Application of medicine composition in preparation of medicine for treating pulmonary fibrosis

Abstract

The invention provides application of a pharmaceutical composition in preparation of a medicine for treating pulmonary fibrosis, and belongs to the field of medicines, the pharmaceutical composition comprises the following traditional Chinese medicinal materials: ginseng, radix ophiopogonis, schisandra chinensis, poria cocos, rhizoma pinelliae preparata, radix scrophulariae powder, bran-fried rhizoma atractylodis, pericarpium citri reticulatae, liquorice, radix bupleuri, rhizoma cimicifugae, semen coicis, scutellaria baicalensis, verbena, rhizoma phragmitis and lophatherum gracile. It is found for the first time that the pharmaceutical composition can delay the development of pulmonary fibrosis, and pulmonary fibrosis can be treated through anti-inflammation and anti-fibrosis.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to an application of a pharmaceutical composition in preparing a medicine for treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is a chronic, progressive lung disease characterized by fibrosis and structural destruction of lung tissue. Its main symptoms include progressively worsening dyspnea, persistent dry cough, extreme fatigue, weight loss, and clubbing. It may even cause serious complications such as respiratory failure and heart failure. There is currently no cure for pulmonary fibrosis, but drugs such as pirfenidone and nintedanib can slow the progression of the disease.

[0003] During the process of pulmonary fibrosis, the number of macrophages increases significantly and is accompanied by over-activation; macrophages polarize into pro-inflammatory / cytotoxic M1 and anti-inflammatory / wound repair M2 macrophages under different local stimuli. M1 macrophages mainly secrete pro-inflammatory cytokines such as IL-6 and TNF-α, while M2 macrophages mainly secrete anti-inflammatory cytokines such as IL-10 and TGF-β. M1 macrophages are first recruited to the site of injury to promote inflammatory response, and then M2 macrophages are recruited to the site of injury to repair lung tissue.

[25] When M1 macrophages are overactivated, the secretion of proinflammatory factors increases, inhibiting the process of pulmonary fibrosis; when M2 macrophages are overactivated, they can induce the phenotypic transformation of lung interstitial cells to lung myofibroblasts, thereby promoting the deposition of ECM in lung tissue and accelerating the process of pulmonary fibrosis: suggesting that the balance of the ratio of M1 and M2 plays an important role in the process of pulmonary fibrosis.

[0004] Pulmonary fibrosis has become one of the main causes of chronic respiratory failure and death. Research and treatment of pulmonary fibrosis still face many challenges, requiring further investment and R&D efforts. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an application of a pharmaceutical composition in preparing a drug for treating pulmonary fibrosis.

[0006] Specifically, the pharmaceutical composition includes the following Chinese medicinal materials: ginseng, ophiopogon japonicus, schisandra chinensis, tuckahoe, pinellia ternata, scrophularia ningpoensis, atractylodes macrocephala, tangerine peel, licorice, bupleurum, cimicifuga heracleifolia, coix seed, scutellaria baicalensis, verbena, reed root and lophatherum gracile.

[0007] Further specifically, the pharmaceutical composition includes the following Chinese medicinal materials in parts by weight: 2-4 parts of ginseng, 5-7 parts of ophiopogon japonicus, 2-4 parts of schisandra chinensis, 7-9 parts of poria cocos, 7-9 parts of pinellia ternata, 5-7 parts of scrophularia ningpoensis, 4-6 parts of atractylodes lancea with bran, 5-7 parts of tangerine peel, 2-4 parts of licorice, 5-7 parts of bupleurum, 2-4 parts of cimicifuga, 8-12 parts of coix seed, 8-12 parts of scutellaria baicalensis, 8-12 parts of verbena, 13-17 parts of reed root, and 1-3 parts of lophatherum gracile.

[0008] In certain specific embodiments of the present invention, the pharmaceutical composition includes the following Chinese medicinal materials in parts by weight: 3 parts of ginseng, 6 parts of ophiopogon japonicus, 3 parts of schisandra chinensis, 8 parts of poria, 8 parts of pinellia ternata, 6 parts of scrophularia ningpoensis, 5 parts of atractylodes macrocephala, 6 parts of tangerine peel, 3 parts of licorice, 6 parts of bupleurum, 3 parts of cimicifuga, 10 parts of coix seed, 10 parts of scutellaria baicalensis, 10 parts of verbena, 15 parts of reed root and 2 parts of lophatherum gracile.

[0009] In some specific embodiments of the present invention, the preparation method of the pharmaceutical composition is as follows: accurately weigh 3 parts of ginseng, 6 parts of ophiopogon, 3 parts of schisandra, 8 parts of tuckahoe, 8 parts of pinellia, 6 parts of scrophularia, 5 parts of atractylodes, 6 parts of dried orange peel, 3 parts of licorice, 6 parts of bupleurum, 3 parts of cimicifuga, 10 parts of coix seeds, 10 parts of scutellaria, 10 parts of verbena, 15 parts of reed rhizome and 2 parts of lophatherum gracile by weight; after mixing, decoct twice in water, the first decoction for 60 minutes, the solid-liquid ratio is 1g:8mL; the second decoction for 40 minutes, the solid-liquid ratio is 1g:6mL; filter, concentrate the filtrate to a relative density of 1.02-1.10 (60°C); spray dry to obtain extract powder, add lactose and mannitol as excipients, wherein the mass ratio of extract powder to lactose and mannitol is 7:2:1; mix well, dry press and granulate to obtain.

[0010] Specifically, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0011] More specifically, the auxiliary materials include but are not limited to: lactose, mannitol, methylcellulose, benzoic acid, sorbic acid, food coloring and / or talc.

[0012] Preferably, the auxiliary material comprises lactose and mannitol, and the mass ratio of lactose to mannitol is (1-3):1.

[0013] Specifically, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, and the excipient includes: a binder, a filler, a disintegrant, a lubricant, a preservative, an antioxidant, a flavoring agent, an aromatic, a solubilizer, an emulsifier, a solubilizer or an osmotic pressure regulator.

[0014] Specifically, the pharmaceutical composition is a tablet, capsule, pill, suppository, aerosol, oral liquid preparation, granule, powder, injection, syrup, wine, tincture, dew, film or a combination thereof.

[0015] Specifically, the administration of the pharmaceutical composition includes oral administration, injection, implantation, external application, spraying, inhalation or a combination thereof.

[0016] Specifically, the pulmonary fibrosis includes idiopathic, primary, immune and / or physicochemical pulmonary fibrosis.

[0017] More specifically, the pulmonary fibrosis is idiopathic pulmonary fibrosis induced by bleomycin.

[0018] Specifically, the mechanism of action of the pharmaceutical composition is to delay the development of pulmonary fibrosis by inhibiting collagen formation and reducing the degree of inflammation.

[0019] Compared with the prior art, the present invention has the following advantages: The present invention finds that a pharmaceutical composition containing ginseng, ophiopogon japonicus, schisandra chinensis, tuckahoe, pinellia ternata, scrophularia ningpoensis, stir-fried atractylodes lancea, tangerine peel, liquorice, bupleurum, cimicifuga heracleifolia, coix seed, scutellaria baicalensis, verbena, reed root and loblolly leaf can significantly improve the body weight and lung index of rats with bleomycin-induced pulmonary fibrosis; can significantly improve the changes in the appearance and pathological structure of lung tissue caused by BLM, and reduce collagen deposition in lung tissue; can reduce the expression levels of α-SMA, collagen type Ⅰ, TGF-β and TNF-α mRNA in lung tissue and the expression of α-SMA and Vimentin protein in lung tissue; can reduce the content of IL-4 and TNF-α in serum; can reduce CD68 in lung tissue sections. + M0 macrophages, CD64 + M1 macrophages and CD206 + The proportion of M2 macrophages can delay the development of pulmonary fibrosis and treat pulmonary fibrosis through anti-inflammatory and anti-fibrotic methods, providing a basis for further research. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure shows the effect of the drug composition on the body weight of rats with pulmonary fibrosis.

[0021] Figure 2 The effects of the pharmaceutical composition on the lung tissue morphology (a) and lung index (b) of rats with pulmonary fibrosis are shown in FIG. ** P <0.01, * P <0.05.

[0022] Figure 3 The figure shows the effect of the drug composition on the pathological morphology of lung tissue in rats with pulmonary fibrosis as shown in HE staining.

[0023] Figure 4 The results of Masson staining experiment.

[0024] Figure 5 The effect of the pharmaceutical composition on macrophage polarization in lung tissue of rats with pulmonary fibrosis, ** P <0.01, * P <0.05.

[0025] Figure 6 The effect of the pharmaceutical composition on the levels of IFN-γ and IL-4 in the serum of rats with pulmonary fibrosis is shown in FIG. ** P <0.01, * P <0.05.

[0026] Figure 7 The quantitative analysis of the expression of α-SMA and Vimentin proteins in lung tissue of rats with pulmonary fibrosis by the drug composition is ** P <0.01, * P <0.05.

[0027] Figure 8 The effect of the pharmaceutical composition on the content of α-SMA and Collagen type Ⅰ mRNA in the lung tissue of rats with pulmonary fibrosis, ** P <0.01, * P <0.05.

[0028] Fig. 9 The effect of the dosage of the drug composition on (a) MRC-5 cell viability and (b) cytotoxicity, ** P <0.01, * P <0.05.

[0029] Fig.10 The effect of the drug composition on the contractile function of serum-stimulated MRC-5 cells, ** P <0.01, * P <0.05.

[0030] Fig.11 The effect of the pharmaceutical composition on the mRNA expression of α-SMA and Collagen type Ⅰ in MRC-5 cells stimulated by TGF-β1, ** P <0.01, * P <0.05.

[0031] Fig.12 The effect of the drug composition on the expression of α-SMA protein in MRC-5 cells stimulated by TGF-β1 was compared with that of Control. ** P <0.01; compared with TGF-β1 # P <0.05, ## P <0.01.

[0032] Fig.13 Effects of the drug composition on polarization of THP-1 derived M0 to (a) M1 and (b) M2 macrophages, compared with M0 ** P <0.01; compared with M2 ## P <0.01.

[0033] Fig.14 Effects of the drug combination and Lianhua Qingwen capsule on the mRNA expression of α-SMA and Collagentype Ⅰ in MRC-5 cells stimulated by TGF-β1, compared with the control ** P <0.01; compared with TGF-β1 # P <0.05, ## P <0.01. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0035] Example 1 Pharmaceutical composition inhibits bleomycin-induced pulmonary fibrosis in rats Table 1 Experimental reagents

[0036] 1. Preparation of Drug Solution Preparation of extract: The preparation method of the drug combination (YWZH) is referenced to: Pang W, J Evid Based Med. 2022 Mar; 15(1): 30-38. After preparation, soak and extract twice, each time with 10 times the volume of boiling water, soak for 40 minutes, filter. Below 60°C, concentrate, spray dry to obtain the extract, and the paste yield is 18%.

[0037] Preparation of YWZH solution: Weigh 71.4 g of the extract, add 210 mL of pure water to make a 0.34 g / mL solution, store at 4°C, and shake well before use.

[0038] 2. Establishment of pulmonary fibrosis rat model, grouping, medication and sampling Preparation of bleomycin (BLM) solution: Weigh 10 mg of BLM and add 4 mL of saline to dissolve to obtain a 2.5 mg / mL BLM solution.

[0039] Sixty male SD rats weighing 220±20g were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (License No.: SCXK(Jing)2016-0006). They were kept in Tianjin Yishengyuan Biotechnology Co., Ltd. (Ethics No.: YSY-DWLL-2021172) and randomly divided into 6 groups, namely 14-day control group (Ctrl-14d), 14-day model group (BLM-14d), 14-day YWZH group (YWZH-14d), 21-day control group (Ctrl-21d), 21-day model group (BLM-21d), and 21-day YWZH group (YWZH-21d), with 10 rats in each group.

[0040] Model establishment and drug administration: Rats were anesthetized by intraperitoneal injection of chloral hydrate, fixed, and the trachea was exposed in the neck. Except for the Ctrl-14d and Ctrl-21d groups, the rats in the other groups were given a single tracheal instillation of BLM (2.5 mg / mL) solution at 0.1 mL / 100 g body weight. The Ctrl-14d and Ctrl-21d groups were injected with a single tracheal injection of normal saline at 0.1 mL / 100 g body weight. The trachea was quickly sutured and the rats were placed upright for 15 seconds to allow the drug solution to be evenly distributed in the lungs. The Ctrl-14d, BLM-14d, and YWZH-14d groups began to receive medication on the second day after modeling. The YWZH-14d group was intragastrically administered with YWZH at 3.4 g / kg, and the Ctrl-14d and BLM-14d groups were given pure water. The Ctrl-21d, BLM-21d, and YWZH-21d groups began to receive medication on the eighth day after modeling. The YWZH-21d group was intragastrically administered with YWZH at 3.4 g / kg, and the Ctrl-21d and BLM-21d groups were given pure water. The medication was continued for 14 days at 10 mL / kg body weight, and the last weighing was done 24 hours after the last medication.

[0041] The changes in rat body weight Figure 1 As shown, YWZH can improve the body weight of rats with pulmonary fibrosis to some extent.

[0042] 3. Detection of pulmonary fibrosis related indicators The rats were weighed 24 hours after the last administration and anesthetized with intraperitoneal injection of chloral hydrate. The rats were fixed on the experimental platform, blood was collected from the abdominal aorta and stored in a 5 mL coagulant tube, and then the lung tissue was separated and removed, the wet weight of the lung tissue was accurately weighed, and the lung index was calculated; the lungs were divided into left and right lungs, the left lung was fixed in tissue fixative for paraffin embedding and sectioning, the right lung was divided into three parts and stored in 1.5 mL centrifuge tubes at -80°C for subsequent RNA extraction and tissue protein extraction of tissue samples, and the mid-end, thymus and liver were placed in tissue fixative and fixed at room temperature.

[0043] The experimental data were analyzed by GraphPad Prism 8.0.1 software, and the data were expressed as mean ± standard deviation, and one-way ANOVA was used for analysis of variance.

[0044] 3.1 Lung Index The rats were fasted the day before sampling. On the day of sampling, the rats were weighed and anesthetized. The lung tissues were separated and placed in a plate filled with physiological saline. The wet weight of the lung tissues was accurately weighed after removing the fat tissue around the organs. The lung index was calculated according to the following formula:

[0045] like Figure 2 As shown in (a), the lungs of rats in the control group showed no abnormal appearance, the lung surface was ruddy and shiny, and there were no nodules or hemorrhages. Compared with the control group, the lungs of rats in the model group showed obvious dark red, nodules and hemorrhages of different sizes were seen in the interstitial space, and edema occurred; the hemorrhages in the lungs of rats in the YWZH group were reduced, and the edema was significantly improved. Compared with the BLM-14d group, the lung tissue surface of rats in the BLM-21d group was rougher, the hemorrhages increased, and the pulmonary edema was more obvious. Compared with the YWZH-14d group, the lung tissue of rats in the YWZH-21d group was more ruddy and shiny, and the hemorrhages were reduced.

[0046] like Figure 2 As shown in (b), the lung index of the model rats was significantly increased compared with the control group; the lung index of the YWZH group was significantly decreased compared with the model group. The lung index of the BLM-21d rats was also significantly increased compared with the BLM-14d group.

[0047] 3.2. Analysis of histopathological changes 3.2.1 Preparation of tissue paraffin sections After the dissected tissue is flattened, it is placed in a dehydration box. The tissue is placed in a dehydrator for dehydration with gradient ethanol. After wax immersion, it is placed in an embedding machine for embedding. Finally, the tissue sections are cut into 4 μm sections and flattened on 55°C warm water. After being picked up with a slide, they are placed in a 60°C oven for 3 hours to dry, and stored at room temperature for later use.

[0048] 3.2.2 HE staining The paraffin sections were dewaxed in xylene I, II, gradient ethanol and tap water, then stained with hematoxylin solution for 5 minutes, washed with tap water, differentiated for 30 seconds after washing, rinsed with tap water, rinsed with running water after blueing, and then dehydrated in gradient ethanol. After that, they were placed in eosin solution, and the stained sections were dehydrated in gradient ethanol and xylene, sealed with neutral gum, and placed in a fume hood for 24 hours before image acquisition under a microscope to analyze the pathological changes of the tissue.

[0049] Figure 3 HE staining results show that the lung tissue structure of the control group was normal, the lung tissue surface was ruddy and elastic, and there were no bleeding spots. Compared with the control group, the pathological specimens of the model group rats showed a large number of inflammatory cells filling the alveolar spaces, thickening of the alveolar walls, septal edema, and a large number of red blood cells (hemorrhage) infiltration. Compared with the model group, the lung tissue structure of the YWZH group was significantly improved, with less bleeding, less inflammatory cell infiltration, and intact alveolar structure. Compared with the BLM-14d group, more severe alveolar wall thickening and increased inflammatory cell and red blood cell infiltration were observed in the lung tissue sections of the BLM-21d group rats.

[0050] 3.2.3 Masson staining The paraffin sections were dewaxed in xylene I, II, gradient ethanol and tap water, stained with Weigert iron hematoxylin, rinsed with distilled water, differentiated for 10 seconds after rinsing, rinsed with distilled water, added with Masson blue solution to return to blue for 2 minutes), rinsed with tap water for 1 minute, stained with Ponceau fuchsin for 10 minutes, rinsed with weak acid working solution for 10 seconds, differentiated with phosphomolybdic acid for 30 seconds, rinsed with weak acid working solution for 10 seconds, rinsed with distilled water for 10 seconds, stained with aniline blue working solution for 30 seconds, rinsed with weak acid working solution for 10 seconds, and the sections were placed in gradient ethanol and xylene I, II, and sealed with neutral gum. After sealing, the sections were placed in a fume hood for 24 hours and then images were collected under a microscope.

[0051] Figure 4 This is the result of Masson staining. Compared with the control group, a large number of blue collagen fibers and red blood cell infiltration were observed in the pathological specimens of the model group. Compared with the model group, the degree of collagen fiber proliferation in the YWZH group was reduced.

[0052] 3.2.4 IHC staining After the paraffin sections were dewaxed in xylene I, II, gradient ethanol and tap water, they were placed in an antigen retrieval box at 98°C for 15 minutes, and then taken out and dried to room temperature (about 30 minutes); washed with PBS 3 times, 3 minutes each time, covered with 3% hydrogen peroxide, covered with film, and reacted at room temperature for 10 minutes; washed with PBS 3 times, 3 minutes each time to block endogenous peroxidase, covered with BSA, covered with film, blocked at room temperature for 40 minutes, and blocked specific proteins. Add primary antibody, cover with film, and incubate at 4°C overnight. The dilution ratio of the primary antibody used in this experiment is shown in Table 2. Place the wet box at room temperature for 30 minutes, add secondary antibody to cover the tissue, cover with film, and incubate at 37°C for 30 minutes; wash with PBS 4 times, 5 minutes each time. Add SABC to cover the tissue, cover with film, and incubate at 37°C for 30 minutes; wash with PBS 4 times, 5 minutes each time. Add DAB working solution to cover the tissue, let it stand for 10-15 minutes, and observe under a microscope; soak in tap water after positive results, and rinse for 10 minutes. Add hematoxylin to cover the tissue, incubate at room temperature for 1 min, and then rinse with tap water. Place the slices in gradient ethanol and xylene I and II, seal with neutral gum, and place in a fume hood for 24 hours before collecting images under a microscope and performing quantitative analysis.

[0053] Table 2 Dilution volume ratio of primary antibody

[0054] Figure 5 The results of IHC staining show that compared with the Ctrl-14d group, the CD68 + 、CD64 + and CD206 + The number of macrophages increased significantly after administration of YWZH. + 、CD64 + and CD206 + The number of macrophages in the BLM-21d group was significantly reduced compared with the Ctrl-21d group. + and CD206 + The number of macrophages was significantly upregulated, CD64 + The number of macrophages also increased, but not significantly. + and CD206 + The number of macrophages was significantly reduced, and CD64 + The number of macrophages was slightly decreased. + 、CD206 + and CD64 +There was no significant difference in the number of macrophages. Comparison of the changes in the number of macrophages between the BLM-14d and BLM-21d groups revealed that compared with the BLM-21d group, the number of CD64 + The number of macrophages increased more significantly; compared with the BLM-14d group, the number of CD68 + and CD206 + The increase in the number of macrophages was more obvious.

[0055] 3.3 Detection of serum cytokines Blood was collected from the abdominal aorta, and the supernatant was divided into seven sets of centrifuge tubes after centrifugation and stored in a refrigerator at -80°C. Before the experiment, the serum was thawed on ice, and the levels of IFN-γ and IL-4 in the serum were detected by ELISA.

[0056] Determination of cytokine levels in serum: Prepare the corresponding concentration gradient standard according to the instructions of the kit, dilute the serum according to the sample amount, add 50 μL of the corresponding solution to each well, cover the membrane, incubate at 37°C for 30 min, add 350 μL of 1× washing solution for 30 s and discard, repeat 5 times, add 50 μL of HRP to cover the membrane and incubate at 37°C for 30 min, repeat once, add 50 μL of color developer A and 50 μL of color developer B to each well, mix well, incubate at 37°C in the dark for 10 min, add 50 μL of stop solution to each well, measure OD value at 450 nm, draw standard curve, and calculate sample concentration.

[0057] The experimental results are as follows Figure 6 As shown, (a) is the result of IFN-γ content determination: compared with Ctrl-14d group, the IFN-γ content in serum of rats in BLM-14d group was significantly increased; compared with BLM-14d group, the IFN-γ content in serum of rats in YWZH-14d group was significantly decreased. Compared with Ctrl-21d group, the IFN-γ content in serum of rats in BLM-21d group was slightly increased; compared with BLM-21d group, the IFN-γ content in serum of rats in YWZH-21d group was slightly decreased.

[0058] (b) is the result of IL-4 content determination. Compared with the control group, the IL-4 content in the serum of the rats in the model group was slightly increased; compared with the model group, the IL-4 content in the serum of the rats in the YWZH group was significantly decreased.

[0059] 3.4 Detection of α-SMA and Vimentin protein expression 3.4.1 Extraction of total protein from lung tissue Prepare an appropriate amount of protein lysis buffer (RIPA: PIC: PMSF = 100: 10: 1) according to the sample volume, take an appropriate amount of lung tissue into a 2 mL centrifuge tube containing 220 μL of protein lysis buffer, ultrasonically break the lung tissue, repeat several times until the homogenate is free of particles, stand on ice for 30 minutes after the end of the ultrasonication, and then centrifuge for 20 minutes. The supernatant is the total protein. The protein concentration is measured by BCA method. The final quantitative concentration of 14-day rat lung tissue protein is 2 μg / μL, and the final quantitative concentration of 21-day rat lung tissue protein is 5 μg / μL. The protein sample and the loading buffer solution are mixed evenly according to the proportion. After heating at 100℃ for 10 minutes, store at -20℃ for later use.

[0060] 3.4.3 Western Blotting Add the marker and protein sample to the sample loading wells in turn, perform electrophoresis at a constant voltage of 160 V for 30 min, cut a PVDF membrane of appropriate area and activate it in methanol, and soak the thick and thin filter papers in 1× TransBuffer.

[0061] Cut the separation gel part where the target protein is located according to the molecular weight indicated by the marker. After the filter paper, gel and PVDF membrane are fixed, put them into the transfer tank and transfer the membrane under the condition of constant current of 300mA. The transfer time is determined by the molecular weight of the protein. The transfer time is 1min for a molecular weight of 1KD.

[0062] After the transfer, rinse the PVDF membrane with 1×TBST solution, add skim milk powder to the blocking buffer and block for 90 min at room temperature, discard the skim milk powder, wash off the skim milk powder on the surface of the PVDF membrane with 1×TBST solution, and then roll the PVDF membrane inward with the front side facing inward and place it in a 50mL centrifuge tube containing the primary antibody solution and incubate it overnight on a roller at 4°C. The dilution ratio of the primary antibody is shown in Table 3.

[0063] Remove the PVDF membrane and add appropriate amount of 1×TBST solution to wash the membrane 5 times, with the time being 1min, 3min, 5min, 7min, and 7min respectively. Add 5mL of secondary antibody solution and incubate at room temperature for 1h. Wash the membrane 5 times with 1×TBST solution according to the above time. The dilution ratio of the secondary antibody is shown in Table 4.

[0064] Prepare ECL luminescent reagents A and B in a 1:1 ratio and mix them evenly in a culture dish. Soak the PVDF membrane face down in the ELC luminescent working solution for about 2 minutes. After complete immersion, place the membrane face up in the dark box of the multifunctional imaging system to take pictures and analyze the electrophoresis bands.

[0065] Table 3 Dilution volume ratio of primary antibody

[0066] Table 4 Dilution volume ratio of secondary antibody

[0067] Western Blotting results are as follows Figure 7 As shown in the figure, compared with the Ctrl-14d group, the expression levels of α-SMA and Vimentin proteins in the lung tissue of the rats in the BLM-14d group were significantly increased; compared with the BLM-14d group, the expression levels of α-SMA and Vimentin proteins in the lung tissue of the rats in the YWZH-14d group were significantly decreased. Compared with the Ctrl-21d group, the expression level of α-SMA protein in the lung tissue of the rats in the BLM-21d group was significantly increased, and the expression level of Vimentin protein was slightly increased; compared with the BLM-21d group, the expression levels of α-SMA and Vimentin proteins in the lung tissue of the rats in the YWZH-21d group were significantly decreased.

[0068] 3.5 Detection of mRNA expression of α-SMA and collagen type Ⅰ Sample pretreatment: Take an appropriate amount of lung tissue into a 2 mL centrifuge tube, add 1 mL LB to grind, place at room temperature for 5 min, add 200 μL chloroform and place at room temperature for 3 min, centrifuge, take 400 μL of the upper aqueous phase, add 200 μL anhydrous ethanol, and mix well.

[0069] Extraction of total RNA: The mixture of the aqueous phase and anhydrous ethanol was transferred to an RNA adsorption column, and the total RNA in the tissue was extracted and its concentration was determined according to the instructions of the YEASEN RNA extraction kit.

[0070] mRNA expression detection: ① gDNA digestion: prepare the reaction system according to Table 5 and incubate at 42°C for 3 min.

[0071] Table 5 gDNA system

[0072] ②RNA reverse transcription: prepare the reaction system according to Table 6, 25℃ for 5 min, 55℃ for 15 min, and 85℃ for 5 min.

[0073] Table 6 RNA reverse transcription system

[0074] ③ Real-time quantitative PCR: Prepare the PCR reaction system according to Table 7 for RT-qPCR experiment. In this experiment, the reaction conditions of RT-PCR are: 95℃ 2min, 95℃ 15s, 62℃ 60s, 72℃ 30s for 40 cycles. Melting curve: between 65℃ and 95℃. Obtain the Ct value of each sample, calculate its ΔCt value, and take 2 -ΔΔCtThe primer sequences in this experiment are shown in Table 8.

[0075] Table 7 PCR reaction system

[0076] Table 8 Primer sequences

[0077] like Figure 8 As shown in the figure, compared with the Ctrl-14d group, the expression levels of α-SMA and Collagen type Ⅰ in the lung tissue of rats in the BLM-14d group were significantly increased; after administration of YWZH, the expression levels of α-SMA and Collagen type Ⅰ in the lung tissue of rats were significantly decreased. Compared with the Ctrl-21d group, the expression level of Collagen type Ⅰ in the lung tissue of rats in the BLM-21d group was significantly increased, and the expression level of α-SMA was slightly increased; after administration of YWZH, the expression levels of α-SMA and Collagen type Ⅰ in the lung tissue of rats were significantly decreased. Compared with the BLM-14d group, the expression level of α-SMA mRNA in the lung tissue of rats in the BLM-21d group was slightly increased, and the expression level of Collagen type Ⅰ mRNA was significantly increased.

[0078] YWZH can significantly improve the body weight and lung index of rats with bleomycin-induced pulmonary fibrosis; pathological results show that YWZH can significantly improve the changes in the appearance and pathological structure of lung tissue caused by BLM, reduce collagen deposition in lung tissue; reduce the expression levels of α-SMA, Collagen type Ⅰ, TGF-β and TNF-α mRNA in lung tissue, as well as the expression of α-SMA and Vimentin protein in lung tissue; reduce the levels of IL-4 and TNF-α in serum; reduce the expression of CD68 in lung tissue sections. + M0 macrophages, CD64 + M1 macrophages and CD206 + The proportion of M2 macrophages. It shows that YWZH can delay the development of pulmonary fibrosis, revealing that YWZH can play an anti-inflammatory and anti-fibrotic role in the treatment of COVID-19-induced pulmonary fibrosis, providing a basis for further follow-up research.

[0079] Example 2 Study on the anti-fibrotic effect of the pharmaceutical composition at the cellular level Table 9 Experimental reagents

[0080] 1. Preparation of experimental solution 1.1 Preparation of TGF-β1 Citric acid was added to prepare a 100 μg / mL TGF-β1 stock solution.

[0081] Add 100 μL of PBS containing 5% w / w trehalose to the mother solution to dilute it to a 50 μg / mL solution, aliquot it, and store it at -20°C until use.

[0082] 1.2 Preparation of complete cell culture medium Complete culture medium for culturing MRC-5 cells: MEM culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) by volume.

[0083] Complete culture medium for culturing THP-1 cells: RPMI-1640 culture medium containing 15% fetal bovine serum and 1% penicillin-streptomycin by volume.

[0084] 2. Cell experiments 2.1 MRC-5 cell proliferation assay MRC-5 cells were seeded at 5000 cells / well in a 96-well plate. After the cells adhered, different concentrations of YWZH (100, 200, 400, 500 μg / mL) were added, and a blank control group was set up. Six replicate wells were set up for each concentration. After the addition of drugs, the plates were placed in a 37°C cell culture incubator and cultured for 48 hours.

[0085] After 48 hours, CCK-8 was added and the cells were incubated at 37°C in the dark for 40 minutes. The OD value was measured at 450 nm, and the data were analyzed graphically to determine the optimal drug concentration for the MRC-5 cells.

[0086] The experimental results are shown in Fig. 9 In (a), 100-500 μg / mL of the drug composition had no significant effect on the viability of MRC-5 cells.

[0087] 2.2 MRC-5 cytotoxicity assay MRC-5 cells were seeded at 5000 cells / well in a 96-well plate. After the cells adhered, different concentrations of YWZH (100, 200, 400, 500 μg / mL) were added, and a blank control group was set up. Six replicate wells were set up for each concentration. After the addition of drugs, the plates were cultured in a 37°C cell culture incubator for 48 h.

[0088] After 48 h, LDH was added and incubated at 37°C in the dark for 30 min. The OD value was measured at 490 nm and the data were analyzed graphically.

[0089] The experimental results are shown in Fig. 9 In (b), 100-500 μg / mL of the drug composition was non-toxic to MRC-5 cells.

[0090] 2.3 MRC-5 cell collagen gel contraction experiment Observation of MRC-5 cells at 25 cm 2 When the cell culture flask confluence reached more than 80%, discard the cell supernatant, add 1 mL D-PBS to wash twice, add 1 mL 0.25% trypsin to digest the cells for 2 min, and transfer the cell suspension to two centrifuge tubes after the cells are completely digested. Centrifuge and discard the supernatant. Resuspend the cells in serum-containing culture medium and serum-free culture medium respectively and adjust the concentration to 1×10 6 cells / mL, prepare the cell suspension containing rat tail collagen according to the ratio in Table 10. After mixing, add 500μL of rat tail collagen cell suspension to each well immediately, incubate the 24-well plate in a 37℃ incubator for 20min, and then add: YWZH prepared with 1% PS-MEM, 5% FBS-1% PS-MEM or 5% FBS-1% PS-MEM by volume. Use a 200μL pipette tip to separate the gel from the well plate to ensure that the gel can move freely in the well plate, and observe and take pictures after giving 1μL 100μg / mL YWZH at 0h and 72h respectively.

[0091] Table 10 Collagen gel system

[0092] Note: The order of adding samples is 1M NaOH, type I rat tail collagen, 10× PBS, and cell suspension. Adjust the volume of the pipette to be used. Once started, it cannot be stopped midway and needs to be completed quickly in one go.

[0093] The experimental results are shown in Fig.10 The results of the collagen gel contraction experiment showed that 5% FBS could induce the contraction of MRC-5 cells in the collagen gel. After treatment with the drug composition for 72 h, the contraction ability of MRC-5 cells was significantly inhibited.

[0094] 2.4 Extraction and detection of total RNA from MRC-5 cells In a 6-well plate, 2 × 10 5 cells / well, and the cell adhesion was observed every other day. After more than 80% of the cells were attached, 100 μg / mL YWZH was added, and the culture was continued for 4 h. After that, 10 ng / mL TGF-β1, M1 CM and M2 CM were added. The cells were placed in an incubator at 37°C and cultured for 48 h. Total cell RNA was extracted according to Experiment 3.5. The primer sequences involved in the experiment are shown in Table 11. The PCR system and amplification procedure were the same as the real-time quantitative PCR step in 3.5 of Example 1.

[0095] Table 11 Primer sequences

[0096] PCR results are as follows Fig.11 As shown in the results, TGF-β1 induced an increase in the expression of α-SMA mRNA and Collagen type Ⅰ mRNA in MRC-5 cells. After administration of YWZH, the increase in the expression of α-SMA and Collagen type Ⅰ mRNA in MRC-5 cells induced by TGF-β1 was significantly inhibited.

[0097] 2.5 Extraction and detection of total protein from MRC-5 cells In a 6-well plate, 2 × 10 5 cells / well, and observe the cell adhesion every other day. When more than 80% of the cells adhered, add 200μg / mL YWZH or 100μg / mL YWZH, and continue to culture for 4h. Then add 10ng / mL TGF-β1, and continue to culture in a 37℃ incubator for 48h. Then refer to experiment 3.4.1 to extract total protein in the cells.

[0098] Western Blotting results are as follows Fig.12 As shown, TGF-β1 induced an increase in the expression of α-SMA protein in MRC-5 cells, and administration of YWZH could reduce the increase in the expression of α-SMA protein in MRC-5 cells induced by TGF-β1.

[0099] 2.6 Extraction and detection of total RNA from THP-1 cells THP-1 cells were grown in 6-well plates at 2 × 10 6 cells / well plate, and phorbol ester (PMA) was added to induce THP-1 cells to transform into M0 macrophages. After 24 hours, 200 μg / mL YWZH was added, and 20 ng / mL IFN-γ and 1 μg / mL LPS, 20 ng / mL IL-4 were added after continuing to culture for 4 hours. After continuing to culture in a 37°C incubator for 24 hours, total cell RNA was extracted according to 3.5 of Example 1. The primer sequences involved in the experiment are shown in Table 12. The PCR system and amplification procedure are the same as the real-time quantitative PCR step 3.5 in Example 1.

[0100] Table 12 Primer sequences

[0101] The experimental results are shown in Fig.13, PCR results showed that YWZH could significantly reduce the increase of CD206 mRNA expression in M2 macrophages derived from THP-1 cells, and slightly reduce the increase of IL-10 mRNA expression. YWZH had no significant effect on the polarization of M0 to M1 macrophages derived from THP-1 cells.

[0102] YWZH can inhibit the contraction of MRC-5 cells stimulated by serum, reduce the increase of α-SMA and Collagen type Ⅰ mRNA expression and α-SMA protein expression in MRC-5 cells stimulated by TGF-β1, reduce the increase of α-SMA and Collagen type Ⅰ mRNA expression in MRC-5 cells stimulated by M1 CM and M2 CM, inhibit the polarization of THP-1-derived M0 to M2 macrophages, but have no significant effect on the polarization of THP-1-derived M0 to M1 macrophages. This suggests that YWZH may inhibit pulmonary fibrosis by regulating the polarization of macrophages from M1 to M2.

[0103] Comparative Example 1 Lianhua Qingwen Lianhua Qingwen Capsule (LHQW) was used as a negative control to explore the anti-fibrosis effect of YWZH. The experimental procedures were exactly the same as those of the YWZH experimental group in 2.4.

[0104] The experimental results are shown in Fig.14 , PCR results showed that the expression levels of α-SMA and Collagen type Ⅰ mRNA increased significantly after TGF-β1 induction. After administration of YWZH, the increase in the expression levels of α-SMA and Collagen type Ⅰ mRNA could be significantly inhibited, while LHQW had no significant inhibitory effect, suggesting that YWZH may have the effect of inhibiting fibrosis, while LHQW has no technical effect of inhibiting fibrosis.

[0105] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for treating pulmonary fibrosis, characterized in that: The pharmaceutical composition comprises the following Chinese medicinal materials: ginseng, ophiopogon japonicus, schisandra chinensis, tuckahoe, pinellia ternata, scrophularia ningpoensis, stir-fried atractylodes lancea with bran, tangerine peel, liquorice, bupleurum, cimicifuga heracleifolia, coix seed, scutellaria baicalensis, verbena, reed root and lophatherum gracile.

2. The use according to claim 1, characterized in that: The pharmaceutical composition comprises the following Chinese medicinal materials in parts by weight: 2-4 parts of ginseng, 5-7 parts of ophiopogon japonicus, 2-4 parts of schisandra chinensis, 7-9 parts of tuckahoe, 7-9 parts of pinellia ternata, 5-7 parts of scrophularia ningpoensis, 4-6 parts of atractylodes lancea with bran, 5-7 parts of tangerine peel, 2-4 parts of licorice, 5-7 parts of bupleurum, 2-4 parts of cimicifuga, 8-12 parts of coix seed, 8-12 parts of scutellaria baicalensis, 8-12 parts of verbena, 13-17 parts of reed root and 1-3 parts of lophatherum gracile.

3. The use according to claim 2, characterized in that: The pharmaceutical composition also includes pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that: The auxiliary materials include lactose and mannitol, and the mass ratio of lactose to mannitol is (1-3):

1.

5. The use according to claim 2, characterized in that: The pharmaceutical composition further comprises a pharmaceutically acceptable excipient, which includes: a binder, a filler, a disintegrant, a lubricant, a preservative, an antioxidant, a flavoring agent, an aromatic agent, a solubilizer, an emulsifier, a solubilizer or an osmotic pressure regulator.

6. The use according to claim 2, characterized in that: The pharmaceutical composition is in the form of tablets, capsules, pills, suppositories, aerosols, oral liquid preparations, granules, powders, injections, syrups, wine preparations, tinctures, lotions or films.

7. The use according to claim 2, characterized in that: The administration method of the pharmaceutical composition includes one or more of oral administration, injection, implantation, external application, spraying or inhalation.

8. The use according to claim 1, characterized in that: The pulmonary fibrosis includes idiopathic, primary, immune and / or physicochemical pulmonary fibrosis.

9. The use according to claim 8, characterized in that: The pulmonary fibrosis is idiopathic pulmonary fibrosis induced by bleomycin.

10. The use according to claim 1, characterized in that: The mechanism of action of the pharmaceutical composition is to delay the development of pulmonary fibrosis by inhibiting collagen formation and reducing the degree of inflammation.

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