Application of ubiquitin specific protease USP4 in treating acute lung injury / acute respiratory distress syndrome

Abstract

The invention discloses application of ubiquitin specific protease USP4 in treatment of acute lung injury / acute respiratory distress syndrome. The GEO database analysis finds that USP4 is increased in patients with acute respiratory distress syndrome; the expression level of USP4 is increased by stimulating human neutrophils in vitro with LPS, and experiments prove that the USP4 inhibitor Vialinin A can effectively treat acute lung injury and effectively improve the survival rate of mice; acute lung injury can be effectively relieved by using adenovirus to inhibit expression of USP4 in lung; the USP4 defect of the hematopoietic system can effectively relieve acute lung injury. The application of the USP4 serving as the target spot in screening the medicine for treating the acute lung injury or the acute respiratory distress syndrome is provided for the first time, a new target spot is provided for treating the acute lung injury or the acute respiratory distress syndrome, and a new treatment strategy is provided for clinical treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of ubiquitin-specific protease USP4 in treating acute lung injury / acute respiratory distress syndrome. Background Art

[0002] Acute lung injury (ALI) is a serious respiratory disease caused by various direct and indirect factors that damage alveolar epithelial cells and capillary endothelial cells, resulting in diffuse pulmonary interstitial and alveolar edema, leading to acute hypoxic respiratory insufficiency. As the disease 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 and is usually caused by common Gram-negative or Gram-positive bacteria, respiratory viruses, and mycobacteria worldwide.

[0004] Although there are many methods for clinical treatment of acute lung injury and great efforts have been made to control inflammatory response and improve clinical symptoms, the incidence and mortality of acute lung injury are still high.

[0005] Ubiquitin specific protease 4 (USP4) belongs to the USP family and is the first deubiquitinase (DUB) discovered in mammalian cells. It mainly participates in regulating many signaling pathways by deubiquitinating multiple substrates, such as 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 injuries and inflammation.

[0006] Vialinin A (CAS No.: 858134-23-3) is a triphenyl compound isolated from Chinese mushrooms and is an effective inhibitor of TNF-α, USP4, USP5 and SENP1 specific protease 1. Vialinin A has been reported many times in recent years and has strong anti-inflammatory and antioxidant properties. It has pharmacological activity against a variety of diseases such as cancer, Kawasaki disease, asthma and pathological scars. However, the effect of Vialinin A on acute lung injury is still unclear. Summary of the invention

[0007] The purpose of the present invention is to address the above problems and provide an application of a ubiquitin-specific protease USP4 in treating acute lung injury / acute respiratory distress syndrome.

[0008] In order to achieve its purpose, the present invention adopts 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 treating 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 concentration of inflammatory factors, chemokines and BALF total protein, thereby improving the inflammatory condition and alleviating the degree of lung damage.

[0012] The second aspect of the present invention provides the use of a ubiquitin-specific protease USP4 inhibitor in the preparation of a drug 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 inhibitors relieve dyspnea and improve low tidal volumes.

[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:

[0017] TGGTCACTGATGTATATAA.

[0018] The compounds include VialininA.

[0019] The 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 the present invention are:

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

[0022] The present invention provides a new potential target for treating acute lung injury / acute respiratory distress syndrome and a new treatment strategy for clinical treatment, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 2 It shows that USP4 inhibitor (VialininA) can effectively treat acute lung injury and improve the survival rate of mice: a. Research model; bc. Inflammatory cytokine concentration and total protein concentration in BALF; d. HE staining of lung tissue; e. Quantification according to acute lung injury scoring criteria; f. Survival rate results of the control group and the drug-treated group of mice; g. Lung function-respiratory rate test results of mice with acute lung injury; h. Lung function-tidal capacity test results of mice with acute lung injury.

[0025] Figure 3 The results show that adenoviral RNA alleviated acute lung injury in mice by inhibiting the expression of lung USP4: a. Research model; bc. Inflammatory cytokine concentrations and total protein concentrations in BALF; d. HE staining of lung tissue; e. Quantification according to the acute lung injury scoring criteria.

[0026] Figure 4 It shows that hematopoietic system USP4 deficiency effectively alleviates the severity of acute lung injury and improves survival rate: a. Research model; bc. Inflammatory cytokine concentrations and total protein concentrations in BALF; d. HE staining of lung tissue; e. Quantification according to acute lung injury scoring criteria; f. Survival rate results of mice. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0028] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0029] Example 1: Analyze the expression level of USP4 protein in ARDS patients using public database data. Changes in protein levels in granulocytes verified in vitro

[0030] Using data from a public database (NCBI GEO database), we searched for RNA tests in the peripheral blood of ARDS patients in the database and found the following two databases:

[0031] GSE172114: The expression level of USP4 protein in samples of 46 critically ill patients (ARDS) and 23 non-critically ill patients was compared and analyzed in human ARDS sample data. The results are as follows Figure 1 As shown in a, the expression level of USP4 protein was significantly increased in human critically ill patients (ARDS).

[0032] GSE32707: The total RNA of whole blood of subjects with sepsis only, sepsis + ARDS (se / ARDS), systemic inflammatory response syndrome (SIRS), and subjects without sepsis, SIRS or ARDS was tested in the database, and the expression level of USP4 in human samples without the above diseases and patients was compared and analyzed. The results are as follows Figure 1 As shown in b, it also suggests that USP4 expression is increased in patients with sepsis or ARDS.

[0033] In view of the results of the above databases, and because the most important effector cells of acute lung injury are neutrophils, we then extracted neutrophils from human peripheral blood, stimulated them with lipopolysaccharide (LPS) in vitro, and lysed the cells into proteins after 6 hours of stimulation. The expression of proteins was detected by 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 of the database.

[0034] These results suggest that elevated USP4 protein levels may play an important role in the development of human ARDS, and that inhibiting USP4 activity may be used to treat human ARDS.

[0035] Example 2: USP4 inhibitor (VialininA) can effectively treat acute lung injury and improve the survival of mice Rate

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

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

[0038] Lung tissue was stained with HE and quantified according to the acute lung injury scoring criteria published by the American Thoracic Society, such as Figure 2 As shown in d and e: the alveolar structure of the VialininA-treated group was relatively intact compared with the control group, and the infiltration of inflammatory cells was less.

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

[0040] Lung function tests were performed on mice with acute lung injury. Figure 2 As shown in g,h, mice with acute lung injury given drug treatment can significantly alleviate dyspnea and improve low tidal volume.

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

[0042] Adeno-associated virus AAV-SiUSP4 for knocking down USP4 was provided by Shanghai GeneCare 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 instilled with AAV-SiUSP4 virus through the trachea once a week for four consecutive weeks to achieve the effect of knocking down USP4 in the lungs. Four weeks later, LPS was used to establish a mouse acute lung injury model, as shown in the following figure. Figure 3 a.

[0045] Then, the bronchoalveolar lavage fluid (BALF) and lung tissues of mice were collected, and the concentrations of inflammatory cytokines and total protein in BALF were detected, as shown in Figure 2. Figure 3 As shown in b and c, similar to the effect of drug treatment, the concentrations of various inflammatory factors and chemokines in mice after knockdown of USP4 were significantly decreased, and the total protein concentration in BALF also decreased significantly, indicating that the overall inflammatory condition of the mice was improved.

[0046] Lung tissue was stained with HE and quantified according to the acute lung injury scoring criteria published by the American Thoracic Society, such as Figure 3 As shown in d and e, it can be seen that knockdown of USP4 in the lung also alleviates the degree of lung damage.

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

[0048] Bone marrow transplantation (BMT): donor (wild-type C57BL / 6 or Usp4 - / - Bone marrow cells were extracted from the donor mouse and transplanted into wild-type C57BL / 6 mice that had been pretreated with a lethal dose of whole-body radiation by intravenous injection, ultimately completely replacing the recipient mouse's hematopoietic system with that of the donor mouse. Four weeks after the chimeric mice were constructed, an acute lung injury model was established in mice using LPS, and then the bronchoalveolar lavage fluid (BALF) and lung tissues of the mice were collected. The model is shown in the figure below. Figure 4 As shown in a.

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

[0050] Lung tissue was stained with HE and quantified according to the acute lung injury scoring criteria published by the American Thoracic Society, such as Figure 4 As shown in d and e, knockout of immune cell USP4 reduced the degree of lung damage.

[0051] The 14 pairs of chimeric mice were given sufficient amounts of LPS to establish an acute lung injury model, and the survival rate of the mice was then observed. Figure 4 As shown in f, knockout of USP4 in the hematopoietic system can significantly improve 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 use 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 use according to claim 1, characterized in that: The drug reduces the concentration of inflammatory factors, chemokines and BALF total protein, thereby improving the inflammatory condition and alleviating the degree of lung damage.

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

5. The use 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 use according to claim 4, characterized in that: The USP4 inhibitors relieve dyspnea and improve low tidal volumes.

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

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

9. The use according to claim 7, characterized in that: The compounds include VialininA.

10. A pharmaceutical composition for treating acute lung injury or acute respiratory distress syndrome, characterized in that: The invention contains a USP4 inhibitor as an active ingredient and a pharmaceutically acceptable carrier.

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