Application of CsV based on NLRP6 inflammasome regulation in diabetic vascular endothelial injury

Abstract

The invention relates to the technical field of drugs for treating diabetes, and discloses application of CsV based on NLRP6 inflammasome regulation in vascular endothelial injury of diabetes, the CsV is used as an NLRP6 inflammasome inhibitor for preparing drugs for treating vascular endothelial injury of diabetes, and the dosage of the CsV is 50-200mg / kg. The triterpenoid saponin compound Chikusetsuaponin V (CsV) adopted in the scheme serves as a natural active component extracted from compositae plants, assembling and activation of inflammasomes are prevented from the source by inhibiting phase separation of NLRP6, the method is a major breakthrough for a traditional strategy, and the innovative mechanism is verified through a mutant experiment.

Description

Technical Field

[0001] The present invention relates to the technical field of diabetes therapeutic drugs, and in particular to an application of CsV based on NLRP6 inflammasome regulation in treating diabetic vascular endothelial damage. Background Art

[0002] Globally, diabetes has become a highly prevalent chronic metabolic disease. The vascular complications it causes, such as diabetic nephropathy, diabetic retinopathy, and peripheral vascular disease, severely impair organ function, significantly reduce quality of life, and even threaten patients' lives. With the deepening of research into disease mechanisms, screening natural ingredients with clear bioactivity from traditional medicinal plants for the treatment of diabetes and its complications has become an important research direction in the biopharmaceutical field due to its advantages such as high safety and multi-target regulation.

[0003] CsV (Chenosanol V), a triterpenoid saponin, is a natural active ingredient extracted from plants in the Asteraceae family. Recent studies have shown its potential medicinal value in improving metabolic disorders and inhibiting inflammatory responses, particularly in the pathological processes associated with diabetic vascular disease. Furthermore, the NLRP6 (NOD-like receptor pyrin domain-associated protein 6) inflammasome, a key molecular complex in the inflammatory response regulatory network, has been increasingly revealed to play an important role in maintaining endothelial cell homeostasis and inhibiting excessive inflammatory responses, making it a potential new target for intervention in diabetic endothelial injury. Further investigation into the regulatory mechanism of CsV on the NLRP6 inflammasome may provide new insights and approaches for the treatment of diabetic endothelial injury.

[0004] Currently, treatments for diabetic vascular complications remain limited. Existing clinical drugs primarily target metabolic markers such as blood sugar, blood pressure, and lipid profiles, but these efforts struggle to effectively block the "metabolic memory" effect. Even with long-term stable blood sugar control, endothelial damage can persist. Furthermore, aberrant inflammasome activation is a core mechanism contributing to chronic inflammation, oxidative stress, and dysfunction in the diabetic endothelium. However, existing drugs have limited targeted inhibition of inflammasomes and are unable to fundamentally curb the inflammatory response's damage to the endothelium. Conventional treatments generally lack specific protection for endothelial cells, making it difficult to precisely improve the endothelial cell pathological microenvironment and effectively reverse key pathological states such as endothelial dysfunction and alleviate oxidative stress and inflammatory damage. Despite research exploring new therapeutic targets and drug candidates, effective solutions to these core issues have yet to be found. Therefore, developing a therapeutic strategy that can specifically target the NLRP6 inflammasome, effectively block the metabolic memory effect, and protect endothelial cells is of significant clinical and scientific interest, highlighting the necessity and innovative nature of this invention. Summary of the Invention

[0005] The present invention aims to provide the application of CsV based on NLRP6 inflammasome regulation in diabetic vascular endothelial injury, so as to solve the technical problem that the existing technology lacks drugs that can specifically target NLRP6 inflammasome to inhibit its abnormal activation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: the application of CsV based on NLRP6 inflammasome regulation in diabetic vascular endothelial damage, wherein the CsV is used as an NLRP6 inflammasome inhibitor to prepare a drug for treating diabetic vascular endothelial damage, the complete amino acid sequence of NLRP6, the core protein of the NLRP6 inflammasome, is shown in SEQ ID NO. 1, and the dosage of the CsV is 50-200 mg / kg.

[0007] Preferably, as an improvement, the CsV acts by inhibiting the phase separation of NLRP6, thereby preventing the assembly and activation of inflammasomes from the source.

[0008] Preferably, as an improvement, the binding site of the CsV and the NLRP6 inflammasome is the 284-354 region of the core protein NLRP6.

[0009] Preferably, as an improvement, the drug is a freeze-dried powder injection containing CsV.

[0010] Preferably, as an improvement, the lyophilized powder injection comprises the following raw materials in a mass volume ratio w / v: mannitol 2-10%, dextran 40 1-5%, surfactant 0.01-0.1%, and the balance is water for injection QS, and a citric acid-sodium citrate buffer pair is used to adjust the pH to 5.0-7.5.

[0011] Preferably, as an improvement, the mass ratio of mannitol to dextran 40 is 3-4:1.

[0012] Preferably, as an improvement, the drug is an enteric-coated sustained-release tablet containing CsV.

[0013] Preferably, as an improvement, the enteric-coated sustained-release tablets comprise the following raw materials in parts by mass: 15-30 parts of hypromellose, 5-15 parts of ethyl cellulose, 20-40 parts of microcrystalline cellulose, 10-25 parts of lactose monohydrate, 0.5-1.5 parts of magnesium stearate, 1-3 parts of talc and enteric coating material.

[0014] The principles and advantages of this solution are: Traditional inflammasome inhibition strategies focus on blocking inflammatory signaling pathways, but lack precise intervention at the critical stage of inflammasome assembly. This approach utilizes the triterpenoid saponin CsV, a natural active ingredient extracted from plants in the Asteraceae family, to prevent inflammasome assembly and activation at the source by inhibiting the phase separation of NLRP6. This represents a significant breakthrough compared to traditional strategies. Mutant experiments have also validated this innovative mechanism.

[0015] First, the inventors used the optogenetic platform (optoDroplets) software to analyze the NLRP6 amino acid sequence and found that its amino acid sequences 86-190 and 284-354 are intrinsically disordered regions, indicating that these two amino acid sequences may be the structural basis for the phase separation of NLRP6.

[0016] Subsequently, the inventors constructed plasmids with alanine mutations at the conserved lysine or arginine residues in the NLRP6 amino acid sequence 86-190 and 284-354.

[0017] In a 293T cell (Hek-293t human embryonic kidney cell) transfection experiment using the two plasmids mentioned above, the inventors further discovered that: based on the application of CsV regulated by the NLRP6 inflammasome in diabetic vascular endothelial injury, wild-type NLRP6 will undergo obvious phase separation under high glucose stimulation, and the addition of CsV can inhibit the occurrence of phase separation; only NLRP6 with amino acid mutations at 284~354 will not undergo phase separation under high glucose stimulation, indicating that the NLRP6 amino acid sequence 284~354 is the structural basis for its phase separation.

[0018] More importantly, molecular docking experiments revealed that CsV formed a stable interaction with NLRP6 amino acid sequence 284–354, with a binding energy of -8.5 kcal / mol, indicating a high affinity between the two. This specific binding further demonstrates that CsV, by binding to NLRP6 amino acid sequence 284–354, inhibits phase separation, thereby preventing inflammasome assembly and activation at the source. These data demonstrate the advanced and specific mechanism of CsV's regulation of NLRP6 phase separation, providing a novel molecular target and mechanism of action for the treatment of diabetic endothelial damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figures 1 and 2 show the results of CsV alleviating vascular inflammation and restoring endothelial function in diabetic mice in Experimental Example 1 of the present invention (A, chemical structure of CsV; B, fasting blood glucose levels of mice in the control group (Con), diabetic group (DM), and CsV-treated groups (DM+CsV-L: low dose; DM+CsV-H: high dose) (* p<0.05 vs. control group); C-E, qPCR analysis of proinflammatory cytokine (IL-1β, TNF-α, IL-6) mRNA levels in aortic tissue (* p<0.05 vs. control group; # p<0.05 vs. diabetic group)).

[0020] Figure 2 The endothelium-dependent vasodilation response induced by acetylcholine (ACh) and the endothelium-independent vasodilation response induced by sodium nitroprusside (SNP) in Experimental Example 1 of the present invention (A, endothelium-dependent vasodilation response induced by acetylcholine (ACh) (* indicates p < 0.05 compared with the Con group; # indicates p < 0.05 compared with the DM group); B, endothelium-independent vasodilation response induced by sodium nitroprusside (SNP)).

[0021] Figure 3 Western blot analysis of eNOS (endothelial nitric oxide synthase) and iNOS (inducible nitric oxide synthase) protein expression in Experimental Example 1 of the present invention (* indicates p < 0.05 compared with the Con group; # indicates p < 0.05 compared with the DM group).

[0022] Figure 4CsV inhibited NLRP6-mediated inflammasome activation and NF-κB (nuclear factor-activated B-cells kappa-light-chain enhancer) signaling pathway in Experimental Example 2 of the present invention (A, Western blot analysis of NLRP6 and NLRP3 (NOD-like receptor pyrin domain-associated protein 3) expression in aortic tissue (* p<0.05 vs. control group (Con); # p<0.05 vs. diabetic group (DM)); B, NF-κB pathway activation (assessed by p-p65 and IκB-α levels) (* p<0.05 vs. control group; # p<0.05 vs. diabetic group)).

[0023] Figure 5 This is experimental example 2 of the present invention, in which CsV inhibits NLRP6-mediated inflammasome activation and NF-κB signaling pathway (immunofluorescence staining of NLRP6 and GSDMD (scrotalin D) in aortic sections (* p < 0.05 vs. control group; # p < 0.05 vs. diabetic group)).

[0024] Figure 6 This is the molecular docking of CsV and NLRP6 in Experimental Example 2 of the present invention (residues 284-354 (NBD domain) of NLRP6 are marked in purple).

[0025] Figure 7 The predicted binding sites of NLRP6 in Experimental Example 2 of the present invention (residues 97-188 and 284-354; the black frame amplifies the red circle area to show the details of the interaction between CsV and the NLRP6 functional binding pocket (284-354)).

[0026] Figure 8 Figure 2 shows that CsV disrupts NLRP6 phase separation to inhibit inflammasome assembly in Experimental Example 2 of the present invention (A, expression of NLRP6 in wild-type (WT) and Δ284–354 mutant 293T cells (added with CsV (10 μM)) under high glucose (30 mM) conditions (* p < 0.05 vs. control group; # p < 0.05 vs. high glucose group); B, in vitro liquid phase separation (LLPS) assay showing NLRP6 aggregate formation; C–F, qPCR and Western blot analysis of NLRP6, GSDMD, IL-1β, and IL-18 in WT and mutant cells (* p < 0.05 vs. control group; # p < 0.05 vs. high glucose group)).

[0027] Figure 9CsV rescues angiogenesis and vascular integrity in the zebrafish model in Experimental Example 3 of the present invention (A, typical images of intersegmental vessels (ISVs) in Tg(kdrl:mcherry;lyve1b:GFP) zebrafish under control, high glucose (DM-mimicking), and CsV-treated conditions (blue arrows: excessive sprouting; yellow asterisks: branches without lumen formation); B, vascular leakage assay in Tg(kdrl:GFP;gata1:mcherry) zebrafish (shown is the fluorescence intensity of extravascular red blood cells)). DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available.

[0029] Example This protocol provides the application of CsV, a NLRP6 inflammasome-regulated drug, in treating diabetic vascular endothelial injury. CsV, as an NLRP6 inflammasome inhibitor, is used to develop a drug for treating diabetic vascular endothelial injury. The CsV dose ranges from 50 to 200 mg / kg. CsV works by inhibiting NLRP6 phase separation, preventing the assembly and activation of the inflammasome. Specifically, the binding site of CsV to the NLRP6 inflammasome is located at regions 284–354 of the core protein NLRP6.

[0030] For reference, the drug is a lyophilized powder injection containing CsV. The lyophilized powder injection comprises the following raw materials in a w / v ratio: mannitol 2-10%, dextran 40 1-5%, a surfactant 0.01-0.1%, and the balance water for injection (QS). The pH is adjusted to 5.0-7.5 using a citric acid-sodium citrate buffer. The mass ratio of mannitol to dextran 40 is further limited to 3-4:1.

[0031] For reference, the drug is an enteric-coated sustained-release tablet containing CsV. The enteric-coated sustained-release tablet comprises the following raw materials by weight: 15-30 parts of hypromellose, 5-15 parts of ethyl cellulose, 20-40 parts of microcrystalline cellulose, 10-25 parts of lactose monohydrate, 0.5-1.5 parts of magnesium stearate, 1-3 parts of talc, and an enteric coating material.

[0032] Among them, the chemical structure of CsV is as follows Figure 1 As shown in A.

[0033] The complete amino acid sequence of NLRP6, the core protein of the NLRP6 inflammasome, is as follows (SEQ ID NO. 1): MDAAGASCSSVDAVARELLMATLEELSQEQLKRFRHKLRDAPLDGRSIPWGRLERSDAVDLVDKLIEFYEPVPAVEMTRQVLKRSDIRDVASRLKQQQLQKLGPTSVLLSVSAFKKKYREHVLRQHAKVKERNARSVKINKRFTKLLIAPGTGAVEDELLGPLGEPEPERARRSDTHTFNRLFRGNDEESSQPLTVVLQGPAGIGKTMAAKKILYDWAAGKLYHSQVDFAFFMPCGELLERPGKRSLADLVLDQCPDRAWPVKRILAQPNRLLFILDGADELPTLPSSEATPCKDPLEATSGLRVLSGLLSQELLPGARLLVTTRHAATGRLQGRLCSPQCAEIRGFSDKDKKKYFFKFFRDERKAERAYRFVKENETLFALCFVPFVCWIVCTVLQQQLELGRDLSRTSKTTTSVYLLFITSMLKSAGTNGPRVQGELRTLCRLAREGILDHHKAQFSEEDLEKLKLRGSQVQTIFLNKKEIPGVLKTEVTYQFIDQSFQEFLAALSYLLEAERTPGTPAGGVQKLLNSDAELRGHLALTTRFLFGLLNTEGLRDIGNHFGCVVPDHVKKDTLRWVQGQSHPKGPPVGAKKTAELEDIEDAEEEEEEEEDLNFGLELLYCLYETQEEDFVRQALSSLPEIVLERVRLTRMDLEVLNYCVQCCPDGQALRLVSCGLVAAKEKKKKKKSLVKRLKGSQSTKKQPPVSLLRPLCETMTTPKCHLSVLILSHCRLPDAVCRDLSEALKVAPALRELGLLQSRLTNTGLRLLCEGLAWPKCQVKTLRMQLPDLQEVINYLVIVLQQSPVLTTLDLSGCQLPGVIVEPLCAALKHPKCSLKTLSLTSVELSENSLRDLQAVKTSKPDLSIIYSK。

[0034] The functional domains of NLRP6 inflammasome are shown in Table 1.

[0035] Table 1 Functional domains of NLRP6 inflammasome

[0036] Experimental Example 1: Animal Model Efficacy Verification System To comprehensively evaluate the therapeutic effects of CsV on diabetic vascular endothelial damage, we used db / db mice as a diabetic model. The db / db mouse is a widely used animal model of hereditary type 2 diabetes and obesity. It holds a crucial position in biomedical research, particularly in the study of diabetes and its complications (vascular disease). The db / db mice used in this study (half male and half female, SPF-grade, 8-10 weeks old, weighing 50-60 g) were housed in the animal room of the Animal Experimental Center of Chongqing Medical University at a room temperature of 20-25°C and a relative humidity of 40%-60%. They were housed on a 12h:12h light-dark cycle, had free access to water, and were fed a standard diet for one week.

[0037] The tail vein blood glucose of db / db mice meets the diagnostic criteria for diabetes (fasting blood glucose levels of db / db mice are as follows Figure 1 B).

[0038] Under normal physiological conditions, vascular endothelial cells release NO (nitric oxide) to promote vasodilation. However, in diabetic vascular lesions, this vasodilation function is impaired. By comparing the vasodilation rates of mice in different treatment groups, the improvement effect of CsV on vascular endothelial function can be intuitively reflected. The experimental animals were divided into a normal control group (control group), a diabetic model group (DM+CsV 0 mg / kg group), and a CsV treatment group (CsV-L group: DM+CsV 50 mg / kg group; CsV-H group: DM+CsV 200 mg / kg group). The normal control group was given a regular diet; the diabetic model group and the CsV treatment group were both db / db mice, and the CsV treatment group was intraperitoneally injected with a certain dose of CsV solution every day, and the model group was given an equal amount of normal saline. The administration period was 8 weeks. This design was intended to highlight the direct repair effect of CsV on vascular endothelial function (such as Figure 1 C~ Figure 1 E), independent of blood sugar control, fully demonstrating its unique therapeutic effect characteristics that are unrelated to blood sugar.

[0039] In this model, a precise quantitative evaluation system for endothelial function repair was established using the DMT-620 multi-channel microvascular tension meter. Acetylcholine-induced vasodilation is a key evaluation indicator. By stimulating mouse thoracic aortic rings with acetylcholine, the degree of vasodilation was precisely recorded using the vascular tension measurement system.

[0040] The experimental process is as follows: The db / db mice were then sacrificed with pentobarbital (70 mg / kg) and fixed to a dissecting table. The thoracic cavity was opened along the midline, exposing the thoracic and abdominal aorta. The thoracic aorta was transected along the spine from the superior border of the diaphragm to the aortic arch using ophthalmic scissors. The aorta was pinned to a petri dish containing physiological saline solution and resin. The connective tissue surrounding the vessels was gently pulled with ophthalmic forceps and carefully removed using ophthalmic scissors (avoiding direct contact with the arterial wall). After removal, the aorta was cut into vascular loops of equal length (2–3 mm). The vascular tension measurement system was set to 37°C and aerated with a mixture of 5% carbon dioxide and 95% oxygen. Five milliliters of preheated PSS solution, pre-flowed with the mixture at 37°C, was added to the vascular tension chamber. After adjusting the instrument tension to "0," ophthalmic forceps were used to carefully secure the vascular ring to steel pins (two pins inserted into the vessel lumen serve as anchors and tension transmitters, respectively). Appropriate preload (20 mN for the thoracic aorta and 3.5 mN for the mesenteric artery) was applied, and equilibration began. After preload application, the ring was gently pulled counterclockwise with a micrometer screw every 5 minutes to maintain preload. The PSS solution was replaced every 15 minutes. Equilibration lasted 70 minutes, and vascular tension was recorded using the DMT sensor system and LabChart software. After equilibration, adjust the tension to "0" in the LabChart software. Add 5 mL of high potassium saline solution to each test tank to stimulate vasoconstriction for 10-15 minutes, and wait until the tension of the vascular ring stabilizes to the plateau phase. Drain the high potassium saline solution, wash the vasoconstriction curve to the baseline level with saline solution, and then perform high potassium pre-stimulation again. The stimulation time should be the same as the first time. If the difference in the vasoconstriction peak value produced by the two high potassium stimulations is within 10%, the structure and function of the vascular ring are considered normal. After the second pre-stimulation, the test tank should contain 5 mL of saline solution. Add norepinephrine solution to the test tank to make the final norepinephrine concentration reach 10 -6 mol / L to exert the effect of vasoconstriction; after the vascular ring contraction reaches the plateau phase, 3.5 uL, 10 uL, 10 -8 ~10 -5 mol / L acetylcholine solution (the maximum final concentration of acetylcholine solution is 10 -5 mol / L); the final concentration of acetylcholine solution reached 10 -5 When the vasodilation rate is more than 70% at 40 mol / L, it is considered that the vasodilation function is normal and the experimental results are credible.

[0041] like Figure 2 As shown in the results, the thoracic aorta vasodilation curve of the CsV-treated group was significantly shifted to the left compared with that of db / db mice, and a significant decrease in the half-effective dose was also observed, indicating that CsV can partially reverse the endothelium-dependent vasodilation dysfunction of the thoracic aorta caused by diabetes.

[0042] In addition, the balance between eNOS (endothelial nitric oxide synthase) and iNOS (inducible nitric oxide synthase) in aortic tissue is also a key indicator. eNOS promotes physiological NO production and maintains vascular homeostasis; iNOS is overexpressed under inflammatory stimulation, produces excessive NO, and triggers oxidative stress to damage blood vessels. By detecting the protein expression levels and activity of the two in aortic tissue and calculating the eNOS / iNOS ratio, we can gain a deeper understanding of the regulatory effect of CsV on the balance of nitric oxide metabolism in vascular endothelial cells. Western blot analysis showed that CsV reversed the diabetes-induced inhibition of eNOS (endothelial nitric oxide synthase) (p<0.05) and inhibited the overexpression of iNOS (inducible nitric oxide synthase) (p<0.05) ( Figure 3 ), which indicates that CsV protects the endothelium-dependent vasodilation function of the thoracic aorta in db / db mice by inhibiting the inflammatory state of the thoracic aorta.

[0043] Experimental Example 2: Verification of cellular and molecular mechanisms In order to further explore the regulatory mechanism of CsV on NLRP6 protein expression, the HUVECs (human umbilical vein endothelial cells) high glucose stimulation model was used to simulate the pathophysiological state of diabetic thoracic aortic endothelial cells. First, HUVECs cells were divided into normal control group, high glucose model group, and CsV intervention group. HUVECs were purchased from the American Type Culture Collection (ATCC) and cultured in 199 medium containing 20% ​​fetal bovine serum, 20 μg / mL endothelial cell growth supplement, 100 μg / mL heparin, 2 mM L-glutamine, and 1% penicillin-streptomycin double antibody solution. They were cultured under 5% CO2, 37°C, and 95% humidity. The HUVECs used in the experiment were passaged between 6 and 10 generations. For high glucose (HG) stimulation, cells were seeded in 6-well plates (1×10 per well). 5 cells) and allowed to adhere for 24 hours. Subsequently, the culture medium was replaced with fresh medium containing 30 mM D-glucose to simulate a high-sugar environment. To control the osmotic pressure effect, a parallel group was set up in normal glucose medium (5.5 mM D-glucose) and 24.5 mM mannitol was added (osmotic pressure control group). The cells were exposed to high-sugar conditions for 24 hours. HUVECs were stimulated with CsV: CsV (final concentration 10 μM) was added to the cell culture medium and the effect lasted for 24 hours (results are shown in Figure 4 (as shown in A).

[0044] After 24 hours of culture, the expression level of NLRP6 protein was detected by Western blot. Total cell protein was extracted, separated by SDS-PAGE electrophoresis, and transferred to a PVDF membrane. Immunoblotting was performed with a specific NLRP6 antibody. The grayscale value of the bands was analyzed by chemiluminescence to accurately determine the changes in NLRP6 protein expression. Figure 4 As shown in A, the results showed that diabetes significantly upregulated the expression of NLRP6 in aortic tissue, while CsV treatment inhibited this upregulation in a dose-dependent manner. Figure 4 As shown in Figure 2 (B), CsV also alleviated the activation of the NF-κB pathway, as evidenced by reduced levels of p-p65 (phosphorylated p65) and stabilized IκB-α (inhibitory protein κB-α) protein.

[0045] At the same time, immunofluorescence technology was used for localization and semi-quantitative analysis. After the cells were fixed and permeabilized, they were incubated with NLRP6 antibodies, and then fluorescently labeled secondary antibodies were added. The distribution and fluorescence intensity of NLRP6 protein in the cells were observed under a fluorescence microscope to visually present its expression changes. Figure 5 As shown, immunofluorescence results confirmed that the expression of NLRP6 and GSDMD was reduced in the CsV-treated group, indicating that inflammatory cell death was inhibited.

[0046] Subsequently, NLRP6 plasmid and mutant transfection experiments were conducted in 293T cells to explore the mechanism of CsV inhibition of NLRP6. Molecular docking identified a high-affinity binding site of CsV within the NLRP6 nucleotide binding domain, and this site overlapped with the amino acid sequences 97-188 and 284-354 of the NLRP6 liquid-liquid phase separation (LLPS) site (e.g. Figure 6 、 Figure 7 As shown). To further clarify the mechanism by which CsV inhibits NLRP6 and exerts its protective effect, we transfected the wild-type NLRP6 plasmid, the amino acid sequence 97~188 mutant (Δ97~188) NLRP6 plasmid, and the amino acid sequence 284~354 mutant (Δ284~354) NLRP6 plasmid into 293T cells, respectively. After transfection, high glucose stimulation was given, and then CsV was added for treatment. By detecting the LLPS of NLRP6 in the cells and the expression of related inflammatory factors, the effect of the mutation on the effect of CsV was clarified. The experiment showed that compared with the transfection of the wild-type NLRP6 plasmid, CsV failed to inhibit the high glucose-induced upregulation of NLRP6 expression in the Δ284–354 mutant, but had a significant inhibitory effect on cells transfected with the Δ97–188 NLRP6 plasmid ( Figure 8C). In vitro LLPS experiments showed that under high glucose conditions, CsV could disrupt the formation of aggregates between wild-type NLRP6 protein and Δ97~188 NLRP6 protein, but this effect did not occur in cells transfected with Δ284~354 NLRP6 plasmids ( Figure 8 B). qPCR confirmed that CsV lost the ability to inhibit NLRP6 and its downstream pyroptosis markers (GSDMD, IL-1β, IL-18) in cells transfected with Δ97~188NLRP6 plasmid ( Figure 8 D~ Figure 8 F). By examining intracellular NLRP6 phase separation and related inflammatory cytokine expression, we first identified amino acid sequence 284–354 as the LLPS site of NLRP6. We then demonstrated that CsV inhibits high-glucose-induced NLRP6 inflammasome activation by inhibiting LLPS. This series of experimental protocols is highly reproducible and highly specific, strongly confirming the unique molecular mechanism by which CsV regulates NLRP6.

[0047] Experimental Example 3: Clinical Translation Simulation - Zebrafish Zebrafish models of vascular regeneration hold significant application value in this process. Zebrafish embryos develop rapidly, and their vasculature is transparent and highly similar to human vasculature. Transgenic zebrafish strains, such as Tg(kdrl:mcherry;lyve1b:GFP), allow for the direct observation of vascular morphology and function, providing visual evidence for drug efficacy and accelerating the process of new drug screening. Transgenic zebrafish strains Tg(kdrl:mcherry;lyve1b:GFP) and Tg(kdrl:GFP;gata1:mcherry) were used to assess vascular morphogenesis and integrity. Embryos were continuously exposed to 150 mM glucose and 1 μM CsV from 24 hours post-fertilization (24 hpf) to 7 days post-fertilization (dpf). Control embryos were cultured in standard embryonic culture medium. At 7 dpf, the development of intersegmental vessels (ISVs) was analyzed using a Leica M205 FA stereofluorescence microscope.

[0048] In the transgenic zebrafish strain Tg (kdrl:mcherry;lyve1b:GFP), high glucose environment (HG: simulated diabetic conditions) led to a significant reduction in the number of functional tubularized intersegmental vessels (ISVs) compared with the control group, accompanied by pathological phenomena such as excessive sprouting (marked by blue arrows) and non-tubularized branches (marked by yellow asterisks). CsV treatment (HG+CsV-H) effectively restored the normal development of ISVs in a dose-dependent manner. Vascular leakage experiments conducted using the Tg (kdrl:GFP;gata1:mcherry) strain showed that the diabetic model group had abnormal accumulation of red blood cell fluorescence signals into the extravascular space, and high-dose CsV significantly alleviated this leakage phenotype ( Figure 9 ).

[0049] Experimental Example 4: The drug is a freeze-dried powder injection containing CsV In the development of the pharmaceutical composition, mannitol and dextran were selected as fillers for the lyophilized powder injection. These agents not only ensure the physical stability of the formulation during the lyophilization process but also prevent aggregation or denaturation of CsV during the process. Furthermore, an appropriate amount of citric acid-sodium citrate buffer was added to precisely adjust the pH of the solution to 6.5-7.0 to maintain the chemical stability of CsV. The core formula (using a single vial as an example) is shown in Table 2.

[0050] Table 2 Formulation of lyophilized powder injection containing CsV

[0051] Note: Dextran selection: Low molecular weight (dextran 40, MW ≈ 40 kDa) is preferred to balance protection and solubility. The above formula further defines a ratio of mannitol to dextran 40 of 3-4:1 by mass, which synergistically improves product appearance and prevents collapse.

[0052] It is prepared by sterile freeze-drying method, and the preparation process is as follows: Step 1: Solution preparation 1-1. Preheat 80% water for injection to 50±5°C → add mannitol and buffer salt (citric acid-sodium citrate buffer pair), stir to dissolve; 1-2. Cool to 25±5°C → Add dextran 40 and stir until clear. 1-3. Add active drug and surfactant → Fill to final volume with water for injection; 1-4. Adjust pH (target ±0.1) → Sterile filter through a 0.22 μm filter membrane and lyophilize the filtrate.

[0053] Step 2: Filling and semi-stoppering Filling accuracy: ±1% filling volume error (e.g. 2.00±0.02 mL); Vials: neutral borosilicate glass (ISO 8362-1 standard); Rubber stopper: Fluorinated butyl rubber stopper (pre-cleaned, siliconized); Step 3: Freeze-drying, refer to the process in Table 3.

[0054] Table 3 Freeze-drying process control

[0055] *Key Controls: Mannitol crystallization: pre-freezing rate ≤ 1℃ / min to ensure the formation of crystalline structure Dextran protection: primary drying temperature < eutectic point + 5°C (to avoid collapse) Step 4: Plug and seal After filling with nitrogen, fully press the plug (residual oxygen ≤ 2%) and seal with aluminum cap (torque 6-15 Ncm).

[0056] Step 5: Quality Control Detect and control critical quality attributes (CQAs), such as:

Appearance

[0057]

Redissolution time

[0058]

Residual moisture

[0059]

Insoluble particles

[0060]

Dextran degradation

[0061] Experimental Example 5: The drug is an enteric-coated sustained-release tablet containing CsV Enteric-coated sustained-release tablets use matrix materials such as hydropropyl methylcellulose and ethyl cellulose. These materials form a stable skeleton structure in the gastrointestinal tract, enabling slow drug release. Cellulose acetate phthalate is also added as an enteric coating material to ensure the tablets remain insoluble in the acidic environment of the stomach while rapidly disintegrating and releasing the drug in the alkaline environment of the intestine. The core formula (per tablet) is shown in Table 4.

[0062] Table 4 Core formula of enteric-coated sustained-release tablets containing CsV

[0063] Note: 1. HPMC selection: K4M (viscosity 4000 cP) provides a sustained-release matrix, while alternative models (such as K15M) can adjust the release time; 2. EC selection: N10 (viscosity 10 cP) to avoid excessive release retardation; 3. CAP alternative: HPMCAS or Eudragit L100 can be used as an alternative (plasticizer ratio needs to be adjusted) The preparation process is as follows: Step 1: Matrix tablet preparation (wet granulation and tableting) (1) Mixing: main drug + HPMC + EC + MCC + lactose → V-type mixer for 20 minutes (speed 15 rpm); (2) Granulation: Add binder (5% PVPK30 ethanol solution) → high-speed shear granulation (cutter 2000 rpm, stirring paddle 500 rpm); end point: the product forms a ball when held and falls apart when touched; (3) Drying: fluidized bed drying (inlet air temperature 50°C, material temperature ≤40°C, moisture ≤3%, LOD method); (4) Granulation: crush with a 16-mesh sieve → add magnesium stearate and talc → mix for 5 minutes; (5) Tablet pressing: rotary tablet press (hardness 50-80N, friability ≤0.8%, tablet weight variation ±5%); Step 2: Enteric coating (high-efficiency coating pan) (1) Preparation of coating solution: CAP + triethyl citrate → dissolved in anhydrous ethanol (concentration 8%-10% w / v) → passed through a 0.3mm sieve (2) Preheating: Preheat the core to 35±2℃ (pot speed 5rpm, air volume 200m 3 / h) (3) Coating procedure is shown in Table 5: Table 5 Coating procedure

[0064] (4) Curing: -40℃ hot air circulation for 2h → reduce the brittleness of CAP film Step 3: Quality control as shown in Table 6: Table 6 Quality Control

[0065] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. Application of CsV based on NLRP6 inflammasome regulation in treating diabetic vascular endothelial injury, characterized by: The CsV is used as an NLRP6 inflammasome inhibitor to prepare a drug for treating diabetic vascular endothelial damage. The complete amino acid sequence of the core protein NLRP6 of the NLRP6 inflammasome is shown in SEQ ID NO. 1; the dosage of the CsV is 50~200 mg / kg.

2. The use according to claim 1, characterized in that: The CsV acts by inhibiting the phase separation of NLRP6, preventing the assembly and activation of inflammasomes from the source.

3. The use according to claim 2, characterized in that: The binding site of CsV and NLRP6 inflammasome is the 284~354 region of the core protein NLRP6.

4. The use according to claim 1, characterized in that: The medicine is a freeze-dried powder injection containing CsV.

5. The use according to claim 4, characterized in that: The freeze-dried powder injection comprises the following raw materials in a mass-to-volume ratio (w / v): 2-10% mannitol, 1-5% dextran 40, 0.01-0.1% surfactant, and the balance being water for injection (QS). A citric acid-sodium citrate buffer pair is used to adjust the pH to 5.0-7.

5.

6. The use of CsV based on NLRP6 inflammasome regulation in diabetic vascular endothelial injury according to claim 5, characterized in that: The mass ratio of the mannitol to dextran 40 is 3-4:

1.

7. The use of CsV based on NLRP6 inflammasome regulation in diabetic vascular endothelial injury according to claim 1, characterized in that: The drug is an enteric-coated sustained-release tablet containing CsV.

8. The use of CsV based on NLRP6 inflammasome regulation in diabetic vascular endothelial injury according to claim 7, characterized in that: The enteric-coated sustained-release tablets comprise the following raw materials in parts by mass: 15-30 parts of hypromellose, 5-15 parts of ethyl cellulose, 20-40 parts of microcrystalline cellulose, 10-25 parts of lactose monohydrate, 0.5-1.5 parts of magnesium stearate, 1-3 parts of talc and an enteric coating material.

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