Application of HTR2b gene as target spot in preparation of medicine for treating NETs-driven diseases

Abstract

The invention relates to the technical field of medicines, in particular to application of an HTR2b gene as a target spot in preparation of a medicine for treating NETs-driven diseases. The NETs driven diseases are sepsis and / or psoriasis. The invention proposes that the HTR2b receptor stimulant can specifically inhibit the formation of NETs for the first time, effectively alleviates inflammatory response and tissue damage of sepsis and psoriasis, shows a better treatment effect, and does not have side effects. Compared with existing methods for inhibiting ROS generation, inhibiting PAD4 and the like, the HTR2b receptor agonist has higher specificity, serious interference on basic functions of neutrophils is avoided, and adverse reactions are reduced. Therefore, the HTR2b receptor agonist can be used for preparing the medicine for treating NETs driven diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a... HTR2b Application of genes as targets in the preparation of drugs to treat NETs-driven diseases. Background Technology

[0002] Neutrophil extracellular traps (NETs) are reticular structures released by neutrophils in response to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), playing a crucial role in infectious and autoimmune diseases. Neutrophils and platelets work together as motile sentinel cells, maintaining host homeostasis and protecting against aseptic and infectious damage. However, under pathological conditions, this coordinated action can exacerbate harmful effects and persistently trigger inflammatory cascades, ultimately disrupting host homeostasis. These reticular structures, composed of decondensed chromatin, citrullinated histones, and excessive production of cytotoxic granule proteins (such as neutrophil elastase and myeloperoxidase (MPO)), disrupt immune homeostasis. Excessive neutrophil extracellular traps can damage tissues, release cytotoxic mediators, trigger coagulation, and amplify excessive inflammatory responses by enhancing pro-inflammatory signaling cascades. This pathological process is involved in the development of various diseases, including sepsis, psoriasis, and thrombotic inflammatory diseases. Sepsis is a systemic inflammatory response syndrome caused by infection. Uncontrolled release of NETs can damage tissues, exacerbate cytokine storms, and form immune thrombosis, seriously threatening the patient's life. Psoriasis is a chronic inflammatory skin disease in which NETs can promote epithelial cell proliferation and mediate the prolongation of inflammation.

[0003] Current treatments for NET abnormalities are limited, such as inhibiting ROS generation, suppressing PAD4, or using DNases. However, these methods suffer from serious adverse reactions or limited therapeutic effects. For example, inhibiting the NADPH oxidase complex (NOX2) may lead to basic neutrophil dysfunction, exacerbating autoimmune diseases or triggering infections; drugs that inhibit PAD4 lack specificity and may interfere with the function of other proteins, leading to prolonged infections; DNases can only degrade DNA in NETs and cannot reduce damaging mediators such as proteases and toxic histones. Therefore, there is an urgent need to develop new therapeutic targets and drugs. Given the central role of the neutrophil extracellular trap in diseases such as sepsis and psoriasis, identifying the signals that regulate the formation of the neutrophil extracellular trap is crucial for developing precision targeted therapies.

[0004] Research results indicate that platelet-5-hydroxytryptamine- HTR2b The axon serves as a physiological inhibitory mechanism for neutrophil extracellular traps (NETs), therefore, HTR2b It could serve as a potential target for treating acute and chronic diseases primarily driven by NETs. Summary of the Invention

[0005] The purpose of this invention is to provide the application of HTR2b receptor agonists in the preparation of drugs for treating NETs diseases, thereby reducing inflammatory response and tissue damage, improving therapeutic efficacy and reducing adverse reactions by regulating NET formation.

[0006] The present invention achieves the above objectives through the following technical solutions: HTR2b Application of genes as targets in the preparation of drugs to treat NETs-driven diseases.

[0007] As a further improvement of the present invention, through drug regulation HTR2b Gene expression is used to suppress the formation of extracellular traps (NETs) in neutrophils.

[0008] As a further improvement of the present invention, the drug is HTR2b Receptor agonists.

[0009] As a further improvement of the present invention, the... HTR2b Receptor agonists are incorporated into pharmaceutically acceptable carriers to create clinically usable drug formulations.

[0010] As a further improvement of the present invention, the HTR2b The receptor agonist is BW723C86.

[0011] As a further improvement of the present invention, the NETs-driven disease is sepsis.

[0012] As a further improvement of the present invention HTR2b Genes are used as targets in the development of drugs to treat sepsis, aiming to reduce lung damage caused by sepsis.

[0013] As a further improvement of the present invention, the NETs-driven disease is psoriasis.

[0014] As a further improvement of the present invention HTR2b When genes are used as targets in the preparation of drugs for treating psoriasis, the administration method is topical application.

[0015] As a further improvement of the present invention, the drug is used to reduce the area of ​​psoriasis, epidermal thickness, and inflammatory response.

[0016] The beneficial effects of this invention are as follows: This invention discovers HTR2b It can serve as a potential therapeutic target for NETs-driven pathology. HTR2bThe agonist BW723C86 reduced sepsis-induced mortality, alleviated lung injury, and decreased NET deposition and inflammatory cytokine levels. This organ-specific protective effect may stem from the significant lung damage caused by NETs during sepsis. In psoriasis, NETs, ​​triggered by cytokines and saturated fatty acids, release LL37 and NET-associated RNA to amplify the inflammatory response; topical application of BW723C86 reduced psoriasis-induced inflammation, decreased epidermal hyperplasia, NET deposition, and the psoriasis area and severity index score.

[0017] Compared with existing methods such as inhibiting ROS generation and PAD4 inhibition, HTR2b receptor agonists have higher specificity, avoid serious interference with the basic functions of neutrophils, and reduce the occurrence of adverse reactions.

[0018] In the treatment of sepsis, it can be administered via subcutaneous injection; in the treatment of psoriasis, it can be administered via topical application. It is convenient to use and suitable for different disease scenarios. Attached Figure Description

[0019] Figure 1 Platelets limit the formation of extracellular traps (NETs) for neutrophils.

[0020] (A) Observation of the formation of extracellular neutrophil traps (NETs) during PMA-induced co-culture of resting platelets using time-lapse imaging; (B) Time-lapse imaging (A) showing neutrophil nuclear disintegration time (left), percentage of surviving neutrophils (middle), and relative mean fluorescence intensity (MFI) of SytoxGreen (SG) / DRAQ5 (right); (C) Schematic diagram of the neutrophil-platelet Transwell co-culture system; (D) Representative immunofluorescence images of NETs, ​​labeled with DNA (DAPI, blue), myeloperoxidase (MPO, red), and citrullinated histone H3 (CitH3, pink) (n=6); (E) Representative bright-field images and mean fluorescence intensity of SytoxGreen fluorescence in the lower chamber.

[0021] Figure 2 Platelet-derived serotonin inhibits the formation of extracellular traps (NETs) on neutrophils.

[0022] (A) Soluble components of human primary neutrophils stimulated with PMA and platelets, showing the relative fluorescence intensity (RFI) of SytoxGreen relative to T0 (n=9); (B) Human primary neutrophils stimulated with PMA and co-cultured with gradient concentrations of serotonin (ranging from 100 μM to 0.1 μM) for 4 hours, showing the RFI of SytoxGreen relative to T0 (n=10); (C) Observation of PMA-induced NET formation by time-lapse imaging (during co-culture with 100 mM serotonin); (D) Time-lapse imaging (B) showing neutrophil nuclear disintegration time (left), percentage of surviving neutrophils (middle), and relative mean fluorescence intensity (MFI) of SytoxGreen (SG) / DRAQ5 (right); (E) Human platelets co-cultured with different stimulants, showing the residual serotonin concentration in platelet pellet (n=3); (F) Wild-type or TPH1- / - Schematic diagram of platelet-bone marrow (BM)-derived neutrophil co-culture experiment; (G) shows representative images and mean fluorescence intensity of bright field and SytoxGreen fluorescence.

[0023] Figure 3 For 5-hydroxytryptamine HTR2b Limit the formation of extracellular traps (NETs) on neutrophils.

[0024] (A) Primary human neutrophils stimulated by PMA were co-cultured with 100 μM 5-hydroxytryptamine and gradient concentrations of selective 5-HT receptor antagonists for 4 hours. The figure shows the relative fluorescence intensity (RFI) of SytoxGreen relative to T0 (n=7); (B) Schematic diagram of the isolation and culture process of BMDNs containing 100 μM 5-hydroxytryptamine in vitro; (C) From HTR2bf / f and HTR2b- / - Mouse BMDNs were co-cultured with and without 5-hydroxytryptamine after PMA stimulation, and the images show representative bright-field images and SytoxGreen fluorescence; (D) relative fluorescence intensity (RFI).

[0025] Figure 4 for HTR2b As a protective target against sepsis.

[0026] (A) administered via intraperitoneal injection HTR2bf / f or HTR2b- / -(a) The median lethal dose (LD50) of 100 μL of cecal serous fluid (CS) in mice was determined, and survival at 48 hours was recorded (n=10 mice per group); (b) Lung tissue was collected 12 hours after administration of CS or PBS, and pathological changes and damage were assessed by hematoxylin-eosin (HE) staining (n=6); (c) Representative immunofluorescence images of NETs in lung tissue were shown, displaying staining results for DNA (DAPI, blue), myeloperoxidase (MPO, green), and citrullinated histone H3 (CitH3, red), as well as mean fluorescence intensity of neutrophils and NET formation rate; (d) Bronchoalveolar lavage fluid (BALF) was collected 12 hours after administration of CS, and quantitative results of total cell count in BALF sediment were shown (n=8); (eH) Quantitative results of total protein (E) and specified cytokine (FH) concentrations in cell-free BALF were shown (n=8).

[0027] Figure 5 BW723C86 was used to improve experimental sepsis-related lung injury.

[0028] (A) After inducing sepsis by intravenous injection of 200 μL of CS, the 5-hydroxytryptamine level in platelets of 106 mice was measured at 0, 6, and 12 hours (n=7 mice at each time point); (B) After administration of 200 μL of CS, wild-type mice were subcutaneously injected with the solvent or BW723C86 (1 mg / kg body weight) at 0, 6, and 12 hours, and the survival rate was recorded for 48 / 72 hours (n=6 mice at each time point); (C) Wild-type mice were injected with CS at 0 hours, BW723C86 at 6 hours, and euthanized at 12 hours. Lung tissues were collected from septic mice, and pathological changes and damage were assessed by HE staining (n=6); (D) Representative immunofluorescence images of NETs in lung tissues are shown, displaying the staining results of DNA (DAPI, blue), myeloperoxidase (MPO, green), and citrullinated histone H3 (CitH3, red); (E) Bronchoalveolar lavage fluid (BALF) was collected 12 hours after CS administration, showing the quantitative results of total cell count in BALF sediment (n=8); (FI) The concentrations of total protein (F) and specified cytokines (GI) in cell-free BALF were quantified (n=8).

[0029] Figure 6 Topical application of BW723C86 can alleviate IMQ-induced psoriasis and improve physiological outcomes.

[0030] (A) Phenotypic features of the ears of mice treated with IMQ cream in combination with DMSO control or 0.5% (w / v) BW723C86; (B) Ear thickness of mice in the DMSO control group or BW723C86 treatment group measured daily according to the specified method (n=12); (C) Representative histopathological images of the ears and quantitative results of epithelial thickness (n=7); (D) Representative immunofluorescence images of NETs in the ears, showing the staining results of DNA staining (DAPI, blue), myeloperoxidase (MPO, green), and citrullinated histone H3 (CitH3, red); (E) Immunoblot analysis of ear tissue for CitH3 expression was performed simultaneously (n=3), with each band representing a different animal, and GADPH used as a loading control; (F) The figure shows the phenotypic features of the dorsal skin of mice after 5 days of IMQ treatment compared with the DMSO control group or BW723C86 group; (G) Line graph showing the dynamic changes of PASI score of mouse dorsal skin lesions with the number of days of modeling (1-5 days); (H) showing representative histopathological images of the ear and quantitative results of epithelial thickness (n=5); (IJ) Immunofluorescence imaging of mouse dorsal tissue and immunoblotting analysis of H3Cit expression (n=3), with each band representing different animals, and GADPH as a sample loading control. Detailed Implementation

[0031] The following specific embodiments are only used to further illustrate this application and should not be construed as limiting the scope of protection of this application. Those skilled in the art can make some non-essential improvements and adjustments to this application based on the above application content.

[0032] At least one embodiment of the present invention discloses HTR2b The application of genes as targets in the preparation of drugs to treat NETs-driven diseases, through drug regulation. HTR2b Gene expression is used to suppress the formation of extracellular traps (NETs) in neutrophils.

[0033] At least one embodiment of the present invention discloses HTR2b Receptor agonists are used in the preparation of treatments for NETs-driven diseases.

[0034] HTR2b Receptor agonists are substances that can bind to and activate serotonin receptors. HTR2b Small molecule compounds or biological agents, such as BW723C86.

[0035] In this invention, NETs drive the diseases sepsis and / or psoriasis.

[0036] HTR2bWhen HTR2b receptor agonists are used to prepare drugs for treating sepsis, the preferred route of administration is subcutaneous injection. The dosage of the HTR2b receptor agonist in the drug is 0.5-5 mg / kg, preferably 1 mg / kg. The drug dosage form includes injections or suspensions.

[0037] HTR2b When receptor agonists are used to prepare drugs for treating psoriasis, the preferred method of administration is topical application. HTR2b The dose of the receptor agonist is 0.5% concentration, and the drug dosage form includes suppositories, ointments, or topical preparations.

[0038] The present invention HTR2b Receptor agonists may also include pharmaceutically acceptable additives, such as starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silica, and hydrogen phosphate. Other additives include calcium, lactose, mannitol, syrup, rubber arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, omega-4, sodium glycolate starch, carnauba starch, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, and may also include glucose, sorbitol, and talc.

[0039] In actual clinical application HTR2b Receptor agonists can be administered in a variety of oral and parenteral dosage forms. When formulating them, diluents or excipients are used, such as commonly used fillers, expanders, binders, wetting agents, disintegrants, and surfactants.

[0040] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms contain at least one excipient, such as starch, calcium carbonate, and water, in the mixed extract of the present invention. It can be prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Oral liquid dosage forms include suspensions, liquid solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavorings, and preservatives, can also be included.

[0041] Preparations intended for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous and suspension solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As a base for suppositories, Witepsol, polyethylene glycol, Tween 61, cocoa butter, lauric acid, and glycerin gelatin can be used.

[0042] The present invention HTR2bReceptor agonists can be administered orally or parenterally as desired. When administered parenterally, the injection method can be selected from topical application or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrapleural injection. The dosage can vary depending on the patient's or study subject's weight, age, sex, health status, diet, time of administration, method of administration, excretion rate, and disease severity.

[0043] The present invention HTR2b The dosage of receptor agonists varies depending on the patient's or test subject's weight, age, sex, health status, diet, timing of administration, method of administration, excretion rate, and severity of disease.

[0044] The present application will now be described in further detail with reference to the embodiments and accompanying drawings. Unless otherwise specified, the methods used in this invention are conventional methods known to those skilled in the art; the reagents and materials used are commercially available products; and the instruments used are conventional instruments known to those skilled in the art.

[0045] Example 1: HTR2b Therapeutic effects of receptor agonists on animal models of sepsis 1. Myeloid specificity HTR2b Gene knockout mice and Tph1 Construction of gene knockout mice HTR2b The flox / flox mouse was developed by the Shanghai Model Organisms Center, Inc. (Shanghai, China). HTR2b The gene is located on mouse chromosome 1 and contains four exons. Exon 2 was selected as the conditional knockout region; deletion of this exon will result in… HTR2b Loss of function. By HTR2b By crossing flox / flox mice with transgenic mice expressing the S100A8 promoter-driven Cre recombinase, myeloid-specific HTR2b knockout mice were successfully constructed.

[0046] Tph1 Gene knockout mice were prepared by Cyagen Biosciences (Suzhou) Co., Ltd. Tph1 The gene is located on mouse chromosome 7, and exons 3 to 10 were selected as the target region, which contains a 1043 bp coding sequence.

[0047] The experiment used 8-10 week old control mice ( HTR2b flox / floxS100A8-Cre, abbreviated as HTR2b fl / fl), myeloid specificity HTR2b Gene knockout mice ( HTR2bflox / floxS100A8-Cre+, abbreviated as flox / floxS100A8-Cre+ HTR2b - / -) TPH1 Gene knockout mice and wild-type C57BL6J mice were used. All experimental procedures involving animals were approved by the Ethics Committee of Southern Medical University.

[0048] A double staining strategy was employed. First, isolated human neutrophils were stained with the cell-permeable DNA dye DRAQ5. Then, 2.5 × 10⁻⁶ cells were... 5 Neutrophils were co-cultured with 500 µL of RPMI 1640 medium (containing 4% fetal bovine serum) without phenol red, with the above medium added separately at 5 × 10⁻⁶ mg / L. 7 Platelets or PBS were mixed with 500 µL of serum, along with the cell-impermeable DNA dye SytoxGreen and the NETs agonist PMA.

[0049] Data are expressed as mean ± standard deviation (SD) and analyzed using GraphPad Prism 8.0.2 software (GraphPad Software Corporation). Normality of the data was assessed using the Shapiro-Wilk test. Normally distributed data were analyzed using the Student's t-test, one-way or two-way ANOVA. P < 0.05 was considered statistically significant.

[0050] 2. Platelet-restricted neutrophil extracellular trap formation Live-cell imaging was used to continuously observe the formation of extracellular neutrophil traps (NETs) and investigate the regulatory role of resting platelets in the kinetics of NET formation. This method can quantify single-cell morphological changes in real time, including nuclear expansion, altered plasma membrane permeability, and extracellular chromatin release; these changes all occurred after stimulation with the classic NET-inducing stimulant phorbol 12-myristate 13-acetate (PMA). Co-culture with resting platelets significantly delayed NET formation. Figure 1 A), characterized by delayed neutrophil nuclear rupture, slowed cell death dynamics, and reduced extracellular chromatin decondensation ( Figure 1 B), indicating that platelets have an inhibitory effect on NETs. PMA activates platelets through the protein kinase C-protein kinase D2 pathway. After platelet activation, its interaction with neutrophils involves cell secretion and the formation of platelet-neutrophil complexes (PNCs). Giemsa staining was performed on samples co-cultured for 30 minutes to elucidate the mechanism of platelet-neutrophil interaction and to identify free neutrophils and PNCs (B). Figure 1B). To differentiate between direct cell-to-cell contact and soluble factor-mediated effects, a Transwell co-culture system with a pore size of 0.4-μm was used. This system physically blocks platelet transmembrane migration but allows soluble factor exchange. Figure 1 C). In both the co-culture system and the Transwell system, platelets inhibited nuclear disaggregation (C). Figure 1 D) and reduce extracellular DNA release ( Figure 1 E). These results indicate that the platelet-induced anti-NETsotic phenotype is primarily mediated by soluble factors released from activated platelets, rather than by direct PNC formation.

[0051] 3. Platelet-derived serotonin limits the formation of extracellular traps on neutrophils. We systematically evaluated key components of platelet secretion in PMA-stimulated human primary neutrophils: 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, ATP, ADP, and platelet factors.

[0052] SYTOX Green quantitative PCR analysis revealed that, under the same experimental conditions, serotonin significantly inhibited NETs-related extracellular DNA release, while 5-hydroxyindoleacetic acid, ATP, ADP, and platelet factor 4 had minimal effects on NET formation. Figure 2 A); 5-hydroxytryptamine dose-dependently inhibits NET formation ( Figure 2 (B) However, serotonin failed to inhibit NOX-independent NET formation triggered by HMGB1, P-selectin, and nigerixin. These results indicate that serotonin is a key inhibitor of NOX-dependent NET formation, highlighting its role in regulating NET formation dynamics.

[0053] Live-cell imaging also showed that, consistent with platelet-mediated NET inhibition, serotonin (100 μM) treatment delayed PMA-induced nuclear membrane rupture, reduced terminal cell death, and attenuated chromatin extrusion compared to the untreated control group. Figure 2 C and D), whose biological activity is similar to that of intact platelets. The highest serotonin levels in the body (approximately 95%) are generated by enterochromaffin cells in the gastrointestinal mucosa via the rate-limiting enzyme tryptophan hydroxylase 1 (C, D). TPH1 Synthesis. It used a method that could not synthesize serotonin. TPH1 Platelets from gene knockout mice. Wild-type ( WT Bone marrow-derived neutrophils (BMDNs) and WT or TPH1 - / - Platelet co-culture ( Figure 2 F). In TPH1 - / - In the presence of platelets, SYTOX Green signaling is enhanced, revealing the effect of using... WTPlatelets show increased ambivalence compared to NETs ( Figure 2 G). This genetic evidence establishes serotonin as a key molecular switch in platelet-mediated NETs regulation.

[0054] The comprehensive results indicate that platelet-derived serotonin (5-HT) acts as a key physiological regulator of neutrophil extracellular trap (NET) formation by delaying nuclear membrane disintegration and chromatin efflux. The differential effects between wild-type and 5-HT-deficient platelets reveal a bidirectional regulatory axis in the platelet-neutrophil interaction, where 5-HT availability directly determines the balance between NET induction and inhibition.

[0055] 4. 5-Hydroxytryptamine via HTR2b Limiting the formation of extracellular traps for neutrophils Serotonin, as a neurotransmitter, plays a crucial role in peripheral tissues and regulates the immune system. However, the specific receptor on neutrophils that mediates serotonin-induced inhibition of neutrophil extracellular trap (NET) formation remains unclear. Currently, seven serotonin receptor families (5-HT1–7) and various serotonin receptor subtypes have been reported. To accurately identify the specific receptor subtype that inhibits NET formation, primary human neutrophils were pharmacologically screened using selective antagonists. HTR2b Receptor antagonists eliminated the inhibitory effect of serotonin on NETs, ​​indicating that... HTR2b (rather than) HTR1a , HTR1b / d , HTR2a / c , HTR3 , HTR4 , HTR5a , HTR6 or HTR7 ) is a key membrane receptor that mediates the inhibition of NETs by serotonin ( Figure 3 A).

[0056] An HTR2b- / - mouse model was constructed to achieve myeloid cell lineage. HTR2b Conditional knockout ( Figure 3 B). In HTR2bf / f In mouse-isolated BMDNs, serotonin significantly reduced PMA-induced neutrophil extracellular trapping web (NET) formation. However, in HTR2b- / - No such inhibitory effect was observed in mice. Figure 3 (C, D). These results strongly suggest that, HTR2b The role of serotonin in delaying NET formation is indispensable.

[0057] 5. HTR2b As a protective target against sepsis For evaluation HTR2bEstablishing myeloid-specific correlations in pathophysiological relationships of NETs-driven diseases. HTR2b Gene knockout mice were subjected to cecal lavage fluid-induced multimicrobial sepsis. Myeloid HTR2b The defect significantly increased sepsis-related mortality. Figure 4 A), which highlights the protective role of neutrophil-derived HTR2b in systemic infection. To further elucidate... HTR2b The impact on sepsis-related organ damage focused on the lungs—a major target organ for NET-mediated damage in sepsis. Among sepsis-related injuries, the lungs are most significantly affected by NETs. We observed... HTR2b The defect exacerbated lung injury and inflammatory cell infiltration in the lungs. Figure 4 B), accompanied by increased NETs deposition and increased neutrophil infiltration ( Figure 4 C), but HTR2bfl / fl and HTR2b- / - (There was no significant difference in BALF cell count between groups) Figure 4 D). Accompanying other pathological changes is, HTR2b- / - In mice, bronchoalveolar lavage fluid (BALF) parameters deteriorated, including increased protein leakage and elevated levels of tumor necrosis factor α, interleukin (IL)-6, and IL-1β. Figure 4 E–H), and HTR2bfl / fl Compared to mice. In contrast, serotonin -HTR2b The axis has limited protective effects on other organs (such as liver, kidney and circulatory system) in cecal lavage fluid-induced lung injury.

[0058] 6. Targeted HTR2b It can reduce sepsis-related lung damage Based on previous findings, serotonin... HTR2b A study to reduce the formation of extracellular traps (NETs) of neutrophils. Subcutaneous administration of cecal lavage fluid at 0, 6, and 12 hours after sepsis induction. HTR2b The agonist BW723C86 (1 mg / kg) showed the best efficacy when administered at 6 hours, reducing the mouse mortality rate from 90% to 50% compared to the solvent control group (equal volume of physiological saline containing 1% DMSO). Figure 5 A).

[0059] To investigate the role of BW723C86 in sepsis-related lung injury, lung tissue was harvested 12 hours later for HE staining to observe the degree of damage, including alveolar septal thickening, inflammatory cell infiltration, and hyaline membrane formation. The NETs markers citrullinated histone H3 and myeloperoxidase (MPO) were also labeled using immunofluorescence to detect the fluorescence intensity of NETs in the lung tissue.

[0060] Histological analysis showed that BW723C86 significantly reduced sepsis-related lung injury. Figure 5 C). Immunofluorescence staining showed that the expression of NETs-related markers (including citrullinated histone H3 and myeloperoxidase MPO) was decreased in the lung tissue of mice in the treatment group. Figure 5 D). BALF analysis showed that BW723C86 effectively reduced the total cell count ( Figure 5 E) and protein concentration ( Figure 5 F). Inflammatory cytokines (including tumor necrosis factor α, IL-6, and IL-1β) were also significantly reduced in BALF. Figure 5 (GI). However, BW723C86 did not significantly reduce markers of damage to other organs.

[0061] These results demonstrate that BW723C86 provides protection in septic mice by inhibiting the formation of neutrophil extracellular traps (NETs), thereby alleviating sepsis-related lung injury. These findings highlight the therapeutic potential of targeting the platelet-5-hydroxytryptamine signaling pathway in regulating NETs-dependent tissue damage in severe inflammatory diseases.

[0062] Example 2. HTR2b Therapeutic effects of receptor agonists on animal models of psoriasis Psoriasis is a chronic inflammatory systemic disease with a genetic basis, characterized by symmetrical erythematous skin lesions covered with silvery-white scales. Its exact cause remains unclear, but current research indicates that neutrophils are among the first cells to infiltrate newly developed psoriatic plaques, their epidermal accumulation is a hallmark of the disease, and NETs play an important role in the pathogenesis of psoriasis.

[0063] Given the safety concerns surrounding systemic HTR2b agonists and their potential adverse events related to valvular heart disease, the efficacy of topical application of BW723C86 in the treatment of psoriasis was investigated.

[0064] Female C57BL6J mice, aged 8-10 weeks and weighing 20-25g, were randomly divided into two groups: a solvent control group (a mouse psoriasis model induced by imiquimod (IMQ) and treated with 1% DMSO) and an experimental group (a mouse psoriasis model induced by imiquimod (IMQ) and treated with BW723C86).

[0065] Administration: Apply imiquimod ointment containing 0.5% BW723C86 to the ear of mice daily. The solvent control is imiquimod ointment containing 1% DMSO. The administration was repeated for 7 days.

[0066] Testing indicators: (1) Ear thickness: The thickness of the mouse ear was measured daily and the degree of swelling was calculated.

[0067] (2) Histopathology: Ear tissue was taken for HE staining to observe Munr microabscesses, thickening of the stratum corneum and stratum spinosum, inflammatory cell infiltration and capillary dilation.

[0068] (3) NETs level: Immunofluorescence dual-color labeling of NETs markers citrullinated histone H3 and MPO, and detection of NETs fluorescence intensity in ear tissue.

[0069] (4) Inflammatory factors: The levels of TNF-α, IL-6, IL-1α and IL-1β in ear tissue were detected by rt-qPCR.

[0070] Experimental results: In an imiquimod (IMQ)-induced mouse model of psoriasis, topical application of BW723C86 significantly reduced ear desquamation and thickness. Figure 6 A, B). Hematoxylin-eosin staining histological analysis showed that, compared with the solvent control group, the degree of epithelial hyperplasia was reduced in the treatment group mice ( Figure 6 C). Immunofluorescence imaging showed that NETs markers, citrullinated histone H3, and myeloperoxidase (MPO) were localized in the dermis and Munro microabscesses of psoriatic lesions, consistent with findings in human plaque psoriasis tissue. Notably, BW723C86 treatment reduced NETs deposition in these lesions. Figure 6 D, E). BW723C86 reduced the Psoriasis Area and Severity Index score (D, E). Figure 6 F, G), epidermal thickness ( Figure 6 G, H) and NETs accumulation on the dorsal skin ( Figure 6 I, J).

[0071] These findings suggest that topical application of BW723C86 can alleviate psoriatic IMQ by inhibiting the inflammatory response mediated by neutrophil extracellular traps (NETs), thereby establishing [therapeutic efficacy / response]. HTR2b As a potential therapeutic target for psoriasis.

[0072] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the protection scope of the present invention.

Claims

1. HTR2b Application of genes as targets in the preparation of drugs to treat NETs-driven diseases.

2. The application according to claim 1, characterized in that, Through drug regulation HTR2b Gene expression is used to suppress the formation of extracellular traps (NETs) in neutrophils.

3. The application according to claim 1, characterized in that, The drug is HTR2b Receptor agonists.

4. The application according to claim 3, characterized in that, The HTR2b Receptor agonists are incorporated into pharmaceutically acceptable carriers to create clinically usable drug formulations.

5. The application according to claim 3, characterized in that, The HTR2b The receptor agonist is BW723C86.

6. The application according to claim 1 or 2, characterized in that, The disease driven by the NETs is sepsis.

7. The application according to claim 6, characterized in that, HTR2b Genes are used as targets in the development of drugs to treat sepsis, aiming to reduce lung damage caused by sepsis.

8. The application according to claim 1 or 2, characterized in that, The disease driven by the NETs mentioned is psoriasis.

9. The application according to claim 8, characterized in that, HTR2b When genes are used as targets in the preparation of drugs for treating psoriasis, the administration method is topical application.

10. The application according to claim 8, characterized in that, The drug is used to reduce the area of ​​psoriasis, epidermal thickness, and inflammatory response.

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