Use of h151 in the preparation of a medicament for treating lymphatic vessel dysfunction

Abstract

The application belongs to the technical field of biological medicine, and provides an application of H151 in preparation of a medicine for treating lymphatic vessel dysfunction, wherein the application uses the STING protein antagonist H151 to treat the lymphatic vessel dysfunction, shows that H151 can significantly inhibit the activation of the cGAS-STING signal path, and then inhibit the secretion of inflammatory factors in the lymphatic endothelial cell, and effectively improve the function of the lymphatic endothelial cell. Therefore, the application uses the STING protein antagonist H151 to treat the lymphatic vessel dysfunction related diseases, opens a new way and method, and effectively solves the problem that the lymphatic vessel dysfunction lacks effective treatment means at present.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of H151 in the preparation of drugs for treating lymphatic vessel dysfunction. Background Technology

[0002] Lymphatic vessel dysfunction, including lymphatic hyperplasia, leakage, chylous reflux obstruction, and congenital mixed lymphovascular malformations, is closely related to the development of multiple systemic diseases, including cardiovascular, respiratory, digestive, immune, and neurological disorders. In recent years, the incidence of lymphatic vessel dysfunction-related diseases has been increasing year by year, with a higher incidence in secondary lymphatic vessel dysfunction following malignant tumor surgery. However, due to the insidious pathogenesis of lymphatic vessel dysfunction, research on the early diagnosis and specific pathogenesis of lymphatic lesions is still incomplete, and molecular-level therapeutic approaches targeting effective sites are lacking.

[0003] Immune inflammatory alterations are considered a major component of lymphatic vessel dysfunction. Cyclic GMP-AMP synthase (cGAS), a classic cytoplasmic DNA sensor, is a central hub connecting mitochondrial homeostasis imbalance and the innate immune response. When cells encounter mitochondrial stress, mitochondrial DNA (mtDNA) from damaged mitochondria is released into the cytoplasm, activating cGAS enzyme activity. This, in turn, activates the adaptor protein STING on the endoplasmic reticulum, ultimately initiating a strong inflammatory cytokine expression program, amplifying the intracellular signals of mitochondrial damage into systemic immune signals. Studies have confirmed that inflammation driven by the cGAS-STING signaling pathway is associated with various diseases, including aortic aneurysm and myocardial infarction. However, the role of the cGAS-STING signaling pathway in lymphatic endothelial dysfunction and lymphatic vessel dysfunction remains undetermined. Summary of the Invention

[0004] The purpose of this invention is to provide the application of H151 in the preparation of drugs for treating lymphatic vessel dysfunction, in order to solve the problems raised in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The application of H151 in the preparation of drugs for treating lymphatic vessel dysfunction, wherein H151 can inhibit the secretion of inflammatory factors in lymphatic vessel endothelial cells and improve the function of lymphatic vessel endothelial cells.

[0007] Furthermore, the lymphatic vessel dysfunction includes one or more of the following: lymphatic vessel hyperplasia, leakage, chylous reflux obstruction, congenital mixed lymphatic vessel malformation, and secondary lymphedema.

[0008] Furthermore, the H151 works by inhibiting the activation of the cGAS-STING signaling pathway.

[0009] Furthermore, the inhibition of cGAS-STING signaling pathway activation manifests as inhibition of IRF3 protein translocation to the nucleus and / or inhibition of STING protein translocation to the perinuclear region.

[0010] Furthermore, the inflammatory factor is one or more of TNF-α, IL-6, and IFNβ.

[0011] Furthermore, the functions include cell proliferation capacity and / or tube formation capacity.

[0012] This invention utilizes the STING protein antagonist H151 to treat lymphatic vessel dysfunction. The results show that H151 can significantly inhibit the activation of the cGAS-STING signaling pathway, thereby suppressing the secretion of inflammatory factors in lymphatic vessel endothelial cells and effectively improving lymphatic vessel endothelial cell function. Therefore, this invention uses the STING protein antagonist H151 to treat lymphatic vessel dysfunction-related diseases, opening up new avenues and methods and effectively addressing the current lack of effective treatments for lymphatic vessel dysfunction. Attached Figure Description

[0013] Figure 1 The results show the expression of interferon-responsive genes and cGAS-STING signaling pathway-related factors in inflamed hLECs; where A represents the mRNA expression results of interferon-responsive genes such as IFI35, IFIT1, and ISG15 in inflamed hLECs; and B represents the mRNA expression results of cGAS-STING signaling pathway-related factors in inflamed hLECs.

[0014] Figure 2 The results show the activation of the cGAS-STING signaling pathway in inflammatory hLECs; where A represents the subcellular localization immunofluorescence results of STING protein; and B represents the subcellular localization immunofluorescence results of IRF3 protein.

[0015] Figure 3 The results show the effects of H151 on inhibiting the cGAS-STING signaling pathway in inflammatory hLECs; where A represents the effect of H151 on inhibiting IRF3 expression in the cell nucleus; and B represents the effect of H151 on inhibiting IRF3 nuclear translocation.

[0016] Figure 4 The results show the effects of H151 on the expression of inflammatory factors in inflammatory hLECs; where A represents the inhibition of IFNβ expression at the mRNA level by H151 in inflammatory hLECs; B represents the inhibition of TNF-α expression at the mRNA level by H151 in inflammatory hLECs; and C represents the inhibition of IL-6 expression at the mRNA level by H151 in inflammatory hLECs.

[0017] Figure 5 The results of H151 improving the function of inflammatory hLECs; where A and B are the results of H151 promoting the proliferation of inflammatory hLECs; and C and D are the results of H151 promoting the formation of inflammatory hLEC ducts.

[0018] In the figure: LPS represents the lipopolysaccharide-induced group; H151 represents the H151-only treatment group; LPS+H151 represents the combined treatment group of lipopolysaccharide and H151; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] H151 (CAS No.: 941987-60-6) is a STING protein antagonist that has attracted much attention in biomedical research. It mainly works by binding to the Cys91 site on the STING protein, blocking its palmitoylation modification, thereby inhibiting the production of downstream type I interferon and inflammatory factors. It is widely used in research on the protective mechanisms of inflammatory diseases, autoimmune diseases and organ damage. Based on this, in one embodiment of the present invention, the application of the STING protein antagonist H151 in the preparation of a drug for treating lymphatic dysfunction is provided. H151 mainly works by inhibiting the activation of the cGAS-STING signaling pathway, specifically by inhibiting the translocation of IRF3 protein to the cell nucleus and inhibiting the translocation of STING protein to the perinuclear region.

[0021] It should be noted that lymphatic vessel dysfunction includes one or more of the following: lymphatic vessel hyperplasia, leakage, chylous reflux obstruction, congenital mixed lymphatic vessel malformation, and secondary lymphedema.

[0022] In this embodiment of the invention, qRT-PCR experiments revealed that the expression of interferon response genes (IFI35, IFIT1, ISG15) and cGAS-STING signaling pathway-related factors (IRF3, PERK) was significantly upregulated in a human lymphovascular endothelial cell (hLECs) inflammation model.

[0023] Furthermore, immunofluorescence staining revealed enhanced nuclear translocation of IRF3 protein and enhanced nuclear peripheral translocation of STING protein in inflamed hLECs, confirming the activation of the cGAS-STING signaling pathway in inflamed hLECs and identifying cGAS-STING as a potential target for lymphatic vessel dysfunction.

[0024] Based on the above findings, this embodiment of the invention utilizes the STING protein antagonist H151 to treat inflammatory hLECs. The results show that H151 can significantly inhibit the activation of the cGAS-STING signaling pathway, reduce the secretion of inflammatory factors such as TNF-α, IL-6 and IFNβ in lymphatic endothelial cells, and effectively improve the cell proliferation and tube formation capabilities of inflammatory hLECs.

[0025] It should be noted that, unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art. Unless otherwise specified, all reagents and materials used are commercially available.

[0026] Example 1: This example is an experiment to detect the activation of the cGAS-STING signaling pathway in inflamed hLECs, as detailed below:

[0027] To validate the cGAS-STING signaling pathway as a potential target for treating lymphatic vessel dysfunction, the following in vitro experiments were conducted in this embodiment:

[0028] Cell model construction: An inflammatory hLEC model was established by inducing hLECs with lipopolysaccharide (LPS, 2 μg / mL) for 24 hours, and this model was used as the inflammatory hLEC model group (Model group); in addition, hLECs induced without LPS were used as the blank control group (Control group).

[0029] Expression validation: The expression of interferon-responsive genes and cGAS-STING signaling pathway-related factors in LPS-induced hLECs was detected by qRT-PCR. The results showed that the expression of interferon-responsive genes (IFI35, IFIT1, ISG15) and cGAS-STING signaling pathway-related factors (IRF3, PERK) was significantly upregulated at the transcriptional level (e.g., ...). Figure 1 (As shown in A and B).

[0030] Detection of cGAS-STING signaling pathway activation: Immunofluorescence staining was used to detect the nuclear perinuclear translocation of STING protein and IRF3 protein in inflamed hLECs. hLECs were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and blocked with serum. They were then incubated overnight at 4°C with STING (or IRF3) primary antibody, followed by incubation with fluorescein-labeled secondary antibody for signal amplification. Finally, the cell nuclei were counterstained with DAPI. Results showed that STING protein in inflamed hLECs translocated to the perinuclear region (e.g., nuclear translocation of STING protein to the perinuclear region). Figure 2 As shown in A), the IRF3 protein translocates to the cell nucleus (as shown in A). Figure 2 (as shown in B in the figure); the above experimental results show that LPS can activate the cGAS-STING signaling pathway of hLECs.

[0031] Example 2: This example is a detection experiment of H151 inhibiting the activity of the cGAS-STING signaling pathway, as detailed below:

[0032] To verify the potential of H151 in treating lymphatic dysfunction by inhibiting the activity of the cGAS-STING signaling pathway, this example used an LPS (2 μg / mL)-induced inflammatory hLECs model and treated with H151 (3 μM). The experiment was divided into four groups: a blank control group; an LPS-induced group (LPS group, 2 μg / mL LPS stimulation for 24 hours); an H151-only treatment group (H151 group, 3 μM H151 stimulation for 24 hours); and a combined LPS and H151 treatment group (LPS+H151 group, 2 μg / mL LPS and 3 μM H151 stimulation simultaneously for 24 hours). The activity of the cGAS-STING signaling pathway was detected by the following experiments:

[0033] 1. Expression Validation: Western blotting was used to detect the activity of the cGAS-STING signaling pathway in the LPS+H151 group. The results showed that the expression of IRF3 protein in the nucleus of inflamed hLECs was significantly upregulated, and H151 could inhibit the expression of IRF3 protein in the nucleus (e.g., Figure 3 (As shown in A).

[0034] 2. Immunofluorescence staining: Immunofluorescence staining was used to detect the nuclear translocation of IRF3 protein after H151 intervention. Results showed that IRF3 protein translocated to the nucleus in inflammatory hLECs, while H151 inhibited this nuclear translocation, allowing it to be distributed in the cytoplasm (e.g., hLECs). Figure 3 (As shown in Figure B); the above experimental results confirm that H151 can inhibit the activation of the cGAS-STING signaling pathway in inflammatory hLECs.

[0035] Example 3: This example is an experiment to detect the inhibition of hLEC inflammation by H151, as detailed below:

[0036] To further verify that H151 can alleviate hLEC inflammation by inhibiting the cGAS-STING signaling pathway, the following in vitro experiments were conducted in this example (experimental groups were the same as in Example 2):

[0037] The level of inflammation in hLECs treated with H151 was detected using qRT-PCR. Figure 4 As shown in Figures A, B, and C, the results indicate that the transcriptional levels of inflammatory factors such as IFNβ, TNF-α, and IL-6 in inflamed hLECs are significantly upregulated, while H151 can inhibit the transcriptional expression of inflammatory factors such as IFNβ, TNF-α, and IL-6. The above experimental results confirm that H151 can inhibit the inflammatory level of hLECs.

[0038] Example 4; This example is an hLECs functional testing experiment, as detailed below:

[0039] To verify the effect of H151 on hLEC function, the following cell function assays were performed in this example (experimental groups were the same as in Example 2):

[0040] 1. Cell proliferation assay: Cell proliferation was assessed using an EdU proliferation assay. Specifically, 10 μM EdU was added to the cell culture medium of each group and incubated for 2 hours, followed by fixation with 4% paraformaldehyde, Triton X-100 permeabilization, and Click-iT reaction, and finally counterstained with DAPI. Cells were observed and counted under a fluorescence microscope, and the percentage of EdU-positive cells was calculated. Figure 5 As shown in Figures A and B, the results indicate that LPS induction significantly inhibited the proliferation of hLECs, while H151 significantly restored cell proliferation activity.

[0041] 2. Tube-forming ability test: The in vitro tube-forming ability of cells was assessed using an in vitro tube-forming assay. Specifically, pre-cooled Matrigel was diluted 1:1 with ECM, plated in 24-well plates, solidified at 37°C for 1 hour, and then seeded with hLECs. After incubation for 3 hours, tube formation was observed under a microscope, and quantitative analysis was performed using ImageJ software. Figure 5 As shown in C and D, the results show that LPS induction leads to sparse and broken hLECs tube formation and significantly impairs their tube-forming ability, while H151 can effectively restore their tube-forming ability.

[0042] In summary, the embodiments of this invention demonstrate that interferon-responsive genes are significantly upregulated in inflamed hLECs, accompanied by a significant upregulation of factors related to the cGAS-STING signaling pathway. This clarifies the activation state of the cGAS-STING signaling pathway in disease states and proposes that the cGAS-STING signaling pathway is a potential effective target for treating lymphatic dysfunction. Furthermore, there are currently no effective drugs for treating lymphatic dysfunction (such as secondary lymphedema). The embodiments of this invention apply the STING protein antagonist H151 to the treatment of inflamed hLECs. Experimental results show that H151 can significantly inhibit the abnormal activation of the cGAS-STING signaling pathway, suppress the secretion of inflammatory factors, and improve the function of inflamed hLECs, demonstrating its ability to treat lymphatic dysfunction and giving it a new application in the field of lymphatic dysfunction repair therapy.

[0043] Therefore, this invention applies the STING protein antagonist H151, which can be used to inhibit the cGAS-STING signaling pathway, to the treatment of lymphatic vessel dysfunction, opening up new ideas and methods for the treatment of lymphatic vessel dysfunction and effectively solving the problem of the lack of effective treatments for lymphatic vessel dysfunction.

[0044] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. The application of H151 in the preparation of drugs for treating lymphatic vessel dysfunction, characterized in that, The H151 can inhibit the secretion of inflammatory factors in lymphatic endothelial cells and improve the function of lymphatic endothelial cells.

2. The application according to claim 1, characterized in that, The lymphatic vessel dysfunction includes one or more of the following: lymphatic vessel hyperplasia, leakage, chylous reflux obstruction, congenital mixed lymphatic vessel malformation, and secondary lymphedema.

3. The application according to claim 1, characterized in that, The H151 works by inhibiting the activation of the cGAS-STING signaling pathway.

4. The application according to claim 3, characterized in that, The inhibition of cGAS-STING signaling pathway activation manifests as inhibition of IRF3 protein translocation to the nucleus and / or inhibition of STING protein translocation to the perinuclear region.

5. The application according to claim 1, characterized in that, The inflammatory factors are one or more of TNF-α, IL-6, and IFNβ.

6. The application according to claim 1, characterized in that, The functions include cell proliferation capacity and / or tube formation capacity.

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