Use of demethylliberine as an nlrp3 inhibitor in the prevention of inflammation-related diseases

Abstract

The application provides application of demethylated berberine as an NLRP3 inhibitor in prevention of inflammation-related diseases, relates to the technical field of biological medicines, and discloses that the demethylated berberine DMB is used as an NLRP3 inflammasome inhibitor, the affinity of the DMB in direct combination with NLRP3 protein is utilized, the assembly and activation of the NLRP3 inflammasome are blocked from the source, caspase-1 activation, inflammatory factor maturation and release and pyroptosis are inhibited, and multi-level intervention on the NLRP3 related inflammation pathway is realized.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically the application of desmethylene berberine as an NLRP3 inhibitor in the prevention of inflammation-related diseases. Background Technology

[0002] NOD-like receptor protein 3 (NLRP3) inflammasomes are involved in the pathophysiological processes of various diseases. Abnormal activation of NLRP3 is closely related to a variety of diseases, including cardiovascular disease, sepsis, gout, stroke, and tumors, seriously threatening human health.

[0003] Structurally, the NLRP3 inflammasome is a multimeric cytoplasmic protein complex assembled from NLRP3, apoptosis-associated speckle-like protein (ASC), and cystein-asparate protease-1 (caspase-1). After assembly, the NLRP3 inflammasome undergoes autocatalysis, activating caspase-1. Activated caspase-1 mediates the maturation and release of IL-1β and IL-18, and cleaves gasdermin D (GSDMD) to produce its N-terminal domain, GSDMD-N. This domain can insert into the cell membrane and form micropores approximately 10-20 nm in diameter, prompting the release of intracellular inflammatory factors such as IL-1β, IL-18, and TNF-α, as well as lactate dehydrogenase (LDH), into the extracellular space. This amplifies the inflammatory response and ultimately leads to cell swelling, lysis, and even death.

[0004] Currently, inhibitors targeting the NLRP3 inflammasome are mainly small molecule compounds, but most are difficult to translate into clinical applications due to poor selectivity, significant toxicity, and low bioavailability. Therefore, developing highly effective, low-toxicity compounds that can effectively inhibit NLRP3 inflammasome activation is an important strategy for combating inflammatory diseases. Summary of the Invention

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides the application of demethylene berberine as an NLRP3 inhibitor in the prevention of inflammation-related diseases, solving the problem of the need for an inhibitor of the NLRP3 inflammasome with low toxicity and side effects.

[0006] (II) Technical Solution To achieve the above objectives, the present invention is implemented through the following technical solution: the application of demethylene berberine as an NLRP3 inhibitor in the prevention of inflammation-related diseases.

[0007] Preferably, the NLRP3 inflammasome-related diseases for the prevention of inflammation-related diseases are selected from cardiovascular diseases, sepsis, gout, stroke, or tumors.

[0008] Preferably, the demethylene berberine binds directly to the NLRP3 protein and inhibits the assembly and / or activation of the NLRP3 inflammasome.

[0009] Preferably, the demethylene berberine inhibits NLRP3 protein expression, inhibits the autocatalytic activation of caspase-1, inhibits the maturation and release of IL-1β and / or IL-18, and inhibits the cleavage of GSDMD.

[0010] Preferably, the binding of demethylene berberine to NLRP3 protein is concentration-dependent, with an equilibrium dissociation constant KD of 36.73 μM.

[0011] Preferably, the dosage form of the demethylene berberine is an injection, an oral solid dosage form, or an oral liquid dosage form.

[0012] Preferably, the oral solid dosage form is a tablet, capsule, or granule.

[0013] (III) Beneficial Effects This invention provides the application of desmethylene berberine as an NLRP3 inhibitor in the prevention of inflammation-related diseases. It has the following beneficial effects: 1. By using demethylene berberine (DMB) as an inhibitor of the NLRP3 inflammasome, and taking advantage of its affinity for the direct binding of the NLRP3 protein, we can block the assembly and activation of the NLRP3 inflammasome at its source, thereby inhibiting caspase-1 activation, maturation and release of inflammatory factors and pyroptosis, thus achieving multi-level intervention on the NLRP3-related inflammatory pathway.

[0014] 2. The demethylene berberine (DMB) used is a natural derivative and in vivo metabolite of berberine. It has significant advantages in terms of high safety and low toxicity and side effects, overcoming the shortcomings of existing small molecule NLRP3 inhibitors such as poor selectivity, high toxicity and side effects, and low bioavailability. It has good prospects for clinical translation.

[0015] 3. It provides specific applications of demethylene berberine (DMB) in the preparation of NLRP3 inflammasome inhibitors and drugs for the prevention and / or treatment of NLRP3-mediated inflammation-related diseases, offering new drug options for the prevention and treatment of cardiovascular diseases, sepsis, gout, stroke, and tumors. Attached Figure Description

[0016] Figure 1 Image showing the fluorescence signal of NLRP3 induced by DMB reversing LPS / Nigericin stimulation; Figure 2 Immunoblot analysis of DMB against LPS / Nigericin combined stimulation of AML-12 cells to induce NLRP3 protein expression; Figure 3 A graph showing the NLRP3 protein expression level induced in AML-12 cells by DMB in response to combined stimulation with LPS / Nigericin. Figure 4 The inhibitory effect of DMB on NLRP3 inflammasome activation and inflammatory factor release in J774A.1 macrophages induced by LPS / ATP combined stimulation; Figure 5 This demonstrates the inhibitory effect of DMB on LPS-induced pyroptosis in a rat model. Figure 6 DMB interacts with the NLRP3 protein in a concentration-dependent manner. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] like Figure 1-6 As shown in the embodiments of the present invention, desmethylene berberine is provided as an NLRP3 inhibitor in the prevention of inflammation-related diseases: 1. Experimental materials: Mouse mononuclear macrophage J774A.1 cells (catalog number: CL-0370) and their dedicated culture medium (catalog number: CM-0370) were purchased from Wuhan Pronosei Biotechnology Co., Ltd.; normal mouse hepatocytes AML-12 (catalog number: HTX2091) and their dedicated culture medium (catalog number: HTX2091M) were purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd.; Opti-MEM culture medium was purchased from Gibco (catalog number: 31985070); LPS (catalog number: L2880) was purchased from Sigma-Aldrich; and Nigericin (catalog number: N102401) was purchased from Shanghai Aladdin Biotechnology Co., Ltd. Recombinant mouse NLRP3 protein (catalog number: CSB-EP823181MO) was purchased from Huamei Biotechnology; and the fluorescent secondary antibody Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (A-11034) was purchased from Invitrogen. The LDH kit (catalog number: ab65391) and NLRP3 antibody were purchased from Abcam (catalog number: ab270449). The nuclear and cytoplasmic protein extraction kit (P0028) and BeyoGold™ 35mm glass-bottom confocal culture dishes (FCFC016-150pcs) were purchased from Beyotime Biotechnology Co., Ltd. The mouse IL-1b ELISA kit (catalog number: VAL601) was purchased from Novus, and the mouse TNF-a ELISA kit (catalog number: EMC102a.96) was purchased from Neobioscience.

[0019] 2. Experimental methods: 2.1 Detection of NLRP3 fluorescence intensity in AML-12 cells using immunofluorescence assay AML-12 cells were seeded at a density of 1.0 × 10⁵ cells / ml in confocal culture dishes. After cell adhesion the following day, the cells were stimulated with LPS at a final concentration of 1 μg / ml for 22 hours, followed by treatment with 10 μM Nigericin for 2 hours. The cells were then fixed with 4% paraformaldehyde for 15 minutes and permeabilized with buffer containing 0.1% Triton X-100. Next, the cells were blocked with 3% BSA for 1 hour, then co-incubated overnight at 4 °C with NLRP3 primary antibody, and finally reacted with the corresponding secondary antibody at room temperature for 60 minutes. The cell nuclei were counterstained with DAPI, and finally, fluorescence images were captured under a confocal microscope.

[0020] 2.2 Western blot analysis of NLRP3 protein expression in AML-12 cells AML-12 cells were seeded at a density of 1.5 × 10⁵ cells / ml in 6-well plates, following the same drug administration protocol as before. After LPS treatment, the culture supernatant was aspirated, and RIPA lysis buffer was added to each well. Lysis was performed on ice for 30 minutes, followed by centrifugation at 14000g for 20 minutes. The supernatant was collected, and protein concentration was determined. The lysate was added to 5× loading buffer and boiled for 5 minutes to denature the protein. After separation by SDS-PAGE gel electrophoresis, the protein samples were transferred to a PVDF membrane. After transfer, the membrane was blocked with 5% skim milk at room temperature for 1 hour, and then incubated overnight with NLRP3 primary antibody at 4°C. The next day, the membrane was washed three times with TBST buffer, reacted with secondary antibody at room temperature for 1 hour, and finally developed using ECL chemiluminescence solution. The grayscale values ​​were analyzed using Image software.

[0021] 2.3 LDH assay for J774A.1 cell membrane integrity J774A.1 cells were seeded in 96-well plates at a density of 1.5 × 10⁵ cells / ml. The following day, the culture medium was replaced with Opti-MEM containing 1% fetal bovine serum, and different concentrations of DMB were added for pre-incubation for 30 minutes, followed by stimulation with 1 μg / ml LPS for 5 hours. Then, 3 mM ATP was added for further treatment for 1 hour, and the results were analyzed using an Abcam kit. After drug administration, the 96-well plates were centrifuged at 4°C and 250 × g for 10 minutes. 50 μl of the supernatant was carefully transferred to a new 96-well plate, and 50 μl of the reaction solution provided in the kit was added. After mixing, the absorbance was measured at 490 nm.

[0022] 2.4 PCR method for detecting gene expression of NLRP3 inflammasome components in J774A.1 cells J774A.1 cells were seeded at a density of 1.5 × 10⁵ cells / ml in 24-well plates, with the drug addition protocol as described above. The culture medium was removed from each well (for ELISA detection), and 350 μl of RL lysis buffer was added to each well. Following the procedure outlined in the Magen RNA Extraction Kit (R4111), RNA was finally eluted with 80 μl of water. Total RNA extraction was performed using the Magen kit (R4111), with on-column DNase digestion kit (R4911) used to remove genomic DNA contamination. Quantitative PCR was performed using Toyobo's RNA-direct™ SYBR® Green Realtime PCR Master Mix. Each sample (including test and standard samples) was divided into three replicate wells, with a template-free control (NTC). The thermal cycling program was set as follows: 90℃ for 30 seconds, 61℃ for 20 minutes, and 95℃ for 60 seconds for reverse transcription; followed by 40 cycles of amplification: 95℃ for 15 seconds and 60℃ for 30 seconds, with signal acquisition. Using GAPDH as an internal reference gene, the relative expression levels of each target gene mRNA were calculated using the ΔΔCt method. The primers used are as follows:

[0023] 2.5 ELISA method for detecting the release of intracellular inflammatory factors in J774A.1 cells The release of IL-1β and TNF-a was determined using a double-antibody sandwich method. Cell supernatant from a 24-well plate was transferred into the wells, and after washing away unbound material, enzyme-linked polyclonal antibodies specifically targeting mouse IL-1β and TNF-a were added to the wells. After washing again to remove any unbound antibody-enzyme reagent, substrate solution was added to the wells, and OD values ​​were measured at 450 nm.

[0024] 2.6 Surface plasmon resonance (SPR) technique for detecting the direct interaction between DMB and NLRP3 NLRP3 was immobilized on a CM5 chip (Cytiva). A series of gradient-diluted DMB solutions were injected onto the chip surface at a flow rate of 30 μL / min. Each injection consisted of a 300-second binding phase followed by a 300-second dissociation phase, with real-time binding responses recorded throughout. The equilibrium dissociation constants, reflecting the binding affinity of each interaction, were calculated using BIAcore T200 evaluation software (Cytiva).

[0025] 2.7 Data Statistics Data were derived from at least three independent biological replicates and presented as mean ± standard error (mean ± SEM). Statistical analysis was performed using SPSS 25.0 (IBM), with p < 0.05 defined as significant. Differences among multiple groups were assessed using one-way ANOVA, with different post-hoc tests selected based on homogeneity of variance: LSD test for homogeneous data and Dunnett's T3 test for non-homogeneous data.

[0026] 3. Experimental Results 3.1 DMB reverses LPS / Nigericin-induced increases in NLRP3 fluorescence intensity and protein expression. LPS / Nigericin combined stimulation significantly enhanced the fluorescence intensity of NLRP3 in AML-12 cells, while treatment with 100 μM DMB effectively attenuated the fluorescence signal. Figure 1 Western blot results further confirmed this trend. Figure 2 , Figure 3 Compared with the control group (0.08±0.01), the NLRP3 protein expression level in the stimulation group was significantly increased to 0.61±0.07; after treatment with 100 μM DMB, the value dropped back to 0.45±0.04.

[0027] 3.2 DMB inhibits NLRPL3 inflammasome activation and the maturation and release of IL-1β and TNF-α. like Figure 4As shown, in the J774A.1 macrophage model, which was damaged by combined stimulation with LPS and ATP, which had a higher specificity, the release of LDH after LPS / ATP combined stimulation increased significantly from (100±1.50)% in the control group to (248.80±18.05)%. 30, 100, and 200 μM DMB reduced LDH to (177.89±10.67)%, (151.75±6.86)%, and (139.51±7.62)%, respectively. In addition, after LPS / ATP combined stimulation, the mRNA expression levels of NLRP3, caspase-1, IL-1β and IL-18 increased from (1.00±0.06), (1.00±0.01), (1.00±0.01) and (1.00±0.01) in the control group to (8.03±0.15), (2.39±0.10), (1296.20±56.40) and (4.16±0.15), respectively. DMB (200 μM) could decrease the above mRNA values ​​to (6.38±0.18), (1.42±0.05), (803.55±53.72) and (2.47±0.04), respectively, suggesting that DMB can inhibit the activation of the NLRP3 inflammasome. Regarding the release of inflammatory factors, 100 and 200 μM DMB significantly reversed the increase in IL-1β and TNF-α release induced by combined LPS / ATP stimulation.

[0028] 3.3 DMB inhibits LPS-induced pyroptosis in a rat model like Figure 5 As shown, after 18 hours of treatment with 6 mg / ml LPS, the expression of NLRP3, ASC, cleaved-caspase-1, and activated N-GSDMD proteins significantly increased in the model group. Both the low-dose and high-dose DMB groups significantly downregulated the expression levels of these proteins, with stronger efficacy than the positive control group. This suggests that DMB inhibits the activation of the NLRP3 inflammasome by regulating the NLRP3 / Caspase-1 / GSDMD-mediated pyroptosis pathway.

[0029] 3.4 DMB interacts with NLRP3 protein in a concentration-dependent manner.

[0030] like Figure 6 As shown, SPR analysis confirmed the existence of a direct and concentration-dependent interaction between DMB and NLRP3, with an equilibrium dissociation constant (KD) of 36.73 μM, indicating that the two have a binding affinity at the micromolar level, suggesting that the binding between DMB and NLRP3 is relatively stable.

[0031] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Application of desmethylene berberine as an NLRP3 inhibitor in the prevention of inflammation-related diseases.

2. The application of demethylene berberine as an NLRP3 inhibitor in the prevention of inflammation-related diseases according to claim 1, characterized in that: NLRP3 inflammasomes are used to prevent inflammation-related diseases, including cardiovascular disease, sepsis, gout, stroke, or cancer.

3. The application of demethylene berberine as an NLRP3 inhibitor in the prevention of inflammation-related diseases according to claim 1 or 2, characterized in that: The demethylene berberine binds directly to the NLRP3 protein and inhibits the assembly and / or activation of the NLRP3 inflammasome.

4. The application of demethylene berberine as an NLRP3 inhibitor in the prevention of inflammation-related diseases according to claim 3, characterized in that: The demethylene berberine inhibits NLRP3 protein expression, inhibits the autocatalytic activation of caspase-1, inhibits the maturation and release of IL-1β and / or IL-18, and inhibits GSDMD cleavage.

5. The application of demethylene berberine as an NLRP3 inhibitor in the prevention of inflammation-related diseases according to claim 4, characterized in that: The binding of demethylene berberine to the NLRP3 protein is concentration-dependent, with an equilibrium dissociation constant KD of 36.73 μM.

6. The application of demethylene berberine as an NLRP3 inhibitor in the prevention of inflammation-related diseases according to claim 5, characterized in that: The dosage form of the demethylene berberine is injection, oral solid dosage form, or oral liquid dosage form.

7. The application of demethylene berberine as an NLRP3 inhibitor in the prevention of inflammation-related diseases according to claim 6, characterized in that: The oral solid dosage form is a tablet, capsule, or granule.

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