Radix ophiopogonis decoction anti-pulmonary fibrosis active component separated based on centrifugation-membrane dialysis method and application of radix ophiopogonis decoction anti-pulmonary fibrosis active component

Abstract

The invention discloses a radix ophiopogonis decoction anti-pulmonary fibrosis active component separated based on a centrifugation-membrane dialysis method and application of the radix ophiopogonis decoction anti-pulmonary fibrosis active component. Belongs to the field of ophiopogon root soup. The invention aims to solve the problem that the research on the ophiopogon root decoction mostly focuses on component analysis, pharmacological effect and the like in the prior art. The precipitation phase state, CP-I, CP-II and true solution phase state of the ophiopogon root decoction are successfully separated by adopting a centrifugation-membrane dialysis method. The results of qualitative and quantitative analysis of chemical components show that CP-I is closer to D, D, CP-I can more effectively improve PF symptoms compared with CP-II, and D, CP-I and CP-II do not cause damage to A549 cells and inhibit proliferation of the A549 cells induced by TGF-beta1 to different degrees. Compared with CP-II, the D and CP-I can more remarkably inhibit phosphorylation of protein Smad2 / 3, and the D, CP-I and CP-II can weaken the EMT of A549 cells induced by TGF-beta1 by blocking a TGF-beta1 / Smad pathway. CP-I is an effective phase state of the ophiopogon root decoction.

Description

Technical Field

[0001] This invention belongs to the field of Ophiopogon japonicus decoction, specifically, it relates to an anti-pulmonary fibrosis active component of Ophiopogon japonicus decoction separated by centrifugation-membrane dialysis and its application. Background Technology

[0002] Mai Men Dong Tang (Ophiopogon japonicus decoction) has the effects of clearing and nourishing the lungs and stomach, relieving nausea and vomiting, and regulating qi.

[0003] Traditional Chinese medicine decoctions are complex multiphase systems, with their active ingredients forming different dispersed phases in the dispersion medium. Increasing research suggests that phase composition may be an important mechanism for the delivery of pharmacological effects in traditional Chinese medicine decoctions. Mai Men Dong Tang (Ophiopogon Decoction) is a classic formula for treating pulmonary atrophy. It heavily utilizes Ophiopogon japonicus as the principal ingredient because its sweet and cold properties nourish the yin of the lungs and stomach, and clear deficiency fire. Pinellia ternata is used as the assistant ingredient to relieve nausea and phlegm. It is further combined with rice, jujube, and licorice to tonify stomach qi, and ginseng to tonify the middle qi. However, research on this decoction has largely focused on component analysis and pharmacological effects, with relatively little in-depth study of its phase composition, macroscopic chemical properties, structural characteristics, and activity. Summary of the Invention

[0004] This invention provides an active component of Ophiopogon japonicus decoction for anti-pulmonary fibrosis separated by centrifugation-membrane dialysis and its application.

[0005] This invention takes Mai Men Dong Tang (Ophiopogon japonicus decoction) as the research object, and performs phase separation of the decoction. Through phase structure characterization, qualitative and quantitative analysis of chemical components, and in vivo and in vitro anti-PF studies, the separated phases are compared with the original decoction. This comprehensive and multi-faceted exploration of the phase composition and characteristics of Mai Men Dong Tang aims to screen out the "effective phase" of Mai Men Dong Tang, providing a preliminary foundation for the application of the classic formula Mai Men Dong Tang in modern Chinese medicine preparations. This invention uses ultracentrifugation sedimentation and membrane dialysis, with particle size and PDI as indicators, to separate Mai Men Dong Tang into different phases, determining the optimal method for phase separation of Mai Men Dong Tang.

[0006] To address the aforementioned technical problems, the present invention adopts the following technical solution: The purpose of this invention is to provide an anti-pulmonary fibrosis active component of Ophiopogon japonicus decoction separated by centrifugation-membrane dialysis, comprising the following steps: Step 1: Soak 42 g of Ophiopogon japonicus, 6 g of Pinellia ternata, 6 g of ginseng, 6 g of licorice, 6 g of japonica rice, and 4 g of jujube in 2400 mL of purified water for 0 min. Bring to a boil over high heat for 38 min, then simmer over low heat until reduced to 1200 mL. Filter the hot liquid through gauze to obtain the original Ophiopogon japonicus decoction. Step 2: Centrifuge at 40 ℃ and 3500 rpm for 10 min, and take the lower layer of precipitate as the precipitate phase; Step 3, the supernatant of the precipitated phase was centrifuged at a temperature of 20℃ and a speed of 10000 rpm for 10 min, and the obtained supernatant was in a suspension phase (CP-I); Step 4, the suspension phase was split by dialysis, the dialysis was performed once, the dialysis bag had a specification MW: 7000 Da, and the dialysis temperature was 37℃, and the dialysis bag inside which was successfully split was a colloidal phase (CP-II), and the dialysis bag outside was a true solution phase.

[0007] In addition, the use of the precipitated phase, the suspension phase (CP-I), and the colloidal phase (CP-II) prepared by the method of the application in the preparation of a drug for improving the symptoms of bleomycin-induced pulmonary fibrosis is provided.

[0008] The use of the precipitated phase, the suspension phase (CP-I), and the colloidal phase (CP-II) prepared by the method of the application in the preparation of a drug for inhibiting the proliferation of A549 cells induced by TGF-β1 is also provided.

[0009] The use of the precipitated phase, the suspension phase (CP-I), and the colloidal phase (CP-II) prepared by the method of the application in the preparation of a drug for inhibiting the phosphorylation of protein Smad2 / 3 is also provided.

[0010] The use of the precipitated phase, the suspension phase (CP-I), and the colloidal phase (CP-II) prepared by the method of the application in the preparation of a drug for weakening the EMT of A549 cells induced by TGF-β1 by blocking the TGF-β1 / Smad pathway is also provided.

[0011] The results of particle size and PDI of the precipitated phase were 2444.73 ± 10.63 nm and 0.742 ± 0.01, respectively. The results of particle size and PDI of CP-I were 116.97 ± 0.75 nm and 0.329 ± 0.04, respectively. The results of particle size and PDI of CP-II were 96.63 ± 0.49 nm and 0.442 ± 0.01, respectively. The salinity of D was 0.66 ± 0.01 ppt, and the conductivity was 1.409 ± 0.00 ms / cm. The salinity of the precipitated phase was 0.66 ± 0.01 ppt, and the conductivity was 1.372 ± 0.01 ms / cm. The salinity of CP-I was 0.37 ± 0.00 ppt, and the conductivity was 0.721 ± 0.01 ms / cm. The salinity of CP-II was 0.36 ± 0.01 ppt, and the conductivity was 0.775 ± 0.01 ms / cm. The salinity of the true solution phase was 0.08 ± 0.00 ppt, and the conductivity was 0.291 ± 0.00 ms / cm. The results showed that the D and the precipitated phase system were complex and unstable, while the CP-I and CP-II systems were stable. The in vitro release results showed that the release models of CP-I, CP-II, and D in artificial gastric juice and artificial intestinal juice release media all conformed to the first-order rate equation. UPLC-Q-TOF-MS technology was used for qualitative analysis. In the positive ion mode, 119 compounds were identified in D, 106 compounds in CP-I, and 97 compounds in CP-II. In the negative ion mode, 70 compounds were identified in D, 59 compounds in CP-I, and 60 compounds in CP-II. HPLC was used to establish the quantitative analysis method of adenine, glycyrrhizin, glycyrrhizic acid, methyl Ophiopogon high flavonoids A, and ginsenoside Rg1. The results of the content determination of each component showed that the content of the five components in CP-I was closer to that in D than in CP-II. The results of pharmacodynamics experiments showed that the lung tissue morphology results, HE, and Masson pathological sections all showed that D and CP-I could better improve the lung tissue damage in rats than CP-II, and the fibrocyte and collagen deposition were reduced. Lipidomics results showed that through multivariate statistics PCA, PLS-DA, and OPLS-DA analysis, there was a clear separation trend between the M group and the C group in the positive and negative ion modes, and the results of the representative lipid molecules were distinct, which proved that the PF group was successfully modeled. The D group, CP-I group, and CP-II group were significantly separated from the M group and approached the C group, indicating that Maidong decoction and each phase could improve the symptoms of PF. The differential metabolites of the D, CP-I, and CP-II groups and the M group were mainly lipids and lipid analogs, and the differential lipid subclasses were mainly PC, SM, and PE. Twenty-five PF-related differential lipids were identified, mainly GP, and it was speculated that Maidong decoction and each phase might improve PF by affecting the level of GP lipids. The in vitro TGF-β1-induced A549 cell experiment showed that EMT was successfully established.CCK-8 experiment shows that D, CP-I and CP-II do not cause damage to A549 cells at a concentration of 18.75 mg / mL, and can inhibit the proliferation of A549 cells induced by TGF-β1. D and CP-I can effectively improve the growth morphology of A549 cells induced by TGF-β1, and better improve EMT. The cell migration experiment shows that the CP-II group has the weakest effect on inhibiting the migration speed of A549 cells induced by TGF-β1, and D and CP-I can significantly inhibit the migration of A549 cells induced by TGF-β1. The Western blot experiment results show that D and CP-I can more significantly inhibit protein Smad2 / 3 phosphorylation than CP-II, and D, CP-I and CP-II can weaken the EMT of A549 cells induced by TGF-β1 by blocking the TGF-β1 / Smad pathway.

[0012] The present application adopts TGF-β1 to induce A549 cell EMT, and evaluates EMT by cell morphology and cell migration experiment. The toxicity of different drug groups to A549 cells is investigated, and the inhibition of different drug groups on the proliferation of A549 cells induced by TGF-β1 is investigated. The inhibition of each drug group on the migration ability of cells is analyzed by cell migration experiment, and the mechanism of action of Maidong decoction and different phase states in treating EMT is explored by Western blot.

[0013] The present application adopts centrifugal-membrane dialysis method to successfully split Maidong decoction precipitate phase, CP-I, CP-II and true solution phase. The qualitative and quantitative analysis results of chemical components show that CP-I is closer to D, D and CP-I can more effectively improve the PF symptoms than CP-II, D, CP-I and CP-II do not cause damage to A549 cells, and inhibit the proliferation of A549 cells induced by TGF-β1 to different degrees. D and CP-I can more significantly inhibit protein Smad2 / 3 phosphorylation than CP-II, and D, CP-I and CP-II can weaken the EMT of A549 cells induced by TGF-β1 by blocking the TGF-β1 / Smad pathway. CP-I is an effective phase state of Maidong decoction.

[0014] For a further understanding of the features and technical contents of the present application, please refer to the detailed description and drawings attached. It should be noted that the drawings are provided for illustrative purposes only, and are not used to limit the present application. DETAILED DESCRIPTION

[0015] Figure 1Figure 2 is the effect of different concentrations of TGF-β1 on the morphology of A549 cells (10x) for 24 h, A: normal A549 cells B: A549 cells induced by TGF-β1 with a final concentration of 2.5 ng / mL C: A549 cells induced by TGF-β1 with a final concentration of 5 ng / mL D: A549 cells induced by TGF-β1 with a final concentration of 10 ng / mL; Figure 2 Figure 3 is the effect of different concentrations of TGF-β1 on the morphology of A549 cells (10x) for 48 h, A: normal A549 cells B: A549 cells induced by TGF-β1 with a final concentration of 2.5 ng / mL C: A549 cells induced by TGF-β1 with a final concentration of 5 ng / mL D: A549 cells induced by TGF-β1 with a final concentration of 10 ng / mL; Figure 3 Figure 4 is the effect of different concentrations of D, CP-I and CP-II on the activity of A549 cells, A: 24 h, B: 48 h, the cell experiment was repeated 3 times, * compared with the control group P <0.05, ** compared with the control group P <0.01, *** compared with the control group P <0.001; Figure 4 Figure 5 is the effect of different concentrations of D, CP-I and CP-II on the activity of A549 cells treated by TGF-β1, A: 24 h, B: 48 h, the cell experiment was repeated 3 times, ** compared with group C P <0.01, the difference was extremely statistically significant; ## compared with the TGF-β1 group P <0.01, the difference was extremely statistically significant; Figure 5 Figure 6 is the effect of TGF-β1 and D, CP-I and CP-II on the morphology of A549 cells (10x), A: normal cell morphology B: A549 cells induced by TGF-β1 with a final concentration of 5 ng / mL C: cell morphology with a final concentration of 18.75 mg / mL of D and 5 ng / mL of TGF-β1 D: cell morphology with a final concentration of 18.75 mg / mL of CP-I and 5 ng / mL of TGF-β1 E: cell morphology with a final concentration of 18.75 mg / mL of CP-II and 5 ng / mL of TGF-β1; Figure 6Scratch picture, C group: control group, M group: model group (TGF-β1 final concentration is 5 ng / mL), D group: (add final concentration of 18.75 mg / mL D, final concentration of 5 ng / mL TGF-β1 solution), CP-I group: (add final concentration of 18.75 mg / mL CP-I, make the final concentration of 5 ng / mL TGF-β1 solution), CP-II group: (add final concentration of 18.75 mg / mL CP-II, final concentration of 5 ng / mL TGF-β1 solution); Figure 7 Scratch migration rate quantitative analysis chart, difference analysis chart is represented by Mean ± SD, P <0.05, P <0.01, P <0.001; Figure 8 Effect of D, CP-I and CP-II phase on TGF-β1 induced A549 cell fibrosis TGF-β1 / smad pathway (n=3),##Compared with the control group P <0.01, P <0.01, P <0.05. DETAILED DESCRIPTION

[0016] The application will be described in detail below with specific examples. These examples help those skilled in the art to further understand the application, but should not be regarded as limiting the application. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made. These all belong to the protection scope of the present application.

[0017] Example 1, the anti-pulmonary fibrosis active component of Mai Men Dong decoction separated by centrifugation-membrane dialysis method is carried out according to the following steps: Step 1, add Omin, boil under the condition of 38min, and transfer to a slow fire to cook until 1200mL. Filter the hot liquid through gauze to obtain Mai Men Dong decoction stock solution; Step 2, centrifuge at 40℃ and 3500rpm for 10min, and take the lower precipitate as the precipitate phase; Step 3, centrifuge the supernatant of the precipitate phase at 20℃ and 10000rpm for 10min, and the obtained supernatant is the suspension phase (CP-I); Step 4, the suspended phase state is split by dialysis, the dialysis frequency is 1 time, the dialysis bag specification MW is 7000Da, the dialysis temperature is 37 DEG C, the dialysis bag inside that is successfully split is colloidal phase state (CP-II), and the dialysis bag outside is true solution phase state.

[0018] UPLC-Q-TOF-MS technology was used for qualitative analysis of Maidongtang and each phase state. 119 compounds were identified under positive ion mode, and 70 compounds were identified under negative ion mode; 106 compounds were identified under positive ion mode, and 59 compounds were identified under negative ion mode; 97 compounds were identified under positive ion mode, and 60 compounds were identified under negative ion mode. Through the above analysis, the chemical composition of D, CP-I and CP-II was preliminarily determined, and through the comparison of the number of compounds contained in each group of positive and negative ion modes, it was concluded that CP-I contained more chemical components closer to D.

[0019] PF is a progressive lung disease caused by genetic and environmental factors or other lung diseases. Lung coefficient is one of the indicators reflecting the degree of PF. During the formation of PF, the lung weight of the model group rats increased significantly due to various factors such as inflammatory cell infiltration, cell swelling, and capillary hyperemia. At the same time, the rats were in a disease state after modeling, leading to weight loss. Therefore, the lung index of the model group rats increased significantly. The histopathological changes of lung tissue are the most intuitive and important indicators for determining PF. The pathological section results show that the alveolar structure of the model group rats is severely damaged, the alveolar septum is thickened, and a large amount of collagen fibers are deposited in the alveolar septum, which indicates that we have successfully replicated the PF model in rats. After treatment of Maidongtang and each phase state, the degree of alveolar inflammation and PF in rats decreased significantly, suggesting that D, CP-I and CP-II can improve BLM-induced PF in rats.

[0020] Lung surfactant lipids play an important role in stabilizing lung function, mainly composed of phospholipids (Phospholipid, PL) and a small amount of neutral lipids including cholesterol. More and more studies have shown that the development process of PF is closely related to the changes in lipid metabolism, therefore, the present application can effectively distinguish D, CP-I and CP-II from PF model by LC-MS technology. The main difference metabolites between D, CP-I and CP-II group and M group are lipids and lipid analogues, and the main difference lipid subclasses are PC, SM and PE. 25 lung fibrosis-related differential lipids mainly containing GP were identified.

[0021] The PF model was established by intratracheal instillation of BLM. The rats showed increased respiratory rate, dyspnea, and emaciation. The effects of Maidong Decoction and its different phases on the improvement of PF were evaluated by the changes in the rats' physical signs, HE and Masson staining pathological sections, and lipidomics. Compared with the M group, the D and CP-I groups were better than the CP-II group in improving the weight loss of the rats and effectively improving the morphological structure of the lung tissue, making the lung tissue luster increase and the texture soft. The histopathological section results showed that the alveolar wall was occasionally thickened, fibroblasts and collagen deposition were reduced, and inflammatory cell infiltration was reduced in the D, CP-I, and CP-II groups compared with the M group. This indicated that Maidong Decoction and its different phases could effectively improve the symptoms of BLM-induced PF in rats.

[0022] Lipid components play a crucial role in the maintenance of lung function, and the development of lipidomics technology has facilitated the understanding of PF from the perspective of lipid metabolism. The differential lipid analysis of different phase groups was performed, and the data acquisition and method stability were investigated, indicating that the method was stable. A total of 220 lipid metabolites were identified from the rat lung tissue, which were composed of GP, FA, SP, and ST. Multivariate statistical analysis, such as PCA, PLS-DA, and OPLS-DA, showed that there was a clear separation trend between the M and C groups in the positive and negative ion modes, and the results of the representative lipid molecules were well-differentiated, proving that the PF group was successfully modeled. Moreover, the D, CP-I, and CP-II groups were significantly separated from the M group and approached the C group, indicating that Maidong Decoction and its different phases could improve the symptoms of PF. Through differential metabolite screening, 25 PF-related differential lipids were identified. The study showed that GP decreased significantly and then rebounded in the serum of BLM-induced PF mice as the disease progressed. Therefore, it was speculated that Maidong Decoction and its different phases might improve PF by affecting the level of GP lipids.

[0023] Effect of Maidong Decoction and its different phases on TGF-β1-induced EMT of A549 cells Cell line: human non-small cell lung cancer cell A549 (purchased from Wuhan Pnuo Life Science Co., Ltd., resource number: CL-0016) Instruments Materials

[0024] Preparation of common reagents (1) Preparation of complete culture medium: basic culture medium (F-12K) + 10% FBS + 1% penicillin-streptomycin solution, mix well, and store at 4 ℃.

[0025] (2) TGF-β1 (10 μg powder) was purchased from the United States PeproTech company, and TGF-β1 (10 μg powder) was centrifuged at a speed of 3000 rpm for 5 min before opening the cover. After centrifugation, 1 mL of citrate buffer was added to dissolve it thoroughly, and a 10 ng / mL TGF-β1 stock solution was obtained. It was divided into sterile centrifuge tubes and stored at -20 ℃ for later use.

[0026] (3) D preparation: the yield of the prepared freeze-dried powder was 1.493 g / g of crude drug. When used, the required amount of freeze-dried powder was weighed and dissolved in complete medium, and filtered with a 0.22 μmol / L filter for subsequent cell experiments, and the concentration was calculated according to the crude drug amount.

[0027] (4) CP-I preparation: the yield of the prepared freeze-dried powder was 1.431 g / g of crude drug. When used, the required amount of freeze-dried powder was weighed and dissolved in complete medium, and filtered with a 0.22 μmol / L filter for subsequent cell experiments, and the concentration was calculated according to the crude drug amount.

[0028] (5) CP-II preparation: the yield of the prepared freeze-dried powder was 1.241 g / g of crude drug. When used, the required amount of freeze-dried powder was weighed and dissolved in complete medium, and filtered with a 0.22 μmol / L filter for subsequent cell experiments, and the concentration was calculated according to the crude drug amount.

[0029] Experimental method

[0030] Cell culture method (1) Resuscitation of cells: start the constant temperature water bath box and preheat to 37 ℃, hydrolyze the A549 cells and put them into the clean bench; add an appropriate amount of complete culture medium to the centrifuge tube, add the dissolved cell suspension, and centrifuge at a speed of 100 r / min for 5 min. After the cells adhere, discard the supernatant, add 1 mL of complete culture medium, and repeatedly blow the cell suspension into the culture bottle, and then add 4 mL of complete culture medium and put it into the incubator.

[0031] (2) Change of cell liquid: after the resuscitation of cells is successful, observe the cell growth under the microscope (such as state, density), if a lot of floating objects (such as dead cells, secretions) are observed in the culture medium, the culture medium should be changed in time. Discard the old culture medium, add 2 mL of PBS, shake it at an appropriate force and angle, discard the PBS after cleaning, add new culture medium, hold the culture bottle steadily with both hands and put it into the incubator for continuous culture.

[0032] (3) Cell passage: Observe the state of A549 cells. When the cell density reaches 80-90%, passage can be performed. Discard the complete culture medium in the flask, add 2 mL of PBS to wash the culture flask, repeat twice, then add 1 mL of trypsin. After placing the flask in an incubator for 1-2 min, observe large areas of cells detached under a microscope, and then quickly add complete culture medium to stop digestion. Repeatedly pipette the cell suspension, mix well, and transfer to a 15 mL centrifuge tube. Centrifuge at 1000 rpm / min for 5 min, discard the supernatant, add 2 mL of complete culture medium, and repeatedly pipette. Pass the cells at a 1:3 ratio, aliquot into culture flasks, add up to 4 mL of complete culture medium per flask, and place in an incubator.

[0033] (4) Cell cryopreservation: Select A549 cells in good logarithmic growth phase that have filled 70-80% of the bottom area of ​​the culture flask. Prepare cell suspensions using the above method by adding 3 mL of cryopreservation solution to each flask of cells. Add 1 mL of cryopreservation solution containing cells to each cryopreservation tube and freeze at -80 °C for storage.

[0034] (5) Cell counting: After digesting the cells with trypsin, shake the culture flask at various angles with appropriate force to mix the cells and trypsin. Place the cells in the incubator and count for 1 minute. When the cells change from long to round and detach from the cell wall and float under the microscope, quickly add 2 mL of 10% complete culture medium to end the digestion. Use appropriate force to pipette the cells and collect the suspension in a centrifuge tube. Centrifuge at 1000 rpm / min for 5 minutes and aspirate the supernatant. Drop 10 μL of the diluted cell suspension onto a counting plate covered with a glass slide, taking care not to generate air bubbles or allow the cell suspension to leak out. Count the cells under high magnification. The total number of cells = total number of cells in 4 grids / 4 × 10 5 ×Number of ml of stock solution.

[0035] Morphological changes in EMT induced by different concentrations of TGF-β1 in A549 cells Cell plating: A549 cells in logarithmic growth phase were digested with trypsin, and 1 mL of complete culture medium was added to form a cell suspension. Cells were counted using a cell counting chamber, and the cell density was adjusted to 2 mL of complete culture medium. The cells were then cultured in a CO2 incubator for 24 h.

[0036] Cell treatment: Culture medium containing 1% serum was used. The cells were divided into two groups: a normal control group (Group C, TGF-β1 final concentration of 0 ng / mL) and TGF-β1 groups (Group M, TGF-β1 final concentrations of 2.5, 5, and 10 ng / mL). Each group was divided into three replicates and cultured in a CO2 incubator. Cell morphology changes were recorded at 24 h and 48 h.

[0037] Images obtained through microscopic observation revealed that A549 cells in the normal control group appeared as well-defined, pebble-like structures.Figure 1 -A and Figure 2 -A). After TGF-β1 treatment, with increasing concentration, cell viability increases, morphology becomes elongated, exhibiting a spindle-like transformation, with larger gaps and looser connections, resembling fibroblast characteristics. The changes in cell morphology become more pronounced over time, such as ( Figure 1 -B, C, D and Figure 2 As shown in -B, C, and D). The optimal conditions for inducing EMT in A549 cells were determined by an action time of 48 h and a final concentration of 5 ng / mL TGF-β1.

[0038] Cell viability and drug toxicity assay Cells were collected and counted using a cell counting chamber. The cell density was adjusted to 8000 cells / well, and the cells were evenly seeded in 96-well plates and incubated at 37 °C for 24 h. D, CP-I, and CP-II were diluted with medium containing 10% serum and TGF-β1 (5 ng / mL) and added to 96-well plates for further incubation for 24 h and 48 h, respectively. Cells were washed twice with phosphate-buffered saline (PFS), and CCK-8 solution was added to each well at a 1:10 ratio. The cells were incubated at 37 °C for 1–2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated and analyzed for each drug group.

[0039] To investigate the potential toxicity of D, CP-I, and CP-II to A549 cells, the effects of different concentrations of D, CP-I, and CP-II on cell viability were detected using the CCK-8 assay. The results showed... Figure 3 Treatment of A549 cells with D, CP-I, and CP-II at a concentration of 18.75 mg / mL for 24 h and 48 h did not decrease cell viability. The results indicate that D, CP-I, and CP-II at a concentration of 18.75 mg / mL did not cause cell damage.

[0040] Effects of Ophiopogon japonicus decoction and its various phases on TGF-β1-induced proliferation of A549 cells (1) Experimental grouping: A549 cells were randomly divided into the following groups: blank group (serum-free culture medium), control group (C, cells + serum-free culture medium), model group (M, cells + serum-free culture medium + TGF-β1 solution, with a final TGF-β1 concentration of 5 ng / mL), D group (five concentrations of D were added, with a final concentration of 18.75 mg / mL, 9.3 mg / mL, 4.68 mg / mL, 2.34 mg / mL, and 0.3 mg / mL in the culture medium, and TGF-β1 solution was added to each, with a final concentration of 5 ng / mL), CP-Ⅰ group (five concentrations of CP-Ⅰ were added, with a final concentration of 18.75 mg / mL, 9.3 mg / mL, 4.68 mg / mL, 2.34 mg / mL, and 0.3 mg / mL in the culture medium, and TGF-β1 solution was added to each, with a final concentration of 5 ng / mL), and CP-Ⅱ group. Group (five concentrations of CP-II were added to achieve final concentrations of 18.75 mg / mL, 9.3 mg / mL, 4.68 mg / mL, 2.34 mg / mL, and 0.3 mg / mL in the culture medium, and TGF-β1 solution was added to each group to achieve a final concentration of 5 ng / mL).

[0041] (2) Experimental procedure: ① Seeding: In a clean bench, A549 cells in good growth condition were prepared into a cell suspension. After cell counting, the cell concentration was adjusted to 8000 cells / well, and the edge wells were filled with sterile PBS. After all cells were added, the cells were observed under an inverted optical microscope to check whether the cells were evenly distributed, and then placed in an incubator. The cells were cultured in complete medium for 24 h. After the cells adhered, the medium was replaced with serum-free medium to starve the cells and synchronize the cell cycle.

[0042] ② Drug addition: Discard the serum-free culture medium and add different concentrations of drug and TGF-β1 culture medium (final TGF-β1 concentration is 5 ng / mL) according to the experimental groups. Continue to incubate in a constant temperature incubator for 24 h and 48 h.

[0043] ③ Microplate reader detection: After 24 h and 48 h of incubation, remove the wells. Replace with 100 µL of medium containing 10 µL of CCK-8, incubate for 2 h in a constant temperature incubator, and measure the absorbance (OD) at 450 nm using a microplate reader. Calculate the inhibition rate of different concentrations of D, CP-Ⅰ, and CP-Ⅱ on TGF-β1-induced A549 cell proliferation according to the formula, repeating 3 times.

[0044] To investigate the effects of D, CP-I, and CP-II on the TGF-β1-induced EMT transformation model of A549 cells, A549 cells treated with 5 ng / mL TGF-β1 were treated with D, CP-I, and CP-II at concentrations of 0.3, 2.34, 4.68, 9.3, and 18.75 mg / mL, as determined in the "Results of the Cytotoxicity of Ophiopogon japonicus Decoction and Different Phases to A549 Cells". The results are as follows: Figure 4 As shown in the figure. Compared with group C, the inhibition rate of A549 cells in the TGF-β1 group was decreased. Compared with the TGF-β1 group, the inhibition rate of A549 cells treated with 5 ng / mL TGF-β1 gradually increased with increasing concentrations of D, CP-Ⅰ, and CP-Ⅱ, and all differences were highly significant. P <0.01).

[0045] Morphological observation of A549 cells induced by TGF-β1 induced by Mai Men Dong decoction and different phases The morphological changes of A549 cells after 48 h of treatment with D, CP-Ⅰ, CP-Ⅱ and TGF-β1 were observed and photographed using an inverted phase contrast microscope.

[0046] Figure 5 -A represents normal A549 cells, which are flattened or cobblestone-shaped. They are indistinguishable from healthy cells and grow well. Under the influence of the inducing factor TGF-β1, the cells gradually elongate, becoming spindle-shaped, and the connections between cells become loose, such as... Figure 5 -As shown in B. When D was added to a final concentration of 18.75 mg / mL, its morphology resembled pebbles, and the cells became more compact, as shown in B. Figure 5 As shown in -C; the addition of CP-Ⅰ to a final concentration of 18.75 mg / mL, while not as effective as in D, was significantly better. Figure 5 -B; Add CP-II to a final concentration of 18.75 mg / mL, its state is superior. Figure 5 -B.

[0047] Effects of Ophiopogon japonicus decoction and its various phases on TGF-β1-induced migration of A549 cells A549 cells in good growth condition were prepared into a cell suspension, and after cell counting, the cell concentration was adjusted to 3×10⁻⁶. 5 A549 cells were seeded per well in 6-well plates. When the cells reached approximately 90% confluence as observed under a microscope, a 100 μL pipette tip was used to make a vertical scratch on the bottom of the 6-well plate. The cells were washed three times with PBS, and 2 mL of serum-free culture medium was added for microscopic photography. A specific concentration of D, CP-I, CP-II, and TGF-β1 was added (TGF-β1 final concentration was 5 ng / mL). After culturing for 24 h and 48 h, the migration of A549 cells was observed under a microscope and photographed.

[0048] The effects of 18.75 mg / mL D, CP-I, and CP-II on TGF-β1-induced migration and invasion abilities of A549 cells were examined using a scratch assay. The results showed that ( Figures 6 to 6 -7), after 48 h of cell treatment, compared with group C, TGF-β1 significantly enhanced the migration ability of A549 cells. The migration ability of cells treated with different drug groups was inhibited to some extent. Among them, the inhibitory effect of CP-II group was the weakest. D and CP-I significantly inhibited the migration ability of A549 cells induced by TGF-β1.

[0049] The effects of Mai Men Dong decoction and its various phases on the TGF-β1 / Smad signaling pathway (1) Extraction of cell proteins Cellular protein extraction: Cells treated with D, CP-Ⅰ and CP-Ⅱ for 48 h were collected. Each well was lysed with 1% PMSFRIPA lysis buffer in an ice bath for 35 min. After centrifugation at 14000 r / min for 15 min at 4 ℃, the supernatant was collected and the protein concentration was determined using a BCA kit.

[0050] (2) Solution preparation Preparation of 5× electrophoresis buffer: Place 15.15 g of Tris, 72 g of Glycine, and 5 g of SDS in a graduated cylinder, add triple-distilled water to bring the volume to 900 mL, mix thoroughly, and bring the volume to 1000 mL. Store at room temperature and dilute to 1× before use.

[0051] Preparation of 1× transfer buffer: Place 3.03 g of Tris and 14.4 g of Glycine in a graduated cylinder, bring the volume to 800 mL, mix well, add 200 mL of methanol and bring the volume to 1000 mL, then store in a sealed container at 4 ℃ for later use.

[0052] Preparation of TBST washing solution: Weigh 2.42 g of Tris-base and 8 g of NaCl into a graduated cylinder and bring the volume to 800 mL. Add 1 mL of Tween and stir well. Adjust the pH to 7.4 and bring the volume to 1000 mL. Mix well and store at room temperature.

[0053] (3) Rubber preparation ① Rinse the glass plates thoroughly with triple-distilled water and then stand them upright to air dry; ② Leak test: Align two glass plates and place them in the gel preparation rack, clamp them tightly, and fill with triple-distilled water. A significant drop in the liquid level within 2 minutes is desirable; ③ Prepare a 10% separating gel: Add 4.0 mL of deionized water, 3.3 mL of 30% acrylamide, 2.5 mL of Tris-HCl (pH 8.8), 0.1 mL of 10% SDS, 0.1 mL of 10% ammonium persulfate, and 0.004 mL of TEMED to a 15 mL centrifuge tube. Vortex thoroughly, and use a 1 mL pipette to pour the gel along the edge of the glass plate until it reaches three-quarters of its height. Seal with anhydrous ethanol; ④ Let stand for 30 minutes, discard the ethanol, and prepare a 4% stacking gel: Add 2.7 mL of deionized water, 0.67 mL of 30% acrylamide, 0.5 mL of Tris-HCl (pH 6.8), and 0.04 mL of 10% SDS. Add 0.04 mL of 10% ammonium persulfate and 0.004 mL of TEMED to a centrifuge tube, mix well, pour into a glass plate, and insert a comb (to avoid air bubbles). Since the gel will shrink in volume during solidification, it is necessary to add gel to both sides during the solidification process of the concentrated gel. Let stand for 20 minutes to allow the gel to solidify.

[0054] (4) Electrophoresis Fix the gel plate in the electrophoresis tank, add electrophoresis buffer (fill the inner tank completely, and the outer tank to one-third of its height), and steadily pull out the comb vertically upwards. Add protein samples and markers to the sample wells. Set the voltage to 80 V for electrophoresis. When the marker shows a red line, change the voltage to 120 V. Stop electrophoresis when the bromophenol blue reaches the bottom of the separating gel.

[0055] (5) Transfer membrane Cut the gel according to the molecular weight indicated by the marker, and cut the corresponding PVDF membrane according to the size of the gel. Mark the corners and activate it in methanol for 10 seconds. Prepare the "sandwich" by stacking the gel in the following order: blackboard - sponge pad - three layers of filter paper - gel - PVDF membrane - three layers of filter paper - sponge pad - whiteboard. Remove air bubbles, place the "sandwich" in the transfer tank, add an ice box and pre-cooled transfer solution, and transfer the membrane for 90 minutes at a current of 130 mA.

[0056] (6) Closed 5% skim milk powder was sealed at 37°C for 90 min.

[0057] (7) Antibody incubation Primary antibody incubation: Discard the blocking buffer, add primary antibody (diluted at 1:1000), and incubate overnight at 4 ℃. Secondary antibody incubation: The next day, recover the antibody, wash the membrane with TBST for 5 min / 4 times, add secondary antibody (1:10000), shake on a shaker at room temperature for 30 min, and then incubate at 37 ℃ for 1 h.

[0058] (8) Exposure ①Development: Remove the membrane and wash it three times in 1×TBST solution, 10 min each time; ② After immersing the membrane in the prepared ECL colorimetric solution for about 1 minute, remove it and expose and develop it under a chemiluminescence imaging system. Calculate the integrated optical density and compare the gray value of the target protein with that of GAPDH.

[0059] All experimental data are expressed as mean ± standard deviation (x ± s), and data analysis was performed using GraphpadPrism 9.0 software. One-way ANOVA was used for comparisons among multiple groups of data. P A value <0.05 is considered statistically significant; like Figure 8 As shown, the expression levels of pSMAD2 and pSMAD3 proteins were significantly increased in group M, and the difference was statistically significant. P <0.01). In groups D and CP-Ⅰ, compared to groups CP-Ⅱ and M, the expression levels of pSMAD2 and pSMAD3 proteins were significantly lower, and the results were statistically significant. P <0.01). In summary, D, CP-I, and CP-II can all attenuate TGF-β1-induced EMT in A549 cells by blocking the TGF-β1 / Smad pathway.

[0060] Pulmonary fibroblasts (PF) are a chronic disease of unknown cause, with a continuous decline in lung function leading to respiratory failure and eventual death. Extracellular matrix (EMT) plays a crucial role in PF formation, with approximately one-third of lung fibroblasts identified as epithelial in origin. It leads to the accumulation of fibroblasts and myofibroblasts, as well as the production of large amounts of extracellular matrix, thus resulting in PF. Therefore, inhibiting EMT to reduce EMT deposition is a key focus of PF treatment research. A549 cells belong to the human lung adenocarcinoma cell line, originating from type II alveolar epithelial cells (AT-II cells). AT-I cells, as a type of pluripotent stem cell, possess considerable plasticity. Studies have found that when alveolar epithelial cells are damaged, AT-II cells transform into fibroblasts and myofibroblasts through the EMT process. A549 cells, possessing both the typical malignant characteristics of lung adenocarcinoma cells and the morphological characteristics of AT-II cells, are widely used in basic and clinical research on lung diseases.

[0061] Traditional Chinese medicine decoctions, as a classic dosage form, are mostly mixed-phase solution systems. Due to their complex composition and special decoction process, they are often used in large doses in clinical applications. Studies have shown that in the Shenbai Jiedu Decoction, the concentration of raw herbs at 8 or 16 mg / mL can regulate EMT-related molecules, thereby helping to reverse EMT. In the study of the effects of Xiaoyan Decoction and its components on apoptosis in A549 cell lines, it was found that the whole Xiaoyan Decoction group and the mass concentrations of each component group at 50 mg / mL-80 mg / mL had a certain inhibitory effect on A549 cells. Therefore, this experiment, combining the clinical dosage, raw herb amount, and cytotoxicity test results of Mai Men Dong Decoction, selected a concentration within 18.75 mg / mL for in vitro cell experiments of Mai Men Dong Decoction and its various phases.

[0062] The TGF-β / Smads signaling pathway is the most important signaling pathway leading to pulmonary fibrosis (PF). Transforming growth factor-β (TGF-β) is mainly divided into three types: TGF-β1, TGF-β2, and TGF-β3. Among the cytokines that cause pulmonary fibrosis, TGF-β1 is the most important regulator. TGF-β1 can induce a large number of fibroblasts to proliferate and differentiate into myofibroblasts. Myofibroblasts can resist apoptosis and accumulate in the active fibrotic sites of fibroblast lesions, ultimately leading to excessive deposition of ECM and collagen. It exerts its biological activity by activating both Smad-dependent (primary) and Smad-independent pathways. The Smad protein family can be divided into three classes: receptor-activated (R-Smads, including Smads 1, 2, 3, 5, and 8), universal (Co-Smads, including Smad4), and inhibitory (I-Smads, including Smad6 and 7). When TGF-β1 receptor kinase is activated, it recruits and phosphorylates Smad proteins for signal transduction. In recent years, research on this pathway has deepened, making it a relatively complete and mature pathway in PF research. Many traditional Chinese medicines and their active ingredients, as well as traditional Chinese medicine compound formulas, can exert certain anti-PF effects by intervening in TGF-β1 / Smad. Therefore, this invention uses TGF-β1 to induce EMT in A549 cells to explore the effects of Mai Men Dong Tang and different phases on TGF-β1-induced EMT in A549 cells.

[0063] This invention successfully induced EMT in A549 cells using TGF-β1 and investigated the effects of Mai Men Dong decoction and different phases on TGF-β1-induced EMT in A549 cells. Under an inverted microscope, after 48 hours of treatment with D, CP-I, CP-II, and TGF-β1, normal A549 cells were flattened or cobblestone-shaped. Under the influence of the inducing factor TGF-β1, the cells gradually elongated, becoming spindle-shaped, and the connections between cells became loose. After adding D, CP-I, and CP-II to a final concentration of 18.75 mg / mL, the morphology became closer to cobblestone, and the cells became more compact. This indicates that D, CP-I, and CP-II can all improve EMT. The CCK-8 assay showed that D, CP-I, and CP-II did not damage A549 cells and could inhibit TGF-β1-induced A549 cell proliferation to varying degrees. Cell migration assays showed that CP-II had the weakest inhibitory effect on TGF-β1-induced A549 cell migration, while D and CP-I significantly inhibited TGF-β1-induced A549 cell migration. Western blot results showed that D, CP-I, and CP-II could inhibit Smad2 / 3 phosphorylation to varying degrees, suggesting that D, CP-I, and CP-II can all attenuate TGF-β1-induced EMT in A549 cells by blocking the TGF-β1 / Smad pathway.

[0064] The specific embodiments of the present invention have been described in detail above. It should be noted that the present invention is not limited to the specific embodiments described above. Various modifications or alterations can be made by those skilled in the art without departing from the scope of protection defined by the claims, and all such modifications or alterations fall within the scope of the present invention.

Claims

1. An anti-pulmonary fibrosis active component of Mai Men Dong Tang isolated based on a centrifugation-membrane dialysis method, characterized in that, The method comprises the following steps: Step 1, add Maidong 42 g, Jiangbanxia 6 g, Renshen 6 g, Gancao 6 g, Jingmi 6 g, and Dazao 4 g into purified water 2400 mL, soak for 0 min, boil under strong fire for 38 min, and then transfer to weak fire and continue to boil until 1200 mL; filter the mixture through gauze while hot to obtain Maidong soup stock solution; Step 2, centrifuge at 40 ℃ and 3500 rpm for 10 min, and take the lower precipitate as the precipitate phase; Step 3, take the supernatant of the precipitate phase, centrifuge at 20 ℃ and 10000 rpm for 10 min, and take the obtained supernatant as the suspension phase (CP-I); Step 4, use the dialysis method to split the suspension phase, the dialysis frequency is 1 time, the dialysis bag specification MW is 7000 Da, the dialysis temperature is 37 ℃, the dialysis bag inside which is successfully split is the colloidal phase (CP-II), and the dialysis bag outside is the true solution phase.

2. Application of the precipitate phase, the suspension phase (CP-I), and the colloidal phase (CP-II) prepared by the method of claim 1 in the preparation of a drug for improving the symptoms of bleomycin-induced pulmonary fibrosis.

3. Application of the precipitate phase, the suspension phase (CP-I), and the colloidal phase (CP-II) prepared by the method of claim 1 in the preparation of a drug for inhibiting the proliferation of A549 cells induced by TGF-β1.

4. Application of the precipitate phase, the suspension phase (CP-I), and the colloidal phase (CP-II) prepared by the method of claim 1 in the preparation of a drug for inhibiting the phosphorylation of protein Smad2 / 3.

5. Application of the precipitate phase, the suspension phase (CP-I), and the colloidal phase (CP-II) prepared by the method of claim 1 in the preparation of a drug for weakening the EMT of A549 cells induced by TGF-β1 by blocking the TGF-β1 / Smad pathway.

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