Application of mitochondrial respiratory chain complex V in diagnosis of idiopathic pulmonary fibrosis

Abstract

The present application finds that the activity of mitochondrial respiratory chain complex V of patients with idiopathic pulmonary fibrosis is significantly lower than that of healthy people, and finds that the increase of TGF-β is related to the decrease of the activity of mitochondrial respiratory chain complex V of patients with pulmonary fibrosis, thereby proving that the mitochondrial respiratory chain complex V is a molecular marker for the diagnosis of idiopathic pulmonary fibrosis. Further, the present application discloses the use of a reagent for detecting the activity of mitochondrial respiratory chain complex V in the preparation of a composition or kit for assisting in the diagnosis of idiopathic pulmonary fibrosis, and further discloses a composition for assisting in the diagnosis of idiopathic pulmonary fibrosis, which comprises a reagent for detecting the activity of mitochondrial respiratory chain complex V, and a kit for assisting in the diagnosis of idiopathic pulmonary fibrosis, and a method for screening an agent for treating idiopathic pulmonary fibrosis in vitro.

Description

TECHNICAL FIELD BACKGROUND

[0001] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fibrotic interstitial lung disease (Lederer and Martinez, 2018). The prognosis of IPF is poor, with an average survival time of 2.5 to 5 years, and only 20% to 30% of subjects are still alive 5 years after diagnosis (Noble et al., 2011; Raghu, 2017; Raghu et al., 2011). So far, the exact cause of IPF is still unknown, and only two approved drugs, nintedanib and pirfenidone, are available for treatment. These two drugs have been shown to slow the progression of the disease, but cannot stop it (Raghu, 2017). Therefore, how to effectively diagnose and treat in the early stage of the disease has always been the key to the problem of IPF disease. Mitochondrial function and behavior are the physiological core of humans, so mitochondrial dysfunction can lead to the occurrence of various diseases (Nunnari and Suomalainen, 2012). Recent studies have shown that the changes in bioenergetics and mitochondrial function with aging may be involved in the pathogenesis of IPF (Bueno et al., 2020; Morita et al., 2017).

[0002] Complex V is the ATP synthase in the oxidative phosphorylation (OXPHOS) system of mitochondria. It converts the proton flux with ADP and inorganic phosphate into ATP using the proton electrochemical gradient generated on the mitochondrial membrane by respiratory chain complexes I-IV (Meulemans et al., 2010). In human cells, 90% or more of cellular ATP is synthesized by Complex V (Jonckheere et al., 2012). Meanwhile, studies have shown that Complex V dimerization collaborates with the mitochondrial contact site and cristae organizing system (MICOS) to establish the inner membrane curvature of mitochondria, initiating the formation of cristae, thus determining the morphology of mitochondria (Baker et al., 2019; Cogliati et al., 2016; De los Rios Castillo et al., 2011; Stephan et al., 2020). Therefore, Complex V has two main functions, ATP synthase and cristae organizer, which play a decisive role in mitochondrial biogenesis and cell biology. Functional defects related to Complex V have been reported in cardiovascular diseases (Kwong and Molkentin, 2015), cancer (Bonora et al., 2015), and neurodegenerative diseases (Golpich et al., 2017). Therefore, it is of great significance to study the functional mechanism of mitochondrial respiratory chain Complex V in IPF disorders. SUMMARY

[0003] The technical problems existing in the prior art are solved by the following technical solutions of the present application.

[0004] 1. Use of a reagent for detecting the activity of mitochondrial respiratory chain Complex V in the preparation of a composition or kit for aiding the diagnosis of idiopathic pulmonary fibrosis.

[0005] 2. The use of item 1, wherein the reagent is an antibody against Complex V, an antigen-binding fragment thereof, a probe or primer binding to a nucleic acid encoding Complex V, or an aptamer.

[0006] 3. The use of item 2, wherein the antibody is a monoclonal antibody.

[0007] 4. A composition for aiding the diagnosis of idiopathic pulmonary fibrosis, comprising a reagent for detecting the activity of mitochondrial respiratory chain Complex V.

[0008] 5. The composition of item 4, wherein the reagent is an antibody against Complex V, an antigen-binding fragment thereof, a probe or primer binding to a nucleic acid encoding Complex V, or an aptamer.

[0009] 6. The composition of item 5, wherein the antibody is a monoclonal antibody.

[0010] 7. A kit for aiding in the diagnosis of idiopathic pulmonary fibrosis, comprising the composition of any one of items 4-6 and instructions for use.

[0011] 8. A method of screening for an agent for treating idiopathic pulmonary fibrosis in vitro, comprising administering the agent to a TGF-βl -induced idiopathic pulmonary fibrosis cell model and determining the activity of mitochondrial respiratory chain complex V before and after the administration of the agent, wherein an increase in the activity of complex V after the administration of the agent compared to the activity of complex V before the administration of the agent indicates that the agent is a potential therapeutic agent for idiopathic pulmonary fibrosis.

[0012] 9. The method of item 8, wherein the method further comprises determining the activity of mitochondrial respiratory chain complex I, the amount of subunit protein NDUFA9, the amount of ATP5a, the amount of ROS, the amount of ATP, and / or the mitochondrial membrane potential before and after the administration of the agent, and determining the activity of mitochondrial respiratory chain complex V before and after the administration of the agent, wherein an increase in the activity of complex I, an upregulation in the amount of subunit protein NDUFA9, an upregulation in the amount of ATP5a, a decrease in the amount of ROS, an increase in the amount of ATP, and a decrease in the mitochondrial membrane potential after the administration of the agent compared to the activity of complex I, the amount of subunit protein NDUFA9, the amount of ATP5a, the amount of ROS, the amount of ATP, and the mitochondrial membrane potential before the administration of the agent, respectively, indicates that the agent is a potential therapeutic agent for idiopathic pulmonary fibrosis.

[0013] 10. The method of item 8 or 9, wherein the idiopathic pulmonary fibrosis cell model is an idiopathic pulmonary fibrosis cell model constructed with A549 cells.

[0014] The agent for detecting the activity of mitochondrial respiratory chain complex V described herein is not particularly limited as long as it is capable of specifically detecting the level of mitochondrial respiratory chain complex V activity and changes in the level. It is contemplated that an antibody or an antibody antigen-binding fragment that specifically binds to complex V can be used to detect the activity of mitochondrial respiratory chain complex V.

[0015] As used herein, the term "antibody" includes an antibody or an antigen-binding fragment thereof that specifically binds to its target and is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody, unless otherwise specified. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgGl antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is a scFv antibody. In some embodiments, such an antibody or an immunologically active fragment thereof that binds to its target is a mouse, chimeric, humanized, or fully human monoclonal antibody.

[0016] An aptamer refers to a nucleic acid molecule having binding activity for a particular target molecule. An aptamer can inhibit the activity of a particular target molecule by binding to the particular target molecule. The aptamer of the present application can be RNA, DNA, a modified nucleic acid, or a mixture thereof. The aptamer of the present application can also take a linear or circular form.

[0017] The aptamer of the present application can be chemically synthesized as disclosed herein and by methods known in the art per se. Aptamers bind to target molecules in a wide variety of binding modes, such as ionic bonds based on the negative charge of phosphate groups, hydrophobic bonds and hydrogen bonds based on ribose, and hydrogen bonds and stacking interactions based on nucleic acid bases. In particular, ionic bonds based on the negative charge of phosphate groups, which exist in the same number as the number of constituent nucleotides, are strong, and bind to lysine and arginine, which exist on the positively charged surface of proteins. For this reason, nucleic acid bases not involved in direct binding to the target molecule can be substituted. In particular, because the region of the stem structure has already formed base pairs, and faces the inside of the double helix structure, nucleic acid bases are less likely to be directly bound to the target molecule. Therefore, even when a base pair is replaced with another base pair, the activity of the aptamer is generally not reduced. In structures in which base pairs are not formed, such as loop structures, base substitution is possible if the nucleic acid bases are not involved in direct binding to the target molecule. With respect to modification of the 2'-position of ribose, the functional group at the 2'-position of ribose rarely directly interacts with the target molecule, but in many cases, it is irrelevant, and can be substituted with another modified molecule. Therefore, unless the functional group involved in direct binding to the target molecule is substituted or deleted, the aptamer generally retains its activity. It is also important that the overall three-dimensional structure not be widely changed.

[0018] Aptamers can be prepared by utilizing the SELEX method or improved versions thereof (e.g., Ellington et al. (1990) Nature, 346, 818-822; Tuerk et al. (1990) Science, 249, 505-510). In the SELEX method, aptamers showing stronger binding potential for the target molecule are enriched and selected by increasing the number of rounds or using a competitor substance. Therefore, by adjusting the number of rounds of SELEX and / or changing the competition conditions, in some cases, aptamers having different binding forces, aptamers having different binding modes, and aptamers having the same binding force or binding mode but different base sequences can be obtained. The SELEX method includes an amplification process by PCR; by causing mutations during the process by using manganese ions or the like, SELEX can be performed with higher diversity. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 .TGF-β elevation is associated with decreased mitochondrial respiratory chain complex V activity in patients with pulmonary fibrosis.

[0020] Figure 1 a shows the complex V activity of patients with pulmonary fibrosis and healthy people (HC). Figure 1 b shows the complex V activity of IPF, CTD-ILD and healthy people (HC). Figure 1 c shows the correlation analysis of complex V activity and serum TGF-β1 level. Figure 1 d shows the ROC curve analysis of complex V activity (solid line) and combined level of complex V activity and serum TGF-β1 (dashed line) of patients with pulmonary fibrosis and healthy people (HC).

[0021] Figure 2 .TGF-β induces A549 cells to change cell morphology and mitochondrial morphology.

[0022] Figure 2 a shows that A549 cells are treated with different concentrations of TGF-β1 for 48h, and inverted fluorescence microscope, transmission electron microscope and OMX mode structured light illumination microscope are used to take pictures. DIC images show the morphological changes of cells under different treatment conditions, and the images are taken using Nikon Ts2 inverted microscope 10x objective lens, scale: 100 μm. TEM images show the changes of mitochondrial cristae under different experimental conditions, which are taken using Tecnai Spirit (120kV), scale: 500nm. OMX images show the 3D changes of mitochondrial (white) morphology under different treatment conditions, which are taken using SIM-OMX and 60x oil immersion objective lens (scale: 10 μm). TGF-β1 induces dynamic changes of mitochondria in lung epithelial cells. Figure 2 b shows that the Mitochondria Analyzer plug-in (default parameters) in the Image J software is used to analyze the mitochondrial surface area, branches and branch nodes in each cell in the 3D image of the cell, and the data is statistically analyzed, and the single factor variance analysis of Turkey multiple comparison test is carried out. *: P<0.05.

[0023] Figure 3 .TGF-β induces A549 cells to change mitochondrial function.

[0024] Figure 3 a shows the method of IGA is used to detect the activities of respiratory chain complex I, complex II, complex IV, complex V in the purified mitochondrial protein. Figure 3 b shows the protein expression levels of NDUFA9, SDHA, UQCRC2, COX4, ATP5a, ATP6, ATP6V0D1, ATP8, TOM20 and GAPDH in A549 under different treatment conditions are detected by Western blot. The protein loading amount of all lanes is consistent.Figure 3 c shows the detection of mitochondrial membrane potential in A549 cells induced by different concentrations of TGF-β1 by TMRE. Images were taken using OLYMPUS FV3000 with 60x oil immersion objective under the same parameter settings. Scale bar: 50 pm. Figure 3 d shows the statistical analysis of the data from Image J Figure 3 c shows the mean fluorescence intensity and the fold change of mitochondrial membrane potential, which was plotted using graphPad. Figure 3 e, f shows the mean fluorescence intensity of DCF and MitoSOx in A549 cells induced by TGF-β1, which was measured by flow cytometry, and the fold change of ROS and MitoSOx in A549 cells. Figure 3 d, e, f Data are expressed as mean with 95% CI and one-way ANOVA with Turkey’s multiple comparison test. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

[0025] Figure 4 .TGF-β-induced fibrosis in A549 cells was recovered after PFD treatment.

[0026] Figure 4 a shows the images of A549 cells treated with different treatment conditions for 48 hours, which were taken using inverted fluorescence microscope and laser confocal microscope. Different treatment conditions: T = TGF-β 15 ng / ml, P1 = PFD 250 pg / ml, P2 = PFD 500 pg / ml. DIC images show the morphological changes of cells under different treatment conditions, which were taken using Nikon Ts2 inverted microscope, scale bar: 100 pm. Confocal images show the morphological changes of mitochondria (white) under different treatment conditions, which were taken using OLYMPUS FV1000 60x oil immersion objective, scale bar: 50 pm, and the scale bar of the magnified image: 20 pm. Figure 4 b shows the detection of protein activity of mitochondrial respiratory chain complex I, complex II, complex IV, complex V in purified mitochondrial proteins using IGA method. Figure 4 c shows the results of immunoblotting experiment of NDUFA9, SDHA, UQCRC2, COX4, ATP5a, TOM20, β-actin and GAPDH protein expression levels in A549 protein extracts under different treatment conditions. The amount of protein loaded in all lanes is consistent. Figure 4 d shows the fold change of ROS in A549 cells under different treatment conditions. The mean fluorescence intensity of DCF in A549 cells was measured by flow cytometry. Figure 4 e shows the ATP content in A549 cells under different treatment conditions.Figure 4 f shows the changes of TMRE (mitochondrial membrane potential) in A549 cells under different treatment conditions. The mean fluorescence intensity was obtained from Image J and statistical analysis was performed. Figure 4 g shows the changes of mitochondrial membrane potential in A549 cells under different treatment conditions by laser confocal using TMRE detection. These images were taken using OLYMPUS FV3000 with a 60x oil immersion objective under the same parameter settings, scale bar: 50 pm. Figure 4 d, e, f Data are expressed as mean values with 95% CI and one-way ANOVA with Turkey’s multiple comparison test. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. DETAILED DESCRIPTION

[0027] In order to make the aims, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application with reference to specific examples and the accompanying drawings.

[0028] Example 1. TGF-β elevation is associated with decreased mitochondrial respiratory chain complex V activity in patients with pulmonary fibrosis

[0029] In order to study the relationship between IPF disease and mitochondrial respiratory chain complex V, we collected blood samples of patients diagnosed with IPF, CD-PF and HCs (healthy people), and detected the activities of mitochondrial respiratory chain complex I, complex II, complex III, complex IV and complex V by biochemical kits. Figure 1 It is shown that the decrease of mitochondrial respiratory chain complex V activity is associated with the elevation of TGF-β in patients with pulmonary fibrosis. Figure 1 a, b It is shown that the mitochondrial respiratory chain complex V activity of patients with pulmonary fibrosis is significantly decreased compared with healthy people. Figure 1 c It is shown that the decrease of mitochondrial respiratory chain complex V activity is significantly associated with the elevation of TGF-β. Figure 1 d shows the receiver operating characteristic (ROC) analysis of the complex V activity level of ILD patients and healthy people, and the result shows that the area under the curve (AUC) value is 74.44%.

[0030] Specific methods

[0031] Obtaining of human blood samples

[0032] Blood samples of adult IPF patients (N = 9), connective tissue disease-related PF (CTD-PF) patients (N = 41) and age- and gender-matched healthy controls (HCs) (N = 9) were collected from Tianjin Medical University General Hospital.

[0033] Detection of activity of mitochondrial respiratory chain complex in human blood sample

[0034] The detection of activity of mitochondrial respiratory chain complex in human blood sample was performed by using Micro Mitochondrial Respiratory Chain Complex Activity Assay Kit (Solarbio) according to its instructions.

[0035] Example 2. TGF-β induces A549 cells to change cell morphology and mitochondrial morphology

[0036] In order to study the relationship between TGF-β and mitochondria, we used TGF-β to induce A549 cells to construct an interstitial cell model, and observed the mitochondrial morphology and cristae condition. Figure 2 a The cell morphology, mitochondrial morphology and cristae condition of TGF-β induced A549 cells were observed by ordinary inverted fluorescence microscope, transmission electron microscope (TEM) and structured light illumination microscope (SIM, OMX mode), which showed that TGF-β induced A549 cells to change to interstitial cell morphology, mitochondrial morphology was broken, and mitochondrial network structure and cristae structure were damaged. Figure 2 b The intracellular mitochondrial surface area and branch node of the 3D picture taken by SIM were counted, which reflected the damage of mitochondrial network structure. The results showed that with the increase of TGF-β concentration, the mitochondrial structure damage gradually increased.

[0037] Specific method

[0038] Cell culture and grouping treatment

[0039] A549 cells were cultured in DMEM medium containing 10% FBS, 1% penicillin and 1% streptomycin in a 5% CO2, 37°C incubator.

[0040] TGF-β1 induction treatment method: first, A549 cells were cultured in DMEM medium without 10% FBS for 24 h for starvation treatment, then TGF-β1 was added to a final concentration of 0, 1, 2, 5, 10 ng / ml, and induced for 48 h.

[0041] Cell morphology observation: after TGF-β1 treatment, ordinary inverted fluorescence microscope was used to take pictures under objective lens 10X, bright field.

[0042] Transmission electron microscope observation of mitochondria morphology and cristae structure: After TGF-β1 treatment, cells were trypsinized, washed with PBS, and collected by centrifugation. Appropriate amount of 2.5% glutaraldehyde was added to fix the cells, and then 3% osmium tetroxide was used to re-fix the cells. The sample was dehydrated with ethanol, then embedded with Epon resin, sectioned, and then imaged with a transmission electron microscope.

[0043] SIM OMX mode was used to observe the 3D structure of mitochondria: After A549 cell treatment, MitoTracker Green and DAPI staining were used, and fresh medium was added after PBS washing for image acquisition on SIM.

[0044] Mitochondrial surface area, branch, and branch node statistics: First, the image obtained by SIM was used to construct the mitochondrial surface using imaris software, add the color bar of the surface area, and perform statistics and data output on the mitochondrial surface area. ImageJ software was used to count the mitochondrial branches and branch nodes.

[0045] Example 3, TGF-β induces mitochondrial dysfunction in A549 cells

[0046] To further study the effect of TGF-β on mitochondrial function, we used A549 cell model to study the mitochondrial oxidative phosphorylation system. Figure 3 a represents Blue native PAGE and In gel activity analysis of mitochondrial respiratory chain complexes, the results show that the activities of complex I and complex V decrease with the increase of TGF-β1 treatment concentration. Figure 3 b is the western blot result of mitochondrial complex subunit protein, NDUFA9 and ATP5a decrease, and there is no significant change in other quantities. Figure 3 c, d results show that the mitochondrial membrane potential (TMRE) decreases with the increase of TGF-β1 treatment concentration. Figure 3 e, f results show that the content of cellular ROS and mitochondrial ROS is significantly up-regulated. The results show that the increase of TGF-β concentration causes mitochondrial dysfunction.

[0047] Specific methods

[0048] Blue Native PAGE: collect the induced cells (Example 2), resuspend the cells with 200 μΐ pre-cooled PBS, then add equal volume of 4 mg / ml Digitonin (dissolved in PBS), mix well by inverting the tube for 2 times, incubate on ice for 10 min; then, add 1.5 ml pre-cooled PBS, centrifuge at 4°C, 10000 g for 10 min; discard the supernatant, then resuspend the pellet with 1 ml PBS, centrifuge at 4°C, 10000 g for 10 min to remove the residual Digitonin; add 50 μΐ BN sample buffer, 5 μΐ 10% DDM, resuspend the pellet by pipetting 10-20 times, make sure no foam is generated, fix on ice for 20 min; centrifuge at 4°C, 20000 g for 20 min, take the supernatant to a new EP tube, take 5 μΐ for protein quantification by BCA method, the rest add 2 μΐ 5% Coomassie Brilliant Blue G-250, mix well. Take 10 μg sample for Blue-native PAGE separation of mitochondrial complex.

[0049] Complex I activity: incubate the Blue-native PAGE separation gel in 50 mM Tris-Hcl, pH 7.4 buffer (containing 0.5 mM NBT (Nitroblue tetrazolium chloride), 5 mM NADH (Nicotinamide adenine dinucleotide)), at room temperature for 1 hour.

[0050] Complex II activity: incubate the Blue-native PAGE separation gel in 50 mM Tris-Hcl, pH 7.4 buffer (0.2 mM PMS (Methyl phenazinium methyl sulfate), 84 mM succinate, 50 mM NBT), at room temperature for 1 hour.

[0051] Complex IV activity: incubate the Blue-native PAGE separation gel in 50 mM Tris-Hcl, pH 7.4 buffer (containing 0.1% diaminobenzidine, 24 units / ml catalase, 0.1% cytochrome c), at 37 degrees for 3-6 hours.

[0052] Complex V activity: The Blue-native PAGE separation gel was rinsed in water for 10 minutes and placed in 0.1 M glycine buffer (pH 8.6) for 1 hour. The separation gel was then placed in a buffer containing the following: 35 mM Tris base, 270 mM glycine, 14 mM magnesium sulfate, 5 mM ATP, 0.2% silver nitrate, at 37 degrees for 3-6 hours.

[0053] Western blot: The induced cells (Example 2) were collected and added with 200 μl RIPA lysis buffer containing protease inhibitor cocktail (Roche), placed on ice for 15 minutes, centrifuged at 14000 rpm for 15 minutes, and the supernatant was used to determine the protein concentration with a BCA kit. A 15% SDS-PAGE gel was prepared, and the sample with the measured protein concentration was added with a certain volume of protein loading buffer, heated at 95 degrees for 15 minutes, and a certain amount of protein was added to a 12% SDS-PAGE gel, and electrophoresis was performed at 120 v for about 2 hours. The protein sample on the gel was transferred to a PVDF membrane, which was blocked with 5% skim milk at room temperature for 3 hours, and then different primary antibodies were diluted according to the instructions, and incubated overnight. The membrane was washed with TBST (150 mM NaCl, 20 mM Tris-HCl, pH 7.4, Tween-20 0.05%) for 3 times, 10 minutes each time. The secondary antibody was diluted at 1:3000, and incubated at room temperature for 3 hours. After washing the membrane with TBST for three times, hypersensitive ECL chemiluminescence reagent (Bi Yun Tian) was added, and the dark box was exposed.

[0054] Mitochondrial membrane potential detection: After the A549 cells were seeded into a confocal culture dish for TGF-β1 induction treatment, TMRE was diluted with PBS to 500 nM, incubated at 37 degrees for 10 minutes in the dark, washed with PBS, and then fresh culture medium was added. The different treated cells were photographed under the same conditions by a confocal microscope, and the fluorescence intensity was counted by ImageJ software.

[0055] Cell ROS detection: The treated cells were collected by trypsin digestion, treated according to the operation instruction of the reactive oxygen species detection kit (Bi Yun Tian), and detected by a flow cytometer.

[0056] Mitochondrial ROS detection: The treated cells were collected by trypsin digestion, treated according to the operation instruction of MitoSOX TM Red Mitochondrial Superoxide Indicator (Invitrogen), and detected by a flow cytometer.

[0057] Example 4. Mitochondrial function recovery after PFD treatment of TGF-β induced A549 cell fibrosis

[0058] To investigate the effect of PFD, a current IPF therapeutic drug, on the treatment of mitochondrial dysfunction and prevention of TGF-β-induced fibrosis, we used an A549 cell model to study this. Figure 4 a PFD can prevent TGF-β-induced cell morphological mesenchymal transition and mitochondrial morphological structure destruction. Figure 4 b Blue native PAGE and in gel activity analysis of mitochondrial respiratory chain complexes showed that the activities of complex I and complex V were significantly up-regulated after PFD treatment compared with the TGF-β-induced group. Figure 4 c Western blot results of mitochondrial complex subunit proteins showed that the amounts of subunit proteins NDUFA9 and ATP5a were significantly up-regulated after PFD treatment compared with the TGF-β-induced group, and there was no significant change in other amounts. Figure 4 d The results showed that the ROS content in the cells was significantly inhibited after the addition of PFD. Figure 4 e The results of ATP detection after PFD treatment showed that the ATP content was restored after the addition of PFD compared with the TGF-β-induced group. Figure 4 f, h The results showed that the mitochondrial membrane potential (TMRE) was significantly reduced after PFD treatment compared with the TGF-β-induced group. The results showed that the increase in TGF-β concentration caused mitochondrial dysfunction.

[0059] Specific methods

[0060] Cell treatment: First, the A549 cells were subjected to serum-free starvation for 24 h, and then 5 ng / ml TGF-β1 (T group), 5 ng / ml TGF-β1 + 250 μg / ml PFD (T-P1 group), 5 ng / ml TGF-β1 + 500 μg / ml PFD (T-P2 group), and 500 μg / ml PFD (P2 group) were added for 48 h induction treatment. The normally cultured cells were set as the control group.

[0061] The specific methods of cell morphology observation, Blue Native PAGE, western blot, ROS, and mitochondrial membrane potential detection were the same as those in the operation methods of Example 2 and Example 3 above.

[0062] ATP detection: The treated cells were collected by trypsin digestion, and the operation instructions of the ATP detection kit (Bi Yun Tian) were followed. The detection was performed by flow cytometry.

[0063] References

[0064] Baker, N., Patel, J., and Khacho, M. (2019). Linking mitochondrial dynamics, cristae remodeling and supercomplex formation: How mitochondrial structure can regulate bioenergetics. Mitochondrion 49, 259-268.

[0065] Bonora, M., Wieckowski, M. R., Chinopoulos, C., Kepp, O., Kroemer, G., Galluzzi, L., and Pinton, P. (2015). Molecular mechanisms of cell death: central implication of ATP synthase in mitochondrial permeability transition. Oncogene 34, 1475-1486.

[0066] Bueno, M., Calyeca, J., Rojas, M., and Mora, A. L. (2020). Mitochondrial dysfunction and metabolic reprogramming as drivers of idiopathic pulmonary fibrosis. Redox Biol 33, 101509.

[0067] Cogliati, S., Enriquez, J. A., and Scorrano, L. (2016). Mitochondrial Cristae: Where Beauty Meets Functionality. Trends Biochem Sci 41, 261-273.

[0068] De los Rios Castillo, D., Zarco-Zavala, M., Olvera-Sanchez, S., Pardo, J.P., Juarez, O., Martinez, F., Mendoza-Hernandez, G., Garcia-Trejo, J.J., and Flores-Herrera, O. (2011). Atypical cristae morphology of human syncytiotrophoblast mitochondria: role for complex V. J Biol Chem 286, 23911-23919.

[0069] Golpich, M., Amini, E., Mohamed, Z., Azman Ali, R., Mohamed Ibrahim, N., and Ahmadiani, A. (2017). Mitochondrial Dysfunction and Biogenesis in Neurodegenerative diseases: Pathogenesis and Treatment. CNS neuroscience & therapeutics 23, 5-22.

[0070] Jonckheere, A.I., Smeitink, J.A., and Rodenburg, R.J. (2012). Mitochondrial ATP synthase: architecture, function and pathology. Journal of inherited metabolic disease 35, 211-225.

[0071] Kwong, J.Q., and Molkentin, J.D. (2015). Physiological and pathological roles of the mitochondrial permeability transition pore in the heart. Cell Metab 21, 206-214.

[0072] Lederer, D.J., and Martinez, F.J. (2018). Idiopathic Pulmonary Fibrosis. The New England journal of medicine 378, 1811-1823.

[0073] Meulemans, A., Seneca, S., Pribyl, T., Smet, J., Alderweirldt, V., Waeytens, A., Lissens, W., Van Coster, R., De Meirleir, L., di Rago, J.P., et al. (2010). Defining the pathogenesis of the human Atp12p W94R mutation using a Saccharomyces cerevisiae yeast model. J Biol Chem 285, 4099-4109.

[0074] Morita, M., Prudent, J., Basu, K., Goyon, V., Katsumura, S., Hulea, L., Pearl, D., Siddiqui, N., Strack, S., McGuirk, S., et al. (2017). mTOR Controls Mitochondrial Dynamics and Cell Survival via MTFP1. Mol Cell 67, 922-935e925.

[0075] Noble, P.W., Albera, C., Bradford, W.Z., Costabel, U., Glassberg, M.K., Kardatzke, D., King, T.E., Jr., Lancaster, L., Sahn, S.A., Szwarcberg, J., et al. (2011). Pirfenidone in patients with idiopathic pulmonary fibrosis (CAPACITY): two randomised trials. Lancet (London, England) 377, 1760-1769.

[0076] Nunnari, J., and Suomalainen, A. (2012). Mitochondria: in sickness and in health. Cell 148, 1145-1159.

[0077] Raghu, G. (2017). Idiopathic pulmonary fibrosis: lessons from clinical trials over the past 25 years. The European respiratory journal 50.

[0078] Raghu, G., Collard, H.R., Egan, J.J., Martinez, F.J., Behr, J., Brown, K.K., Colby, T.V., Cordier, J.F., Flaherty, K.R., Lasky, J.A., et al. (2011). An official ATS / ERS / JRS / ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. American journal of respiratory and critical care medicine 183, 788-824.

[0079] Stephan, T., Bruser, C., Deckers, M., Steyer, A.M., Balzarotti, F., Barbot, M., Behr, T.S., Heim, G., Hubner, W., Ilgen, P., et al. (2020). MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation. The EMBO journal 39, e104105.

[0080] The above-described specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application, and it should be understood that the above-described is only a specific embodiment of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of an agent for detecting the activity of mitochondrial respiratory chain complex V in the manufacture of a composition or kit for aiding the diagnosis of idiopathic pulmonary fibrosis.

2. The use of claim 1, wherein the agent is an antibody against complex V, an antigen-binding fragment thereof, a probe or primer that binds to a nucleic acid encoding complex V, or an aptamer.

3. The use of claim 2, wherein the antibody is a monoclonal antibody.

4. A composition for aiding the diagnosis of idiopathic pulmonary fibrosis, comprising an agent for detecting the activity of mitochondrial respiratory chain complex V.

5. The composition of claim 4, wherein the agent is an antibody against complex V, an antigen-binding fragment thereof, a probe or primer that binds to a nucleic acid encoding complex V, or an aptamer.

6. The composition of claim 5, wherein the antibody is a monoclonal antibody.

7. A kit for aiding the diagnosis of idiopathic pulmonary fibrosis, comprising the composition of any one of claims 4-6 and instructions for use.

8. A method for screening an agent for treating idiopathic pulmonary fibrosis in vitro, comprising administering the agent to a TGF-βl-induced idiopathic pulmonary fibrosis cell model, and determining the activity of mitochondrial respiratory chain complex V before and after the administration, wherein an increase in the activity of complex V after the administration of the agent compared to the activity of complex V before the administration of the agent indicates that the agent is a potential therapeutic agent for idiopathic pulmonary fibrosis; the idiopathic pulmonary fibrosis cell model is an idiopathic pulmonary fibrosis cell model constructed using A549 cells.

9. The method of claim 8, wherein the method further comprises determining the activity of mitochondrial respiratory chain complex I, the amount of subunit protein NDUFA9, the amount of ATP5a, the ROS content, the ATP content, and / or the mitochondrial membrane potential before and after the administration, and wherein an increase in the activity of complex I, an up-regulation in the amount of subunit protein NDUFA9, an up-regulation in the amount of ATP5a, a decrease in the ROS content, an increase in the ATP content, and a decrease in the mitochondrial membrane potential after the administration compared to the activity of complex I, the amount of subunit protein NDUFA9, the amount of ATP5a, the ROS content, the ATP content, and the mitochondrial membrane potential before the administration, respectively, indicates that the agent is a potential therapeutic agent for idiopathic pulmonary fibrosis.

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