Application of tetrahydroxybenzoic acid in inflammation inhibition

Abstract

The invention relates to application of tetrahydroxybenzoic acid in inhibition of inflammation, the structural formula of the tetrahydroxybenzoic acid is shown in the specification, the invention provides a novel natural product Nlrp3 inflammasome inhibitor, and the Nlrp3 inflammasome is inhibited from being started and activated by using the natural product, so that the inflammatory reaction mediated by the Nlrp3 inflammasome is inhibited, and the application of the Nlrp3 inflammasome inhibitor in the preparation of medicines for treating inflammation is realized. The natural product can be used as a potential drug for treatment or adjuvant treatment of inflammation and inflammation-related diseases.

Description

Technical Field

[0001] This invention relates to the application of 4-Hydroxybenzoic acid (4-HBA) in inhibiting inflammation, and particularly to the inhibition of inflammation by 4-HBA through the inhibition of inflammasome activation, belonging to the field of biomedicine. Background Technology

[0002] Excessive inflammatory response is a key factor contributing to infections, poisoning, tissue damage, and poor prognosis in many chronic degenerative diseases. Therefore, inhibiting excessive inflammatory responses is an important means of treating or adjuvant therapy for these diseases. Inflammasomes, especially the Nucleotide-binding domain-like receptor family pyrindomain containing 3 (Nlrp3), are activated in various inflammatory responses, and Nlrp3 activation has been shown to be one of the main causes of exacerbating inflammation. Inhibiting Nlrp3 activation has become an important measure for treating inflammation and inflammation-related diseases. Currently, the only mainstream Nlrp3 inhibitor used clinically is Tranilast. Tranilast limits Nlrp3 activation by inhibiting Nlrp3-Nlrp3 interactions, exhibiting good anti-inflammatory effects. However, tranilast may cause side effects on the liver, gastrointestinal tract, and nervous system during use, and its use in combination with the anticoagulant warfarin can affect the efficacy of warfarin, which limits the use of this drug to some extent. Developing novel Nlrp3 inflammasome inhibitors with mechanisms of action different from tranilast will be of significant practical importance for alleviating Nlrp3 inflammasome-driven inflammatory responses and related diseases. Complete activation of the Nlrp3 inflammasome involves two aspects: initiation and activation. The initiation phase mainly involves the transcription of genes encoding inflammasome components such as Nlrp3, caspase 1 (Casp1), interleukin 1β (Il1β), and interleukin 18 (Il18). In the lipopolysaccharide (LPS)-induced inflammatory response, LPS induces the initiation of the Nlrp3 inflammasome by activating the Tlr4 / Md2 / Cd14 complex, and downregulating Tlr4 / Md2 / Cd14 expression can effectively inhibit this process. Complete activation of Nlrp3 also requires post-initiation activation, which mainly involves the assembly of the Nlrp3 inflammasome and the activation of Casp1. Activated Casp1 cleaves the precursors of Il1β and Il18 into mature Il1β and Il18, inducing inflammation. Simultaneously, Casp1 can also cleave Gasdermin-D; the released N-terminal domain of Gasdermin-D mediates pyroptosis and promotes the release of Il1β and Il18, thus exacerbating the inflammatory response. Excessive intracellular reactive oxygen species (ROS) are a key trigger for the activation of Nlrp3 inflammasomes, and neutralizing intracellular ROS is an effective strategy to inhibit the activation of Nlrp3 inflammasomes.Therefore, theoretically, a substance that can both inhibit the initiation and activation of the Nlrp3 inflammasome would possess superior anti-inflammatory effects. Currently reported compounds and drugs rarely simultaneously inhibit both inflammasome initiation and activation. Even tranilast, the mainstream Nlrp3 inflammasome inhibitor currently used clinically, primarily works by inhibiting inflammasome activation. Therefore, the search for new Nlrp3 inflammasome inhibitors, especially safe and effective natural product inhibitors, holds great promise in the medical field. Summary of the Invention

[0003] The present invention aims to provide a novel natural product-based Nlrp3 inflammasome inhibitor. This natural product inhibits the initiation and activation of the Nlrp3 inflammasome, thereby suppressing the Nlrp3 inflammasome-mediated inflammatory response. This natural product can serve as a potential drug for the treatment or adjunctive treatment of inflammation and inflammation-related diseases. The application of tetrahydroxybenzoic acid in inhibiting inflammation is also discussed. The structural formula of the tetrahydroxybenzoic acid (4-HBA) is shown below: This invention reveals that 4-HBA can downregulate transcription by interfering with the binding of transcription factor PU.1 to the promoters of Tlr4 and Md2. Tlr4 and Md2 Transcription, thereby inhibiting LPS-induced Nlrp3 inflammasome initiation; this invention also demonstrates that 4-HBA itself has antioxidant activity, capable of neutralizing excessive intracellular ROS, thereby inhibiting Nlrp3 inflammasome activation. 4-HBA reduces the release of pro-inflammatory cytokine Il1β and decreases pyroptosis by inhibiting Nlrp3 inflammasome initiation and activation, thus suppressing excessive inflammation. To achieve the above effects, this invention provides the following technical solution: Animal experiments: 4-HBA was injected intraperitoneally at a dose of 50 mg / kg body weight, while the control group received an equal volume of solvent. Two hours later, a lethal dose (50 mg / kg body weight) of LPS was injected intraperitoneally to simulate the systemic inflammatory response induced by endotoxin and subsequent death due to the cytokine storm. Survival curves of the experimental animals were recorded. Alternatively, tissue samples were taken at different time points after LPS injection to detect the transcription and levels of pro-inflammatory cytokines. Western blot was used to detect the activation of the pyroptosis marker protein Gasdmd, and tissue HE staining was used to evaluate tissue damage. Cell experiments: Bone marrow-derived macrophages (BMDMs) were stimulated with 100 ng / ml LPS to prepare a BMDMs Nlrp3 inflammasome initiation model, with or without 4-HBA (2 mM). Six hours later, qPCR was used to detect inflammasome initiation-related genes (…). Nlrp3, Casp1 , Il1β Transcription, detection Tlr4 , Md2 , Cd14 Transcription. BMDMs were stimulated with 100 ng / ml LPS for 3 hours (with or without 2 mM 4-HBA) followed by 10 μM Nigeriamycin for another 3 hours to establish a fully activated Nlrp3 inflammasome model. Cell supernatant was collected to detect LDH release to evaluate cell death, ELISA was used to detect the release of the pro-inflammatory cytokine Il1β, and the number of Asc speck cells formed was collected by immunofluorescence to evaluate Nlrp3 inflammasome activation. Western blot was used to detect the activation of the pyroptosis marker protein Gasdmd. Beneficial effects

[0004] This invention provides a novel anti-inflammatory natural product, offering a new potential drug for suppressing inflammatory responses based on inhibiting Nlrp3 inflammasome activation. Using the 4-HBA involved in this invention, it is possible to disrupt the transcription factor PU.1 and... Tlr4 / Md2 The promoter binding inhibits LPS-induced Nlrp3 initiation, while the antioxidant activity of 4-HBA itself neutralizes excess intracellular ROS, thereby inhibiting Nlrp3 inflammasome activation. Therefore, the 4-HBA involved in this invention can both inhibit Nlrp3 inflammasome initiation and activation, suppressing the inflammatory response driven by the Nlrp3 inflammasome. Furthermore, the 4-HBA involved in this invention is a natural phenolic acid, abundant in various foods such as nuts, seeds, leafy green vegetables, and tea. Reports have also confirmed that 4-HBA can even be used during pregnancy, thus exhibiting higher safety compared to other non-natural, chemically synthesized drugs. Attached Figure Description

[0005] Figure 1 This is a graph showing the results of how tetrahydroxybenzoic acid (4-HBA) of the present invention inhibits excessive inflammatory response and alleviates death induced by lethal doses of endotoxin (LPS); wherein Figure 1 A represents the protective effect of 4-HBA against lethal doses of LPS-induced death in experimental mice, with the vertical axis representing survival rate and the horizontal axis representing time after LPS injection. Figure 1 B is HE staining showing the protective effect of 4-HBA against LPS-induced lung tissue damage in experimental mice; Figure 1 C represents the effect of qPCR detection of 4-HBA pretreatment on LPS-induced transcription of pro-inflammatory cytokines in lung tissue. The vertical axis represents the relative transcription fold, and the horizontal axis represents different pro-inflammatory cytokines. Figure 1D represents the effect of ELISA detection of 4-HBA pretreatment on serum and lung tissue pro-inflammatory cytokine Ilβ levels. The vertical axis represents Ilβ content, and the horizontal axis represents the sample type after LPS treatment. Figure 2 This is a diagram showing the results of the present invention, which uses tetrahydroxybenzoic acid (4-HBA) to inhibit the activation of the Nlrp3 inflammasome in BMDMs, inhibit the release of pro-inflammatory cytokines, and inhibit pyroptosis; in which... Figure 2 A shows the inhibitory effect of 4-HBA on the formation of Asc speck, a marker of Nlrp3 inflammasome activation, as demonstrated by immunofluorescence. Figure 2 B represents the inhibitory effect of 4-HBA on the release of the pro-inflammatory cytokine Il1β from BMDMs as detected by ELISA. The vertical axis represents Ilβ content, and the horizontal axis represents different experimental groups. Figure 2 C represents the protective effect of 4-HBA against cell death as detected by the LDH release assay. The vertical axis represents the cell death rate, and the horizontal axis represents the experimental group. Figure 2 D represents the effect of 4-HBA on GsdmD activation detected by Western blot, where different lanes represent different samples in different groups, and β-Actin is an internal reference. Figure 3 The present invention relates to tetrahydroxybenzoic acid (4-HBA), which interferes with transcription factor PU.1 and... Tlr4 / Md2 The resulting diagram showing the inhibition of Nlrp3 inflammasome activation of BMDMs by promoter binding; in which... Figure 3 A represents the Nlrp3 inflammasome initiation status detected by qPCR, with the ordinate representing the relative transcription fold and the abscissa representing the marker genes for Nlrp3 inflammasome initiation. Figure 3 B represents the molecular docking technique used to predict the binding of 4-HBA to the ETS domain of transcription factor PU.1; Figure 3 C represents the effect of 4-HBA on Tlr4 and Md2 transcription by qPCR after intervention with PU.1 inhibitor. The vertical axis represents the relative transcription fold and the horizontal axis represents the gene name. Figure 3 D represents the effect of 4-HBA on the initiation of Nlrp3 inflammasomes by qPCR after intervention with PU.1 inhibitors. The vertical axis represents the relative transcription fold, and the horizontal axis represents the marker genes for the initiation of Nlrp3 inflammasomes. Figure 4 This diagram illustrates the effect of tetrahydroxybenzoic acid (4-HBA) in neutralizing intracellular ROS and inhibiting Nlrp3 inflammasome activation. Figure 4 A represents the antioxidant activity of 4-HBA itself, with the vertical axis representing cell viability and the horizontal axis representing experimental groups; Figure 4 B represents the flow cytometry analysis of 4-HBA's clearance of intracellular ROS, with the vertical axis representing cell number and the horizontal axis representing the fluorescence intensity of the ROS probe. Figure 4C represents the effect of 4-HBA on Il1β release after Nlrp3 inflammasome activation by ELISA under the condition of inhibiting Nlrp3 inflammasome initiation. In other words, it evaluates whether 4-HBA has an inhibitory effect on Nlrp3 inflammasome activation when Nlrp3 inflammasome initiation is inhibited. The vertical axis represents the Il1β content in cell supernatant, and the horizontal axis represents the experimental group. Detailed Implementation

[0006] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments to enable those skilled in the art to understand the present invention, but the scope of protection of the present invention is not limited thereto. The hydroxybenzoic acid (4-HBA) mentioned in the embodiments was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. The experimental animals mentioned in the embodiments were male C57BL / 6J mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The bone marrow-derived macrophages mentioned in the embodiments were isolated from male C57BL / 6J mice and cultured using IMDM medium. Example 1:

[0007] Determining the ability of 4-HBA to inhibit excessive inflammatory response and alleviate mortality in experimental mice induced by lethal dose of endotoxin (LPS). (1) Six- to eight-week-old male C57BL / 6J mice were intraperitoneally injected with 4-HBA at a dose of 50 mg / kg body weight two hours before LPS stimulation, while the control group was injected with an equal volume of solvent. (2) Two hours later, the experimental mice were intraperitoneally injected with a lethal dose (50 mg / kg body weight) of LPS to establish an endotoxin shock mortality model. (3) The mortality of mice in each group was recorded, and survival curves were plotted. (4) Sixteen hours after LPS injection, mouse lung tissue was collected, fixed with paraformaldehyde, and subjected to pathological analysis by HE staining. (5) Four hours after LPS injection, mouse serum and lung tissue were collected, and qPCR was used to detect inflammatory cytokines in lung tissue. Tnf , Il6 , Il1β Transcription, and ELISA detection of Il1β content in serum and lung tissue. Example 2

[0008] 4-HBA inhibits the activation of Nlrp3 inflammasomes in bone marrow-derived macrophages (BMDMs), reduces the release of pro-inflammatory cytokine Il1β, and decreases the determination of pyroptosis capacity. (1) Take the tibia and femur of 6-8 week old male C57BL / 6J mice, cut off the epiphyseal end, and flush out the bone marrow with sterile PBS (containing 1% penicillin-streptomycin) under aseptic conditions. Collect the bone marrow, blow it apart, and centrifuge at 450 g for 5 minutes to collect bone marrow cells. (2) Use red blood cell lysis buffer (purchased from Beyotime Biotechnology Co., Ltd.) to lyse the red blood cells. Wash the remaining cells twice with sterile PBS and filter them through a 200-mesh nylon net to collect the filtrate. (3) After centrifugation, discard the supernatant and resuspend the precipitated cells with an appropriate volume of IMDM (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to a cell concentration of 210. 6 (4) The resuspended cells were seeded in a 10 cm cell culture dish and M-CSF was added to a final concentration of 20 ng / ml to induce BMDMs formation. After three days of culture, the medium was replaced and cultured for another six days. The adherent cells were then BMDMs. (5) Construction of the Nlrp3 inflammasome initiation model: BMDMs were treated with 100 ng / ml LPS for 6 hours to complete the Nlrp3 inflammasome initiation. If 4-HBA treatment was required, 4-HBA at a final concentration of 2 mM was added to the medium along with LPS. (6) Construction of the Nlrp3 inflammasome activation model: BMDMs were treated with 100 ng / ml LPS for 3 hours and then treated with 10 μM Nigeriamycin for another 3 hours to complete the Nlrp3 inflammasome activation. If 4-HBA treatment was required, 4-HBA at a final concentration of 2 mM was added to the medium along with LPS. (7) Construction of Nlrp3 inflammasome activation model: BMDMs were treated with the Myd88 inhibitor TJ-M2010-5 at a final concentration of 10 μM for 48 hours before LPS stimulation. Then, Nlpr3 inflammasome activation was induced according to the steps in (6) (at this time, the initiation of Nlpr3 inflammasome was inhibited). Cell supernatant was collected and Il1β release was detected by ELISA to evaluate the activation of Nlrp3 inflammasome.

Claims

1. The application of tetrahydroxybenzoic acid in inhibiting inflammation, wherein the structural formula of the p-hydroxybenzoic acid is shown below:

2. The use of tetrahydroxybenzoic acid as described in claim 1 as a potential medicine in the prevention and treatment of inflammation and inflammation-related diseases.

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