Use of yap agonists for the preparation of a medicament for the treatment of intestinal lymphatic vessel dysfunction

Abstract

The present application is suitable for the field of biological medicine technology, and provides application of a YAP agonist in preparation of a drug for treating intestinal lymphatic vessel dysfunction. The present application analyzes a molecular mechanism of the YAP agonist in improving high-fat diet induced lymphatic vessel dysfunction through targeted regulation of a VEGFR3 / YAP / mTOR signal pathway and a therapeutic application thereof, the YAP agonist antagonizes excessive phosphorylation of YAP, restores the nuclear translocation ability of YAP, activates the mTOR signal pathway, significantly improves the proliferation, migration and tube formation functions of LECs, and up-regulates VE-cadherin expression to repair endothelial barrier integrity; the YAP agonist can effectively relieve intestinal lymphatic vessel dysfunction and structural abnormalities induced by HFD.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a YAP agonist in preparation of a drug for treating intestinal lymphatic vessel dysfunction. BACKGROUND

[0002] The wide prevalence of high-fat diet is closely related to various metabolic diseases. As a key channel for dietary fat absorption and transport, the intestinal lymphatic vessel is the first to bear metabolic pressure under the condition of high-fat diet. Long-term high-fat intake can cause increased lipid uptake of intestinal epithelial cells and excessive synthesis of chylomicrons, so that the lymphatic vessel is in an overloading state for a long time. This sustained load can cause structural changes such as abnormal connection of lymphatic endothelial cells, lumen expansion and basement membrane thickening, and at the same time, damage the rhythmic contraction function of the lymphatic vessel, leading to lymphatic backflow disorder. More seriously, the high-fat environment can damage the lymphatic endothelial barrier function and increase the permeability, so that a large number of chylomicrons leak into the surrounding tissues, forming a vicious cycle. These pathological changes not only cause abnormal deposition of lipids in the intestinal tract, but also cause elevated circulating lipid levels by affecting systemic lipid transport, which is closely related to the occurrence and development of metabolic diseases such as obesity and atherosclerosis. It is worth noting that the dysfunction of the intestinal lymphatic vessel often occurs before obvious metabolic abnormalities, suggesting that it may be an early event of metabolic disorder induced by high-fat diet. Therefore, in-depth exploration of the damage mechanism of high-fat diet to the intestinal lymphatic vessel not only provides a new perspective for understanding the pathogenesis of metabolic diseases, but also lays a theoretical foundation for developing intervention strategies targeting the lymphatic vessel, which has important significance for the prevention and treatment of high-fat diet related diseases.

[0003] At present, there are many significant limitations in the treatment of intestinal lymphatic vessel dysfunction caused by high-fat diet: first, traditional therapy mainly focuses on regulating lipid metabolism through lipid-lowering drugs or inhibiting inflammatory response through anti-inflammatory drugs. This symptomatic intervention strategy can alleviate symptoms to a certain extent, but it fails to touch the deep pathological changes caused by high-fat diet to the intestinal lymphatic system, especially the destruction of the growth state and functional integrity of lymphatic endothelial cells; second, the existing drug targets generally lack tissue specificity, which often causes systemic adverse reactions including liver function damage, gastrointestinal discomfort and the like while exerting therapeutic effect, seriously affecting the medication compliance and long-term treatment effect of patients; more importantly, there is currently a lack of targeted treatment means that can specifically protect the function of lymphatic endothelium, which is an important bottleneck restricting the improvement of treatment effect. SUMMARY

[0004] The purpose of the embodiments of the present application is to provide the application of the YAP agonist in the preparation of the drug for treating the intestinal lymphatic vessel dysfunction, and aims to solve the problems raised in the background.

[0005] The application embodiment is implemented in this way, the YAP agonist is applied to the preparation of a drug for treating intestinal lymphatic vessel dysfunction.

[0006] Preferably, the YAP agonist is XMU-MP-1.

[0007] Preferably, the intestinal lymphatic vessel dysfunction is high-fat diet-induced intestinal lymphatic vessel dysfunction.

[0008] Preferably, the YAP agonist improves high-fat diet-induced lymphatic vessel dysfunction by targeting regulation of the VEGFR3 / YAP / mTOR signal pathway.

[0009] Another purpose of the application embodiment is to provide a drug for treating intestinal lymphatic vessel dysfunction, which comprises a YAP agonist.

[0010] Preferably, the YAP agonist is XMU-MP-1.

[0011] Preferably, the intestinal lymphatic vessel dysfunction is high-fat diet-induced intestinal lymphatic vessel dysfunction.

[0012] The application embodiment analyzes and studies that high-fat diet hinders YAP nuclear translocation and formation of YAP-TEAD complex by inhibiting VEGFR3 expression and promoting YAP phosphorylation, and then down-regulates expression of endothelial junction proteins such as VE-cadherin, resulting in lymphatic endothelial cell (LEC) dysfunction, while the YAP agonist XMU-MP-1 significantly improves the proliferation, migration and tube formation functions of LECs by antagonizing excessive phosphorylation of YAP, restoring its nuclear translocation ability, and activating the mTOR signal pathway, while up-regulating the expression of VE-cadherin to repair the integrity of the endothelial barrier; it is proved through animal experiments that XMU-MP-1 can effectively relieve intestinal lymphatic vessel dysfunction and structural abnormalities induced by HFD. Therefore, the application embodiment proves the application of XMU-MP-1 in the preparation of high-fat-induced lymphatic vessel dysfunction drugs, and its specific regulation effect on the YAP / VEGFR3 / mTOR signal axis. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 A schematic diagram for analyzing the influence of palmitic acid on the function of lymphatic endothelial cells provided for the application embodiment 1;

[0014] Figures 2 to 5 Results of analyzing the influence of palmitic acid on the function of lymphatic endothelial cells provided for the application embodiment 1, wherein: Figure 2 A is the palmitic acid concentration suitable for treating lymphatic endothelial cells measured by CCK8 experiment; Figure 2 B-E are the results of tube formation experiment of palmitic acid-treated lymphatic endothelial cells; Figure 3A-D are the results of cell proliferation measured by EdU assay and the fluorescence intensity of VE-cadherin between cells; Figure 3 E-F are the results of Western blotting to detect the protein expression changes of Bax and Bcl2 in palmitic acid-treated lymphatic endothelial cells; Figure 4 A-C are the protein levels of VEGFR3 detected by western blot after palmitic acid treatment of lymphatic endothelial cells; Figure 4 D-F are the nuclear translocation of YAP detected by western blot after palmitic acid treatment of lymphatic endothelial cells; Figure 5 A is the result of immunofluorescence staining to observe the subcellular localization of YAP, wherein the YAP protein presents red fluorescent signal, and the nucleus is stained with DAPI showing blue fluorescence; Figure 5 B-C are the expression levels of VEGFR3 / YAP / mTOR signaling pathway in the presence or absence of palmitic acid and VEGFC detected by western blot;

[0015] Figure 6 The schematic diagram for analyzing the YAP agonist XMU-MP-1 regulating high-fat-induced lymphatic endothelial cell dysfunction provided in Example 2 of the present application;

[0016] Figures 7 to 10 The results of YAP agonist XMU-MP-1 regulating high-fat-induced lymphatic endothelial cell dysfunction provided in Example 2 of the present application, wherein: Figure 7 A is the effect of different concentrations of XMU-MP-1 on the activity of lymphatic endothelial cells; Figure 7 B-E are the results of XMU-MP-1 regulating the tube formation ability of PA-treated lymphatic endothelial cells; Figure 8 A-B are the results of detecting the proliferation rate of lymphatic endothelial cells by EdU staining method; Figure 8 C-D are the fluorescence intensity determination of VE-cadherin; Figure 8 E is the transcellular endothelial permeation experiment; Figure 9 A-D are the results of Western blotting to detect the protein expression levels of VEGFR3, p-YAP, YAP, p-mTOR, mTOR, Bax, Bcl2, Cleaved-Caspase3 and Caspase3; Figure 10 A-D are the results of Western blotting to further verify the relationship between VEGFR3 and YAP;

[0017] Figure 11 The schematic diagram for targeted intervention research on intestinal chyliferous duct dysfunction based on YAP provided in Example 3 of the present application;

[0018] Figures 12 to 14 Results of the study on the targeted intervention of intestinal chylomicron duct dysfunction based on YAP provided in Example 3 of the present application, wherein: Figure 12 Figure 1A is a schematic diagram of the construction of an animal model of high-fat diet-induced intestinal lymphatic duct dysfunction and treatment intervention by intraperitoneal injection of XMU-MP-1 at the 13th week of high-fat feeding. Figure 12 Figure 1B is the result of regular (every two weeks) monitoring of changes in body weight of rats. Figure 12 Figure 1C is the fat accumulation in the mesentery, liver and epididymis of rats. Figure 12 Figures 1D-G are the results of serum low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol (TC) and triglyceride (TG) levels of rats, respectively. Figure 13 Figures 1A-D are the results of evaluation of pathological changes in intestinal chylomicron duct by hematoxylin-eosin (H&E) staining, quantitative analysis of aortic lipid deposition by oil red O staining, and measurement and statistics of intestinal villus morphological parameters (length and width). Figure 13 Figures 1E-F are the results of immunohistochemical detection of YAP expression in intestinal chylomicron duct. Figure 14 Figures 1A-D are the results of Western blotting detection of the expression levels of VEGFR3, YAP, p-YAP, mTOR, p-mTOR, LYVE1, Bax, Bcl2, Cleaved-Caspase3 and Caspase3 proteins in intestinal villus lymphatic duct.

[0019] In the drawings, p < 0.05, p < 0.01, p < 0.001 vs control. DETAILED DESCRIPTION

[0020] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in conjunction with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0021] The specific implementation of the present application will be described in detail below in conjunction with specific examples.

[0022] Example 1, as Figure 1 shown, analyzes the effect of palmitic acid (PA, Sigma, P5585) on the function of lymphatic endothelial cells (LECs), and the results are shown in Figures 2-5

[0023] ​Firstly, the activity of lymphatic endothelial cells was measured by CCK8 experiment with different concentrations of palmitic acid, and the activity of lymphatic endothelial cells was measured, as shown in Figure 2 Fig. 1A; then the angiogenesis ability was detected by tube formation experiment, 200 μl Matrigel matrix gel (Corning) was formed into gel in a 24-well plate, and LECs were inoculated at 5×10 4 cells per well, and the formation of tubular structure was observed after 2 hours of treatment, and the results are shown in Figure 2 Fig. 1B-E.

[0024] To evaluate cell proliferation, 3×10 4 cells per well of LECs were treated for 24 hours, then EdU (Beyotime, C0075S) reagent was added and cultured for another two hours, then fixed with paraformaldehyde, and the positive rate was counted under fluorescence microscope, and the results are shown in Figure 3 Fig. 2A, C, and the fluorescence intensity was analyzed by immunofluorescence experiment on VE-cadherin, and the results are shown in Figure 3 Fig. 2B, D; in terms of molecular mechanism, the expression of key proteins was analyzed by Western Blot, and 1.5×10 6 cells per well of cells were inoculated in a 6-well cell culture plate, and the above grouping was added with drugs, and after 24 hours of culture, the culture medium was gently aspirated, and the cells were scraped with pre-cooled cell scraper and centrifuged at 16 000 r / min for 10 min in a pre-cooled centrifuge at 4℃, and the supernatant was transferred to a new centrifuge tube, and the protein content was detected by BCA method, and 20 μg of protein was electrophoretically transferred to a membrane, and the expression changes were detected by anti-apoptotic protein Bcl-2 (Santa Cruz Biotechnology, sc-7382) and pro-apoptotic protein Bax (Santa Cruz Biotechnology, sc-70407) β-actin (Bioworld, BS42679) antibody 4℃ incubation overnight, and the results are shown in Figure 3 Fig. 3E-F;

[0025] In addition, the expression level of mRNA of lymphatic endothelial cells was detected by RT-qPCR, and the results showed that the mRNA level of VEGFR3 of lymphatic endothelial cells treated with palmitic acid was significantly reduced, and the Western blot results showed that the protein level of VEGFR3 (Santa Cruz Biotechnology, sc-365748) was also significantly reduced, as shown in Figure 4 Fig. 4A-C; the results of Western Blot are shown in Figure 4As shown in Figs. 1D-1F, it is shown that palmitic acid treatment increases the accumulation of YAP in the cytoplasm.

[0026] Different treatments were performed on the palmitic acid treated lymphatic endothelial cells in the presence or absence of VEGFC, and the cell localization of YAP was observed by immunofluorescence staining, using specific antibodies to label YAP protein (red fluorescence) and DAPI to stain the nucleus (blue fluorescence), and the fluorescence microscope results are shown in Figs. 2A-2C. Figure 5 As shown in Fig. 2A, the analysis shows that the red fluorescence signal of YAP in the nucleus is significantly enhanced in the VEGFC treatment group based on the palmitic acid treatment, indicating that VEGFC can promote the nuclear translocation of YAP; the Western Blot results are shown in Fig. 2B-2C. Figure 5 As shown in Figs. 2B-2C, it is shown that palmitic acid treatment increases the accumulation of p-YAP (Bioss, bsm-52214R) in the cytoplasm, reduces the expression level of p-mTOR and CTGF, and the use of VEGFC significantly increases the level of YAP (Bioss, bs-3605R) in the nucleus and up-regulates the expression of p-mTOR and CTGF proteins;

[0027] In summary, the present application illustrates the effects of PA on the migration, tube formation and proliferation of LECs and the possible signal pathway mechanism through functional experiments combined with molecular detection. It can be seen that PA significantly inhibits the proliferation, migration and tube formation ability of LECs.

[0028] As shown in Fig. 2D, the YAP specific agonist XMU-MP-1 (MCE, HY-100526) was used to regulate the function of palmitic acid treated lymphatic endothelial cells, and the results are shown in Figs. 3A-3E. Figure 6 Figures 7-10

[0029] CCK8 experiment was used to evaluate the cytotoxicity of XMU-MP-1, and the lymphatic endothelial cells were seeded in a 96-well plate at a density of 5x10 3 / well, and control group (Ctrl), solvent control group (DMSO) and 0.5-7 μM gradient concentration of XMU-MP-1 treatment group were set. After 24 hours of intervention, 10 ul of CCK8 reagent was added to each well for incubation for 90 min, and the absorbance was detected at 450 nm wavelength using a microplate reader. It was found that there was no significant difference in cell activity between each concentration group, and the results are shown in Fig. 4A. Figure 7 As shown in Fig. 4A, it can be seen that the effect is best at a concentration of 5 μM, so the concentration of 5 μM is selected for subsequent experiments; the above grouping was used for functional experiments, and the tube formation experiment results are shown in Figs. 5A-5E. Figure 7 As shown in Figs. 5B-5E, it is shown that XMU-MP-1 can significantly improve the cell tube formation ability inhibited by palmitic acid.

[0030] The fluorescence microscope results are shown in Figs. 6A-6C. Figure 8 ​​As shown in FIGS. 11A-11D, observation confirmed that XMU-MP-1 could promote cell proliferation; in order to study the effect of XMU-MP-1 on the expression of lymphatic endothelial cell junction protein VE-cadherin, immunofluorescence staining experiment was performed, XMU-MP-1, palmitic acid and combined treatment were used, cell fixation was performed using 4% paraformaldehyde, and then 10% goat serum was used for blocking for 90 minutes to block non-specific binding, the primary antibody was incubated using VE-cadherin antibody (product number #2500) produced by Cell Signaling Company, and incubated overnight at 4°C; the next day, Cy3-labeled goat anti-rabbit IgG (H+L) secondary antibody (product number A0516) produced by Beyotime Company was used for incubation in the dark for 60 minutes, and finally DAPI was used for nuclear staining, and the confocal microscope results are as shown in FIGS. 11A-11D. Figure 8 As shown in FIGS. 11C-11D, compared with the palmitic acid treatment group alone, the XMU-MP-1 treatment group significantly increased the expression level of VE-cadherin, indicating that XMU-MP-1 could reverse the down-regulation of VE-cadherin expression induced by palmitic acid, suggesting that XMU-MP-1 could maintain the lymphatic endothelial barrier function by regulating the expression of cell junction proteins; at the same time, the results of the transcellular endothelial permeability experiment are as shown in FIGS. 11E-11F. Figure 8 As shown in FIG. 11E, under the action of XMU-MP-1, the permeability of lymphatic endothelial cells was reduced;

[0031] The molecular mechanism research showed that the Western Blot detection results are as shown in FIGS. 12A-12D. Figure 9 As shown in FIGS. 12A-12D, it was found that XMU-MP-1 treatment significantly up-regulated p-mTOR (Bioworld, BS43025), CTGF (Wanlei, WL02602), and reduced the protein expression level of p-YAP, and increased the expression of anti-apoptotic protein Bcl-2 (Santa Cruz Biotechnology, sc-7382), while reducing the levels of pro-apoptotic proteins Bax (Santa Cruz Biotechnology, sc-70407) and Cleaved-Caspase (Bioss, bsm-61090R).

[0032] Based on the above experimental conclusions, it was speculated that VEGFR3 and YAP existed in positive feedback regulation; Western Blot experiment was performed, and the results are as shown in FIGS. 13A-13D. Figure 10 As shown in FIGS. 13A-13D, it was proved that the lymphatic endothelial cells treated with palmitic acid would reduce the expression level of VEGFR3 and increase the expression of p-YAP, and after using VEGFC and YAP agonist XMU-MP-1, the expression of VEGFR3 was alleviated, and the nuclear translocation of YAP was increased.

[0033] Based on the above results, the embodiment of the present application verifies the specific mechanism of palmitic acid in promoting the survival and function maintenance of lymphatic endothelial cells by activating the VEGFR3 / YAP / mTOR signal axis, and confirms that the regulation of YAP can significantly promote the key biological processes of lymphatic endothelial cells such as proliferation, migration and lumen formation; in addition, it is also revealed that the signal network mediated by YAP plays an important role in maintaining the homeostasis and functional integrity of lymphatic endothelial cells, and its transcriptional activity directly regulates the endothelial barrier function by regulating the expression and distribution of intercellular junction proteins.

[0034] Example 3, as shown in Figure 11 , based on the targeted intervention of YAP on intestinal chyliferous duct dysfunction, the results are shown in Figures 12-14 .

[0035] Six-week-old SD rats (Liaoning Changsheng Biotechnology Co., Ltd.) were fed with high-fat diet (60% fat energy supply) to construct an intestinal lymphatic vessel injury model, and at the same time, the experimental group rats were given YAP specific agonist XMU-MP-1 (0.5 mg / kg, intraperitoneal injection, once every two days) intervention until the 16th week, as shown in Figure 12 A; XMU-MP-1 treatment significantly improved the intestinal lymphatic vessel injury model induced by high-fat diet, and compared with the high-fat diet group, the body weight of the rats in the XMU-MP-1 treatment group increased significantly, and the fat tissue deposition was reduced, as shown in Figure 12 B-C. At the same time, the serum lipid metabolism indicators were significantly improved, showing that the levels of low-density lipoprotein (LDL-C), total cholesterol (TC) and triglyceride (TG) were reduced, while the level of high-density lipoprotein (HDL-C) was increased (Comin, TC-1-W, TG-1-W, LDL-C-1-G, HDL-C-1-G), as shown in Figure 12 D-G.

[0036] The results of histopathological analysis are shown in Figure 13 A-D, it can be seen that the small intestine proximal tissue structure of the XMU-MP-1 treatment group is significantly improved, the HE staining shows that the intestinal morphology is restored well, and the oil red O staining confirms that the lipid deposition is reduced; the results of immunohistochemistry are shown in Figure 13 E-F, the expression amount of YAP protein in the intestinal villus lymphatic vessels of the high-fat fed SD rats is significantly lower than that of the normal diet group, and this process is reversed after injection of XMU-MP-1.

[0037] The results of further molecular mechanism research are shown in Figure 14As shown in A-D, XMU-MP-1 treatment promoted the nuclear translocation of YAP, significantly up-regulated the expression of lymphatic vessel markers VEGFR3 and LYVE1 (Bioss, bs-20120R), reduced the protein content of p-YAP, activated the mTOR signaling pathway, manifested as the expression level of p-mTOR was increased, while the expression of Cleaved-Caspase3 and pro-apoptotic protein Bax was reduced, and the expression of anti-apoptotic protein Bcl2 protein was increased;

[0038] In summary, the whole phenotype, histopathology to the molecular level system reveals the key role of XMU-MP-1 in VEGFR3 / YAP / mTOR signaling pathway in regulating the intestinal lymphatic vessel injury model.

[0039] In summary, the present embodiment discloses the molecular mechanism of inhibition of VEGFR3 / YAP / mTOR signaling pathway by palmitic acid in high-fat diet leading to dysfunction of lymphatic endothelial cells, clarifies the core role of YAP in regulating lymphatic permeability and lipid transport, and finds that YAP agonists (such as XMU-MP-1) can reverse the abnormal nuclear translocation of YAP induced by high-fat environment, restore VE-cadherin expression and endothelial barrier function, and significantly improve the lymphatic leakage, adipose tissue stasis and metabolic disorders (such as reducing serum triglyceride and total cholesterol levels) caused by high-fat diet; the present embodiment also highlights the therapeutic value of YAP for lymphatic function, and effectively repairs the damaged lymphatic structure, enhances the stability of endothelial cell connection, and restores normal lipid transport function by activating the YAP signaling pathway; in addition, the positive feedback regulation mechanism between VEGFR3 and YAP is also disclosed, which provides a new strategy for targeted intervention of lymphatic dysfunction, and the present embodiment not only fills the research gap of the molecular mechanism of high-fat diet-induced lymphatic dysfunction, but also reveals the core regulatory role of YAP signaling pathway in the pathological process, and provides a new idea for developing intervention strategies targeting lymphatic repair and treating metabolic disease-related lymphatic dysfunction.

[0040] The above only describes the preferred embodiments of the present application and should not be used to limit the present application, and any modifications, equivalent replacements and improvements made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of a YAP agonist for the preparation of a medicament for the treatment of intestinal lymphatic vessel dysfunction, characterized in that, The YAP agonist is XMU-MP-1, and the intestinal lymphatic dysfunction is high-fat diet-induced intestinal lymphatic dysfunction.

2. The use of the YAP agonist according to claim 1 for the preparation of a medicament for the treatment of intestinal lymphatic vessel dysfunction, characterized in that, The YAP agonist improves high-fat diet-induced lymphatic dysfunction by targeting the regulation of the VEGFR3 / YAP / mTOR signaling pathway.

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