Application of ubiquitin-specific protease USP4 in the treatment of acute lung injury / acute respiratory distress syndrome

Abstract

This invention discloses the application of ubiquitin-specific protease USP4 in the treatment of acute lung injury / acute respiratory distress syndrome (ARDS). Analysis of the GEO database revealed elevated USP4 levels in ARDS patients; in vitro stimulation of human neutrophils with LPS showed increased USP4 expression levels; experiments confirmed that the USP4 inhibitor Vialinin A effectively treats ARDS and significantly improves mouse survival; adenovirus inhibition of USP4 expression in the lungs effectively alleviates ARDS; and USP4 deficiency in the hematopoietic system effectively alleviates ARDS. This invention is the first to propose the application of USP4 as a target in screening drugs for the treatment of ARDS, providing a new target and a novel treatment strategy for clinical practice.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a ubiquitin-specific protease USP4 in the treatment of acute lung injury / acute respiratory distress syndrome. Background Technology

[0002] Acute lung injury (ALI) is a serious respiratory condition caused by various direct and indirect damaging factors that lead to damage to alveolar epithelial cells and capillary endothelial cells, resulting in diffuse interstitial and alveolar edema and causing acute hypoxic respiratory failure. As the condition worsens, it may progress to acute respiratory distress syndrome (ARDS).

[0003] Pneumonia, the most common cause of acute lung injury and acute respiratory distress syndrome, is a lower respiratory tract infection that affects the lung parenchyma. It is usually caused by common Gram-negative or Gram-positive bacteria, respiratory viruses, and mycobacteria worldwide.

[0004] Despite the availability of various clinical treatments for acute lung injury and significant efforts made to control inflammatory responses and improve clinical symptoms, the incidence and mortality rates of acute lung injury remain high.

[0005] Ubiquitin-specific protease 4 (USP4) belongs to the USP family and was the first deubiquitinating enzyme (DUB) discovered in mammalian cells. It mainly participates in the regulation of many signaling pathways by deubiquitinizing various substrates, such as the p53, TGF-β, Wnt / β-catenin, and nuclear factor κB (NF-κB) signaling pathways. USP4 also affects other pathological environments, such as viral and bacterial infections, cancer, acute injury, and inflammation.

[0006] Viialin A (CAS No.: 858134-23-3) is a para-triphenyl compound isolated from *Fungiformis chinensis*, and is an effective inhibitor of TNF-α, USP4, USP5, and SENP1-specific protease 1. In recent years, Viialin A has been reported to possess strong anti-inflammatory and antioxidant properties, exhibiting pharmacological activity against various diseases including cancer, Kawasaki disease, asthma, and pathological scarring. However, the effect of Viialin A on acute lung injury remains unclear. Summary of the Invention

[0007] The purpose of this invention is to address the above-mentioned problems by providing an application of ubiquitin-specific protease USP4 in the treatment of acute lung injury / acute respiratory distress syndrome.

[0008] To achieve its objective, the present invention employs the following technical solution:

[0009] The first aspect of the present invention provides the use of ubiquitin-specific protease USP4 as a target in screening drugs for the treatment of acute lung injury or acute respiratory distress syndrome.

[0010] The drug inhibits the expression of USP4 and / or inhibits the catalytic activity of USP4 deubiquitinase.

[0011] The drug reduces the concentrations of inflammatory factors, chemokines, and total protein in BALF, thereby improving the inflammatory condition and reducing the degree of lung damage.

[0012] A second aspect of the present invention provides the use of a ubiquitin-specific protease USP4 inhibitor in the preparation of a medicament for treating acute lung injury or acute respiratory distress syndrome.

[0013] The USP4 inhibitor refers to a substance that can downregulate USP4 expression and / or inhibit the catalytic activity of USP4 deubiquitinase.

[0014] The USP4 inhibitor relieves dyspnea and improves low tidal volume.

[0015] The USP4 inhibitors include nucleic acid molecules and compounds.

[0016] The nucleic acid molecule includes siRNA, and the nucleotide sequence of the siRNA is as follows:

[0017] TGGTCACTGATGTATATAA.

[0018] The compound includes Vialinin A.

[0019] A third aspect of the present invention provides a pharmaceutical composition for treating acute lung injury or acute respiratory distress syndrome, comprising a USP4 inhibitor as an active ingredient and a pharmaceutically acceptable carrier.

[0020] The beneficial effects of this invention are:

[0021] This invention, through GEO database analysis, found that USP4 is elevated in patients with acute respiratory distress syndrome; by stimulating human neutrophils with LPS in vitro, it was found that USP4 expression level was elevated; experiments confirmed that the USP4 inhibitor Vialinin A can effectively treat acute lung injury and effectively improve the survival rate of mice; using adenovirus to inhibit USP4 expression in the lungs can effectively alleviate acute lung injury; and USP4 deficiency in the hematopoietic system can effectively alleviate acute lung injury.

[0022] This invention provides a new potential target for the treatment of acute lung injury / acute respiratory distress syndrome, offers a new treatment strategy for clinical treatment, and has broad application prospects. Attached Figure Description

[0023] Figure 1 The results of analyzing the expression level of USP4 protein in ARDS patients using public database data (a, b) and the results of validating changes in USP4 protein levels in vitro using extracted human neutrophils (c).

[0024] Figure 2 The results showed that the USP4 inhibitor (VialininA) could effectively treat acute lung injury and improve the survival rate of mice: a. Study mode; b. and c. Concentrations of inflammatory cytokines and total protein in BALF; d. HE staining of lung tissue; e. Quantification according to the acute lung injury scoring criteria; f. Survival results of the control group and the treated group; g. Lung function-respiratory rate test results of mice with acute lung injury; h. Lung function-tidal volume test results of mice with acute lung injury.

[0025] Figure 3 The results showed that adenoviral RNA alleviated acute lung injury in mice by inhibiting the expression of USP4 in the lungs: a. Study model; b. and c. Concentrations of inflammatory cytokines and total protein in BALF; d. HE staining of lung tissue; e. Quantification according to the acute lung injury scoring criteria.

[0026] Figure 4 The study demonstrated that USP4 deficiency in the hematopoietic system effectively alleviated the severity of acute lung injury and improved survival: a. Study model; b. and c. Concentrations of inflammatory cytokines and total protein in BALF; d. HE staining of lung tissue; e. Quantification according to the acute lung injury scoring criteria; f. Survival results of mice. Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0028] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0029] Example 1: Analysis of USP4 protein expression levels in ARDS patients using public database data. Human samples were extracted... Validation of protein level changes in neutrophils in vitro

[0030] Using data from public databases (NCBI GEO database), the following two databases were found by searching for data on peripheral blood RNA testing of ARDS patients:

[0031] GSE172114: The expression levels of USP4 protein in samples from 46 critically ill patients (ARDS) and 23 non-critically ill patients were compared and analyzed to evaluate the expression levels of USP4 in human ARDS samples. The results are as follows: Figure 1 As shown in figure a, the expression level of USP4 protein was significantly increased in human patients with acute respiratory distress syndrome (ARDS).

[0032] GSE32707: Total RNA was detected in whole blood samples from subjects with sepsis, sepsis + ARDS (se / ARDS), systemic inflammatory response syndrome (SIRS), and those without sepsis, SIRS, or ARDS. The expression levels of USP4 in human samples from patients without these diseases were compared and analyzed. Results are as follows: Figure 1 As shown in b, this also suggests elevated USP4 expression in patients with sepsis or ARDS.

[0033] Based on the results from the above database, and considering that neutrophils are the most important effector cells in acute lung injury, we extracted neutrophils from human peripheral blood and cultured them in vitro with lipopolysaccharide (LPS). After 6 hours of stimulation, the cells were lysed into proteins, and their expression was detected using Western blot. The results are as follows: Figure 1 As shown in c, compared with the control group, LPS treatment significantly increased the expression of USP4 in peripheral blood neutrophils, which is consistent with the results in the database.

[0034] Elevated USP4 protein levels may play an important role in the development of acute respiratory distress syndrome in humans, and inhibiting USP4 activity may be used to treat ARDS in humans.

[0035] Example 2: The USP4 inhibitor (Vialinin A) can effectively treat acute lung injury and improve the survival rate of mice. Rate

[0036] To verify whether inhibiting USP4 can effectively treat acute lung injury, we established a mouse model of acute lung injury using lipopolysaccharide (LPS). Twenty wild-type C57BL / 6 mice were randomly divided into a control group and a treatment group. The treatment group was treated with the USP4 inhibitor (Vialinin A) via the following administration route: Figure 2 As shown in a: Each mouse was administered 50 μL of the USP4 inhibitor (Vialinin A, 5 μM) via intranasal drip at 1 h and 12 h after modeling.

[0037] Then, bronchoalveolar lavage fluid (BALF) and lung tissue from mice were collected, and the concentrations of inflammatory cytokines and total protein in BALF were detected, as shown in Figures 2b and 2c. After drug treatment, the levels of various inflammatory factors (TNF-α, IL-1β, IL-6) and important neutrophil chemokines (CXCL-1, CXCL-2) in mice were significantly reduced, and the total protein concentration in BALF was also significantly reduced, indicating that the overall inflammatory condition of the mice had improved.

[0038] Lung tissue was stained with hematoxylin and eosin (HE) and quantified according to the American Thoracic Society's Acute Lung Injury Scoring Criteria, such as... Figure 2 As shown in d and e: Compared with the control group, the VialininA treatment group had relatively intact alveolar structure and less inflammatory cell infiltration.

[0039] Mouse survival results are as follows Figure 2 As shown in f: the USP4 inhibitor (VialininA) significantly improved the survival rate of mice.

[0040] Lung function was tested in mice with acute lung injury, such as... Figure 2 As shown in g,h, the drug treatment significantly relieved respiratory distress and improved low tidal volume in mice with acute lung injury.

[0041] Example 3: Adenovirus RNA alleviates acute lung injury in mice by inhibiting the expression of USP4 in the lungs.

[0042] The adeno-associated virus AAV-SiUSP4 used to knock down USP4 was provided by Shanghai Jikai Gene Medical Technology Co., Ltd. (China). The siRNA sequence of USP4 (SEQ ID NO.1) is as follows:

[0043] Usp4-RNAi(101348-11): TGGTCACTGATGTATATAA.

[0044] Wild-type C57BL / 6 mice were intratracheally instilled with the viral AAV-SiUSP4 once a week for four consecutive weeks to achieve USP4 knockdown in the lungs. After four weeks, an acute lung injury model was established in mice using LPS, as shown in the model below. Figure 3 a.

[0045] Then, bronchoalveolar lavage fluid (BALF) and lung tissue were collected from mice, and the concentrations of inflammatory cytokines and total protein in the BALF were measured, such as... Figure 3 As shown in b and c, similar to the effects of drug treatment, the concentrations of various inflammatory factors and chemokines in mice after USP4 knockdown were significantly reduced, and the total protein concentration of BALF was also significantly reduced, indicating that the overall inflammatory condition of the mice improved.

[0046] Lung tissue was stained with hematoxylin and eosin (HE) and quantified according to the American Thoracic Society's Acute Lung Injury Scoring Criteria, such as... Figure 3 As shown in d and e, knockdown of USP4 in the lungs also reduces the degree of lung damage.

[0047] Example 4: USP4 deficiency in mouse hematopoietic system effectively alleviates the severity of acute lung injury and improves survival rate

[0048] Bone marrow transplantation (BMT): This involves placing a donor (wild-type C57BL / 6 or Usp4) in the bone marrow. - / - Bone marrow cells were extracted from mice and intravenously transplanted into wild-type C57BL / 6 mice that had undergone lethal dose whole-body radiation pretreatment, ultimately replacing the hematopoietic system of the recipient mice with that of the donor mice. Four weeks after chimeric mouse construction, an acute lung injury model was established using LPS. Bronchoalveolar lavage fluid (BALF) and lung tissue were then collected from the mice. A schematic diagram is shown below. Figure 4 As shown in a.

[0049] The concentrations of inflammatory factors and total protein in BALF are as follows: Figure 4 As shown in b and c, it can be seen that in mice after the hematopoietic system was re-established, BM-Usp4, compared to BM-WT, - / - The levels of inflammatory factors and chemokines were significantly reduced, and the total protein concentration in BALF was also significantly decreased, indicating that knockout of USP4 in mouse immune cells can effectively reduce inflammation.

[0050] Lung tissue was stained with hematoxylin and eosin (HE) and quantified according to the American Thoracic Society's Acute Lung Injury Scoring Criteria, such as... Figure 4 As shown in d and e, knocking out the immune cell USP4 reduced the degree of lung damage.

[0051] Fourteen pairs of chimeric mice were given sufficient LPS to establish an acute lung injury model. The survival rate of the mice was then observed. Figure 4 As shown in f, knockout of USP4 in the hematopoietic system significantly improves the survival rate of mice.

Claims

1. Application of ubiquitin-specific protease USP4 as a target in screening drugs for the treatment of acute lung injury or acute respiratory distress syndrome.

2. The application according to claim 1, characterized in that: The drug inhibits the expression of USP4 and / or inhibits the catalytic activity of USP4 deubiquitinase.

3. The application according to claim 1, characterized in that: The drug reduces the concentrations of inflammatory factors, chemokines, and total protein in BALF, thereby improving the inflammatory condition and reducing the degree of lung damage.

4. Application of ubiquitin-specific protease USP4 inhibitors in the preparation of drugs for the treatment of acute lung injury or acute respiratory distress syndrome.

5. The application according to claim 4, characterized in that: The USP4 inhibitor refers to a substance that can downregulate USP4 expression and / or inhibit the catalytic activity of USP4 deubiquitinase.

6. The application according to claim 4, characterized in that: The USP4 inhibitor relieves dyspnea and improves low tidal volume.

7. The application according to claim 4, characterized in that: The USP4 inhibitors include nucleic acid molecules or compounds.

8. The application according to claim 7, characterized in that: The nucleic acid molecule includes siRNA, and the nucleotide sequence of the siRNA is: TGGTCACTGATGTATATAA.

9. The application according to claim 7, characterized in that: The compound includes Vialinin A.

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