Use of mitochondrial complex I inhibitor and lactate dehydrogenase A inhibitor in synergistically anti-myocardial fibrosis

Abstract

The present invention discloses the use of mitochondrial complex I inhibitor and lactate dehydrogenase A inhibitor in synergistically anti-myocardial fibrosis. The present invention discovers that there is a synergistic effect between mitochondrial complex I inhibitor and lactate dehydrogenase A inhibitor in anti-myocardial fibrosis, and a therapeutic effect of 1 + 1 > 2 can be achieved, which is unexpected to those skilled in the art.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, relates to combination drug use, and specifically relates to the use of a mitochondrial complex I inhibitor and a lactate dehydrogenase A inhibitor in synergistically anti-myocardial fibrosis. Background Art

[0002] Myocardial fibrosis is one of the common pathological bases for poor prognosis of cardiovascular diseases such as myocardial infarction and hypertension, and is mainly characterized by fibroblast activation and extracellular matrix deposition. Activated myofibroblasts synthesize and secrete collagen and the like to cause extracellular matrix deposition, and the deposited matrix components can also be cleared or remodeled through degradation, indicating that the fibrotic response is a reversible process. Drug intervention in fibrosis has positive significance for delaying the occurrence of heart failure. Activation of pathways such as TGF-β1 or Galectin-3 can trigger fibroblast activation, but the clinical transformation of the corresponding single-target drugs developed so far is not ideal, suggesting that there may be limitations in only intervening in a single pathological factor in the treatment of complex diseases. Summary of the Invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the medical use of a mitochondrial complex I inhibitor and a lactate dehydrogenase A inhibitor in combination for preparing a drug for anti-myocardial fibrosis.

[0004] The above object of the present invention is achieved by the following technical solutions:

[0005] Use of a mitochondrial complex I inhibitor and a lactate dehydrogenase A inhibitor in combination for preparing a drug for anti-myocardial fibrosis.

[0006] Preferably, the mitochondrial complex I inhibitor is metformin, IACS-010759 or dihydrotanshinone I.

[0007] Preferably, the lactate dehydrogenase A inhibitor is sodium oxamate, GNE-140 or salvianolic acid A.

[0008] Advantageous Effects:

[0009] The present invention discovers that there is a synergistic effect between a mitochondrial complex I inhibitor and a lactate dehydrogenase A inhibitor in anti-myocardial fibrosis, and a therapeutic effect of 1 + 1 > 2 can be achieved, and this technical effect is unexpected to those skilled in the art. Description of the Drawings

[0010] Figure 1 Immunofluorescence results of the regulatory effect of metformin (Met) and sodium oxamate (Oxa) alone or in combination on α-SMA in TGF-β1-induced cardiac fibroblasts (scale bar: 20 μm);

[0011] Figure 2Immunoblotting results of the regulatory effects of metformin (Met) and sodium oxamate (Oxa) alone or in combination on the expression of fibrosis marker proteins α-SMA, Collagen I, and Collagen III in TGF-β1-induced cardiac fibroblasts (the reference protein is α-Tubulin);

[0012] Figure 3 QPCR results of the regulatory effects of metformin (Met) and sodium oxamate (Oxa) alone or in combination on the expression of the fibrosis marker gene Acta2 in TGF-β1-induced cardiac fibroblasts (the reference gene is 18s RNA);

[0013] Figure 4 QPCR results of the regulatory effects of metformin (Met) and sodium oxamate (Oxa) alone or in combination on the expression of the fibrosis marker gene Col1a1 in TGF-β1-induced cardiac fibroblasts (the reference gene is 18s RNA);

[0014] Figure 5 QPCR results of the regulatory effects of metformin (Met) and sodium oxamate (Oxa) alone or in combination on the expression of the fibrosis marker gene Postn in TGF-β1-induced cardiac fibroblasts (the reference gene is 18s RNA);

[0015] Figure 6 In-Cell Western fluorescence imaging results of the regulatory effects of orthogonal use of different gradient concentrations of metformin (Met) and sodium oxamate (Oxa) on the expression of the fibrosis marker protein α-SMA in TGF-β1-induced cardiac fibroblasts;

[0016] Figure 7 In-Cell Western quantitative results of the regulatory effects of orthogonal use of different gradient concentrations of metformin (Met) and sodium oxamate (Oxa) on the expression of the fibrosis marker protein α-SMA in TGF-β1-induced cardiac fibroblasts. Taking the control group (untreated group) as 100, the other drug treatment groups are shown in the form of relative values to the control group;

[0017] Figure 8 Heat map of the calculation of Loewe synergy score for the regulatory effects of orthogonal use of different gradient concentrations of metformin (Met) and sodium oxamate (Oxa) on the expression of the fibrosis marker protein α-SMA in TGF-β1-induced cardiac fibroblasts. The scores are marked above the heat map; it is generally considered that there is a significant synergistic effect between active ingredient combinations with a Loewe synergy score greater than 5;

[0018] Figure 9Results of Masson staining and quantitative statistical data of the fibrotic area in the myocardial tissue of mice induced by acute myocardial infarction with metformin (Met) and sodium oxamate (Oxa) used alone or in combination;

[0019] Figure 10 Immunofluorescence results of the regulatory effect of IACS-010759 and sodium oxamate GNE-140 used alone or in combination on α-SMA in TGF-β1-induced cardiac fibroblasts (scale bar: 20 μm);

[0020] Figure 11 Immunoblotting results of the regulatory effect of IACS-010759 and sodium oxamate GNE-140 used alone or in combination on the expression of fibrosis marker proteins α-SMA, Collagen I, and Collagen III in TGF-β1-induced cardiac fibroblasts (loading control protein: α-Tubulin);

[0021] Figure 12 QPCR results of the regulatory effect of IACS-010759 and sodium oxamate GNE-140 used alone or in combination on the expression of fibrosis marker gene Acta2 in TGF-β1-induced cardiac fibroblasts (reference gene: 18s RNA);

[0022] Figure 13 QPCR results of the regulatory effect of IACS-010759 and sodium oxamate GNE-140 used alone or in combination on the expression of fibrosis marker gene Col1a1 in TGF-β1-induced cardiac fibroblasts (reference gene: 18s RNA);

[0023] Figure 14 QPCR results of the regulatory effect of IACS-010759 and sodium oxamate GNE-140 used alone or in combination on the expression of fibrosis marker gene Postn in TGF-β1-induced cardiac fibroblasts (reference gene: 18s RNA);

[0024] Figure 15 In-Cell Western quantitative results of the regulatory effect of orthogonal use of different gradient concentrations of IACS-010759 and sodium oxamate GNE-140 on the expression of fibrosis marker protein α-SMA in TGF-β1-induced cardiac fibroblasts. Taking the control group (untreated group) as 100, the other drug treatment groups are shown in the form of relative values to the control group;

[0025] Figure 16Heat map for calculating the Loewe synergy score of the regulatory effect of different gradient concentrations of IACS-010759 and sodium oxamate GNE-140 used orthogonally on the expression of the intracellular fibrotic marker protein α-SMA induced by TGF-β1. The scores are marked above the heat map. It is generally considered that there is a significant synergistic effect between the combinations of active ingredients with a Loewe synergy score greater than 5;

[0026] Figure 17 Masson staining results and quantitative statistical data of the fibrotic area of the myocardial tissue of mice induced by acute myocardial infarction for IACS-010759 and sodium oxamate GNE-140 used alone or in combination;

[0027] Figure 18 Immunofluorescence results (scale bar: 20 μm) of the regulatory effect of dihydrotanshinone I (DT) and salvianolic acid A (SAA) used alone or in combination on α-SMA in TGF-β1-induced cardiac fibroblasts;

[0028] Figure 19 Immunoblotting results (the internal reference protein is α-Tubulin) of the regulatory effect of dihydrotanshinone I (DT) and salvianolic acid A (SAA) used alone or in combination on the expression of the intracellular fibrotic marker proteins α-SMA, Collagen I, and Collagen III induced by TGF-β1;

[0029] Figure 20 QPCR results (the internal reference gene is 18s RNA) of the regulatory effect of dihydrotanshinone I (DT) and salvianolic acid A (SAA) used alone or in combination on the expression of the intracellular fibrotic marker gene Acta2 induced by TGF-β1;

[0030] Figure 21 QPCR results (the internal reference gene is 18s RNA) of the regulatory effect of dihydrotanshinone I (DT) and salvianolic acid A (SAA) used alone or in combination on the expression of the intracellular fibrotic marker gene Col1a1 induced by TGF-β1;

[0031] Figure 22 QPCR results (the internal reference gene is 18s RNA) of the regulatory effect of dihydrotanshinone I (DT) and salvianolic acid A (SAA) used alone or in combination on the expression of the intracellular fibrotic marker gene Postn induced by TGF-β1;

[0032] Figure 23Quantification results of In-Cell Western for the regulatory effect of orthogonal use of different gradient concentrations of dihydrotanshinone I (DT) and salvianolic acid A (SAA) on the expression of the fibrosis marker protein α-SMA in TGF-β1-induced cardiac fibroblasts. Taking the control group (untreated group) as 100, other drug treatment groups are shown in the form of relative values to the control group;

[0033] Figure 24 Heat map of Loewe synergy score calculation for the regulatory effect of orthogonal use of different gradient concentrations of dihydrotanshinone I (DT) and salvianolic acid A (SAA) on the expression of the fibrosis marker protein α-SMA in TGF-β1-induced cardiac fibroblasts. The scores are marked above the heat map. It is generally considered that there is a significant synergistic effect between active ingredient combinations with a Loewe synergy score greater than 5;

[0034] Figure 25 Masson staining results and quantitative statistical data of the fibrosis area for the effect of single or combined use of dihydrotanshinone I (DT) and salvianolic acid A (SAA) on myocardial tissue fibrosis in acute myocardial infarction-induced mice;

[0035] Figure 26 Immunofluorescence results (scale bar: 20 μm) for the regulatory effect of single or combined transfection of siRNA targeting Ndufs4 or Ldha on α-SMA in TGF-β1-induced cardiac fibroblasts;

[0036] Figure 27 QPCR results and calculated Jin Zhengjun Q values (with 18s RNA as the internal reference gene) for the regulatory effect of single or combined transfection of siRNA targeting Ndufs4 or Ldha on the expression of the fibrosis marker gene Acta2 in TGF-β1-induced cardiac fibroblasts;

[0037] Figure 28 QPCR results and calculated Jin Zhengjun Q values (with 18s RNA as the internal reference gene) for the regulatory effect of single or combined transfection of siRNA targeting Ndufs4 or Ldha on the expression of the fibrosis marker gene Postn in TGF-β1-induced cardiac fibroblasts. It is generally considered that there is a significant synergistic effect between combinations with a Jin Zhengjun Q value greater than 1.15. Detailed implementation manners

[0038] The following specifically introduces the substantial content of the present invention in combination with examples, but does not limit the protection scope of the present invention hereby.

[0039] I. Experimental materials

[0040] 1. Experimental animals

[0041] C57BL / 6 mice: 200 (SPF grade, male, 8 weeks old), purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (License No.: SCXK(Su)2020-0009).

[0042] 2. Experimental reagents and consumables

[0043] Metformin (Met): purchased from Aladdin, catalog number M107827;

[0044] Sodium oxamate (Oxa): purchased from Aladdin, product number S123221;

[0045] IACS-010759: purchased from MedChemExpress, catalog number HY-112037;

[0046] GNE-140: purchased from MedChemExpress, catalog number HY-100742;

[0047] Nicotinamide nucleotide (NMN): purchased from Aladdin, catalog number N131850;

[0048] Dihydrotanshinone I (DT): purchased from Chengdu Mansite Biotechnology Co., Ltd., CAS No. 87205-99-0, with a purity of more than 95% after inspection;

[0049] Salvianolic acid A (SAA): purchased from Yuanye Biotechnology, CAS No. 96574-01-5, purity tested to be above 98%;

[0050] TGF-β1: purchased from Cell signalling technology, catalog number 5231LF;

[0051] DMEM high-glucose medium (containing double antibodies): purchased from Keygene Biotechnology, catalog number: KGM12800-500;

[0052] Pancreatin: purchased from Gibco, Thermo Fisher Scientific, catalog number 25200072;

[0053] Fetal bovine serum (FBS): purchased from Gibco, Thermo Fisher Scientific, catalog number 30044333;

[0054] PBS: purchased from Solebio Biotechnology, catalog number P1010;

[0055] Collagenase type II: Worthington, LS004176;

[0056] Type Ⅳ Collagenase: Worthington, LS004188;

[0057] 4% Paraformaldehyde: purchased from Biosharp, product number BL539A;

[0058] Bovine Serum Albumin: purchased from Solarbio, product number A8020;

[0059] Goat Serum: purchased from Jackson ImmunoResearch, product number 005-000-121;

[0060] DMSO: purchased from Sigma Aldrich, product number D2650;

[0061] Triton X-100: purchased from Biosharp, product number BS084;

[0062] DAPI: purchased from Sigma Aldrich, product number D9542;

[0063] SteadyPure Quick RNA Extraction Kit: purchased from Aikerui Biotech, product number AG21023;

[0064] Evo M-MLV Reverse Transcription Premix Kit: purchased from Aikerui Biotech, product number AG11728;

[0065] RNase free Double Distilled Water: purchased from Sangon Biotech, product number B541018;

[0066] SYBR Green Pro Taq HS Premix qPCR Kit: purchased from Aikerui Biotech, product number AG11701;

[0067] Loading buffer: purchased from Cell signalling technology, product number 7723;

[0068] NC Membrane: purchased from Millipore, product number: ISEQ00010;

[0069] Protein Prestained Marker: purchased from Bio-Rad, product number 161-0394;

[0070] α-SMA Antibody: purchased from Abways, product number CY5295;

[0071] α-SMA Antibody: purchased from abcam, product number ab7817;

[0072] α-Tubulin antibody: Purchased from Proteintech, catalog number 11224-1-AP;

[0073] CollagenⅠ: Purchased from proteintech, catalog number 14695-1-AP;

[0074] CollagenⅢ: Purchased from proteintech, catalog number 22734-1-AP;

[0075] IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody: Purchased from LI-COR, catalog number 926-32211;

[0076] IRDye 680RD Goat anti-Mouse IgG Secondary Antibody: Purchased from 926-68070, catalog number 926-68070;

[0077] Xfect RNA Transfection Reagent: Purchased from Takara, catalog number 631450.

[0078] 3. Experimental instruments and equipment

[0079] CO2 incubator (SANYO, Japan); biological safety cabinet (Thermo Fisher Scientific, USA); Eclipse Ti-inverted microscope (NIKON, Japan); ice machine (Scotsman, Italy); 1 / 100000 electronic balance (Sartorius, Germany); vortex shaker (Qilin Bell Instrument Manufacturing Co., Ltd., Haimen, China); HH-2 digital display constant temperature water bath (Guohua, Changzhou, China); pH meter (Sartorius, Germany); 4°C refrigerator (Haier, Qingdao, China); 0421-1 low-speed desktop centrifuge (Shanghai Medical Instrument Co., Ltd., Shanghai, China); 5810R multi-function desktop centrifuge (Eppendorff, Germany); single-channel and multi-channel pipettes (Eppendorff, Germany); KH-500DB CNC ultrasonic cleaner (Hechuang Ultrasonic Instrument Co., Ltd., Kunshan, China); Eclipse Ti-inverted microscope (NIKON, Japan); Ts2R inverted fluorescence microscope (Nikon, Japan); EVOS FLoid live cell imaging workstation (ThermoFisher Scientific, USA); CM3050S cryostat (Leica, Germany); fume hood (KeiPu Laboratory Equipment Development Co., Ltd., Guangdong, China); Nano-100 micro-volume spectrophotometer (Aosheng Instrument, Hangzhou, China); protein electrophoresis system (Biorad, USA); protein transfer system (Biorad, USA); PowerPac universal electrophoresis power supply (Biorad, USA); standard digital controller cooling / heating circulating water bath (Polyscience, USA); -20℃ refrigerator (Haier, Qingdao, China); -80℃ refrigerator (Thermo Fisher Scientific, USA); Odyssey Near-Infrared Western Detection System (LICOR Bioscience, USA); horizontal constant speed shaker (Qilin Bell Instrument Manufacturing Co., Ltd., Haimen, China); multi-purpose rotary shaker (Qilin Bell Instrument Manufacturing Co., Ltd., Haimen, China); plastic film sealer (Baoqin Ouke Tools Co., Ltd., Hangzhou, China); Nano-100 micro-volume spectrophotometer (Aosheng Instrument, Hangzhou, China); 2720 Thermal Cycler reverse transcription instrument (Applied Biosystems, Thermo Fisher Scientific, USA); PCR instrument (Light Cycler 480, Roche); HX-101E small animal ventilator (Taimeng, Chengdu, China);37°C Small Animal Constant Temperature Heating Pad (Tongjian Electric Appliance Technology Co., Ltd., Shenzhen, China).;

[0080] II. Experimental Methods

[0081] 1. Isolation and Culture of Adult Mouse Cardiac Fibroblasts

[0082] The hearts of C57BL / 6 mice were isolated using the four-step shearing method and placed in a 100 mm petri dish containing pre-cooled PBS solution. The hearts were cut into 1×1×1 cm 3 sized pieces with a curved scissors and transferred to a stoppered Erlenmeyer flask. The supernatant containing blood and other tissue residues was discarded, and the tissue was washed once with PBS and the supernatant was discarded. A mixed enzyme solution (collagenase type II and collagenase type IV) was added to the stoppered Erlenmeyer flask, and the flask was shaken at 100 rpm in a 37°C water bath for 2 minutes, and the supernatant was discarded. Four 15 mL centrifuge tubes were prepared, each containing 7 mL of 10% FBS-DMEM solution, for collecting the myocardial tissue digestive fluid. A 50 mL centrifuge tube T1 was prepared, with a 100 μm cell sieve pre-laid at the mouth of the tube to filter out tissue clumps when filtering the cell suspension. 7 mL of the mixed enzyme solution was added to the stoppered Erlenmeyer flask, and the flask was shaken at 100 rpm in a 37°C water bath for 5 minutes. The supernatant myocardial tissue digestive fluid was collected and added to the pre-prepared 15 mL centrifuge tube. Centrifugation was carried out at 1500 rpm for 3 minutes, and the supernatant was discarded. The precipitate below was resuspended with 2 mL of 10% FBS-DMEM solution, pipetted evenly, and filtered through the cell sieve and added to T1. The myocardial tissue was digested repeatedly according to the above steps until most of the myocardial tissue in the stoppered Erlenmeyer flask showed a white viscous state, indicating that most of the myocardial parenchymal cells had been digested and eluted. All the myocardial tissue digestive fluids were collected into T1. The myocardial tissue digestive fluid in T1 was evenly spread onto petri dishes at a ratio of one 100 mm petri dish for every 5 mouse hearts, and an appropriate amount of 10% FBS-DMEM solution was added to make the total volume of the solution in each petri dish approximately 8 mL. The cells were cultured in an incubator for 2 - 3 hours. Taking advantage of the characteristic that cardiac fibroblasts adhere to the wall preferentially, cardiac muscle cells and cardiac fibroblasts were separated. After 2 - 3 hours of culture, the non-adherent cardiac muscle cells in the petri dish were gently pipetted with a 1 mL pipette gun, and the supernatant containing the cardiac muscle cells was discarded. In addition, 2 mL of 10% FBS-DMEM solution was added to each petri dish to wash the petri dish once again, and the washing fluid was discarded together. The next experiment could be carried out after the cardiac fibroblasts in the petri dish were completely adhered to the wall.

[0083] 2. Cellular Immunofluorescence

[0084] After fixing cells with 4% paraformaldehyde at room temperature for 15 minutes, add PBS solution containing 0.1% Triton X-100, place it on a horizontal shaker (rotation speed about 60 revolutions per minute) and rinse 2 times, 5 minutes each time. Prepare a PBS solution containing 5% goat serum and 0.3% Triton X-100 as the blocking buffer, and block the cells in the blocking buffer for 1 hour. According to the dilution ratio recommended in the antibody instruction manual, prepare the primary antibody with a PBS solution containing 1% BSA and 0.3% Triton X-100. After sucking dry the blocking buffer, add the diluted primary antibody, place it in a refrigerator at 4°C, and incubate overnight. After the incubation of the primary antibody is completed, discard the primary antibody solution, rinse with 0.1% Triton X-100 PBS solution 3 times, 5 minutes each time. Dilute the secondary antibody labeled with fluorescent substance with a PBS solution containing 1% BSA and 0.3% Triton X-100, and incubate in the dark at room temperature for 2 hours. To label the cell nucleus, add DAPI staining solution and stain for 10 minutes. Subsequently, discard the DAPI solution, add PBS solution and rinse the cells 3 times, 5 minutes each time, and transfer to a live cell workstation for microscopic photography.

[0085] 3. Immunoblotting experiment

[0086] After treating cells with culture medium containing different drugs for 24 hours, discard it and wash once with pre-cooled PBS. Place the 6-well plate on ice, add 100 μL of 1X Loading buffer to each well to lyse the cells, immediately scrape the cells from the plate and transfer them to a microcentrifuge tube. Ultrasonically treat for 15 seconds to completely lyse the cells, then heat at 100°C for 10 minutes to denature the protein, and place on ice to cool.

[0087] Add an appropriate amount of protein sample and protein prestained marker to the corresponding lanes of the SDS-PAGE gel. Electrophorese at a constant voltage of 80V for 35 minutes, then increase the voltage to 120V and continue electrophoresis until the loading buffer is close to the bottom of the gel, then stop electrophoresis. Subsequently, perform wet transfer at a constant current of 500 mA in a low-temperature environment for 2 hours. After the sample is wet-transferred to the nitrocellulose membrane, block the membrane with 5% milk at room temperature for 1 hour. Dilute the primary antibody according to the dilution ratio recommended in the antibody instruction manual, place the membrane in the primary antibody, and incubate overnight on a shaker at 4°C. When the incubation of the primary antibody is completed, recover the primary antibody, wash the membrane with TBST solution, and then place it in the secondary antibody and incubate in the dark at room temperature for 2 hours. After washing the membrane again, the target protein band can be detected using a near-infrared laser imaging system.

[0088] 4. Extraction of total cellular RNA and RT-PCR experiment

[0089] Refer to the instruction manuals of the SteadyPure Quick RNA Extraction Kit and the Evo M-MLV Reverse Transcription Premix Kit to extract total RNA from the drug-stimulated cells and perform reverse transcription. Prepare a PCR reaction system containing 10 μL AceQ qPCR SYBR Green Master Mix, 1 μL primer, 2 μL cDNA, and 7 μL RNase free ddH 2 O, and use the LightCycler480 RT-PCR system to perform subsequent RT-PCR experiments. Using 18S as the internal reference, the expression levels of the target genes in each group are presented as the fold change relative to the expression level in the control group. The primer sequences of genes such as Acta2, Col1a1, Postn, Ndufs4, Ldha, and 18S are shown in Table 1.

[0090] Table 1

[0091]

[0092] 5. ICW

[0093] Carefully aspirate the liquid culture medium, wash once with PBS, add 150 μL of methanol pre-cooled to -20 °C to each well, and fix the cells at room temperature for 20 minutes. Carefully aspirate the fixing solution, add 200 μL of PBS solution, and place it on a horizontal shaker (rotation speed about 60 rpm) for rinsing 2 times, 5 minutes each time. Prepare a PBS solution containing 5% goat serum and 0.3% Triton X-100 as the blocking buffer, add 150 μL of the blocking buffer to each well, and block on the horizontal shaker for 90 minutes. While blocking, prepare the primary antibody with a PBS solution containing 1% BSA and 0.3% Triton X-100 according to the dilution ratio recommended in the antibody instruction manual. After blocking, aspirate the blocking buffer, add 150 μL of 0.1% Triton X-100 PBS to each well, wash 2 times, 5 minutes each time. Discard the washing solution, add 50 μL of the diluted primary antibody to each well, cover with a layer of plastic wrap, and incubate overnight at 4 °C. Carefully recover the primary antibody, add 200 μL of 0.1% Tween20-PBS to each well, and rinse on the horizontal shaker four times, 5 minutes each time. Add 50 μL of the diluted secondary antibody to each well, and incubate at 4 °C in the dark for 6 - 8 hours. Carefully recover the secondary antibody, add 200 μL of 0.1% Tween20-PBS to each well, and rinse four times, 5 minutes each time. Aspirate the liquid in the wells, measure the expression level of the target protein using a near-infrared imaging system, and calculate the fluorescence intensity ratio of α-SMA and α-Tubulin in each group using Image Pro Plus (IPP, MediaCybernetics, USA) 6.0 software. Present the data of each group as a percentage of the control group.

[0094] 6. Method for Establishing Mouse Acute Myocardial Infarction Model

[0095] Mice were fasted and water-deprived for 8 hours before surgery. After anesthesia, the left chest was depilated, and the mice were placed supine with their four limbs fixed on a circulating heating operating table. The trachea was isolated and intubated with a 20-gauge intravenous catheter with a blunt tip, and then connected to a small animal ventilator to maintain respiration. It could be observed that the respiratory undulation of the mice was consistent with the ventilator frequency.

[0096] The skin of the surgical area was disinfected with iodophor. The skin was incised between the 3rd and 4th intercostal spaces on the left side of the sternum, and the subcutaneous tissue and muscle were bluntly dissected layer by layer. The chest cavity was opened to expose the heart, and the pericardium was incised. A pink blood vessel could be seen at the lower edge of the auricle or on the left side, which was the left anterior descending branch (LAD) of the left coronary artery. It was permanently ligated with 6-0 suture. The whitening of the myocardial tissue indicated complete occlusion of the left anterior descending branch (LAD) artery. After ligation, the chest wall, muscle layer, and skin layer were sutured. After removing the tracheal intubation, the mice were placed on a 37°C warming pad to recover until they woke up. The sham-operated group of mice underwent the same surgery without ligating the coronary artery.

[0097] 7. Transfection

[0098] The siRNA sequences of Ldha and Ndufs4 genes used in the siRNA transfection experiment are shown in Table 2. For a single well of a 6-well plate, 200 μL of Xfect reaction buffer, 100 pmol of siRNA, and 10 μL of Xfect RNA transfection polymer were thoroughly mixed and allowed to stand at room temperature for 10 minutes to form the transfection complex. The transfection complex was added dropwise to the culture medium and gently shaken to mix. Then, the 6-well plate was incubated in an incubator for 4 hours. After transfection, the culture medium needed to be changed, and the transfection efficiency was checked 24 hours later or the next experiment was carried out.

[0099] Table 2

[0100]

[0101] 8. Calculation of Synergy Score

[0102] Calculation of Loewe synergy score: Using the signal intensity of the fibroblast activation marker α-SMA as an index, calculate the relative signal value of each drug treatment group compared to the model group (presented as a percentage). Apply SynergyFinder software (https: / / synergyfinder.fimm.fi) to calculate the Loewe synergy score to evaluate the strength of the synergistic effect between the two drugs (it is generally considered that there is a significant synergistic effect between the active ingredient combinations with a Loewe synergy score greater than 5). Finally, determine the synergistic component combination based on the principle of combination optimization.

[0103] Calculation of Kim's synergy index: E A represents the effect when only intervention A is used, E B represents the effect when only intervention B is used, E A+B represents the effect after combining interventions A and B, which is the expected value of the combined effect of the two interventions. When evaluating the synergy effect, it is calculated according to the following formula: Q = E A+B / (E A +E B -E A ×E B ), where: Q < 0.85 is the antagonistic effect, 0.85 ≤ Q < 1.15 is the additive effect, and Q ≥ 1.15 is the synergistic effect.

[0104] 9. Statistical analysis

[0105] Data are expressed as mean ± standard deviation (mean ± SD), and statistical analysis was performed using GraphPad Prism 8.0 software. One-way ANOVA was used for comparison of means among multiple groups. The results of statistical analysis: *p < 0.05 indicates significant difference, **p < 0.01 indicates very significant difference, and ***p < 0.001 indicates extremely significant difference.

[0106] III. Experimental results

[0107] 1. Synergistic anti-myocardial fibrosis effect of mitochondrial complex I inhibitor Met and LDHA inhibitor Oxa

[0108] There is a phenomenon of hyperactive aerobic glycolysis in activated myofibroblasts. At the same time, the level of mitochondrial oxidative phosphorylation will also be upregulated synchronously. Due to the flexibility of cell metabolism, the coupling mechanism between aerobic glycolysis and mitochondrial oxidative phosphorylation is of great significance for metabolic intervention in fibroblast activation. Single inhibition of LDH or mitochondrial complex I may compensate through another pathway. Based on the fibroblast activation model, we applied the inhibitor Met targeting mitochondrial complex I and the inhibitor Oxa of LDH, and confirmed the feasibility of the synergistic mode by comparing the effects of single intervention and combined intervention on fibroblast activation.

[0109] First, TGF-β1 stimulates quiescent cardiac fibroblasts to establish an activated myofibroblast model. The establishment of the model can be confirmed by measuring α-SMA through immunofluorescence assay, measuring fibrotic marker proteins α-SMA, CollagenⅠ, and CollagenⅢ by Western blotting, and measuring the expression of key fibrotic genes Acta2, Col1a1, and Postn by PCR. According to the results of immunofluorescence assay, we found that the use of mitochondrial complex Ⅰ inhibitor Met or LDH inhibitor Oxa alone only partially reduced the level of the fibroblast activation marker protein α-SMA, while the combination of Met and Oxa more significantly inhibited the formation of α-SMA filaments( Figure 1 ). Western blotting also confirmed that the use of Met or Oxa alone partially reduced the expression of myofibroblast fibrosis-related proteins α-SMA, CollagenⅠ, and CollagenⅢ, while the inhibitory effect was more significant when the two were combined( Figure 2 ). The results of PCR experiments also showed that the use of Met or Oxa alone partially inhibited the expression of the myofibroblast fibrosis marker genes Acta2( Figure 3 , Table 3), Col1a1( Figure 4 , Table 4), and Postn( Figure 5 , Table 5), and the inhibitory effect was more significant after combination. Subsequently, In-Cell Western technology was used to examine the efficacy of different concentration orthogonal groups of Met and Oxa with the fibroblast activation marker α-SMA as an index( Figure 6 ), and the percentage of the signal value of the drug administration group compared to the signal value of the model group was calculated( Figure 7 , Table 6). Accordingly, the Loewe synergy score calculated by SynergyFinder software was 21.224( Figure 8 ), demonstrating that the combination of Met and Oxa can synergistically block the occurrence of fibroblast activation. Immediately, a mouse acute myocardial infarction model was used to examine the in vivo anti-myocardial fibrosis effects of Met and Oxa. After preparing myocardial tissue sections, Masson staining was used to evaluate the degree of myocardial tissue fibrosis. The results showed that the use of Met and Oxa alone both partially reduced the myocardial fibrosis area in MI mice, and the anti-myocardial fibrosis efficacy was more significant after the two were combined( Figure 9 , Table 7). The above results jointly demonstrated the synergistic anti-myocardial fibrosis effect of mitochondrial complex Ⅰ inhibitor Met and LDHA inhibitor Oxa, and explored the existence of metabolic flexibility during the activation process of fibroblasts.

[0110] Table 3 (corresponding to Figure 3 )

[0111] Control TGF-β1 Met Oxa Met+Oxa 1.03 11.86 10.71 6.73 2.89 0.96 11.68 10.53 7.26 2.87 1.01 11.24 10.53 7.17 3.15

[0112] Table 4 (corresponding toFigure 4 )

[0113] Control TGF-β1 Met Oxa Met+Oxa 1.05 2.20 1.91 1.27 0.92 0.95 2.27 1.94 1.32 0.98 1.01 2.17 1.89 1.30 1.06

[0114] Table 5 (corresponding to Figure 5 )

[0115] Control TGF-β1 Met Oxa Met+Oxa 0.99 4.00 2.98 2.64 1.00 1.00 3.13 2.97 2.70 1.04 1.01 3.35 3.05 2.60 1.07

[0116] Table 6 (corresponding to Figure 7 )

[0117]

[0118] Table 7 (corresponding to Figure 9 )

[0119] Sham MI Met Oxa Met+Oxa 1.11 23.38 11.13 12.17 8.73 1.60 21.36 20.10 17.22 10.98 1.38 23.52 13.33 17.29 6.73 0.82 18.84 15.56 16.97 8.22 0.64 17.55 14.63 12.50 5.93 0.79 17.45 14.64 9.55 4.36

[0120] 2. Synergistic anti-myocardial fibrosis effect of specific mitochondrial complex I inhibitor IACS-010759 and LDHA inhibitor GNE-140

[0121] We also investigated whether the use of specific mitochondrial complex I inhibitor IACS-010759 (a phase I clinical drug, Average IC 50 = 5.6 nM in mouse cell lines) and LDHA inhibitor GNE-140 (a phase I clinical drug, IC 50 = 3 nM for LDHA) could reproduce the Met / Oxa pharmacodynamic effect.

[0122] The results of immunofluorescence assay showed that treatment with IACS-010759 or GNE-140 alone only partially reduced the level of α-SMA in fibroblasts, while the combination of IACS-010759 and GNE-140 significantly inhibited the expression of α-SMA ( Figure 10 ). The results of Western blotting also confirmed that treatment with IACS-010759 or GNE-140 alone could partially reduce the expression of fibrosis-related proteins α-SMA, Collagen I, and Collagen III in myofibroblasts, and the inhibitory effect was more significant when the two drugs were used in combination ( Figure 11 ). PCR assay also showed that treatment with IACS-010759 or GNE-140 alone could partially inhibit the expression of Acta2 ( Figure 12 , Table 8), Col1a1 ( Figure 13 , Table 9), Postn ( Figure 14 , Table 10) and other genes in myofibroblasts, and the inhibitory effect was more significant when the two drugs were used in combination. Subsequently, In-Cell Western technology was used to evaluate the pharmacodynamic effects of different concentration orthogonal groups of IACS-010759 and GNE-140 with α-SMA as the index.Figure 15 , Table 11), and the Loewe synergy score calculated by SynergyFinder software was 16.649( Figure 16 ), demonstrating that the combination of IACS-010759 and GNE-140 has a synergistic inhibitory effect on the activation of cardiac fibroblasts. In addition, through a mouse acute myocardial infarction model, the in vivo anti-myocardial fibrosis effects of IACS-010759 and GNE-140 were verified. The results of Masson staining showed that either IACS-010759 or GNE-140 alone could partially reduce the myocardial fibrosis area in MI mice, and the anti-myocardial fibrosis efficacy was more significant after the combination of the two( Figure 17 , Table 12).

[0123] Table 8 (corresponding to Figure 12 )

[0124] Control TGF-β1 Met Oxa Met+Oxa 1.12 11.75 9.55 8.70 6.46 0.95 10.99 8.87 6.89 4.95 0.93 10.06 9.13 7.11 5.20

[0125] Table 9 (corresponding to Figure 13 )

[0126] Control TGF-β1 Met Oxa Met+Oxa 0.87 4.71 3.45 3.23 1.97 1.09 4.81 3.62 2.93 1.98 1.04 4.19 3.29 2.75 1.93

[0127] Table 10 (corresponding to Figure 14 )

[0128] Control TGF-β1 Met Oxa Met+Oxa 0.99 4.89 3.01 3.98 1.96 1.01 5.39 3.11 3.46 1.68 1.00 5.14 2.75 3.40 1.90

[0129] Table 11 (corresponding to Figure 15 )

[0130]

[0131] Table 12 (corresponding to Figure 17 )

[0132] Sham MI IACS-010759 GNE-140 IACS-010759+GNE-140 2.64 19.64 14.62 12.36 11.11 1.82 19.44 14.55 11.21 5.65 2.94 23.18 16.30 11.01 8.22 5.00 17.52 14.07 14.95 6.40 1.70 20.38 12.48 15.27 12.71 1.26 17.78 9.14 9.72 4.75

[0133] This part of the results reproduced the efficacy of the combination of Met and Oxa with the specific mitochondrial complex I inhibitor IACS-010759 and the LDHA inhibitor GNE-140, demonstrating the feasibility of the synergistic mode of synchronously intervening in LDHA and mitochondrial complex I against myocardial fibrosis.

[0134] 3. Synergistic anti-myocardial fibrosis of mitochondrial complex I inhibitor dihydrotanshinone I (DT) and LDHA inhibitor salvianolic acid A (SAA)

[0135] Previous studies by our research group found that dihydrotanshinone I in Salvia miltiorrhiza could inhibit the activity of mitochondrial complex I, and salvianolic acid A could inhibit the activity of LDH and lactate synthesis, suggesting a potential synergistic effect between the two active ingredients. In addition, we have confirmed in previous work that the inhibition of mitochondrial complex I by dihydrotanshinone I is reversible, similar to metformin, which may reversibly inhibit NADH dehydrogenase by regulating the conformational changes of subunits of mitochondrial complex I such as ND3, thus avoiding possible myocardial toxicity. Therefore, we then studied the synergistic effect of the mitochondrial complex I inhibitor dihydrotanshinone I (DT) and the LDHA inhibitor salvianolic acid A (SAA) against myocardial fibrosis. As Figure 18 shown, the formation of α-SMA myofilaments in myofibroblasts was partially reduced by either DT or SAA alone, and the inhibitory effect was significantly enhanced when DT and SAA were used in combination. In addition, the expression of α-SMA, Collagen I, and Collagen III in myofibroblasts was partially reduced by either DT or SAA alone ( Figure 19 ), and the inhibitory effect was significantly enhanced after combination. PCR experiments showed that the expression of the myofibroblast activation marker genes Acta2( Figure 20 , Table 13), Col1a1( Figure 21 , Table 14), and Postn( Figure 22 , Table 15) was partially inhibited by either DT or SAA alone, and the effect of inhibiting myofibroblast activation was more significant after combination. In addition, through In-Cell Western experiments, the pharmacodynamic effects of different concentration orthogonal groups of DT and SAA were measured( Figure 23 , Table 16), and the Loewe synergy score was calculated to be 20.216( Figure 24 ). In addition, through a mouse acute myocardial infarction model, the in vivo pharmacodynamic effects of DT and SAA were investigated. The results of Masson staining showed that either DT or SAA alone could partially reduce the myocardial fibrosis area in MI mice, and the downregulation of the myocardial fibrosis area was more significant when the two were used in combination( Figure 25 , Table 17). The above results confirmed that the active ingredient combination DT / SAA of natural origin, which inhibits mitochondrial complex I / LDHA, could synergistically inhibit fibroblast activation, and the anti-myocardial fibrosis effect was enhanced when the two were used in combination.

[0136] Table 13 (corresponding to Figure 20 )

[0137] Control TGF-β1 DT SAA DT+SAA 1.04 10.93 4.89 3.97 3.05 1.03 10.11 4.65 4.13 3.03 0.93 9.86 5.28 4.14 3.16

[0138] Table 14 (corresponding to Figure 21 )

[0139] Control TGF-β1 DT SAA DT+SAA 1.09 4.72 2.62 3.50 1.52 0.95 4.68 2.47 3.29 1.44 0.96 4.70 2.31 3.35 1.50

[0140] Table 15 (corresponding to Figure 22 )

[0141] Control TGF-β1 DT SAA DT+SAA 0.99 7.61 6.20 3.91 2.81 0.97 6.58 5.32 3.97 3.01 1.04 6.71 5.42 4.29 2.87

[0142] Table 16 (corresponding to Figure 23 )

[0143]

[0144] Table 17 (corresponding to Figure 25 )

[0145] Sham MI DT SAA DT+SAA 2.31 20.30 13.56 11.70 6.50 0.98 15.98 16.63 11.00 6.73 2.90 16.87 13.46 16.57 10.20 2.64 19.52 12.38 11.60 12.60 3.08 20.91 13.39 16.19 8.61 1.37 16.50 15.53 15.05 9.61

[0146] The results in this part demonstrated the synergistic effect of dihydrotanshinone I and salvianolic acid A in Salvia miltiorrhiza Bunge in treating myocardial fibrosis, and also provided a demonstration for a new strategy of anti-myocardial fibrosis by traditional Chinese medicine metabolic intervention.

[0147] 4. Gene knockdown proved that inhibiting mitochondrial complex I and lactate dehydrogenase A synergistically reversed myocardial fibrosis

[0148] In this part of the experiment, we studied the synergistic reversal of myocardial fibrosis by gene intervention of mitochondrial complex I / lactate dehydrogenase A by knocking down Ndufs4 (a key subunit of mitochondrial complex I) and Ldha (lactate dehydrogenase A) alone or synchronously. As shown in the figure, knocking down Ndufs4 or Ldha alone could partially inhibit the formation of α-SMA, a marker of fibroblast activation, while the inhibitory effect was significantly enhanced after synchronous knockdown ( Figure 26 ). The results of PCR experiments showed that synchronous knockdown of Ndufs4 and Ldha could reduce the expression of Col1a1 ( Figure 27 , Table 18), Postn ( Figure 28 , Table 19), markers of myofibroblast activation in fibroblasts. The Jin Zhengjun Q value method was used to evaluate the synergistic inhibitory effect between the two. The results showed that the Jin Zhengjun Q values of combined knockdown of Ndufs4 and Ldha on Col1a1 and Postn genes (Q = Ea+b / (Ea+Eb-Ea×Eb), where Ea+b is the inhibition rate of the combined drug, and Ea and Eb are the inhibition rates of drug A and drug B alone) were 1.43 ( Figure 27 ) and 17.18 ( Figure 28 ) (both greater than 1.15), indicating a synergistic effect. The above results showed that there was a synergistic effect of inhibiting mitochondrial complex I and lactate dehydrogenase A in reversing myocardial fibrosis.

[0149] Table 18 (corresponding to Figure 27 )

[0150] Control TGF-β1 Ndufs4siRNA LdhasiRNA Ndufs4siRNA+LdhasiRNA 1.01 3.56 2.54 2.12 0.68 0.98 3.49 2.55 2.17 0.66 1.01 3.50 2.58 2.05 0.68

[0151] Table 19 (corresponding to Figure 28 )

[0152] Control TGF-β1 Ndufs4siRNA LdhasiRNA Ndufs4siRNA+LdhasiRNA 1.02 4.06 3.72 4.31 0.53 1.01 4.02 3.71 4.39 0.52 0.96 4.11 3.65 4.01 0.53

[0153] The function of the above embodiments is to specifically introduce the substantial content of the present invention. However, those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. Use of a mitochondrial complex I inhibitor and a lactate dehydrogenase A inhibitor in the combined preparation of a drug for anti-myocardial fibrosis; wherein, the mitochondrial complex I inhibitor is metformin, and the lactate dehydrogenase A inhibitor is sodium oxamate.

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