CN116115622B - Use of mitochondrial complex I inhibitor and lactate dehydrogenase A inhibitor in synergistic anti-myocardial fibrosis - Google Patents

Abstract

The present invention discloses the use of mitochondrial complex I inhibitors and lactate dehydrogenase A inhibitors to synergistically resist myocardial fibrosis. The present invention finds that mitochondrial complex I inhibitors and lactate dehydrogenase A inhibitors have a synergistic effect in resisting myocardial fibrosis, and can achieve a therapeutic effect of 1+1>2. This technical effect is unexpected by those skilled in the art.

Description

Application of mitochondrial complex I inhibitor and lactate dehydrogenase A inhibitor in synergistic anti-myocardial fibrosis

Technical Field

The invention belongs to the field of medicines, relates to combined medicines, and in particular relates to application of a mitochondrial complex I inhibitor and a lactate dehydrogenase A inhibitor in synergistic anti-myocardial fibrosis.

Background

Myocardial fibrosis is one of the common pathological bases of poor prognosis of cardiovascular diseases such as myocardial infarction, hypertension and the like, 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 deposited matrix components can be cleared or remodeled through degradation, which indicates that fibrosis reaction is a reversible process, and drug intervention fibrosis has positive significance for delaying heart failure. Activation of pathways such as TGF-beta 1 or Galectin-3 may trigger fibroblast activation, but the corresponding single-target drugs developed at present are not ideal in clinical transformation, suggesting that there may be limitations in the treatment of complex diseases in which only a single pathological factor is interfered.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the medical application of the combination of the mitochondrial complex I inhibitor and the lactic dehydrogenase A inhibitor in preparing the anti-myocardial fibrosis medicament.

The above object of the present invention is achieved by the following technical scheme:

Use of a mitochondrial complex I inhibitor in combination with a lactate dehydrogenase a inhibitor for the preparation of a medicament for the treatment of myocardial fibrosis.

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

Preferably, the lactate dehydrogenase A inhibitor is sodium oxalate, GNE-140 or salvianolic acid A.

The beneficial effects are that:

The invention discovers that the mitochondrial complex I inhibitor and the lactate dehydrogenase A inhibitor have synergistic effect on the aspect of resisting myocardial fibrosis, and can realize the treatment effect of 1+1>2, which is unexpected to the person skilled in the art.

Drawings

FIG. 1 shows immunofluorescence results (scale: 20 μm) of metformin (Met) alone or in combination with sodium oxamate (Oxa) for TGF-. Beta.1 induction of modulation of alpha. -SMA in myocardial fibroblasts;

FIG. 2 shows the immunoblotting results of the regulatory effect of metformin (Met) on the induction of the expression of the cardiac fibrosis marker protein α -SMA, collagen I and Collagen III in myocardial fibrosis cells by TGF- β1 alone or in combination with sodium oxamate (Interreference protein α -Tubulin);

FIG. 3 shows the result of QPCR of the modulation of the expression of the fibrotic marker gene Acta2 in myocardial fibroblasts induced by TGF-. Beta.1 (18 s RNA as internal reference) with metformin (Met) and sodium oxamate (Oxa) alone or in combination;

FIG. 4 shows the result of QPCR of the regulatory effect of metformin (Met) on the induction of the expression of the fibrotic marker gene Col1a1 in myocardial fibroblasts by TGF-. Beta.1 alone or in combination with sodium oxamate (18 s RNA as an internal reference gene);

FIG. 5 shows the result of QPCR of the regulatory effect of metformin (Met) on the induction of the expression of the fibrotic marker gene Postn in myocardial fibroblasts by TGF-. Beta.1 alone or in combination with sodium oxamate (18 s RNA as an internal reference gene);

FIG. 6 is a graph showing the results of In-CELL WESTERN fluorescence imaging of metformin (Met) at various gradient concentrations In quadrature with sodium oxalate (Oxa) using modulation of TGF- β1 induced expression of the fibrotic marker protein α -SMA In myocardial fibroblasts;

FIG. 7 shows quantitative results of the use of In-CELL WESTERN of different gradient concentrations of metformin (Met) In orthogonalization with sodium oxalate (Oxa) for TGF-. Beta.1 induction of regulation of the expression of the fibrotic marker protein α -SMA In myocardial fibroblasts, based on 100 control (non-dosed) and relative values of the other drug treatment groups;

FIG. 8 is a graph of the calculated heat of metformin (Met) at various gradient concentrations in quadrature with sodium oxamate (Oxa) using Loewe synergy score for the modulation of TGF- β1 induced expression of the fibrotic marker protein α -SMA in myocardial fibroblasts, with scores marked above the graph, and with a significant synergistic effect between combinations of active ingredients with Loewe synergy score greater than 5 being generally considered;

FIG. 9 is quantitative statistical data of Masson staining results and areas of fibrotic regions for acute myocardial infarction-induced mice myocardial tissue fibrosis, with metformin (Met) alone or in combination with sodium oxamate (Oxa);

FIG. 10 shows immunofluorescence results of IACS-010759 and sodium oxamate GNE-140 alone or in combination for TGF-beta 1 induction of modulation of alpha-SMA in myocardial fibroblasts (scale: 20 μm);

FIG. 11 shows the immunoblotting results of IACS-010759 and sodium oxalate GNE-140, alone or in combination, for the modulation of TGF-beta 1-induced expression of the cardiac muscle fibroblast internal fibrosis marker protein alpha-SMA, collagen I and Collagen III (reference protein alpha-Tubulin);

FIG. 12 shows the result of QPCR of IACS-010759 and sodium oxalate GNE-140 alone or in combination for TGF-. Beta.1 induction of the regulation of the expression of the fibrosis marker gene Acta2 in cardiac myocytes (reference gene 18s RNA);

FIG. 13 shows the result of QPCR of IACS-010759 and sodium oxalate GNE-140 alone or in combination for TGF-. Beta.1 induction of the regulatory effect of the expression of the fibrotic marker gene Col1a1 in cardiac myocytes (reference gene 18s RNA);

FIG. 14 shows the result of QPCR of IACS-010759 and sodium oxamate GNE-140 alone or in combination with each other for the modulation of TGF-. Beta.1-induced expression of the fibrotic marker gene Postn in cardiac myocytes (18 s RNA as an internal reference gene);

FIG. 15 shows the results of quantification of In-CELL WESTERN of different gradient concentrations IACS-010759 In quadrature with sodium oxalate GNE-140 for the modulation of TGF- β1 induced expression of the fibrotic marker protein α -SMA In myocardial fibroblasts, based on 100 control (non-dosed) groups, with other drug treatment groups shown as relative values to control groups;

FIG. 16 is a graph of the calculated heat of the orthogonal use of IACS-010759 and sodium oxalate GNE-140 at different gradient concentrations using Loewe synergy score for the modulation of TGF- β1 induced expression of the fibrotic marker protein α -SMA in myocardial fibroblasts, with scores marked above the heat graph, and with a significant synergistic effect between combinations of active ingredients of Loewe synergy score > 5 being generally considered;

FIG. 17 is quantitative statistics of Masson staining results and areas of fibrotic regions for acute myocardial infarction-induced mice myocardial tissue fibrosis, with IACS-010759 alone or in combination with sodium oxamate GNE-140;

FIG. 18 shows immunofluorescence results of dihydrotanshinone I (DT) and Salvianolic Acid A (SAA) alone or in combination for TGF-. Beta.1 induction of modulation of alpha. -SMA in myocardial fibroblasts (scale: 20 μm);

FIG. 19 shows immunoblotting results of the regulatory effect of dihydrotanshinone I (DT) and Salvianolic Acid A (SAA) on TGF-beta 1 induction of myocardial fibrosis marker protein alpha-SMA, collagen I and Collagen III expression (reference protein alpha-Tubulin);

FIG. 20 shows the result of QPCR of the regulation of the expression of the fibrosis marker gene Acta2 in myocardial fibrosis cells induced by TGF-. Beta.1 (18 s RNA as an internal reference gene) using dihydrotanshinone I (DT) and Salvianolic Acid A (SAA) alone or in combination;

FIG. 21 shows the result of QPCR of the regulation of expression of the TGF-. Beta.1 induced myocardial fibrosis marker gene Col1a1 in fibroblasts by dihydrotanshinone I (DT) and Salvianolic Acid A (SAA) alone or in combination (18 s RNA as an internal reference gene);

FIG. 22 shows the result of QPCR of the regulation of TGF-. Beta.1-induced expression of the fibrotic marker gene Postn in myocardial fibroblasts (18 s RNA as an internal reference gene) alone or in combination with dihydrotanshinone I (DT) and Salvianolic Acid A (SAA);

FIG. 23 shows quantitative results of using In-CELL WESTERN of modulating actions of TGF-. Beta.1 on induction of the expression of the fibrotic marker protein α -SMA In myocardial fibroblasts In various gradient concentrations of dihydrotanshinone I (DT) orthogonal to Salvianolic Acid A (SAA) based on 100 In the control group (non-dosed group) and relative values of the other drug treatment groups In the control group;

FIG. 24 is a graph of the calculated heat of the differential gradient concentrations of dihydrotanshinone I (DT) orthogonal to Salvianolic Acid A (SAA) using Loewe synergy score for the modulation of TGF-. Beta.1 induced expression of the fibrotic marker protein α -SMA in myocardial fibroblasts, with scores marked above the heat graph, and with a significant synergistic effect between combinations of active ingredients with Loewe synergy score greater than 5 being generally considered;

FIG. 25 is quantitative statistical data of Masson staining results and areas of fibrotic regions of acute myocardial infarction-induced mice with dihydrotanshinone I (DT) and Salvianolic Acid A (SAA) alone or in combination;

FIG. 26 is immunofluorescence results (scale: 20 μm) of siRNA transfected individually or in combination with targeted Ndufs or Ldha for TGF-. Beta.1 induction of modulation of alpha-SMA in myocardial fibroblasts;

FIG. 27 shows the result of QPCR of siRNA individually or in combination transfected to Ndufs or Ldha for the regulation of TGF-. Beta.1-induced expression of the fibrosis marker gene Acta2 in myocardial fibrosis cells and the calculated positive average Q value (18 s RNA as reference gene);

FIG. 28 shows the result of QPCR of siRNA transfected individually or in combination with target Ndufs or Ldha for the modulation of TGF-. Beta.1 induced expression of the fibrotic marker gene Postn in myocardial fibroblasts and the calculated golden average Q value (18 s RNA as reference gene), which is generally believed to have a significant synergistic effect between combinations of golden average Q values greater than 1.15.

Detailed Description

The following describes the essential aspects of the present invention in detail with reference to examples, but is not intended to limit the scope of the present invention.

1. Experimental materials

1. Experimental animal

C57BL/6 mice, 200 (SPF grade, male, 8 week old), were purchased from Jiangsu Hua Xinnuo pharmaceutical technologies Co., ltd (license number: SCXK (Su) 2020-0009).

2. Experimental reagent and consumable

Metformin (Metformin, met) commercially available from aladine under the designation M107827;

Sodium oxamate (Sodium oxamate, oxa) available from Allatin under the designation S123221;

IACS-010759, commercially available from MedChemExpress under the accession number HY-112037;

GNE-140, commercially available from MedChemExpress under the trade designation HY-100742;

nicotinamide nucleotides (NMN) purchased from Alatine under the accession number N131850;

Dihydrotanshinone I (DT) from Chengdoman Biotechnology Co., ltd., CAS number 87205-99-0, purity over 95% as tested;

salvianolic Acid A (SAA) is obtained from source leaf organism, CAS number 96574-01-5, and has purity of above 98% by inspection;

TGF-. Beta.1 available from CELL SIGNALLING technology under the accession number 5231LF;

DMEM high sugar culture medium (containing double antibody) purchased from Kaiki group organism, with the product number of KGM12800-500;

Pancreatin, commercially available from Gibco, thermo FISHER SCIENTIFIC, cat No. 25200072;

Fetal bovine serum (Fetal Bovine Serum, FBS) from Gibco, thermo FISHER SCIENTIFIC under the accession number 30044333;

PBS, available from Soy Cork, cat# P1010;

Type II collagenase, worthington, LS004176;

collagenase type IV, worthington, LS004188;

4% paraformaldehyde from Biosharp under the trade designation BL539A;

bovine SerumAlbumin available from Solarbio under the designation A8020;

goat serum purchased from Jackson ImmunoResearch under the number 005-000-121;

DMSO available from SigmaAldrich under the designation D2650;

TritonX-100, available from Biosharp under the trade designation BS084;

DAPI, available from SigmaAldrich under the designation D9542;

SteadyPure Rapid RNA extraction kit purchased from Ai Kerui organism under the accession number AG21023;

evo M-MLV reverse transcription premix kit purchased from Ai Kerui organisms under the accession number AG11728;

RNASE FREE double distilled water is purchased from a manufacturer, and the product number is B541018;

SYBR Green Pro Taq HS premix qPCR kit purchased from Ai Kerui organism under the accession number AG11701;

loadingbuffer available from CELL SIGNALLING technology company under the trade designation 7723;

NC membrane purchased from Millipore company, cat# ISEQ00010;

protein pre-dyeing Marker purchased from Bio-Rad company under the product number 161-0394;

alpha-SMA antibody, available from Abways company under the trade designation CY5295;

alpha-SMA antibody from abcam under the accession number ab7817;

alpha-Tubulin antibody commercially available from Proteintech under the trade designation 11224-1-AP;

CollagenI, available from proteintech under the trade designation 14695-1-AP;

CollagenIII available from proteintech under the accession number 22734-1-AP;

IRDye 800CW Goat anti-Rabbit IgG SecondaryAntibody, available from LI-COR under the number 926-32211;

IRDye 680RD Goat anti-Mouse IgG Secondary Antibody, commercially available from 926-68070 under the number 926-68070;

Xfect RNATransfection Reagent available from Takara under the trade designation 631450.

3. Laboratory instrument and apparatus

Carbon dioxide incubator (SANYO, japan); biosafety cabinet (Thermo FISHER SCIENTIFIC, USA); ECLIPSE TI-inverted microscope (NIKON, japan); ice maker (Scotsman, italy), one ten thousandth electronic balance (Sartorius, germany), vortex shaker (manufactured by linbell instruments, sea, china), HH-2 digital constant temperature water bath (national, constant state, china), pH meter (Sartorius, germany), 4 ℃ refrigerator (hail, peninsula, china), 0421-1 low speed bench centrifuge (Shanghai medical instruments, shanghai, china), 5810R multifunctional bench centrifuge (eppendorf, germany), single channel and multichannel pipettor (eppendorf, germany), KH-500DB digital controlled ultrasonic cleaner (manufactured by wound ultrasonic instruments, kunski, china), ECLIPSE TI-inverted microscope (NIKON, japan), ts2R inverted fluorescence microscope (NIKON, japan), EVOS FLoid living cell imaging workstation (thermal FISHER SCIENTIFIC, USA), 0421-1 low speed bench centrifuge (eppendorf, germany), refrigerator (light-controlled system (eppendorf, germany), single channel and multichannel pipettor (eppendorf, germany), KH-500DB digital controlled system (applied to electrophoresis system, USA, refrigerator, 4, water bath system, refrigerator, 4, water-vessel, refrigerator, water-transfer system, refrigerator, 4, water-vessel, water-cooled system, water-supply system, water supply system, water, ice, water, water, peninsula, china), an 80 ℃ refrigerator (Thermo FISHER SCIENTIFIC, USA), odyssey Near-INFRAREDWESTERN DETECTION SYSTEM (LICOR Bioscience, USA), a horizontal constant speed shaker (manufactured by Linbell instruments, inc., haimen, china), a multipurpose rotary shaker (manufactured by Linbell instruments, inc., haimen, china), a plastic film sealer (manufactured by Bao qin Europe tool Co., hangzhou, china), a Nano-100 micro-spectrophotometer (ao Cheng Yiqi, hangzhou, china), 2720Thermal Cycler reverse transcription instrument (Applied Biosystems, thermo FISHER SCIENTIFIC, USA), a PCR instrument (LIGHT CYCLER 480, roche), an HX-101E small animal ventilator (Taban, chengdu, china), a 37 ℃ small animal constant temperature heating mat (manufactured by Homek electronics technologies Co., deep, china).

2. Experimental method

1. Isolation and culture of adult mouse myocardial fibroblasts

C57BL/6 mouse hearts are separated by adopting a four-part shearing method, placed in a 100mm culture dish containing precooled PBS solution, sheared into fragments with the size of 1 multiplied by 1cm ³ by using bent shears, transferred into a triangular flask with a stopper grinding mouth, removed the supernatant containing blood and other tissue residues, washed once by PBS, and removed the supernatant. A mixed enzyme solution (type II collagenase and type IV collagenase) was added to the stopper-milled Erlenmeyer flask, and the mixture was shaken in a constant temperature water bath at 37℃for 2 minutes at 100rpm, and the supernatant was discarded. 415 mL centrifuge tubes were prepared, and 7mL of 10% FBS-DMEM solution was added to the tubes for collection of myocardial tissue digests. A50 mL centrifuge tube T1 was prepared, and a 100 μm cell screen was pre-laid on the tube port in preparation for filtration of the cell suspension to remove tissue aggregates. To the stopper-milled Erlenmeyer flask, 7mL of the mixed enzyme solution was added, and the mixture was shaken in a thermostatic waterbath at 37℃for 5 minutes at 100rpm, and the supernatant myocardial tissue digestion solution was collected and added to a 15-mL centrifuge tube prepared in advance. Centrifugation at 1500rpm for 3 minutes, the supernatant was discarded, the lower pellet was resuspended in 2mL of 10% FBS-DMEM solution, blown down evenly, and filtered through a cell screen and added to T1. Repeatedly digesting the myocardial tissue according to the steps until most of the myocardial tissue in the triangular flask with the stopper grind is white and viscous, which indicates that most of myocardial parenchymal cells are digested and eluted, and collecting all myocardial tissue digestive juice until T1 is reached. The T1 myocardial tissue digestion solution was spread evenly into a 100mm dish of 5 mouse hearts, and an appropriate amount of 10% FBS-DMEM solution was added to make the total volume of the solution in each dish about 8mL. Culturing in incubator for 2-3 hr, and separating myocardial cell and myocardial fibroblast with the characteristic of preferential adhesion of myocardial fibroblast. After 2-3 hours of incubation, the non-adherent cardiomyocytes in the petri dish were gently blown with a 1mL pipette and the supernatant containing the cardiomyocytes was discarded. In addition, each dish was washed once again by adding 2mL of 10% FBS-DMEM solution, and the washings were discarded. After the myocardial fibroblasts in the culture dish are completely adhered, the next experiment can be performed.

2. Cellular immunofluorescence

After fixing the cells with 4% paraformaldehyde at room temperature for 15 minutes, a PBS solution containing 0.1% Triton X-100 was added and rinsed 2 times for 5 minutes on a horizontal shaker (rotation speed about 60 rpm). PBS solution containing 5% goat serum and 0.3% Triton X-100 was prepared as a blocking buffer, and cells were blocked in the blocking buffer for 1 hour. The primary antibody was formulated with PBS containing 1% BSA and 0.3% Triton X-100, in accordance with the dilution ratio recommended in the antibody specification. After blotting the blocking buffer, diluted primary antibody was added and placed in a4 ℃ refrigerator and incubated overnight. After the end of the primary antibody incubation, the primary antibody solution was discarded and rinsed 3 times for 5 minutes with 0.1% Triton X-100 PBS. After dilution of the fluorescent-labeled secondary antibody with PBS containing 1% BSA and 0.3% Triton X-100, the secondary antibody was incubated at room temperature for 2 hours in the absence of light. To label the nuclei, DAPI dye was added for 10 minutes. The DAPI solution was then discarded, the cells were rinsed 3 times with PBS solution for 5 minutes each and transferred to a live cell workstation for photomicrograph acquisition.

3. Immunoblotting experiments

After 24 hours of treatment of the cells with the medium containing the different drugs, they were discarded and washed once with pre-chilled PBS. The 6-well plate was placed on ice and 100mL 1X Loading buffer was added to each well to lyse the cells, which were immediately scraped from the plate and transferred to a microcentrifuge tube. Sonication for 15 seconds allowed the cells to lyse completely, followed by heating at 100 ℃ for 10 minutes to denature the proteins, and cooling on ice.

A proper amount of protein sample and protein pre-staining marker are added in the corresponding lanes of SDS-PAGE gel. After electrophoresis at a constant voltage of 80V for 35 minutes, the voltage is adjusted to 120V, electrophoresis is continued until the loading buffer approaches the bottom of the gel, electrophoresis is stopped, and then the gel is wet-turned for 2 hours at a constant current of 500mA in a low-temperature environment. After the samples were wet transferred to nitrocellulose membranes, the membranes were blocked with 5% milk for 1 hour at ambient temperature. The primary antibody was diluted with reference to the dilution ratio recommended in the antibody specification, the membrane was placed in the primary antibody and incubated overnight on a 4 ℃ shaker. At the end of the primary antibody incubation, the primary antibody was recovered, and the membrane was washed with TBST solution, placed in the secondary antibody, and incubated at room temperature for 2 hours in the dark. After the membrane is washed again, a near infrared laser imaging system can be used for detecting the target protein band.

4. Extraction of Total RNA from cells and RT-PCR experiments

Referring to SteadyPure quick RNA extraction kit and Evo M-MLV reverse transcription premix kit instructions, total RNA was extracted from cells after drug stimulation and reverse transcribed. A PCR reaction system containing 10. Mu. LACEQ QPCR SYBR GREEN MASTER Mix, 1. Mu.L of primer, 2. Mu.L of cDNA and 7. Mu. L RNASE FREE DDH ₂ O was prepared and a subsequent RT-PCR experiment was performed using the LightCycler480 RT-PCR system. With 18S as an internal control, the expression level of the target gene in each group was expressed as a multiple of the expression level in the control group. Primer sequences of genes such as Acta2, col1a1, postn, ndefs 4, ldha, and 18S can be seen in Table 1.

TABLE 1

5、ICW

The liquid culture was carefully blotted dry, washed once with PBS, and 150. Mu.L-20℃precooled methanol was added to each well, and the cells were fixed at room temperature for 20 minutes. The fixative was carefully aspirated, 200. Mu. LPBS of the solution was added and rinsed 2 times for 5 minutes on a horizontal shaker (rotation speed about 60 rpm). A PBS solution containing 5% goat serum and 0.3% Triton X-100 was prepared as a blocking buffer, 150. Mu.L of blocking buffer was added to each well, and blocking was performed for 90 minutes on a horizontal shaker. At the same time as blocking, the primary antibody was formulated with PBS solution containing 1% BSA and 0.3% Triton X-100, in accordance with the dilution ratio recommended in the antibody specification. After blocking, the blocking buffer was blotted dry, and 150. Mu.L of 0.1% Triton X-100PBS was added to each well and washed 2 times for 5 minutes. The washes were discarded, 50 μl of diluted primary antibody was added to each well, and a fresh-keeping film was applied and incubated overnight at 4 ℃. The primary antibody was carefully recovered, 200. Mu.L of 0.1% Tween20-PBS was added to each well, and rinsed four times for 5 minutes on a horizontal shaker. 50 μl of the diluted secondary antibody was added to each well and incubated at 4℃for 6-8 hours in the dark. The secondary antibody was carefully recovered, and 200. Mu.L of 0.1% Tween20-PBS was added to each well, and rinsed four times for 5 minutes. The liquids in the wells were blotted and the target protein expression levels were measured using a near infrared imaging system, and the ratio of the α -SMA to α -Tubulin fluorescence intensities for each group was calculated using Image Pro Plus (IPP, media Cybernetics, USA) 6.0 software and the data for each group was presented as a percentage of the control group.

6. Method for establishing acute myocardial infarction model of mice

The mice were kept on water for 8 hours before surgery, left chest was dehaired after anesthesia, and the mice were supine with their limbs fixed on a cyclic heating operating table. The trachea was isolated and connected to a small animal ventilator to maintain respiration after catheterization using a blunt ended number 20 intravenous catheter, and the mice were observed to breathe with a ventilator frequency.

The skin in the operation area is disinfected by iodophor, the skin is cut off between the 3 rd rib and the 4 th rib at the left side of the sternum, and subcutaneous tissues and muscles are separated layer by layer in a blunt manner. The chest was opened, the heart was exposed, and the pericardium was cut, and a pink blood vessel was seen at the lower edge or left side of the auricle, i.e., anterior descending branch (LAD) of the left coronary artery, and permanent ligation was performed with 6-0 sutures. Myocardial tissue whitening indicates complete occlusion of the anterior left descending (LAD) artery. After ligation, the chest wall, muscle layer and skin layer are sutured. After the tracheal cannula is removed, the tracheal cannula is placed in a 37 ℃ heat preservation pad to recover until the tracheal cannula is recovered. Sham mice underwent the same procedure without ligating the coronary arteries.

7. Transfection

The siRNA sequences of Ldha and Ndufs genes used in the siRNA transfection experiments are shown in Table 2. For individual wells of a 6-well plate, 200. Mu. LXfect reactionbuffer, 100pmol siRNA, 10. Mu. LXfect RNAtransfectionpolymer were thoroughly mixed and allowed to stand at room temperature for 10 minutes to allow the transfection complex to form. The transfection complex was added drop-wise to the culture broth, gently swirled to mix, and then the 6-well plate was incubated in the incubator for 4 hours. After the transfection, the culture medium needs to be replaced, and the transfection efficiency is checked after 24 hours or the next experiment is performed.

TABLE 2

8. Calculation of synergy scores

Loewe synergy score calculation, namely calculating relative signal values (presented in percentage form) of each administration group compared with a model group by taking the signal intensity of a fibroblast activation marker alpha-SMA as an index, calculating Loewe synergy score by applying SYNERGYFINDER software (https:// synergyfnder. Fimm. Fi) to evaluate the synergy intensity between the two medicines (generally, loewe synergy score is considered to have obvious synergy between active ingredient combinations with the value larger than 5), and finally determining the synergy ingredient combinations based on the principle of combination optimization.

Golden average synergy index calculation E _A represents the effect when intervention A alone is used, E _B represents the effect when intervention B alone is used, E _A+B represents the effect when both A and B stem prognosis are used in combination, which is the expected value of the combined effect of both interventions. The synergistic effect is evaluated by the following formula Q=E _A+B/(E_A+E_B-E_A×E_B), wherein Q <0.85 is antagonistic effect, Q <1.15 is antagonistic effect, and Q is synergistic effect, Q is equal to or greater than 1.15.

9. Statistical analysis

Data are expressed as mean ± standard deviation (mean ± SD) and statistically analyzed using GRAPHPAD PRISM 8.0.0 software. The comparison between the sets of averages uses a one-way analysis of variance, with a statistical analysis result of p <0.05 being significantly different, p <0.01 being particularly significantly different, and p <0.001 being extremely significantly different.

3. Experimental results

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

The activated myofibroblasts have the phenomenon of aerobic glycolysis hyperactivity, and simultaneously, the mitochondrial oxidative phosphorylation level is also synchronously up-regulated. Because of the flexibility of cellular metabolism, the coupled engagement mechanism of aerobic glycolysis and mitochondrial oxidative phosphorylation is of great significance for metabolic intervention in fibroblast activation, and it is possible for a single inhibition of LDH or mitochondrial complex I to be compensatory back through another pathway. We confirmed the feasibility of the synergy pattern by comparing the effect of the interventions alone with the combined interventions on fibroblast activation, based on the fibroblast activation model, using the inhibitors Oxa targeting the inhibitors Met and LDH of mitochondrial complex I.

Firstly, TGF-beta 1 stimulates resting myocardial fibroblasts, an activated myofibroblast model can be established, alpha-SMA is measured through a cell immunofluorescence experiment, expression of fibrosis key genes Acta2, col1a1 and Postn is measured through an immunoblotting method for measuring fibrosis marker proteins alpha-SMA, collagenI and CollagenIII, PCR, and establishment of the model can be confirmed. From the results of the immunofluorescence experiments, we found that either the mitochondrial complex I inhibitor Met or the LDH inhibitor Oxa alone was only able to partially reduce the level of the fibroblast activation marker protein α -SMA, whereas the combination of Met and Oxa inhibited the formation of α -SMA myofilaments more significantly (fig. 1). Immunoblotting experiments also demonstrated that Met or Oxa alone partially reduced the expression of myofibroblast fibrosis-associated protein α -SMA, collagenI, collagenIII, whereas the combined inhibition was more pronounced (fig. 2). The PCR experiment results also prove that Met or Oxa alone can partially inhibit the expression of fibrotic marker genes Acta2 (figure 3, table 3), col1a1 (figure 4, table 4) and Postn (figure 5, table 5) of myofibroblasts, and the inhibition effect is more obvious after the combination. Subsequently, using In-CELL WESTERN technology, the efficacy of orthogonal groups of different concentrations of Met and Oxa was examined using the fibroblast activation marker α -SMA as an indicator (fig. 6), and the percentage of the signal values of the dosing group compared to the signal values of the model group was calculated (fig. 7, table 6), whereby the Loewe synergy score calculated by SYNERGYFINDER software was 21.224 (fig. 8), demonstrating that the Met and Oxa combination synergistically blocked the onset of fibroblast activation. Immediately, the acute myocardial infarction model of mice is adopted, the in-vivo anti-myocardial fibrosis effect of Met and Oxa is examined, after myocardial tissue sections are prepared, the myocardial tissue fibrosis degree is evaluated by adopting a Masson staining method, and the results show that the myocardial fibrosis area of MI mice can be partially reduced by using both Met and Oxa alone, and the anti-myocardial fibrosis effect of the mice is more remarkable after the combination of the Met and the Oxa (figure 9 and table 7). Together, the above results demonstrate the synergistic anti-myocardial fibrosis effect of the mitochondrial complex I inhibitor Met with the LDHA inhibitor Oxa and confirm the presence of metabolic flexibility during fibroblast activation.

Table 3 (corresponding to FIG. 3)

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

Table 4 (corresponding to FIG. 4)

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

Table 5 (corresponding to FIG. 5)

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

Table 6 (corresponding to FIG. 7)

Table 7 (corresponding to FIG. 9)

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

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

We further investigated whether Met/Oxa efficacy can be reproduced using specific mitochondrial complexes IIACS-010759 (phase I clinical, AVERAGE IC ₅₀ =5.6 nM in mouse cell lines) and the LDHA inhibitor GNE-140 (phase I clinical, IC ₅₀ =3 nM for LDHA).

The results of the cellular immunofluorescence experiments showed that either IACS-010759 or GNE-140 alone only partially reduced the level of α -SMA in the fibroblasts, whereas IACS-010759 and GNE-140 combined inhibited the expression of α -SMA more significantly (FIG. 10). Immunoblotting experiments also demonstrate that IACS-010759 or GNE-140 alone can partially reduce the expression of myofibroblast fibrosis-related protein alpha-SMA, collagenI, collagenIII, and the combined inhibition effect of the two can be more remarkable (figure 11). PCR experiments also show that IACS-010759 or GNE-140 can partially inhibit the expression of genes such as myofibroblast Acta2 (figure 12, table 8), col1a1 (figure 13, table 9) and Postn (figure 14, table 10) and the like, and the inhibition effect is more obvious after combined use. Subsequently, the efficacy of IACS-010759 and GNE-140 In different concentrations of orthogonal groups was examined using the In-CELL WESTERN technique and alpha-SMA as an index (FIG. 15, table 11), and the Loewe synergy score calculated by SYNERGYFINDER software was 16.649 (FIG. 16), demonstrating that IACS-010759 and GNE-140 combined use had a synergistic inhibitory effect on myocardial fibroblast activation. In addition, through a mouse acute myocardial infarction model, the in vivo anti-myocardial fibrosis effect of IACS-010759 and GNE-140 is verified, and the Masson staining result shows that the IACS-010759 or GNE-140 alone can partially reduce the myocardial fibrosis area of MI mice, and the anti-myocardial fibrosis effect is more remarkable after the IACS-010759 or GNE-140 is combined (figure 17 and table 12).

Table 8 (corresponding to FIG. 12)

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

Table 9 (corresponding to FIG. 13)

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

Watch 10 (corresponding to FIG. 14)

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

Table 11 (corresponding to FIG. 15)

Watch 12 (corresponding to FIG. 17)

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

The partial results reproduce the combined curative effect of Met and Oxa through the specific mitochondrial complex I inhibitor IACS-010759 and the LDHA inhibitor GNE-140, and prove the feasibility of the synergistic mode of synchronously interfering with the anti-myocardial fibrosis of the LDHA and the mitochondrial complex I.

3. Mitochondrial complex I inhibitor dihydrotanshinone I (DT) and LDHA inhibitor Salvianolic Acid A (SAA) cooperate to resist myocardial fibrosis

Earlier studies of the subject group found that dihydrotanshinone I in salvia miltiorrhiza can inhibit the activity of mitochondrial complex I, salvianolic acid A can inhibit the activity of LDH and the synthesis of lactic acid, and the potential synergistic effect between the two active ingredients is suggested. In addition, we have demonstrated in earlier work that inhibition of mitochondrial complex I by dihydrotanshinone I is reversible, as with metformin, possibly by modulating conformational changes in subunits of mitochondrial complex I such as ND3, etc., inhibiting NADH dehydrogenase, thus avoiding possible myocardial toxicity. Therefore, we next studied the synergistic anti-myocardial fibrosis effect of the mitochondrial complex I inhibitor dihydrotanshinone I (DT) and the LDHA inhibitor Salvianolic Acid A (SAA). As shown in fig. 18, both DT and SAA alone partially reduced myofibroblast α -SMA myofilament formation, and inhibition by the combination of DT and SAA was significantly enhanced. In addition, intervention of DT or SAA alone in myofibroblast fraction reduced expression of α -SMA, collagenI, collagenIII (fig. 19), and inhibition was significantly enhanced in combination. PCR experiments show that the expression of myofibroblast activation marker genes Acta2 (figure 20, table 13), col1a1 (figure 21, table 14) and Postn (figure 22, table 15) are partially inhibited by DT or SAA alone, and the combined inhibition effect of myofibroblast activation is more obvious. In-CELL WESTERN experiments were also performed to determine the efficacy of orthogonal groups of DT and SAA at different concentrations (FIG. 23, table 16), and Loewe synergy score was calculated as 20.216 (FIG. 24). In addition, the in vivo efficacy of DT and SAA is examined through a mouse acute myocardial infarction model, and the Masson staining results show that DT or SAA alone can partially reduce myocardial fibrosis area of MI mice, and the myocardial fibrosis area is reduced more remarkably by combining the DT and the SAA (FIG. 25, table 17). The above results demonstrate that the combination of DT/SAA, which is a naturally derived active ingredient having an inhibitory effect on mitochondrial complex I/LDHA, synergistically inhibits fibroblast activation, and that the combination of both has an enhanced anti-myocardial fibrosis effect.

Table 13 (corresponding to FIG. 20)

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

Watch 14 (corresponding to FIG. 21)

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

Table 15 (corresponding to FIG. 22)

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

Table 16 (corresponding to FIG. 23)

Table 17 (corresponding to FIG. 25)

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

The partial results prove that the dihydrotanshinone I and the salvianolic acid A in the red sage root have the synergistic effect of treating the myocardial fibrosis, and provide demonstration for a new strategy of resisting the myocardial fibrosis by the metabolic intervention of the traditional Chinese medicine.

4. Gene knockout has proved that inhibiting mitochondrial complex I and lactic dehydrogenase A synergistically reverse myocardial fibrosis

In this part of the experiment, we studied the gene intervention mitochondrial complex I/lactate dehydrogenase a to synergistically reverse myocardial fibrosis by knocking down Ndufs4 (mitochondrial complex I key subunit) and Ldha (lactate dehydrogenase a) alone or simultaneously. As shown, knockdown Ndufs or Ldha alone partially inhibited the formation of the marker α -SMA for fibroblast activation, whereas after simultaneous knockdown, the inhibition was significantly enhanced (fig. 26). The PCR experiment results show that synchronous knockdown Ndufs and Ldha can reduce the expression of fibroblastic myofibroblast activation marker genes Col1a1 (fig. 27, table 18) and Postn (fig. 28, table 19), and the synergistic inhibition effect between the two is evaluated by using a positive average Q value method, and the results show that the positive average Q values (q=ea+b/(ea+eb-ea×eb) of the combined knockdown Ndufs and Ldha on the inhibition Col1a1 and Postn genes, where ea+b is the inhibition rate of the combined administration, ea and Eb is the inhibition rate of the individual administration of the a and B drugs respectively) are 1.43 (fig. 27) and 17.18 (fig. 28) (both are greater than 1.15), indicating that the synergistic effect exists. The above results indicate that there is a synergistic reversal of myocardial fibrosis effect in inhibiting mitochondrial complex I and lactate dehydrogenase a.

Watch 18 (corresponding to FIG. 27)

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

Table 19 (corresponding to FIG. 28)

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

The above-described embodiments serve to describe the substance of the present invention in detail, but those skilled in the art should understand that the scope of the present invention should not be limited to this specific embodiment.

Claims (1)

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