阅读说明：本技术 人参皂苷Rg5在制备抗肺纤维化药物中的用途 (Application of ginsenoside Rg5 in preparation of anti-pulmonary fibrosis drugs ) 是由 窦德强 韩雪莹 于 2021-01-07 设计创作，主要内容包括：本发明涉及一种中药单体化合物及其应用,具体是人参皂苷Rg5单独或与其他药物组合在制备预防和治疗肺纤维化病药物中的应用。本发明分别采用博来霉素诱导的肺纤维化病动物模型与TGF-β诱导的体外肺纤维化病模型,证明了人参皂苷Rg5具有抗肺纤维化的药理活性。人参皂苷Rg5能够抑制炎症发生与胶原纤维形成并促进胶原纤维的降解及肺成纤维细胞向肌成纤维细胞转分化等多方面阻碍肺纤维化发展进程,发挥抗肺纤维化病药理作用。通过研究人参皂苷Rg5抗肺纤维化病的在体内外的生物活性,为抗肺纤维化药物的研发和生产提供了新的思路和方案。(The invention relates to a traditional Chinese medicine monomeric compound and application thereof, in particular to application of ginsenoside Rg5 alone or in combination with other medicines in preparation of a medicine for preventing and treating pulmonary fibrosis. The invention respectively adopts a bleomycin-induced pulmonary fibrosis animal model and a TGF-beta-induced in vitro pulmonary fibrosis model, and proves that the ginsenoside Rg5 has pharmacological activity of resisting pulmonary fibrosis. The ginsenoside Rg5 can inhibit inflammation and collagen fiber formation, promote collagen fiber degradation and lung fibroblast to myofibroblast transdifferentiation, inhibit pulmonary fibrosis development process, and exert pharmacological effect of resisting pulmonary fibrosis. By researching the in vivo and in vitro biological activity of the ginsenoside Rg5 for resisting pulmonary fibrosis, a new idea and scheme are provided for the research, development and production of anti-pulmonary fibrosis drugs.)

1. The application of the ginsenoside Rg5 in the preparation of drugs for preventing and treating pulmonary fibrosis alone or in combination with other drugs.

2. The use of claim 1, wherein the ginsenoside Rg5 is prepared by extracting and separating black ginseng.

3. The application of claim 1, wherein the pharmacological activity of ginsenoside Rg5 in resisting pulmonary fibrosis is proved by respectively adopting a bleomycin-induced pulmonary fibrosis animal model and a TGF-beta-induced in vitro pulmonary fibrosis model.

4. The use of claim 1, wherein the ginsenoside Rg5 can inhibit inflammation and collagen fiber formation, promote collagen fiber degradation and lung fibroblast transdifferentiation into myofibroblast, hinder pulmonary fibrosis development process, and exert pharmacological effect against pulmonary fibrosis.

5. The use of claim 1, wherein said medicament is in any pharmaceutically acceptable dosage form.

6. The use of claim 1, wherein the dosage form is a medicament or pharmaceutical composition containing ginsenoside Rg5 as main active ingredient with optional pharmaceutically acceptable carrier or excipient, including but not limited to tablet, capsule, syrup for oral administration.

Technical Field

The invention relates to a traditional Chinese medicine monomeric compound and application thereof, in particular to application of ginsenoside Rg5 in preparation of anti-pulmonary fibrosis drugs.

Background

Ginsenoside is the main active ingredient in ginseng, and ginsenoside Rg5 is rare ginsenoside extracted from black ginseng, and belongs to protopanaxadiol type ginsenoside. At present, the extraction process of ginsenoside Rg5 is quite mature, and hydrolysis is usually carried out by taking acid as a catalyst, wherein the acid comprises citric acid, phosphoric acid, hydrochloric acid and the like. Research shows that the compound has the functions of resisting cancer, resisting platelet aggregation, resisting radiation, resisting inflammation, improving memory and the like.

Pulmonary fibrosis is a serious respiratory disease that is difficult to reverse, due to abnormal lung injury self-repair that may form local fibroblastic foci of the lung, leading to alveolar damage, excessive deposition of extracellular matrix, leading to lung tissue architecture reconstruction, which can lead to respiratory failure with mortality rates as high as 50% to 70%. There are many factors inducing pulmonary fibrosis, and there are environmental, occupational, physical, chemical and disease factors. To date, the main clinical treatment means for pulmonary fibrosis are alveolar lavage and lung transplantation, and no specific treatment method exists.

Disclosure of Invention

In view of the problems in the prior art, the invention aims to provide a new application of ginsenoside Rg5 in preparation of anti-pulmonary fibrosis drugs, and provides a new idea and scheme for research, development and production of anti-pulmonary fibrosis drugs by researching in vivo and in vitro biological activity of ginsenoside Rg5 in resisting pulmonary fibrosis.

In order to achieve the above object, the present invention adopts the following technical solutions.

The application of the ginsenoside Rg5 in the preparation of drugs for preventing and treating pulmonary fibrosis alone or in combination with other drugs.

Further, the ginsenoside Rg5 is prepared by extracting and separating black ginseng.

Furthermore, a bleomycin-induced pulmonary fibrosis animal model and a TGF-beta-induced in vitro pulmonary fibrosis model are respectively adopted to prove that the ginsenoside Rg5 has pharmacological activity of resisting pulmonary fibrosis.

Furthermore, the ginsenoside Rg5 can inhibit inflammation and collagen fiber formation, promote collagen fiber degradation and lung fibroblast to myofibroblast transdifferentiation, etc., hinder pulmonary fibrosis development process, and play a pharmacological role in resisting pulmonary fibrosis.

Further, the medicament is any pharmaceutically acceptable dosage form.

Furthermore, the dosage form is a medicament or a pharmaceutical composition which takes the ginsenoside Rg5 as a main active ingredient by utilizing a selective pharmaceutically acceptable carrier or excipient, and the dosage form comprises but is not limited to tablets, capsules, syrups and the like which are orally administrated.

The in vivo anti-pulmonary fibrosis effect of the ginsenoside Rg5 is as follows: in vivo pharmacological experiments, a rat pulmonary fibrosis model is established by using bleomycin induction through a tracheal intubation method, and pharmacological activity of ginsenoside Rg5 on resisting pulmonary fibrosis is proved. By observing the influence of the ginsenoside Rg5 on the lung coefficient, the levels of NF-kappa B, MMP-1, TGF-beta and HYP in serum and the pathological observation of lung tissues, the ginsenoside Rg5 can inhibit inflammation generation and collagen fiber formation, promote the degradation of the collagen fiber and the like to block the development process of pulmonary fibrosis in various aspects, and can be applied to the preparation of the medicine for resisting the pulmonary fibrosis.

In-vitro anti-pulmonary fibrosis effect of ginsenoside Rg5 in-vitro pharmacological experiments adopt TGF-beta to induce human embryonic lung fibroblasts to establish an in-vitro pulmonary fibrosis model, and prove the effect of the ginsenoside Rg5 on the transdifferentiation of the lung fibroblasts into myofibroblasts. By investigating the influence of the ginsenoside Rg5 on the expression of the alpha-SMA protein, the ginsenoside Rg5 is proved to be capable of effectively inhibiting the transdifferentiation of lung fibroblasts to myofibroblasts and delaying the pulmonary fibrosis process, and can be applied to the preparation of drugs for resisting pulmonary fibrosis.

Compared with the prior art, the invention has the following beneficial effects.

The invention discloses a new application of a traditional Chinese medicine monomer compound ginsenoside Rg5 in preparation of anti-pulmonary fibrosis drugs for the first time. In vivo and in vitro pharmacological studies find that the ginsenoside Rg5 has stronger anti-pulmonary fibrosis activity, and the effective amount of the ginsenoside Rg5 which is used as an active ingredient is applied to the prevention and treatment of pulmonary fibrosis alone or in combination with other medicines. The anti-pulmonary fibrosis drug dosage form is a selective pharmaceutically acceptable carrier or excipient, and comprises oral liquid, tablets, granules, syrup, capsules, pills and the like which are taken by oral administration.

The invention discloses an anti-pulmonary fibrosis action mechanism of ginsenoside Rg5 for the first time, and the ginsenoside Rg5 can inhibit inflammation and collagen fiber formation, promote degradation of collagen fiber and the like to block the development process of pulmonary fibrosis and play a pharmacological role in anti-pulmonary fibrosis.

Drawings

Fig. 1 is a lung tissue morphology observation, wherein a is a blank group, B is a model group, C is a positive drug group, D is a ginsenoside Rg5 low dose group, and E is a ginsenoside Rg5 high dose group.

Fig. 2 is a pathological section observation of lung tissues, wherein A is a blank group, B is a model group, C is a positive drug group, D is a low-dose group of ginsenoside Rg5, and E is a high-dose group of ginsenoside Rg 5.

Fig. 3 shows the effect of ginsenoside Rg5 on lung coefficients.

FIG. 4 is a graph showing the effect of ginsenoside Rg5 on serum HYP levels.

FIG. 5 is a graph of the effect of ginsenoside Rg5 on serum NF- κ B levels.

FIG. 6 is a graph of the effect of ginsenoside Rg5 on MMP-1 levels in serum.

FIG. 7 is a graph of the effect of ginsenoside Rg5 on serum TGF- β levels.

FIG. 8 shows the cytotoxicity of ginsenoside Rg 5.

FIG. 9 is a graph of α -SMA protein expression.

FIG. 10 shows the effect of ginsenoside Rg5 on the expression level of alpha-SMA protein.

Detailed Description

The present invention is further described with reference to the following examples and the accompanying drawings, which are included to illustrate the invention and are not to be construed as limiting the scope of the invention, and various equivalent modifications of the invention will occur to those skilled in the art upon reading the present invention and are within the scope of the appended claims.

Example 1 ginsenoside Rg5 study on the resistance of bleomycin-induced pulmonary fibrosis in rats.

1. Materials and instruments.

1.1 animals.

The male Wistar rat of SPF grade healthy adult, weighing 180-.

1.2 reagents.

Ginsenoside Rg5 (prepared by extraction, separation and purification from black ginseng in the laboratory, with purity > 95%); bleomycin hydrochloride (Haizingrey pharmaceutical Co., Ltd., production lot: 16032311); pirfenidone (bulk drug, Wuhan JiangMin Huatai pharmaceutical chemical Co., Ltd., production batch No. 203819); hydroxyproline kit (Nanjing institute of bioengineering, production lot 20190610), TGF-beta ELISA kit (Nanjing institute of bioengineering, production lot 201906), NF-kappa BELISA kit (Nanjing institute of bioengineering, production lot 201906), and MMP-1ELISA kit (Nanjing institute of bioengineering, production lot 201906). The reagents used in the experiment are analytically pure and purchased from Tianjin Damao reagent factory.

1.3 instruments.

YQ-A3-SS-048 high-speed refrigerated centrifuge (Eppendorf Co.); FA-1004 electronic balance (shanghaienkogaku balance factory); UV-2100 ultraviolet spectrophotometer (UNICO, Shanghai); adjustable pipettes (RAININ, usa); the Caretium enzyme marker (Kate biomedical electronics Co., Ltd., Shenzhen).

1.4 preparation of main reagent.

Bleomycin hydrochloride solution: the bleomycin hydrochloride is prepared into a solution of 5mg/mL by using normal saline and is prepared for use.

2. Experimental methods.

2.1 pulmonary fibrosis model building method.

After the experimental rats are weighed, 10% chloral hydrate (3mL/kg) is injected into the abdominal cavity, the rats are subjected to oral tracheal intubation under aseptic operation after entering an anesthetic state, physiological saline or bleomycin solution (5mg/kg, about 0.2mL) is injected, the intubation is rapidly pulled out after the administration, the rats are massaged, and the bodies of the rats are appropriately shaken to promote the uniform distribution of the drugs in the lungs. After the rat breathes stably, the rat is placed on a bracket with a gentle slope, the head is at a high position, and the rat is put back to the rat cage after waking up.

2.2 animal groups and methods of administration.

50 SPF-level Wistar rats (provided by Liaoning Biotechnology Limited, the qualification number: SCXK (Liaoning) 2015-0001) with a weight of 180-220g are divided into a blank control group, a model group, a positive drug group, a ginsenoside Rg5 high-dose group and a ginsenoside Rg5 low-dose group, and 10 rats are respectively divided into 10 groups. A pulmonary fibrosis model of rats is constructed according to the method under item 2.1, and rats in a control group are injected with physiological saline with the same volume as the rat pulmonary fibrosis model. The following doses were administered to each group of animals on the next day after molding, 1 time per day, and 30 days after continuous gavage. Weighing and recording the weight of the rats before the rats are killed on the 31 th day, taking blood from abdominal aorta after anesthesia, dissecting after blood loss and death, centrifuging the collected blood by a low-temperature centrifuge at 4 ℃ for 10min (3000rpm/min), taking supernatant, subpackaging and storing in an ultra-low temperature refrigerator at-80 ℃ for measuring the HYP, NF-kappa B, MMP-1 and TGF-beta levels in serum.

Table 1 the doses administered in each group.

2.3 index measurement.

2.3.1 general case observations.

The rat conditions including skin, mental state, diet, respiratory rate and mode were observed daily.

2.3.2 pulmonary coefficient.

Dissecting and taking out the lung, washing bloodstain and removing connective tissue, sucking dry water, and weighing the lung to obtain the total lung wet weight. The lung coefficient [ total lung wet weight (mg)/body weight (g) ] was calculated.

2.3.3HE staining.

The left lung of the rat was fixed in 4% paraformaldehyde and HE stained as follows.

(1) Washing: the tissue blocks were placed in the embedding cassette in running water for 24 h.

(2) And (3) dehydrating: 70% alcohol overnight, 80% alcohol for 2h, 90% alcohol for 1h, 95% alcohol for 40min, anhydrous ethanol I for 40min, and anhydrous ethanol II for 30 min.

(3) And (3) transparency: the dehydrated tissue was immersed in xylene I for 10min and xylene II for 10 min.

(4) Wax dipping: immersing the tissue into soft wax I for about 1h, and then immersing the tissue into soft wax II for 40 min; hard wax I for 40min and hard wax II for 1 h.

(5) Embedding: and (3) injecting the wax liquid into an embedding hard tool, horizontally placing the tissue block into the wax liquid, righting and paving the tissue block, and then moving the tissue block to a cold table.

(6) And (6) slicing.

(7) Dyeing: xylene I5-10 min → xylene II 5-10min → absolute ethyl alcohol I1-3 min →

Absolute ethyl alcohol II 1-3min → 90% ethyl alcohol 1min → 80% ethyl alcohol 1min → 70% ethyl alcohol 1min → water washing → hematoxylin 3-5min → water washing → hydrochloric acid ethyl alcohol 1-3s → water washing → eosin 3-5min → water washing → 70% ethyl alcohol 1-2s → 80% ethyl alcohol 3-5min → 90% ethyl alcohol 3-5min → 95% ethyl alcohol 3-5min → absolute ethyl alcohol 5-10min → xylene I3-5 min → xylene II 3-5min → neutral gum sealant.

2.3.4 serum NF-. kappa. B, MMP-1 and TGF-. beta.levels.

The levels of NF-kappa B, MMP-1 and TGF-beta were measured by ELISA, the procedure was as follows, according to the kit instructions.

(1) The kit was equilibrated at room temperature for 20min in advance, the required strip was removed, the remaining strip sealed with a stopcock and placed back at 4 ℃.

(2) And arranging standard substance holes and sample holes, and respectively adding 50 mu L of standard substances with different concentrations into each standard substance hole.

(3) And (3) adding 10 mu L of the sample to be detected into each sample hole, then adding 40 mu L of the sample diluent into each sample hole, and not adding blank holes.

(4) In addition to blank wells, 100. mu.L of detection antibody labeled with horseradish peroxidase (HRP) was added to each of the standard wells and the sample wells, and then the reaction wells were sealed with a sealing plate film and incubated in an incubator at 37 ℃ for 60 min.

(5) Discarding liquid, drying on absorbent paper, filling washing solution into each hole, standing for 1min, discarding washing solution, drying on absorbent paper, and washing the plate for 5 times.

(6) Adding 50 μ L of color-developing agent A into each well, adding 50 μ L of color-developing agent B, and incubating at 37 deg.C in dark for 15 min.

(7) Stop solution (50. mu.L) was added to each well, and the OD value of each well was measured at a wavelength of 450nm within 15 min.

2.3.6 determination of HYP content in serum.

The measurement method was carried out according to the kit instructions, and the specific method was as follows.

(1) Taking serum and hydrolysate in a test tube 1: 2 and mixing uniformly. Hydrolyzing at 95 deg.C for 20min, and adjusting pH to 6.0-6.8.

(2) Adding appropriate amount of active carbon (about 20mg) into diluted hydrolysate, mixing, and centrifuging at 3500rpm/min for 10 min.

(3) Taking 1mL of the supernatant for detection, sequentially adding three reagents provided in the reagent kit, uniformly mixing, and carrying out water bath at 60 ℃ for 15 min.

(4) Cooling, centrifuging at 3500rpm/min for 10min, collecting supernatant with wavelength of 550nm and optical path of 1cm, adjusting to zero with double distilled water, and measuring absorbance of each tube.

Hydroxyproline content (μ g/mL) ═ measurement OD-blank OD value)/(standard OD-blank OD value) × 5 μ g/mL × total hydrolysate volume (mL)/sample volume (mL).

3. And (6) data processing.

SPSS 17.0 statistical software is used, and the One-Way ANOVA method is adopted for group comparison. LSD tests are used when variances are uniform and Dunnett's T3 tests are used when variances are irregular. (Note: P <0.05, # P <0.01 as compared to the model group; P <0.05, # P <0.01 as compared to the control group).

4. And (5) experimental results.

4.1 general signs observations in rats.

Compared with the control group, the rats after molding have different degrees of bradykinesia, cachexia, accelerated respiratory frequency, obvious respiratory sound, poor spirit, hair withered, diet reduction, weight loss and other symptoms.

4.2 visual inspection of lung tissue.

The macroscopic observation results of lung tissues are shown in fig. 1, and the lung tissues of the rats in the blank group are pink, soft, smooth and full. The lungs of the model group rats are dark red, hard and poor in elasticity, and bleeding points and scar-like lines can be seen by naked eyes. The lung tissues of other groups of rats are light red, have good elasticity, no obvious fibrous lines and less bleeding spots.

4.3 pathological section observation of lung tissue.

The results are shown in fig. 2, lung histology section observations (10 × 10); note: a represents blank group, B represents model group, C represents positive drug group, D represents ginsenoside Rg5 low dose group, E represents ginsenoside Rg5 high dose group, and the scale size in the figure is 100 μm.

The blank group of rats has normal lung tissue structure, clear alveolar structure, no change of congestion, edema, acute and chronic inflammation and the like. The original structure of the lung tissue of the model group is damaged, the alveolar space is widened, the lung interstitial inflammatory cells are infiltrated, and a great amount of fibroblasts are proliferated. The lung tissue of the positive group is occasionally interstitial and mildly edematous, inflammatory cell infiltration is less compared with the model group, and a part of more obvious alveolar structures can be observed. The ginsenoside Rg5 high-dose group and low-dose group have thinner alveolar walls, a small part of alveolar cells have slight edema, inflammatory cells have less infiltration, and alveolar structures are clearer.

4.4 pulmonary factor.

The increase of the lung coefficient is probably related to the abnormal remodeling of the lung tissue structure, the lung fibroblasts are converted into the myofibroblasts under the catalysis of a plurality of cytokines to promote the synthesis and the secretion of collagen, the deposition of extracellular matrix is increased, thereby the lung weight and the lung coefficient are increased, and the determination of the lung coefficient can indirectly reflect the degree of the lung tissue fibrosis.

As shown in fig. 3, the lung coefficients of the model group were significantly increased (P <0.01) compared to the blank group. Compared with the model group, the lung coefficient of the high-dose group of the ginsenoside Rg5 is obviously reduced (P is less than 0.01), while the lung coefficient of the low-dose group of the ginsenoside Rg5 is in a descending trend, but has no statistical difference.

4.5 serum HYP levels.

HYP is involved in the synthesis of collagen, is the main component of collagen, and the content of HYP is as high as 13.4%, so that HYP can be used as a scale for evaluating the collagen content in organism tissues. As shown in fig. 4, HYP levels in the serum of the model group were significantly increased compared to the blank group, and the difference was statistically significant (P < 0.01). Compared with the model group, the serum HYP level of the high-dose group and the low-dose group of the ginsenoside Rg5 and Rg5 are obviously reduced, and the difference has significant statistical significance (P is less than 0.05 and P is less than 0.01).

4.6 serum NF-. kappa.B levels.

After the NF-kappa B is activated, various cytokines can be regulated and controlled, the proinflammatory and fibrosis promoting effects are exerted, and the oxidation and oxidation resistance systems of organisms can be influenced, so that the activation of the NF-kappa B can be an important link for the occurrence of pulmonary fibrosis. In addition, NF-kappa B can promote cytokines such as TNF-alpha, IL-8, TGF-beta and the like to mediate alveolitis in a transcription mode so as to develop fibrosis.

As shown in FIG. 5, NF-. kappa.B levels in the sera of the model group were significantly elevated compared to the blank group, and the difference was statistically significant (P < 0.01). Compared with the model group, the serum NF-kB levels of the positive group, the ginsenoside Rg5 high-dose group and the ginsenoside Rg5 low-dose group are all obviously reduced, and the differences have significance (P is less than 0.01, and P is less than 0.01).

4.7 MMP-1 levels in serum.

MMP-1 is a key enzyme in MMPs that degrades type I collagen, and decreased type I collagen degradation results in excessive ECM deposition leading to fibrosis. As shown in fig. 6, MMP-1 levels in the serum of the model group were significantly reduced compared to the blank group, and the difference was statistically significant (P < 0.01). Compared with the model group, the levels of the positive group, the group with high dosage of ginsenoside Rg5 and the group with low dosage of ginsenoside Rg5 are increased, and the statistical differences are significant (P <0.01 ).

4.8 serum TGF-. beta.levels.

TGF-beta is used as a transforming growth factor, can promote differentiation, growth and proliferation of certain cells such as fibroblasts, plays a key role in the formation and development process of pulmonary fibrosis, and stimulates the synthesis of a large amount of collagen production to promote the deposition of pulmonary extracellular matrix by stimulating the transformation of the fibroblasts and alveolar epithelial cells into myofibroblasts and mesenchymal cells in the formation and development process of the pulmonary fibrosis so as to promote the development and development process of the pulmonary fibrosis.

As shown in fig. 7, TGF- β levels in the model group sera were significantly elevated compared to the blank group, and the differences were statistically significant (P < 0.01). Compared with the model group, the TGF-beta levels of the positive group, the ginsenoside Rg5 high-dose group and the ginsenoside Rg5 low-dose group are obviously reduced, and the difference has significant statistical significance (P is less than 0.01, and P is less than 0.01).

The experimental results show that the ginsenoside Rg5 can inhibit the development process of pulmonary fibrosis through multiple aspects such as inhibiting inflammation generation and collagen fiber formation and promoting collagen fiber degradation, play a pharmacological role in resisting pulmonary fibrosis, and can be applied to the treatment of pulmonary fibrosis.

Example 2 ginsenoside Rg5 study of the anti-TGF-beta induced model effect of pulmonary fibrosis in vitro.

1. Test materials.

1.1 cells.

Human embryonic lung fibroblast cell line (MRC-5) was purchased from the institute of basic medicine, national academy of medical sciences.

1.2 instruments and reagents.

High-glucose DMEM medium (Gibco, USA, batch No. 8120246); fetal bovine serum (Gibco, USA, lot number: 8166872); penicillin, streptomycin (Hyclone, U.S.A., lot number J160035); PBS phosphate buffer (Solambio, Beijing, Lot. No. 1015M 023); MTT (Solarbio, Beijing, lot 303H 0525); DMSO (Sigma, USA, 520C0313 batch); sodium lauryl sulfate (batch No. J0719A; Dalian Melam Biotechnology Ltd.); glycine (batch No. S0812A; Dalian Meiren Biotechnology Ltd.); tris (hydroxymethyl) aminomethane (batch No. S0721A; Dalian Meiren Biotech Co., Ltd.); sodium chloride (batch No. 20190428; Baishi chemical Co., Tianjin); RIPA lysate (batch No. 072219190802; Biyuntian Biotechnology Co., Ltd.); PMSF (batch number: 042819190617; Biyuntian Biotechnology Co., Ltd.); BCA protein concentration assay kit (batch No. 011819190819; Biyuntian Biotechnology Co., Ltd.); SDS-PAGE gel rapid formulation kit (batch No. 022219190801; Biyuntian Biotechnology Co., Ltd.); rainbow 180 broad-spectrum protein marker (batch: PR 1910)(ii) a Beijing solibao science and technology limited); SDS-PAGE protein loading buffer (5X) (batch No. 081519190903; Biyuntian Biotechnology Co., Ltd.); ponceau staining solution (batch No. 030419190826; Biyuntian Biotechnology Co., Ltd.); anti-alpha-SMA rabbit polyclonal antibody (batch No. I09012510; Shenyang Wan Biotech Co., Ltd.); anti-beta-actin rabbit polyclonal antibody (batch: 2127; Absci, USA); HRP-labeled goat anti-rabbit IgG antibody (batch No.: 26A 069; Shenyang Wan class Biotech Co., Ltd.); hypersensitive ECL chemiluminescence kit (batch No. 041019190725; Biyuntian Biotechnology Co., Ltd.); western primary antibody removal solution (strong alkaline) (batch No. 032719190814; Biyuntian Biotechnology Co., Ltd.); NU-4750 CO₂Incubators (NUAIRE, usa); HD₂-BCN-1360B biological cleaning bench (beijing tokyo haar instruments ltd); AE31EF model inverted microscope (Motic corporation); a Caretium enzyme-labeling instrument (Kate biomedical electronics Co., Ltd., Shenzhen); u570-86 Premium series ultra low temperature refrigerator (-80 deg.C) (NBS corporation, USA); TDZ4-WS low speed desk centrifuge (Hunan instrument centrifuge); filter (0.22 μm) (millex. gp); cell culture flasks (Corning corporation); a 96-well sterile plate (Corning corporation); 6-well sterile plates (Corning Corp.); BXM-30R type vertical pressure steam sterilizer (shanghai bosch industries, ltd); ChemiScope6600Touch ultrasensitive multifunctional fluorescence chemiluminescence imaging system (Shanghai Shang Xiang scientific instruments Co., Ltd.).

2. And (4) preparing a main reagent.

(1) High-glucose DMEM complete medium: taking a proper amount of high-glucose DMEM medium, adding 10% fetal calf serum inactivated at 56 ℃ and 1% double antibody, filtering by a 0.22-micron filter membrane, and storing in a refrigerator at 4 ℃ for later use.

(2) MTT solution: 50mg of MTT was weighed, dissolved in 10mL of PBS (pH 7.4, 0.01mol/L), sterilized by filtration through a 0.22 μm microporous filter, and stored at-20 ℃ in the dark for further use.

3. Experimental methods.

3.1 cytotoxicity of ginsenoside Rg 5.

Taking MRC-5 cells in logarithmic growth phase, adjusting cell concentration to 3 × 10⁴100 mu per mLAnd L/well is inoculated in a 96-well plate, after cells adhere to the wall, culture solution containing ginsenoside Rg5 and positive drugs with final concentrations of 0.001 mu mol/mL, 0.01 mu mol/mL, 0.1 mu mol/mL, 1 mu mol/mL and 10 mu mol/mL and TGF-beta 110 ng/mL are cultured for 24 hours, a blank control group, a model group (TGF-beta 110 ng/mL) and a zero adjusting well are simultaneously set, 3 multiple wells are formed in each group, and the cytotoxicity of the ginsenoside Rg5 is determined by adopting an MTT method.

Growth inhibition ratio (%) - (1- (a)_{Experiment of}-A_{Zero setting})/(A_{Blank space}-A_{Zero setting}))×100％。

3.2 action of ginsenoside Rg5 on transdifferentiation of lung fibroblasts into myofibroblasts.

Taking MRC-5 cells in logarithmic growth phase, and adjusting cell concentration to 1.5x10⁵And (2) inoculating the mixture in a 6-well plate, culturing for 24h, removing the culture solution, changing into serum-free culture solutions containing medicines with different concentrations, respectively arranging a blank control group, a model group, a positive medicine group and a ginsenoside Rg5 group, and adding TGF-beta 1 to stimulate the rest groups except the blank control group (the final concentration is 10 ng/mL). After 48h incubation, the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1mM in accordance with the RIPA lysate protocol, and the culture was removed and washed once with PBS. Add 200 microliter lysis solution into each hole of 6-hole plate, blow and beat several times with the rifle, make lysis solution and cell fully contact. After full cracking, centrifuging at 4 ℃ for 5min at 12000r/min, and taking the supernatant. Detecting protein concentration by BCA method, adjusting sample loading amount according to protein concentration measurement value, filling volume of each histone with lysis solution to prepare equal-concentration equal-volume protein solution, making each histone concentration be 2.5mg/mL, subpackaging and storing at-80 deg.C. According to the volume ratio of 4: adding protein solution and 5 Xloading buffer solution to 1, heating at 100 deg.C for 5min for denaturation, loading, performing SDS-PAGE electrophoresis, transferring membrane, sealing in 5% skimmed milk at 4 deg.C for 2 hr, incubating with antibody, and detecting expression level of alpha-SMA protein by WB method.

4. And (6) data processing.

SPSS 17.0 statistical software is used, and the One-Way ANOVA method is adopted for group comparison. LSD tests are used when variances are uniform and Dunnett's T3 tests are used when variances are irregular. (Note: P <0.05, # P <0.01 as compared to the model group; P <0.05, # P <0.01 as compared to the control group).

5. And (5) experimental results.

5.1 cytotoxicity.

The following results were obtained according to the experimental procedures described above. The proliferation inhibition rate of ginsenoside Rg5 on MRC-5 cells after TGF-beta 1 stimulation is shown in figure 8, when the concentration of ginsenoside Rg5 is 10 mu mol/mL, the inhibition rate is (30.349 +/-2.125)%, and the ginsenoside Rg5 has significant statistical differences (P <0.01 and P <0.01) compared with a blank control group or a model group and shows obvious cytotoxicity. Therefore, 1 mu mol/mL is selected as the detection concentration of the subsequent experiment.

5.2 expression of α -SMA.

The alpha-SMA is an important mark for myofibroblast activation, the expression degree of the alpha-SMA is in positive correlation with the severity degree of pulmonary fibrosis, and the alpha-SMA can be used for evaluating the pulmonary fibrosis progress.

The protein expression result of alpha-SMA protein level detected by Western-blot method is shown in FIG. 9 and FIG. 10. Compared with a blank control group, the alpha-SMA protein expression strip of the model group is enhanced, and the relative gray value is increased (P < 0.01). After administration, compared with a model group, the ginsenoside Rg5 group has reduced alpha-SMA protein expression level, the degree of reduction of relative gray value has statistical difference (P <0.05), and the positive drug has reduced trend but no obvious statistical difference to the alpha-SMA protein expression, which shows that the ginsenoside Rg5 has better reduction effect on the alpha-SMA protein expression level than the positive drug.

The experimental results show that the ginsenoside Rg5 can reduce the expression of alpha-SMA in myofibroblasts, and that the ginsenoside Rg5 has a certain inhibiting effect on the transdifferentiation and differentiation of TGF-beta 1-induced lung fibroblasts into myofibroblasts, and can be applied to the treatment of pulmonary fibrosis.

Example 3 study of formulations containing ginsenoside Rg 5.

1. Instruments and reagents.

The instrument is a TDP-5T single-punch tablet press (Shanghai Guanlian pharmaceutical Equipment Co., Ltd.); electronic balance of FA-1004 type (Shanghaienke balance plant); a water bath (Jiangyi Provisioning instruments, Inc.); DHG-9146A type electric heating constant temperature air blast drying oven. All reagents used in the experiment were analytically pure and purchased from Yuwang reagent company.

2. And (4) preparing tablets.

Taking ginsenoside Rg510 g, optionally pressing starch 30g, preparing into soft material with 7mL 80% ethanol, kneading into a group, kneading, dispersing, molding, oven drying at 60 deg.C for 12h, sieving with 10 mesh sieve, and sieving to obtain granule; weighing the prepared granules, adding magnesium stearate in an amount of 0.5% of the weight of the granules, tabletting by tabletting machines of different specifications to respectively prepare tablets with tablet weights of 200 mg/tablet and 100 mg/tablet, and the pressure is 16-18 kg/tablet; each 200mg of the tablet contains 50mg of phenylethanoid glycosides. Administration in combination with the patient's particular symptoms is recommended twice daily, 2 tablets (50 mg/tablet) each, 2 times daily.

3. And (4) preparing capsules.

And (3) filling the prepared granules into capsules by a capsule machine to prepare capsules of 200 mg/capsule. The administration is combined with the specific symptoms of the patients, and the administration dosage is recommended to be 2 capsules each time and 2 times a day.

4. And (4) preparing oral liquid.

Dissolving ginsenoside Rg520g in 300mL of water, adding hesperidin 4mL, and adding simple syrup to 1000mL to obtain syrup. The administration is combined with the specific symptoms of the patients, and the administration dose is recommended to be 15mL each time and 2 times per day.