CN116159047A

CN116159047A - Application of fucoxanthin in preparation of medicines for treating arterial vascular endothelial injury diseases guided by cells Jiao Wangjie

Info

Publication number: CN116159047A
Application number: CN202211088867.8A
Authority: CN
Inventors: 徐林; 崔胜宇; 吴浩亮; 陶波; 韩丹翔
Original assignee: Demeter Biotechnology Zhuhai Co ltd
Current assignee: Demeter Biotechnology Zhuhai Co ltd
Priority date: 2022-06-30
Filing date: 2022-09-07
Publication date: 2023-05-26

Abstract

The invention relates to an application of fucoxanthin in preparing a medicament for treating arterial vascular endothelial injury diseases guided by cells Jiao Wangjie. Experimental research shows that the fucoxanthin can directly act on arterial endothelial cells, and can achieve the effects of reducing arterial vascular cell apoptosis and vascular endothelial injury by regulating and controlling the upstream signal pathway of cell apoptosis (including activating cell PI3K/AKT signal pathway and inhibiting the activation degree of cell TLR4/NF kappa B signal pathway), thereby improving vascular pathological remodeling, and therefore, the fucoxanthin can be used as an optional and effective treatment means for arterial vascular endothelial injury diseases mediated by cell apoptosis.

Description

Application of fucoxanthin in preparation of medicines for treating arterial vascular endothelial injury diseases guided by cells Jiao Wangjie

Technical Field

The invention relates to the technical field of natural medicines, in particular to application of fucoxanthin in preparing a medicine for treating arterial vascular endothelial injury diseases guided by cells Jiao Wangjie.

Background

Fucoxanthin is considered to be the most abundant natural carotenoid, about 10% of the total amount of carotenoids in nature, and it is mainly present in large algae (e.g., brown algae, etc.) and microalgae (e.g., diatom, golden algae, etc.), and plays a key role in algal photosynthesis. Fucoxanthin is a small molecular compound with molecular formula of C ₄₂ H ₅₈ O ₆ The molecular weight is 658.91. The ingested fucoxanthin is hydrolyzed into fucoxanthin under the action of digestive enzymes (such as lipase and cholesterol esterase) in gastrointestinal tract, and then enters the systemic circulation through lymph, and part of fucoxanthin is degraded into Amarouciaxanthin A in liver. Fucoxanthin and Amarouciaxanthin A are subsequently further metabolized by enzymatic reactions such as isomerization, dehydrogenation, deacetylation, oxidation and demethylation. Fucoxanthin has a unique chemical structure comprising allene bonds, 5, 6-monoepoxides, and acetyl groups. Previous studies have shown that fucoxanthin has various biological properties, such as excellent antioxidant effect, which can prevent the cytotoxicity of oxidants, and also has the function of resisting ultraviolet beta radiation and gamma radiation. In addition, according to a plurality of research results, the compound also has anti-inflammatory, anti-obesity, anticancer and other biological characteristics, and has potential protection effects on various diseases, such as: fucoxanthin exerts nuclear translocation induction effect on NRF2 through PI3K/AKT signals, so that macrophage inflammation induced by lipopolysaccharide is reduced; at the position ofIn obesity, fucoxanthin can induce the expression of uncoupling protein 1 in the mitochondria of the white adipose tissue of the abdomen, so that fatty acid oxidation and heat generation in white fat are caused, and further, the energy conversion of the fat is induced, so that the fat-reducing effect is realized.

Pyrodeath, also known as cell inflammatory necrosis, is a newly discovered apoptosis. The process first recognizes the intracellular and extracellular stimulus signals by intracellular pattern recognition receptors and constitutes an inflammatory small complex with procaspase zymogen (pro-caspase) by ASC (apoptosis-associated speck-like protein CARD). The complex promotes pro-caspase to be sheared into caspase with enzymatic activity, so that pro-IL-1 beta and pro-IL-18 are IL-1 beta and IL-18 on the one hand; on the other hand, the Gasderm protein family (GSDMs) is cut into an active configuration and polymerized and perforated on the cell membrane, so that proteins such as IL-1 beta, IL-18 and the like and small ions pass through, and the cell permeation barrier is broken, na ⁺ And water molecules enter the cell in large quantities, causing the cell to expand so that the cell membrane breaks down, and intracellular inflammatory factors and contents are rapidly released in large quantities, thereby causing cell death. NLRP3 (NOD-like receptor family, pyrin domain containing) is the most studied pattern recognition receptor at present, and the classical focal apoptotic inflammation complex consists of NLRP3, ASC and pro-caspase-1. The pathological process of focal death has been shown to play an important role in a number of diseases: current researches prove that in tumors such as lung cancer, gastric cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer and the like, the scorch can play a role in inhibiting abnormal proliferation of tumor cells and promoting death of the tumor cells; in autoimmune diseases, focal death is an important factor that contributes to disease onset and progression, such as systemic lupus erythematosus, inflammatory bowel disease, rheumatoid arthritis, and the like, has been shown to be closely related to cell focal death. In recent years, research shows that the scorch is also involved in the progress of various cardiovascular diseases, such as myocardial infarction, ischemia/reperfusion injury, myocardial hypertrophy and the like. Some molecular drugs capable of blocking the downstream of NLRP3 inflammatory body-mediated apoptosis show potential in treating related diseases, such as antibody drug Anakinra, canakinumab, rilonacept antagonizing IL-1β, which is currently used for treating inflammatory diseases, but may have allergic and off-target effects.

As a "first line of defense" of the blood vessel against blood, the role of vascular endothelial cells is critical. As a primary regulator of vascular homeostasis, endothelial cells exert a variety of vascular protective effects, while endothelial dysfunction is an early marker of arterial vascular injury, detectable prior to angiographic or ultrasound display of changes in vessel wall structure. Recent studies have shown that alleviating endothelial cell injury can ameliorate vascular diseases caused by a variety of causes, such as myocardial infarction, cerebral infarction, peripheral vascular disease, and the like. The death and destruction of endothelial cells have a significant promoting effect on arterial vascular damage. It is thought that endothelial cells are associated with elevated levels of apoptosis during the pathological process of injury. For example, kawasaki disease is the most common cause of heart disease in children in developed countries, and can lead to permanent coronary artery injury, coronary aneurysm and other long-term sequelae, while coronary artery endothelial cell apoptosis is proved to be widely existing in Kawasaki disease and plays an important role in promoting coronary artery injury caused by the Kawasaki disease. In addition, in oxidative stress induced endothelial cell dysfunction, the endothelial cell dysfunction is obviously improved after inhibiting key molecules of a focal death signal path by using small interfering RNA, which is also accompanied with the occurrence of focal death of endothelial cells. Therefore, the method can improve the scorching level of endothelial cells in the injury process, and can play a role in relieving arterial vascular injury, thereby improving disease prognosis.

However, there is currently no specific drug that protects vascular endothelial cells. Although some researchers have found that statins have some endothelial cell protection, their side effects limit use in some cases. If statin can change mevalonate pathway in muscle cells to induce cell death, it can affect the production of coenzyme Q10 to cause rhabdomyolysis, and can cause adverse reactions such as abnormal increase of liver enzymes. While fucoxanthin is taken as a natural component, has few side effects, and some researches at present disclose that the fucoxanthin has certain effects of resisting oxidation, resisting radiation, resisting inflammation, resisting obesity, resisting cancer, inducing fat energy conversion and the like, and has obvious therapeutic effects in various pathological injury models, but the biological activity of Guan Yanzao flavins for inhibiting cell apoptosis is not yet reported.

Disclosure of Invention

First, the technical problem to be solved

In view of the above-mentioned shortcomings and drawbacks of the prior art, the present invention proposes the use of fucoxanthin in the preparation of a medicament for treating arterial vascular endothelial injury diseases mediated by cells Jiao Wangjie. Experiments prove that the fucoxanthin has the activity of regulating and controlling the upstream signal channel of the scorching to reduce the level of the scorching of cells, and can be used for improving arterial vascular endothelial injury and related diseases of cells Jiao Wangjie; compared with the existing medicine molecules, the fucoxanthin not only can play a role in treating arterial vascular endothelial injury diseases mediated by pyrosis, but also can avoid anaphylaxis and off-target effects of antibody medicines or side effects of statin medicines and the like.

(II) technical scheme

In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:

use of fucoxanthin in the preparation of a medicament for treating arterial endothelial injury diseases mediated by cells Jiao Wangjie.

Preferably, the arterial vascular endothelial injury disease mediated by cells Jiao Wangjie includes, but is not limited to: myocardial infarction, cerebral infarction, myocardial ischemia/reperfusion injury, peripheral arterial vascular disease, etc.

Preferably, the use comprises use in combination with or as a supplement to a statin to reduce the amount of statin.

(III) beneficial effects

According to the research results of the invention, the fucoxanthin can directly act on arterial endothelial cells, and the effects of relieving arterial vascular apoptosis and vascular endothelial injury are achieved mainly by regulating and controlling the upstream signal pathway of cell apoptosis (including activating cell PI3K/AKT signal pathway and inhibiting the activation degree of cell TLR4/NF kappa B signal pathway), so that the fucoxanthin can improve vascular pathological remodeling, and can be used as an alternative and effective treatment means for arterial vascular endothelial injury diseases guided by endothelial cells Jiao Wangjie. The fucoxanthin can replace or partially replace statin drugs, and brings good news to people who are intolerant to statin drugs or need to resist endothelial injury. Fucoxanthin is a natural compound, has no anaphylaxis and off-target effect of antibody medicines, and can play a therapeutic potential in diseases related to pyrosis.

Drawings

FIG. 1 shows the experimental results of activation of the PI3K/AKT signaling pathway in vascular endothelial cells in mice upon intervention with fucoxanthin: wherein, the A graph is a Western Blot protein expression graph; panel B shows statistics of P-P13K/P13K protein expression for different treatment groups in panel A, and panel C shows statistics of P-AKT/AKT protein expression for different treatment groups in panel A (P < 0.05).

FIG. 2 shows the expression levels of NLRP3, caspase1, IL-1β, IL-18 that inhibit the levels of apoptosis reflected by vascular endothelial cells in mice upon intervention with fucoxanthin; panel A is a Western Blot protein expression diagram; the B-F diagram is a statistical diagram of the expression of p-AKT/AKT, NLRP3, caspase1, IL-1β and IL-18 proteins (p < 0.05) of different treatment groups in the A diagram in sequence; wherein the expression levels of NLRP3, caspase1, IL-1 beta, IL-18 are used to characterize the level of arterial endothelial cell apoptosis.

FIG. 3 shows the results of experiments that reduced activation of TLR 4/NFkB signaling in vascular endothelial cells in mice using fucoxanthin intervention: wherein, the A graph is a Western Blot protein expression graph; the expression statistical graphs of TLR4 protein, p-NFkB/NFkB protein, NLRP3 protein, caspase1 protein, 1L-1 beta protein and IL-18 protein in different treatment groups in the A graph are shown in sequence (p is less than 0.05); wherein the expression levels of p-nfκb/nfκ B, NLRP3, caspase1, IL-1β, IL-18 are used to characterize the level of apoptosis of arterial vascular endothelial cells.

FIG. 4 is a flow cytometry (Annexin V-APC/7AAD staining) for detecting endothelial cell apoptosis in each group; panel A shows the results of fluorescent staining assays in CTL, OA, OA+FUCO, OA+DMSO groups, and Panel B shows the statistics of Panel A (p < 0.05).

FIG. 5 shows the experimental results of the destruction of the fucoxanthin anti-pyrosis effect due to the overexpression of TLR4 gene after the TLR4 gene overexpression plasmid is transferred into the arterial vascular endothelium of mice: wherein, the A graph is a Western Blot protein expression graph; panel B-F shows statistics of expression of p-NFkB/NFk B, NLRP3, caspase1, 1L-1β, and IL-18 after TLR4 gene is over-expressed in cells (p < 0.05)

FIG. 6 is an experimental result of fucoxanthin being able to delay lipid-induced arterial vascular injury; figures a-C show results of red staining of the aortic row of mice and HE staining of the horizontal row of heart aortic valves at 1, 2, 3 months of molding, respectively.

Detailed Description

The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.

1. Preparation work and experimental method

1. Experimental animal and cell grouping treatment conditions:

the fucoxanthin dry powder is dissolved in DMSO and diluted to a safe concentration with physiological saline or PBS buffer. Fucoxanthin (HPLC > 90% purity) was extracted from large scale cultured marine diatom phaeodactylum tricornutum, offered by the company de merter biotechnology (zhuhai) limited.

In vivo experiments of animals were carried out by selecting 18C 57BL/6 normal mice, 54 ApoE-/-gene mice (purchased from Venlhua laboratory animal technologies Co., ltd. (Beijing)), and dividing the mice into 4 groups of 18 animals:

group a (normal group): c57BL/6 normal mice+normal diet;

group B (high fat group): apoE-/-mice+high fat diet;

group C (high fat + fucose Huang Suzu): apoE-/-mice + high fat diet + fucoxanthin (0.0018 mg/day) intragastric group;

group D (high fat + DMSO group): apoE-/-mice+high fat diet+DMSO gavage control group.

The cell ex vivo experiments used aortic endothelial cells, grouped as follows:

control (CTL) group: normally culturing the cells without any treatment;

oleic Acid (OA) stimulated group: stimulation with 200. Mu. Mol/L oleic acid was given for 48 hours;

oleic acid + fucoxanthin (OA + FUCO) treated group: stimulation with 200. Mu. Mol/L oleic acid was given for 48 hours while intervention with 0.25. Mu. Mol/L fucoxanthin was given;

oleic acid+dmso (oa+dmso) control: the oleic acid 200. Mu. Mol/L stimulation was given for 48 hours while DMSO was given in an amount equivalent to that of the OA+FUCO group in which FUCO was dissolved;

oa+fuco+ly294002 (PI 3K/AKT signaling pathway inhibitor) treated group: adding PI3K/AKT signal path inhibitor based on OA stimulation and FUCO intervention;

oa+fuco+tlr4 over-expression Plasmid (Plasmid-TLR 4) panel: the TLR4 over-expression plasmid is additionally given on the basis of OA stimulation and FUCO intervention;

oa+fuco+negative Control Plasmid (Plasmid-Control) group: a negative control plasmid was additionally administered on the basis of OA stimulation and FUCO intervention.

2. Animal model construction

All C57BL/6, apoE-/-mice were fed with normal feed for one week after entering a protective environment without specific pathogens. Group A is fed by general feed in the whole course; after the group B normal feed feeding is finished, high-fat feed feeding is carried out (until the experiment is finished); after the group C adaptive feeding is finished, high-fat feed feeding is carried out (until the experiment is finished), and the gastric lavage treatment of 0.0018mg/1 day of fucoxanthin (dissolved in physiological saline dissolved in DMSO) is carried out (until the experiment is finished); after the group D adaptive feeding is finished, high-fat feed feeding is carried out (until the experiment is finished), and the group C is subjected to gastric lavage treatment (until the experiment is finished) with DMSO (within a safe dosage range) at the same dosage, and the administration period and other conditions are completely consistent with those of the group C.

3. Experimental material selection and detection:

a. every 4 weeks, 6 mice were randomly harvested and euthanized;

b. taking out the rat aorta, preparing pathological sections, and detecting the influence of fucoxanthin on the occurrence and progress of arterial vascular injury caused by lipid.

2. Experimental results

1. Fucoxanthin inhibits endothelial cell apoptosis by activating PI3K/AKT signaling pathway

In the experiment, the protein detection of PI3K/AKT signal path is carried out on the endothelial cells stimulated by oleic acid by a western blot method, and the experimental result is shown in figure 1: it can be seen from panel B of FIG. 1 that the phosphorylated PI3K (p-PI 3K) expression level of the OA+FUCO group is close to that of the control group CTL, and that the phosphorylated AKT (p-AKT) expression level of the OA+FUCO group is only slightly lower than that of the control group CTL, both higher than that of the OA group and the OA+DMSO group, as can be seen from panel C of FIG. 1.

Thus, it was demonstrated that p-PI3K and p-AKT expression were significantly down-regulated after oleic acid stimulation in endothelial cells (fig. 1), demonstrating that PI3K/AKT signaling pathway was inhibited after oleic acid stimulation, whereas this inhibition of PI3K/AKT by oleic acid was alleviated in endothelial cells with concomitant administration of fucoxanthin.

To further verify that the PI3K/AKT signaling pathway is one of the possible pathways for fucoxanthin to exert protective effects. The experiment uses the inhibitor LY294002 of the PI3K/AKT signal path as a control simultaneously with the interference of fucoxanthin, and the related protein expression condition reflecting the apoptosis is detected, and the experimental result is shown in figure 2. As can be seen from FIG. 2B, the p-AKT expression level was slightly lower in the OA+FUCO group than in the control group CTL, and higher in the OA group, the OA+DMSO group and the OA+FUCO+LY294002 group. Thus, it was demonstrated that both FUCO (fucoxanthin) and the inhibitor LY294002 act on the PI3K/AKT signaling pathway. The C-F statistical response of FIG. 2 shows that NLRP3, caspase1, IL-1. Beta. And IL-18 expression levels reflecting the level of apoptosis were inhibited when fucoxanthin was used for intervention, whereas the effect of fucoxanthin was destroyed after simultaneous administration of LY294002 and the expression levels of NLRP3, caspase1, IL-1. Beta. And IL-18 reflecting the level of apoptosis were again increased.

The results show that the fucoxanthin can inhibit the scorch of endothelial cells and promote the survival of the endothelial cells by activating PI3K/AKT signal channels, thereby playing the role of resisting arterial vascular injury caused by lipid.

2. Fucoxanthin improves the level of oleic acid activated TLR 4/NFkB and pyrosis in endothelial cells

In the experiment, the expression level and the scorching degree of the arterial endothelial cells TLR 4/NFkB between the treatment groups are compared by a western blot method, and the experimental result is shown in figure 3. FIG. 3A is a western Blot protein expression pattern, as can be seen from the B-G pattern of FIG. 3: compared with the CTL group, the TLR 4/NFkB signal path and the apoptosis related indexes NLRP3, caspase1, IL-1 beta and IL-18 in the OA group and the OA+DMSO group have higher expression level, and the TLR 4/NFkB signal path and the apoptosis related indexes NLRP3, caspase1, IL-1 beta and IL-18 in the OA+FUCO group have obviously lower expression level. Thus, it is demonstrated that FUCO (fucoxanthin) intervention can significantly reduce activation of TLR 4/nfkb signaling pathway in arterial vascular endothelium of mice and reduce expression level of pyro-related proteins.

Meanwhile, the degree of the scorching of the arterial endothelial cells is also detected by flow cytometry. As a result, as shown in fig. 4, the degree of scorch was significantly increased in the oleic acid-treated group (OA group and oa+dmso group), while the rate of scorch of arterial endothelial cells was significantly decreased in the mice given the fucoxanthin-treated group (oa+fuco group). These results demonstrate that: fucoxanthin can obviously lighten the scorching level of arterial endothelial cells by inhibiting TLR4/NF kappa B signal channels, thereby lightening the damage degree of arterial vascular endothelium.

Further, in the experiment, a TLR4 over-expression Plasmid (Plasmid-TLR 4) and a corresponding negative Control Plasmid (Plasmid-Control) were constructed and transferred into endothelial cells, and the detection results are shown in FIG. 5. After TLR4 is over-expressed, the expression level of p-NF kappa B/NF kappa B signaling pathway, NLRP3, caspase1, 1L-1 beta and IL-18 protein in the OA+FUCO+plasmid-TLR4 group is obviously higher than that of the OA+FUCO+plasmid-Control group. This suggests that when TLR4 gene is overexpressed in vascular endothelial cells, fucoxanthin is inhibited against endothelial cell apoptosis, which is manifested as a re-elevation of the apoptosis-related protein. This suggests that fucoxanthin achieves the effect of reducing vascular endothelial cell apoptosis by inhibiting the TLR 4/NFkB signaling pathway.

3. Fucoxanthin has activity of relieving arterial vascular endothelial injury induced by high fat diet

In the experiment, the change of the serum level of the mice is monitored, and the aortic materials of each treatment group are subjected to oil red staining, in particular to section HE staining of the aortic valve level. The experimental results are shown in fig. 6: at the first month (panel a of fig. 6), there was no significant difference in oil red staining positive rate between the fucoxanthin treated group and its negative DMSO control group due to the low degree of aortic plaque formation, but plaque formation was more pronounced at aortic valve level, and the visible plaque area was significantly reduced in the fucoxanthin treated group. In the second and third months (B-C diagram of fig. 6), the effect of fucoxanthin on delaying arterial injury was more pronounced, and oil red staining showed a significant decrease in the positive rate for group C (high fat + fucose Huang Suzu) compared to both group B (high fat) and group D (high fat + DMSO), and also a significant improvement in the level of aortic valve plaque formation in the fucoxanthin treated group. From this it follows that: fucoxanthin can reduce damage to arterial blood vessels caused by lipids and the resulting diseases.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. Use of fucoxanthin in the preparation of a medicament for treating arterial endothelial injury diseases mediated by cells Jiao Wangjie.

2. The use according to claim 1, wherein the arterial vascular endothelial injury disease mediated by cells Jiao Wangjie includes, but is not limited to: myocardial infarction, cerebral infarction, myocardial ischemia/reperfusion injury, peripheral arterial vascular disease.

3. The use according to claim 1, wherein the use comprises use in combination with or as a supplement to a statin.