Application of aryl sulfatase K in preparation of medicine for preventing, delaying or treating chronic obstructive pulmonary disease

Abstract

The invention belongs to the technical field of biomedicine, and discloses application of aryl sulfatase K in preparation of a medicine for preventing, delaying or treating chronic obstructive pulmonary disease. According to the application disclosed by the invention, the effect of ARSK (arylsulfatase K) on inhibiting mitochondrial autophagy and delaying airway epithelial cell senescence is found for the first time, the key effect of ARSK in occurrence and development of COPD is further determined, and a new target spot can be provided for prevention and treatment of COPD. The invention determines that ARSK can alleviate the mitochondrial autophagy process, inhibit airway epithelial cell senescence and further slow down the lung senescence process, so that a new treatment strategy is provided for COPD (chronic obstructive pulmonary disease).

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to the application of arylsulfatase K in preparing medicines for preventing, delaying or treating chronic obstructive pulmonary disease. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic, progressive lung disease. It is a common respiratory disease with airway (bronchitis, bronchiolitis) and / or alveolar abnormalities (emphysema) as the main pathological features. The disease causes persistent airflow limitation and corresponding respiratory symptoms (dyspnea, cough, sputum). The occurrence of COPD is usually related to exposure to harmful particles or gases, and cigarette smoke exposure is the main factor. In addition, many host factors such as genetic susceptibility, abnormal inflammatory response and abnormal lung development are involved in the pathogenesis.

[0003] In recent years, COPD has been considered a disease of accelerated lung aging. Cellular senescence, especially airway epithelial cell senescence, is a driving mechanism for the onset of COPD. Cellular senescence is an irreversible state of cell cycle arrest, usually caused by factors such as telomere attrition, DNA damage, and oxidative stress. Senescent cells secrete a series of inflammatory mediators, called the senescence-associated secretory phenotype (SASP), which can further promote aging through autocrine and paracrine pathways, leading to the occurrence and development of COPD. As the first line of defense for lung tissue to respond to external stimuli, the airway epithelium plays a key role in the pathogenesis of COPD. More and more evidence indicates that the stimulation of cigarette smoke causes airway epithelial cells to show severe accelerated senescence, which in turn participates in the destruction of lung tissue structure and the occurrence of airway inflammation in COPD.

[0004] Currently, the clinical treatment options for COPD are still very limited. The treatment of COPD includes smoking cessation, inhaled bronchodilators and / or glucocorticoids, and supportive treatment (such as oxygen therapy and pulmonary rehabilitation). The main purpose of treatment is to relieve symptoms and reduce the risk and severity of acute exacerbations, but it cannot fundamentally cure the disease. Therefore, it is urgent to conduct in-depth research on the pathogenesis of COPD airway epithelial cell aging, find intervention targets for lung aging, and fully combine the frontier progress of molecular biology to provide a theoretical basis for the development of new and effective therapeutic drugs.

[0005] Aryl sulfatase K (ARSK) is a member of the arylsulfatase family that hydrolyzes sulfate groups on sulfated substrates, thereby promoting sulfate metabolism. Deficiency or mutations in arylsulfatase family members have been associated with the development of several age-related diseases, such as Alzheimer's disease and Parkinson's disease. Previous studies have reported that sulfate metabolites may play a role in the senescence of human colonic epithelial cells. In addition, altered expression of a member of the same family, arylsulfatase A (ARSA), was observed in senescent fibroblasts and promoted the generation of a senescence-associated secretory phenotype. However, the expression and function of ARSK in chronic obstructive pulmonary disease (COPD) have not been reported. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide the use of arylsulfatase K in the preparation of drugs for preventing, delaying or treating chronic obstructive pulmonary disease, and to provide a new intervention target for the development of new and effective therapeutic drugs for chronic obstructive pulmonary disease.

[0007] In order to achieve the above purpose, this application adopts the following technical solutions: In a first aspect, the present invention provides a biomarker for diagnosing or assisting in the diagnosis of chronic obstructive pulmonary disease in a subject, wherein the biomarker is arylsulfatase K.

[0008] In the above technical solution, the chronic obstructive pulmonary disease is chronic obstructive pulmonary disease induced by cigarette smoke or cigarette smoke extract.

[0009] In a second aspect, the present invention provides the use of arylsulfatase K in the preparation of products for diagnosing chronic obstructive pulmonary disease, wherein the products include foods, health products or medicines.

[0010] In a third aspect, the present invention provides the use of arylsulfatase K in the preparation of a drug for preventing, delaying or treating chronic obstructive pulmonary disease.

[0011] In the above technical solution, the agent that enhances or promotes the expression of arylsulfatase K can inhibit the aging of airway epithelial cells, thereby preventing, delaying or treating chronic obstructive pulmonary disease.

[0012] In a fourth aspect, the present invention provides the use of arylsulfatase K in the preparation of a product for inhibiting mitochondrial autophagy, wherein the product includes a food, a health product or a medicine.

[0013] In the above technical solution, the reagent that enhances or promotes the expression of arylsulfatase K can reduce the expression of mitochondrial autophagy proteins PINK1, Parkin, LC3, and p-DRP1, thereby inhibiting mitochondrial autophagy.

[0014] In a fifth aspect, the present invention provides the use of arylsulfatase K in the preparation of a product for delaying the aging of airway epithelial cells, wherein the product includes a food, a health product or a medicine.

[0015] In the above technical scheme, the reagent that enhances or promotes the expression of arylsulfatase K increases the expression of arylsulfatase K, reduces the expression of cell senescence-related proteins p21 and p53, and inhibits mitochondrial autophagy, thereby delaying the senescence of airway epithelial cells.

[0016] In a sixth aspect, the present invention provides a kit for diagnosing chronic obstructive pulmonary disease, comprising a reagent for detecting arylsulfatase K.

[0017] The beneficial effects of the present invention are as follows: the present invention has discovered for the first time the role of ARSK (arylsulfatase K) in inhibiting mitochondrial autophagy and thus delaying the aging of airway epithelial cells, further confirming the key role of ARSK in the occurrence and development of COPD, and providing a new target for the prevention and treatment of COPD. The present invention has determined that ARSK can inhibit the aging of airway epithelial cells by alleviating the mitochondrial autophagy process, thereby slowing down the aging process of the lungs, thereby providing a new treatment strategy for COPD.

[0018] The present invention analyzes the content of ARSK in different populations, different mice and different airway epithelial cells, and determines that the expression of ARSK in the lung tissue of COPD population and COPD model mice is significantly reduced; through experimental analysis of overexpression of ARSK in cells and mice, it is determined that ARSK overexpression can reduce the expression of mitochondrial autophagy proteins PINK1, Parkin, LC3, p-DRP1, inhibit mitochondrial autophagy, thereby inhibiting the level of ROS in cells and mitochondria, alleviating the decrease in mitochondrial membrane potential and ATP, and further reducing the expression of cell senescence-related proteins p21 and p53, and further slowing down the aging of airway epithelial cells; the present invention injects ARSK-AAV into the airways of mice to overexpress ARSK in the lung tissue of mice, alleviates the aging phenotype of COPD airway epithelial cells, successfully improves the pathophysiological state of COPD, and further determines the key role of ARSK in the occurrence and development of COPD. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the basic expression diagram of ARSK in humans, animals and cells; Figure 1 a is the RNA expression of ARSK in lung tissues of non-smokers (n=18), healthy smokers (n=18) and COPD patients (n=20); Figure 1 b is FISH staining of human lung tissue sections to explore the expression of ARSK in airway epithelium; Figure 1c is the expression of ARSK RNA level in lung tissues of control mice and COPD model mice; Figure 1 d is FISH staining of mouse lung tissue sections to explore the expression of ARSK in mouse airway epithelium; Figure 1 e. Figure 1 f is the RNA expression level of ARSK in human airway epithelial cells (HBE) after being stimulated by cigarette smoke extract (CSE) at different concentrations and for different times in 1; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 2 This is a detection diagram of cell senescence after HBE cells were transfected with ARSK overexpression plasmid and stimulated with CSE for 24 hours; wherein: Figure 2 a is a validation diagram for successful overexpression of ARSK protein level; Figure 2 b is the expression of aging marker proteins P21 and P53 in HBE cells. Figure 2 c. Figure 2 d is Figure 2 b’s statistical graph; Figure 2 e. Figure 2 f is the expression of aging-related secretory phenotypic factors IL-6, IL-8 and IL-1β in HBE cells at the RNA and protein levels; Figure 2 g is the staining image of senescent cells; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 3 This is a detection diagram of mitochondrial autophagy in HBE cells after being transfected with ARSK overexpression plasmid and receiving CSE stimulation for 24 hours; wherein: Figure 3 a. Figure 3 b is the expression and statistical diagram of mitochondrial autophagy-related proteins in HBE cells; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 4 This is a detection diagram of mitochondrial autophagy and cell senescence after HBE cells were transfected with ARSK small interfering gene and the mitochondrial autophagy inhibitor liensinine was added; among which: Figure 4 a is the CCK8 graph of cells after adding different concentrations of mitochondrial autophagy inhibitors. Figure 4 b. Figure 4 c is the detection and statistical graph of mitochondrial autophagy and cell senescence after adding mitochondrial autophagy inhibitors to HBE cells; Figure 4 d is the staining image of senescent cells; Figure 4e is the expression of senescence-related secretory phenotypic factors IL-6, IL-8 and IL-1β at the protein level in HBE cells; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 5 To test lung function and lung aging in mice with ARSK overexpression in the lungs after exposure to cigarette smoke (CS); Figure 5 a is a statistical chart of mouse lung function results; Figure 5 b is the HE staining of mouse lung tissue sections and the statistical graph of airway peri-inflammatory scores; Figure 5 c is the expression of aging-related secretory phenotypic factors IL-6, KC and IL-1β at the RNA level in mouse lung tissue; Figure 5 d is a statistical graph of the concentrations of IL-6, KC, and IL-1β proteins contained in the BALF supernatant; Figure 5 e is the result of β-galactosidase staining of frozen lung sections of mice to detect airway epithelial senescence; Figure 5 f shows the expression of aging marker proteins p21 and p53 in mouse lung tissue and the corresponding statistical graph; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0020] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0021] Example 1: Determination of the differential expression of ARSK in COPD humans, smoked mouse lung tissue, and cigarette smoke-stimulated airway epithelial cells 1. Human experiment: COPD patients and healthy volunteers were recruited and divided into three groups according to lung function and smoking history: 18 non-smoking healthy control group (NS group), 18 smoking non-COPD group (HS group) and 20 smoking COPD group (COPD group). Lung tissues of the subjects were collected and the expression of ARSK was detected.

[0022] 1.1. Extract RNA from lung tissue and detect the expression of ARSK by PCR.

[0023] Take an appropriate amount of tissue and place it in a 2ml EP tube. Add 500 μl Trizol to immerse the tissue. Grind the tissue with a grinder (3 gears) (20~30s) until it is crushed. Transfer to a 1.5 ml RNase-free EP tube and add 500 μl Trizol. Place on ice for 5 minutes to lyse, add 200 μl chloroform (trichloromethane), shake vigorously (milky red), and place on ice for 10 minutes. Centrifuge in a pre-cooled centrifuge (4 degrees, 12000 rpm, 15 minutes). After centrifugation, carefully transfer the upper aqueous phase (about 1 / 2 Trizol volume) to a new EP tube, add an equal volume of isopropanol, shake, and let stand at room temperature for 5-10 minutes, then centrifuge at 12000g at 4°C, and add 1ml 75% ethanol (prepared with DEPC-treated water) to wash the precipitate. For every 1ml of Trizol, add at least 1ml of ethanol, centrifuge at 10000rpm for 10 minutes at 4℃, remove the supernatant, leave to dry at room temperature, add appropriate amount of DEPC water (30-100ul water can be added according to experimental needs), blow several times with the tip of the gun to fully dissolve the RNA. Subsequently, reverse transcription was performed using Takara's cDNART PCR kit. Quantitative RT-PCR was performed using Takara's SYBR Premix Ex Taq. The relative expression level of the target gene was based on the β-Actin gene as an internal reference.

[0024] like Figure 1 As shown in a, the RNA expression of ARSK in the lung tissues of COPD people was significantly reduced compared with that of non-smokers and healthy smokers.

[0025] 1.2. FISH staining was performed on lung tissue sections to detect the expression of ARSK.

[0026] Fluorescence in situ hybridization (FISH) was performed at Wuhan Pinofi Biotech (Wuhan, China) to evaluate the expression of ARSK gene in fixed human lung sections and mouse lung sections. A digoxigenin-labeled locked nucleic acid (LNA) probe targeting ARSK was used, with the sequence of UUGUCCACGCUUCCACACGAUUA. The probe / target complex was visualized after a series of horseradish peroxidase (HRP)-mediated tyramide signal amplification (TSA) reactions.

[0027] like Figure 1 As shown in b, the red color in the slice is in situ ARSK, and the blue color is nucleus DAPI. It can be seen that the red fluorescence intensity in the lung slices of COPD people is significantly higher than that in the lung slices of non-smokers and healthy smokers. Therefore, the expression of ARSK is significantly reduced in COPD people.

[0028] 2. Animal experiment: A COPD model was established in mice using passive smoking. After the model was established, the lung function of the mice was tested. RNA was extracted from the lung tissue of the mice and the expression of ARSK was detected by PCR. Lung tissue sections were stained with FISH to detect the expression of ARSK.

[0029] 2.1. Construction of mouse COPD model All mice (8-12 weeks old, male) were housed in SPF animal isolation cages at Tongji Hospital and maintained under a 12-hour light-dark cycle. They were exposed to room air or smoke for approximately 3 hours per day (12 cigarettes burned each time, 45 minutes of exposure, 4 times a day), 5 days a week for 3 months, using Hongjinlong cigarettes.

[0030] 2.2. Extract RNA from mouse lung tissue and detect the expression of ARSK by PCR.

[0031] RNA was extracted from mouse lung tissues and the expression of ARSK was detected by PCR as described previously.

[0032] like Figure 1 As shown in c, the RNA content of ARSK in the lung tissue of COPD model mice was significantly higher than that in control mice, indicating that the expression of ARSK was significantly reduced after being stimulated by cigarette smoke.

[0033] 2.3. FISH staining was performed on lung tissue sections to detect the expression of ARSK.

[0034] FISH experiments were performed to detect the expression of ARSK in mouse lung sections as described previously.

[0035] like Figure 1 As shown in d, the red color in the slice is in situ ARSK, and the blue color is nucleus DAPI. It can be seen that the red fluorescence intensity in the lung slices of COPD model mice is significantly higher than that of control mice, indicating that the expression of ARSK is significantly reduced after being stimulated by cigarette smoke.

[0036] 3. Cell experiment: Human airway epithelial cells HBE4-E6 / E7 (ATCC, CRL-2078) were used as the research object, and different concentrations of cigarette smoke extract (CSE) were used to stimulate different time periods. No CSE was added as the control group for in vitro experimental research. Subsequently, cell RNA was extracted and the expression of ARSK was detected by PCR.

[0037] 3.1 Cell culture Human bronchial epithelial cells HBE4-E6 / E7 (ATCC, CRL-2078) were cultured in medium 1640 containing 10% fetal bovine serum. The cells were maintained in a humidified incubator at 37°C with 5% CO2, seeded in 12-well plates and exposed to different concentrations of CSE (cigarette smoke extract) for different times.

[0038] 3.2. Extracting cellular RNA and detecting ARSK expression by PCR Add 1ml Trizol to each well, pipette repeatedly and transfer to a 1.5ml RNase-free EP tube, add 200μl chloroform (chloroform), shake vigorously (milky red), and let stand on ice for 10 minutes. Centrifuge in a pre-cooled centrifuge (4 degrees, 12000 rpm, 15 minutes). After centrifugation, carefully transfer the upper aqueous phase (about 1 / 2 Trizol volume) to a new EP tube, add an equal volume of isopropanol, shake and let stand at room temperature for 5-10 minutes, then centrifuge at 12000g at 4°C, and add 1ml 75% ethanol (prepared with DEPC-treated water) to wash the precipitate. Add at least 1ml ethanol for every 1ml Trizol used, centrifuge at 4℃ 10000rpm for 10 minutes, remove the supernatant, let dry at room temperature, add an appropriate amount of DEPC water (30-100ul water can be added according to experimental needs), pipette several times with a pipette tip to fully dissolve the RNA. Subsequently, reverse transcription was performed using a cDNA RT PCR kit from Takara. Quantitative RT-PCR was performed using SYBR Premix Ex Taq from Takara. The relative expression level of the target gene was calculated using the β-Actin gene as an internal reference.

[0039] like Figure 1 As shown in e and f, with the increase of CSE stimulation concentration and time, the RNA level of ARSK gradually decreased.

[0040] In summary, the expression of ARSK in the lung tissue of COPD people was significantly reduced compared with that in healthy people, and the expression in the lung tissue of COPD model mice was significantly reduced compared with that in control mice. Similarly, the expression of ARSK in HBE cells stimulated by CSE was significantly reduced.

[0041] Example 2: Effect of overexpression of ARSK on airway epithelial cell senescence HBE cells were transfected with control or ARSK overexpression plasmids to verify overexpression efficiency, and CSE was added for 24 hours before evaluating cell senescence using Western blotting, β-galactosidase staining, PCR, and ELISA. The following steps were included: 1. ARSK overexpression plasmid construction and cell transfection The ARSK gene sequence was obtained from the NCBI gene library, and the pcDNA expression vector was selected. The target sequence and the vector were cut by restriction endonucleases, and then DNA ligase was used to connect them. Finally, the connected recombinant plasmid was transferred into the competent cell DH5α for transformation and plasmid DNA extraction. HBE cells were evenly inoculated in a 12-well plate. When they grew to a density of 80%, the liposome transfection reagent Lipo3000 was used to form a complex with the plasmid DNA, and then added to the cell culture medium. Through the interaction between the liposome and the cell membrane, the plasmid DNA entered the cell. After 24 hours, CSE was added to continue stimulation for 24 hours, and the cell protein was extracted for overexpression verification. Therefore, we divided the cells into four groups: control group, control + CSE group, overexpression group, and overexpression + CSE group.

[0042] like Figure 2 As shown in a, the cells in the overexpression group and overexpression + CSE group had the Flag tag carried by the ARSK plasmid, while the Flag tag expression was not observed in the control group, indicating that the ARSK overexpression plasmid was successfully transfected.

[0043] 2. Western blotting Total protein extraction from HBE cells was performed using RIPA lysis buffer containing phosphatase inhibitors. Proteins were separated by 10% or 12% SDS-PAGE gel, then transferred to PVDF membranes and blocked in 5% milk for 1-2 hours. Subsequently, the membranes were incubated with primary antibodies overnight at 4°C, rinsed three times with TBST, incubated with secondary antibodies for 1 hour, and finally exposed using chemiluminescent solution.

[0044] like Figure 2 As shown in b, c, and d, the expression of senescence proteins p21 and p53 in the control + CSE group was significantly increased compared with the control group, but the expression of p21 and p53 in the overexpression + CSE group was significantly decreased compared with the control + CSE group, indicating that overexpression of ARSK can significantly improve the expression of CSE-induced cellular senescence proteins.

[0045] 3. PCR PCR experiments were performed as described previously.

[0046] like Figure 2 As shown in Figure e, the RNA level expression of IL-6, IL-8 and IL-1β in the control + CSE group was significantly increased compared with the control group, but the RNA level expression of IL-6, IL-8 and IL-1β in the overexpression + CSE group was significantly decreased compared with the control + CSE group, indicating that overexpression of ARSK can significantly improve the CSE-induced aging-related secretory phenotype.

[0047] 4.ELISA Specific IL-6, IL-8 and IL-1β antibodies are immobilized in the wells of an ELISA plate. This is done by diluting the antigen in coating buffer and incubating overnight at 4°C. After coating, unbound sites are blocked with blocking buffer to reduce nonspecific binding. This is usually incubated for 1-2 hours at room temperature. The plate is washed with washing buffer to remove unbound blocking buffer. The sample to be tested and a series of standards of known concentrations are added to the ELISA plate. This is usually incubated for 1-2 hours at 37°C to allow the antigen in the sample to bind to the coated antibody. The plate is washed again with washing buffer, and the specific primary antibody is added and incubated for 1-2 hours at 37°C. The plate is washed again with washing buffer, and the secondary antibody conjugated to HRP is added and incubated for 1 hour at 37°C. Finally, the plate is washed again, and a substrate that can be catalyzed by HRP to produce a color change is added. After a certain period of time, the reaction is stopped by adding stop solution to stabilize the color change, and the results are finally read on a microplate reader.

[0048] like Figure 2 As shown in figure f, the protein expression levels of IL-6, IL-8 and IL-1β in the control + CSE group were significantly increased compared with the control group, but the protein expression levels of IL-6, IL-8 and IL-1β in the overexpression + CSE group were significantly decreased compared with the control + CSE group, indicating that overexpression of ARSK can significantly improve the CSE-induced aging-related secretory phenotype.

[0049] 5. β-Galactosidase staining After washing with PBS and fixing in β-galactosidase fixative for 15 min, we stained HBE cells using the SA-β-galactosidase staining kit (Solarbio, Beijing, China) at 37°C overnight. The staining results were observed and photographed under an optical microscope.

[0050] like Figure 2 As shown in g, the number of senescent cells in the control + CSE group increased significantly compared with the control group, and the number of senescent cells in the overexpression + CSE group decreased significantly compared with the control + CSE group, indicating that overexpression of ARSK can significantly improve the increase in the number of senescent cells induced by CSE.

[0051] In summary, overexpression of ARSK can reduce the expression of cell senescence-related proteins p21 and p53 caused by CSE stimulation, reduce the number of senescent cells, and alleviate the senescence-related secretory phenotype.

[0052] Example 3: Effect of overexpression of ARSK on mitochondrial autophagy in airway epithelial cells HBE cells were transfected with control or ARSK overexpression plasmids, and mitochondrial autophagy was detected by Western blotting 24 h after CSE stimulation.

[0053] 1. ARSK overexpression plasmid construction and cell transfection The experimental process is shown in step 1 of Experimental Example 2. Thus, we divided the cells into four groups: control group, control + CSE group, overexpression group, and overexpression + CSE group.

[0054] 2. Western blotting Experiments were performed as previously described like Figure 3 As shown in a, b, the expression of mitophagy proteins PINK1, Parkin, LC3, and p-DRP1 in the control + CSE group was significantly increased compared with the control group, but the expression of mitophagy proteins PINK1, Parkin, LC3, and p-DRP1 in the overexpression + CSE group was significantly decreased compared with the control + CSE group.

[0055] In summary, overexpression of ARSK can alleviate the expression of mitophagy proteins PINK1, Parkin, LC3, and p-DRP1 induced by CSE stimulation.

[0056] Example 4: Adding mitochondrial autophagy inhibitors to HBE cells after ARSK knockdown to observe the recovery effect on mitochondrial autophagy and cell senescence HBE cells were transfected with control or ARSK small interference gene, and then stimulated with CSE for 24 h after adding mitochondrial autophagy inhibitor. Mitochondrial autophagy and cell senescence were detected by Western blotting.

[0057] 1. Transfect HBE cells with control or ARSK small interfering gene ARSK siRNA and control siRNA were purchased from Ruibo Biotechnology. The ARSK siRNA sequence is GGACTATACTTCAGGACAT. HBE cells were seeded in 12-well plates, and 500ul of culture medium, 60nM siRNA, and 1ul of Lipo3000 were added to each well. The medium was changed after 8-12h, and CSE and mitochondrial autophagy inhibitor liensinine were added after 12h to stimulate for 24h. Thus, the cells were divided into 6 groups: control group, control + CSE group, ARSK knockdown group, ARSK knockdown + CSE group, ARSK knockdown + liensinine group, ARSK knockdown + liensinine + CSE group.

[0058] like Figure 4 As shown in a, the RNA level expression of ARSK in HBE cells of the ARSK knockdown group was significantly decreased compared with that in the control group cells, thereby determining the knockdown efficiency of ARSK.

[0059] 2. CCK8 experiment An appropriate amount of cells was inoculated into a 96-well cell culture plate, usually with a cell density of 1×10^4 to 1×10^5 cells per well. After cell inoculation, the cells were cultured in an environment of 37°C and 5% CO2 for 24 hours to allow the cells to attach to the wall and grow stably. Different concentrations of lianxinine were added to the cell culture plate for 24 hours. Subsequently, 10 μL of CCK8 reagent was added to each well and gently shaken to mix. The cell culture plate was placed in a 37°C incubator and incubated for 1-4 hours. After sufficient reaction, the absorbance of each well was measured at a wavelength of 450 nm using an ELISA reader.

[0060] like Figure 4 As shown in b, when the concentration of liensinine was 40uM, the cells were in good condition. Therefore, we selected 40uM for subsequent treatment.

[0061] 3. Western blotting Method as described above like Figure 4 As shown in Figure c, the expression of mitophagy proteins PINK1, Parkin, LC3, and p-DRP1 in the ARSK knockdown + CSE group was significantly increased compared with the control + CSE group, accompanied by an increase in the expression of senescence proteins p21 and p53, indicating that ARSK knockdown can aggravate the increase in mitophagy and senescence caused by CSE stimulation. The expression of mitophagy proteins PINK1, Parkin, LC3, and p-DRP1 in the ARSK knockdown + liensinine + CSE group was significantly reduced compared with the ARSK knockdown + CSE group, indicating that mitophagy was indeed inhibited. At this time, we found that the expression of senescence proteins p21 and p53 was also significantly reduced, indicating that mitophagy mediates the regulatory effect of ARSK on cell senescence.

[0062] 3. β-Galactosidase staining The method is the same as described above.

[0063] like Figure 4 As shown in Figure d, the positive rate of β-galactosidase staining in the ARSK knockdown + CSE group was significantly higher than that in the control + CSE group, indicating that ARSK knockdown aggravated cell senescence, while the number of senescent cells in the ARSK knockdown + liensinine + CSE group was significantly reduced compared with the ARSK knockdown + CSE group, indicating that under the stimulation of CSE, inhibiting mitochondrial autophagy can alleviate the aggravation of cell senescence caused by ARSK knockdown.

[0064] 4.ELISA The method is the same as described above.

[0065] like Figure 4As shown in Figure e, the levels of IL-6, IL-8 and IL-1β in the cell supernatant of the ARSK knockdown + CSE group were significantly higher than those of the control + CSE group, indicating that ARSK knockdown aggravated the secretory phenotype associated with cell senescence, while the levels of the above factors in the ARSK knockdown + liensinine + CSE group were significantly lower than those in the ARSK knockdown + CSE group. This indicates that under the stimulation of CSE, inhibition of mitochondrial autophagy can alleviate the aggravation of the secretory phenotype associated with cell senescence by ARSK knockdown.

[0066] In summary, if Figure 4 As shown, adding mitophagy inhibitors to ARSK knockdown HBE cells can partially reverse the increase in mitophagy and cell senescence caused by CSE stimulation, indicating that mitophagy mediates the regulatory effect of ARSK on HBE cell senescence.

[0067] Example 5: Smoke-induced modeling of mice with ARSK overexpression in the lungs to observe the preventive and therapeutic effects on COPD lung aging ARSK-AAV was injected into the airways of wild-type C57 mice to construct ARSK-overexpressing mice in the lungs. A blank control AAV was injected into the airways of the control mice. After 21 days, a mouse COPD model was established by passive smoking for 3 months. After modeling, lung function was detected by pulmonary function instrument, lung inflammation was detected by HE staining of lung sections, and lung aging was detected by Western blotting, PCR and β-galactosidase staining. The mice were divided into four groups: control group, control smoking group, ARSK-AAV group, and ARSK-AAV smoking group.

[0068] 1. Construct a mouse COPD model and detect lung function The method for establishing the mouse COPD model was as described above. After the model was established, the lung function was tested by a spirometer. The lung function of all mice was measured under general anesthesia using the FlexiVent system (SCIREQ, Canada). Anesthesia was induced by intraperitoneal injection of 1% sodium pentobarbital at a dose of 10 ml per kg of body weight.

[0069] like Figure 5 a shows the lung function FEV of the mice in the control group compared with that of the mice in the control group. 0.1s , FEV 0.1s The FVC value was significantly reduced, indicating that the COPD model mice were successfully established. The lung function of the ARSK-AAV smoked mice was significantly increased compared with the control smoked mice, indicating that ARSK overexpression in the lungs has a preventive and therapeutic effect on COPD.

[0070] 2. HE staining of lung sections Fresh mouse lung tissue was taken out and fixed with 4% paraformaldehyde for 48 hours, 70% ethanol for 1.5 hours, 80% ethanol for 30 minutes, 95% ethanol for 15 minutes, anhydrous ethanol for 10 minutes, xylene for 2 times, each for 15 minutes, paraffin I for 30 minutes, paraffin II for 90 minutes, paraffin III for 6 hours, embedded and sliced, and then dried at 55-60°C for 1 hour. Then dewaxed to water, stained with hematoxylin and eosin, and dehydrated and sealed after hydrochloric acid alcohol differentiation.

[0071] like Figure 5 As shown in b, the infiltration of inflammatory cells around the airways of mice in the control fumigation group was significantly increased compared with the control group mice, while the inflammation around the airways of mice in the ARSK-AAV fumigation group was significantly reduced compared with the control fumigation group mice. This indicates that ARSK overexpression in the lungs alleviates COPD lung inflammation, which is also a part of lung aging. It also indicates that ARSK overexpression in the lungs alleviates COPD lung aging.

[0072] 3. PCR The method is the same as described above.

[0073] like Figure 5 As shown in Figure c, the expression of IL-6, KC and IL-1β in lung tissue of mice in the control fumigation group was significantly increased compared with that in the control group, while the expression of IL-6, KC and IL-1β in lung tissue of mice in the ARSK-AAV fumigation group was significantly decreased compared with that in the control fumigation group. This indicates that ARSK overexpression in the lung can alleviate COPD lung inflammation, and also indicates that ARSK overexpression in the lung can alleviate COPD lung aging.

[0074] 4.ELISA After anesthesia, the mouse's limbs were fixed, the chest cavity was opened, the trachea was exposed, a small incision was made in the trachea near the upper part of the mouse head, and an lavage needle was inserted into the trachea, which was tied to the trachea with a thin thread. A 1 mL syringe was used to remove the needle and then inserted into the lavage needle to lavage the mouse lungs. Each time, 0.3 mL of pre-cooled saline containing 1% FBS (fetal bovine serum) was drawn with a syringe and slowly infused. After the syringe was pumped three times, the recovered liquid was slowly withdrawn and placed in a 1.5 mL EP tube and placed on ice. After centrifugation, the supernatant was obtained to obtain the bronchoalveolar lavage fluid (BALF) supernatant. ELISA was performed as described above to detect the content of IL-6, KC and IL-1β in BALF.

[0075] like Figure 5As shown in Figure d, the IL-6, KC and IL-1β levels in the BALF of the control fumigation group mice were significantly increased compared with those of the control group mice, while the IL-6, KC and IL-1β levels in the BALF of the ARSK-AAV fumigation group mice were significantly decreased compared with those of the control fumigation group mice. This indicates that ARSK overexpression in the lungs alleviates COPD lung inflammation, and also indicates that ARSK overexpression in the lungs alleviates COPD lung aging.

[0076] 5. β-Galactosidase staining Mouse lung sections were stained as described previously.

[0077] like Figure 5 As shown in Figure e, the positive rate of β-galactosidase staining in the airway epithelium of the mice in the control fumigation group was significantly higher than that in the control group, while the positive rate of β-galactosidase staining in the airway epithelium of the mice in the ARSK-AAV fumigation group was significantly lower than that in the control fumigation group, indicating that ARSK overexpression in the lung alleviates the aging of the airway epithelium in COPD.

[0078] 6. Western blotting Mouse lung tissue proteins were extracted and subjected to Western blotting as described previously.

[0079] like Figure 5 As shown in Figure f, the expression of senescence proteins p21 and p53 in the lung tissue of mice in the control fumigation group was significantly increased compared with that in the control group, while the expression of senescence proteins p21 and p53 in mice in the ARSK-AAV fumigation group was significantly reduced compared with that in the control fumigation group, indicating that ARSK overexpression in the lung alleviates COPD lung senescence.

[0080] In summary, the lung function of ARSK overexpressing mice was significantly improved compared with that of control mice after smoking. HE staining showed that the inflammation around the airways of ARSK overexpressing mice was significantly reduced, and the RNA level of inflammatory factors in lung tissue and the number of inflammatory cells in bronchoalveolar lavage fluid (BALF) were significantly reduced. Western blotting and β-galactosidase staining showed that the lung aging of ARSK overexpressing mice was significantly alleviated.

[0081] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of a reagent for detecting arylsulfatase K in the preparation of products for diagnosing chronic obstructive pulmonary disease, wherein the products include medicines.

2. The application according to claim 1, characterized in that: The chronic obstructive pulmonary disease is chronic obstructive pulmonary disease induced by cigarette smoke or cigarette smoke extract.

3. Use of arylsulfatase K in the preparation of drugs for preventing, delaying or treating chronic obstructive pulmonary disease.

4. The application according to claim 3, characterized in that: Vectors that overexpress arylsulfatase K can inhibit airway epithelial cell senescence, thereby preventing, delaying or treating chronic obstructive pulmonary disease.

5. Use of arylsulfatase K in the preparation of products for delaying airway epithelial cell aging, wherein the products include medicines.

6. The application according to claim 5, characterized in that: The vector overexpressing arylsulfatase K delays the senescence of airway epithelial cells by increasing the expression of arylsulfatase K, reducing the expression of cell senescence-related proteins p21 and p53, and inhibiting mitochondrial autophagy.

7. Use of a kit in the preparation of a product for diagnosing chronic obstructive pulmonary disease, characterized in that: The kit includes a reagent for detecting arylsulfatase K.

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