CN113082015A

CN113082015A - Application of genistein in preventing chronic obstructive pulmonary disease and verification method

Info

Publication number: CN113082015A
Application number: CN202010018070.5A
Authority: CN
Inventors: 张鹏; 曹伟灵; 马婧; 潘明月; 匡海珠
Original assignee: Shenzhen Luohu Peoplel's Hospital
Current assignee: Shenzhen Luohu Peoplel's Hospital
Priority date: 2020-01-08
Filing date: 2020-01-08
Publication date: 2021-07-09

Abstract

The invention discloses a preventive effect of genistein on Chronic Obstructive Pulmonary Disease (COPD). Wherein the molecular formula of the genistein is as follows: c₁₅H₁₀O₅(ii) a Molecular weight: 270.24; CAS number: 446-72-0; the chemical name is: 4',5, 7-Trihydroxyisoflavone. According to the invention, after the pre-action of genistein, a human normal bronchial epithelial cell (BEAS-2B) COPD model and a Sprague Dawley rat COPD model induced by a smoking method are adopted, the genistein pre-action shows that the expression of inflammatory cytokines interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and the like can be remarkably inhibited, the expression level of lipid peroxidation product MDA is reduced, and genistein is found to prevent the generation and development of COPD by regulating and controlling NF-kappa B, ERK1/2, p38-MAPK and other signaling pathways, so that the compound can be used for preventing the COPD field.

Description

Application of genistein in preventing chronic obstructive pulmonary disease and verification method

Technical Field

The invention relates to the technical field of medicines, in particular to a preventive effect of genistein on a smoking-induced human normal bronchial epithelial cell (BEAS-2B) COPD model and a Sprague Dawley rat COPD model, belonging to the fields of functional foods, medicines and other related products.

Background

Chronic Obstructive Pulmonary Disease (COPD) is a pulmonary disease characterized by persistent obstruction of airflow from the lungs. According to the report of the world health organization, COPD is located at the fourth cause of common death and death, the incidence and the death rate of COPD are increased year by year, and 2030 is predicted to become the third approximately death disease in the world. At present, COPD patients in China exceed 1 hundred million people and become the third most common chronic disease in China, which is second to hypertension and diabetes. Conventional drug therapy cannot slow or halt the progression of COPD. The intervention of COPD has become an important and difficult point of contemporary medicine. Therefore, the development of new COPD prevention and treatment medicines and the research of molecular mechanisms have important theoretical significance and clinical application value.

Genistein (4', 5, 7-trihydroxy isoflavone) is a trihydroxy natural isoflavone compound which exists in leguminous and dentate plants and has an aromatic A-ring, has pharmacological effects of anti-inflammation, antioxidation, antitumor and the like, is widely applied to in vitro and in vivo researches on lung and systemic inflammation and various cancers (prostatic cancer, pancreatic cancer, breast cancer and bladder cancer), and becomes the focus of the researches. One clinical study in the united states showed that oral administration of isoflavone supplements significantly reduced the number of severe attacks in asthmatics with a high-expression subtype of plasminogen activator inhibitor 1, revealing the potential role of isoflavones in the treatment of asthma. The study of the subject group taught by Fanti shows that oral administration of isoflavones is effective in ameliorating underlying systemic inflammation in renal failure patients. It can be seen that genistein may play a positive role in the intervention of COPD due to its excellent anti-inflammatory activity. According to the results of an epidemiological study in Japan, oral administration of isoflavones has been shown to significantly improve lung function and reduce the incidence of COPD. The Chinese scholars, namely the professor of the chrysanthemum at dawn, find that genistein can obviously reduce the content of inflammatory factor tumor necrosis factor-alpha (TNF-alpha) in lymphocytes of COPD patients, and simultaneously, the expression levels of nuclear factor kappa B (NF-kappa B) and matrix metalloproteinase-9 (MMP-9) are reduced. The above studies indicate that genistein is expected as a potential drug for preventing COPD.

Disclosure of Invention

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the invention provides a use of genistein in preventing chronic obstructive pulmonary disease, wherein the formula of genistein is: c₁₅H₁₀O₅(ii) a Molecular weight: 270.24; CAS number: 446-72-0; the chemical name is: 4',5, 7-Trihydroxyisoflavone.

In the application provided by the invention, the genistein inhibits the expression of inflammatory cytokines IL-1 beta and IL-6.

In the application provided by the invention, the genistein can down regulate the expression level of the lipid peroxidation product MDA.

In the application provided by the invention, the genistein prevents the occurrence and development of COPD by regulating MAPK and NF-kB signaling pathways.

The invention also provides a method for verifying the application of genistein in preventing chronic obstructive pulmonary disease, which adopts a smoking-induced human normal bronchial epithelial cell (BEAS-2B) COPD model. Wherein the cigarette smoke extract is used at a concentration of 4% and the stimulation time is 24 hours; the action concentration of genistein is 10 μ M, and the pre-action time is 1 hr.

The COPD cell model verification method comprises the following steps:

selecting BEAS-2B cells as a research object, pretreating genistein (10 mu M) for 1 hour, incubating the cells with a cigarette smoke extract (CSM) for 24 hours, and detecting the expression of related inflammatory factors such as interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), lipid peroxide MDA and the like and related signaling pathway proteins such as MAPK and NF-kappa B by adopting methods such as protein immunoblotting (western blotting), enzyme-linked immunosorbent assay (ELISA) and the like.

The invention also provides a method for verifying the application of genistein in preventing chronic obstructive pulmonary disease, wherein the method adopts a smoking-induced Sprague Dawley rat COPD model. Wherein, the rats are continuously stimulated by a smoking method (4%) for 8 weeks, 1 hour per day; the dosage of genistein is 10mg/kg, and the administration method is gastric lavage.

The COPD animal model verification method comprises the following steps:

sprague Dawley rats (from the laboratory animal center of hong Kong university) were selected as study subjects and divided into a fresh air group (0%, v/v) and a smoke group (4%, v/v) for 8 weeks at 1 hour per day, and genistein (10mg/kg, gavage) was administered to both groups, and the rats were sacrificed 24 hours after the last smoke. Blood, BALF and the like are collected, western blotting and the like are adopted to detect the expression of IL-6, IL-1 beta, MDA and other related inflammatory factors, H & E staining and other methods are adopted to observe the pathological change of rat lung tissues, and the result shows that genistein has the in vivo pharmacological activity effect of obviously preventing rat COPD from happening and developing.

Compared with the prior art, the invention has the following beneficial effects: according to the invention, after the pre-action of genistein, a human normal bronchial epithelial cell (BEAS-2B) COPD model and a Sprague Dawley rat COPD model induced by a fumigation method are adopted, the genistein pre-action shows that the expression of inflammatory cytokines IL-1 beta and IL-6 can be obviously inhibited, the expression level of lipid peroxidation product MDA is reduced, and the genistein is found to prevent the occurrence and development of COPD by regulating and controlling NF-kappa B, ERK1/2, p38-MAPK and other signal pathways, so that the compound can be used for preventing the COPD field.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a chemical structural formula of genistein (genistein);

FIG. 2 is the expression levels of (A) the inflammatory cytokine IL-1 β and (B) the lipid peroxidation product MDA in the model of COPD induced by BEAS-2B cells by genistein inhibition CSM in example 1;

FIG. 3 is a graph showing that genistein-regulated CSM induces (A) ERK1/2 signaling pathway, (B) p38-MAPK signaling pathway, (C) ERK1/2 expression level and (D) p38-MAPK expression level in BEAS-2B cells in example 2;

FIG. 4 shows that genistein inhibition CSM induced the nuclear NF-. kappa. B p65 expression level and (B) phosphorylation levels of I.kappa.B.alpha.and p65 in BEAS-2B cells (A) in example 2;

FIG. 5 is a graph showing that genistein significantly down-regulates the expression levels of (A) inflammatory cytokine IL-1 β and (B) lipid peroxidation product MDA in the rat COPD model induced by the fumigation method in example 3;

FIG. 6 is a graph showing the results of genistein significantly improving cigarette smoke induced lung tissue damage in rats in example 4.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.

Example 1

A COPD cell model was established by CSM induction of BEAS-2B cells, where the concentration of CSM was 4% (v/v), with 24 hours stimulation, with significant upregulation of the inflammatory factor IL-1 β and promotion of the production of the lipid peroxidation product MDA. The test group genistein (10. mu.M) was pre-applied to the cells for 1 hour and after 24 hours of incubation with CSM, the expression of IL-1. beta. and MDA was examined. As shown in figure 2, compared with a COPD model group, genistein can remarkably reduce the release of IL-1 beta in the model group, inhibit the generation of MDA, and reveal that the genistein has stronger anti-inflammatory and antioxidant activities and has the potential of becoming a new medicine for preventing COPD.

Example 2

BEAS-2B cells are selected as a research object, after CSM (4%) stimulates the cells for half an hour, western blotting is utilized to detect the expression level of MAPK and NF-kB related pathway proteins. As shown in FIGS. 3 and 4, the results confirmed that the phosphorylation levels of EKR1/2 and p38-MAPK and the expression levels of NF-. kappa. B p65, p-. kappa.B.alpha.and p-p65 in the nucleus of the model group were significantly increased after 30 minutes of the stimulation of BEAS-2B cells by CSM (4%) as compared with the control group. The phosphorylation levels of EKR1/2 and p38 in the group of 1 hour after pretreatment by Genistein (10 mu M) are obviously reduced, the content of p-65 in cell nucleus is obviously reduced, the phosphorylation levels of I kappa B alpha and p65 are inhibited, and the fact that the Genistein can prevent the occurrence and development of COPD by regulating MAPK and NF-kappa B signal pathways is disclosed.

Example 3

Sprague Dawley rats were selected as study subjects and divided into a fresh air group (0%, v/v) and a smoke group (4%, v/v) for 8 weeks at 1 hour per day, genistein (10mg/kg, gavage) was administered to both groups, and 24 hours after the last smoke, rats were sacrificed and IL-1 β, MDA expression in blood or BALF was examined. As shown in FIG. 5, the experimental results confirmed that the IL-1. beta. content in the BALF of the model rat was significantly increased and MDA production in lung tissue was increased, compared to the control group. Compared with a model group, genistein (10mg/kg) can obviously reduce the IL-1 beta and MDA levels in rats in a prevention group, and the fact that the genistein has stronger anti-inflammatory and antioxidant activity in vivo is revealed.

Example 4

According to the embodiment 3, a COPD rat model is established, lung tissue sections are taken after the rat is sacrificed, and pathological changes of lung tissues are observed by adopting an H & E staining method, as shown in fig. 6, the result shows that compared with a control group, the lung tissues of the rat in the model group are seriously damaged, alveolar spaces are enlarged, part of alveoli are ruptured and fused to form pulmonary bullae, the phenomena of rupture and fusion of the alveoli of the rat after the pre-protection of genistein (10mg/kg, intragastric administration) and the like are obviously improved, and the fact that the genistein has the in-vivo pharmacological activity effect of obviously preventing COPD is revealed.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.

Claims

1. Application of genistein in preventing chronic obstructive pulmonary disease is provided.

2. Use according to claim 1, characterized in that genistein inhibits the expression of the inflammatory cytokines IL-1 β and IL-6.

3. Use according to claim 1, characterized in that genistein down-regulates the expression level of lipid peroxidation product MDA.

4. The use according to claim 1, wherein genistein prevents the development and progression of COPD by modulating the MAPK and NF- κ B signaling pathways.

5. A method for verifying the application of genistein in the prevention of chronic obstructive pulmonary disease, which is characterized in that the method adopts a smoking-induced human normal bronchial epithelial cell (BEAS-2B) COPD model.

6. A method according to claim 5, wherein the cigarette smoke extract is used at a concentration of 4% and the stimulation time is 24 hours; the action concentration of genistein is 10 μ M, and the pre-action time is 1 hr.

7. A method for verifying the application of genistein in the prevention of chronic obstructive pulmonary disease, characterized in that the method adopts a smoking-induced Sprague Dawley rat COPD model.

8. The method according to claim 7, characterized in that rats are continuously stimulated for 8 weeks with fumigation (4%) for 1 hour a day; the dosage of genistein is 10mg/kg, and the administration method is gastric lavage.