Application of ubiquitin specific protease 28 inhibitor in preparation of sepsis prevention and treatment medicine

Abstract

The invention discloses application of a ubiquitin specific protease 28 inhibitor in preparation of a sepsis prevention and treatment medicine. The medicine can relieve multi-organ injury caused by sepsis, relieve organ dysfunction caused by sepsis and reduce release of inflammatory factors and injury factors. The USP28 is found to be a key target of sepsis for the first time, it is proved that the USP28 inhibitor can remarkably improve related symptoms such as multi-organ injury caused by sepsis, has a protection effect on lung, intestine, liver and kidney injuries, can be used for preventing and treating sepsis, and has clinical application value and wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of ubiquitin-specific protease 28 inhibitors in the preparation of drugs for preventing and treating sepsis. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by the dysregulation of the host's response to infection (Singer M, et al. JAMA, 2016, 315(8): 801 - 810). The disease progresses rapidly and usually leads to tissue damage, organ failure, and even death. According to statistics, sepsis is one of the main causes of death in patients in the intensive care unit. Without timely and effective intervention, the mortality rate will quickly exceed 30 - 35% (Vincent JL, et al. Crit Care, 2019, 23(1): 196). Its pathogenesis is very complex. In the early stage, it is an overwhelming and systemic pro-inflammatory response to infection, followed by an immunosuppressive stage characterized by immune dysfunction, lymphopenia, and secondary infection (Cecconi M, et al. Lancet, 2018, 392(10141): 75 - 87), which is closely related to the pathophysiological changes of multiple systems and organs in the body. Currently, there is no effective treatment for sepsis, and supportive treatments such as antibiotics and fluid resuscitation are still the mainstream treatment methods for sepsis (Vincent JL. EBioMedicine, 2022, 86: 104318). Therefore, it is of great significance to deeply explore the important pathophysiological mechanisms of sepsis, find new therapeutic targets, and conduct active drug interventions on them.

[0003] Ubiquitin-specific protease 28 (USP28) is a deubiquitinating enzyme discovered due to its homology with USP25. It mainly regulates the ubiquitination and degradation of many proteins. After regulating proteins, it promotes cell proliferation, initiates invasion and metastasis, stimulates cell survival, inhibits cell differentiation, and induces angiogenesis (Ren X, Exp Hematol Oncol, 2023, 12(1): 27), and plays an important physiological role in the occurrence and development of various tumors such as breast cancer, colorectal cancer, and lung cancer (Zhou L, et al. Biochem Pharmacol, 2023, 213: 115624). Currently, the role of USP28 in sepsis has not been reported. Summary of the Invention

[0004] One object of the present invention is to provide the application of USP28 inhibitors in the preparation of drugs for preventing and treating sepsis.

[0005] Further, the USP28 inhibitor is AZ-1.

[0006] Specifically, the applications include: alleviating multiple organ injuries caused by sepsis, including sepsis-induced lung injury, sepsis-induced intestinal injury, sepsis-induced liver injury, and sepsis-induced kidney injury; alleviating organ dysfunction caused by sepsis; increasing the survival rate of sepsis patients or inhibiting body temperature decline; improving the histopathological damage of the lungs, intestines, liver, and kidneys; reducing the expression of serum inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), chemokine (CXCL1), interferon-α (IFN-α), and interferon-β (IFN-β); reducing the expression of damage-related molecules C-reactive protein (CRP), creatine kinase (CK), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), alanine aminotransferase (ALT), and endothelial cell-specific molecule 1 (ESM-1); alleviating vascular leakage in the lungs, intestines, liver, and kidneys or reducing edema in each organ; and improving the expression of lung endothelial function-related proteins E-selectin, intercellular adhesion molecule (ICAM-1), and vascular endothelial cadherin (VE-cadherin).

[0007] The second object of the present invention is to provide an application of a pharmaceutical composition in the preparation of a drug for preventing and treating sepsis. The pharmaceutical composition includes an active ingredient, and the active ingredient is a USP28 inhibitor.

[0008] Further, the USP28 inhibitor is AZ-1.

[0009] In the present invention, the structural formula of the USP28 inhibitor AZ-1 is shown as follows:

[0010]

[0011] AZ1 is an orally effective selective and non-competitive dual ubiquitin-specific protease USP25 / 28 inhibitor, with IC 50 values of 0.7 μM and 0.6 μM, respectively. In the HCT116 colon cancer cell line, AZ1 can downregulate the expression of USP28, promote the degradation of c-Myc through the proteasome-mediated pathway, and induce cell cycle arrest and apoptosis in a dose- and time-dependent manner, thereby exerting an anti-tumor effect (Wrigley J D, Gavory G, Simpson I, et al. Identification and characterization of dual inhibitors of the USP25 / 28 deubiquitinating enzyme subfamily [J]. ACS Chem Biol, 2017, 12(12): 3113-3125). Currently, AZ1 is mainly used to study the physiological functions of USP28.

[0012] In the present invention, the administration mode of the application can be oral administration or injection, specifically intraperitoneal, subcutaneous, intravenous or intramuscular injection; pharmaceutically acceptable carriers such as diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. can also be added to prepare a pharmaceutical composition in a certain form, such as powders, pills, capsules, tablets, microcapsules, soft capsules, films, suppositories, injections, ointments, powders, granules, aerosols and other preparations.

[0013] Compared with the prior art, the present invention has the following remarkable advantages:

[0014] The present invention discovers for the first time that USP28 is a key target for sepsis, and confirms that USP28 inhibitors can significantly improve the related symptoms such as multiple organ damage caused by sepsis, have a protective effect on lung, intestine, liver and kidney injuries, can be used for the prevention and treatment of sepsis, have clinical application value and broad application prospects. Description of the Drawings

[0015] Figure 1 It is a graph showing the results of the effects of USP28 inhibition on the survival rate and body temperature of CLP surgery-induced septic mice. Among them, A is a statistical graph of the survival rate of CLP mice within 72 hours after surgery, n = 20; B is a graph showing the change in body temperature of CLP mice within 72 hours after surgery, n = 6;

[0016] Figure 2 It is the effect of USP28 inhibition on the pathological damage of the lung, intestine, liver and kidney tissues of CLP surgery-induced septic mice. Among them, A is the HE staining map of the liver, kidney, lung and intestine, and B is the statistical graph of the HE staining pathological score (n = 6, expressed as mean ± SD, ## P < 0.01 compared with the sham operation group, ** P < 0.01 compared with the model group);

[0017] Figure 3 It is the effect of USP28 inhibition on the content of serum inflammatory factors in CLP surgery-induced septic mice. Among them, A is interleukin-1β (IL-1β), B is the statistical graph of interleukin-6 (IL-6), C is the statistical graph of tumor necrosis factor α (TNF-α), D is the statistical graph of chemokine (CXCL1), E is the statistical graph of interferon-α (IFN-α), and F is the statistical graph of interferon-β (IFN-β) (n = 8, expressed as mean ± SD, ## P < 0.01 compared with the sham operation group, ** P < 0.01 compared with the model group);

[0018] Figure 4Effect of USP28 inhibition on the levels of damage-related factors in the serum of mice with cecal ligation and puncture (CLP)-induced sepsis. Panel A shows the statistical chart of C-reactive protein (CRP), panel B shows the statistical chart of creatine kinase (CK), panel C shows the statistical chart of lactate dehydrogenase (LDH), panel D shows the statistical chart of blood urea nitrogen (BUN), panel E shows the statistical chart of alanine aminotransferase (ALT), and panel F shows the statistical chart of endothelial cell-specific molecule 1 (ESM-1) (n = 8, expressed as mean ± SD, ## P < 0.01 compared with the sham operation group, ** P < 0.01 compared with the model group);

[0019] Figure 5 Effect of USP28 inhibition on vascular leakage in multiple organs (lungs, intestines, liver, kidneys, etc.) and the wet / dry weight ratio of each organ in mice with CLP-induced sepsis. Panel A shows the gross images of the lungs, intestines, liver, and kidneys, panel B shows the statistical chart of the wet / dry weight ratio of each organ, panel C shows the Evans blue staining images of the lungs, intestines, liver, and kidneys, and panel D shows the statistical chart of the Evans blue leakage rate in each organ tissue (n = 6, expressed as mean ± SD, ## P < 0.01 compared with the sham operation group, ** P < 0.01 compared with the model group);

[0020] Figure 6 Effect of USP28 inhibition on proteins related to lung endothelial function in mice with CLP-induced sepsis. Panel A shows the immunoblot image and statistical chart of E-selectin protein expression, panel B shows the immunoblot image and statistical chart of intercellular adhesion molecule-1 (ICAM-1) protein expression, panel C shows the immunoblot image and statistical chart of VE-cadherin protein expression, and panel D shows the immunoblot image and statistical chart of deubiquitinating enzyme USP28 protein expression (n = 6, expressed as mean ± SD, ## P < 0.01 compared with the sham operation group, ** P < 0.01 compared with the model group). Detailed implementation

[0021] Currently, the treatment methods for sepsis and its related multiple organ injuries mainly focus on antibiotic therapy, fluid resuscitation, and supportive treatment. However, these methods mainly aim to relieve symptoms and support the body's functions, lacking targeted intervention means for pathological mechanisms. Although antibiotics can effectively inhibit pathogen infections, their effects on improving immune dysregulation, inflammatory responses, and multiple organ dysfunction in the late stage of sepsis are limited. In addition, the excessive use of antibiotics may lead to an increase in bacterial drug resistance, further restricting the breadth and effectiveness of their clinical applications. From a pathological mechanism perspective, the onset of sepsis involves complex inflammatory responses, immune dysregulation, and multiple organ failure. Currently, there is no clear treatment target or specific drug available to comprehensively improve the clinical outcomes of patients. For example, certain inflammatory factor-targeted drugs discovered in existing studies, such as inhibitors of IL-1β and TNF-α, although showing certain efficacy in vitro or in animal models, have not significantly improved the survival rate of patients in clinical trials, indicating the limitations of their therapeutic effects. At the same time, although USP28 has been proven to play an important regulatory role in diseases such as tumors, its specific functions and therapeutic potential in sepsis have not been systematically studied. Therefore, it is of great significance to deeply explore the important pathophysiology and action mechanisms of sepsis, find new treatment targets, and actively intervene with drugs to improve sepsis.

[0022] The present invention aims to solve the problems of unclear treatment targets and limited efficacy in existing sepsis treatments, providing new intervention targets and treatment strategies for drug research and development of sepsis and its related diseases, with important clinical application value and broad prospects. By inhibiting the enzymatic activity or protein expression of USP28, the USP28 inhibitor can significantly reduce the release of inflammatory factors and damage-related factors, alleviate vascular leakage, improve multiple organ functions, and thus increase the survival rate of sepsis patients.

[0023] Based on the mechanism of action of ubiquitin-specific protease 28 (USP28) in sepsis and its related organ injuries, the present invention proposes a novel method for intervening in drug targets. USP28 plays a key role in the occurrence and development of multiple organ injuries in sepsis by regulating the process of protein deubiquitination. The present invention for the first time confirms that the USP28 inhibitor can significantly improve the pathological damage of organ tissues such as the lungs, liver, kidneys, and intestines caused by sepsis, providing a new direction for the treatment of sepsis.

[0024] The present invention has found through research that USP28 inhibitors (such as AZ-1) can reduce the release of pro-inflammatory factors (such as IL-1β, IL-6, TNF-α) and the expression of damage-related factors (such as CRP, BUN, LDH) by decreasing the enzymatic activity and protein expression level of USP28, thereby alleviating vascular leakage and tissue edema in sepsis-related organs. AZ-1 can improve the abnormal expression of proteins related to lung endothelial function (such as E-selectin, ICAM-1, VE-cadherin) in the sepsis model and significantly enhance the stability of the vascular endothelial barrier.

[0025] The present invention has verified the therapeutic effect of USP28 inhibitors on sepsis through animal experiments. In a mouse sepsis model induced by cecal ligation and puncture (CLP), the survival rate of mice in the AZ-1 treatment group was significantly increased within 72 hours, and the decrease in body temperature was effectively controlled. The experiment also found that after administration of AZ-1, the pathological scores of HE staining of organs such as the lungs, liver, kidneys, and intestines in mice were significantly reduced, showing an obvious tissue protection effect, suggesting its important role in regulating systemic inflammatory responses.

[0026] The USP28 inhibitors described in the present invention can be administered by oral or injection methods, including routes such as intraperitoneal, subcutaneous, intravenous, or intramuscular injection. In addition, the USP28 inhibitors can be formulated into various dosage forms in combination with excipients, fillers and other auxiliary materials, including tablets, capsules, injections, aerosols, etc., to meet different clinical needs.

[0027] The clinical application prospect of USP28 inhibitors is broad. It significantly improves sepsis and multiple organ injuries through multi-level action mechanisms, not only providing a new target for the treatment of sepsis, but also providing a new research idea for the development of intervention drugs for multiple organ injuries.

[0028] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0029] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0030] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0031] Example 1

[0032] Effect of USP28 Inhibition on the Survival Rate and Body Temperature of Sepsis Mice Induced by CLP Surgery

[0033] I. Experimental Materials

[0034] 1. Instruments and Reagents

[0035] Electronic analytical balance (Shanghai Mettler-Toledo Company), Milli-Q ultrapure water machine (Millipore Company, USA), microplate reader (BioTek Company, USA), refrigerated centrifuge (Thermo Fisher Company, USA), sterilized medical suture needles (Ningbo Medical Suture Co., Ltd.), tissue scissors, surgical forceps (Shanghai Surgical Instruments Factory), USP28 inhibitor AZ-1 (MedChemExpress, USA), sodium pentobarbital (Sigma Company, USA).

[0036] 2. Experimental Animals

[0037] Male C57BL / 6J mice, weighing 22 - 24 g, SPF grade, purchased from Nanjing Anokang Biotechnology Co., Ltd., meeting the quality standards of ordinary experimental animals. License number: SCXK (Su) 2020 - 0009. The mice were housed separately in cages, with free access to water and food. The temperature of the breeding room was 23 - 25 °C, and the relative humidity was 40% - 80%. The experiment started after 2 - 3 days of adaptive breeding.

[0038] II. Experimental Methods

[0039] 1. Animal Grouping and Drug Administration

[0040] Eighty male C57BL / 6J mice were randomly divided into 4 groups (20 mice / group):

[0041] (1) Sham operation group (Sham): Administered an equal volume of normal saline by gavage 1 h before surgery, once.

[0042] (2) Sham operation + AZ-1 group (Sham+AZ-1): Administered 20 mg / kg of AZ-1 by gavage 1 h before surgery, once.

[0043] (3) CLP model group (CLP): Administered an equal volume of normal saline by gavage 1 h before surgery, once.

[0044] (4) CLP model + AZ-1 group (CLP+AZ-1): Administered 20 mg / kg of AZ-1 by gavage 1 h before surgery, once.

[0045] 2. Preparation of Sepsis Model Induced by CLP in Mice

[0046] C57BL / 6J mice were anesthetized intraperitoneally according to body weight with sodium pentobarbital. After verifying the absence of the righting reflex, the animals were placed in the supine position, and their four limbs were fixed on the operating board. The abdominal hair was shaved off. In the midline of the anterior abdomen, an incision about 1 cm long was made along the linea alba, the epidermis and muscle layer were separated, the cecum was found in the abdominal cavity, the cecum and mesentery were separated, and 75% of the cecum was ligated with 5-0 suture. A 20-gauge needle was used to penetrate the cecum once from the avascular side of the mesentery to the non-mesenteric side above the end of the cecum, and a small amount of intestinal contents was extruded from both ends of the needle hole. The cecum was then returned to the abdominal cavity, and the abdomen was sutured layer by layer.

[0047] 3. Survival rate and body temperature detection

[0048] The survival rate of mice was continuously observed and calculated within 72 h, and the body temperature of the surviving mice was measured at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, respectively.

[0049] 4. Data processing

[0050] All data were statistically analyzed using Graphpad Prism 8 software, expressed as mean±SD. For comparisons between two groups, Students’t test was used, and for comparisons among three or more groups, one-way ANOVA was used. The test was performed using Dunnett’s test, and P<0.05 was considered statistically significant.

[0051] III. Experimental results

[0052] As Figure 1 shown, compared with the sham operation group, the survival rate of mice in the CLP model group decreased significantly within 72 h, while the survival rate of mice in the model group increased significantly after administration; the results of body temperature monitoring within 72 h showed that compared with the sham operation group, the body temperature of mice in the model group decreased significantly, while the body temperature of mice in the model group increased significantly after administration. The above results suggest that the USP28 inhibitor AZ-1 can significantly improve the survival rate and body temperature of septic mice.

[0053] Example 2

[0054] Effect of USP28 inhibition on histopathological damage of organs and tissues in septic mice induced by CLP surgery

[0055] I. Experimental materials

[0056] The instruments and experimental animals were the same as those in Example 1.

[0057] II. Experimental methods

[0058] 1. The animal grouping, model preparation, and drug administration methods were the same as those in Example 1.

[0059] 2. HE staining of lung, intestine, liver, and kidney tissues

[0060] After blood collection from each group of mice, the lung, intestine, liver, and kidney tissues were excised, fixed in 4% paraformaldehyde solution for 24 h, embedded in paraffin, and sent to Wuhan Sevier Biotechnology Co., Ltd. for HE staining. The sections were scanned using a digital scanner, and the HE sections were blindly scored by professionals.

[0061] 3. The data processing was the same as in Example 1.

[0062] III. Experimental Results

[0063] The results were as Figure 2 shown. Compared with the sham operation group, the pathological damage of the lung, intestine, liver, and kidney tissues in the CLP model group mice was significantly aggravated ( ## P < 0.01), indicating that sepsis can cause multi-organ tissue damage such as the lung, intestine, liver, and kidney. After administration of 20 mg / kg of AZ-1 and then cecal ligation and puncture, the pathological damage scores of the lung, intestine, liver, and kidney tissues in the mice were significantly decreased ( ** P < 0.01). The above results indicate that USP inhibitors can improve multi-organ damage caused by sepsis.

[0064] Example 3

[0065] Effect of USP28 Inhibition on Circulating Inflammatory Factors in Mice with CLP Surgery-Induced Sepsis

[0066] I. Experimental Materials

[0067] The instruments and experimental animals were the same as in Example 1. The kits for interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), chemokine (CXCL1), interferon-α (IFN-α), and interferon-β (IFN-β) were purchased from Nanjing Jinyibai Biotechnology Co., Ltd.

[0068] II. Experimental Methods

[0069] 1. The animal grouping, model preparation, and administration method were the same as in Example 1.

[0070] 2. Detection of serum IL-1β, IL-6, TNF-α, CXCL1, IFN-α, and IFN-β

[0071] After 24 h of model establishment in each group of mice, blood was collected from the orbital cavity. After standing at room temperature for 30 min, centrifugation was performed (3500 rpm, 10 min), and the supernatant was taken and aliquoted for storage. The determination of the contents of IL-1β, IL-6, TNF-α, CXCL1, IFN-α, and IFN-β was carried out according to the kit instructions.

[0072] 3. The data processing was the same as in Example 1.

[0073] III. Experimental Results

[0074] The results were as Figure 3 shown. Compared with the sham operation group, the levels of serum IL-1β, IL-6, TNF-α, CXCL1, IFN-α and IFN-β in the CLP model group mice were significantly increased ( ## P<0.01). After administration of the USP inhibitor AZ-1 (20 mg / kg) followed by cecal ligation and puncture, the levels of IL-1β, IL-6, TNF-α, CXCL1, IFN-α and IFN-β in the serum of the treatment group mice were significantly improved ( ** P<0.01). The above results suggest that the USP28 inhibitor can effectively inhibit the release of inflammatory factors in CLP-induced septic mice.

[0075] Example 4

[0076] Effect of USP28 Inhibition on Injury-Related Factors in CLP Surgery-Induced Septic Mice

[0077] I. Experimental Materials

[0078] The instruments, reagents and experimental animals were the same as those in Example 1. Kits for C-reactive protein (CRP), creatine kinase (CK), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), alanine aminotransferase (ALT), and endothelial cell-specific molecule 1 (ESM-1) were purchased from Nanjing Jinyibai Biotechnology Co., Ltd.

[0079] II. Experimental Methods

[0080] 1. Animal grouping, modeling and administration were the same as those in Example 1.

[0081] 2. Detection of serum CRP, CK, LDH, BUN, ALT, and ESM-1

[0082] After 24 h of modeling in each group of mice, blood was collected from the orbital cavity. After standing at room temperature for 30 min, centrifugation was performed (3500 rpm, 10 min), and the supernatant was taken and stored in aliquots. The determination of the contents of CRP, CK, LDH, BUN, ALT, and ESM-1 was carried out according to the kit instructions.

[0083] 3. Data processing was the same as that in Example 1.

[0084] III. Experimental Results

[0085] The results were as Figure 4 shown. Compared with the sham operation group, the levels of serum CRP, CK, LDH, BUN, ALT and ESM-1 in the CLP model group mice were significantly increased ( ##P < 0.01). After administration of the USP inhibitor AZ-1 (20 mg / kg) followed by cecal ligation and puncture, the levels of CRP, CK, LDH, BUN, ALT, and ESM-1 in the serum of the mice in the treatment group were significantly improved ( ** P < 0.01). The above results suggest that the USP28 inhibitor can effectively inhibit the release of sepsis injury-related factors induced by CLP.

[0086] Example 5

[0087] Effect of USP28 inhibition on the wet / dry weight ratio and vascular leakage of various organs in septic mice induced by CLP surgery

[0088] I. Experimental materials

[0089] The instruments, reagents, and experimental animals were the same as in Example 3. Evans blue was purchased from Sigma, USA, and formamide was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0090] II. Experimental methods

[0091] 1. Animal grouping, model establishment, and drug administration were the same as in Example 3.

[0092] 2. Determination of the wet / dry weight ratio of each organ

[0093] At 24 h after the surgery, mice were randomly selected from each group for dissection. The lungs, intestines, liver, and kidneys were collected, slightly washed in PBS, and weighed. Then they were placed in an oven at 60 °C for 48 h until constant weight, and the dry weight was measured. The wet / dry weight ratio was calculated.

[0094] 3. Determination of the vascular leakage rate by Evans blue staining

[0095] After the model establishment in each group of mice, Evans blue (20 mg / kg) was injected into the tail vein. After 2 h of blood circulation, each organ was perfused with normal saline, and then the organ tissues were taken out, the surface moisture was blotted dry, and the weight was measured after blotting with filter paper. Formamide (1 mL / 100 mg) was added to the tissue for homogenization. The homogenate was transferred to a 5 mL EP tube, incubated at 60 °C for 18 h, and the homogenate was centrifuged. The supernatant was taken, and the absorbance was measured at a wavelength of 620 nm with an enzyme-labeled instrument. The content of Evans blue was calculated according to the standard curve regression equation, and the unit was μg / g.

[0096] 4. Data processing was the same as in Example 1.

[0097] III. Experimental results

[0098] As Figure 5 shown in A and B below, compared with the sham operation group, the wet / dry weight ratio of the lungs, intestines, liver, and kidneys in the CLP model group of mice was significantly increased ( ##P < 0.01), indicating that multiple organs such as the lungs, intestines, liver, and kidneys of septic mice showed edema. After treatment with 20 mg / kg of the USP28 inhibitor AZ-1, the wet / dry weight ratios of various organs in the mice were significantly decreased ( ** P < 0.01). The above results suggest that USP inhibitors can significantly improve the edema of multiple organs such as the lungs, intestines, liver, and kidneys in septic mice.

[0099] As Figure 5 shown in C and D, compared with the sham operation group, the Evans blue leakage rates of the lungs, intestines, liver, and kidneys in the CLP model group were significantly increased ( ## P < 0.01), indicating vascular leakage in the lung, intestine, liver, and kidney tissues of septic mice. After treatment with 20 mg / kg of the USP28 inhibitor AZ-1, the Evans blue leakage rates of various organs in the mice were significantly decreased ( ** P < 0.01). The above results suggest that USP inhibitors can significantly improve vascular leakage in the lung, intestine, liver, and kidney tissues of septic mice.

[0100] Example 6

[0101] Effect of USP28 Inhibition on Lung Endothelial Function-Related Proteins in CLP Surgery-Induced Septic Mice

[0102] I. Experimental Materials

[0103] The instruments, reagents, and experimental animals were the same as those in Example 3. Gel imaging system (Bio-Rad); primary antibodies against E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular endothelial cadherin (VE-cadherin), and ubiquitin-specific protease 28 (USP28) (all purchased from Proteintech); secondary antibodies of goat anti-rabbit and goat anti-mouse HRP (purchased from Bioworld).

[0104] II. Experimental Methods

[0105] 1. Animal grouping, modeling, and drug administration were the same as those in Example 3.

[0106] 2. Detection of the expression of lung endothelial function-related proteins by Western blotting

[0107] Add an appropriate amount of lysis buffer to the homogenate of mouse lung tissue, grind it thoroughly at 4°C, let it stand for 30 min, add the liquid to the corresponding EP tube, centrifuge at 12,000 rpm and 4°C for 15 min, take the supernatant, and measure the protein content. Add 6×loading buffer to the remaining supernatant. After protein denaturation, load the samples onto an SDS-PAGE gel for analysis. Incubate the bands after wet transfer with the corresponding primary antibody overnight, then incubate with the corresponding secondary antibody, and finally develop the image using an ECL kit and analyze it with a gel imager. The expression level of the target protein is expressed as the relative value of the expression level of the corresponding internal reference protein.

[0108] 3. The data processing is the same as that in Example 1.

[0109] III. Experimental Results

[0110] As Figure 6 shown, compared with the sham operation group, the protein expression levels of E-selectin, ICAM-1, and USP28 in the lung tissue of mice in the CLP model group were significantly increased, and the protein expression level of VE-cadherin was significantly decreased ( ## P<0.01), indicating that sepsis can cause pulmonary endothelial dysfunction in mice. After treatment with 20 mg / kg of AZ-1, the protein expression levels of E-selectin, ICAM-1, and USP28 in the lung tissue of CLP mice were significantly decreased, and the protein expression level of VE-cadherin was significantly increased ( ** P<0.01), indicating that the USP28 inhibitor can significantly improve vascular endothelial injury in the lung tissue of mice.

Claims

1. Application of USP28 inhibitors in the preparation of drugs for the prevention and treatment of sepsis.

2. The use according to claim 1, characterized in that: The drug alleviates sepsis-induced organ damage.

3. The use according to claim 1, characterized in that: The drug alleviates sepsis-induced organ dysfunction.

4. The use according to claim 1, characterized in that: The drug reduces the release of inflammatory factors and damaging factors.

5. The use according to any one of claims 1 to 4, characterized in that: The USP28 inhibitor is AZ-1.

6. Use of a pharmaceutical composition in the preparation of a drug for the prevention and treatment of sepsis, characterized in that: The pharmaceutical composition comprises an active ingredient, and the active ingredient is a USP28 inhibitor.

7. The use according to claim 6, characterized in that: The USP28 inhibitor is AZ-1.

8. The use according to claim 6, characterized in that: The pharmaceutical composition also includes pharmaceutically acceptable excipients.

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