Application of brassica rapa polysaccharide in preparation of medicine for preventing and / or treating pulmonary fibrosis

Abstract

The invention provides application of brassica rapa polysaccharide in preparation of a medicine for preventing and / or treating pulmonary fibrosis, and belongs to the technical field of medicine. It is found that the brassica rapa polysaccharide can effectively prevent and / or treat pulmonary fibrosis, and can also effectively relieve lung injury and inhibit lung inflammation; brassica rapa polysaccharide inhibits fibrosis and migration of human embryonic lung fibroblasts induced by recombinant human transforming growth factors, and improves pulmonary function impairment and pulmonary fibrosis of bleomycin-induced pulmonary fibrosis mice. Bronchoalveolar inflammatory cell infiltration of a pulmonary fibrosis mouse and expression of a lung tissue NOD-like receptor family pyridine structural domain containing protein 3 (NLRP3) are inhibited, so that lung inflammation is inhibited; the Brassica rapa polysaccharide can inhibit Brassica rapa polysaccharide in vitro and in vivo, can inhibit a Hedgehog signal channel in vitro and in vivo, can further enhance the improvement effect of the Brassica rapa polysaccharide on the lung function of a mouse with bleomycin-induced pulmonary fibrosis by assisting with a Hedgehog channel inhibitor, and can enhance the treatment effect of the Brassica rapa polysaccharide on pulmonary fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of chamacarp polysaccharide in the preparation of drugs for preventing and / or treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis (PF), especially idiopathic pulmonary fibrosis (IPF), is a chronic, progressive, and fatal interstitial lung disease with the main pathological features of interstitial inflammatory cell infiltration, massive deposition of extracellular matrix (ECM), proliferation of fibroblasts (FB), and disordered alveolar structure. Clinically, it is mainly manifested as progressive decline in lung function and even respiratory failure. Due to the complex and yet to be fully clarified pathogenesis, IPF treatment has always been a difficult problem in the medical field. In addition to expensive lung transplantation, there is no effective cure. The diagnosis rate of IPF in my country is relatively low. In terms of treatment, although pirfenidone and nintedanib have been approved for marketing, patients still face problems such as poor drug tolerance and large side effects, which seriously affect their quality of life. There is an urgent need for anti-PF drugs with good tolerance and few side effects to improve this situation.

[0003] Natural plants are an important source of new drug discovery. In particular, medicinal and edible plants have attracted much attention from scholars due to their natural, safe, effective and nutritious advantages. Using medicinal and edible plant resources and combining them with modern scientific and technological means to develop effective active ingredients with good tolerance, few side effects, and the ability to prevent and / or treat PF has always been the unremitting pursuit of scientific researchers.

[0004] Chamagu, scientific name Brassica rapa L., called "Chamagu" (transliteration) in Uyghur, belongs to the Brassica rapa subspecies of the Brassica species of the Cruciferae family. It is cultivated in various parts of Xinjiang, my country, and is known as the "holy fruit of longevity" and "small ginseng in the desert". The people of Xinjiang have been eating Chamagu for more than 2,000 years, and the Uyghurs in many areas of southern Xinjiang even regard it as an indispensable daily food. Chamagu is also rich in polysaccharides, glucosinolates, phenols and organic acids and other medicinal components. It has multiple pharmacodynamic activities such as moistening the lungs, relieving cough and relieving asthma, anti-oxidation, anti-tumor, anti-radiation, immune regulation, organ and nerve protection. Uyghur medicine has always regarded it as a plant-based lung-nourishing medicine with no toxic side effects. As a distinctive medicinal and edible plant in ethnic regions, Chamagu has a bright future in the research and development of drugs for the prevention and treatment of lung diseases.

[0005] Polysaccharides of Brassica rapa L. (BRPs) are the main active ingredients extracted from Chamagu, which is both a medicinal and edible plant. Our previous studies have found that BRPs have good antioxidant and immunomodulatory activities, can regulate the polarization of M2 macrophages to M1 phenotypes and thus exert anti-tumor effects, and can also reduce doxorubicin-induced rat cardiomyocyte damage through the Nrf2 / HO-1 signaling pathway. However, there is no prior art on the use of BRPs in drugs for the prevention and / or treatment of PF. Summary of the invention

[0006] The purpose of the present invention is to provide the use of chamagu polysaccharide in the preparation of drugs for preventing and / or treating pulmonary fibrosis (PF), wherein the BRPs can inhibit the levels of fibrosis marker proteins ColIα1, α-SMA and Fibronectin in human embryonic lung fibroblasts induced by recombinant human transforming growth factor (TGF-β1), and inhibit their cell migration; in vivo, BRPs can effectively improve the lung function of PF mice induced by Bleomycin (BLM), improve lung tissue fibrosis, reduce lung damage, and inhibit lung inflammation. BRPs can also inhibit the Hedgehog signaling pathway in vitro and in vivo; Hedgehog inhibitors can further enhance the improvement effect of BRPs on the lung function of PF mice induced by BLM, and enhance the therapeutic effect of BRPs on lung tissue fibrosis of PF mice induced by BLM.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides the use of chamacarp polysaccharide in preparing a medicine for preventing and / or treating pulmonary tissue fibrosis.

[0009] Preferably, the chamagu polysaccharide prevents and / or treats PF by inhibiting lung cell fibrosis and cell migration. The present invention has been proved through experiments that BRPs can inhibit the expression and cell migration of fibrosis marker proteins ColIα1, α-SMA, and Fibronectin in TGF-β1-induced fibrotic human embryonic lung fibroblasts in vitro; in vivo, BRPs can improve the lung function of BLM-induced PF mice, inhibit inflammatory lesions, collagen and collagen fiber production, and lung tissue ColIα1, α-SMA and Fibronectin protein expression in BLM-induced PF mice, and improve lung tissue fibrosis.

[0010] The present invention provides the use of chamagu polysaccharide in the preparation of drugs for resisting lung injury and lung inflammation. The present invention proves through experiments that BRPs can inhibit the infiltration of bronchial alveolar inflammatory cells and inhibit the expression of inflammasome NLRP3 protein.

[0011] The present invention provides the use of chamagu polysaccharide in the preparation of a drug for inhibiting the Hedgehog signaling pathway of lung cells. The present invention proves through experiments that BRPs can inhibit the Hedgehog signaling pathway of TGF-β1-induced fibrotic human embryonic lung fibroblasts and BLM-induced PF mouse lung tissue, and the combination of BRPs and Hedgehog inhibitors can further improve the lung function of BLM-induced PF mice and the fibrosis of lung tissue.

[0012] Preferably, the effective concentration of the chamagu polysaccharide on TGF-β1-induced fibrotic human embryonic 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, it is estimated that the effective dosage for an adult is 0.22-0.55 g / kg.

[0013] Preferably, the chamagu polysaccharide can be used together with a Hedgehog inhibitor; the Hedgehog inhibitor includes Gant 61. The present invention proves through experiments that the Hedgehog inhibitor Gant61 can enhance the improvement effect of BRPs on the total lung volume, deep inspiration volume, forced vital capacity, and quasi-static compliance of BLM-induced PF mice, improve the lung function of PF mice, and enhance the therapeutic effect of BRPs on lung tissue fibrosis of BLM-induced PF mice.

[0014] Preferably, the drug comprises chamacarp polysaccharide and pharmaceutically permitted excipients.

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

[0016] Preferably, the pharmaceutically acceptable dosage form is tablets, granules, capsules, pills, syrups, suspensions, powders, oral solutions, and mixtures.

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

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

[0019] The present invention provides the use of chamacarp polysaccharides (BRPs) in the preparation of drugs for preventing and / or treating pulmonary fibrosis (PF). The BRPs provided by the present invention can inhibit the levels of fibrosis marker proteins ColIα1, α-SMA and Fibronectin in human embryonic lung fibroblasts induced by TGF-β1, and inhibit their cell migration; in vivo, BRPs can effectively improve the lung function of BLM-induced PF mice, improve lung tissue fibrosis, reduce lung damage, and inhibit lung inflammation. BRPs can also inhibit the Hedgehog signaling pathway in vitro and in vivo; Hedgehog inhibitors can further enhance the improvement effect of BRPs on the lung function of BLM-induced PF mice, and enhance the therapeutic effect of BRPs on lung tissue fibrosis of BLM-induced PF mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the standard curve for determining the total polysaccharide content of BRPs using the anthrone sulfate method.

[0021] Figure 2 This is a diagram of the monosaccharide components of BRPs determined by HPLC; A is the internal standard spectrum of the monosaccharide components; and B is the spectrum of the monosaccharide components of BRPs.

[0022] Figure 3 This is a graph showing the effect of BRPs on the proliferation of human embryonic lung fibroblasts detected by CCK8 experiment; A is the light absorption value of the cells at 450nm; B is the cell survival rate.

[0023] Figure 4 The figure shows the effect of BRPs on the morphology of human embryonic lung fibroblasts induced by fibrosis inducer TGF-β1; A is the blank control group; B is the TGF-β1 group (1 ng / mL); C is the TGF-β1+2 mg / mL BRPs group; D is the TGF-β1+4 mg / mL BRPs group; E is the TGF-β1+6 mg / mL BRPs group; F is the TGF-β1+8 mg / mL BRPs group.

[0024] Figure 5 This is a diagram showing the effect of BRPs on the expression levels of ColIα1, α-SMA and Fibronectin in TGF-β1-induced fibrotic human embryonic lung fibroblasts detected by Western blotting; A is a typical ColIα1 protein band; B is a typical α-SMA protein band; C is a typical Fibronectin protein band; D is the relative expression level of ColIα1; E is the relative expression level of α-SMA; and F is the relative expression level of Fibronectin.

[0025] Figure 6The figure shows the effect of BRPs on the migration of TGF-β1-induced fibrotic human embryonic lung fibroblasts; A is a typical cell migration diagram; B is the cell migration rate.

[0026] Figure 7 The figure shows the effect of BRPs on the lung function of BLM-induced PF mice; A is the flow chart of PF mouse modeling and BRPs administration; 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 maximum respiratory flow (PEF); H is the dynamic lung compliance (Cdyn); and I is the quasi-static compliance (Cchord).

[0027] Figure 8 The figure shows the effect of BRPs on the inflammatory lesions of lung tissue in BLM-induced PF mice; A is a typical H&E staining image; B is the H&E staining inflammation score image.

[0028] Fig. 9 The figure shows the effect of BRPs on interstitial fibrosis in lung tissue of BLM-induced PF mice; A is a typical Masson staining image; B is a Masson staining fibrosis degree score image.

[0029] Fig.10 The figure shows the effect of BRPs on the formation of type I collagen fibers in the lung tissue of BLM-induced PF mice.

[0030] Fig.11 The figure shows the effect of BRPs on the collagen content in lung tissue of BLM-induced PF mice.

[0031] Fig.12 The figure shows the effect of BRPs on the expression of ColIα1 protein in lung tissue of BLM-induced PF mice.

[0032] Fig.13 This figure shows the effect of BRPs on COLIA1 gene transcription in lung tissue of BLM-induced PF mice.

[0033] Fig.14 This figure shows the effect of BRPs on the expression of α-SMA protein in lung tissue of BLM-induced PF mice.

[0034] Fig.15 The figure shows the effect of BRPs on the expression of Fibronectin protein in lung tissue of BLM-induced PF mice.

[0035] Fig.16 The figure shows the effect of BRPs on lung injury of PF mice induced by BLM; A is a typical TUNEL staining image; B is the percentage of TUNEL staining positive cells.

[0036] Fig.17The figure shows the effect of BRPs on bronchoalveolar inflammatory cell infiltration in BLM-induced PF mice.

[0037] Fig.18 This figure shows the effect of BRPs on the expression of inflammasome receptor protein NLRP3.

[0038] Fig.19 The figure shows the effect of BRPs on the expression of SHH, Gli1 and Gli2 proteins in the Hedgehog signaling pathway of TGF-β1-induced fibrotic human embryonic lung fibroblasts; 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] Fig. 20 This figure shows the effect of BRPs on SHH protein expression in lung tissue of BLM-induced PF mice.

[0040] Fig.21 This figure shows the effect of BRPs on the expression of Gli1 protein in lung tissue of BLM-induced PF mice.

[0041] Fig. 22 This figure shows the effect of BRPs on the expression of Gli2 protein in lung tissue of BLM-induced PF mice.

[0042] Fig.23 This figure shows the effect of BRPs on the expression of Sufu protein in lung tissue of BLM-induced PF mice.

[0043] Fig.24 The figure shows the effect of Hedgehog inhibitor Gant 61 combined with BRPs on the lung function of BLM-induced PF mice; A is the flow chart of PF mouse 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); and I is quasi-static compliance (Cchord).

[0044] Fig.25 This figure shows the effect of Hedgehog inhibitor Gant 61 combined with BRPs on inflammatory lesions in lung tissue of BLM-induced PF mice.

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

[0046] Fig. 27This figure shows the effect of Hedgehog inhibitor Gant 61 combined with BRPs on interstitial fibrosis in lung tissue of BLM-induced PF mice.

[0047] Fig.28 This figure shows the effect of Hedgehog inhibitor Gant 61 combined with BRPs on the degree of interstitial fibrosis in lung tissue of BLM-induced PF mice.

[0048] Fig.29 This figure shows the effect of Hedgehog inhibitor Gant 61 combined with BRPs on the formation of type I collagen fibers in the lung tissue of BLM-induced PF mice.

[0049] Fig.30 This figure shows the effect of Hedgehog inhibitor Gant 61 combined with BRPs on hydroxyproline production in lung tissue of BLM-induced PF mice. DETAILED DESCRIPTION

[0050] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0051] Example 1

[0052] The preparation of chamagu polysaccharide comprises the following steps:

[0053] 1) Chamagu flakes are vacuum dried and crushed into Chamagu dry powder. 1500-2500 mL of pure water is added to every 100 g of Chamagu dry powder. The mixture is refluxed under an electric heating jacket for 1-6 h. Reflux extraction is performed twice in total. The filtrates are filtered and combined.

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

[0055] 3) Concentrate the Chamagu water extract to 200-300 mL in a 65-70°C water bath, add ethanol to the concentrate to a final concentration of 80%, and place in a 4°C refrigerator overnight.

[0056] 4) Discard the supernatant, add a small amount of distilled water to the chamagu polysaccharide precipitate, transfer it to an evaporating dish, and dry it in a vacuum drying oven at 40-50°C and 0.07Pa to constant weight to obtain BRPs.

[0057] Specifically in the embodiments of the present invention,

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

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

[0060] Example 2

[0061] 1. Methods:

[0062] 1.1 Determination of total polysaccharide content of BRPs by anthrone sulfate method

[0063] Weigh 13.30 mg of D-anhydrous glucose (D-Glucose) reference substance dried to constant weight at 105°C accurately, place it in a 50 mL volumetric flask, dilute to the mark with water, shake well, and prepare a 266 μg / mL D-anhydrous glucose solution. Accurately measure 0 mL, 1.0 mL, 2.0 mL, 4.0 mL, 6.0 mL, and 8.0 mL of the reference solution respectively and place them in different 25 mL volumetric flasks, dilute to the 25 mL mark with water, shake well, and then take 1.0 mL of the fixed-volume reference solution into a 10 mL stoppered graduated test tube, after ice bath, accurately add 4 mL of freshly prepared 2‰ anthrone sulfate solution (take 0.2 g of anthrone, dissolve in a mixture containing 5 mL of water and 95 mL of sulfuric acid, shake, and cool), shake well, heat at 90°C water bath for 10 min, take out, and quickly cool to room temperature. The preparation of BRPs solution is carried out simultaneously with the D-glucose reference substance. After 30 minutes, the first portion was used as the blank control solution, and the absorbance of each reference substance and the test substance BRPs at 610 nm was measured, a D-glucose-absorbance standard curve was drawn, and the total polysaccharide content of BRPs was calculated based on the absorbance of the BRPs solution.

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

[0065] Weigh 10 mg of BRPs sample into an ampoule, add 1 mL of ultrapure water to fully dissolve it, then add an equal volume of 4 M trifluoroacetic acid (TFA), seal the tube with a spray gun, and hydrolyze in an oven at 110 ° C for 4 hours. Open the ampoule, aspirate the sample solution, and neutralize it with NaOH. Take 200 μL of the fixed volume solution, add 200 μL of internal standard, oscillate and mix, and draw 100 μL of the mixed solution into a 1.5 mL centrifuge tube, add 0.3 M NaOH and 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP) 120 μL, mix well, and bathe in 70 ° C light-proof water for 1 hour. Cool to room temperature, add 0.3 M HCl to neutralize to pH = 7. Add 500 μL of chloroform to extract, mix well, and centrifuge at 7000 rpm for 5 minutes. Carefully aspirate the upper solution (aqueous layer) and transfer it to a new centrifuge tube. Repeat the extraction 4 times. Pipette the supernatant from the last extraction and filter it into a liquid phase bottle using a 0.22 μm aqueous phase filter. Detect the monosaccharide components by HPLC.

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

[0067] 1.3 Effects of BRPs on the proliferation of human embryonic lung fibroblasts MRC-5

[0068] MRC-5 cells in the logarithmic growth phase were routinely digested and collected, and the cell concentration was adjusted to 7×10 4 / mL was inoculated in a 96-well plate, 100μL of cell suspension was inoculated in each well, and incubated overnight in a 37°C, 5% CO2 incubator. The next day, BRPs (2, 4, 6 and 8mg / mL) were added for 48h. Cell-free blank control wells and drug-free control wells were set up simultaneously in the experiment. After the drug treatment, the culture medium was discarded, 100μL of serum-free MEM / EBSS culture medium was added to each well, and then 10μL / well of CCK-8 solution was added. The plates were incubated for 1-2h in a 37°C, 5% CO2 incubator. The absorbance value (Optical density, OD) of each well at 450nm was measured by an enzyme reader, and the cell survival rate was calculated.

[0069]

[0070] 1.4 Effects of BRPs on the morphology and growth status of MRC-5 cells induced by TGF-β1

[0071] MRC-5 cells were cultured at 20×10 4 / well were inoculated in a 6-well cell culture plate and cultured overnight in a 37°C, 5% CO2 incubator. Washed twice with PBS, starved for 24 hours with serum-free MEM / EBSS culture medium, added with 1 ng / mL TGF-β1 and treated with different concentrations of BRPs for 48 hours, observed and photographed under an inverted optical microscope. Experimental groups: blank control group, TGF-β1 group (1 ng / mL), TGF-β1+BRPs (2, 4, 6 and 8 mg / mL) group.

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

[0073] Human embryonic lung fibroblast MRC-5 cells were cultured at 20×10 4Cells were inoculated in 6-well plates and cultured overnight. After being starved for 24 hours with serum-free MEM / EBSS culture medium, 1 ng / mL TGF-β1 and different concentrations of BRPs were added to act on the cells for 48 hours. The experimental groups were: Control group, TGF-β1 group, T GF-β1+BRPs (2, 4, 6 mg / mL) group. At the end of the experiment, the cells were washed with PBS twice, and RIPA lysis buffer containing 1× protease and phosphatase inhibitors was added to each well. The cells were lysed on ice for 30 minutes and centrifuged at 14000 rpm for 30 minutes. The supernatant was taken, the protein concentration was determined by the BCA method, and an equal amount of protein was taken for 10% SDS-PAGE, and then wet-transferred to a PVDF membrane at constant pressure. The PVDF membrane was blocked with 5% skim milk powder / PBST at room temperature for 2 hours, and anti-ColIα1 rabbit antibody (1:1000), anti-α-SMA rabbit antibody (1:5000), anti-Fibronectin (1:1000) rabbit antibody and anti-GAPDH mouse antibody (1:4000) were added, respectively, and incubated overnight at 4°C on a shaker. The membrane was washed 3 times with PBST, 10 minutes each time, and horseradish peroxidase-labeled anti-rabbit IgG (1:2000) or anti-mouse IgG (1:2000) was added and incubated at room temperature for 1.5 hours. The membrane was washed 3 times with PBST, and ECL reagent was used for color development to obtain Western blot protein bands to observe the effects of BRPs on the expression levels of fibrosis-related proteins ColIα1, α-SMA and Fibronectin in human embryonic lung fibroblasts induced by TGF-β1.

[0074] Image J software was used to measure the integrated optical density of each band and quantified by comparing it with the internal reference GAPDH.

[0075] Effects of 1.6BRPs on the migration of TGF-β1-induced fibrotic human embryonic lung fibroblasts

[0076] Use a marker pen to draw horizontal and vertical lines evenly on the back of the 6-well plate in advance, with the interval between the lines being about 0.5 to 1 cm, and at least 5 lines should be drawn through each well. 4 Cells were seeded in 6-well culture plates and cultured overnight. The cells were washed twice with PBS, and 2 mL / well serum-free MEM / EBSS was added to starve the cells for 24 hours. A 10 μL pipette tip was used to draw vertical lines in the 6-well cell culture plates containing cells, and the cells were rinsed with PBS three times to remove floating cells caused by scratches. Then, 1 ng / mL TGF-β1 and BRPs were added to treat the cells for 48 hours, and the cells were observed and photographed at 0 hours, 24 hours, and 48 hours. Image J software was used to calculate the migration rate of cells in each group.

[0077]

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

[0079] 1.7.1 Animal grouping and drug administration

[0080] After one week of adaptive feeding of SPF-grade C57BL / 6J mice, a single tracheal instillation of an appropriate amount of BLM was used to establish a mouse PF model; the next day after modeling, BRPs were administered orally once a day for 21 consecutive days. Experimental groups: control group (normal feeding); BLM model group; BLM+BRPs (5g / kg) group.

[0081] 1.7.2 Establishment of mouse PF model

[0082] BLM was diluted to an appropriate concentration with saline. After the mice were anesthetized with isoflurane, the tongue of the mice was gently pulled to compress the tongue ventral part to see the glottis. When the mice were breathing, an appropriate amount of diluted BLM was quickly pushed into the trachea along the palate. After the tracheal instillation was completed, the chest of the mice was gently patted with both hands, and the mice were assisted to rotate so that the drug solution was evenly distributed in the lungs. Under the same conditions, the mice in the normal control group were instilled with an equal volume of saline in the trachea once.

[0083] 1.7.3 Determination of mouse lung function

[0084] After the animal grouping, modeling and drug administration, mice in each group were intraperitoneally injected with 0.8% sodium pentobarbital (dosage: 0.09 mL / 10 g). After the corneal reflex of the mice disappeared, the mice were fixed on the operating board, the skin and hair of the anterior neck area were removed, the incision was about 1 cm, the subcutaneous tissue was bluntly separated, the trachea was exposed, and a small incision was made in the trachea under the cricoid cartilage with surgical scissors, and the tracheal tube was inserted. The tracheal tube was fixed with surgical sutures and connected to the calibrated small animal ventilator to measure various indicators of lung function.

[0085] 1.8 Effect of BRPs on the improvement of lung fibrosis in BLM-induced PF mice

[0086] 1.8.1 Animal grouping and drug administration

[0087] After one week of adaptive feeding of SPF-grade C57BL / 6J mice, a single tracheal instillation of an appropriate amount of BLM was used to establish a mouse PF model; on the next day of modeling, BRPs or positive drug pirfenidone (PFD) were administered orally once a day for 21 consecutive days. Experimental groups: control group (normal feeding); BLM model group; BLM+BRPs (2, 4, 5g / kg) group and BLM+PFD (200mg / kg) group.

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

[0089] After the animals were grouped, modeled, and administered, the mouse heart was surgically exposed. After a large amount of PBS was used to lavage the lungs through the heart, the left lung was removed and fixed in a 4% paraformaldehyde solution overnight. The next day, the sections were sent for paraffin embedding and sectioning. The paraffin sections of the test group were placed in xylene for dewaxing for 10 minutes at room temperature, repeated twice; then, they were soaked in anhydrous ethanol for 5 minutes, repeated twice; followed by soaking in gradient ethanol (95%, 85%, 75%) for 5 minutes in sequence; PBS was added for washing twice, 3 minutes each time. The PBS was gently discarded, and the excess liquid was absorbed with filter paper. H&E staining was performed to observe the pathological changes of the lung tissues in each experimental group and evaluate the degree of inflammation.

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

[0091] After the animals were grouped, modeled, and administered, the lung tissues of each group were embedded in paraffin and sliced. The obtained paraffin sections were dewaxed by xylene, followed by alcohol gradient hydration, and then stained with Masson and Sirius Red to investigate the degree of pulmonary interstitial fibrosis and the production of type I collagen fibers.

[0092] 1.8.4 Sircol assay to determine the effect of BRPs on the collagen content in lung tissue of BLM-induced PF mice

[0093] After the animal grouping, modeling and drug administration, the two middle lobes of the right lung were removed and operated according to the instructions of the Sircol assay kit. First, the two middle lobes of the right lung were weighed and placed in a FACS tube containing an appropriate amount of homogenate lysis solution. The homogenized tissue was transferred to a 2mL EP tube and shaken overnight at 4°C; the next day, centrifuged at 4°C, 14000rpm for 10min, 100uL of the supernatant was taken, 1mL of Sircol dye reagent was added, incubated at room temperature for 1h, and centrifuged at 4°C, 14000rpm for 30min; the supernatant was discarded, and the precipitate was completely dissolved with 1mL of alkaline reagent, and its light absorption value (OD) at 540nm was measured by an enzyme marker 540 ), and calculate the collagen content of the test sample according to the collagen standard curve.

[0094] 1.8.5 Immunohistochemical staining to detect the effect of BRPs on the expression of ColIα1 protein in lung tissue of BLM-induced PF mice

[0095] After the animals were grouped, modeled and administered, the lung tissues of each group were embedded in paraffin and sliced. The obtained paraffin sections were dewaxed by xylene, alcohol gradient and hydrated, immersed in citrate buffer, antigen repaired at 92-98℃ for 15-20min; cooled to room temperature naturally, added 3% H2O2 and incubated for 20min to block endogenous peroxidase; washed with PBS 3 times, added 5% BSA, blocked at 37℃ for 30min; removed the blocking solution, added anti-ColIα1 rabbit antibody, and incubated in a 4℃ wet box overnight. Washed 3 times with PBST, added horseradish peroxidase-labeled anti-rabbit IgG and incubated at room temperature in the dark for 2h. Washed 3 times with PBST, developed with DAB reagent, and stopped color development after a clear positive reaction. The sections were sealed with resin and observed and photographed using an automatic section scanner.

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

[0097] After the animals were grouped, modeled, and administered, the posterior lobe of the right lung cavity was removed and placed in 1 mL of Trizol reagent, homogenized at low speed on ice, and total RNA was extracted using a commercial RNA kit. A260 and A280 were measured by BioDrop to evaluate RNA purity and calculate its concentration. According to the instructions of the reverse transcription kit, 1 μg RNA was reverse transcribed to prepare cDNA, and PCR was performed using the SYBR kit according to the instructions, and GAPDH was used as the internal reference gene. 2 -ΔΔCT The transcription level of COLIA1 gene was evaluated by

[0098] 1.8.7 Effects of BRPs on the expression of fibrosis markers α-SMA and Fibronectin in lung tissue of BLM-induced PF mice

[0099] After paraffin sections were dewaxed with xylene, washed with alcohol gradient and hydrated, they were immersed in citrate buffer and antigen repaired at 92-98℃ for 15-20min. They were cooled to room temperature naturally and incubated with 3% H2O2 for 20min to block endogenous peroxidase. They were washed with PBS 3 times and permeabilized with 0.5% Triton X-100 prepared in PBS for 30min at room temperature. They were washed with PBS 3 times and blocked with 5% BSA at 37℃ for 30min. The blocking solution was removed and anti-α-SMA or anti-Fibronectin rabbit antibody was added and incubated in a humidified box at 4℃ overnight. The next day, they were washed with PBST 3 times and anti-rabbit IgG labeled with Alexa Fluor 555 was added and incubated at room temperature in the dark for 2h. They were washed with PBST 3 times and the nuclei were counterstained with DAPI. The sections were sealed with anti-fluorescence attenuator and observed and photographed with a laser confocal microscope.

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

[0101] After dewaxing with xylene, ethanol gradient and hydration, lung tissue sections were incubated with an appropriate amount of proteinase K solution at room temperature for 20 minutes to permeabilize the cell membrane; washed twice with PBS to remove proteinase K, and excess liquid was removed with filter paper; 50 μL TUNEL working solution / sample was added and incubated at 37°C in the dark for 1 hour. Washed twice with PBS, anti-fluorescence attenuation agent containing DAPI was added to seal the sections, and TUNEL fluorescence signals were observed under a laser confocal microscope.

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

[0103] 1) Count of inflammatory cells in bronchoalveolar lavage fluid (BALF): The mice were anesthetized routinely, placed in a supine position and fixed, the skin and hair of the anterior neck area were removed, a 1 cm vertical incision was made, the subcutaneous tissue was bluntly separated, the trachea was exposed, a cannula was inserted into the mouse trachea and fixed, and 800 μL PBS was drawn with a 1 mL syringe to lavage the bronchoalveoli, and lavage was performed twice in total. The BALF was collected, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended with 100 μL 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 animal grouping, modeling and drug administration, lung tissues of each group were embedded in paraffin and sliced. The obtained paraffin sections were dewaxed by xylene, ethanol gradient and hydrated, immersed in citrate buffer, and antigen repaired at 92-98°C for 15-20min. Naturally cooled to room temperature, 3% H2O2 was added and incubated for 20min to block endogenous peroxidase. Washed 3 times with PBS, 5% BSA was added, and blocked at 37°C for 30min. The blocking solution was removed, anti-NLRP3 rabbit antibody was added, and incubated in a 4°C wet box overnight. The next day, washed 3 times with PBST, anti-rabbit IgG secondary antibody labeled with Alexa Fluor 555 was added, and incubated at room temperature in the dark for 2h. Washed 3 times with PBST, and DAPI counterstained the cell nucleus. The slides were sealed with anti-fluorescence attenuator, and observed and photographed with a 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 fibroblast MRC-5 cells were cultured at 20×10 4Cells were inoculated in 6-well plates and cultured overnight. The next day, the cells were washed twice with PBS and starved for 24 hours with serum-free MEM / EBSS culture medium. The culture medium was discarded, and 1 ng / mL TGF-β1 and different concentrations of BRPs were added to treat the cells for 48 hours. The experimental groups were: normal control group (Control), TGF-β1 (T) group, and T+BRPs (2, 4, 6 mg / mL) group. After drug treatment, the cells were washed twice with PBS, and RIPA lysis buffer containing 1× protease and phosphatase inhibitors was added to lyse the cells on ice for 30 minutes, centrifuged at 14000 rpm for 30 minutes, and the supernatant was retained. The protein concentration of the supernatant was determined by the BCA method, and an equal amount of protein was taken for 10% SDS-PAGE, and then wet-transferred to a PVDF membrane at constant pressure. The PVDF membrane was blocked with 5% skim milk / PBST at room temperature for 2 h, and anti-SHH rabbit antibody, anti-Gli1 mouse antibody, anti-Gli2 rabbit antibody and anti-GAPDH mouse antibody were added respectively and incubated in a shaker at 4°C overnight; washed with PBST three times, 10 min each time, and anti-rabbit IgG or anti-mouse IgG labeled with horseradish peroxidase was added and incubated at room temperature for 1.5 h; washed with PBST three times, developed with ECL reagent, and photographed to observe the effect of BRPs on the expression of Hedgehog signaling pathway proteins in TGF-β1-induced fibrotic human embryonic lung fibroblasts.

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

[0108] After one week of adaptive feeding of SPF-grade C57BL / 6J mice, a single intratracheal instillation of an appropriate amount of BLM was used to establish a mouse PF model; on the next day of modeling, BRPs or anti-PF positive drug pirfenidone (PFD) were gavaged once a day for 21 consecutive days. Experimental groups: control group (normal feeding), BLM model group, BLM+BRPs (5g / kg) group and BLM+PFD (200mg / 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 both lungs through the heart, the left lung was removed and fixed in 4% paraformaldehyde solution overnight, and sent for examination the next day for paraffin embedding and sectioning. The corresponding paraffin sections were placed in xylene for dewaxing for 10 minutes at room temperature, repeated twice; then, soaked in anhydrous ethanol for 5 minutes, repeated twice; followed by soaking in gradient ethanol (95%, 85%, 75%) for 5 minutes in sequence; PBS was added for washing twice, each time for 3 minutes. Gently discard PBS and use filter paper to absorb excess liquid. Immerse the slices in citrate buffer and perform antigen repair at 92-98℃ for 15-20min. Cool naturally to room temperature and add 3% H2O2 for 20min to block endogenous peroxidase. Wash 3 times with PBS and permeabilize the cell membrane with 0.5% Triton X-100 prepared in PBS at room temperature for 30min. Wash 3 times with PBS and add 5% BSA for blocking at 37℃ for 30min. Remove the blocking solution and add 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) respectively, and incubate overnight in a humidified box at 4℃. The next day, the cells were washed three times with PBST, and anti-rabbit IgG labeled with Alexa Fluor 555 or anti-mouse IgG labeled with Alexa Fluor 488 (1:500) was added and incubated at room temperature in the dark for 2 h. The cells were washed three times with PBST, and the cell nuclei were counterstained with 0.5 μg / mL DAPI. The cells were sealed with anti-fluorescence attenuator, and observed and photographed under a laser confocal microscope.

[0109] 1.11 Adding Hedgehog inhibitor to observe the effect of BRPs on lung function and lung fibrosis in BLM-induced PF mice

[0110] 1.11.1 Animal grouping, modeling and drug administration

[0111] After one week of adaptive feeding of SPF-grade C57BL / 6J mice, the animals were divided into control group (normal culture), BLM group, BLM+Gant 61 group, BLM+BRPs group and BLM+Gant 61+BRPs group. During modeling, the control group was given a single intratracheal instillation of an appropriate amount of PBS, and the other groups were given a single intratracheal instillation of an appropriate amount of BLM to construct a mouse PF model. On the second day of modeling, the BLM+BRPs group was given BRPs (3 g / kg) by gavage once a day for 21 consecutive days; starting from the 8th day of modeling, the BLM+Gant 61 group was given an intraperitoneal injection of the 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 medication simultaneously with the corresponding single drug.

[0112] 1.11.2 Adding Hedgehog inhibitors to observe the effects of BRPs on lung function evaluation indicators of BLM-induced PF mice

[0113] After the animal grouping, modeling and drug administration, each group of mice was intraperitoneally injected with 0.8% sodium pentobarbital (0.09 mL / 10 g). After the corneal reflex of the mice disappeared, the mice were fixed on the operating board, the skin and hair of the anterior neck area were removed, a vertical incision of about 1 cm was made, the subcutaneous tissue was bluntly separated, the trachea was exposed, and a small incision was made in the trachea under the cricoid cartilage with surgical scissors, and a tracheal tube was inserted. After the tracheal tube was fixed with surgical sutures, it was connected to the calibrated small animal ventilator to measure various indicators of lung function.

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

[0115] After the animals were grouped, modeled, and administered, the mouse heart was surgically exposed. After a large amount of PBS was used to lavage both lungs through the heart, the left lung was removed and fixed in a 4% paraformaldehyde solution overnight. The sections were sent for examination the next day for paraffin embedding and sectioning. The paraffin sections were dewaxed in xylene for 10 minutes at room temperature, and repeated twice. Then, they were soaked in anhydrous ethanol for 5 minutes, repeated twice; followed by soaking in gradient ethanol (95%, 85%, 75%) for 5 minutes in sequence. PBS was added for washing twice, 3 minutes each time. The PBS was gently discarded, and the excess liquid was absorbed with filter paper. H&E staining was performed to observe the inflammatory lesions of the lung tissues in each experimental group and evaluate the degree of inflammation.

[0116] 1.11.4 Adding Hedgehog inhibitors to observe the effects of BRPs on lung tissue interstitial fibrosis and type I collagen fibril formation

[0117] After the animals were grouped, modeled, and administered, the mouse hearts were surgically exposed, and the lungs were lavaged through the heart with a large amount of PBS. The left lung was removed and fixed in 4% paraformaldehyde solution overnight. The lung tissues of each experimental group were embedded in paraffin, sectioned, dewaxed in xylene, and hydrated in a downward gradient of alcohol. Masson and Sirius Red staining were performed to observe pulmonary interstitial fibrosis and the formation of type I collagen fibers in lung tissue.

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

[0119] The change of hydroxyproline content can reflect the collagen deposition in lung tissue. mg ) Place lung tissue in a test tube, add 1 mL of hydrolysis solution, and mix. After covering, hydrolyze at 95°C for 20 minutes, mixing once every 10 minutes. Cool each test tube with running water, add 10 μL of indicator per tube, shake well, and then add 1.0 mL of pH adjustment solution A per tube and mix well. Then, add pH adjustment solution B dropwise to each tube, mixing while adding until the color of the indicator in the liquid turns yellow-green, and then add double distilled water to 10 mL (V 水解液 ), mix well. Take 3 mL of the diluted hydrolyzate, add an appropriate amount of activated carbon, mix well, centrifuge at 3500 rpm for 10 min, take 1 mL of the supernatant, detect its absorbance value (OD) at 550 nm, and calculate the hydroxyproline content according to the following formula.

[0120]

[0121] 2 Experimental results

[0122] 2.1 Determination of total polysaccharide content of BRPs by anthrone sulfate method

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

[0124] 2.2 Monosaccharide components of BRPs

[0125] The HPLC method was used to detect the monosaccharide composition of BRPs using commercial monosaccharide components as standards. The results showed that the monosaccharide components of BRPs were 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 CCK-8 experiment was used to investigate the effects of different concentrations of BRPs on the proliferation of human embryonic lung fibroblasts (MRC5). The results showed that BRPs did not inhibit the proliferation of MRC5 cells when the concentration was not higher than 6 mg / ml; 8 mg / mL BRPs could partially inhibit 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.

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

[0129] TGF-β1 is one of the main inducing factors of fibrosis, which can promote fibroblast proliferation and collagen deposition, leading to organ fibrosis. To observe the effect of BRPs on the morphology of TGF-β1-induced fibrotic MRC5 cells, we seeded human embryonic lung fibroblasts MRC5 in 6-well culture plates and cultured overnight, starved them with serum-free MEM / EBSS for 24 hours, and then added 1ng / mL TGF-β1 and different concentrations of BRPs for 48 hours. Figure 4 The results showed that the cell bodies of the control group were slender and spindle-shaped with clear boundaries. Compared with the control group, the cell boundaries of the TGF-β1 model group were blurred, the cell bodies became flat and flat, and the fibroblasts differentiated into myofibroblasts. Compared with the model group, 2-6 mg / mL BRPs could reverse the deformation and differentiation of fibrotic MRC5 cells induced by TGF-β1 to varying degrees, and although 8 mg / mL BRPs could reverse the deformation and differentiation of cells induced by TGF-β1, it also had a significant inhibitory effect on cell proliferation.

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

[0131] Western blotting was used to detect the effect 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 could significantly upregulate the expression levels of fibrosis marker proteins ColIα1, α-SMA and Fibronectin, indicating that TGF-β1 could induce MRC5 cell fibrosis; compared with the TGF-β1 model group, BRPs could significantly reduce the levels of ColIα1, α-SMA and Fibronectin proteins in a dose-dependent manner, indicating that BRPs could improve TGF-β1-induced MRC5 cell fibrosis.

[0132] 2.6 BRPs inhibit the migration of TGF-β1-induced fibrotic human embryonic lung fibroblasts

[0133] The effect of BRPs on the migration of TGF-β1-induced fibrotic human embryonic lung fibroblasts MRC5 was evaluated by wound healing assay. Figure 6 The results showed that compared with the control group, TGF-β1 treatment of cells for 24h or 48h could gradually heal the scratch area, indicating that it can promote cell migration; compared with the TGF-β1 group, BRPs could significantly inhibit the healing of the scratch area of ​​TGF-β1-induced fibrotic MRC5 cells, suggesting that BRPs can inhibit the migration of TGF-β1-induced fibrotic human embryonic lung fibroblasts.

[0134] 2.7 BRPs significantly improved lung function in BLM-induced PF mice

[0135] We used BLM to establish a PF model in C57BL / 6J mice and gave them BRPs for 3 weeks ( Figure 7 A), detect the effect of BRPs on the lung function evaluation indexes of mice in each experimental group. Figure 7 As shown in BI, compared with the control group, a single tracheal instillation of BLM could significantly reduce the total lung capacity (TLC), vital capacity (VC), deep inspiratory capacity (IC), tidal volume (TV), forced vital capacity (FVC), maximum respiratory flow (PEF), dynamic lung compliance (Cdyn) and quasi-static compliance (Cchord) of mice, indicating that BLM could lead to decreased lung function in mice; while 5g / kg BRPs could significantly reverse the above lung function indicators of BLM-induced PF mice, indicating that BRPs could improve the lung function of BLM-induced PF mice.

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

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

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

[0139] 1) Masson staining is a classic connective tissue staining method that can stain muscle fibers red and collagen fibers blue. Masson staining can be used to observe the distribution of collagen fibers and muscle fibers in lung tissue. Fig. 9 As shown in the figure, 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 was not only severely damaged in structure, but also had a large number of blue-stained collagen fibers, and the degree of pulmonary interstitial fibrosis was high; compared with the BLM model group, both BRPs and PFD could significantly reduce the blue-stained collagen fibers in the lung interstitium of mice and reduce 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 strong acid anionic dye that reacts with alkaline collagen, causing collagen fibers to produce obvious birefringence when exposed to polarized light. Among them, type I collagen fibers are closely arranged and show strong birefringence, appearing red in tissue sections exposed to polarized light and ordinary light. Through Sirius Red staining, we found that the lung tissue structure of mice in the normal group was relatively complete, and there were fewer red-stained type I collagen fibers; compared with the normal group, a large number of red-stained type I collagen fibers appeared in the lung tissue of mice in the BLM model group; and compared with 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 ( Fig.10 ).

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

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

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

[0144] The results of immunohistochemical staining showed that the ColIα1 staining in the lung tissue of the normal group mice was basically negative, indicating that the expression level of ColIα1 protein was low; compared with the normal control group, the lung tissue of the BLM model group mice showed strong positive staining of ColIα1, indicating that the expression level of ColIα1 was increased; compared with the model group, both BRPs and PFD could make the positive staining of ColIα1 in the lung tissue of mice lighter, indicating that BRPs can inhibit the expression of ColIα1 in the lung tissue of BLM-induced PF mice ( Fig.12 ).

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

[0146] RQ-qPCR results showed that compared with the normal control group, BLM could significantly increase the COLIA1 mRNA level in the lung tissue of mice, indicating that BLM could promote COLIA1 gene transcription; compared with the BLM model group, BRPs could downregulate the COLIA1 mRNA level in the lung tissue of BLM-induced PF mice, and there was no significant difference compared with PFD, suggesting that BRPs could inhibit COLIA1 gene transcription ( Fig.13 ).

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

[0148] Fig.14 and Fig.15 The results of immunofluorescence staining showed that compared with the normal control group, BLM significantly upregulated the expression levels of α-SMA and Fibronectin, markers of fibrosis in mouse lung tissue; compared with the BLM model group, both BRPs and PFD could decrease the expression levels of α-SMA and Fibronectin in mouse lung tissue, indicating that BRPs, like PFD, can also improve BLM-induced pulmonary fibrosis in PF mice by down-regulating α-SMA and Fibronectin.

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

[0150] Most lung tissue damage is accompanied by a certain degree of fibrosis. Based on the characteristic that "endogenous nucleases in apoptotic cells are activated and cut their own chromosomal DNA into gaps or 3'-OH terminal fragments containing 180-200bp", the TUNEL kit can transfer FITC-labeled dUTP to the 3'-OH end through the terminal deoxynucleotidyl transferase-mediated dUTP gap end labeling technology, so that the broken DNA can show the green fluorescence of FITC under a fluorescence microscope. Fig.16As shown, there were few FITC green fluorescent spots in the normal control group during TUNEL staining, indicating that there were few DNA breaks in the lung tissue; compared with the control group, there were more green fluorescent spots in the BLM model group, indicating that there were more DNA breaks; and compared with the model group, BRPs could significantly reduce the green fluorescent spots in the lung tissue of BLM-induced PF mice, and there was no significant difference compared with PFD, suggesting that BRPs can alleviate BLM-induced PF mice lung injury.

[0151] 2.15 BRPs can inhibit the infiltration of inflammatory cells in the bronchoalveolar region of BLM-induced PF mice and reduce lung inflammation

[0152] Lung tissue may also undergo fibrosis when stimulated by inflammation. The bronchoalveolar lavage fluid (BALF) of mice contains inflammatory cells such as macrophages, lymphocytes, neutrophils, and eosinophils. The number of inflammatory cells is an important indicator for measuring inflammatory responses. 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 an increase in bronchial alveolar inflammatory cell infiltration; compared with the BLM group, BRPs could significantly downregulate the number of inflammatory cells, suggesting that BRPs can reduce BLM-induced PF mouse lung tissue inflammation by inhibiting bronchial alveolar inflammatory cell infiltration ( Fig.17 ).

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

[0154] The initiation of inflammatory response is inseparable from inflammasomes. NLRP3 is an important component of NLRP3 inflammasomes. Fig.18 The results of immunofluorescence staining showed that the expression level of NLRP3 in normal lung tissue was extremely low; compared with normal lung tissue, BLM could significantly enhance the red fluorescence of NLRP3 in mouse lung tissue, indicating an enhanced inflammatory response; compared with the BLM model group, both BRPs and PFD could significantly weaken the red fluorescence of NLRP3 in the lung tissue of BLM-induced PF mice, indicating that BRPs and PFD could also effectively inhibit the activation of NLRP3 inflammasomes, thereby inhibiting the inflammatory response.

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

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

[0157] 2.18 BRPs inhibit Hedgehog signaling pathway in lung tissue of BLM-induced PF mice

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

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

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

[0161] 2.20 Hedgehog inhibitor significantly enhanced the inhibitory effect of BRPs on inflammatory lesions in lung tissue of BLM-induced PF mice

[0162] The effect of Hedgehog inhibitor combined with BRPs on inflammatory lesions in lung tissue 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 tissue of mice; compared with the BLM model group, Hedgehog inhibitor Gant 61 (10 mg / kg) and BRPs (3 g / kg) alone could partially inhibit the inflammatory lesions in lung tissue of BLM-induced PF mice, and the combined use of the two had a more significant inhibitory effect on the inflammatory lesions in lung tissue of BLM-induced PF mice ( Fig.25 and Fig.26 ).

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

[0164] Masson staining was used to evaluate the effect of the combination of Hedgehog inhibitors and BRPs on pulmonary interstitial fibrosis in BLM-induced PF mice. The results showed that compared with the control group, a large number of blue-stained collagen fibers appeared in the lung tissue of the BLM group mice, and the fibrosis score was higher, indicating the occurrence of pulmonary interstitial fibrosis; compared with the BLM model group, the Hedgehog inhibitor Gant 61 and BRPs alone could partially inhibit the production of blue-stained collagen fibers in the lung tissue 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 reduce the fibrosis score ( Fig. 27 and Fig.28 ), suggesting that Hedgehog inhibitors can enhance the inhibitory effect of BRPs on pulmonary interstitial fibrosis in BLM-induced PF mice. SiriusRed staining can observe similar inhibitory effects of Hedgehog inhibitors combined with BRPs on type I collagen fibers in the lung tissue of BLM-induced PF mice ( Fig.29 ).

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

[0166] Excessive deposition of collagen is the main feature of fibrotic diseases, and hydroxyproline is an amino acid unique to 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. Fig.30As shown in the results, compared with the control group, BLM significantly increased the total hydroxyproline content in the lung tissue of mice, indicating the occurrence of pulmonary fibrosis; compared with the BLM model group, both Gant 61 and BRPs could partially downregulate the hydroxyproline content in the lung tissue of BLM-induced PF mice; and compared with each single drug, the combination of Gant 61 and BRPs could further reduce the hydroxyproline content in the lung tissue of BLM-induced PF mice and improve pulmonary fibrosis. These results suggest that Hedgehog inhibitors can enhance the therapeutic effect of BRPs on pulmonary fibrosis in BLM-induced PF mice.

[0167] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of Chamaglucoside polysaccharide in the preparation of drugs for preventing and / or treating pulmonary fibrosis.

2. The use according to claim 1, characterized in that: The chamagu polysaccharide prevents and / or treats pulmonary fibrosis by inhibiting pulmonary cell fibrosis and cell migration.

3. Application of Chamaglucoside polysaccharide in the preparation of drugs for preventing lung injury and lung inflammation.

4. Application of Chamaglucoside polysaccharide in the preparation of drugs that inhibit the Hedgehog signaling pathway in lung cells.

5. The use according to any one of claims 1 to 4, characterized in that: The effective concentration of the chamagu polysaccharide on recombinant human transforming growth factor-induced fibrotic human embryonic lung fibroblasts is 2 to 6 mg / mL, and the effective dose on bleomycin-induced pulmonary fibrosis mice is 2 to 5 g / kg.

6. The use according to claim 5, characterized in that: The chamacarotene polysaccharide can be used together with a Hedgehog inhibitor; the Hedgehog inhibitor includes Gant 61.

7. The use according to claim 5, characterized in that: The medicine comprises chamagu polysaccharide and pharmaceutically permitted excipients.

8. The use according to claim 7, characterized in that: The dosage form of the drug is a pharmaceutically acceptable dosage form.

9. The use according to claim 8, characterized in that: The pharmaceutically acceptable dosage forms are tablets, granules, capsules, pills, syrups, suspensions, powders, oral solutions, and mixtures.

10. The use according to any one of claims 7 to 9, characterized in that: The total polysaccharide content of chamagu polysaccharide in the medicine is 35% to 53%.

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