Use of chagmu polysaccharide in the preparation of a drug for preventing and / or treating pulmonary fibrosis

Abstract

The application provides application of chamaecyparis funebris polysaccharide in preparation of a medicine for preventing and / or treating pulmonary fibrosis, and belongs to the technical field of medicines.The application finds that chamaecyparis funebris polysaccharide can effectively prevent and / or treat pulmonary fibrosis, and can effectively reduce lung injury and inhibit lung inflammation; chamaecyparis funebris polysaccharide inhibits fibrosis and migration of human embryo lung fibroblasts induced by recombinant human transforming growth factor, improves lung function injury and pulmonary fibrosis of a bleomycin-induced pulmonary fibrosis mouse, inhibits inflammatory cell infiltration of bronchial alveoli and expression of NOD-like receptor family pyridine domain-containing protein 3 (NLRP3) in lung tissue of the pulmonary fibrosis mouse, and further inhibits lung inflammation; chamaecyparis funebris polysaccharide can also inhibit the Hedgehog signal pathway in vitro and in vivo, and the improvement of lung function of the bleomycin-induced pulmonary fibrosis mouse and the treatment of pulmonary fibrosis by chamaecyparis funebris polysaccharide are further enhanced when assisted by a Hedgehog pathway inhibitor.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of Chamaegu polysaccharide in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis (PF), especially idiopathic pulmonary fibrosis (IPF), is a chronic, progressive, and fatal interstitial lung disease characterized by inflammatory cell infiltration in the lung interstitium, massive deposition of extracellular matrix (ECM), fibroblast (FB) proliferation, and alveolar structural disorder. Clinically, it mainly manifests as a progressive decline in lung function, even respiratory failure. Due to its complex and incompletely understood pathogenesis, IPF treatment remains a challenge in the medical field. Aside from expensive lung transplantation, there are currently no effective radical cures. The diagnosis rate of IPF in my country is relatively low. Regarding treatment, although pirfenidone and nintedanib have been approved for marketing, patients still face problems such as poor drug tolerance and significant side effects, severely impacting their quality of life. There is an urgent need for anti-PF drugs with good tolerability and fewer side effects to improve this situation.

[0003] Natural plants are an important source for the discovery of new drugs. In particular, medicinal and edible plants have attracted much attention from scholars due to their advantages such as being natural, safe, effective, and nutritious. Utilizing medicinal and edible plant resources and combining them with modern scientific methods to develop effective active ingredients with good tolerability, few side effects, and the ability to prevent and / or treat polydipsia (PF) has always been a relentless pursuit of researchers.

[0004] Chamagu, scientifically known as *Brassica rapa* L., is called "Chamagu" in Uyghur (a transliteration). It belongs to the Brassica genus of the Brassicaceae family and is cultivated throughout Xinjiang, my country. It is known as the "longevity fruit" and "desert ginseng." The people of Xinjiang have consumed Chamagu for over two thousand years, and in many areas of southern Xinjiang, Uyghurs consider it an essential daily food. Chamagu is rich in polysaccharides, glucosinolates, phenols, and organic acids, possessing various pharmacological activities including lung-moistening, cough-relieving, and asthma-relieving effects, antioxidant, anti-tumor, anti-radiation, immune-regulating, and organ and neuroprotective properties. Uyghur medicine has long regarded it as a non-toxic, lung-nourishing herbal medicine. As a distinctive medicinal and edible plant native to this ethnic region, Chamagu holds promising prospects for drug development in the prevention and treatment of lung diseases.

[0005] Polysaccharides of Brassica rapa L. (BRPs) are the main active ingredients extracted from Brassica rapa L. which is a kind of medicinal and edible plant. Our previous studies have shown that BRPs have good antioxidant and immunomodulatory activities, can regulate M2 macrophage polarization to M1 phenotype to play an anti-tumor role, and can also alleviate doxorubicin-induced myocardial cell damage in rats through the Nrf2 / HO-1 signaling pathway. However, there is no application of BRPs in the prevention and / or treatment of PF in the prior art. SUMMARY

[0006] The application provides an application of polysaccharides of Brassica rapa L. in the preparation of a drug for preventing and / or treating pulmonary fibrosis (PF), and the BRPs can inhibit the expression of fibrosis marker proteins ColIα1, α-SMA and Fibronectin of fibrosis human embryonic lung fibroblasts induced by recombinant human transformation growth factor (TGF-β1) and inhibit cell migration. In vivo, the BRPs can effectively improve the lung function of PF mice induced by bleomycin (BLM), improve the fibrosis of lung tissue, alleviate lung injury and inhibit lung inflammation. The BRPs can also inhibit the Hedgehog signaling pathway in vitro and in vivo. The Hedgehog inhibitor can further enhance the improvement of the lung function of PF mice induced by BLM and the treatment of the fibrosis of lung tissue of PF mice induced by BLM.

[0007] In order to achieve the above application purposes, the application provides the following technical scheme:

[0008] The application provides an application of polysaccharides of Brassica rapa L. in the preparation of a drug for preventing and / or treating pulmonary fibrosis (PF).

[0009] As a preferred, the polysaccharides of Brassica rapa L. prevent and / or treat PF by inhibiting the fibrosis and cell migration of lung cells. The application proves by experiments that the BRPs can inhibit the expression of fibrosis marker proteins ColIα1, α-SMA and Fibronectin of fibrosis human embryonic lung fibroblasts induced by TGF-β1 and cell migration in vitro. In vivo, the BRPs can improve the lung function of PF mice induced by BLM, inhibit the inflammatory lesions, collagen and collagen fiber formation of lung tissue of PF mice induced by BLM and the expression of ColIα1, α-SMA and Fibronectin proteins of lung tissue, and improve the fibrosis of lung tissue.

[0010] The application provides an application of polysaccharides of Brassica rapa L. in the preparation of a drug for preventing and / or treating pulmonary fibrosis (PF). The application proves by experiments that the BRPs can inhibit the inflammatory cell infiltration of bronchoalveolar inflammation and inhibit the expression of NLRP3 protein of inflammasomes.

[0011] The application provides application of chagmu polysaccharide in preparation of a medicine for inhibiting a Hedgehog signal path of lung cells.

[0012] Preferably, the effective concentration of the chagmu polysaccharide on TGF-β1 induced fibrosis human embryo lung fibroblasts is 2-6 mg / mL. In the experiment, the effective concentration of BRPs on BLM induced PF mice is 2-5 g / kg; in clinical application, the effective dose for an adult is 0.22-0.55 g / kg.

[0013] Preferably, the chagmu polysaccharide can be used together with a Hedgehog inhibitor; the Hedgehog inhibitor includes Gant 61. The application proves through experiments that the Hedgehog inhibitor Gant 61 can enhance the improvement of BRPs on total lung capacity, deep inhalation capacity, forced vital capacity and quasi-static compliance of BLM induced PF mice, improve the lung function of PF mice; and enhance the treatment of BRPs on lung tissue fibrosis of BLM induced PF mice.

[0014] Preferably, the medicine includes chagmu polysaccharide and pharmaceutically acceptable adjuvants.

[0015] Preferably, the dosage form of the medicine is a pharmaceutically acceptable dosage form.

[0016] Preferably, the pharmaceutically acceptable dosage form is tablets, granules, capsules, pills, syrup, suspension, powder, oral liquid, mixture.

[0017] Preferably, the total polysaccharide content of chagmu polysaccharide in the medicine is 35%-53%.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] The application provides application of chagmulose (BRPs) in preparation of a drug for preventing and / or treating pulmonary fibrosis (PF). The BRPs provided by the application can inhibit the fibrosis marker protein ColIa1, a-SMA and Fibronectin levels of fibrosis human embryo lung fibroblasts induced by TGF-β1, and inhibit cell migration; in vivo, the BRPs can effectively improve the lung function of a BLM-induced PF mouse, improve lung tissue fibrosis, reduce lung injury and inhibit lung inflammation. The BRPs can also inhibit the Hedgehog signal pathway in vitro and in vivo; a Hedgehog inhibitor can further enhance the improvement effect of the BRPs on the lung function of a BLM-induced PF mouse, and enhance the treatment effect of the BRPs on the lung tissue fibrosis of a BLM-induced PF mouse. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a standard curve graph for determining the total polysaccharide content of BRPs by anthrone-sulfuric acid method.

[0021] Figure 2 It is a monosaccharide component graph of BRPs determined by HPLC method; wherein A is a monosaccharide component internal standard graph; and B is a BRPs monosaccharide component graph.

[0022] Figure 3 It is a graph for detecting the influence of BRPs on the proliferation of human embryo lung fibroblasts by CCK8 experiment; wherein A is the light absorption value of cells at 450 nm; and B is the cell survival rate.

[0023] Figure 4 It is a graph for the influence of BRPs on the morphology of human embryo lung fibroblasts induced by a fibrosis inducer TGF-β1; wherein A is a blank control group; B is a TGF-β1 group (1 ng / mL); C is a TGF-β1+2 mg / mL BRPs group; D is a TGF-β1+4 mg / mL BRPs group; E is a TGF-β1+6 mg / mL BRPs group; and F is a TGF-β1+8 mg / mL BRPs group.

[0024] Figure 5 It is a graph for detecting the influence of BRPs on the expression levels of ColIa1, a-SMA and Fibronectin of human embryo lung fibroblasts induced by TGF-β1 by Western blotting; wherein A is a typical ColIa1 protein band; B is a typical a-SMA protein band; C is a typical Fibronectin protein band; D is the relative expression level of ColIa1; E is the relative expression level of a-SMA; and F is the relative expression level of Fibronectin.

[0025] Figure 6Figure of the effect of BRPs on the migration of fibrosis-induced human fetal lung fibroblasts by TGF-β1; wherein, A is a typical cell migration graph; B is the cell migration rate.

[0026] Figure 7 Figure of the effect of BRPs on the lung function of PF mice induced by BLM; wherein, A is a flow chart of the modeling of PF mice and the administration of BRPs; B is the total lung capacity (TLC); C is the vital capacity (VC); D is the inspiratory capacity (IC); E is the tidal volume (TV); F is the forced vital capacity (FVC); G is the peak expiratory flow (PEF); H is the dynamic lung compliance (Cdyn); I is the quasi-static compliance (Cchord).

[0027] Figure 8 Figure of the effect of BRPs on the inflammatory lesions of lung tissue of PF mice induced by BLM; wherein, A is a typical H&E staining graph; B is a graph of the inflammation score of H&E staining.

[0028] Figure 9 Figure of the effect of BRPs on the interstitial fibrosis of lung tissue of PF mice induced by BLM; wherein, A is a typical Masson staining graph; B is a graph of the fibrosis score of Masson staining.

[0029] Figure 10 Figure of the effect of BRPs on the formation of type I collagen fibers of lung tissue of PF mice induced by BLM.

[0030] Figure 11 Figure of the effect of BRPs on the collagen content of lung tissue of PF mice induced by BLM.

[0031] Figure 12 Figure of the effect of BRPs on the expression of ColIα1 protein of lung tissue of PF mice induced by BLM.

[0032] Figure 13 Figure of the effect of BRPs on the transcription of COLIA1 gene of lung tissue of PF mice induced by BLM.

[0033] Figure 14 Figure of the effect of BRPs on the expression of α-SMA protein of lung tissue of PF mice induced by BLM.

[0034] Figure 15 Figure of the effect of BRPs on the expression of Fibronectin protein of lung tissue of PF mice induced by BLM.

[0035] Figure 16 Figure of the effect of BRPs on the lung injury of PF mice induced by BLM; wherein, A is a typical TUNEL staining graph; B is the percentage of TUNEL staining positive cells.

[0036] Figure 17Figure for the effect of BRPs on BLM-induced PF mice bronchoalveolar inflammatory cell infiltration.

[0037] Figure 18 Figure for the effect of BRPs on the expression of NLRP3, an inflammasome receptor protein.

[0038] Figure 19 Figure for the effect of BRPs on the expression of SHH, Gli1 and Gli2 proteins in TGF-β1-induced fibrotic human fetal lung fibroblasts Hedgehog signaling pathway; wherein A is a typical SHH protein band; B is a typical Gli1 protein band; C is a typical Gli2 protein band; D is the relative expression level of SHH; E is the relative expression level of Gli1; F is the relative expression level of Gli2.

[0039] Figure 20 Figure for the effect of BRPs on the expression of SHH protein in BLM-induced PF mice lung tissue.

[0040] Figure 21 Figure for the effect of BRPs on the expression of Gli1 protein in BLM-induced PF mice lung tissue.

[0041] Figure 22 Figure for the effect of BRPs on the expression of Gli2 protein in BLM-induced PF mice lung tissue.

[0042] Figure 23 Figure for the effect of BRPs on the expression of Sufu protein in BLM-induced PF mice lung tissue.

[0043] Figure 24 Figure for the effect of BRPs on BLM-induced PF mice lung function combined with Hedgehog inhibitor Gant 61; wherein A is a flow chart of PF mice modeling and drug administration; B is total lung capacity (TLC); C is vital capacity (VC); D is inspiratory capacity (IC); E is tidal volume (TV); F is forced vital capacity (FVC); G is maximum respiratory flow (PEF); H is dynamic lung compliance (Cdyn); I is quasi-static compliance (Cchord).

[0044] Figure 25 Figure for the effect of BRPs on BLM-induced PF mice lung tissue inflammatory lesions combined with Hedgehog inhibitor Gant 61.

[0045] Figure 26 Figure for the effect of BRPs on the degree of inflammation in BLM-induced PF mice lung tissue combined with Hedgehog inhibitor Gant 61.

[0046] Figure 27Figure 1. Effect of BRPs combined with Hedgehog inhibitor Gant 61 on the degree of pulmonary interstitial fibrosis in BLM-induced PF mice.

[0047] Figure 28 Figure 2. Effect of BRPs combined with Hedgehog inhibitor Gant 61 on the degree of pulmonary interstitial fibrosis in BLM-induced PF mice.

[0048] Figure 29 Figure 3. Effect of BRPs combined with Hedgehog inhibitor Gant 61 on the formation of type I collagen fibers in the lung tissue of BLM-induced PF mice.

[0049] Figure 30 Figure 4. Effect of BRPs combined with Hedgehog inhibitor Gant 61 on the generation of hydroxyproline in the lung tissue of BLM-induced PF mice. DETAILED DESCRIPTION

[0050] Various exemplary embodiments of the present application will now be described in detail, which should be considered as non-limiting examples of the present application, and are understood to be a more detailed description of certain aspects, features and embodiments of the present application.

[0051] Example 1

[0052] Preparation of BRPs, including the following steps:

[0053] 1) After vacuum drying, the thin slices of Chagamu are crushed into Chagamu dry powder, 1500-2500 mL of purified water is added to every 100 g of Chagamu dry powder, and the mixture is extracted by reflux for 1-6 h, and the extraction is repeated twice. The filtrate is combined after filtration.

[0054] 2) The combined filtrate is centrifuged at 3000 rpm for 15 min, and the supernatant is the Chagamu water extract.

[0055] 3) The Chagamu water extract is concentrated to 200-300 mL in a water bath at 65-70°C, and ethanol is added to the concentrated solution to a final concentration of 80%, and the mixture is placed in a refrigerator at 4°C overnight.

[0056] 4) The supernatant is discarded, and a small amount of distilled water is added to the Chagamu polysaccharide precipitate, which is then transferred to an evaporating dish and dried in a vacuum drying oven at 40-50°C and 0.07 Pa to constant weight, and BRPs are obtained.

[0057] In the embodiments of the present application,

[0058] The total polysaccharide content of the obtained BRPs is 49.13%.

[0059] The monosaccharide components of the obtained BRPs are mannose, rhamnose, galacturonic acid, glucose, galactose and xylose.

[0060] Example 2

[0061] 1. Method:

[0062] 1.1 Determination of total polysaccharide content of BRPs by anthrone-sulfuric acid method

[0063] Accurately weigh 13.30 mg of D-glucose reference substance dried to constant weight at 105°C, and place it in a 50 mL volumetric flask. Dilute to the mark with water, shake well, and prepare a D-glucose solution of 266 μg / mL. Accurately pipette 0 mL, 1.0 mL, 2.0 mL, 4.0 mL, 6.0 mL, and 8.0 mL of the reference solution into different 25 mL volumetric flasks, dilute to the 25 mL mark with water, shake well, and then pipette 1.0 mL of the diluted reference solution into a 10 mL test tube with a stopper. After ice bath, accurately add 4 mL of freshly prepared 2‰ anthrone-sulfuric acid solution (take anthrone 0.2 g, dissolve in a mixture containing 5 mL water and 95 mL sulfuric acid, shake, and cool), shake well, heat in a 90°C water bath for 10 min, remove, and quickly cool to room temperature. Prepare the BRPs solution synchronously with the D-glucose reference substance. After 30 min, determine the absorbance of each reference and BRPs sample at 610 nm with the first one as the blank control, draw the D-glucose-absorbance standard curve, and calculate the total polysaccharide content of BRPs according to the absorbance of the BRPs solution.

[0064] 1.2 Determination of monosaccharide components of BRPs by HPLC method

[0065] Weigh 10 mg of BRPs sample into an ampoule, add 1 mL of ultrapure water to dissolve it thoroughly, and then add an equal volume of 4M trifluoroacetic acid (TFA). Seal the tube with a spray gun, hydrolyze in a 110°C oven for 4 h. Open the ampoule, and pipette out the sample solution, and neutralize it with NaOH. Take 200 μL of the diluted solution, add 200 μL of internal standard, shake well, and pipette 100 μL of the mixture into a 1.5 mL centrifuge tube. Add 0.3M NaOH and 0.5M 1-phenyl-3-methyl-5-pyrazolone (PMP) 120 μL each, mix well, and incubate in a 70°C dark water bath for 1 h. Cool to room temperature, and neutralize with 0.3M HCl to pH = 7. Add 500 μL of chloroform for extraction, mix well, and centrifuge at 7000 rpm for 5 min. Carefully pipette out the upper layer (water layer) into a new centrifuge tube, repeat the extraction for 4 times, and pipette the supernatant of the last extraction into a liquid phase bottle after filtration with a 0.22 μm water phase filter. Detect the monosaccharide components by HPLC.

[0066] HPLC detection conditions: column: Agilent Eclipse XDB-C18(5 μm, 4.6 x 250 mm), flow rate 1 mL / min, column temperature 30 °C, detection wavelength 254 nm, mobile phase A: 18% acetonitrile triethylamine solution, mobile phase B: 60% acetonitrile triethylamine solution, gradient elution.

[0067] 1.3 Effect of BRPs on proliferation of human embryonic lung fibroblast MRC-5

[0068] After routine digestion and collection of MRC-5 cells in logarithmic growth phase, the cell concentration was adjusted to 7 x 10 4 / mL, 100 μL of cell suspension was inoculated in each well of a 96-well plate, and the plate was incubated overnight in a 37 °C, 5% CO2incubator. The next day, BRPs (2, 4, 6 and 8 mg / mL) were added for 48 h. Cell-free blank control wells and drug-free control wells were set up synchronously. After drug treatment, the culture solution was aspirated, 100 μL of serum-free MEM / EBSS culture solution was added to each well, 10 μL of CCK-8 solution was added to each well, and the plate was further incubated in a 37 °C, 5% CO2incubator for 1-2 h. The optical density (OD) at 450 nm of each well was determined by a microplate reader, and the cell survival rate was calculated.

[0069]

[0070] 1.4 Effect of BRPs on morphology and growth state of MRC-5 cells induced by TGF-β1

[0071] MRC-5 cells were inoculated in 6-well cell culture plates at a concentration of 20 x 10 4 / well, and the plates were incubated overnight in a 37 °C, 5% CO2incubator. After washing twice with PBS, the cells were starved with serum-free MEM / EBSS culture solution for 24 h, 1 ng / mL TGF-β1 was added, and different concentrations of BRPs were added to treat the cells for 48 h. The cells were observed and photographed under an inverted optical microscope. The experimental groups were: blank control group, TGF-β1 group (1 ng / mL), TGF-β1 + BRPs (2, 4, 6 and 8 mg / mL) group.

[0072] 1.5 Effect of BRPs on expression levels of ColIα1, α-SMA and Fibronectin of human embryonic lung fibroblasts induced by TGF-β1

[0073] Human embryonic lung fibroblasts MRC-5 were inoculated in 6-well cell culture plates at a concentration of 20 x 10 4Cells were seeded in 6-well plates and incubated overnight. Cells were washed twice with PBS, and then starved for 24 h in serum-free MEM / EBSS medium. After that, 1 ng / mL TGF-β1 and different concentrations of BRPs were added to the cells for 48 h. The experimental groups were: Control group, TGF-β1 group, TGF-β1 + BRPs (2, 4, 6 mg / mL) group. At the end of the experiment, the cells were washed twice with PBS, and then 1 mL of RIPA lysis buffer containing protease and phosphatase inhibitors was added to each well. The cells were lysed on ice for 30 min, and then centrifuged at 14,000 rpm for 30 min. The supernatant was collected, and the protein concentration was determined by BCA method. Equal amounts of protein were subjected to 10% SDS-PAGE and transferred to PVDF membranes at a constant voltage. The PVDF membranes were blocked with 5% non-fat milk / PBST at room temperature for 2 h. Anti-Collal rabbit antibody (1:1000), anti-a-SMA rabbit antibody (1:5000), anti-Fibronectin rabbit antibody (1:1000), and anti-GAPDH mouse antibody (1:4000) were added, respectively, and incubated overnight at 4°C on a shaker. The membranes were washed with PBST for 10 min three times, and then horseradish peroxidase-labeled anti-rabbit IgG (1:2000) or anti-mouse IgG (1:2000) was added and incubated at room temperature for 1.5 h. The membranes were washed with PBST for 10 min three times, and then ECL reagent was added for color development. Western blot protein bands were obtained, and the effects of BRPs on the expression levels of fibrosis-related proteins Collal, a-SMA, and Fibronectin in TGF-β1-induced human fetal lung fibroblasts were observed.

[0074] The integral optical density of each band was determined by Image J software, and quantified by comparison with the internal reference GAPDH.

[0075] 1.6 Effect of BRPs on the migration of TGF-β1-induced fibrosis human fetal lung fibroblasts

[0076] Marker pens were used to draw horizontal and vertical lines on the back of the 6-well plates, with a spacing of about 0.5-1 cm between lines. At least 5 lines were drawn through each well. Human fetal lung fibroblasts were seeded in 6-well plates at a density of about 30 x 10 4 The cells were seeded in 6-well plates and incubated overnight. The cells were washed twice with PBS, and then starved for 24 h in 2 mL / well serum-free MEM / EBSS medium. A vertical line was drawn in the 6-well cell culture plate containing the cells using a 10 μL gun tip, and the cells were washed with PBS for 3 times to remove the cells that floated due to scratching. Then, 1 ng / mL TGF-β1 and BRPs were added to the cells for 48 h, and the migration rate of each group was calculated by Image J software.

[0077]

[0078] 1.7 Effects of BRPs on lung function of PF mice induced by BLM

[0079] 1.7.1 Animal grouping and administration

[0080] After one week of adaptive feeding, SPF C57BL / 6J mice were used to construct PF models by single intratracheal instillation of an appropriate amount of BLM. The next day after modeling, BRPs were administered by gavage once a day for 21 days of continuous intervention. Experimental grouping: control group (normal feeding); BLM model group; BLM+BRPs (5 g / kg) group.

[0081] 1.7.2 Establishment of PF model in mice

[0082] Dilute BLM with physiological saline to the appropriate concentration. After isoflurane anesthesia of the mouse, gently pull the mouse's tongue to press the tongue belly, and look at the glottis. At the moment of the mouse's breathing, quickly push the appropriate amount of diluted BLM along the upper jaw into the trachea. After the end of tracheal instillation, gently pat the mouse's chest with both hands, and assist the mouse to make a rotating action, so that the liquid is evenly distributed in the lung. The normal control group of mice was intratracheally instilled with the same volume of physiological saline under the same conditions.

[0083] 1.7.3 Measurement of lung function in mice

[0084] After the end of animal grouping, modeling and administration, the mice in each group were intraperitoneally injected with 0.8% sodium pentobarbital (dose used: 0.09 mL / 10 g). After the corneal reflex of the mouse disappeared, the mouse was fixed on the operation board, the skin and hair in the anterior region of the neck were removed, the incision was about 1 cm, the subcutaneous tissue was bluntly separated, the trachea was exposed, a small opening was made at the trachea below the ring cartilage with surgical scissors, a tracheal catheter was inserted, surgical sutures were used to fix the tracheal catheter, which was connected with the calibrated small animal respirator to measure the indicators of lung function.

[0085] 1.8 Improvement effect of BRPs on pulmonary fibrosis in PF mice induced by BLM

[0086] 1.8.1 Animal grouping and administration

[0087] After one week of adaptive feeding, SPF C57BL / 6J mice were used to construct PF models by single intratracheal instillation of an appropriate amount of BLM. The next day after modeling, BRPs or positive drug pirfenidone (PFD) were administered by gavage once a day for 21 days of continuous intervention. Experimental grouping: control group (normal feeding); BLM model group; BLM+BRPs (2, 4, 5 g / kg) group and BLM+PFD (200 mg / kg) group.

[0088] 1.8.2 Effects of BRPs on inflammatory lesions in lung tissue of PF mice induced by BLM

[0089] After the completion of animal grouping, modeling and drug administration, the mouse heart was exposed by surgery, and after the double lungs were perfused with a large amount of PBS, the left lung was removed and fixed in 4% paraformaldehyde solution overnight. The next day, the sample was sent for examination, paraffin embedding and sectioning. The paraffin section of the test group was placed in xylene at room temperature for 10 min, repeated twice; then, it was immersed in anhydrous ethanol for 5 min, repeated twice; then, it was immersed in gradient ethanol (95%, 85%, 75%) for 5 min; and then, PBS was added for washing twice, each for 3 min. The PBS was gently discarded, and the excess liquid was absorbed with filter paper. H&E staining was performed, and the pathological changes of the lung tissue of each group were observed, and the degree of inflammation was evaluated.

[0090] 1.8.3 Effect of BRPs on pulmonary fibrosis and type I collagen fiber formation in BLM-induced PF mice

[0091] After the completion of animal grouping, modeling and drug administration, the lung tissue of each group was taken for paraffin embedding and sectioning. After the obtained paraffin section was deparaffinated with xylene, alcohol gradient and hydrated, Masson staining and Sirius Red staining were performed, respectively, to investigate the degree of pulmonary fibrosis and the production of type I collagen fibers.

[0092] 1.8.4 Effect of BRPs on collagen content in lung tissue of BLM-induced PF mice by Sircol assay

[0093] After the completion of animal grouping, modeling and drug administration, the middle two lobes of the right lung were removed, and the operation was performed according to the Sircol assay kit instruction. First, the middle two lobes of the right lung were weighed and placed in a FACS tube containing a suitable amount of homogenate lysis solution. The tissue was homogenized and transferred to a 2 mL EP tube. The tube was shaken on a 4°C shaking table overnight. The next day, the tube was centrifuged at 4°C and 14000 rpm for 10 min. 100 uL of supernatant was taken, 1 mL of Sircol dye reagent was added, and the tube was incubated at room temperature for 1 h. The tube was then centrifuged at 4°C and 14000 rpm for 30 min. The supernatant was discarded, and the precipitate was completely dissolved with 1 mL of alkaline reagent. The optical absorbance value (OD 540 ) at 540 nm was measured by an enzyme-labeled instrument, and the collagen content of the test sample was calculated according to the collagen standard curve.

[0094] 1.8.5 Effect of BRPs on ColIα1 protein expression in lung tissue of BLM-induced PF mice by immunohistochemical staining

[0095] After the completion of animal grouping, modeling and drug administration, the lung tissues of each group were taken for paraffin embedding and sectioning. After the obtained paraffin sections were dewaxed with xylene, gradiently descended with alcohol and hydrated, they were immersed into a citrate buffer, and antigen was repaired at 92-98°C for 15-20 min. After natural cooling to room temperature, 3% H2O2 was added for incubation for 20 min to block endogenous peroxidase. After PBS washing for 3 times, 5% BSA was added for blocking at 37°C for 30 min. After removing the blocking solution, anti-Collal rabbit antibody was added for incubation in a wet box at 4°C overnight. After PBST washing for 3 times, Alexa Fluor 555-labeled anti-rabbit IgG was added for incubation at room temperature in the dark for 2 h. After PBST washing for 3 times, DAPI was used for re-staining of cell nuclei. An anti-fluorescence decay agent was used for mounting, and a laser confocal microscope was used for observation and photographing.

[0096] 1.8.6 RT-qPCR detection of the effect of BRPs on the transcription of COLIA1 gene in the lung tissues of PF mice induced by BLM

[0097] After the completion of animal grouping, modeling and drug administration, the right posterior lobe of the lung cavity was taken and put into 1 mL Trizol reagent, and homogenized on ice at low speed. Total RNA was extracted by using a commercial RNA kit, A260 and A280 were determined by BioDrop, and the purity of RNA was evaluated and its concentration was calculated. According to the instructions of the reverse transcription kit, 1 μg of RNA was used for reverse transcription to prepare cDNA, SYBR kit was used for PCR according to the instructions, GAPDH was used as an internal reference gene, and the transcription level of COLIA1 gene was evaluated by using 2 -ΔΔCT .

[0098] 1.8.7 Effect of BRPs on the expression of fibrosis marker proteins α-SMA and Fibronectin in the lung tissues of PF mice induced by BLM

[0099] After the paraffin sections were dewaxed with xylene, gradiently descended with alcohol and hydrated, they were immersed into a citrate buffer, and antigen was repaired at 92-98°C for 15-20 min. After natural cooling to room temperature, 3% H2O2 was added for incubation for 20 min to block endogenous peroxidase. PBS was washed for 3 times, and 0.5% Triton X-100 prepared with PBS was used for permeating cell membranes at room temperature for 30 min. PBS was washed for 3 times, 5% BSA was added for blocking at 37°C for 30 min. After removing the blocking solution, anti-α-SMA or anti-Fibronectin rabbit antibody was added for incubation in a wet box at 4°C overnight. The next day, PBST was washed for 3 times, Alexa Fluor 555-labeled anti-rabbit IgG was added for incubation at room temperature in the dark for 2 h. PBST was washed for 3 times, and DAPI was used for re-staining of cell nuclei. An anti-fluorescence decay agent was used for mounting, and a laser confocal microscope was used for observation and photographing.

[0100] 1.8.8. Effects of BRPs on lung tissue injury in BLM-induced PF mice

[0101] After xylene deparaffinization, descending ethanol gradient and hydration, lung tissue sections were incubated with appropriate amount of proteinase K solution at room temperature for 20 min to permeate cell membranes; washed with PBS twice to remove proteinase K, and excess liquid was absorbed with filter paper; 50 μL of TUNEL working solution / sample was added, and incubated at 37°C for 1 h in the dark. Washed twice with PBS, and mounted with anti-fluorescence quenching agent containing DAPI, and observed under laser confocal microscope for TUNEL fluorescence signal.

[0102] 1.8.9. Effects of BRPs on lung inflammation in BLM-induced PF mice

[0103] 1) Inflammatory cell counting in bronchoalveolar lavage fluid (BALF): The mice were routinely anesthetized, made to lie on their back and fixed, the skin and hair in the anterior cervical region were removed, a vertical incision was made 1 cm long, the subcutaneous tissue was bluntly separated, the trachea was exposed, a trocar needle was inserted into the trachea of the mouse and fixed, and 800 μL of PBS was used to lavage the bronchoalveolar with a 1 mL syringe, a total of 2 times. The BALF was collected, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended with 100 μL of PBS, and the number of inflammatory cells in the BALF was determined by a cell counter.

[0104] 2) Immunofluorescence staining of inflammasome protein NLRP3: After the animals were grouped, modeled and dosed, the lung tissues of each group were paraffin-embedded and sectioned. After the obtained paraffin sections were dewaxed with xylene, descending ethanol gradient and hydrated, they were immersed in citrate buffer and antigen-repaired at 92-98°C for 15-20 min. After natural cooling to room temperature, 3% H2O2 was added and incubated for 20 min to block endogenous peroxidase. After washing with PBS for 3 times, 5% BSA was added and incubated at 37°C for 30 min. The blocking solution was removed, anti-NLRP3 rabbit antibody was added, and incubated overnight in a wet box at 4°C. The next day, PBST was washed for 3 times, Alexa Fluor 555-labeled anti-rabbit IgG secondary antibody was added, and incubated at room temperature for 2 h in the dark. PBST was washed for 3 times, and the cell nucleus was counterstained with DAPI. Anti-fluorescence quenching agent was mounted, and observed and photographed under laser confocal microscope.

[0105] 1.9. Effects of BRPs on Hedgehog signaling pathway in TGF-β1-induced fibrotic human embryonic lung fibroblasts

[0106] Human embryonic lung fibroblasts MRC-5 were cultured at 20×10 4The cells were inoculated in 6-well plates and cultured overnight. The next day, the cells were washed twice with PBS and then starved for 24 h in serum-free MEM / EBSS medium. The medium was discarded and the cells were treated with 1 ng / mL TGF-β1 and different concentrations of BRPs for 48 h. The experimental groups were: normal control group (Control), TGF-β1 (T) group, T+BRPs (2, 4, 6 mg / mL) group. After drug treatment, the cells were washed twice with PBS, and the cells were lysed with RIPA lysis buffer containing 1x protease and phosphatase inhibitors on ice for 30 min. The cells were centrifuged at 14,000 rpm for 30 min, and the supernatant was collected. The protein concentration of the supernatant was determined by BCA method, and an equal amount of protein was subjected to 10% SDS-PAGE, and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk / PBST at room temperature for 2 h, and then anti-SHH rabbit antibody, anti-Gli1 mouse antibody, anti-Gli2 rabbit antibody and anti-GAPDH mouse antibody were added and incubated at 4°C overnight. The membrane was washed with PBST for 3 times, each for 10 min, and then horseradish peroxidase-labeled anti-rabbit IgG or anti-mouse IgG was added and incubated at room temperature for 1.5 h. The membrane was washed with PBST for 3 times, and then ECL reagent was added for color development. Photographs were taken to observe the effect of BRPs on the expression of Hedgehog signaling pathway proteins in TGF-β1-induced fibrotic human fetal lung fibroblasts.

[0107] 1.10 Effect of BRPs on Hedgehog signaling pathway in BLM-induced PF mouse lung tissue

[0108] SPF level C57BL / 6J mice were adaptively fed for one week, and then a single intratracheal instillation of an appropriate amount of BLM was performed to construct a mouse PF model. The next day after modeling, BRPs or anti-PF positive drug pirfenidone (PFD) was administered by gavage, once a day, for 21 days of continuous intervention. Experimental grouping: control group (normal feeding), BLM model group, BLM+BRPs (5 g / kg) group and BLM+PFD (200 mg / kg) group. At the end of the experiment, the mouse heart was surgically exposed, and after a large amount of PBS was used to lavage the double lungs through the heart, the left lung was removed and fixed in 4% paraformaldehyde solution overnight. The next day, it was sent for examination, paraffin embedding and sectioning. The corresponding paraffin sections were placed in xylene at room temperature for 10 min, repeated 2 times; then, they were immersed in anhydrous ethanol for 5 min, repeated 2 times; then, they were sequentially immersed in gradient ethanol (95%, 85%, 75%) for 5 min; 2 times of PBS washing was performed, each for 3 min. The PBS was gently discarded, and the excess liquid was absorbed with filter paper. The sections were immersed in citrate buffer, and antigen repair was performed at 92-98°C for 15-20 min. Natural cooling to room temperature, 3% H2O2 was added and incubated for 20 min to block endogenous peroxidase. PBS was washed 3 times, and 0.5% Triton X-100 was used to permeabilize the cell membrane at room temperature for 30 min. PBS was washed 3 times, 5% BSA was added, and the sections were blocked at 37°C for 30 min. The blocking solution was removed, and anti-SHH rabbit antibody (1:50), anti-Gli1 mouse antibody (1:500), anti-Gli2 rabbit antibody (1:200) and anti-Sufu rabbit antibody (1:200) were added, respectively, and incubated in a wet box at 4°C overnight. The next day, PBST was washed 3 times, and Alexa Fluor 555-labeled anti-rabbit IgG or Alexa Fluor 488-labeled anti-mouse IgG (1:500) was added, and the sections were incubated at room temperature in the dark for 2 h. PBST was washed 3 times, and 0.5 μg / mL DAPI was used to restain the cell nucleus. The sections were mounted with an anti-fluorescence quencher, and laser confocal microscopy was used for observation and photography.

[0109] 1.11.1 Animal grouping, modeling and drug administration

[0110] 1.11.1 Animal grouping, modeling and drug administration

[0111] SPF level C57BL / 6J mice were adaptively fed for one week, and then the animals were divided into control group (normal culture), BLM group, BLM+Gant 61 group, BLM+BRPs group and BLM+Gant 61+BRPs group. At the time of modeling, the control group was intratracheally instilled with an appropriate amount of PBS, and the other groups were intratracheally instilled with an appropriate amount of BLM to construct a mouse PF model. The next day after modeling, the BLM+BRPs group was given BRPs (3 g / kg) by gavage, once a day, for 21 days of continuous intervention; from the 8th day of modeling, the BLM+Gant 61 group was injected intraperitoneally with Hedgehog inhibitor Gant 61 (10 mg / kg), once every 2 days, until the end of the experiment. The BLM+Gant 61+BRPs group was given drugs synchronously with the corresponding single drug.

[0112] 1.11.2. Add Hedgehog inhibitor and observe the effect of BRPs on the evaluation index of lung function in BLM-induced PF mice

[0113] After the animal grouping, modeling and drug administration were completed, 0.8% sodium pentobarbital (0.09 mL / 10 g) was injected intraperitoneally into each group of mice. After the corneal reflex of the mice disappeared, the mice were fixed on the operation board, the skin and hair in the anterior neck region were removed, a vertical incision about 1 cm was made, the subcutaneous tissue was bluntly separated, the trachea was exposed, a small opening was made at the trachea below the ring cartilage with a surgical scissors, a tracheal catheter was inserted, and after the tracheal catheter was fixed with surgical sutures, it was connected with a calibrated small animal respirator for determination of various indexes of lung function.

[0114] 1.11.3. Add Hedgehog inhibitor and observe the effect of BRPs on lung tissue inflammatory lesions in BLM-induced PF mice

[0115] After the animal grouping, modeling and drug administration were completed, the mouse heart was surgically exposed, and after the left lung was removed after the double lungs were perfused with a large amount of PBS through the heart, it was fixed in 4% paraformaldehyde solution overnight, and sent for examination the next day for paraffin embedding and sectioning. The paraffin section was placed in xylene at room temperature for 10 min, repeated 2 times. Then, it was immersed in anhydrous ethanol for 5 min, repeated 2 times; then, it was immersed in gradient ethanol (95%, 85%, 75%) for 5 min. Add PBS for washing 2 times, 3 min each time. Gently discard the PBS and absorb the excess liquid with filter paper, perform H&E staining, and observe the inflammatory lesions of the lung tissue in each experimental group and evaluate the degree of inflammation.

[0116] 1.11.4. Add Hedgehog inhibitor and observe the effect of BRPs on pulmonary interstitial fibrosis and type I collagen fiber formation

[0117] After the completion of animal grouping, modeling and drug administration, the mouse heart was exposed by surgery, and after the double lungs were perfused with a large amount of PBS through the heart, the left lung was removed and fixed in 4% paraformaldehyde solution overnight. After the lung tissues of each experimental group were paraffin-embedded, sectioned, deparaffinated with xylene, alcohol gradient and hydrated, Masson and Sirius Red staining were performed respectively to observe the pulmonary interstitial fibrosis and the formation of type I collagen fibers in the lung tissue.

[0118] 1.11.5 Adding a Hedgehog inhibitor to observe the effect of BRPs on the hydroxyproline content of lung tissue

[0119] The change in hydroxyproline content can reflect the collagen deposition in the lung tissue. Briefly, 50 mg (W mg ) of lung tissue was weighed into a test tube, 1 mL of hydrolysis solution was added, and it was mixed well. After being covered, it was hydrolyzed at 95°C for 20 min, and mixed well every 10 min. The test tubes were cooled with running water, 10 μL of indicator was added per tube, and it was shaken well, then 1.0 mL of pH adjusting solution was added per tube, and it was mixed well. Then, the pH adjusting solution was added dropwise to each tube while mixing, until the color of the indicator in the liquid changed to yellow-green, and then double distilled water was added to 10 mL (V 水解液 ), and it was mixed well. 3 mL of the diluted hydrolysis solution was taken, an appropriate amount of activated carbon was added, and it was mixed well, then centrifuged at 3500 rpm for 10 min, 1 mL of supernatant was taken, and the absorbance value (OD) at 550 nm was detected, and the hydroxyproline content was calculated according to the following formula.

[0120]

[0121] 2 Experimental results

[0122] 2.1 Determination of the total polysaccharide content of BRPs by the anthrone-sulfuric acid method

[0123] The total polysaccharide content of BRPs was determined by the anthrone-sulfuric acid method. The results showed that within the range of 2.128-17.024 μg / mL, there was a good linear relationship between the D-glucose control and the absorbance at 610 nm, the regression equation was y = 0.04653x + 0.0821, r = 0.9985( Figure 1 ). The absorbance of BRPs at 610 nm was 0.539, and the total polysaccharide content was calculated to be 49.13% according to the standard curve.

[0124] 2.2 Monosaccharide composition of BRPs

[0125] The monosaccharide composition of BRPs was detected by HPLC method using commercial monosaccharide components as standard. The results showed that the monosaccharide composition of BRPs was mannose, rhamnose, galacturonic acid, glucose, galactose and xylose( Figure 2 ).

[0126] 2.3 Effects of BRPs on the proliferation of human embryonic lung fibroblasts

[0127] The effects of BRPs on the proliferation of human embryonic lung fibroblasts (MRC5) were investigated by CCK-8 assay. The results showed that BRPs did not inhibit the proliferation of MRC5 cells at a concentration of not more than 6 mg / ml. BRPs at a concentration of 8 mg / ml partially inhibited the proliferation of MRC5 cells ( Figure 3 ), affecting cell viability. Therefore, 2, 4 and 6 mg / ml were used as low, medium and high concentrations for subsequent experiments, respectively.

[0128] 2.4 Effects of BRPs on the morphology of TGF-β1-induced human embryonic lung fibroblasts

[0129] TGF-β1 is one of the main inducers of fibrosis, which can promote fibroblast proliferation and collagen deposition, leading to organ fibrosis. To observe the effects of BRPs on the morphology of TGF-β1-induced fibrotic MRC5 cells, human embryonic lung fibroblasts MRC5 were seeded in 6-well plates and cultured overnight. After 24 h of serum-free MEM / EBSS starvation, 1 ng / ml TGF-β1 and different concentrations of BRPs were added simultaneously for 48 h. Figure 4 The results showed that the cell bodies of the control group were elongated and spindle-shaped with clear boundaries. Compared with the control group, the cell boundaries of the TGF-β1 model group were blurred, and the cell bodies were flat and flat, and the fibroblasts were differentiated into myofibroblasts. Compared with the model group, 2-6 mg / mL BRPs could reverse the TGF-β1-induced fibrosis of MRC5 cells to varying degrees, while 8 mg / mL BRPs could reverse the TGF-β1-induced cell shape change and differentiation, and also had a significant inhibitory effect on cell proliferation.

[0130] 2.5 BRPs can improve the fibrosis of TGF-β1-induced fibrotic human embryonic lung fibroblasts

[0131] Western blotting was used to detect the effects of BRPs on the expression levels of fibrosis marker proteins ColIα1, α-SMA and Fibronectin in TGF-β1-induced fibrotic MRC5 cells. Figure 5 The results showed that compared with the control group, TGF-β1 significantly up-regulated the expression levels of fibrosis marker proteins ColIα1, α-SMA and Fibronectin, suggesting that TGF-β1 can induce fibrosis of MRC5 cells. Compared with the TGF-β1 model group, BRPs significantly reduced the protein levels of ColIα1, α-SMA and Fibronectin in a dose-dependent manner, suggesting that BRPs can improve TGF-β1-induced fibrosis of MRC5 cells.

[0132] 2.6 BRPs inhibit the migration of TGF-βl-induced fibrotic human fetal lung fibroblasts

[0133] The effect of BRPs on the migration of TGF-βl-induced fibrotic human fetal lung fibroblasts MRC5 was evaluated by wound healing assay. Figure 6 The results showed that compared with the control group, TGF-βl-treated cells for 24 h or 48 h could gradually heal the scratch area, indicating that it could promote cell migration; compared with the TGF-βl group, BRPs could significantly inhibit the healing of the scratch area of TGF-βl-induced fibrotic MRC5 cells, suggesting that BRPs could inhibit the migration of TGF-βl-induced fibrotic human fetal lung fibroblasts.

[0134] 2.7 BRPs significantly improve the lung function of BLM-induced PF mice

[0135] We used BLM to establish a PF model in C57BL / 6J mice and administered BRPs for a 3-week treatment Figure 7 A), to detect the effect of BRPs on the evaluation index of lung function in mice of each experimental group. As shown in Figure 7 B-I, compared with the control group, a single intratracheal instillation of BLM could significantly reduce the total lung capacity (TLC), vital capacity (VC), inspiratory capacity (IC), tidal volume (TV), forced vital capacity (FVC), peak expiratory flow (PEF), dynamic lung compliance (Cdyn), and quasi-static compliance (Cchord) in mice, suggesting that BLM could cause a decrease in lung function in mice; while 5 g / kg BRPs could significantly reverse the above lung function indicators in BLM-induced PF mice, suggesting that BRPs could improve the lung function of BLM-induced PF mice.

[0136] 2.8 BRPs reduce the inflammatory lesions in the lung tissue of BLM-induced PF mice

[0137] H&E staining is the most basic and widely used technique in pathology teaching and scientific research, which can make the cell nucleus blue and the cytoplasm red. As shown in Figure 8 , the lung tissue structure of the normal control group of mice was intact, the alveolar tissue structure was normal, and there was no inflammatory cell infiltration; compared with the control group, the lung tissue structure of the BLM model group of mice was severely damaged, the alveolar wall was significantly thickened, and was accompanied by a large number of inflammatory cell infiltration, and the inflammatory degree score value was significantly increased; compared with the BLM model group, both BRPs and the positive drug PFD could make the lung tissue structure of mice relatively intact, the alveolar wall was only slightly thickened, and the inflammatory infiltrating cells were relatively few, and the inflammatory degree score value was significantly reduced. These results suggest that BRPs can reduce the inflammatory lesions in the lung tissue of BLM-induced PF mice, and there is no significant difference compared with the positive drug PFD.

[0138] 2.9 BRPs reduce pulmonary fibrosis and inhibit the formation of type I collagen fibers in BLM-induced PF mice

[0139] 1) Masson staining is a classic method for staining connective tissue, which stains muscle fibers red and collagen fibers blue. Masson staining allows observation of the distribution of collagen and muscle fibers in lung tissue. For example... Figure 9 As shown, the lung tissue structure of mice in the normal control group was relatively intact, with fewer blue-stained collagen fibers. Compared with the control group, the lung tissue of mice in the BLM model group not only suffered severe structural damage but also showed a large number of blue-stained collagen fibers, indicating a higher degree of pulmonary interstitial fibrosis. Compared with the BLM model group, both BRPs and PFD significantly reduced the number of blue-stained collagen fibers in the lung interstitium of mice, thus reducing the degree of pulmonary interstitial fibrosis. These results indicate that BRPs can improve pulmonary interstitial fibrosis in BLM-induced PF mice.

[0140] 2) Sirius Red is a strongly acidic anionic dye that reacts with basic collagen, causing collagen fibers to exhibit a significant birefringence when irradiated with polarized light. Type I collagen fibers, being densely packed, exhibit strong birefringence and appear red in tissue sections irradiated by both polarized and normal light. Through Sirius Red staining, we found that the lung tissue structure of normal mice was relatively intact, with fewer red-stained type I collagen fibers. Compared to the normal group, the lung tissue of mice in the BLM model group showed a large number of red-stained type I collagen fibers. Compared to the BLM model group, the number of red-stained type I collagen fibers in the lung tissue of mice in the BLM+BRPs and BLM+PFD groups was significantly reduced, suggesting that BRPs can inhibit the formation of type I collagen fibers in the lung tissue of BLM-induced PF mice. Figure 10 ).

[0141] 2.10 BRPs inhibit collagen deposition in the lung tissue of BLM-induced PF mice

[0142] Lung tissue collagen content can reflect collagen deposition. Sircol assay results showed that, compared with the control group, the lung tissue collagen content of mice in the BLM model group was significantly increased, indicating the occurrence of lung tissue collagen deposition; compared with the model group, both medium and high doses of BRPs and PFD significantly decreased the lung tissue collagen content in mice; and there was no significant difference in lung tissue collagen content between the high-dose BRPs group and the anti-PF positive drug PFD. Figure 11 The results suggest that BRPs can inhibit BLM-induced collagen deposition in the lung tissue of PF mice.

[0143] 2.11 BRPs inhibit ColIα1 protein expression in lung tissue of BLM-induced PF mice

[0144] Immunohistochemical staining results showed that the normal group of mice lung tissue ColIa1 staining was basically negative, indicating that the expression level of ColIa1 protein was low; compared with the normal control group, the lung tissue of BLM model group mice showed strong positive staining of ColIa1, indicating that the expression level of ColIa1 was increased; compared with the model group, BRPs and PFD can make the positive staining of ColIa1 in the lung tissue of mice lighter, suggesting that BRPs can inhibit the expression of ColIa1 in the lung tissue of BLM-induced PF mice Figure 12

[0145] 2.12 BRPs inhibit COLIA1 gene transcription in the lung tissue of BLM-induced PF mice

[0146] RQ-qPCR results showed that compared with the normal control group, BLM can significantly increase the mRNA level of COLIA1 in the lung tissue of mice, indicating that BLM can promote COLIA1 gene transcription; compared with the BLM model group, BRPs can down-regulate the mRNA level of COLIA1 in the lung tissue of BLM-induced PF mice, and there is no significant difference compared with PFD, suggesting that BRPs can inhibit COLIA1 gene transcription Figure 13

[0147] 2.13 BRPs inhibit the expression of fibrosis marker proteins a-SMA and Fibronectin in the lung tissue of BLM-induced PF mice

[0148] Figure 14 and Figure 15 Immunofluorescence staining results showed that compared with the normal control group, BLM significantly up-regulated the expression levels of fibrosis marker proteins a-SMA and Fibronectin in the lung tissue of mice; compared with the BLM model group, BRPs and PFD can reduce the expression levels of a-SMA and Fibronectin in the lung tissue of mice, suggesting that BRPs, similar to PFD, can also improve the fibrosis of the lung tissue of BLM-induced PF mice by down-regulating a-SMA and Fibronectin.

[0149] 2.14 BRPs can reduce lung injury in BLM-induced PF mice

[0150] Most lung tissue injuries are accompanied by a certain degree of fibrosis. The TUNEL kit can transfer FITC-labeled dUTP to the 3'-OH end by terminal deoxynucleotide transferase-mediated dUTP gap end labeling technology according to the feature that "endogenous endonuclease in apoptotic cells is activated to cut their own chromosomal DNA into gaps or 3'-OH end fragments containing 180-200 bp", so that the broken DNA shows green fluorescence of FITC under a fluorescence microscope. For example Figure 16 ​​As shown, the FITC green fluorescence points were very few in the normal control group, indicating that the lung tissue DNA fragmentation was very few; compared with the control group, more green fluorescence points appeared in the BLM model group, indicating that the DNA fragmentation was more; and compared with the model group, BRPs could significantly reduce the green fluorescence points in the lung tissue of BLM-induced PF mice, and there was no significant difference compared with PFD, suggesting that BRPs could reduce the lung injury of BLM-induced PF mice.

[0151] 2.15 BRPs can inhibit BLM-induced PF mouse bronchoalveolar inflammatory cell infiltration and reduce lung inflammation

[0152] Pulmonary fibrosis may also occur when the lung tissue is stimulated by inflammation. Macrophages, lymphocytes, neutrophils, eosinophils and other inflammatory cells are contained in the bronchoalveolar lavage fluid (BALF) of mice, and the number of inflammatory cells is an important indicator for measuring inflammatory response. By collecting BALF and counting cells, it was found that compared with the normal control group, BLM could significantly increase the number of inflammatory cells in BALF, indicating that bronchoalveolar inflammatory cell infiltration increased; compared with the BLM group, BRPs could significantly down-regulate the number of inflammatory cells, suggesting that BRPs could reduce BLM-induced PF mouse lung tissue inflammation by inhibiting bronchoalveolar inflammatory cell infiltration. Figure 17 ).

[0153] 2.16 BRPs can also reduce BLM-induced PF mouse lung inflammation by inhibiting NLRP3 inflammasome activation

[0154] Inflammatory response cannot be started without inflammasome. NLRP3 is an important component of NLRP3 inflammasome. Figure 18 Immunofluorescence staining results showed that the expression of NLRP3 in normal lung tissue was very low; compared with normal lung tissue, BLM could significantly enhance the red fluorescence of NLRP3 in mouse lung tissue, suggesting that the inflammatory response was enhanced; and compared with the BLM model group, BRPs and PFD could significantly weaken the red fluorescence of NLRP3 in BLM-induced PF mouse lung tissue, suggesting that BRPs and PFD could also effectively inhibit the activation of NLRP3 inflammasome, and further inhibit the inflammatory response.

[0155] 2.17 BRPs can inhibit TGF-β1-induced fibrosis in human embryonic lung fibroblasts Hedgehog signaling pathway

[0156] Western blotting results showed that compared with the control group, TGF-β1 could significantly up-regulate the Hedgehog signaling pathway proteins SHH, Gli1 and Gli2 levels in MRC5 cells; while compared with the TGF-β1 model group, BRPs could dose-dependently down-regulate the SHH, Gli1 and Gli2 protein expression, suggesting that BRPs could inhibit the Hedgehog signaling pathway of fibrosis induced by TGF-β1 in human embryonic lung fibroblasts Figure 19

[0157] 2.18 BRPs could inhibit the Hedgehog signaling pathway of BLM-induced PF mouse lung tissue

[0158] Figures 20-22 Immunofluorescence staining results showed that in the normal control group of mice, the Hedgehog signaling pathway SHH, Gli1 and Gli protein expression were low; compared with the normal control group, the BLM model group of mice lung tissue SHH, Gli1 and Gli2 protein expression was significantly increased; compared with the model group, BRPs and PFD could significantly reduce the expression of SHH, Gli1 and Gli2 protein. The expression trend of Sufu protein, a negative regulator of Hedgehog signaling pathway, in the lung tissue of mice in each experimental group was opposite to that of SHH, Gli1 and Gli2 protein Figure 23 ) These results suggest that BRPs can inhibit the Hedgehog signaling pathway of BLM-induced PF mouse lung tissue.

[0159] 2.19 Hedgehog inhibitors enhance the improvement of BRPs on BLM-induced PF mouse lung function

[0160] Based on the regulatory effect of BRPs on the Hedgehog signaling pathway, we explored the effect of BRPs on BLM-induced PF mouse lung function with the help of Hedgehog inhibitor Gant 61. Figure 24 Results showed that compared with the control group, BLM could significantly reduce the total lung capacity (TLC), vital capacity (VC), inspiratory capacity (IC), tidal volume (TV), forced vital capacity (FVC), maximum respiratory flow (PEF), dynamic lung compliance (Cdyn) and quasi-static compliance (Cchord), leading to decreased lung function in mice; Gant 61 (10 mg / kg) and BRPs (3 g / kg) single drug could respectively make the above lung function indexes of BLM-induced PF mice tend to improve, and the combination of the two could significantly reverse the decrease of lung function indexes such as total lung capacity, inspiratory capacity, forced vital capacity and quasi-static compliance in BLM-induced PF mice. These results suggest that Hedgehog inhibitors can further enhance the improvement of BRPs on BLM-induced PF mouse lung function.

[0161] ​2.20 Hedgehog inhibitors significantly enhance the inhibitory effect of BRPs on the inflammatory lesions of lung tissues of BLM-induced PF mice

[0162] The effect of Hedgehog inhibitors combined with BRPs on the inflammatory lesions of lung tissues of BLM-induced PF mice was evaluated by H&E staining. The results showed that compared with the control group, BLM could cause obvious inflammatory lesions in the lung tissues of mice; compared with the BLM model group, both Hedgehog inhibitor Gant 61 (10 mg / kg) and BRPs (3 g / kg) alone could partially inhibit the inflammatory lesions of lung tissues of BLM-induced PF mice, and the combination of the two had a more significant inhibitory effect on the inflammatory lesions of lung tissues of BLM-induced PF mice Figure 25 and Figure 26 ).

[0163] 2.21 Hedgehog inhibitors combined with BRPs can further inhibit pulmonary interstitial fibrosis and type I collagen fiber formation in BLM-induced PF mice

[0164] The effect of Hedgehog inhibitors combined with BRPs on pulmonary interstitial fibrosis in BLM-induced PF mice was evaluated by Masson staining. The results showed that compared with the control group, the BLM group had a large number of blue-stained collagen fibers in the lung tissues of mice, and the fibrosis score was high, indicating the occurrence of pulmonary interstitial fibrosis; compared with the BLM model group, both Hedgehog inhibitor Gant 61 and BRPs alone could partially inhibit the production of blue-stained collagen fibers in the lung tissues of BLM-induced PF mice, and the fibrosis score was lower; compared with Gant 61 or BRPs alone, the combination of the two could further inhibit the production of blue-stained collagen fibers and down-regulate the fibrosis score Figure 27 and Figure 28 ), suggesting that Hedgehog inhibitors can enhance the inhibitory effect of BRPs on pulmonary interstitial fibrosis in BLM-induced PF mice. Similar inhibitory effect of Hedgehog inhibitors combined with BRPs on type I collagen fibers in the lung tissues of BLM-induced PF mice was observed by Sirius Red staining Figure 29 ).

[0165] 2.22 Hedgehog inhibitors enhance the therapeutic effect of BRPs on pulmonary fibrosis in BLM-induced PF mice

[0166] Excessive deposition of collagen is a major feature of fibrotic diseases, and hydroxyproline is a unique amino acid of collagen. The total content of hydroxyproline in tissues has become one of the most commonly used indicators for evaluating the severity of fibrosis, and is the "gold standard" for preclinical evaluation of pulmonary fibrosis. For example Figure 30As shown, compared with the control group, BLM can make the total content of hydroxyproline in lung tissue of mice significantly increased, indicating the occurrence of pulmonary fibrosis; compared with the BLM model group, Gant 61 and BRPs can partially down-regulate the content of hydroxyproline in lung tissue of BLM-induced PF mice; and compared with each single drug, Gant 61 and BRPs in combination can further reduce the content of hydroxyproline in lung tissue of BLM-induced PF mice, and improve the pulmonary fibrosis. These results suggest that Hedgehog inhibitors can enhance the therapeutic effect of BRPs on BLM-induced PF in mice.

[0167] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can also be made, which should be considered as the protection scope of the present application.

Claims

1. Use of a pharmaceutical composition for the manufacture of a medicament for the prevention and / or treatment of pulmonary fibrosis, characterized in that, The pharmaceutical composition consists of chagmalose, a Hedgehog inhibitor; The Hedgehog inhibitor is Gant 61.

Images