TWI818466B - Flavonoids for the treatment of arsenic induced lung damage - Google Patents

Abstract

The present invention relates to flavonoids for the treatment of arsenic-induced lung function damage. The flavonoids can regulate the expression of protein and molecular markers, thereby reducing the lung function damage caused by arsenic.

Description

Flavonoid drugs used to treat arsenic-induced lung function impairment

The present invention relates to a flavonoid drug for treating arsenic-induced lung function damage, and in particular to a flavonoid drug using natural flavonoids for treating arsenic-induced lung function damage.

Arsenic poisoning is a medical condition caused by an increase in arsenic content in the body. It mainly refers to the allosteric regulation of important metabolic enzymes in the animal body under the influence of arsenic. It is also generally regarded as heavy metal poisoning. If arsenic poisoning occurs in a short period of time, symptoms may include headache, dizziness, vomiting, abdominal pain, brain lesions, and bloody, watery diarrhea. Long-term exposure to arsenic can lead to skin thickening, keratosis of the feet, melanin deposition, abdominal pain, diarrhea, cardiovascular disease, peripheral neuropathy, and cancer.

In recent years, excessive arsenic levels in drinking water have been continuously found in various countries, such as India, Japan, Vietnam, China, Bangladesh, Chile, Mexico and Australia [Non-patent Document 1]; survey results show that about 5 billion people in the world have The arsenic content in drinking water exceeds the standard [Non-patent Document 2], and other survey results found that there is a dose-response relationship between the risk of lung cancer and arsenic exposure concentration. When the arsenic concentration is When the arsenic concentration reaches 10-100 μg/L, the risk of lung cancer does not change significantly. However, when the arsenic concentration reaches 100-300 μg/L, the risk of lung cancer increases significantly [Non-patent Document 3].

In addition, some researchers have conducted surveys in Taiwan and found that when the arsenic content in the environment exceeds 0.35mg/L, the incidence of lung cancer increases significantly [Non-patent Document 4]; other researchers have found that occupational chronic arsenic exposure may also cause lung epithelial damage. Cell oncogene activation leads to malignant transformation [Non-patent document 5].

In order to treat arsenic-induced lung function damage, many medical-related scholars and doctors have developed many drugs to treat arsenic-induced lung function damage, especially pulmonary fibrosis, such as pyridine, which is commonly used to treat idiopathic pulmonary fibrosis. Pirfenidone and Nintedanib; however, the side effects of pirfenidone may cause liver insufficiency and liver failure, while the side effects of nintedanib are diarrhea, nausea or abnormal liver function. And there was no obvious improvement in the treatment effect.

In summary, it is necessary to develop a drug that has few or almost no side effects and can significantly improve the lung function damage caused by arsenic, especially the drug that improves the pulmonary fibrosis caused by arsenic.

non-patent literature

1. Blow NS. Arsenic poisoning[J]. Biotechniques, 2011, 51(1): 11.

2. Ravenscroft P. Arsenic pollution of groundwater in Bangladesh[J]. Encyclopedia of Environ Health, 2011: 181-192.

3. Chen CL, Chiou HY, Hsu LI, et al. Ingested arsenic, characteristics of well water consumption and risk of different histological types of lung cancer in northeastern Taiwan[J]. Environl Res, 2010, 110(5): 455- 462. doi: 10.1016/j.envres.2009.08.010

4. Chung YL, Liaw YP, Hwang BF, et al. Arsenic in drinking and lung cancer mortality in Taiwan[J]. J of Asian Earth Sci,, 2013, 15(77): 327-331.

5. Stueckle TA, Lu YJ, Davis ME, et al. Chronic occupational exposure to arsenic induces carcinogenic gene signaling networks and neoplastic transformation in human lung epithelial cells[J]. Toxicol and Appl Pharm, 2012, 2(261): 204- 216.

In view of the above-mentioned problems of the conventional technology, the purpose of the present invention is to provide a flavonoid drug for treating arsenic-induced lung function damage, so as to solve the problem of conventional drugs having many side effects and insignificant therapeutic effect.

Research has found that compounds in flavonoids such as apigenin, naringenin, quercetin, kaempferol, baicalein, etc. have anti-inflammatory, It has the effects of reducing coronary artery disease and inhibiting the growth of cancer cells. These flavonoid compounds are rich in daily vegetables and fruits. They are natural flavonoid compounds, so their consumption basically has no side effects on the human body.

Among the various flavonoid compounds mentioned above, the inventor of the present invention found that flavonoids containing apigenin can reduce the expression of fibrosis-related proteins and molecular markers, such as SNAIL (which can induce epithelial-mesenchymal transition (EMT)). (transcription factor), α-smooth muscle actin (α-SMA), matrix metallopoateinase-2 (MMP-2) and/or fibronectin, etc., and then Improve pulmonary fibrosis induced by arsenic.

Among them, when mammalian bronchial epithelial cells (normal human bronchial epithelial, NHBE) are exposed to arsenic, the expression of the above-mentioned fibrosis-related proteins and molecular markers will be promoted, leading to fibrosis of mammalian bronchial epithelial cells.

Hereinafter, through the examples and drawings, it will be explained that apigenin can reduce the expression of fibrosis-related proteins and molecular markers, thereby improving pulmonary fibrosis induced by arsenic.

In order to make the technical features, content and advantages of the present invention and the effects it can achieve more obvious, the present invention is described in detail as follows in conjunction with the accompanying drawings and in the form of embodiments: Figure 1 shows bronchial epithelial cells ( Schematic diagram of detecting the effect of apigenin on arsenic-induced epithelial to mesenchymal transition (EMT) in NHBE). The second picture is a schematic diagram of apigenin reducing the expression of matrix metalloproteinase-2 induced by arsenic. The third picture is a diagram of apigenin in mice. Schematic diagram of detecting the effect of apigenin on pulmonary fibrosis and expression of interstitial cell marker proteins.

The terms "invention" and "present invention" as used in this patent are intended to refer broadly to all subject matter within the scope of this patent and the following patent applications. Statements containing such terms are to be understood as not limiting the subject matter described herein or limiting the meaning or scope of the patent claims below. The embodiments of the invention covered by this patent are defined by the scope of the following claims and not by this summary. This summary is a high-level overview of various aspects of the invention and introduces some concepts that are further described below in the Description section. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to solely Used to determine the scope of the subject being claimed. The subject matter should be understood by reference to the appropriate portions of the entire specification, any or all drawings, and each claim.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms such as those defined in commonly used dictionaries should be construed to have meanings consistent with their meanings in the context of the relevant technology and the present invention, and are not to be construed as idealistic or excessive Formal meaning, unless expressly so defined herein.

Materials and methods

Cell culture, drugs and reagents

Bronchial epithelial cells (NHBE, purchased from Lonza) were maintained at 37°C and 5% carbon dioxide and supplemented with 5 μg/L human recombinant epithelial growth factor (EGF), 50 mg/L bovine pituitary extract (BPE), and 5 mg/L insulin. and 25 nM hydrogenated cortisol in keratinocyte SFM basal medium.

Sodium arsenite (NaAsO ₂ ), apigenin and other drugs were purchased from Sigma-Aldrich in the United States.

Matrix metalloproteinase-2 (MMP-2) as the primary antibody was purchased from GeneTex (product code: GTX104577), fibronectin was purchased from Sigma (product code: F3648), and SNAIL was purchased from GeneTex (product code: GTX100574) , and α-smooth actin (α-SMA) were purchased from GeneTex (product code: GTX100034).

animal

C57BL/6 mice aged 6 to 8 weeks were purchased from the National Laboratory Animal Center in Taiwan and divided into three groups, including a control group, an arsenic-exposed group, and an apigenin-administered arsenic-exposed group. Mice were exposed to 50 mg/L (equivalent to 30 ppm) sodium arsenite (purchased from Sigma, St Louis, MO, 99% purity) through drinking water. for 12 weeks; apigenin (purchased from Sigma, St Louis, MO, purity 95.0%) was administered by oral gavage at a dose of 10 mg/kg/day for 12 weeks. The control group received no treatment at all.

Lung histology and immunohistology

The lung samples were placed in 10% formalin, fixed and embedded in paraffin, and cut into sections with a thickness of 3 μm and placed on glass slides. Hematoxylin and eosin ( The sections were stained with H&E staining, Masson's trichrome staining and immunohistological staining. Immunohistological analysis was performed using the UltraVision Quanto Detection System Hrp DAB (Thermo, model TL-060-QHD); i.e., lung samples were dewaxed and rehydrated using a hydrogen peroxide block and Tris-EDTA (pH9.0) After antigen retrieval in buffer, immune blockade was performed.

For MMP-2, antibodies were diluted 1:500 in phosphate buffer saline (PBS) and incubated at room temperature for 2 hours; while for SNAIL, antibodies were diluted 1:400 in PBS and incubated overnight at 4°C.

After the sections of the lung samples were washed three times with PBS, the sections were incubated with the above-mentioned primary antibody for hybrid reaction, and then the primary antibody was identified with a rabbit-specific secondary antibody immunostaining reagent, and then stained with DAB chromogenic reagent; Sections were then counterstained with hematoxylin and mounted using xylene-based mounting medium. Finally, a MoticRastScan slide scanner (Motic, purchased from China) was used and the scanned images were captured using Motic DSAssistant software.

result

Referring to Figure 1, Figure 1 is a schematic diagram for detecting the effect of apigenin on arsenic-induced epithelial to mesenchymal transition (EMT) in bronchial epithelial cells (NHBE). In this test, bronchial epithelial cells were grown in a culture dish and allowed to grow for 48 hours to achieve monolayer spreading; 2 hours before administration of sodium arsenite (NaAsO ₂ ) at a concentration of 4 μM, the cells were Apigenin at a concentration of 10 μM was administered, and 12 hours after the wound was formed, images were taken for gap closure (Gap%) analysis. The images and analysis results are as shown in Figure 1 A and Figure 1 B. Shown; the data are expressed as mean + standard deviation (Mean+SEM), * indicates statistical significance compared with the control group, # indicates statistical significance compared with the group given 4 μM sodium arsenite meaning.

In addition, the cells were first given apigenin at a concentration of 10 μM 2 hours before the administration of 4 μM sodium arsenite (NaAsO ₂ ). After 24 hours of culture, the cells were collected for protein extraction and Use antibodies (i.e., the primary and secondary antibodies mentioned above) to perform western blot analysis. The analysis results are shown in Figure 1C; in addition, after 48 hours of culture, the cells were used for Immunostaining analysis, the analysis results are shown in Figure 1D.

Referring to Figure 1A and Figure 1B, significant cell migration can be seen in the arsenic-exposed group exposed to sodium arsenite (NaAsO ₂ ) compared with the untreated control group. The wound area was reduced, and the effect of reducing the wound area was significantly reversed in the group given apigenin.

Referring to Part C of Figure 1, it can be seen from the figure that the expression of interstitial cell marker proteins induced by sodium arsenite, that is, the expression of fibronectin, MMP-2, α-SMA and SNAIL is all attenuated by apigenin.

Referring to Part D of Figure 1, it can be seen from the immunofluorescence diagram (the blue part is the fluorescent dye DAPI, 4',6-diamidino-2-phenylindole, which can strongly bind to DNA), Sodium arsenite caused a significant increase in the expression of fibronectin (green part); while after administration of apigenin, the expression of fibronectin was significantly reduced, confirming that apigenin can indeed reduce the expression of fibronectin.

Next, refer to Figure 2, which is a schematic diagram showing that apigenin reduces the expression of matrix metalloproteinase-2 induced by arsenic. In this experiment, bronchial epithelial cells were treated with apigenin at a concentration of 10 μM 2 hours before the administration of sodium arsenite (NaAsO ₂ ) at a concentration of 4 μM. The cells were cultured for 24 hours and 48 hours respectively. The cells were then collected and subjected to Western blot analysis using antibodies against matrix metalloproteinase-2 (MMP-2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

As can be seen from Figure 2, apigenin at 2 μM or 10 μM can effectively reduce the expression of MMP-2 induced by sodium arsenite in a dose-dependent manner. The higher the concentration, the more obvious the inhibitory effect.

Finally, refer to Figure 3, which is a schematic diagram of detecting the effect of apigenin on pulmonary fibrosis and the expression of interstitial cell marker proteins in mice. In this experiment, mouse lung tissue was exposed to sodium arsenite and apigenin for 12 weeks, and then H&E staining (Figure 3A), Masson's trichrome staining (Figure 3B), and immunohistological staining were performed. method (Figure 3C is MMP-2, Figure 3D is SNAIL), and Western blot analysis was used to analyze TGF-β, SNAIL and α-smooth actin (α-SMA) in mouse lung tissue. A schematic diagram of the analysis (Figure 3E), and Figure 3F is a schematic diagram of the quantitative results of three replicates of Western blot analysis.

Referring to Figure 3A, in the lung tissue of mice exposed to sodium arsenite, it can be seen that the thickness of the alveolar compartments increases; and after oral administration of 10 mg/kg apigenin, the thickness of the alveolar compartments tends to decrease.

Referring to Figure 3B, the lung tissue of mice exposed to sodium arsenite, using Masson's trichrome staining, showed increased deposition of collagen fibers in the peribronchial and alveolar areas, but after administration of apigenin, the collagen fibers The amount of fiber lamination is reduced.

Referring to Figure 3C and Figure 3D, the expression of MMP-2 and SNAIL increased substantially in the lung tissue of mice exposed to sodium arsenite, but after administration of apigenin, the expression of MMP-2 and SNAIL significantly reduced.

Referring to Figure 3 E and Figure 3 F, in the lung tissue of mice exposed to sodium arsenite, the expression of TGF-β, SNIAL and α-smooth actin increased with the dose of sodium arsenite. The expression of TGF-β, SNIAL and α-smooth actin was significantly decreased after administration of apigenin.

In summary, the results obtained from the above experiments clearly show that apigenin has the effect of treating, preventing or alleviating arsenic-induced lung function damage. Patients with lung damage can be given drugs or health foods containing apigenin, which can significantly improve their lung function damage with almost no side effects, especially pulmonary fibrosis.

The above is only illustrative and not restrictive. Any equivalent modifications or changes that do not depart from the spirit and scope of the present invention shall be included in the appended patent scope.

Claims (2)

A flavonoid drug for treating, preventing or alleviating arsenic-induced lung function damage, wherein the flavonoid drug contains apigenin, wherein the arsenic-induced lung function damage is pulmonary fibrosis, and the pulmonary fibrosis It is fibrosis caused by exposure of mammalian bronchial epithelial cells (normal human bronchial epithelial) to arsenic; and the pulmonary fibrosis is caused by the expression of fibrosis-related proteins and molecular markers, and the fibrosis-related proteins And molecular markers include: SNAIL, α-smooth actin and/or matrix metalloproteinase-2. A use of apigenin to reduce the expression of fibrosis-related proteins and molecular markers, including administering apigenin to the fibrosis-related proteins and molecular markers, wherein the fibrosis-related proteins and molecular markers include: SNAIL, α-smooth muscle kinesin and/or matrix metalloproteinase-2.