Use of a casz1b protein

Abstract

The application belongs to the technical field of target drugs, and particularly relates to application of CASZ1b protein; the amino acid sequence of the CASZ1b protein is shown as SEQ ID NO. 1, and the application of the CASZ1b protein in preparation of a drug for treating diseases mediated by mitochondrial function damage; the drug can activate expression of mitochondrial autophagy proteins Beclin1 and P62, improve a mitochondrial membrane potential, inhibit ROS production, and repair mitochondrial function damage.

Description

Technical Field

[0001] This invention belongs to the field of targeted drug technology, and in particular relates to the application of a CASZ1b protein. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease of unknown cause that commonly affects middle-aged and elderly individuals. It is also the most common form of pulmonary fibrosis. Epidemiological studies show that the incidence and mortality rates of IPF are on the rise. IPF has a poor prognosis, with a mortality rate higher than most cancers, and is therefore referred to as a "tumor-like disease." Current treatment strategies are very limited. Drug therapy (such as pirfenidone, nintedanib, N-acetylcysteine, etc.) and non-drug therapy (such as pulmonary rehabilitation, lung transplantation, etc.) can improve patient symptoms to some extent, but their effects on preventing the progression of pulmonary fibrosis and improving its prognosis are not ideal. Alveolar epithelial-mesenchymal transition (EMT) is one of the key steps in the pathogenesis of IPF. Mitophagy is closely related to EMT. Reduced mitophagy is a key factor in the development and progression of IPF. Mitophagy is a crucial mechanism for maintaining intracellular homeostasis; it can clear dysfunctional or damaged mitochondria, reduce oxidative stress, inhibit apoptosis and senescence, thereby combating the occurrence of pulmonary fibrosis. Dysregulation of mitochondrial autophagy leads to an increase in reactive oxygen species (ROS) and a decrease in adenosine triphosphate (ATP), exacerbating the progression of pulmonary fibrosis. Therefore, finding drugs that can repair mitochondrial function is of great application value in inhibiting the occurrence and development of pulmonary fibrosis (IPF). Summary of the Invention

[0003] The purpose of this invention is to provide an application of the CASZ1b protein, using the CASZ1b protein as a drug development target to address the problem of how to repair mitochondrial functional damage, and to provide a target for the development of drugs to treat IPF.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An application of the CASZ1b protein, the amino acid sequence of which is shown in SEQ ID NO. 1 (which can be found in NCBI accession number NP_060236), is disclosed. The application of the CASZ1b protein in the preparation of drugs for treating diseases mediated by mitochondrial dysfunction is described. When using the CASZ1b protein as a drug target in the preparation of drugs for diseases caused by mitochondrial dysfunction, mitochondrial function is repaired by increasing the expression of CASZ1b, for example, through plasmid transfection to overexpress CASZ1b.

[0005] Furthermore, the disease is idiopathic pulmonary fibrosis, and the mitochondrial dysfunction is TGF-β1-induced mitochondrial dysfunction in alveolar epithelial cells.

[0006] Furthermore, the drug can activate the expression of mitochondrial autophagy proteins Beclin1 and P62, increase mitochondrial membrane potential, inhibit ROS production, and repair mitochondrial functional damage.

[0007] Furthermore, the active ingredient of the drug includes a carrier or agonist expressing the CASZ1b protein or its active ingredient; the dosage form of the drug includes tablets, injections, and oral preparations.

[0008] The advantages of this invention are as follows: This application utilizes a bleomycin-induced pulmonary fibrosis mouse model and a TGF-β1-induced A549 cell mitochondrial damage model. It was found that CASZ1b protein was lowly expressed in both models, and mitochondrial function was impaired. Knockdown of CASZ1b caused lung function impairment and abnormal collagen deposition, indicating that decreased CASZ1b protein levels are associated with mitochondrial damage. Furthermore, this invention established animal and cell models overexpressing CASZ1b, finding that overexpression of CASZ1b can inhibit lung pathological damage and abnormal collagen deposition, restore lung function, and activate the expression of mitophagy-related proteins, increase mitochondrial membrane potential, and inhibit ROS production. By establishing a CASZ1b knockout cell model, it was found that CASZ1b knockout exacerbated mitochondrial functional damage. This invention reveals the important role of CASZ1b in repairing mitochondrial functional damage, and further development of related mitochondrial damage repair agents based on this target has significant application value for the treatment of pulmonary fibrosis. Attached Figure Description

[0009] Figure 1 This is a diagram showing mitochondrial functional impairment in mice with bleomycin-induced pulmonary fibrosis in Experiment Example 1 of this invention. Figure A shows the pathological images of pulmonary fibrosis stained with HE and Masson staining; Figure B shows the expression level of CASZ1b protein; Figure C shows the mRNA expression level of CASZ1b; and Figure D shows the expression of the mitophagy-related proteins Beclin1 and P62.

[0010] Figure 2 This is an illustration of lung pathological damage and decreased lung function in mice caused by knockdown of CASZ1b in Experiment Example 1 of this invention. Figure A shows the pathological images of HE and Masson staining and the immunohistochemical image of collagen III; Figure B shows the lung function tests, including tidal volume (TV), minute ventilation (MV), peak expiratory flow (PEF), and mid-expiratory flow (EF50).

[0011] Figure 3This is an example of how overexpression of CASZ1b improved lung pathological damage and pulmonary function in mice with bleomycin-induced pulmonary fibrosis, as shown in Experiment 1 of this invention. Figure A shows the pathological images of pulmonary fibrosis stained with HE and Masson staining; Figure B shows the detection of hydroxyproline (HYP) content; Figure C shows the pulmonary function tests, including tidal volume (TV), minute ventilation (MV), peak expiratory flow (PEF), and mid-expiratory flow (EF50).

[0012] Figure 4 In Experiment 1 of this invention, overexpression of CASZ1b inhibited collagen deposition. Figure A shows the immunohistochemical staining of collagen III (COL III) in lung tissue; Figure B shows the immunofluorescence staining of COL I in lung tissue.

[0013] Figure 5 This is a diagram showing how overexpression of CASZ1b activates mitophagy and inhibits collagen expression in Experiment Example 1 of this invention. Figure A shows the protein expression of CASZ1b, Beclin1, P62, COX IV, COL III, and COL I; Figure B shows the grayscale analysis of protein expression levels; Figure C shows the ROS level; and Figure D shows the ATP concentration detection.

[0014] Figure 6 This invention presents the expression of CASZ1b in different cells and the detection of TGF-β1-induced mitochondrial functional damage in alveolar epithelial cells in Experiment 1. Figure A shows the expression of CASZ1b in human embryonic lung fibrosis cells (MRC-5), human monocytic leukemia cells (THP-1), human alveolar epithelial cells (A549), human lung microvascular endothelial cells (HPMEC), and human bronchial epithelial cells (Beas-2B); Figure B shows mitochondrial ATP levels; Figure C shows ROS levels; Figure D shows mitochondrial membrane potential; and Figure E shows the expression results of CASZ1 and autophagy-related proteins Beclin1 and P62.

[0015] Figure 7 This is a diagram illustrating the aggravated mitochondrial functional impairment caused by CASZ1b knockdown in Experiment Example 2 of this invention. Figure A shows the expression of CASZ1, Beclin1, and P62 proteins; Figure B shows ROS levels; and Figure C shows ATP concentration.

[0016] Figure 8 This is a diagram showing the repair of mitochondrial damage by CASZ1b overexpression in Experiment Example 3 of this invention. Figure A shows the protein expression levels of CASZ1b, Beclin1, and P62; Figure B shows the ROS level; Figure C shows the mitochondrial membrane potential; and Figure D shows the ATP concentration. Detailed Implementation

[0017] Experimental materials: 60 SPF-grade C57BL / 6 mice weighing 20-25g were purchased from Beijing Spaford Biotechnology Co., Ltd.; human alveolar epithelial cells (A549) were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; bleomycin hydrochloride for injection (15 mg / vial, batch number 650427) was purchased from Nippon Kayaku Co., Ltd., with a modeling concentration of 5 mg / ml; siRNA for the CASZ1b gene and negative control siNC; pcDNA3.1 (vector); pcDNA3.1-Flag-CASZ1 plasmid and AAV-mediated knockdown and overexpression viruses were constructed and synthesized by Hanheng Biotechnology Co., Ltd.

[0018] Experimental reagents: RPMI 1640 medium, fetal bovine serum, and PBS buffer were purchased from BI; transforming growth factor TGF-β1 was purchased from Peprotech; HE staining kit, Masson staining kit, immunohistochemistry kit, reactive oxygen species detection kit, mitochondrial membrane potential detection kit (JC-1), and enhanced ATP detection kit were purchased from Beyotime; Gibco Opti-MEM medium and Lipofectamine 3000 Reagent were purchased from Thermo Fisher Scientific; antibodies: GAPDH, Beclin1, P62, CASZ1b, E-cadherin, N-cadherin, COL III, COL I, and Fibronectin were purchased from Proteintech; RIPA protein lysis buffer and BCA protein concentration assay kit were purchased from Solarbio.

[0019] Effect verification and application Experimental Example 1: Function of CASZ1b protein in bleomycin-induced pulmonary fibrosis mice I. Experimental Methods 1. Construction of a mouse model of pulmonary fibrosis 1.1 Mouse model establishment and treatment Sixty mice were randomly divided into four groups using a random number table: normal group (con), model group (model), low-expression CASZ1b group (si-CASZ1b), model + low-expression group (model + si-CASZ1b), model + overexpression CASZ1b group (oe-CASZ1b), and model + pirfenidone treatment group (PFD). Fourteen days before establishing the pulmonary fibrosis model, mice in the low-expression and overexpression CASZ1b groups were anesthetized with tribromoethanol, and 50 μL of AAV virus solution (1 × 10⁻⁶) was injected intratracheally using a microinjector. 11(vg / mouse). Subsequently, a mouse model of pulmonary fibrosis was established by a single intratracheal infusion of bleomycin. Before anesthesia, the mice were weighed, and bleomycin was injected into the trachea at a dose of 5 mg / kg. The mice were immediately upright and rotated to ensure even distribution of the drug in both lungs. The control group received a single injection of an equal volume of physiological saline. Fourteen days after modeling, the PFD group mice were administered pirfenidone suspension by gavage daily. After the administration was completed, the mice were sacrificed and samples were collected for further testing.

[0020] 1.2 Lung tissue sampling Lung tissue was washed with pre-cooled PBS, and the left and right lungs were separated. The left lung was fixed in 10% formaldehyde for 1 hour, dehydrated, fixed, and embedded in paraffin for case observation, immunohistochemistry, and other tests. The right lung was aliquoted into cryovials, flash-frozen in liquid nitrogen, and then transferred to -80°C for storage for protein and gene expression detection.

[0021] 2. Construction of a mitochondrial injury cell model 2.1 Cell Culture Human alveolar epithelial cells A549 were cultured in RPMI-1640 containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator.

[0022] 2.2 Cell Modeling Cells were seeded in 6-well plates and cultured at 37°C with 5% CO2. When the cell density reached about 70%, the medium was replaced with medium containing 0.1% serum and starved for 8 h. The medium was then discarded, and complete medium containing TGF-β1 was added and treated for 48 h to prepare a cell model.

[0023] 3. Indicator Testing 3.1 Observation of lung tissue morphology Paraffin-embedded tissue was cut into 5μm pieces. HE staining was used to observe structural changes in alveoli and bronchioles, as well as the degree of alveolitis. Masson staining was used to observe collagen deposition and interstitial changes. Immunohistochemistry and immunofluorescence staining were used to observe collagen expression.

[0024] 3.2 Lung function test The WBP pulmonary function testing system was used to measure respiratory rate, tidal volume (TV), minute ventilation (MV), maximum expiratory flow (PEF), and mid-expiratory flow (EF50) in mice during active conditions.

[0025] 3.3 Determination of hydroxyproline (HYP) content in lung tissue: Take 20 mg of lung tissue and determine the HYP content according to the instructions of the hydroxyproline assay kit. Calculate the HYP content of the sample by plotting the calculation formula based on the concentration of the standard solution and the absorbance value.

[0026] 3.4 Protein and gene expression detection Total protein and RNA were extracted from lung tissue and cells. The expression levels of CASZ1b, Beclin1, and P62 were detected by Western blotting and RT-qPCR.

[0027] 3.5 Mitochondrial membrane potential detection Remove the cell culture medium, wash twice with PBS, add JC-1 staining working solution to each well, incubate at 37°C in the dark for 30 min, then remove the supernatant, wash the cells twice with JC-1 staining buffer, and observe the changes in red and green fluorescence of the cells under a fluorescence microscope.

[0028] 3.6 ROS Level Detection Remove the supernatant culture medium, wash the cells once with PBS, add trypsin to digest and collect the cells, add DCFH-DA solution, incubate at 37°C in the dark for 30 min, centrifuge at 1200 rpm for 5 min, discard the supernatant, clear the cells 3 times, and then detect them by flow cytometry.

[0029] 3.7 ATP Level Detection Add ATP lysis buffer to the processed cells and collect the supernatant. Add the ATP assay working solution to a 96-well plate and let it stand for 5 minutes. Add the lysed sample or standard to the wells and detect the RLU value using a chemiluminescence analyzer. Calculate the ATP concentration based on the standard curve.

[0030] 4. Data Processing Experimental data were analyzed using IBM SPSS 22.0 statistical software. One-way ANOVA was used to compare the variances between groups of quantitative data. For groups with homogeneous variances, the Least Significant Difference (LSD) method was used. For groups with unequal variances, the LSD method was used after data transformation. The significance level was set at α=0.05. Data were statistically described as mean (x̅) ± standard deviation (s).

[0031] II. Experimental Results 1. Pathological changes in lung tissue and lung function in mice with pulmonary fibrosis like Figure 1 As shown, bleomycin induces alveolar structural damage in mouse lung tissue, including alveolar cavity collapse, alveolar wall thickening, and excessive deposition of collagen fibers. Figure 1 (A). Compared with the normal group, the protein and mRNA expression levels of CASZ1b in the lung tissue of the model group mice were significantly reduced ( Figure 1 In the middle BC), the expression of autophagy-related protein Beclin1 was significantly decreased, and the expression level of P62 was significantly increased ( Figure 1 (D).

[0032] like Figure 2As shown in Figure A, compared with the control group, both the bleomycin-treated model group and the lentivirus-mediated CASZ1b low-expression group (si-CASZ1b) exhibited significant alveolar structural damage, alveolar cavity collapse, alveolar wall rupture and fusion, and massive collagen deposition; co-treatment with bleomycin and low expression further aggravated the above pathological phenomena. The tidal volume (TV), minute ventilation (MV), peak expiratory flow (PEF), and EF50 of the lungs of mice in both the model group and the low-expression CASZ1b group were significantly decreased, with the co-treatment group showing increased lung function impairment. Figure 2 (B)

[0033] 2. Overexpression of CASZ1b activates mitophagy and inhibits collagen deposition. In a mouse model of bleomycin-induced pulmonary fibrosis, CASZ1b overexpression virus (oe-CASZ1b) was used to intervene in lung tissue, with pirfenidone (PFD) as a positive control. HE and Masson staining results showed that, compared with the model group, both the oe-CASZ1b treatment group and the PFD group significantly improved the aforementioned pathological changes and significantly inhibited collagen deposition. Figure 3 (A). Hydroxyproline (HYP) content detection experiments showed that the HYP content in the lung tissue of model group mice was significantly increased, while the HYP content in the treatment groups was significantly decreased. Figure 3 (B) Further pulmonary function tests showed that all pulmonary function indicators in the model group mice were significantly decreased, while CASZ1b overexpression and PFD treatment significantly improved pulmonary function indicators. Figure 3 (C)

[0034] like Figure 4 As shown, immunohistochemical and lung tissue immunofluorescence results indicate that overexpression of CASZ1b can significantly reduce the expression levels of collagen III (COL III) and collagen I (COL I).

[0035] Western blot results further confirmed that, compared with the control group, the expression of CASZ1b and Beclin1 was significantly reduced in the pulmonary fibrosis model group, while the expression of mitophagy-related proteins P62 and COX IV, as well as collagen III and I, was significantly increased. Figure 5 (AB). Furthermore, the level of reactive oxygen species (ROS) was significantly increased in the model group ( ). Figure 5 (C), while ATP production was significantly reduced ( Figure 5 (D), the changes in the above indicators were all restored in the CASZ1b overexpression group.

[0036] The above results suggest that CASZ1b expression is significantly reduced during the progression of pulmonary fibrosis, accompanied by significant mitochondrial dysfunction and abnormal collagen deposition. Further research revealed that overexpression of CASZ1b can significantly improve lung function and mitochondrial dysfunction, effectively inhibiting abnormal collagen deposition, thereby treating pulmonary fibrosis.

[0037] 3. Changes in various indicators in the mitochondrial injury cell model First, in previous studies, the applicant systematically analyzed human single-cell transcriptome datasets GSE122960 and GSE128033 derived from pulmonary fibrosis patients. Single-cell cluster analysis revealed that the known CASZ1b marker signal is mainly distributed in alveolar epithelial cells and ciliated cell populations in human lung tissue. Furthermore, individual t-SNE data for each patient were analyzed, and cell-specific expression of CASZ1b was finely assessed using mean expression level (avg_logFC) and expression ratio (pct.1 / pct.2) as indicators. The results showed that CASZ1b expression was almost entirely confined to alveolar epithelial cells (avg_logFC = 0.285, pct.1 = 0.366, pct.2 = 0.129) (see supplementary table 5 in Associated Data; single-cell sequencing data analysis results can be downloaded from: https: / / pmc.ncbi.nlm.nih.gov / articles / PMC6580683 / #_ad93). It was not expressed in fibroblasts, immune cells, or endothelial cells. Furthermore, we examined the expression level of CASZ1b in different cell types, including human embryonic lung fibroblasts (MRC-5), human monocytic leukemia cells (THP-1), human alveolar epithelial cells (A549), human lung microvascular endothelial cells (HPMEC), and human bronchial epithelial cells (Beas-2B). The results showed that CASZ1b was expressed only in alveolar epithelial cells. Figure 6 (A). Finally, after inducing alveolar epithelial cells with TGF-β1, observation under a light microscope revealed that the morphology of the alveolar epithelial cells changed from irregular polygons to spindle shapes, exhibiting an EMT phenotype. This induced a significant decrease in the expression of CASZ1b and Beclin proteins and a significant increase in the expression of P62 protein. Figure 6 (B) Compared with the normal group, ATP production in the model cell group was significantly reduced ( Figure 6 C), while ROS levels were significantly elevated ( Figure 6 D in the text). JC-1 staining results showed that, compared with the control group, the ratio of red fluorescence to green fluorescence of mitochondrial membrane potential in the model cell group was significantly decreased, indicating a significant decrease in mitochondrial membrane potential (D). Figure 6E in the text). Western blot results showed that, compared with the control group, the expression of CASZ1b and the mitophagy protein Beclin1 was significantly decreased, while the expression of p62 protein was significantly increased (in the text). Figure 6 (F in the middle).

[0038] Experimental Example 2: CASZ1b knockdown exacerbates mitochondrial dysfunction I. Experimental Methods 1. Cell Culture and Grouping The culture conditions for A549 cells were the same as in Method 2.1 of Experiment 1. Cells were divided into four groups: siNC group, siCASZ1b group, TGF-β1+siNC group (transfected with blank siRNA and treated with TGF-β1), and TGF-β1+siCASZ1b group (knocked down CASZ1b and treated with TGF-β1).

[0039] 2. Gene silencing Healthy cells were seeded into 6-well plates until the cell density reached approximately 50%, and then transfected with siRNA. Samples were prepared according to the Lipofectamine™ 3000 instructions. 500 μl of the mixture was added to each well containing 1.5 ml of culture medium and mixed thoroughly. The plates were then incubated at 37°C with 5% CO2 for 48 h. After 48 h, cells were collected for RNA or protein extraction for subsequent experiments.

[0040] 3. The index detection and data processing are the same as in Experiment 1, steps 3 and 4.

[0041] 4. Experimental Results like Figure 7 As shown, CASZ1b knockdown resulted in a significant decrease in the expression of CASZ1 and the autophagy-related protein Beclin1, and a significant upregulation of P62 protein; TGF-β1 induction further exacerbated these effects. Figure 7 (A). Furthermore, compared to the siNC group, CASZ1b knockdown also caused a significant upregulation of ROS ( Figure 7 In the middle B), ATP concentration was significantly downregulated ( Figure 7 The results indicate that CASZ1b knockdown inhibits mitophagy and exacerbates TGF-β1-induced mitochondrial dysfunction.

[0042] Experimental Example 3: Overexpression of CASZ1b can repair mitochondrial functional damage I. Experimental Methods 1. Cell culture and grouping The cell culture method was the same as in Experiment 1, Section 2.1. The cells were divided into four groups: control group (normal cell group), TGF-β1 group, TGF-β1+vector group (transfected with empty vector plasmid + TGF-β1 treatment) and TGF-β1+oe-CASZ1b group (CASZ1b overexpression + TGF-β1 treatment).

[0043] 2. Plasmid transfection Healthy cells were seeded into 6-well plates until the cell density reached approximately 50%, and then transfected with plasmids. Samples were prepared according to the Lipofectamine™ 3000 instructions: 500 μl of the mixture was added to a well containing 1.5 ml of culture medium and mixed thoroughly. The plates were then incubated at 37°C with 5% CO2 for 48 h. After 48 h, cells were collected for RNA or protein extraction for subsequent experiments.

[0044] 3. The index detection and data processing are the same as in Experiment 1, steps 3 and 4.

[0045] II. Experimental Results like Figure 8 As shown, compared with the vector group, the expression level of oe-CASZ1b protein was significantly upregulated, indicating that the overexpression of CASZ1b in A549 cells was successfully achieved. Figure 8 (A). Compared with the TGF-β1 group and the vector group, overexpression of CASZ1b led to upregulation of the autophagy-related protein Beclin1 and downregulation of p62 expression. Figure 8 (A). ROS results showed that CASZ1b overexpression reduced ROS levels ( Figure 8 (B). JC-1 staining results showed that CASZ1b overexpression caused a significant increase in the ratio of red fluorescence to green fluorescence, indicating an increase in mitochondrial membrane potential (B). Figure 8 (C). ATP results showed that, compared with the TGF-β1 group, CASZ1b overexpression restored ATP levels ( Figure 8 (D). The above results indicate that overexpression of CASZ1b can restore mitophagy and thus repair mitochondrial functional damage.

[0046] The above experimental examples demonstrate for the first time the therapeutic effect of CASZ1b protein in pulmonary fibrosis mouse and cell models. In animal experiments, CASZ1b expression was significantly reduced in pulmonary fibrosis mice, accompanied by mitochondrial dysfunction. Knockdown of CASZ1b induced lung pathological damage, collagen deposition, and decreased lung function in mice; while overexpression of CASZ1b inhibited lung pathological damage and abnormal collagen deposition in fibrotic mice, activated mitophagy, and restored lung function. In cell experiments, knockdown of CASZ1b expression exacerbated mitochondrial dysfunction; while overexpression of CASZ1b activated mitophagy, reduced ROS production, and increased ATP, thereby repairing mitochondrial function. Therefore, CASZ1b protein can serve as a target for screening and preparing drugs to repair mitochondrial dysfunction, and CASZ1b protein activators show promise for development into drugs for the treatment of pulmonary fibrosis.

Claims

1. An application of the CASZ1b protein, characterized in that: The amino acid sequence of the CASZ1b protein is shown in SEQ ID NO.

1. The application of the CASZ1b protein in the preparation of drugs for treating diseases mediated by mitochondrial dysfunction.

2. The application of the CASZ1b protein as described in claim 1, characterized in that: The disease is idiopathic pulmonary fibrosis, and the mitochondrial dysfunction is TGF-β1-induced mitochondrial dysfunction in alveolar epithelial cells.

3. The application of the CASZ1b protein as described in claim 2, characterized in that: The drug can activate the expression of mitochondrial autophagy proteins Beclin1 and P62, increase mitochondrial membrane potential, inhibit ROS production, and repair mitochondrial functional damage.

4. The application of the CASZ1b protein as described in claim 3, characterized in that: The active ingredient of the drug includes a carrier or agonist expressing the CASZ1b protein or its active ingredient; the dosage form of the drug includes tablets, injections, and oral preparations.

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