Application of DPP3 as target spot in preparation of traditional Chinese medicine for treating and / or preventing pyemia sleepiness
By using DPP3 as a target, DPP3 inhibitors were developed to solve the drowsiness problem of sepsis patients, and the effect of effectively reducing narcolepsy symptoms was achieved, which has important clinical application value.
Patent Information
- Application Number
- CN202510276225.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2025-06-20
AI Technical Summary
Patients with sepsis often experience problems such as insomnia, excessive drowsiness, and sleep cycle disorders, which affect the recovery and prognosis of the disease. The existing technology has not yet effectively solved this problem.
By using DPP3 as a target, DPP3 inhibitors or other drugs that interfere with DPP3 are developed for the treatment and prevention of sepsis.
Studies have shown that DPP3 knockout mice have decreased sleep during the dark period after sepsis, while the awake period increases, suggesting that DPP3 inhibitors can effectively alleviate the symptoms of drowsiness in sepsis and have significant clinical application value.
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Figure CN120168637A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biopharmaceutical technology, and more specifically, it relates to the use of DPP3 as a target in the preparation of drugs for the treatment and / or prevention of sepsis-related somnolence. Background Art
[0002] Patients with sepsis generally experience changes in sleep patterns, especially significant changes in the quality and structure of sleep, which often affect the recovery and prognosis of the patients' conditions. Recent studies have shown that patients with sepsis often have problems such as insomnia, excessive somnolence, and sleep cycle disorders, and these problems are closely related to factors such as mortality and organ function recovery. Sleep is crucial for maintaining aspects such as the immune system, metabolism, and cardiovascular health. In addition, somnolence is also one of the most common symptoms in patients with sepsis, and its incidence in neonatal sepsis is 78.33%. Thus, sleep disorders may become a major challenge in the recovery process of patients with sepsis. In recent years, studies have revealed that sleep disorders caused by sepsis may be closely related to various factors such as inflammatory responses and neurotransmitter balance disorders. Therefore, finding effective treatment targets and means for sepsis-related somnolence is an urgent problem to be solved.
[0003] Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent peptidase widely distributed in various tissue cells throughout the body. It can regulate the renin-angiotensin-aldosterone system, oxidative stress, and inflammatory levels by cleaving polypeptides such as angiotensin II, endorphin, and enkephalin, and is involved in various physiological processes. Many studies have confirmed that an increase in the content of DPP3 in the circulation is associated with a poor prognosis in patients with severe diseases such as sepsis and septic shock, indicating that the level of DPP3 in the circulation may be used as a biomarker for the prognosis of critically ill patients. However, in addition to being a biomarker, whether the elevated DPP3 itself can produce biological effects and affect sepsis-related sleep is not clear. Based on this, the present invention explores the role of elevated DPP3 in sepsis in sepsis-related somnolence. Summary of the Invention
[0004] The purpose of the present invention is to provide the use of DPP3 as a target in the preparation of drugs for the treatment and / or prevention of sepsis-related somnolence, providing a new molecular target and intervention means for the treatment of sepsis-related somnolence, and having important clinical application value.
[0005] The above technical purpose of the present invention is achieved through the following technical solutions:
[0006] The first aspect of the present invention provides the use of DPP3 as a target in the preparation of drugs for the treatment and / or prevention of sepsis-related somnolence.
[0007] In a second aspect of the present invention, there is also provided the use of a DPP3 inhibitor in the preparation of a medicament for treating and / or preventing sepsis-induced somnolence.
[0008] In combination with the above, the present invention is further configured such that: the medicament includes any one or more of a DPP3 antagonist, DPP3 interfering RNA, and a DPP3 gene knockout drug.
[0009] In summary, the present invention has the following beneficial effects:
[0010] The present invention explores the role of elevated DPP3 in sepsis-induced narcolepsy. It is found that the content of DPP3 in plasma increases after sepsis, and somnolence occurs. After inducing sepsis in DPP3 knockout mice, the sleep time in the dark period decreases, while the wakefulness time increases. In addition, after intravenous injection of DPP3 into the mouse tail vein, the non-rapid eye movement sleep in the dark period increases, and the wakefulness duration decreases. The present invention uses DPP3 as a therapeutic and / or preventive target for sepsis-induced somnolence, providing a key basis for the drug development of sepsis. The treatment method of the present invention has a targeted molecular intervention strategy, which can effectively alleviate the clinical symptoms of sepsis-induced somnolence. The method for treating and / or preventing sepsis-induced narcolepsy of the present invention is applicable to various sepsis patients, especially suitable for the treatment of sepsis-induced narcolepsy induced by lipopolysaccharide (LPS), and has significant clinical application value and broad market prospects. Description of the Drawings
[0011] Figure 1 It is the verification of the sepsis model in Example 1 of the present invention. (Figure A is a schematic diagram of LPS modeling, Figure B is the result of measuring the body temperature of mice 24 hours after LPS modeling, Figure C is the content of IL-6 in plasma 24 hours after LPS modeling, and Figure D is the content of IL-1β in plasma 24 hours after LPS modeling. n = 3 - 5, *P < 0.05 vs. PBS);
[0012] Figure 2 It is the graph showing the change in the content of DPP3 in plasma detected by taking blood from control group / sepsis group mice at 6h, 12h, 18h, and 24h after inducing sepsis in the examples of the present invention (the content of DPP3 in mice induced with sepsis by intraperitoneal injection of lipopolysaccharide (LPS) increases significantly at 6h, 12h, 18h, and 24h, n = 5 - 6, *P < 0.05 vs. PBS);
[0013] Figure 3It is the change of the sleep-wake cycle after inducing sepsis in WT mice in the embodiments of the present invention (Figure A is a schematic diagram of sleep monitoring, Figure B is a schematic diagram of the administration method and monitoring time, and Figure C is a spectrogram and phase diagram of sleep monitoring. Figure D is the change of the waking period within 24 hours before and after administration, Figure E is the change of the non-rapid eye movement sleep period within 24 hours before and after administration, and Figure F is the change of the rapid eye movement sleep period within 24 hours before and after administration. n = 5, *P < 0.05 vs. PBS);
[0014] Figure 4 It is the change of the sleep-wake cycle after inducing sepsis in DPP3 knockout mice in the embodiments of the present invention (Figure A is a schematic diagram of sleep monitoring, Figure B is a schematic diagram of the administration method and monitoring time, and Figure C is a spectrogram and phase diagram of sleep monitoring. Figure D is the change of the waking period within 24 hours before and after administration, Figure E is the change of the non-rapid eye movement sleep period within 24 hours before and after administration, and Figure F is the change of the rapid eye movement sleep period within 24 hours before and after administration. n = 6, *P < 0.05 vs. WT);
[0015] Figure 5 It is the change of the sleep-wake cycle after tail vein administration of DPP3 to WT mice in the embodiments of the present invention (Figure A is a schematic diagram of sleep monitoring, Figure B is a schematic diagram of the administration method and monitoring time, Figure C is a spectrogram and phase diagram of sleep monitoring, Figure D is the change of the waking period within a total of 6 hours before and after administration, Figure E is the change of the non-rapid eye movement sleep period within a total of 6 hours before and after administration, and Figure F is the change of the rapid eye movement sleep period within a total of 6 hours before and after administration. n = 5, *P < 0.05 vs. PBS). Detailed implementation manners
[0016] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.
[0017] Experimental animals
[0018] The experimental animals in this study mainly included C57BL / 6J and DPP3 knockout mice (purchased from Shanghai Bikai Biotechnology Co., Ltd. and Jiangsu Jicui Yakang Biotechnology Co., Ltd. respectively, with an age of 8 - 12 weeks and a body weight of 25 ± 2.5 g). The animal breeding environment was kept at a constant temperature and humidity and relatively quiet, with a light environment of 12 h / 12 h light-dark alternation. The mice were housed separately in cages and had free access to water and food. All surgical operations or intervention treatments on the experimental animals strictly complied with the relevant regulations of the Ethics Committee of Naval Medical University, and full attention was paid to ensuring animal welfare during the experiment.
[0019] Experimental materials
[0020] Name of reagent and consumables Company Female header Jiangsu Yige Biotechnology Co., Ltd. Insulated silver wire A-MSystems, USA Tissue bioadhesive 3M Company, USA Isoflurane Reword Life Science Co., Ltd. Normal saline Website of Shandong Qidu Pharmaceutical Co., Ltd. Iodophor disinfectant Hangzhou Langsu Medical Disinfectant Co., Ltd. 75% Alcohol Hangzhou Langsu Medical Disinfectant Co., Ltd. Dental cement New Century Dental Materials Co., Ltd. Urethane Sigma-Aldrich (Shanghai) Trading Co., Ltd. PBS buffer Wuhan Sevier Biotechnology Co., Ltd. Chlortetracycline eye ointment Beijing Shuangji Pharmaceutical Co., Ltd. Mouse DPP3 ELISA Kit 96T Shanghai Ruixiu Biotechnology Co., Ltd. Lipopolysaccharide Sigma-Aldrich (Shanghai) Trading Co., Ltd. Human DPP3 protein MedChemExpress (China) Mouse interleukin-1β ELISA Kit Thermo Fisher Scientific Mouse interleukin-6 ELISA Kit Thermo Fisher Scientific
[0021] Experimental instruments
[0022] Name of instrument Company Stereotaxic instrument Shanghai Alcott Biotechnology Co., Ltd. Skull drill Shanghai Alcott Biotechnology Co., Ltd. Flux Pro'sKit Industries Co., Ltd., Chinese Taipei Soldering tin Pro'sKit Industries Co., Ltd., Chinese Taipei Constant temperature soldering iron Pro'sKit Industries Co., Ltd., Chinese Taipei Operating microscope Shanghai Medical Instruments Co., Ltd. Small animal anesthesia machine Shanghai Alcott Biotechnology Co., Ltd. Absorbent cotton balls, cotton swabs Beijing Lanjieke Technology Co., Ltd. Disposable sterile syringe Beijing Lanjieke Technology Co., Ltd. Codos Pet Electric Hair Clipper Shenzhen Codos Electric Appliance Co., Ltd. Three-dimensional operating arm Reword Life Science Co., Ltd. Medusa Mouse & Rat EEG & EMG Recording System Jiangsu Yige Biotechnology Co., Ltd. Microplate reader Thermo Fisher Scientific Intelligent constant temperature magnetic stirrer Heraeus GmbH, Germany Constant temperature oven Heraeus GmbH, Germany Low-speed centrifuge Wuhan Sevier Biotechnology Co., Ltd. Low-temperature centrifuge SCILOGEX, USA Electronic balance Shanghai Liangping Instrumentation Co., Ltd. Cold light source Bobei Optoelectronic Technology Co., Ltd. Ultra-pure water instrument Shanghai Lichen Bangxi Instrument Technology Co., Ltd.
[0023] Example 1: Construction of sepsis model and model validation
[0024] like Figure 1 In Figure A, mice were intraperitoneally injected with LPS (Sigma, L2630, 10 mg / kg) to establish an LPS-induced sepsis mouse model. 24 hours later, the body temperature of mice in the WT group / model group was measured, and blood was taken. The content of inflammatory factors was measured by enzyme-linked immunosorbent assay (ELISA) technology, and the level of inflammatory factors in plasma was detected to verify whether the model construction was successful. The specific experimental plan is as follows:
[0025] 1. Coating: Dilute the antibody to a protein content of 1-10 μg / ml with carbonate coating buffer. Add 100 μl to each well of the polystyrene ELISA plate and incubate at 4°C overnight. The next day, discard the solution in the wells and wash three times with washing buffer, 3 minutes each time.
[0026] 2. Blocking: Add 200ul of blocking solution to each well and incubate at 37℃ for 1-2h.
[0027] 3. Washing: Carefully remove the sealing film, put into the plate washer, and wash 3-5 times. You can also wash the plate manually: discard the liquid, add 300ul of washing solution to each well, soak for 1-2 minutes, pat dry on absorbent paper, and repeat 3-5 times.
[0028] 4. Sample addition: Add 100 μl of appropriately diluted sample to be tested into the above coated reaction wells. (At the same time, make blank wells, standard sample wells with multiple dilutions, and negative control wells and positive control wells as quality control points if conditions permit).
[0029] 5. Incubation: Seal the plate with a sealing film and incubate at 37°C for 1-2 hours.
[0030] 6. Washing: Same as step 3.
[0031] 7. Add antibody: Add 100 μl of diluted biotinylated antibody working solution to each well.
[0032] 8. Incubation: Seal the plate with a sealing film and incubate at 37°C for 1 hour.
[0033] 9. Washing: Same as step 3.
[0034] 10. Add enzyme conjugate: Add 100 μl of diluted enzyme conjugate working solution to each well.
[0035] 11. Incubation: Seal the plate with a sealing film and incubate it at 37°C in the dark for 30 min.
[0036] 12. Washing: The same as step 3.
[0037] 13. Adding chromogenic substrate: Add 100 μl of TMB substrate solution to each well and react at 37°C in the dark for 10 - 30 min until an obvious color gradient appears in the wells of the serially diluted standards.
[0038] 14. Terminating the reaction: Add 100 μl of 2 M sulfuric acid to each reaction well, and the color changes from blue to yellow.
[0039] 15. Result determination: Within 10 min, measure the OD value of each well at 450 nm on an ELISA reader after zeroing with the blank control well.
[0040] The results are as Figure 1 shown in Figure B, C, and D. It was found that the body temperature of the mice decreased significantly and the levels of inflammatory factors increased significantly after modeling, indicating that the LPS-induced concentration of blood model was effective. In addition, the control group was intraperitoneally injected with PBS, and other conditions were the same.
[0041] Example 2: Determining the change in DPP3 content in septic mice
[0042] Construct an LPS-induced septic mouse model. At 6 h, 12 h, 18 h, and 24 h, collect blood, centrifuge it, take the plasma, and use ELISA technology to detect the change in DPP3 content in the plasma.
[0043] The experimental protocol is as follows:
[0044] 1. Take out the required strip from the aluminum foil bag that has been equilibrated at room temperature for 20 min, and seal the remaining strips with a self-sealing bag and return them to 4°C.
[0045] 2. Set up the standard wells and sample wells, and add 50 μL of standards with different concentrations to each standard well.
[0046] 3. First add 10 μL of the sample to be tested to the sample wells, then add 40 μL of sample diluent, and do not add anything to the blank wells.
[0047] 4. Add 100 μL of the detection antibody labeled with horseradish peroxidase (HRP) to each well in the standard wells and sample wells except the blank well. Seal the reaction wells with a sealing film and incubate them in a 37°C water bath or incubator for 60 min.
[0048] 5. Discard the liquid, pat it dry on absorbent paper, fill each well with washing solution, let it stand for 1 min, discard the washing solution, and pat it dry on absorbent paper. Repeat the washing process 5 times (or use a plate washer).
[0049] 6. Add 50 μL of substrate A and 50 μL of substrate B to each well, and incubate in the dark at 37 °C for 15 min.
[0050] 7. Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min.
[0051] It was found that the content of DPP3 in mice with sepsis induced by intraperitoneal injection of lipopolysaccharide (LPS) was significantly increased at 6 h, 12 h, 18 h, and 24 h after sepsis induction. The specific data are as Figure 2 shown.
[0052] Example 3: Determine the sleep changes in sepsis
[0053] One week before intraperitoneal injection of LPS to induce sepsis, electroencephalogram and electromyogram electrodes were implanted. After intraperitoneal injection of LPS, the changes in the 24-hour sleep-wake cycle were monitored;
[0054] The specific experimental protocol for sleep monitoring is as follows:
[0055] 1. Place the mice in the anesthesia chamber of the Orcot small animal anesthesia machine and induce anesthesia with isoflurane at a flow rate of 5% for 3 min. After induction of anesthesia, adjust the flow rate to 2% to maintain anesthesia. Shave the hair on the head, fix the head using a stereotaxic apparatus, ensure that the ear canal is aligned with the ear rod, the nose is in the correct position, and fix with a incisor clamp.
[0056] 2. Apply erythromycin eye ointment, disinfect the head skin with povidone-iodine, and then wipe with 75% alcohol.
[0057] 3. Skin incision and skull exposure: Cut open the head skin, fix with a vascular clamp, remove the connective tissue on the skull, and expose the anterior and posterior fontanelles. Mark the electrode implantation site, and drip physiological saline to reduce the drilling resistance.
[0058] 4. Use a 0.5 mm drill bit to drill holes at the marked sites without penetrating the skull. Screw the sterilized silver wire lead screw into the skull and fix it with 3M bioadhesive.
[0059] 5. Remove the insulation layer of the silver wire, insert it into the neck muscle, and drip bioadhesive to fix it. Prepare dilute dental cement, fix the screw and electromyogram electrode, remove the excess cement, and smooth it.
[0060] 6. Weld the silver wire to the six-hole female header, avoid short circuits, and check the connection with a multimeter. Apply dental cement to fix the female header. After it solidifies, clean the wound, remove the vascular clamp, and place the mice on a warming pad to wait for recovery.
[0061] 7. After more than one week of postoperative recovery, connect the monitor after 1-2 days of environmental adaptation before sleep-wake cycle monitoring, and record the sleep-wake cycle.
[0062] In addition, the control group was intraperitoneally injected with PBS, and other conditions were the same. The results are asFigure 3 As shown, the content of DPP3 in plasma increases, and drowsiness occurs.
[0063] Example 4: To clarify the involvement of DPP3 in sepsis-induced drowsiness
[0064] DPP3 knockout mice were bred. One week before intraperitoneal injection of LPS to induce sepsis in DPP3 knockout mice, electroencephalogram and electromyogram electrodes were implanted. The experimental protocol was the same as above. After intraperitoneal injection of LPS, the changes in the 24-hour sleep-wake cycle were monitored, with WT mice as the control. The results are as Figure 4 shown. The sleep time of DPP3 knockout mice decreased during the dark period, while the wake time increased.
[0065] Example 5: To clarify that DPP3 has a sleep-promoting effect
[0066] To verify the effect of DPP3 on increasing sleep, WT mice were injected with DPP3 via the tail vein, and the sleep-wake changes before and after administration were monitored. The results are as Figure 5 shown. The non-rapid eye movement sleep of WT mice increased during the dark period, and the wake duration decreased.
[0067] In summary, this study confirmed through molecular and animal experiments that DPP3 has a sleep-promoting effect and is involved in the occurrence of sepsis-induced drowsiness, thus providing a new therapeutic target for the treatment of sepsis-induced drowsiness clinically.
[0068] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. Application of DPP3 as a target in the preparation of drugs for the treatment and / or prevention of sepsis-induced lethargy.
2. Use of DPP3 inhibitors in the preparation of drugs for treating and / or preventing sepsis-induced drowsiness.
3. The use according to claims 1-2, characterized in that: The drug includes any one or more of a DPP3 antagonist, a DPP3 interfering RNA, and a DPP3 gene knockout drug.