CN108210492B

CN108210492B - Mundoserone inhibits zebrafish embryonic angiogenesis

Info

Publication number: CN108210492B
Application number: CN201611162550.9A
Authority: CN
Inventors: 王长谦; 陈侃; 范虞琪
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2016-12-15
Filing date: 2016-12-15
Publication date: 2019-12-31
Anticipated expiration: 2036-12-15
Also published as: CN108210492A

Abstract

The invention relates to Mundoserone for inhibiting angiogenesis of zebra fish embryos. The method comprises the steps of firstly putting the zebra fish into Mundoselone medicated baths with different concentrations, taking a medicament with a definite anti-angiogenesis effect as a positive control and a solvent as a negative control, finding that the Mundoselone has the effect of inhibiting the angiogenesis of the zebra fish embryo, and the inhibition rate is related to the concentration of the medicament. Therefore, Mundoserone can be used for preparing related medicaments for treating diseases which are characterized by angiogenesis as pathological features, such as tumors, diabetic retinopathy, hemangioma, psoriasis, rheumatoid arthritis, menstrual disorder and the like.

Description

Mundoserone inhibits zebrafish embryonic angiogenesis

Technical Field

The invention relates to the technical field of biomedicine, in particular to an effect of Mundoserone on inhibiting angiogenesis of zebra fish embryos and an application of Mundosone in preparing a medicine for treating diseases with angiogenesis as a pathological characteristic.

Background

Angiogenesis plays an important role in the progression of malignant tumors. The new blood vessels provide nutrients for the tumor cells, so that the tumor cells can continuously proliferate; conversely, if angiogenesis is inhibited and the tumor cells do not receive sufficient nutrients, proliferation and even necrosis may cease. Therefore, inhibition of angiogenesis is a method for treating malignant tumors, and drugs for inhibiting angiogenesis or potential anti-tumor drugs can be used.

Chinese patent document CN100577180C discloses an application of gambogic acid in preparing angiogenesis inhibiting medicines, and the invention provides an application of gambogic acid in preparing angiogenesis inhibiting medicines, in particular an application in preparing medicines for inhibiting angiogenesis of lesion tissues of diseases such as tumor, arthritis, retinopathy, hemangioma, psoriasis and the like; chinese patent document CN102048727A discloses an application of formononetin in preparation of angiogenesis inhibiting medicines, and the invention discovers for the first time that formononetin has an anti-angiogenesis effect and can be used as an angiogenesis inhibitor to be applied to preparation of angiogenesis medicines for treating angiogenesis-dependent and angiogenesis-related diseases such as tumors, arthritis, psoriasis, ophthalmic diseases, atherosclerosis and the like.

The zebra fish embryo angiogenesis condition can be clearly observed under a fluorescence microscope by enabling vascular endothelial cells of the zebra fish to express green fluorescent protein (fli1a-EGFP strain) by a transgenic method, wherein the zebra fish embryo is fertilized in vitro, and the embryo develops in vitro and is transparent, and various changes in vivo can be easily observed. 24-48 hours after fertilization is the main period of zebra fish embryo angiogenesis, and in the period, the embryo is treated by the medicament, so that whether the medicament can inhibit the zebra fish embryo angiogenesis or not and has the effect of resisting the angiogenesis can be visually observed.

The Mundoserone has a molecular weight of 342.352, and a molecular structural formula shown in figure 1. There is no report of a model animal-based zebrafish to confirm the relationship between Mundoserone and angiogenesis.

Disclosure of Invention

The invention aims to provide a new application of Mundoserone aiming at the defects in the prior art.

In a first aspect, the present invention provides the use of Mundoserone for the preparation of a medicament for the treatment of a disease characterised by angiogenesis as a pathological feature.

The disease with angiogenesis as pathological characteristic can be tumor, diabetic retinopathy, hemangioma, psoriasis, rheumatoid arthritis or menstrual disorder, etc.

In a second aspect, the present invention provides the use of Mundoserone for the preparation of a reagent for the study of the molecular mechanism of angiogenesis.

In a third aspect, the invention provides an application of Mundoserone in preparing a reagent for inhibiting angiogenesis of zebra fish embryos.

In a fourth aspect, the present invention provides the use of Mundoserone for the preparation of a reagent for:

a) generating zebra fish embryo ISV or DLAV;

b) inhibiting the expression of slit3, robo1, robo2, fgfr2, or fgfr 3; or

c) Increasing the expression of notch1 a.

The invention has the advantages that:

the method comprises the steps of firstly putting the zebra fish into Mundoselone medicated baths with different concentrations, taking PTK787 with a definite anti-angiogenesis effect as a positive control and taking a solvent DMSO as a negative control, finding that the Mundoselone has the effect of inhibiting the angiogenesis of the zebra fish embryo, wherein the inhibition rate is related to the drug concentration, and the low-concentration Mundoselone shows a remarkable inhibition effect. On the basis, the influence of Mundoserone on related signal paths in the process of angiogenesis of zebra fish embryos is researched through Real-time PCR, and the mechanism of inhibiting angiogenesis is determined.

Based on the research results of the invention, Mundoserone can be used for preparing related medicaments for treating diseases with angiogenesis as pathological characteristics. Herein, the term "angiogenesis" as a pathological feature means a biological process in which an excessive angiogenesis occurs during the development of the disease, or an angiogenesis must be generated during the development of the disease although the excessive angiogenesis does not exist. The above diseases can be tumor, diabetic retinopathy, hemangioma, psoriasis, rheumatoid arthritis, menstrual disorder, etc.

Drawings

FIG. 1 shows the molecular structure of Mundoserone.

Figure 2.Mundoserone inhibits zebrafish embryonic angiogenesis.

FIG. 3-FIG. 5.Real time PCR results.

FIG. 6. mechanism by which Mundoserone inhibits angiogenesis.

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings.

Example 1

Materials and methods

1. Zebra fish breeding and reproduction

Fla 1a-EGFP transgenic zebrafish, adult zebrafish, juvenile zebrafish and embryos expressing green fluorescent protein using vascular endothelial cells were all raised in culture water at 28 ℃ and pH7.0, and the room was routinely set to 14 hours light and 10 hours dark to simulate day and night cycles. When in propagation, 5-6 pairs of zebra fishes are mated each time, and each pair of zebra fishes can produce 200-300 eggs at one time. The zebra fish breeding equipment and the breeding method both meet the requirements of the evaluation and approval committee (AAALAC) of the international experimental animals.

2. Drug formulation and use

Mundoserone was purchased from Microsource, the positive control drug PTK787 was purchased from Selleckchem, and the solvent dimethyl sulfoxide (DMSO) was purchased from Sigma. During preparation, Mundoserone and PTK787 are dissolved in DMSO firstly, and then the DMSO in which the medicines are dissolved is added into culture water, so that the final DMSO concentration in the culture water of different medicines and different concentration groups is ensured to be 0.1%. The working concentration of Mundoserone was set at three concentrations of 2.5. mu.M, 5. mu.M, and 10. mu.M, with 5. mu.M PTK787 as a positive control and 0.1% DMSO as a negative control, and the prepared drugs were placed in 12-well plates (BD Falcon) for study, respectively.

Effect of Mundosemione on angiogenesis in zebrafish embryos

The 24-hour (24hpf) fertilized zebrafish embryos are put into a 12-hole plate, 30 embryos are placed in each hole, 10 embryos are randomly taken out from each hole after 24-hour bath in the different medicines, the embryos are anesthetized by 0.016% MS-222 and the result is observed under a fluorescence microscope, and the number of the complete ISV of each zebrafish is counted by taking the internodal blood vessels (ISV) as an observation index. The complete ISV is defined as: ISV connecting the dorsal longitudinal vessel (DLAV) and the Dorsal Aorta (DA). The inhibition rate of the medicine on the angiogenesis of the zebra fish embryo is calculated according to the following formula: inhibition (%) ═ 100% (1-number of intact ISVs in drug group/intact ISVs in negative control group).

4. Taking pictures of microscopes

Zebrafish were photographed using an SMZ1500 fluorescence microscope (Nikon). Zebrafish were anesthetized and laid on their side in methylcellulose (Sigma) and placed in the same position, i.e. head on the left side of the photograph, abdomen down. The microscope magnification was 40 times for the whole fish and 112.5 times for the trunk vessels.

5.Real-time PCR

The 24hpf zebrafish embryos were divided into three groups of 90 and each was bathed in 10. mu.M Mundoserone, 5. mu.M PTK787, 0.1% DMSO, 24 hours later, 30 random aliquots were removed from each group for Real-time PCR. Total RNA was extracted from embryos using trizol (roche). RNA reverse transcription was performed using PrimeScript RT kit (Takara). Gene expression was quantified using the Bio-rad iQ SYBR Green Supermix (Bio-rad) and Realplex system (Eppendorf). Relative quantitative analysis of gene expression is based on the comparative Ct method (2)^-ΔΔCt) And beta actin was used as an internal control. The primer sequences are shown in Table 1. The entire experiment was repeated three times.

TABLE 1 primer sequences

6. Statistical analysis

The measured data are expressed as mean + -standard deviation, the comparison between two sets of measured data is performed by t-test, and the comparison between three or more sets is performed by variance analysis. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad). Differences of P <0.05 were statistically significant. In the results, P <0.05, P <0.01, and P <0.001 were indicated.

Second, result in

Mundoserone inhibits zebrafish embryonic angiogenesis

The vascularity of the negative control group of zebrafish is shown in fig. 2A, F, K, with intact DA, ISV and DLAV. The positive control group (PTK787) zebrafish vascularity is shown in FIG. 2E, J, O, and the formation of ISV and DLAV was completely inhibited. Mundoserone inhibits zebrafish embryonic angiogenesis and is concentration dependent: the ISV of zebrafish incubated with 2.5 μ M Mundoserone remained essentially intact, but significantly attenuated compared to the negative control group (FIG. 2B, G, L); ISV of zebrafish bathed with 5 μ M Mundosenone was partially incomplete except for significant thinning (FIG. 2C, H, M); zebrafish ISV, which were medicated with 10 μ M Mundoserone, became attenuated and most of the ISV was incomplete, with DLAV production also inhibited (fig. 2D, I, N). A comparison of the number of intact ISVs of zebra fish in each treatment group is shown in FIG. 2P, and the inhibition rate of ISV in each group is shown in FIG. 2Q.

Mechanism of Mundoserone inhibition of zebrafish embryonic angiogenesis

Real time PCR results showed that Mundoserone increased notch1a expression (fig. 3), inhibited slit3, robo1, robo2 expression (fig. 4), inhibited fgfr2, fgfr3 expression (fig. 5), and inhibited angiogenesis by altering the expression of the above-mentioned key genes in the angiogenic signaling pathway.

Third, conclusion

Mundosone inhibits the angiogenesis of zebra fish embryos, and 10 mu M of Mundosone can obviously inhibit the generation of ISV and DLAV of the zebra fish embryos in relation to concentration.

The mechanism by which Mundoserone inhibits angiogenesis is that it alters the expression of key genes in the angiogenic signalling pathway: increasing the expression of notch1a, inhibiting the expression of slit3, robo1, robo2, fgfr2, fgfr3 (FIG. 6).

In conclusion, Mundoserone has a remarkable regulation effect on the expression of key genes in an angiogenesis signal pathway, can remarkably inhibit angiogenesis, and can be used for preparing related medicaments for treating diseases with angiogenesis as a pathological feature. Herein, the term "angiogenesis" as a pathological feature means a biological process in which an excessive angiogenesis occurs during the development of the disease, or an angiogenesis must be generated during the development of the disease although the excessive angiogenesis does not exist. The above diseases can be tumor, diabetic retinopathy, hemangioma, psoriasis, rheumatoid arthritis, menstrual disorder, etc.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

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<120> Mundoserone inhibits zebrafish embryonic angiogenesis

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Claims

The application of Mundoserone in preparing a reagent for inhibiting angiogenesis of zebra fish embryos.
Use of Mundoserone for the preparation of a reagent for:

a) generating zebra fish embryo ISV or DLAV;

b) inhibiting the expression of slit3, robo1, robo2, fgfr2, or fgfr 3; or

c) Increasing the expression of notch1 a.