CN114601825B

CN114601825B - Application of intestinal flora metabolite indole-3-formaldehyde in preventing and treating atherosclerosis

Info

Publication number: CN114601825B
Application number: CN202210149432.3A
Authority: CN
Inventors: 葛均波; 陆仪静; 杨文龙; 孙爱军
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2022-02-18
Filing date: 2022-02-18
Publication date: 2023-08-04
Anticipated expiration: 2042-02-18
Also published as: CN114601825A

Abstract

The invention discloses an application of intestinal flora metabolite indole-3-formaldehyde in preventing and treating atherosclerosis. According to the invention, an apoE-/-mouse is used for constructing an atherosclerosis model, and the effect of indole-3-formaldehyde on preventing and treating atherosclerosis is detected through indexes such as the size of aortic plaque, the condition of aortic root plaque and activation inhibition of related signal molecules, and the result shows that the indole-3-formaldehyde can obviously reduce the weight of a high-fat fed mouse, and meanwhile, the size of aortic plaque can be reduced. The indole-3-formaldehyde provided by the invention can protect intestinal mucosa barriers, has the effects of resisting oxidation and inflammation and protecting aortic endothelium, is a human endogenous metabolite, is safe for organisms, can be used together with probiotics prebiotics, reduces the contradiction emotion of patients to medicines, and has good clinical application prospect.

Description

Application of intestinal flora metabolite indole-3-formaldehyde in preventing and treating atherosclerosis

Technical Field

The invention relates to an application of intestinal flora metabolite indole-3-formaldehyde in preventing and treating atherosclerosis, belonging to the field of biological medicine.

Background

A large number of microorganisms are lodged in the human body. In the gut, bacteria, viruses, protozoa, archaebacteria and fungi constitute the major commensal microbiota. Although the bacterial species and number vary from person to person, individual differences are small at higher classification levels (e.g., phylum). The intestinal microbiota is mainly dominated by two bacteroides or firmicutes, which occupy more than 90% of the taxa in the human intestinal tract, followed by actinomycetes, cyanobacteria, fusobacterium, proteus, etc.

Humans have long evolved with these microorganisms in a reciprocal and reciprocal manner. Microorganisms benefit from a sustained supply of substrate by humans, while human hosts benefit from microbial activity. Such as the production of essential vitamins (e.g., vitamins B and K), the digestion of carbohydrates, the "line of defense" of the immune system, and the production of important signaling molecules including short chain fatty acids and secondary bile acids. The number of bacterial genomes in the gut is at least two orders of magnitude higher than human genes. The microbiota can affect host physiology, but external factors such as genetics, diet, pharmacological compounds, lifestyle and hygiene can also affect the microbial composition.

More and more studies have demonstrated that dysbacteriosis is associated with many diseases in humans. Any factor that interferes with the homeostasis balance of the host-gut flora may be the initiating or enhancing factor in the pathogenesis of the disease. The current surrounding flora metabolites are mainly focused on short chain fatty acid, bile acid, tryptophan metabolites. Tryptophan is an essential aromatic amino acid for the human body, and although the least abundant amino acid in proteins and cells, it is a biosynthetic precursor for a large number of microorganisms and host metabolites. Since animal cells cannot produce tryptophan, humans rely on exogenous tryptophan, in natural foods such as oats, bananas, dairy products, tunas, and the like. Tryptophan metabolism in the gut includes molecules such as indole and its derivatives that are directly converted by intestinal bacteria. Several studies have demonstrated that indole-3-carbaldehyde has a remarkable anti-inflammatory effect while having a protective effect on intestinal epithelial cells.

Previous studies have found that coliform bacteria greatly exceed the standard in patients with atherosclerosis compared to healthy individuals, where eubacterium and rogowski bacteria are dominant in numbers. It has also been found that a number of inflammation-related genes are very active in patients. In recent years, scientists have also shifted their eyes to metabolites of the intestinal flora, and several studies have demonstrated that trimethylamine oxide exacerbates the inflammatory levels in plaques by inhibiting lipid metabolism. The short chain fatty acid mainly from vegetables and fruits has a certain protection effect on cardiovascular diseases. However, little has been discussed about the role of tryptophan and its derivatives in the cardiovascular field

Tryptophan is one of the essential amino acids of human body and is a precondition of various metabolites, and the influence of the deficiency of tryptophan on cardiovascular diseases is worth discussing. Although statins have been seen with a good eye in their ability to treat atherosclerosis, with the increasing level of living, unhealthy eating habits destroy a large secretory organ, i.e., the intestinal flora, in humans. Once the flora homeostasis is disrupted, it is a challenge to the intestinal barrier function, and as harmful substances enter the circulatory system, they pose a considerable health hazard.

Disclosure of Invention

The purpose of the invention is that: a metabolite capable of protecting intestinal barrier function and thus slowing down atherosclerosis occurrence and development is provided, and the purpose of improving atherosclerosis is achieved by remodelling intestinal flora steady state.

In order to achieve the above purpose, the invention provides an application of intestinal flora metabolite indole-3-formaldehyde in preparing medicines for preventing and/or treating atherosclerosis.

The invention also provides application of the intestinal flora metabolite indole-3-formaldehyde combined probiotics and/or prebiotics in preparation of medicines for preventing and/or treating atherosclerosis.

The invention also provides a medicament for preventing and/or treating atherosclerosis, which is characterized by comprising a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient comprises indole-3-carbaldehyde.

Preferably, the active ingredient further comprises probiotics and/or prebiotics.

Compared with the prior art, the invention has the beneficial effects that:

1. according to the invention, an apoE-/-mouse is used for constructing an atherosclerosis model, and the effect of indole-3-formaldehyde on preventing and treating atherosclerosis is detected through indexes such as the size of aortic plaque, the condition of aortic root plaque and activation inhibition of related signal molecules, and the result shows that the indole-3-formaldehyde can obviously reduce the weight of a high-fat fed mouse, can reduce the size of aortic plaque, can inhibit endothelial cells from secreting inflammatory factors such as IL6 and CCL2, and can effectively reduce the oxidative stress level induced by oxidized low-density lipoprotein;

2. the indole-3-formaldehyde provided by the invention can protect intestinal mucosa barriers, has the effects of resisting oxidation and inflammation and protecting aortic endothelium, is a human endogenous metabolite, is safe for organisms, can be used together with probiotics prebiotics, reduces the contradiction emotion of patients to medicines, and has good clinical application prospect.

Drawings

FIG. 1 shows the results of weight measurement of mice in the 9 th to 20 th week control group (CON), high fat group (HFD) and experimental group (HFD+ICA);

FIG. 2 is the results of evaluation of atherosclerosis by oil red staining in each experimental mouse after 12 weeks of feeding;

FIG. 3 is a graph showing the evaluation of the protective effect of indole-3-carbaldehyde on atherosclerosis at the cellular level;

FIG. 4 shows that the expression levels of indole-3-carbaldehyde receptor AhR, and AhR receptor-associated signal pathways CYP1a1, arnt and Ahrr were significantly up-regulated after indole-3-carbaldehyde treatment;

FIG. 5 shows that the protection effect of indole-3-carbaldehyde on endothelial cells disappeared after interfering with the indole-3-carbaldehyde receptor AhR using siRNA;

FIG. 6 is a graph showing that AhR downstream gene Nrf2 is found to regulate a plurality of antioxidant genes by cut & tag technology;

FIG. 7 shows that interfering Nrf2, a dual luciferase reporter gene, and chip verifies that indole-3-carbaldehyde activates the receptor AhR, ahR modulates and activates the downstream gene Nrf2, promoting the expression of antioxidant genes;

FIG. 8 shows the interference with Nrf2 downstream gene HO-1, verifying that indole-3-carbaldehyde activation AhR-Nrf2-HO-1 pathway reduces endothelial oxidative stress levels, and inhibits inflammatory levels, protecting endothelial function and barrier;

in the above figures, ns represents that there is no significant difference between the two groups through statistical analysis; * Indicating that there was a significant difference between the two groups, P <0.05, after statistical analysis; * Representing that there was a significant difference between the two groups, P <0.01, after statistical analysis; * Representing that there was a significant difference between the two groups, P <0.001, after statistical analysis; * The expression was statistically analyzed to show significant differences between the two groups, P <0.0001.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.

Example 1

This example provides the use of the intestinal flora metabolite indole-3-carbaldehyde to slow atherosclerosis (experimental protocols approved by the committee for animal management and ethics at the university of double denier):

(1) Experimental animals and animal model preparation: SPF-class male C57BL/6 background apoE-/-mice (purchased from Nanjing Seiko Biochemical Co., ltd.) were selected, with a week-old of 8-weeks and a body weight of 20-25g. And feeding high-fat feed at 9 weeks, and constructing an atherosclerosis model after 12 weeks of high-fat feeding.

(2) Grouping and processing: mice were grouped into a control group (CON), a high fat group (HFD), an experimental group (hfd+ica), and a sham group (ICA). The animals of the experimental group and the sham group were fed with the high-fat diet for 12 weeks, and the mice of the experimental group and the sham group were fed with 150mg/kg of indole-3-carbaldehyde daily.

(3) Mice were evaluated for body weight: the body weights of the control group (CON), the high fat group (HFD) and the experimental group (hfd+ica) mice were measured at the 9 th to 20 th weeks (the weeks of age of the mice), respectively, and the results are shown in fig. 1, and comparing the body weights of the HFD group and the hfd+ica group in fig. 1, indicate that the indole-3-carbaldehyde can significantly reduce the body weight of the high fat fed mice.

(4) Assessment of the severity of atherosclerosis: after 12 weeks of feeding (weeks of feeding counted from the start of feeding with high-fat diet and/or indole-3-carbaldehyde), the mice were sacrificed under anesthesia. The mouse aorta was taken and the aortic gross plaque number and size was observed by oil red staining. The joint of the heart and the aortic root is taken, a frozen section is manufactured, the thickness and the area of the plaque are observed through oil red staining, the plaque stability such as necrotic nuclei and lipid vesicles are observed, the occurrence and development degree of the atherosclerosis is estimated, and the result is shown in a figure 2, and the number, the size, the thickness and the area of the plaque are effectively reduced after the atherosclerosis experimental mouse takes indole-3-formaldehyde, so that the indole-3-formaldehyde can effectively improve atherosclerosis.

(5) The protective effect of indole-3-carbaldehyde on atherosclerosis on the cellular level was evaluated: human umbilical vein cells were treated with oxidized low density lipoprotein in experiments and divided into four groups: the expression levels of the intercellular adhesion molecule ICAM, vascular cell adhesion molecule VCAM and endothelin ET-1 of endothelial cell function, the expression of secreted inflammatory factor levels IL6, CCL2, the expression of intracellular oxidative stress levels eNOS, NRF2, HO-1 and NQO1 were observed in the control group (CON), indole-3-carbaldehyde treated group (ICA), oxidized low density lipoprotein treated group (OXLDL) and experimental group (OXLDL+ICA, treated with oxidized low density lipoprotein and indole-3-carbaldehyde, respectively. It was observed in the experiment that indole-3-carbaldehyde inhibits the secretion of inflammatory factors such as IL6 and CCL2 by endothelial cells while effectively reducing oxidative stress levels induced by oxidized low density lipoproteins, and the results are shown in fig. 3.

Example 2

This example provides the protection of atherosclerosis against molecular mechanisms by indole-3-carbaldehyde:

(1) The expression levels of the indole-3-carbaldehyde receptor AhR and the AhR receptor-related signal pathways CYP1a1, arnt and Ahrr were significantly up-regulated after treatment with indole-3-carbaldehyde, as shown in FIG. 4, and the indole-3-carbaldehyde receptor AhR was interfered with by siRNA, and the effect of indole-3-carbaldehyde on endothelial cell was found to be lost, as shown in FIG. 5.

(2) Finding out AhR downstream gene Nrf2 through cut & tag technology, finding out that a plurality of antioxidant genes such as HO-1 can be regulated and controlled as shown in figure 6, and after indole-3-formaldehyde activates receptor AhR through interference of Nrf2 and dual-luciferase reporter gene and chip verification, ahR regulates and activates downstream gene Nrf2, and promoting expression of antioxidant genes, and the result is shown in figure 7.

(3) Interfering with Nrf2 downstream gene HO-1, verifying that indole-3-carbaldehyde activates AhR-Nrf2-HO-1 pathway to reduce oxidative stress level of endothelium, and inhibit inflammation level, and finally protecting endothelial function and endothelial barrier, and the result is shown in FIG. 8.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be limiting in any way and in nature, and it should be noted that several modifications and additions may be made to those skilled in the art without departing from the invention, which modifications and additions are also intended to be construed as within the scope of the invention.

Claims

1. The application of indole-3-formaldehyde as an active ingredient in preparing a medicament for preventing and/or treating atherosclerosis.