Application of tetrandrine citrate and analogue thereof in preparation of medicine for treating pulmonary fibrosis

Abstract

The invention relates to the technical field of medicine, in particular to application of tetrandrine citrate and analogues thereof in preparation of medicine for treating pulmonary fibrosis, safety is improved, a tetrandrine citrate solution is dosed through airway atomization, and the weight loss degree of pulmonary fibrosis rats is relieved; the lung coefficient of the pulmonary fibrosis rats is reduced; the phenomena of alveolar gap reduction, alveolar wall thickening, inflammatory cell infiltration and the like are relieved; the contents of IL-1beta, TNF-alpha, alpha-SMA and Vimentin in lung tissues, alveolar lavage fluid and serum of the rats with pulmonary fibrosis are reduced, and the content of SOD (superoxide dismutase) is increased; the proliferation of A549 cells is inhibited; the contents of COL-I, FN and Vimentin in a pulmonary fibrosis cell model are reduced.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of tetrandrine citrate and analogs thereof in preparing medicines for treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is a respiratory disease that continues to develop and eventually endangers life. In recent years, the number of patients with pulmonary fibrosis has continued to increase, and the incidence rate has also continued to rise with age, mainly concentrated in people over 60 years old, and the average survival time of patients after diagnosis is 3-5 years. At present, the etiology and pathogenesis of pulmonary fibrosis have not been fully elucidated. Studies have confirmed that one of the pathogenesis of pulmonary fibrosis is the epithelial-mesenchymal transition of alveolar epithelial cells, in which transforming growth factor can promote the occurrence of EMT by mediating Smads and ERK pathways, overexpression of type I and type IV collagen in cells, and overexpression of serine / threonine protein kinases, ultimately leading to an increase in the content of adhesion proteins and smooth muscle actin α. Currently, the FDA-approved drugs for the treatment of pulmonary fibrosis are pirfenidone and nintedanib, but the above drugs have strong toxic side effects: it is reported that the use of nintedanib ethanesulfonate capsules can induce nephrotic syndrome, and the use of pirfenidone causes more than 73.44% of patients to have adverse reactions to varying degrees. Therefore, it is urgent to explore the relationship between a low-toxic drug and pulmonary fibrosis. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides the use of tetrandrine citrate and its analogs in the preparation of drugs for treating pulmonary fibrosis.

[0004] Application of tetrandrine citrate and its analogs in the preparation of drugs for treating pulmonary fibrosis.

[0005] Preferably, the drug is tetrandrine citrate solution.

[0006] Preferably, the preparation method of the tetrandrine citrate solution is: dissolving citric acid in water to obtain a citric acid solution;

[0007] adding tetrandrine to the citric acid solution and dissolving it to obtain the tetrandrine citrate solution;

[0008] Among them, the solid-liquid ratio of tetrandrine to citric acid solution is 100mg-300mg:5mL-20mL.

[0009] Preferably, the mass fraction of the citric acid solution is 7% to 9%.

[0010] Preferably, the drug is a solution of an analog of tetrandrine citrate.

[0011] Preferably, the drug contains tetrandrine citrate as an active ingredient.

[0012] Preferably, the drug is in the form of a spray.

[0013] Preferably, the pulmonary fibrosis includes primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary interstitial fibrosis and interstitial pneumonia.

[0014] In the present invention, when treating rats with pulmonary fibrosis by airway atomization, tetrandrine citrate solution was administered at 5 mg / kg and 10 mg / kg, once a day, for 5 days; pirfenidone solution was administered by intraperitoneal injection at a dose of 10 mg / kg, once a day, for 5 days. When administering to the A549 pulmonary fibrosis model, tetrandrine solutions at concentrations of 5 μM and 10 μM were used, and TGF-β1 was added after incubation for 3 hours and incubated for 24 hours.

[0015] Using a rat model of pulmonary fibrosis and a TGF-β1-induced pulmonary fibrosis cell model, administration of tetrandrine citrate solution to the rats with pulmonary fibrosis and the pulmonary fibrosis cell model can effectively alleviate the progression of pulmonary fibrosis, specifically: the administration of tetrandrine citrate solution by airway atomization can alleviate the weight loss of rats with pulmonary fibrosis; the lung coefficient of rats with pulmonary fibrosis is reduced; the shrinkage of alveolar spaces, thickening of alveolar walls, and inflammatory cell infiltration are alleviated; the levels of IL-1β, TNF-α, α-SMA and Vimentin in the lung tissue, bronchoalveolar lavage fluid and serum of rats with pulmonary fibrosis are decreased, and the SOD content is increased; the proliferation of A549 cells is inhibited; the levels of COL-Ⅰ, FN and Vimentin in the pulmonary fibrosis cell model are decreased.

[0016] Compared with the prior art, the present invention is beneficial in that:

[0017] Citric acid is approved by the FDA as a natural, non-toxic organic acid that can be used in the production of food and medicine. Studies have shown that when citric acid is used in combination with other drugs, it can promote its penetration effect in the affected area, and citric acid itself has a certain inhibitory effect on the growth of cancer cells and fibroblasts. Therefore, the beneficial effects of the tetrandrine citrate solution of the present invention are reflected in: 1. The efficacy and safety of the drug are improved at the same time; 2. Low cost, pollution-free, and cheap. No external energy is required, TET is converted from fat-soluble to water-soluble, and can enter the systemic circulation by atomization inhalation, reducing the dosage of the drug and reducing the first-pass effect and toxic side effects of the drug.

[0018] Fangji is a traditional Chinese medicine that first appeared in Shennong's Classic of Materia Medica. Its production areas are mainly distributed in Jiangsu, Zhejiang, Guangdong and Guangxi regions of China. Figure 1As shown, it is a dibenzylisoquinoline alkaloid, the main biologically active substance in Stephania tetrandra, generally found in the roots of plants of the Menispermaceae family, and has certain analgesic, diuretic, hypertension treatment and anti-inflammatory effects.

[0019] The present invention provides the preparation of tetrandrine citrate solution and its application in anti-pulmonary fibrosis. The bleomycin-induced rat pulmonary fibrosis model is used for research. Different doses of tetrandrine citrate solution are administered by airway atomization to rats with pulmonary fibrosis. The dosage can be reduced while the progression of pulmonary fibrosis is effectively alleviated. Specifically, the preparation of tetrandrine citrate can improve the solubility of tetrandrine so that it can act on the body by airway atomization; the tetrandrine citrate solution can alleviate the pulmonary fibrosis caused by BLM. The results showed that the weight loss trend of rats was reduced; the lung coefficient of rats was effectively reduced; the alveolar space narrowing, alveolar wall thickening and inflammatory cell infiltration caused by BLM were alleviated; the increase of interleukin-1β, tumor necrosis factor, α-SMA and vimentin caused by pulmonary fibrosis was alleviated; the superoxide dismutase caused by pulmonary fibrosis; the tetrandrine citrate solution had a certain inhibitory effect on human lung adenocarcinoma cell A549; the COL-Ⅰ, FN and Vimentin contents in pulmonary fibrosis cells induced by TGF-β1 decreased. Therefore, the tetrandrine citrate solution can be used as a new treatment for pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the molecular structure and relative molecular mass of tetrandrine.

[0021] Figure 2 Schematic diagram of the preparation process of tetrandrine citrate solution.

[0022] Figure 3 The figures show the therapeutic effect of tetrandrine citrate solution on rats with pulmonary fibrosis, where A is a schematic diagram of the weight changes of rats with pulmonary fibrosis; B is a record of the lung coefficient of rats with pulmonary fibrosis; C is a schematic diagram of the SOD concentration in the serum of each group of rats determined by ELISA experiment; D and E are schematic diagrams of the IL-1β and TNF-α concentrations in the bronchoalveolar lavage fluid of each group of rats determined by ELISA experiment; F and G are schematic diagrams of the α-SMA and Vimentin concentrations in the lung tissue of each group of rats determined by RT-PCR experiment; H and I are schematic diagrams and quantitative analysis results of the Vimentin concentration in the lung tissue of each group of rats determined by Western Blot experiment.

[0023] Figure 4 These are Masson-stained sections of each group of rats with pulmonary fibrosis treated with tetrandrine citrate solution, where A is a lung tissue section of rats with pulmonary fibrosis 8 days after modeling; B is a lung tissue section of rats with pulmonary fibrosis 14 days after modeling.

[0024] Figure 5 Figure 3 shows the effect of tetrandrine citrate solution on TGF-β1-induced A549 cell therapy, where A is a schematic diagram of the MTT data results of different concentrations of TGF-β1 in A549 cell line; B is a schematic diagram of the MTT data results of different concentrations of tetrandrine citrate solution in A549 cell line; C is a schematic diagram of the results of ELISA experiment to determine the effect of different concentrations of TGF-β1 on the expression concentration of COL-Ⅰ in A549 cells; D and E are schematic diagrams of ELISA experiment to determine the concentrations of COL-Ⅰ and FN in the supernatant of each group of cell culture; F and G are schematic diagrams and quantitative analysis results of Western Blot experiment to determine the concentration of Vimentin in each group of cells. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0026] Table 1 Abbreviation comparison table

[0027]

[0028]

[0029] Example 1

[0030] Preparation of tetrandrine citrate solution, such as Figure 2 As shown:

[0031] Weigh 80 mg of citric acid powder using an electronic balance, add 10 mL of pure water to dissolve, mix thoroughly to obtain an 8% citric acid solution, and store at 4°C.

[0032] Weigh 200 mg of tetrandrine powder using an electronic balance, add 10 mL of 8% citric acid solution to dissolve, mix thoroughly and store at 4°C, and dilute with a suitable solvent before use.

[0033] Example 2

[0034] Construction of rat model of pulmonary interstitial fibrosis and administration of tetrandrine citrate solution

[0035] 7-week-old male Wistar rats weighing 200-230 g were selected and raised in an SPF-grade experimental animal room. Bleomycin was administered by airway atomization to establish a pulmonary fibrosis model. The rats were divided into 5 groups:

[0036] Normal control group n=4: Sterile PBS was nebulized in the airway, and one day later, 8% citric acid solution was nebulized in the airway, once a day, for 5 days.

[0037] Negative control group n=4: 10 mg / kg bleomycin was nebulized in the airway, and one day later, 8% citric acid solution was nebulized in the airway, once a day, for 5 days.

[0038] Tet low-dose group n=4: 10 mg / kg of bleomycin was nebulized in the airway, and one day later, 5 mg / kg of tetrandrine citrate solution was nebulized in the airway, once a day for 5 days.

[0039] Tet high-dose group n=4: 10 mg / kg of bleomycin was nebulized in the airway, and one day later, 10 mg / kg of tetrandrine citrate solution was nebulized in the airway, once a day, for 5 days.

[0040] Positive control group n=4: 10 mg / kg of bleomycin was administered by airway aerosolization, and 1 day later, 10 mg / kg of pirfenidone solution was intraperitoneally injected once a day for 5 days.

[0041] Corresponding samples were collected from each group of rats in batches after 8 days and 14 days for testing.

[0042] (1) Pathological staining sections

[0043] The right upper lung was fixed in 4% paraformaldehyde for 24 hours, dehydrated, embedded in paraffin, and sliced ​​for Masson staining. The degree of fibrosis was evaluated according to the Aschroft score. The slices were observed and counted under an optical microscope.

[0044] (2) Lung coefficient measurement

[0045] The right middle lung was removed and its mass was measured by differential weight method. It was frozen at -80℃ for 4h and freeze-dried at -50℃ for 12h before its dry weight was measured. The lung coefficient was calculated and analyzed. The calculation formula of the lung coefficient was lung wet weight / lung dry weight.

[0046] (3) ELISA experiment

[0047] ELISA kits were used to detect the SOD content in rat serum and the IL-1β and TNF-α contents in rat BALF.

[0048] (4) RT-PCR experiment

[0049] RNA was extracted from the lung tissues of rats in all groups, and cDNA was obtained using a reverse transcription kit. RT-PCR experiments were performed using the HyperScriptSYBR Green One-Step RT-qPCR kit. The primers used are shown in Table 2. The experimental results were collected and analyzed. The reverse transcription kit model was R102-02, purchased from XINBEI, Shanghai, China, and the RT-qPCR kit model was Q301-01, purchased from XINBEI, Shanghai, China.

[0050] Table 2 Primer sequences

[0051]

[0052] (5) Western Blot experiment

[0053] Proteins from the right lower lung of rats in all groups were extracted. 12% polyacrylamide gel was prepared, and the proteins were loaded and electrophoresed at 80V for 20min; the voltage was adjusted to 100V and continued until the end of electrophoresis. PVDF membrane was used for transfer, QuickBlock TM Western blocking solution was used for 15 min, and QuickBlock TM The model of Western blocking solution is P0252-500 mL, which was purchased from Beyotime, Shanghai, China. The primary antibodies of Vimentin and β-actin diluted at 1:1000 were incubated at 4°C overnight, and the secondary antibodies were incubated at room temperature for 90 min. Finally, the experimental results were developed and analyzed using chemiluminescence.

[0054] result

[0055] The results are as follows Figure 3 , Figure 4 As shown, administration of tetrandrine citrate solution to rats with pulmonary fibrosis can effectively alleviate the progression of pulmonary fibrosis. Administration of tetrandrine citrate solution alleviated the weight loss of rats with pulmonary fibrosis, reduced lung coefficient, restored alveolar space, alveolar wall thickness, and inflammatory cell infiltration, and decreased IL-1β, TNF-α, α-SMA, and Vimentin levels in lung tissue, alveolar lavage fluid, and serum, and increased SOD content. The preparation of tetrandrine citrate solution is simple, and has a good therapeutic effect on pulmonary fibrosis. It can reduce the dosage without affecting the efficacy by airway atomization.

[0056] Example 3

[0057] Application of tetrandrine citrate solution in cellular pulmonary interstitial fibrosis model

[0058] (1) In vitro cytotoxicity assay

[0059] This experiment used the MTT method to determine the in vitro cytotoxicity of tetrandrine citrate solution and TGF-β1 on human lung adenocarcinoma cell A549. The specific steps are as follows:

[0060] Cell plating: Take cells in logarithmic growth phase and dilute them with DMEM medium containing 10% PBS and 1% double antibody, then plate them into 96-well plates at a concentration of 5000 cells / well, and add double volume of sterile PBS to the surrounding wells, and culture them in a 37°C CO2 incubator overnight.

[0061] Drug configuration, dosing and detection: After passing through a 0.22μm water filter, the tetrandrine citrate solution was diluted to 0nM, 2.5nM, 5nM, 10nM, 20nM, and 40nM using DMEM medium containing 10% FBS and 1% double antibody, where 1% double antibody refers to 100μg / mL penicillin and 50μg / mL streptomycin; TGF-β1 was diluted to 0ng / mL, 2.5ng / mL, 5ng / mL, and 10ng / mL using the same dilution method. After removing the original culture medium in the 96-well plate, the above diluted liquids were added to different wells and cultured for 24h. After discarding the original culture medium, each well was added with a culture medium containing 500μg / mL MTT, incubated at 37℃ in the dark for 4h, and then the MTT liquid was discarded. After adding DMSO, the absorbance was measured at a wavelength of 570nm and data analysis was performed.

[0062] (2) ELISA experiment

[0063] Cell model establishment and sample extraction: After culturing A549 in the logarithmic phase in DMEM medium containing TGF-β1 for 24 hours, the cell culture supernatant was collected and centrifuged at 4°C 2000 rpm for 20 minutes. The supernatant was taken and the concentrations of TGF-β1 were 0 ng / mL, 2.5 ng / mL, 5 ng / mL, and 10 ng / mL, respectively.

[0064] ELISA detection: COL-Ⅰ in the above samples was detected using an ELISA kit to verify the establishment of the TGF-β1-induced A549 cell pulmonary fibrosis model.

[0065] Cell administration and sample extraction: A549 cells in the logarithmic phase were cultured in DMEM medium containing 5 μM and 10 μM tetrandrine citrate solution for 3 h, and then DMEM medium containing TGF-β1 was added. After incubation for 24 h, sample extraction was performed, and the extraction steps were the same as above.

[0066] ELISA detection: Use ELISA kit to detect the COL-Ⅰ and FN content in the above samples.

[0067] (3) Western Blot experiment

[0068] Cell administration and sample extraction: A549 cells in the logarithmic phase were cultured in DMEM medium containing 5 μM and 10 μM tetrandrine citrate solution for 3 h, then DMEM medium containing TGF-β1 was added and cultured for another 24 h. The liquid in the wells was discarded, and the cells were washed three times with PBS before protein extraction.

[0069] Western Blot experiment: Use Western Blot method to detect the content of Vimentin in the above protein samples.

[0070] result

[0071] The results are as follows Figure 5 As shown in the results, administration of tetrandrine citrate solution to the TGF-β1-induced human lung adenocarcinoma cell A549 pulmonary fibrosis model can effectively alleviate the progression of pulmonary fibrosis. The abnormal high expression of COL-Ⅰ and FN in cells induced by TGF-β1 was alleviated, and the intracellular Vimentin content decreased. The above results prove that tetrandrine citrate solution has a good therapeutic effect on the in vitro model of pulmonary fibrosis.

[0072] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0073] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0074] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of tetrandrine citrate and its analogs in the preparation of drugs for the treatment of pulmonary fibrosis.

2. The use according to claim 1, characterized in that: The drug is tetrandrine citrate solution.

3. The use according to claim 2, characterized in that: The preparation method of the tetrandrine citrate solution is as follows: dissolving citric acid in water to obtain a citric acid solution; adding tetrandrine to the citric acid solution and dissolving it to obtain the tetrandrine citrate solution; Among them, the solid-liquid ratio of tetrandrine to citric acid solution is 100mg-300mg:5mL-20mL.

4. The use according to claim 3, characterized in that: The mass fraction of the citric acid solution is 7% to 9%.

5. The use according to claim 1, characterized in that: The drug is a solution of an analog of tetrandrine citrate.

6. The use according to claim 1, characterized in that: The medicine takes tetrandrine citrate as an effective ingredient.

7. The use according to claim 1, characterized in that: The dosage form of the medicine is a spray.

8. The use according to claim 1, characterized in that: The pulmonary fibrosis includes primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary interstitial fibrosis and interstitial pneumonia.

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