CN108721306B

CN108721306B - Application of arenobufagin in medicine for preventing/treating asthma

Info

Publication number: CN108721306B
Application number: CN201810752467.XA
Authority: CN
Inventors: 李宝晶; 黄丰; 何红平; 王亭; 孙赟; 杨竹雅
Original assignee: Yunnan University of Traditional Chinese Medicine TCM
Current assignee: Yunnan University of Traditional Chinese Medicine TCM
Priority date: 2018-07-10
Filing date: 2018-07-10
Publication date: 2020-07-14
Anticipated expiration: 2038-07-10
Also published as: CN108721306A

Abstract

Experiments prove that the bufogenin can reduce inflammatory cell infiltration and airway mucus high secretion in lung tissues of mice in asthma models, and can play a role in treating asthma by inhibiting the expression of Eotaxin, I L-13 and I L-8 in bronchoalveolar lavage fluid.

Description

Application of arenobufagin in medicine for preventing/treating asthma

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of arenobufagin in medicines for preventing/treating asthma.

Background

Bronchial Asthma, Asthma (Asthma), is a recurrent chronic inflammatory disease of the airways characterized by airway hyperresponsiveness, airway inflammation and airway remodeling, which is involved by a variety of inflammatory cells. The World Health Organization (WHO) reports that "the harm and economic burden of asthma to human health exceeds the sum of tuberculosis and aids". With the aggravation of environmental pollution, the incidence and mortality of asthma increase year by year. At present, about 3 hundred million patients with asthma exist in the whole world, wherein about 3000 million patients with asthma exist in China, and the number of patients with asthma is obviously increased year by year. Therefore, the research and development of asthma medicines have important social significance and value.

A variety of inflammatory cells are involved in the course of asthma, including T lymphocytes (T cells), B lymphocytes (Bcells), Eosinophils (Eos), neutrophils (PMNs), and the like.

Eosinophils (Eos) are highly activated and are a typical hallmark of asthma airway inflammation. Eotaxin is a powerful chemotactic factor of eosinophils, and can promote the development, activation and proliferation of Eos, promote the massive infiltration of Eos at airway inflammation sites, and the activated Eos induces airway hyperresponsiveness and airway remodeling by releasing harmful substances such as main basic protein, cationic protein, leukotriene and the like.

The dominant reaction of type 2 auxiliary lymphocyte T cells (T helper 2 cells, Th2) is presented in bronchial alveolar lavage Fluid (BA L F) of asthma patients Th2 cells secrete a large amount of Th2 cytokine I L-13, I L-13 can mediate airway smooth muscle spasm to cause airway hyperreactivity, cup cell hyperplasia to cause airway mucus hypersecretion, and Eos can be recruited to enter the site of allergic inflammation by stimulating the generation of Eotaxin.

Clinical studies indicate that over 50% of asthma patients have neutrophil-infiltrated inflammation, I L-8 is a chemotactic factor and activator of neutrophils, which chemotactic neutrophils enter the airway and participate in airway inflammation and airway hyperreactivity.

The Chinese medicinal cutis Bufonis is dried cutis of Bufo bufogargarizans Cantor or Bufo melanostictus Schneider of Bufo animal belonging to Bufo of Bufo, has effects of clearing away heat and toxic materials, inducing diuresis and relieving flatulence, and can be used for treating carbuncle, pyogenic infections, scrofula, etc. The chemical components of toad skin mainly comprise toad venom lactones, indole alkaloids and the like. Wherein the bufogenin compounds are the main active components of cutis Bufonis. The bufogenin compounds are cardenolide compounds with diene lactone structure derived from Bufo siccus animal.

Arenobufagin is a bufogenin compound with high content in cutis Bufonis, and its structural formula is shown in figure 1. At present, the research on the biological activity focuses on the cardiotonic effect and the anti-tumor effect. The research shows that the arenobufagin has obvious curative effect on liver cancer and gastric cancer, and has better curative effect on heart failure, viral hepatitis B and other diseases.

In the research process, we unexpectedly find that the bufogenin has a good treatment effect on asthma. The invention relates to the effect and efficacy of arenobufagin in the aspect of anti-asthma, and no relevant report is found.

Disclosure of Invention

The invention provides an application of a arenobufagin, namely an application of the arenobufagin in preparing a medicine for preventing/treating asthma diseases, wherein the arenobufagin has the following structural formula:

。

the preparation of the arenobufagin is oral preparation or intravenous injection.

The oral dosage form is tablet, hard capsule, soft capsule, powder, pill, or granule.

The intravenous injection is solution injection, suspension injection, emulsion injection, or sterile powder for injection.

The dosage is 1-3 times daily, 4-12mg each time.

The recommended dose is 3 times daily, 4mg each time.

The invention relates to a series of biological experiments of anti-asthma of bufogenin, which provides a basis for developing new application of the bufogenin in anti-asthma.

The research conclusion of the specific biological experiment comprises the following aspects:

a BA L B/c mouse asthma model is established through Ovalbumin (OVA) induction, the influence of the arenobufagin on a model animal is researched, a pathological section of a mouse lung tissue is made, and the airway inflammation and inflammatory cell infiltration conditions, goblet cell proliferation and airway mucus high secretion conditions are observed.

The invention uses OVA to induce BA L B/c mice to establish asthma model animals, and researches the influence of arenobufagin on inflammatory cytokine I L-13 generated by Th2 cells in bronchoalveolar lavage fluid, the influence of Eotaxin and the influence of neutrophil chemotactic factor I L-8.

The invention has the following effects:

the invention has the advantages and positive effects that the toad venom has obvious treatment effect on experimental asthma through biological research, can improve the lung tissue inflammatory infiltration and goblet cell proliferation of mice, and the action mechanism of the toad venom is probably related to the inhibition of the expression of asthma mice Eotaxin, I L-8 and I L-13, thereby prompting that the toad venom has the prospect of developing new anti-asthma medicaments.

Drawings

FIG. 1 shows the chemical structure of Arenobufagin.

FIG. 2 is HE staining showing changes in lung histology of asthmatic mice by arenobufagin.

FIG. 3 is the PAS staining to observe the pathological changes of the arenobufagin on the lung tissue of asthmatic mice.

FIG. 4 shows the effect of bufogenin on Eotaxin Eotaxin in asthmatic mouse BA L F, wherein C is a control group, M is a model group, D is a dexamethasone group (1mg/kg), S is a bufogenin drug group (2mg/kg), n =10, Note:^##p < 0.05vs control group; p < 0.05vs model group.

FIG. 5 shows the effect of bufogenin on inflammatory cytokine I L-13 produced by Th2 in asthmatic mouse BA L F, wherein C is control group, M is model group, D is dexamethasone group (1mg/kg), S is bufogenin drug group (2mg/kg), n =10, Note:^##p < 0.05vs control group; p < 0.05vs model group.

FIG. 6 shows the effect of bufogenin on neutrophil chemokine I L-8 in asthmatic mouse BA L F, C as control, M as model, D as dexamethasone (1mg/kg), S as bufogenin drug (2mg/kg), n =10, Note:^##p < 0.05vs control group; p < 0.05vs model group.

Detailed Description

The following examples are presented only to assist those skilled in the art in a more complete understanding of the present invention, and are not intended to limit the invention in any way.

The application of the bufogenin in the preparation of the medicine for preventing/treating asthma diseases is that the structural formula of the bufogenin is as follows:

。

The dosage is 1-3 times daily, 4-12mg each time.

The recommended dose is 3 times daily, 4mg each time.

Example 1 observation of the efficacy of bufogenin in OVA-induced mouse asthma model

1. Test method

1.1 grouping and modeling

40 healthy BA L B/c female mice are taken and randomly divided into 4 groups, and each group comprises 10 mice, namely a control group, a model group, a dexamethasone group and a toad venom drug group.

Dexamethasone and arenobufagin drug groups were injected with 1mg of aluminum hydroxide (emulsified with 20 μ g of OVA) intraperitoneally on day 1 of the experiment, treated with the same method on day 7 and day 15, treated with OVA (50mg/m L normal saline) for 30min on day 21-23, and treated with gastric lavage on day 1, and arenobufagin drug group (2mg/kg) after OVA sensitization, control group was injected with normal saline instead of OVA solution into mice intraperitoneally on days 1, 7, and day 15, and treated with normal saline for atomization on day 21, and the method and time were the same as those of the model group, which was only subjected to OVA sensitization.

1.2 Lung tissue specimen Collection

Mice were sacrificed on day 28 and the second lobe of the right lung of the mice was harvested on a clean bench. After flushing the blood on the lung tissue surface with pre-cooled PBS, the sections were fixed in 10% formalin for pathological sectioning.

1.3 HE staining for pathological changes in mouse lung tissue

(1) Taking a paraffin embedded tissue section with the thickness of 5 mu m, and dewaxing by a conventional method;

(2) staining sappan wood semen for 5 min;

(3) slightly washing the sappan wood semen for 1-3s with running water;

(4) 1% hydrochloric acid ethanol for 1-3 s;

(5) slightly washing with water for 10-30 s;

(6) slightly washing with distilled water for 1-2 s;

(7) dyeing with 0.5% eosin solution for 1-3 min;

(8) slightly washing with distilled water for 1-2 s;

(9) and (4) dehydrating conventionally, enabling dimethylbenzene to be transparent, and sealing by using a neutral resin.

Pathological changes in mouse lung tissue were observed under a microscope and × 200 photographs were taken.

1.4 PAS staining to observe mouse lung tissue bronchial goblet cell proliferation

(2) slightly washing with distilled water for 1-2 min;

(3) dyeing with periodic acid and ethanol for 10 min;

(4) washing with tap water for 10 min;

(5) schiff's solution for 10 min;

(6) flushing with running water for 5 min;

(7) staining nuclei with harzilein for 3 min;

(8) flushing with running water for 5 min;

Images were collected using an orthostatic fluorescence microscope in which positive goblet cells were stained purple red, pathological changes in mouse lung tissue were observed, and × 200 photographs were taken.

Test results

2.1 HE staining for pathological changes in mouse Lung tissue

The bronchial epithelium of the mice in the normal group is intact, inflammatory cells are not infiltrated in the alveoli and the periphery of the bronchi, and the bronchial wall is not proliferated and reconstructed (as shown in figure 2A).

There was a large amount of inflammatory cell infiltration around the alveoli and bronchi in the model group mice, the bronchial walls were hypertrophic, the lumen was narrowed, and the airway integrity was destroyed (see fig. 2B).

The symptoms are obviously improved after the intervention treatment of dexamethasone positive medicament (1mg/kg) and arenobufagin (2 mg/kg). The dexamethasone group and the bufogenin group can significantly improve airway inflammation and reduce infiltration of inflammatory cells (see fig. 2C and 2D).

2.2 PAS staining to observe the effects of arenobufagin on airway goblet cell proliferation

High secretion of airway mucus during the pathological process of asthma is associated with goblet cells. Goblet cells secrete mucin, and sugar chains in mucin can be stained purple red, so that cells represented by purple red are goblet cells.

The normal group of goblet cells was rare (as can be seen in FIG. 3A).

Goblet cell proliferation was evident in the model group mice (seen in fig. 3B).

After the intervention treatment of dexamethasone positive drug (1mg/kg) and arenobufagin (2mg/kg), the goblet cell proliferation is obviously inhibited (see the details in fig. 3C and 3D).

Example 2 mechanism of action of bufogenin in the amelioration of asthma in mice

1. Test method

1.1 grouping and modeling

Dexamethasone and arenobulin group mice were injected intraperitoneally with 1mg of aluminum hydroxide (emulsified with 20 μ g of OVA) on day 1 of the experiment, and treated with the same method on day 7 and day 15. After 21-23 days, mice were treated with OVA (50mg/ml saline) for 1 time daily for 30min in the airway for 7 consecutive days. Dexamethasone (1mg/kg) and arenobufagin drug (2mg/kg) were administered by gavage after OVA sensitization. The control group was injected into the abdominal cavity of mice with normal saline instead of OVA solution on days 1, 7 and 15, and the mice were administered with normal saline for atomization excitation on day 21, in the same manner and time as the model group. The model group was only treated with OVA sensitization.

1.2 Collection of bronchial cell lavage fluid (BA L F)

Mice were sacrificed on day 28, the thoracic cavity aseptically cut in a super clean bench, the right main bronchus was occluded by a blood vessel, and-4 ℃ PBS0.5m L was injected intratracheally into the left lung BA L F (total volume 1.5m L) was collected by three consecutive breaths, the supernatant was centrifuged with BA L F to obtain bronchoalveolar lavage fluid (BA L F), which was stored at-80 ℃.

1.3 cytokine detection

The expression of the inflammatory factors Eotaxin, I L-8 and I L-13 in bronchoalveolar lavage fluid (BA L F) is detected by adopting an E L ISA kit.

1.4 statistical methods

Data analysis was performed using the SPSS17.0 statistical software package, significant differences were analyzed using one-way anova and t-test, all experimental data are expressed as mean ± standard deviation (± s), test level α = 0.05.

Test results

2.1 Effect of arenobufagin on Eotaxin Eotaxin in BA L F

As can be seen from fig. 4, the Eotaxin concentration in model group BA L F was higher (n 10,^##P<0.05vs control group), Eotaxin was known to be highly expressed in asthmatic mice. Dexamethasone (1mg/kg) and bufotoxin (2mg/kg) were used to reduce Eotaxin levels (n 10, P)<0.05vs model set).

2.2 Effect of arenobufagin on Th2 cytokine I L-13 in BA L F

As can be seen from fig. 5, the concentration of the cytokine I L-13 secreted from Th2 was higher in the model group BA L F compared to the control group (n-10,^##P<0.05vs control group), it was known that I L-13 was highly expressed in asthmatic mice, dexamethasone (1mg/kg) and bufalin (2mg/kg) were able to cause a decrease in I L-13 levels (n-10, P)<0.05vs model set).

2.3 Effect of bufogenin on neutrophil chemokine I L-8 in BA L F

As can be seen from fig. 6, the concentration of neutrophil chemokine I L-8 in model group BA L F was increased compared to the control group (n-10,^##P<0.05vs control group), it was known that I L-8 was highly expressed in asthmatic mice, dexamethasone (1mg/kg) and bufalin (2mg/kg) were able to cause a decrease in I L-8 levels (n-10, P)<0.05vs model set).

In the mouse asthma model test, the dose of the bufogenin is 2 mg-kg^-1d^-1. Converted into human (calculated by adult weight 60 kg) dosage of 12 mg/d. In view of the differences in body weight and various other pathological conditions, the recommended dose is 1-3 times daily, 4-12mg each time. The most common dose is 3 times daily, 4mg each time.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims

1. The application of the bufogenin serving as the only active ingredient in preparing the medicine for treating the asthma disease is characterized in that the bufogenin inhibits the expression of inflammatory cytokines I L-13, Eotaxin and neutrophil chemotactic factors I L-8 in bronchoalveolar lavage fluid, and the structural formula of the bufogenin is as follows:

。

2. the use according to claim 1, wherein said bufogenin is in the form of an oral dosage form or an intravenous injection.

3. Use according to claim 2, characterized in that the oral dosage form is a tablet, hard capsule, soft capsule, powder, pill or granule.

4. The use according to claim 2, wherein the intravenous injection is a solution injection, a suspension injection, an emulsion injection or a sterile powder for injection.

5. The use according to claim 1, wherein the dose of said bufogenin is 1-3 times daily, 4-12mg each time.

6. The use according to claim 5, wherein the dose of said bufogenin is 3 times daily, 4mg each time.