μM和10μM分别为高、中、低剂量，同时我们也采用阳性药物吡非尼酮(PFD)高、中、低三种剂量，分别为0.1mg/ml，0.03mg/ml与0.01mg/ml(相当于540μM，162μM与54μM)。如图1与图2(C为不添加药物和TGF-β)所示，在HFL-1细胞与MRC-5细胞进行的CCK-8检测中，显示除了PAP高剂量在一定程度上降低了细胞的活性之外，其余包括PAP的中、低剂量和PFD三种剂量均对细胞活性无明显影响。
2)TGF-β1诱导人肺成纤维细胞HFL-1肌化指标α-SMA和collagen I的检测
首先建立TGF-β1诱导肌成纤维细胞活化的体外模型，当细胞达60％左右的融合 度时进行0.4％FBS饥饿处理，24h后换液后，外源性给予TGF-β1(10ng/ml)，同时采用同时间点的细胞为对照，分别刺激2、3、4与7天后收集细胞，采用Trizol进行裂解提取RNA，进行实时定量PCR检测成纤维细胞肌化指标α-SMA和collagen I，如图3所示，TGF-β1在2、3、4与7天可明显刺激α-SMA和collagen I的mRNA表达，且都在第二天达最高值。因此，在之后的实验中，我们均采用48h的时间点来进行细胞的收获以及后续的实验。
另外，本发明也检测了PDE10A在TGF-β1的刺激下的表达情况，也显示了在第2天时mRNA表达最高。
进一步我们采用免疫印记WB的方式对PDE10A的表达进行验证，在TGF-β1刺激HFL-1细胞48小时之后收集蛋白检测PDE10A的含量，如图4所示，48h时刺激组PDE10A的蛋白水平显著增加。
3)罂粟碱对TGF-β1诱导的成纤维细胞肌化的影响
接下来，本发明用HFL-1细胞中来进行罂粟碱对TGF-β1诱导的成纤维细胞肌化的影响的探究。本发明中首先给予罂粟碱与吡非尼酮不同剂量处理30分钟后，再给予10ng/ml的TGF-β1刺激48小时，我们用α-SMA分子作为成纤维细胞肌化的指标。采用细胞免疫荧光实验法检测TGF-β1刺激HFL-1细胞48小时后的α-SMA的蛋白表达，如图5所示，刺激组α-SMA的表达在48小时明显增加，而罂粟碱的高剂量明显降低了α-SMA的表达，另外，中剂量的罂粟碱与高剂量吡非尼酮均显示出了对HFL-1的肌化现象的抑制。但吡非尼酮显示出了对肌化现象的活化趋势。
为了进一步验证罂粟碱能够抑制成纤维肌化现象，我们采用蛋白印记的方式来检测TGF-β1刺激HFL-1细胞48小时后的肌化指标α-SMA的蛋白表达，同时检测在成纤维细胞活化过程中产生的两种促纤维化的蛋白分子Fibronectin与collagen I。如图6所示，罂粟碱高剂量可明显抑制由TGF-β1诱导的α-SMA、Fibronectin与collagen I蛋白水平的增高，且呈剂量依赖性，其中collagen I蛋白水平在罂粟碱中剂量时抑制效果最明显。另外，本发明还采用了另一个常用的成纤维化细胞肌化的体外模型细胞，MRC-5细胞。实验结果显示与罂粟碱在HFL-1细胞的影响结果一致。采用了细胞免疫荧光实验法来检测TGF-β1刺激MRC-5细胞48小时后的α-SMA的蛋白表达，如图7所示，显示出TGF-β1刺激的α-SMA的表达明显增高，罂粟碱高剂量能显著的降低由TGF-β1在MRC-5上诱导的成纤维细胞肌化。图8采用免疫印迹法检测法确认TGF-β1刺激MRC-5细胞48小时后的肌化指标α-SMA、Fibronectin与collagen I蛋白水平，显示罂粟碱高剂量可明显抑制由TGF-β1诱导的α-SMA蛋白水平，但罂粟碱对Fibronectin与collagen I蛋白水平的降低没有显著性意义，即在图8上也显示出了罂粟碱的高剂量能够明显抑制由TGF-β1诱导的肌化指标的增加。
PF-2545920是一种有效的，高选择性的PDE10A抑制剂，IC50为0.37nM，比作用于PDE选择性高1000倍以上。图9采用了高剂量PF(10μM)或/和低剂量PF(3μM)对TGF-β1刺激HFL-1细胞48小时后的肌化指标α-SMA、Fibronectin的蛋白表达进行了检测，同时检测在成纤维细胞活化过程中信号通路蛋白分子GSK-3β和β-catenin。结果显示PF-2545920高剂量可明显抑制由TGF-β1诱导的α-SMA蛋白水平，比罂粟碱高剂量的效果更明显，也明显优于吡非尼酮。因此，以上结果提示PDE10A抑制剂 能够明显抑制由TGF-β1诱导的肌成纤维细胞的活化，涉及β-catenin的上游调节通路，如GSK-3β。
2、罂粟碱对smad2/3活化的影响
为探讨罂粟碱对TGF-β1有诱导的经典的Smad通路的作用，首先用TGF-β1(10ng/ml)刺激HFL-1细胞(不添加罂粟碱)，在0.5，1，2，3小时分别采用Western印记检测磷酸化Smad3和Smad2以及其各自的总smad3和总smad2的表达程度。如图10所示，在TGF-β1(10ng/ml)的作用下成纤维细胞内磷酸化的smad3与smad2表达明显增加，且呈时间依赖性，于刺激后0.5小时达到高峰。
在给予不同剂量的罂粟碱和吡非尼酮30分钟之后加入TGF-β1(10ng/ml)刺激0.5小时，分别检测smad3和smad2的磷酸化情况，如图11所示，显示罂粟碱与吡非尼酮均对TGF-β1刺激的smad3和smad2的磷酸化升高无降低作用。而罂粟碱不能抑制smad3/smad2的磷酸化的增加，提示罂粟碱可能不通过影响smad2/3的活化来影响成纤维细胞的肌化过程。
3、罂粟碱对β-catenin通路的影响
TGF-β1除了通过经典的smad通路来促进成纤维细胞的肌化，还可能通过一些非经典的通路例如wnt/β-catenin来影响成纤维细胞的肌化。为了探究罂粟碱在TGF-β1诱导的成纤维细胞活化过程中β-catenin的作用，我们采用与之前实验一致的给药方法，在HFL-1细胞中，加入不同剂量的罂粟碱和吡非尼酮30分钟之后，加入TGF-β1(10ng/ml)刺激48小时，利用Western免疫印迹来检测PDE10A以及β-catenin的蛋白表达情况。如图12，显示了48小时后罂粟碱能明显降低由TGF-β1诱导的β-catenin的含量增加情况，且与罂粟碱的剂量呈负相关，与之前检测的成纤维细胞肌化的蛋白指标趋势呈现一致性，确认高剂量的罂粟碱能降低PDE10A的表达量，而罂粟碱对TGF-β1刺激升高的β-catenin有明显的下降趋势。罂粟碱作为PDE10A的抑制剂能明显抑制PDE10A的表达情况。
经典的wnt/β-catenin通路是通过Wnt与受体结合之后激活蓬乱蛋白(Dvl)，Dvl能破坏β连环蛋白降解复合物，从而使未磷酸化的β-连环蛋白在胞质中积累，β-连环蛋白进入细胞核内，调节靶基因的表达。因此，接下来为明确罂粟碱是否对β-catenin的入核环节有作用，在HFL-1细胞中，用不同剂量的罂粟碱和吡非尼酮处理30分钟后，加入TGF-β1(10ng/ml)刺激48小时，我们采用免疫荧光的方法检测了β-catenin在胞内的表达情况，如图13所示，吡非尼酮对β-catenin的胞内含量也有一定的降低趋势，显示了罂粟碱不能抑制β-catenin的核转移，但能明显降低β-catenin在胞质的含量，且呈剂量依赖性。
因此，该结果提示罂粟碱可能通过抑制β-catenin在胞质中的表达，从而对TGF-β1诱导的成纤维细胞肌化过程起抑制作用。
4、罂粟碱在胎鼠分离的成纤维细胞中的作用
在体外实验中，本发明还检测了罂粟碱在胎鼠中分离的原代成纤维细胞MEF中的作用，采用胚胎小鼠中分离的成纤维细胞(6代以内)，加入罂粟碱和吡非尼酮处理后30分钟再加入外源性TGF-β1(10ng/ml)进行刺激，48小时之后进行细胞免疫荧光检测肌化指标α-SMA，如图14所示，我们以检测α-SMA作为成纤维细胞肌化 的指标，TGF-β1明显增加了小鼠原代成纤维细胞的肌化现象，而罂粟碱呈剂量依赖性逆转了成纤维细胞肌化这一过程，显示了在小鼠分离的成纤维中加入罂粟碱能明显抑制由TGF-β1刺激引起的α-SMA的升高。因此，提示罂粟碱能抑制由TGF-β1诱导的原代小鼠成纤维细胞肌化过程。
5、罂粟碱在博来霉素造模的小鼠中的作用
在体内实验中，本发明采用气管滴注的方式注射C57BL/6小鼠博来霉素(2.5mg/kg)来制备肺纤维化的小鼠模型，按实验方法中的介绍于造模21天时处理小鼠，分别采用3.0mg/kg高剂量组，0.3mg/kg中剂量组与0.1mg/kg低剂量组的罂粟碱进行预防或治疗处理。用Masson三色染色法来检测经过不同处理的21天小鼠的肺组织中胶原纤维来观察纤维化程度。如图15所示，在博来霉素造模组，肺组织Masson三色染色中胶原纤维呈蓝色，在肺间质有大量胶原纤维沉积，尤其在气管周围。与治疗组的罂粟碱相对比，罂粟碱高剂量的预防组可以看到显著抑制了肺间质胶原的沉积，而中剂量组虽然肺泡间隔有增宽，但也明显抑制了胶原沉积。结果均显示博来霉素组明显增加了气管周围间质的胶原纤维的积聚。阳性对照药显著降低了胶原纤维的积聚，另外，预防组的高剂量与中剂量罂粟碱均明显降低了胶原的产生，而在治疗组，高剂量的罂粟碱也显示了降低胶原产生的效果。提示罂粟碱高剂量组在体内实验中能显著抑制由博来霉素诱导的肺纤维化。

Claims (5)

  1. 一种PDE10A抑制剂在制备成纤维细胞活性抑制药物中的应用。
  2. 如权利要求1所述的应用，其特征在于所述成纤维细胞为人胚肺成纤维细胞HFL-1、人胚肺成纤维细胞MRC-5或胚胎成纤维细胞MEF。
  3. 如权利要求1所述的应用，其特征在于所述PDE10A抑制剂为罂粟碱或PF-2545920。
  4. 如权利要求1所述的应用，其特征在于所述药物为制备预防或治疗纤维化的药物。
  5. 如权利要求4所述的应用，其特征在于所述预防或治疗纤维化的药物为预防或治疗肺纤维化的药物。
PCT/CN2019/114644 2018-11-02 2019-10-31 一种pde10a抑制剂在制备成纤维细胞活性抑制药物中的应用 WO2020088575A1 (zh)

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