Application of YAP agonist in preparation of medicine for treating intestinal lymphatic vessel dysfunction

Abstract

The invention is applicable to the technical field of biological medicines, and provides application of a YAP agonist in preparation of a medicine for treating intestinal lymphatic vessel dysfunction. The invention analyzes the molecular mechanism of improving high fat diet induced lymphatic vessel dysfunction through targeted regulation of VEGFR3 / YAP / mTOR signaling pathways and the treatment application of the YAP agonist, the YAP agonist recovers the nuclear translocation ability through antagonism of YAP excessive phosphorylation, and activates the mTOR signaling pathways, so that the proliferation, migration and tube formation functions of LECs are remarkably improved, and the treatment effect of the YAP agonist on the lymphatic vessel dysfunction induced by high fat diet is improved. Meanwhile, VE-cadherin expression is up-regulated so as to repair the integrity of the endothelial barrier; the YAP agonist can effectively relieve intestinal lymphatic vessel dysfunction and structural abnormality induced by HFD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of YAP agonists in the preparation of drugs for the treatment of intestinal lymphatic vessel dysfunction. Background Technology

[0002] The widespread prevalence of high-fat diets is closely related to various metabolic diseases. As a key channel for the absorption and transport of dietary fat, the intestinal lymphatic vessels bear the brunt of metabolic stress under high-fat dietary conditions. Long-term high-fat intake leads to increased lipid uptake by intestinal epithelial cells and excessive synthesis of chylomicrons, causing the lymphatic vessels to be in a state of chronic overload. This sustained overload can trigger structural changes such as abnormal lymphatic endothelial cell connections, lumen dilation, and basement membrane thickening, while also impairing their rhythmic contraction function, leading to lymphatic drainage obstruction. More seriously, the high-fat environment can damage the lymphatic endothelial barrier function, increasing permeability and allowing a large amount of chylomicrons to leak into surrounding tissues, forming a vicious cycle. These pathological changes not only cause abnormal local lipid deposition in the intestine but also lead to 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 noteworthy that dysfunction of the intestinal lymphatic vessels often precedes obvious metabolic abnormalities, suggesting that it may be an early event in the metabolic disorders induced by a high-fat diet. Therefore, in-depth exploration of the damage mechanism of high-fat diet to intestinal lymphatic vessels not only provides a new perspective for understanding the pathogenesis of metabolic diseases, but also lays a theoretical foundation for developing intervention strategies targeting lymphatic vessels, which is of great significance for the prevention and treatment of diseases related to high-fat diet.

[0003] Current treatments for intestinal lymphatic vessel dysfunction caused by a high-fat diet have several significant limitations: First, traditional therapies primarily focus on regulating lipid metabolism with lipid-lowering drugs or suppressing inflammatory responses with anti-inflammatory drugs. While this symptomatic approach can alleviate symptoms to some extent, it fails to address the deep-seated pathological changes caused by a high-fat diet to the intestinal lymphatic system, particularly the disruption of the growth state and functional integrity of lymphatic endothelial cells—a crucial pathological aspect. Second, existing drugs generally lack tissue specificity in their targets, often triggering systemic adverse reactions, including liver damage and gastrointestinal discomfort, while exerting their therapeutic effects. This severely impacts patient adherence and long-term treatment outcomes. More critically, there is currently a lack of targeted therapies that can specifically protect lymphatic endothelial function, which has become a major bottleneck restricting the improvement of treatment effectiveness. Summary of the Invention

[0004] The purpose of this invention is to provide the application of YAP agonists in the preparation of drugs for the treatment of intestinal lymphatic vessel dysfunction, thereby addressing the problems mentioned in the background art.

[0005] The present invention is implemented in the application of YAP agonists in the preparation of drugs for the treatment of intestinal lymphatic vessel dysfunction.

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

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

[0008] Preferably, the YAP agonist improves high-fat diet-induced lymphatic dysfunction by targeting and regulating the VEGFR3 / YAP / mTOR signaling pathway.

[0009] Another objective of this invention is to provide a therapeutic drug for intestinal lymphatic vessel dysfunction, the drug comprising a YAP agonist.

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

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

[0012] This invention analyzes and studies how a high-fat diet inhibits VEGFR3 expression and promotes YAP phosphorylation, hindering YAP nuclear translocation and the formation of the YAP-TEAD complex, thereby downregulating the expression of endothelial junction proteins such as VE-cadherin, leading to lymphatic endothelial cell (LEC) dysfunction. The YAP agonist XMU-MP-1, by antagonizing YAP hyperphosphorylation, restoring its nuclear translocation ability, and activating the mTOR signaling pathway, significantly improves LEC proliferation, migration, and tube formation, while upregulating VE-cadherin expression to repair endothelial barrier integrity. Animal experiments confirm that XMU-MP-1 can effectively alleviate HFD-induced intestinal lymphatic vessel dysfunction and structural abnormalities. Therefore, this invention demonstrates the application of XMU-MP-1 in the preparation of drugs for high-fat-induced lymphatic vessel dysfunction and its specific regulatory effect on the YAP / VEGFR3 / mTOR signaling axis. Attached Figure Description

[0013] Figure 1 This is a schematic diagram illustrating the effect of palmitic acid on lymphatic endothelial cell function as provided in Example 1 of the present invention. Figures 2 to 5 The results of the analysis of the effect of palmitic acid on the function of lymphatic endothelial cells provided in Example 1 of the present invention, wherein: Figure 2 In the figure, A represents the palmitic acid concentration suitable for treating lymphatic endothelial cells, as determined by the CCK8 assay. Figure 2 The results of the BE experiment on the formation of lymphatic endothelial cells treated with palmitic acid are shown in the middle. Figure 3The results of the EdU assay for cell proliferation and the fluorescence intensity of VE-cadherin between cells are shown in the figure. Figure 3 The results of Western blotting in the middle section (EF) show the changes in protein expression of Bax and Bcl2 in palmitic acid-treated lymphatic endothelial cells. Figure 4 The AC method involves detecting the protein level of VEGFR3 in lymphatic endothelial cells after palmitic acid treatment via western blot. Figure 4 The middle DF is the detection of YAP nuclear translocation after palmitic acid treatment of lymphatic endothelial cells by Western blot. Figure 5 Image A shows the results of immunofluorescence staining to observe the subcellular localization of YAP, where YAP protein shows a red fluorescent signal and cell nuclei show blue fluorescence after DAPI staining; Figure 5 The expression levels of the VEGFR3 / YAP / mTOR signaling pathway in the presence or absence of palmitic acid and VEGFC were detected by Western blot in BC. Figure 6 This is a schematic diagram illustrating the regulation of high-lipid-induced lymphatic endothelial cell dysfunction by the YAP agonist XMU-MP-1, as provided in Example 2 of the present invention. Figures 7 to 10 The results of the YAP agonist XMU-MP-1 provided in Example 2 of this invention regulating high-lipid-induced lymphatic endothelial cell dysfunction, wherein: Figure 7 In Figure A, different concentrations of XMU-MP-1 are shown to have an effect on the activity of lymphatic endothelial cells. Figure 7 The results of BE in the study indicate that XMU-MP-1 regulates the tube-forming ability of PA-treated lymphatic endothelial cells. Figure 8 In the middle section, AB represents the results of detecting the proliferation rate of lymphatic endothelial cells using the EdU staining method; Figure 8 The fluorescence intensity of CD (corticosteroid) was measured for VE cadherin. Figure 8 E in the middle represents the transendothelial permeation assay; Figure 9 The results of Western blotting analysis of the expression levels of VEGFR3, p-YAP, YAP, p-mTOR, mTOR, Bax, Bcl2, Cleaved-Caspase3, and Caspase3 proteins are shown in the figure. Figure 10 The results of Western blotting in AD represent further verification of the relationship between VEGFR3 and YAP. Figure 11 This is a schematic diagram of a targeted intervention study on intestinal lacteal dysfunction based on YAP provided in Embodiment 3 of the present invention; Figures 12 to 14The results of the targeted intervention study on intestinal lacteal dysfunction based on YAP provided in Embodiment 3 of the present invention are as follows: Figure 12 Figure A is a schematic diagram of constructing an animal model of intestinal lymphatic vessel dysfunction induced by a high-fat diet and administering XMU-MP-1 via intraperitoneal injection during the 13th week of high-fat feeding. Figure 12 Figure B shows the results of rat body weight changes monitored regularly (every two weeks); Figure 12 C represents the fat accumulation in the rat's mesentery, liver, and epididymis; Figure 12 The results of serum low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol (TC), and triglyceride (TG) levels in rats were shown in the figures. Figure 13 The results of AD in the study were obtained by assessing the pathological changes of intestinal lacteals through hematoxylin-eosin (H&E) staining, quantitatively analyzing aortic lipid deposition using Oil Red O staining, and measuring and statistically analyzing the morphological parameters (length and width) of intestinal villi. Figure 13 The expression of YAP in the intestinal lacteals was detected by immunohistochemistry in the middle EF. Figure 14 The results of Western blotting analysis of the expression levels of VEGFR3, YAP, p-YAP, mTOR, p-mTOR, LYVE1, Bax, Bcl2, Cleaved-Caspase3, and Caspase3 proteins in intestinal villous lymphatic vessels are shown in the image. In the attached image, * p < 0.05、** p < 0.01、*** p < 0.001 vs control. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0015] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0016] Example 1, such as Figure 1 As shown, the effects of palmitic acid (PA, Sigma, P5585) on the function of lymphatic endothelial cells (LECs) were analyzed, and the results are as follows. Figure 2-5 As shown: First, lymphatic endothelial cells were treated with different concentrations of palmitic acid using the CCK8 assay, and the activity of the lymphatic endothelial cells was measured. Figure 2As shown in Figure A; subsequently, angiogenesis capacity was assessed using a tube formation assay. 200 μl of Matrigel (Corning) was deposited in a 24-well plate to form a gel, and then subjected to a 5 × 10⁻⁶ ppm hydrogel. 4 LECs were inoculated per well, and the formation of tubular structures was observed 2 hours after treatment. The results are as follows: Figure 2 As shown in BE.

[0017] To assess cell proliferation, 3 × 10⁻⁶ cells were used. 4 After treating LECs per well for 24 hours, EdU (Beyotime, C0075S) reagent was added and the cells were cultured for another 2 hours. The cells were then fixed with paraformaldehyde, and the positive rate was statistically analyzed under a fluorescence microscope. The results are as follows: Figure 3 As shown in Figures A and C, VE-cadherin was stained using an immunofluorescence assay, and its fluorescence intensity was analyzed. The results are as follows. Figure 3 In terms of molecular mechanisms, the expression of key proteins was analyzed by Western blotting at a concentration of 1.5 × 10⁻⁶. 6 Cells were seeded at a density of [number] cells / well in 6-well cell culture plates, and drugs were added according to the above grouping. After culturing for 24 h, the culture medium was gently aspirated, and the cells were lysed on ice for 5 min with RIPA (Solarbio, R0010) lysis buffer containing 1 mmol / L PMSF (Solarbio, P0100). Cells were scraped off with a pre-chilled cell scraper and centrifuged at 16,000 r / min for 10 min in a pre-chilled centrifuge at 4 °C. The supernatant was transferred to a new centrifuge tube, and the protein content was detected by BCA method. 20 μg of protein was taken for electrophoresis and transferred to a membrane. The expression changes were detected by incubation at 4 °C overnight with antibodies against anti-apoptotic protein Bcl-2 (Santa Cruz Biotechnology, sc-7382) and pro-apoptotic proteins Bax (Santa Cruz Biotechnology, sc-70407) and β-actin (Bioworld, BS42679). The results are as follows: Figure 3 As shown in EF; In addition, RT-qPCR was used to detect the expression level of mRNA in lymphatic endothelial cells. The results showed that the mRNA level of VEGFR3 in lymphatic endothelial cells treated with palmitic acid was significantly reduced. Western blot results also showed a significant decrease in the protein level of VEGFR3 (Santa Cruz Biotechnology, sc-365748). Figure 4 AC; Western Blot results as follows Figure 4 As shown in the DF diagram, palmitic acid treatment increased the accumulation of YAP in the cytoplasm.

[0018] Lymphatic endothelial cells treated with palmitic acid were subjected to different treatments, with and without VEGFCs. Immunofluorescence staining was used to label YAP protein with a specific antibody (red fluorescence) and the nuclei were stained with DAPI (blue fluorescence) to observe the cellular localization of YAP. The fluorescence microscopy results are shown below. Figure 5 As shown in Figure A, the analysis revealed that, in addition to palmitic acid treatment, the VEGFC-treated group exhibited significantly enhanced red fluorescence signal of YAP in the cell nuclei, indicating that VEGFC can promote the nuclear translocation of YAP; Western blotting results are shown below. Figure 5 As shown in BC, palmitic acid treatment increased the accumulation of p-YAP (Bioss,bsm-52214R) in the cytoplasm and decreased the expression levels of p-mTOR and CTGF, while the use of VEGFC significantly increased the level of YAP (Bioss,bs-3605R) in the nucleus and upregulated the expression of p-mTOR and CTGF proteins. In summary, the embodiments of the present invention, through functional experiments combined with molecular detection, elucidate the effects of PA on LEC migration, tube formation and proliferation and its possible signaling pathway mechanisms. It can be seen that PA significantly inhibits the proliferation, migration and tube formation ability of LECs.

[0019] Example 2, as follows Figure 6 As shown, the regulatory effect of the YAP-specific agonist XMU-MP-1 (MCE, HY-100526) on the function of PA-treated lymphatic endothelial cells was obtained as follows. Figure 7-10 As shown: The CCK8 assay was used to evaluate the cytotoxic effects of XMU-MP-1, in which lymphatic endothelial cells were injected at a concentration of 5 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of 10 μL in 96-well plates, with control (Ctrl), solvent control (DMSO), and XMU-MP-1 treatment groups at gradient concentrations of 0.5–7 μM. After 24 hours of intervention, 10 μL of CCK8 reagent was added to each well for 90 min of incubation. Absorbance was measured at 450 nm using a microplate reader. No significant difference in cell viability was found among the concentration groups. The results are as follows: Figure 7 As shown in Figure A, it can be seen that a concentration of 5 μM yields the best results; therefore, a concentration of 5 μM was selected for subsequent experiments. Functional experiments were conducted using the above groupings, and tube formation experiments were performed. The results are shown below. Figure 7 As shown in the BE study, XMU-MP-1 significantly improved palmitic acid-inhibited cell tube formation.

[0020] Fluorescence microscopy results as follows Figure 8As shown in the AD diagram, observation confirmed that XMU-MP-1 can promote cell proliferation. To investigate the effect of XMU-MP-1 on the expression of VE-cadherin, a junctional protein in lymphatic endothelial cells, immunofluorescence staining was performed. XMU-MP-1, palmitic acid, and a combination of these treatments were used. Cells were fixed with 4% paraformaldehyde, followed by blocking with 10% goat serum for 90 minutes to prevent non-specific binding. Primary antibody incubation was performed using VE-cadherin antibody (Cell Signaling, catalog number #2500) overnight at 4°C. The following day, secondary antibody (Cy3-labeled goat anti-rabbit IgG (H+L) secondary antibody, catalog number A0516, from Beyotime) was used for 60 minutes in the dark. Finally, the nuclei were stained with DAPI. Confocal microscopy results are shown below. Figure 8 Mid-CD showed that, compared with the palmitic acid-only treatment group, the XMU-MP-1 treatment group significantly increased the expression level of VE-cadherin, indicating that XMU-MP-1 can reverse the palmitic acid-induced downregulation of VE-cadherin expression, suggesting that XMU-MP-1 may maintain lymphatic endothelial barrier function by regulating cell junction protein expression; further evidence from transendothelial permeability assays also supported this finding. Figure 8 As can be seen from the middle E, the permeability of lymphatic endothelial cells is reduced under the action of XMU-MP-1; Molecular mechanism studies have shown that Western blotting results are as follows: Figure 9 As shown in the AD diagram, XMU-MP-1 treatment significantly upregulated p-mTOR (Bioworld, BS43025) and CTGF (Wanlei, WL02602), while decreasing the protein expression level of p-YAP. It also increased the expression of the anti-apoptotic protein Bcl-2 (Santa Cruz Biotechnology, sc-7382) and decreased the levels of the pro-apoptotic proteins Bax (Santa Cruz Biotechnology, sc-70407) and Cleaved-Caspase (Bioss, bsm-61090R).

[0021] Based on the above experimental conclusions, it is hypothesized that VEGFR3 and YAP exhibit positive feedback regulation; Western blotting experiments were conducted, and the results are as follows: Figure 10 As shown in the AD diagram, palmitic acid treatment of lymphatic endothelial cells reduced the expression level of VEGFR3 and increased the expression of p-YAP. However, the expression of VEGFR3 was alleviated and the nuclear translocation of YAP increased after the use of VEGFC and the YAP agonist XMU-MP-1.

[0022] In summary, the embodiments of this invention verify the specific mechanism by which palmitic acid promotes the survival and functional maintenance of lymphatic endothelial cells by activating the VEGFR3 / YAP / mTOR signaling axis. They also confirm that regulating YAP can significantly influence key biological processes such as lymphatic endothelial cell proliferation, migration, and lumen formation. Furthermore, the invention reveals that the YAP-mediated signaling network plays a crucial role in maintaining lymphatic endothelial cell homeostasis and functional integrity, and its transcriptional activity directly regulates endothelial barrier function by modulating the expression and distribution of intercellular junction proteins.

[0023] Example 3, such as Figure 11 As shown in the figure, a targeted intervention study based on YAP for intestinal lacteal dysfunction yielded the following results: Figure 12-14 As shown: A six-week-old SD rat model of intestinal lymphatic vessel injury was established by feeding them a high-fat diet (60% fat for energy) (using Synergy Biotech). At week 13, the experimental group rats were treated with the YAP-specific agonist XMU-MP-1 (0.5 mg / kg, intraperitoneal injection, every two days) until week 16. Figure 12 As shown in Figure A; XMU-MP-1 treatment significantly improved the high-fat diet-induced intestinal lymphatic vessel injury model. Compared with the high-fat diet group, the XMU-MP-1 treatment group showed significantly slower weight gain and reduced adipose tissue deposition, as shown in Figure A. Figure 12 As shown in BC. Simultaneously, serum lipid metabolism indicators significantly improved, manifested as decreased levels of low-density lipoprotein (LDL-C), total cholesterol (TC), and triglycerides (TG), while increased levels of high-density lipoprotein (HDL-C) (Comin, TC-1-W, TG-1-W, LDL-C-1-G, HDL-C-1-G), as shown in BC. Figure 12 As shown in DG.

[0024] Histopathological analysis results as follows Figure 13 As shown in the AD diagram, the proximal small intestine tissue structure of the XMU-MP-1 treatment group was significantly improved. HE staining showed good restoration of intestinal morphology, and Oil Red O staining confirmed reduced lipid deposition. Immunohistochemical results are as follows: Figure 13 As shown in the middle EF, the expression level of YAP protein in the intestinal villi lymphatic vessels of high-fat-fed SD rats was significantly lower than that in the normal diet group, and this process was reversed after injection of XMU-MP-1.

[0025] Further molecular mechanism research results, such as Figure 14As shown in the AD diagram, XMU-MP-1 treatment promoted nuclear translocation of YAP and significantly upregulated the expression of lymphatic markers VEGFR3 and LYVE1 (Bioss,bs-20120R), reduced the protein content of p-YAP, activated the mTOR signaling pathway, as evidenced by increased p-mTOR expression levels, while simultaneously reducing the expression of Cleaved-Caspase3 and the pro-apoptotic protein Bax, and increasing the expression of the anti-apoptotic protein Bcl2. In summary, this study systematically revealed the key role of XMU-MP-1 in regulating the VEGFR3 / YAP / mTOR signaling pathway through an intestinal lymphatic vessel injury model, from the overall phenotype and histopathology to the molecular level.

[0026] In summary, the embodiments of this invention reveal the molecular mechanism by which palmitic acid inhibits the VEGFR3 / YAP / mTOR signaling pathway in a high-fat diet, leading to lymphatic endothelial cell dysfunction. They clarify the core role of YAP in regulating lymphatic permeability and lipid transport, and discover that YAP agonists (such as XMU-MP-1) can reverse high-fat environment-induced abnormal YAP nuclear translocation, restore VE-cadherin expression and endothelial barrier function, and significantly improve high-fat diet-induced lymphatic leakage, adipose tissue accumulation, and metabolic disorders (such as reducing serum triglyceride and total cholesterol levels). These embodiments also highlight the role of YAP in… The therapeutic value of this invention for lymphatic vessel function lies in its ability to effectively repair damaged lymphatic vessel structures, enhance endothelial cell connectivity stability, and restore normal lipid transport function by activating the YAP signaling pathway. Furthermore, it reveals the positive feedback regulatory mechanism between VEGFR3 and YAP, providing a new strategy for targeted intervention in lymphatic vessel dysfunction. This invention not only fills the research gap on the molecular mechanisms of high-fat diet-induced lymphatic vessel dysfunction but also reveals the core regulatory role of the YAP signaling pathway in this pathological process, providing a novel approach for developing targeted lymphatic vessel repair intervention strategies and treating lymphatic vessel dysfunction associated with metabolic diseases.

[0027] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of YAP agonists in the preparation of drugs for the treatment of intestinal lymphatic vessel dysfunction.

2. The application of the YAP agonist according to claim 1 in the preparation of a therapeutic drug for intestinal lymphatic vessel dysfunction, characterized in that, The YAP agonist is XMU-MP-1.

3. The application of the YAP agonist according to claim 1 in the preparation of a therapeutic drug for intestinal lymphatic vessel dysfunction, characterized in that, The intestinal lymphatic vessel dysfunction mentioned is an intestinal lymphatic vessel dysfunction induced by a high-fat diet.

4. The application of the YAP agonist according to claim 1 in the preparation of a therapeutic drug for intestinal lymphatic vessel dysfunction, characterized in that, The YAP agonist improves high-fat diet-induced lymphatic dysfunction by targeting and regulating the VEGFR3 / YAP / mTOR signaling pathway.

5. A drug for treating intestinal lymphatic vessel dysfunction, characterized in that, The drug includes YAP agonists.

6. The medicament for treating intestinal lymphatic vessel dysfunction according to claim 5, characterized in that, The YAP agonist is XMU-MP-1.

7. The medicament for treating intestinal lymphatic vessel dysfunction according to claim 5, characterized in that, The intestinal lymphatic vessel dysfunction mentioned is an intestinal lymphatic vessel dysfunction induced by a high-fat diet.

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