Application of MT-CO2 gene inhibitor in preparation of medicine for treating organ injury diseases

Abstract

The invention relates to the technical field of biological medicines, in particular to application of an MT-CO2 gene inhibitor in preparation of a medicine for treating organ injury diseases. Experimental evidences show that MT-CO2 mRNA can be actively secreted to extracellular gaps and whole body circulatory systems, is widely distributed among tissues and organs and plays a unique regulation function. This discovery establishes the status of mitochondria as a novel endocrine organelle, which endows it with the ability to generate and release hereditary endocrine media into systemic circulation, thereby redefining all mitochondrial-containing cells as potential endocrine cells. The endocrine-like function of the mRNA outside the mitochondria reveals an important mechanism of interorgan communication and cell reprogramming, and opens up a brand new view for endocrine biology research. MT-CO2 is associated with various organ injury diseases, and expression of DYNC2H1 is interfered by an MT-CO2 gene inhibitor, so that the organ injury diseases are treated. The invention opens up a brand new target for treatment of various human diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of MT-CO2 gene inhibitors in the preparation of drugs for organ damage diseases. Background Technology

[0002] The central dogma of molecular biology describes the flow of genetic information from DNA to mRNA and then to proteins, and has long served as a fundamental principle guiding biological research. Based on the central dogma, mRNA resides within the cell and serves as a template for protein translation. Once released from the cell, cell-free mRNA is rapidly degraded by RNases and immediately loses its biological function.

[0003] Around 1.5 billion years ago, a pivotal evolutionary event occurred—the fusion of primitive non-mitochondrial eukaryotes with bacterial prokaryotes (the precursors of mitochondria) gave rise to modern eukaryotic cells. This evolutionary revolution from non-mitochondrial eukaryotes to modern mitochondrial eukaryotes profoundly impacted all cellular functions and is the direct cause of the complexity of life on Earth. This evolutionary leap profoundly influenced cellular complexity, metabolic processes, and signal transduction mechanisms, laying the foundation for diverse life forms.

[0004] In modern eukaryotes, mitochondria and host cells each retain independent genetic systems—including their own genomes and gene expression mechanisms—derived from different ancestors. Therefore, efficient cellular function depends on precise communication and coordination between the nuclear genome and the mitochondrial genome. Traditional research has primarily focused on protein communication between mitochondria and host cells, while the question of whether mitochondrial-derived mRNA can cross cell membranes and participate in intercellular and even inter-organ communication mechanisms remains largely unexplored. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention proposes the following technical solution: This invention relates, in one aspect, to the application of MT-CO2 gene inhibitors in the preparation of drugs for organ-related diseases. The sequence of the MT-CO2 gene is shown in SEQ ID NO.1.

[0006] SEQ ID NO.1:

[0007] In a preferred embodiment of the present invention, the organ-damaging diseases include acute myocardial infarction, sepsis, and atherosclerosis.

[0008] There are no particular limitations on the MT-CO2 gene inhibitors described in this invention, but preferably the MT-CO2 gene inhibitors are selected from small interfering RNA and MT-CO2 gene knockout reagents.

[0009] The drug also includes pharmaceutically acceptable excipients.

[0010] Another aspect of the present invention relates to the application of MT-CO2 gene inhibitors in reducing cell apoptosis in vitro.

[0011] In a preferred embodiment of the present invention, the cells are cardiomyocytes or vascular endothelial cells.

[0012] In a preferred embodiment of the present invention, the apoptosis is apoptosis induced by hypoxia / reoxygenation, serum starvation, or H2O2 treatment.

[0013] Another aspect of the present invention relates to the application of MT-CO2 gene inhibitors in inhibiting the transcription of the downstream gene DYNC2H1 in in vitro cardiomyocytes.

[0014] The present invention has the following beneficial effects: This invention reveals a groundbreaking discovery: mitochondrial-encoded mitochondrial cytochrome c oxidase II (MT-CO2) mRNA can detach from mitochondria, cross cell boundaries, and spread across cells and organs in the extracellular space and systemic circulation. Notably, extramitochondrial MT-CO2 does not retain its traditional protein-coding function but plays a crucial role as a novel transcellular and transorganic regulatory RNA. Experimental data show that extramitochondrial mRNA mediates interorgan communication, significantly influencing physiological and pathological processes in multiple tissues and organ systems. The findings reveal a new function of mRNA in the central dogma—mitochondrial-secreted extracellular mRNA has a significant regulatory effect on other genes and biological functions. This discovery not only provides a new perspective on the classical central dogma and modern endocrine theory but also has the potential to trigger revolutionary breakthroughs in the life sciences. Attached Figure Description

[0015] Figure 1 The results show the detection of MT-CO2 from mitochondria into host cells, the extracellular space, and circulating blood. a: Fluorescence in situ hybridization (FISH) image of MT-CO2 in human arterial tissue using a probe. Red: MT-CO2; Green: Mitochondrial MT-CO2 protein; Blue: Cell nucleus. MT-CO2 is distributed within mitochondria, cytoplasm, and the nucleus, as well as in the extracellular space. b: Results of RT-PCR detection of extracellular MT-CO2 and mitochondrial cytochrome b (MT-CYB) in circulating blood using specific primers. c: Complete sequence of MT-CO2 mRNA as shown by RACE sequencing. d: Expression levels of MT-CO2 mRNA at different time points in both microvesicle and non-microvesicle (supermembrane) regions. n=9; *p<0.01, compared to 0-hour level; #p<0.01, compared to microvesicle level.

[0016] Figure 2The results show the detection of MT-CO2 actively released into the extracellular space via microvesicles. a: MT-CO2 content in the culture medium of human cardiomyocytes (top), mouse cardiomyocytes (middle), and mouse vascular endothelial cells (bottom). b: Working model of mitochondrial DNA deletion experiment. cd: Expression level of D-loop encoded by mtDNA before and after mitochondrial DNA deletion. **P<0.01. ef: Mitochondrial DNA deletion significantly reduced MT-CO2 levels in human cardiomyocytes. *P<0.05, **P<0.01. gh: Mitochondrial DNA deletion significantly reduced MT-CO2 levels in human cardiomyocyte culture medium. **P<0.01. ik: Typical Western Blot results of CD36 and CD9 in human cardiomyocytes treated with GW4869. **P<0.01. l: GW4869 significantly reduced MT-CO2 levels in human cardiomyocyte culture medium. **P<0.01.

[0017] Figure 3 Extramitochondrial CO2 (MT-CO2) released by cells can enter other cells and organs. a: The specificity of the human MT-CO2 (h-only-MT-CO2) primers was verified by RT-PCR. b: The specificity of the mouse MT-CO2 (m-only-MT-CO2) primers was verified by RT-PCR. c: When mouse cardiomyocytes were co-cultured with human cardiomyocytes, MT-CO2 released by mice could enter human cardiomyocytes. d: When human cardiomyocytes were co-cultured with mouse cardiomyocytes, human MT-CO2 could enter mouse cardiomyocytes. e: After injecting human cardiomyocyte culture medium into mouse cardiomyocytes, human MT-CO2 was detected in the mouse cardiomyocytes. f: After injecting mouse cardiomyocyte culture medium into human cardiomyocytes, mouse MT-CO2 was detected in the mouse cardiomyocytes. g: Schematic diagram of injecting human cardiomyocyte culture medium into the left ventricle of a mouse. h: Results of detecting human MT-CO2 in mouse organs using h-only-MT-CO2 primers after injecting human cardiomyocyte culture medium into the left ventricle of a mouse. i: Schematic diagram of injecting human cardiomyocyte culture medium into the jugular vein of a mouse. j: After injecting human cardiomyocyte culture medium into the jugular vein of a mouse, human MT-CO2 was detected in the mouse organ using the h-only-MT-CO2 primer.

[0018] Figure 4To investigate the function of extramitochondrial MT-CO2 as a non-coding RNA via its target gene DYNC2H1. a: Freshly cultured human cardiomyocytes (human CM) were cultured for 24 hours after treatment with supernatant containing extramitochondrial MT-CO2 or control medium. b: q-PCR showed a significant increase in MT-CO2 mRNA levels in freshly cultured human CM treated with extramitochondrial MT-CO2 (**P<0.01). c: Western blot comparison of MT-CO2 protein levels in human CM treated with extramitochondrial MT-CO2 and in the control group. d: No significant change in MT-CO2 protein levels was observed in cells with increased extramitochondrial MT-CO2 concentration. e: Treatment with supernatant containing extramitochondrial MT-CO2 significantly increased DYNC2H1 mRNA levels in human CM (**P<0.01). f: Western blot comparison of DYNC2H1 protein levels in human CM treated with extramitochondrial MT-CO2 and in the control group. g: The level of DYNC2H1 protein was significantly increased in the extramitochondrial MT-CO2 treatment group, *P<0.05. h: After MT-CO2 knockdown with siRNA, the level of MT-CO2 mRNA in human cardiomyocytes was significantly decreased, **P<0.01. i: After MT-CO2 knockdown with siRNA, the expression levels of both DYNC2H1 mRNA and protein in human cardiomyocytes were significantly decreased, **P<0.01.

[0019] Figure 5MT-CO2, an extramitochondrial RNA, is used as a novel regulatory RNA to promote apoptosis. a: Representative TUNEL staining micrographs of human cardiomyocytes after treatment with negative control, siRNA-MT-CO2, and ischemia / re-tension (H / R). b: siRNA-MT-CO2 reduces H / R-induced apoptosis in human cardiomyocytes. **P<0.01. c: Representative TUNEL staining micrographs of human cardiomyocytes after treatment with negative control, siRNA-DYNC2H1, and ischemia / re-tension. d: siRNA-DYNC2H1 reduces H / R-induced apoptosis in human cardiomyocytes. *P<0.05, **P<0.01. e: Western blot micrographs showing that siRNA-MT-CO2 and siRNA-DYNC2H1 have a protective effect against HR-induced apoptosis in cardiomyocytes. f: Regulation of MT-CO2 in a co-culture system of human cardiomyocytes and HCAECs. g: MT-CO2 levels in the HCAECs culture medium in the co-culture system. h: MT-CO2 level in HCAECs in the co-culture system. i: Representative TUNEL-stained micrographs of human cardiomyocytes co-cultured with HCAECs, including negative control, siRNA-MT-CO2 treatment, and starvation treatment. j: Co-culture of human cardiomyocytes treated with siRNA-MT-CO2 reduced starvation-induced apoptosis in HCAECs. *P<0.05(k) Regulatory role of DYNC2H1 in the co-culture system of human cardiomyocytes and HCAECs. l: MT-CO2 level increased in the medium for HCAECs co-cultured with human cardiomyocytes. m: MT-CO2 level increased in human cardiomyocytes co-cultured with HCAECs. n: Co-culture with human cardiomyocytes treated with siRNA-DYNC2H1 reduced starvation-induced apoptosis in both cardiomyocytes and HCAECs. N*P<0.05, **P<0.01. o: Representative TUNEL-stained micrographs of HCAECs co-cultured with blank controls, human cardiomyocytes, negative controls, cells treated with siRNA-DYNC2H1, and cells subjected to starvation. Note: Red indicates TUNEL-stained apoptotic cells; blue indicates DAPI-stained cell nuclei.

[0020] Figure 6Changes in the expression levels of extramitochondrial MT-CO2 in damaged cells, organs, animals, and healthy human patients. a: In a hypoxia model, MT-CO2 levels were elevated in both human cardiomyocytes (left) and human cardiomyocytes in culture medium (right). **P<0.01. b: Effect of hydrogen peroxide on MT-CO2 levels in cultured human cardiomyocytes (left) and human cardiomyocytes in culture medium (right). *P<0.005, **P<0.01. c: In starvation injury, MT-CO2 levels were elevated in both extramitochondrial cardiomyocytes (MAECs) (left) and cardiomyocytes in culture medium (right). *P<0.05, **P<0.01. d: Effect of hydrogen peroxide on MT-CO2 levels in extramitochondrial cardiomyocytes (MAECs) (right) and human cardiomyocytes in culture medium (right). *P<0.05, **P<0.01. e: Working models of acute myocardial infarction and sepsis. f: Changes in MT-CO2 levels in the heart, liver, spleen, lung, kidney, aorta, and serum of mice 3 hours after ischemic myocardial infarction (AMI). *P<0.05, **P<0.01. g: Changes in MT-CO2 levels in the heart, liver, spleen, lung, kidney, and serum of mice 6 hours after LPS injection. *P<0.05, **P<0.01. h: Circulating serum MT-CO2 levels in patients were significantly higher than those in healthy controls. i: Schematic diagram of the intervention mechanism of MT-CO2 on atherosclerosis in a mouse model of acute atherosclerosis. j: Serum and arterial MT-CO2 levels were significantly reduced by injection of the siRNA-siRNA / in vivo-jetPEI complex. **P<0.01. k: Representative images of HE-stained carotid artery sections show that knockdown of MT-CO2 significantly inhibited atherosclerotic lesions. l: siRNA-MT-CO2 successfully reduced the intima / media thickness ratio of atherosclerotic carotid arteries. n=12; **P<0.01. m: Representative vascular ultrasound images show that knocking down MT-CO2 can inhibit carotid artery thickness.

[0021] Figure 7 This study describes the detection of MT-CYB in human arterial tissue using in situ fluorescence hybridization (FISH) with a probe targeting the 14763-15766 bp region of the human mitochondrial cytochrome b (MT-CYB) gene (product number 531591, Advanced Cell Diagnostics, USA). Red: MT-CYB; Green: Mitochondrial MT-CYB protein; Blue: Cell nucleus. MT-CYB is located in the mitochondria, cytoplasm, and nucleus of cells, as well as in the extracellular space.

[0022] Figure 8 The microvesicles were confirmed by electron microscopy images (circular).

[0023] Figure 9 To determine human MT-CO2 expression in organs of mice that received human cardiomyocytes via jugular vein infusion and those that did not, using a human MT-CO2 expression-specific primer (H-only-MT-CO2).

[0024] Figure 10 The effect of MT-CO2 siRNA (50 nM) on the level of MT-CO2 released from mitochondria (sample number = 9, P value < 0.01).

[0025] Figure 11 This study validates the protective effect of MT-CO2 siRNA against serum starvation-induced apoptosis in vascular endothelial cells. a: MT-CO2 expression in mouse vascular endothelial cells. b: MT-CO2 expression in mouse vascular endothelial cell culture medium. c: TUNEL-positive cells (%). Starvation led to increased apoptosis. siRNA-MT-CO2 reduced apoptosis in mouse vascular endothelial cells. Note: Data are expressed as mean ± standard error (error bar). **P < 0.01, one-way ANOVA. d: Representative TUNEL-stained micrographs from mouse vascular endothelial cells treated with negative control, siRNA-MT-CO2, control, and starvation. Note: Red represents TUNEL staining of apoptotic cells; blue represents DAPI-stained cell nuclei. Scale bar = 100 μm. We averaged the results from three independent experiments.

[0026] Figure 12 Validation results of the protective effect of MT-CO2 siRNA against hydrogen peroxide (H2O2)-induced apoptosis in vascular endothelial cells. a: TUNEL-positive cells (%). H2O2 led to increased apoptosis. SiRNA-MT-CO2 reduced apoptosis in mouse vascular endothelial cells. Note: Data are expressed as mean ± standard error (error bar). *P<0.05, **P<0.01, one-way ANOVA. b: Representative TUNEL-stained micrographs of mouse vascular endothelial cells treated with negative control, siRNA-MT-CO2, control, and H2O2. Note: Red represents TUNEL staining of apoptotic cells; blue represents DAPI-stained cell nuclei. Scale bar = 100 μm. We averaged the results of three independent experiments.

[0027] Figure 13This section presents the results of detecting the involvement of extracellular mitochondrial MT-CO2 in apoptosis after entering mouse aortic endothelial cells (MAECs). a: Working model of co-culturing MT-CO2 knockdown mouse cardiomyocytes and starvation-induced apoptosis in MAECs. b: TUNEL-positive cells (%). Starvation leads to increased apoptosis. Co-culturing with mouse cardiomyocytes treated with siRNA-MT-CO2 reduced apoptosis in MAECs. Note: Data are expressed as mean ± standard error (error bar). **P < 0.01, one-way ANOVA. c: Representative TUNEL-stained micrographs of MAECs co-cultured with mouse cardiomyocytes treated with negative control, siRNA-MT-CO2, control, and starvation. Note: Red indicates TUNEL staining of apoptotic cells; blue indicates DAPI-stained cell nuclei. Scale bar = 100 μm. We averaged the results from three independent experiments.

[0028] Figure 14 The circulating serum MT-CYB level in 100 patients was significantly higher than that in 50 healthy controls. **P<0.01. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0030] The nucleic acid sequence information involved in the following examples is shown below: Table 1 siRNA sequence information Note: All siRNAs in Table 1 have a TT appended to the end.

[0031] Table 2 PCR primer sequence information Table 3 RACE primer sequence information Example 1 I. Experimental Materials and Methods Laboratory Mice: All laboratory mice were bred and housed at the University of Alabama at Birmingham's animal facility, which is accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC) and maintained a pathogen-free environment throughout. All mice were housed in a temperature-controlled (23±1℃) environment with a 12-hour circadian rhythm and had free access to drinking water and feed, in accordance with the animal research protocol approved by the institution's Animal Care and Use Committee. Male mice aged 5-7 weeks were used in the experiments; both C57BL / 6 wild-type mice and Apo E knockout mice with a C57BL / 6 background were purchased from the Jackson Laboratory.

[0032] Fluorescence in situ hybridization and immunofluorescence: In situ hybridization of MT-CO2 and MT-CYB miR-145 was performed on 5-µm human arterial tissue sections. Tissue sections were cut using a cryostat and transferred to SuperFrost / plus slides (Fisher). Vascular sections were fixed in 4% paraformaldehyde and acetylated with acetic anhydride / triethanolamine, followed by washing with PBS. Sections were then prehybridized for 30 min at 25°C below the predicted Tm value of the LNA probe in hybridization solution (50% formamide, 5×SSC, 0.5 mg / mL yeast tRNA, 1×Denhardt's solution). Probes (3 pmol) against MT-CO2 or MT-CYB (Exiqon) were labeled with DIG (DIG Oligonucleotide 3′ Tailing Kit; Roche Applied Sciences) and hybridized with the sections for 1 h at the same temperature as prehybridization. After post-hybridization washing with 0.1×SSC at 55°C, ISH signals were detected using a tyramine signal amplification system (PerkinElmer) according to the manufacturer's instructions. Slides were mounted with DAPI-containing Prolong Gold mounting medium (Invitrogen). MT-CO2 or MT-CYB proteins were immunofluorescently stained with their antibodies (Sigma). All fluorescence images were analyzed using a Nikon microscope equipped with a CCD camera and imaging software.

[0033] Rapid amplification of cDNA ends (RACE) technology: In this study, the SMARTerRACE 5′ / 3′ kit from Clontech was used to complete the full-sequence sequencing of the MT-CO2 transcript according to the manufacturer's instructions. The specific procedure is as follows: Total RNA was extracted from serum, cell culture medium, or cytoplasm, and 3′- and 5′-RACE-ready cDNAs were synthesized using SMARTScribe reverse transcriptase. After purifying the PCR amplification products by 1% agarose gel electrophoresis, the purified PCR fragments were cloned into a linearized pRACE vector for sequencing. 3′- and 5′-RACE-specific primers (GSPs) were designed based on the sequences obtained from RNA-seq. The relevant primer sequences are detailed in Table 3.

[0034] Cell Culture: 1) Primary neonatal mouse cardiomyocytes: 1-3 day old neonatal mice were used, and their surfaces were quickly rinsed and disinfected with 75% ethanol solution. The hearts were removed from the body and placed in a culture dish containing 1×DPBS (without Ca²⁺ or Mg²⁺). Ice bath solution containing 20 mM BDM (Merck Millipore, USA) was added to remove blood. The cleaned tissue blocks were pulverized and transferred to conical tubes. Separation medium (DPBS containing 20 mM BDM and 0.0125% trypsin) was added, and the cells were incubated overnight at 4°C with gentle shaking. The next day, the supernatant was discarded, and digestion solution (DMEM / F12 medium containing 1.5 mg / ml type II collagenase and 20 mM BDM) and an equal volume of F12 medium were added to the tissue blocks. The cells were incubated at 37°C with gentle shaking for 25 minutes. The tissue blocks were then gently pipetted, and the supernatant was collected and centrifuged at 100×g for 5 minutes. After discarding the supernatant, the cells were resuspended in plating medium (containing 65% high-glucose DMEM, 19% DMEM / F12 medium, 10% horse serum, 5% fetal bovine serum, and 1% penicillin / streptomycin). After removing fibroblasts using the differential adhesion method, primary mouse cardiomyocytes were plated at 1 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 12-well gelatin-coated culture plates. 1) After 24-48 hours of culture, the medium was replaced with maintenance medium (78% DMEM high-glucose medium, 17% DMEM / F12 medium, 4% horse serum, 1% penicillin / streptomycin) for further treatment. 2) Human cardiomyocytes (HCM) (ABM, BC, Canada) were cultured in Prigrow I medium (ABM, BC, Canada) supplemented with 10% (v / v) fetal bovine serum and antibiotics. 3) Mouse aortic endothelial cells (MAEC) (Thermo Fisher Scientific, MA, USA) were cultured using a pre-packaged kit (M1168, CellBio, IL, USA) in complete mouse endothelial cell culture medium. 4) Human primary coronary endothelial cells (HCAEC) (ATCC) were cultured in vascular cell basal medium (ATCC®PCS-100-030™) supplemented with endothelial cell growth kit-VEGF (ATCC®PCS-100-041™).

[0035] Co-culture model. Human cardiomyocytes (A cells) were first seeded into 0.4 μm Transwell plates (Corning®, USA) and cultured adherently for 24 hours. After removing the culture medium, the cells were washed three times thoroughly with PBS. The Transwell plates were then placed in primary neonatal mouse cardiomyocyte (B cell) seeding plates and cultured for another 24 hours. B cells were then collected for subsequent assays.

[0036] HCM ischemic injury (induced by hypoxia and reoxygenation (H / R) in serum-free and glucose-free medium). Hypoxia was achieved by placing cells in a hypoxic chamber filled with 5% CO2 and 95% nitrogen at 37°C for 120 minutes.

[0037] Treatment was performed using hydrogen peroxide. Cultured human cardiomyocytes and mouse aortic endothelial cells were treated for 48 hours with either solvent or hydrogen peroxide (Thermo Fisher Scientific, Massachusetts, USA) (50-1000 μM).

[0038] Cell transfection. siRNA (50 nM) was transfected into cells using TransIT-X2 transfection reagent (Mirus Bio, Wisconsin, USA). The siRNA targeted human MT-CO2, human DYNC2H1, mouse MT-CO2 (custom-made, GenePharma, China), or a negative control (see Table 1). Cells were analyzed 48 hours after transfection.

[0039] Mitochondrial DNA deletion model. Human heart cell lines were exposed to medium containing 50 ng / ml ethidium bromide (EtBr) (Millibosigma, MO), with additional additions of 100 μg / ml pyruvate (Millibosigma, MO) and 50 μg / ml uridine (Millibosigma, MO) for 5 and 7 days, respectively. DNA deletion was determined by PCR detection of the D-loop and MT-CO2 genes encoded by MT-DNA. A control experiment for the h-18S gene was also performed. The PCR program consisted of initial denaturation for 5 minutes and 35 cycles, specifically: denaturation at 94°C for 35 seconds, annealing at 58°C for 35 seconds, and extension at 72°C for 50 seconds. Electrophoresis was performed on a 2.5% agarose gel at 100V for 60 minutes, and the bands were visualized and photographed using the ChemiDoc system. Primer sequences are detailed in Table 2.

[0040] Microvesicle isolation. The methods for isolating serum microvesicles from circulating blood and microvesicles from cell culture media were as described in our previous study 15. Briefly, the supernatant of human cardiomyocytes treated with serum-free medium for 24 hours (with or without GW4869 treatment) was collected on ice and centrifuged sequentially: 300g for 10 min to remove cells, 2000g for 30 min to remove dead cells, and 10,000g for 30 min to remove cell debris. The supernatant was then transferred to fresh tubes. The microvesicle pellet was washed once with sterile PBS to remove any secreted proteins, then centrifuged at 100,000g for 70 min, and resuspended in 30 μL of PBS. All centrifugation was performed at 4°C. The protein content of the microvesicles was then determined using the Bradford method.

[0041] RNA extraction and qRT-PCR experimental procedure. Total RNA was extracted from cells and mouse tissues using TRIzol reagent (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer's instructions. Mouse blood samples were collected and incubated at room temperature for 2 hours, followed by centrifugation at 3000 rpm for 10 minutes to separate serum. After centrifugation to thoroughly remove cell debris, total RNA was extracted from serum and supernatant using TRIzol LS reagent (Thermo Fisher Scientific, USA). RNA quality was assessed using a Nanodrop 2000 nanoparticle size analyzer (Thermo Fisher Scientific, USA). All RNA samples were treated with RQ1 RNase-Free DNase (Promega, USA) according to the manufacturer's instructions. cDNA was synthesized from 1 μg of RNA and transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, USA). qPCR was performed using Bullseye Evagreen qPCR premix (Midwest Scientific, USA) on a Quant Studio 3 instrument (Bio-Rayet, USA). A two-step reaction program was used: 95°C denaturation for 15 seconds, followed by annealing and extension at 60°C for 60 seconds, for a total of 40 cycles. The 18S transcript was used as an internal reference gene to assess relative mRNA expression levels, and the enrichment fold was calculated using Equation 2 (−ΔCt).

[0042] Western blotting assay. Total cell or exosome lysates were separated by SDS-PAGE electrophoresis and then analyzed using Western blotting. The specific steps were as follows: Total cell or exosome lysates were processed according to the specified method, washed, and placed on ice for lysis with SDS lysis buffer (10 mM Tris-HCl, 1% SDS, 5 mM EDTA) for 30 minutes. After brief sonication, the mixture was centrifuged at 12,000 rpm for 20 minutes at 4°C, and the supernatant was collected as the protein extract. Protein concentration was determined using the Qubit protein quantification kit (Thermo Fisher Scientific, Massachusetts). Equal volumes of the protein extract were separated by 4%–20% SDS-PAGE gel (Bio-Rad Laboratories, California) and transferred to 0.2 / 0.45 μm polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Massachusetts). The PVDF membranes were blocked in Tris-buffered saline containing 5% skim milk powder at room temperature for 2 hours. The antibodies used in the experiment included: CD9 (1:500, Santa Cruz Biotechnology, USA), CD63 (1:500, Santa Cruz Biotechnology, USA), β-actin (1:5000, Merck Millipore, USA), MT-CO2 (1:1000, Thermo Fisher Scientific, USA), DYNC2H1 (1:2000, Biomatik, Germany), BAX (1:1000, Cell Signaling Technologies, USA), caspase3 (1:1000, Cell Signaling Technologies, USA), cleaved caspase3 (1:1000, Cell Signaling Technologies, USA), and Bcl-2 (1:500, Santa Cruz Biotechnology, USA).

[0043] Animal model of acute myocardial infarction. We used a mature mouse AMI model with left coronary artery ligation. Briefly, mice were anesthetized with inhaled isoflurane (2%), orally intubated, and connected to a rodent ventilator. Sham-operated mice served as a control group; the sham operation involved the same surgical procedures, except that the sutures were bypassed around the blood vessel without occlusion of the left anterior descending coronary artery.

[0044] Apoptosis detection. Apoptosis in cultured cells was detected using the terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL) staining method. A simplified procedure is as follows: First, cells were fixed with 4% paraformaldehyde, followed by TUNEL staining according to the Roche in situ cell death assay kit instructions. Finally, the number of TUNEL-positive cells in the infarcted area and surrounding areas was statistically analyzed against the total cell count using fluorescence microscopy.

[0045] Acute mouse atherosclerosis model and mouse siRNA transfection. Acute atherosclerosis was induced in ApoE knockout mice (ApoE− / − strain, C57BL / 6 background, Jackson Laboratory) for 3 weeks via partial left carotid artery ligation (PLCA) and a Western diet (17.5% protein, 20% fat, 0.15% cholesterol, TestDiet formula). The model was established by ligating three of the four branches of the left carotid artery (left lateral carotid artery, internal carotid artery, and occipital artery) using 6-0 sutures. To regulate MT-CO2 levels, 200 μl of mouse MT-CO2 siRNA / in vivo sprayed PEI complex (Polyplus Transfection Company, France) was administered via jugular vein injection, or the control group was given.

[0046] H&E-stained vascular sections and morphometric analysis. Morphometric analysis of neointimal lesion formation. Morphometric analysis was performed using a computerized image analysis system (Scion Image CMS-800), analyzing H&E-stained sections as described in reference 13. Six sections (5µm thick) were taken at equal intervals from the injured carotid artery. The average I / M of the six segments was taken as the I / M of the animal.

[0047] Carotid artery ultrasound examination. On postoperative days 3, 5, and 21, percutaneous vascular ultrasound of both carotid arteries was performed using an M700 (50MHz) probe (Vevo2100, Fujifilm Vision Acoustics, Canada). Mice were anesthetized with a gas mixture containing 3% isoflurane at a rate of 1-2 liters / minute of oxygen. Brief description: Animals were maintained in a gas mixture containing 1% isoflurane at a rate of 1-2 liters / minute of oxygen. Carotid artery diameter and blood flow were assessed by ultrasound.

[0048] Study participants included 100 patients with atherosclerotic coronary artery disease (CAD) from the Department of Cardiology, Guangdong Provincial People's Hospital. All CAD patients were diagnosed via angiography. A healthy control group of 50 individuals, matched for age and sex, was also included. These participants were from a health checkup center and had no history of coronary artery disease or cerebrovascular disease. Detailed characteristics of the participants are shown in Table 4.

[0049] Table 4 Detailed characteristics of the subjects CAD: Coronary artery disease; SAP: Stable angina; UAP: Unstable angina; BMI: Body mass index. All tests were two-tailed, and a p-value less than 0.05 was considered statistically significant. Data are presented as mean ± standard deviation or n / n.

[0050] Statistical explanation. All data are expressed as mean ± standard error (SEM) or mean ± standard deviation (SD). In the calculation of relative gene expression levels, the mean of the solvent control group was set as the baseline of 1 or 100%. Statistical evaluation was performed using two-tailed unpaired t-tests and analysis of variance (ANOVA). Data analysis was performed using SPSS 22.0 software. The statistical significance criterion was p-value < 0.05.

[0051] II. Results 1. Mitochondrial mRNA is located in mitochondria, extracellular space, circulation, and distal organs. We first investigated whether mitochondrial DNA-encoded mRNA can detach from the mitochondria and diffuse beyond its site of origin. Using a fluorescence in situ hybridization (FISH) probe specifically targeting human mitochondrial cytochrome C oxidase II mRNA (MT-CO2), we found that although MT-CO2 is transcribed within the mitochondria, it is not only present in the mitochondria but can also be localized in the cytoplasm, nucleus, and extracellular space of human arterial tissue. Figure 1 a). These observations suggest that MT-CO2 in mitochondria can transition to a cell-free state, thereby gaining the ability to traverse cellular compartments.

[0052] To detect the presence of free mitochondrial CO2 (MT-CO2) in circulating blood, we analyzed serum samples from 50 healthy subjects. Using RT-PCR and qRT-PCR techniques, we detected a large amount of extracellular MT-CO2 in human serum. Figure 1 b). Notably, its concentration far exceeded that of GAPDH (Ct ~26 vs 33), indicating that this substance is widely present throughout the body. To confirm the molecular integrity of the circulating mRNA transcript, we used rapid 5′ and 3′ cDNA end amplification (RACE) technology for verification, and the results confirmed that the detected MT-CO2 was completely identical to the intact MT-CO2 mRNA. Figure 1 c).

[0053] To verify whether this phenomenon exists only in mitochondrial MT-CO2, we used specific fluorescence in situ hybridization (FISH) and PCR techniques to detect another mitochondrial transcript—mitochondrial cytochrome b mRNA (MT-CYB). Similar to MT-CO2, MT-CYB mRNA is also present in the cytoplasm, nucleus, extracellular space, and circulatory system. Figure 7 This suggests that the export mechanism of mitochondrial mRNA may be a behavioral characteristic that is universal among different types of mitochondrial mRNA.

[0054] 2. MT-CO2 is actively transported into the extracellular space via microvesicles. Given the presence of abundant microvesicle-coated MT-CO2 in human serum, we hypothesized that it might remain stable by binding to an extracellular carrier. Serum was fractionated into microvesicle and non-vesicle components (supernatant) by ultracentrifugation. Electron microscopy confirmed the isolated vesicle structures. Figure 8 Although trace amounts of MT-CO2 are also present in non-vesicular components, they are most abundant in microvesicular components. Time-series analysis shows that the degradation rate of vesicular-bound MT-CO2 is slower than that of non-vesicular forms. Figure 1 d) This indicates that the vesicle packaging mechanism confers stronger extracellular stability.

[0055] To directly investigate extracellular MT-CO2 derived from cells, we cultured human cardiomyocytes, mouse cardiomyocytes, and mouse vascular endothelial cells in serum-free medium. The experiment revealed that MT-CO2 was not detected in the cell-free medium, but it was clearly detectable immediately after cell seeding. Figure 2 a) This indicates that cells actively release MT-CO2 into the extracellular space. Notably, because cardiomyocytes have a much larger number of mitochondria than vascular endothelial cells, the concentration of MT-CO2 in their culture medium is significantly higher in cardiomyocytes than in vascular endothelial cells.

[0056] To provide the source and mechanism of MT-CO2 release from cells, we employed two methods. First, we depleted mitochondrial DNA using ethidium bromide (50 ng / ml), pyruvate (100 μg / ml), and uridine (50 μg / ml) for 5 and 7 days, respectively. Figure 2 b), as shown by the decrease in the level of D-loop encoded by mtDNA ( Figure 2 c and 2d). We found that in the MT-DNA depletion group, the levels of MT-CO2 in cells and culture medium were significantly reduced ( Figure 2 e-2h). The results showed that the extracellular MT-CO2 in the culture medium did indeed originate from the cell mitochondria. Secondly, we used GW4869 (10 μg / ml) to inhibit the release of cell microvesicles for 24 hours (e-2h). Figure 2 The MT-CO2 level in the culture medium was significantly reduced (ik). Figure 2 This indicates that microvesicles are key carriers for cells to release MT-CO2 into the extracellular space. These findings demonstrate that the release of MT-CO2 mRNA is an active, mitochondrial-derived, vesicle-mediated process, rather than a passive consequence of cell damage.

[0057] 3. Extramitochondrial MT-CO2 acts as a transcellular and transorganic molecule. To investigate whether extramitochondrial MT-CO2 released from a single cell can enter neighboring cells, we used a multi-well co-culture system. Human and mouse cardiomyocytes were placed in separate culture chambers and co-cultured using a shared culture medium while avoiding direct contact. We designed species-specific primers for human and mouse MT-CO2 and verified their specificity (and specificity) using RT-PCR. Figure 3 ab). After 24 hours of co-culture, RNA extracted from each cell population showed that mouse cells contained MT-CO2 from human mitochondria, while human cells also contained MT-CO2 from mouse mitochondria (ab). Figure 3 (cd), indicating the presence of bidirectional transcellular MT-CO2 transfer.

[0058] To verify whether culture medium containing extramitochondrial MT-CO2 possesses transfer capability, we conducted a conditioned medium exchange experiment. Human cardiomyocyte culture medium was cross-added with mouse cardiomyocyte culture medium. RT-PCR results showed that cross-species extramitochondrial MT-CO2 was successfully transferred in the culture medium. Figure 3 This strongly supports the hypothesis that "extramitochondrial RNA can be transmitted across cells via extracellular pathways without direct contact."

[0059] To further verify whether extramitochondrial MT-CO2 released by cells can reach distant organs, we conducted an in vivo transfer experiment. First, 100 μl of human cardiomyocytes (~1×10⁻⁶) were transferred... 6 (Number of cells) were injected into the jugular vein of mice. The results showed that human mitochondrial MT-CO2 was detected in multiple organs, confirming its systemic distribution characteristics. Figure 9 Secondly, after injecting 1 ml of human cardiomyocyte conditioned medium into the left ventricle of six mice, the substance was also detected in serum, heart, liver, spleen, lungs, kidneys, and aorta. Figure 3 It is noteworthy that the distribution of exogenous mitochondrial MT-CO2 varies significantly across different organs: the highest concentration is found in the heart, while the lowest concentration is found in the kidneys.

[0060] In another experiment, researchers found accumulation of human MT-CO2 in mouse tissues after injecting 100 μl of conditioned culture medium of human cardiomyocytes into the jugular vein, with the highest content in the lungs and the lowest in the aorta. Figure 3 These results strongly demonstrate that extramitochondrial MT-CO2 not only has the ability to migrate across cells, but also to be transported across organs, thereby enabling long-distance mitochondrial CO2 transfer between different organs.

[0061] 4. Extramitochondrial MT-CO2 is a novel regulatory RNA. Abundant mitochondrial outer membrane extended MT-CO2 mRNA was detected in the extracellular space and systemic circulation, suggesting that its biological significance may extend beyond its intramitochondrial protein-coding function. To verify whether extracellular MT-CO2 still retains its classical protein-coding function, we added human cardiomyocyte culture medium rich in mitochondrial outer membrane extended MT-CO2 to untreated human cardiomyocytes and cultured them for 24 hours. Figure 4 a). This treatment significantly increased MT-CO2 levels in cardiomyocytes ( Figure 4 b), but no corresponding increase in MT-CO2 protein expression was observed ( Figure 4 These results indicate that MT-CO2 ceases to function as a protein translator once it leaves the mitochondria.

[0062] We hypothesize that released extramitochondrial CO2 (MT-CO2) may have a regulatory function on RNA. This MT-CO2, which accumulates in the cytoplasm, nucleus, extracellular space, and circulating blood outside the mitochondria, may affect gene expression in distant cells and organs through transcellular and transorganic regulation. Bioinformatics analysis revealed an MT-CO2 binding site in the DYNC2H1 protein, suggesting it may be a potential target gene of extramitochondrial MT-CO2. To verify this hypothesis, we examined the effect of extramitochondrial MT-CO2 on DYNC2H1 expression in human cardiomyocytes. The results showed that extramitochondrial MT-CO2 treatment significantly increased DYNC2H1 expression at both the mRNA and protein levels. Figure 4 e.g., however, after knocking down MT-CO2 with specific siRNA, the expression level of DYNC2H1 decreased significantly. Figure 4 Hi). It is worth noting that siRNA typically cannot enter mitochondria, and its main effects occur in the cytoplasm and nucleus. Nevertheless, we found that a concentration of 50 nM MT-CO2 siRNA reduced total cellular MT-CO2 by approximately 25%, but the inhibitory effect on mitochondrial-released MT-CO2 was even more significant, with a reduction of up to 50%. Figure 10 ).

[0063] These results indicate that DYNC2H1 is a key target gene of extramitochondrial MT-CO2, and that extramitochondrial MT-CO2 mRNA may regulate cellular function by modulating the expression of other genes. This is an unprecedented discovery in cellular mRNA.

[0064] 5. Extramitochondrial MT-CO2 induces apoptosis through its target gene DYNC2H1. Bioinformatics analysis suggests that the target gene DYNC2H1 may be involved in the apoptosis process. To investigate the biological role of extramitochondrial MT-CO2 in apoptosis, we conducted a series of experiments. First, we found that knocking down extramitochondrial MT-CO2 significantly reduced hypoxia / reoxygenation (H / R)-induced cardiomyocyte apoptosis. Figure 5 a–b, 5e). Secondly, knocking down the MT-CO2 target gene DYNC2H1 can also reduce cardiomyocyte apoptosis ( Figure 5 Furthermore, we found that the protective effect of MT-CO2 siRNA against apoptosis was not limited to cardiomyocytes; it also showed a significant protective effect against apoptosis induced by serum starvation or hydrogen peroxide (H2O2) in vascular endothelial cells. Figure 11 – Figure 12 ).

[0065] To directly verify that extramitochondrial MT-CO2 enters other cells and participates in apoptosis, we used a co-culture system for the experiment. Figure 5 f). In this experiment, human cardiomyocytes were transfected with either negative control siRNA or MT-CO2 siRNA for 24 hours. After washing three times with PBS, the cardiomyocytes were co-cultured with human primary coronary endothelial cells (HCAECs) for 24 hours (MT-CO2 knockdown group and control group were placed in different culture chambers). After 48 hours, we assessed the apoptosis of HCAECs treated with serum starvation or untreated cells. The results showed that the MT-CO2 level in both HCAECs and their culture medium was significantly reduced in the MT-CO2 siRNA group (f). Figure 5 Furthermore, in HCAECs co-cultured with MT-CO2-deficient cardiomyocytes, serum starvation-induced apoptosis was significantly reduced (gh). Figure 5 These results were also validated in parallel experiments with mouse cells: when mouse vascular endothelial cells (mouse ECs) were co-cultured with MT-CO2-deficient mouse cardiomyocytes, their apoptosis levels were significantly reduced (ij). Figure 13 ).

[0066] To investigate whether extramitochondrial MT-CO2 participates in apoptosis by regulating DYNC2H1, we designed a co-culture experiment: human cardiomyocytes or DYNC2H1 knockout human cardiomyocytes were placed in one culture chamber, while human pulmonary artery endothelial cells (HCAECs) were placed in another chamber. Figure 5 The results showed that in the co-culture system containing cardiomyocytes, the MT-CO2 level in HCAECs and its culture medium was significantly increased (k). Figure 5 lm). Compared with cardiomyocytes without DYNC2H1 deficiency, HCAECs co-cultured with DYNC2H1-deficient cardiomyocytes showed a significantly lower incidence of serum starvation-induced apoptosis. Figure 5 no).

[0067] 6. Extramitochondrial MT-CO2 increases in injured cells and organs in animals with damaged cells and organs, as well as in human patients with strong functional cells. The role of extramitochondrial MT-CO2 under pathological conditions has not been investigated. To explore this question, we first examined the MT-CO2 levels in damaged cells. The study found that under various injury stimuli, including hypoxia / reoxygenation (H / R), serum starvation, and H2O2 treatment, the MT-CO2 levels in human cardiomyocytes and vascular endothelial cells (ECs) and their culture medium were significantly elevated. Figure 6 ad).

[0068] To investigate the association between extramitochondrial MT-CO2 and disease state, we selected two classic mouse models of organ injury: acute myocardial infarction (AMI) and sepsis. AMI was induced by ligation of the left anterior descending coronary artery, while sepsis was induced by intraperitoneal injection of lipopolysaccharide (LPS; 20 mg / kg). Compared with control mice, the circulating serum MT-CO2 level in these diseased mice was significantly elevated. Figure 6 e.g.

[0069] We also measured extramitochondrial MT-CO2 levels in human subjects by collecting serum samples from 150 patients with atherosclerotic coronary artery disease (CAD) and 50 age- and sex-matched healthy controls. All CAD patients were diagnosed by coronary angiography. Notably, serum MT-CO2 levels in CAD patients were significantly higher than those in healthy controls. Figure 6 In addition, the level of another extramitochondrial RNA—MT-CYB—also showed an elevated trend in CAD patients. Figure 14 ).

[0070] Finally, we investigated the effects of extramitochondrial MT-CO2 on disease in an ApoE knockout mouse model of acute atherosclerosis induced by partial ligation of the left carotid artery (PLCA) and a Western diet. ApoE- / - mice were injected with either a PEI complex-based control siRNA or a mouse MT-CO2 siRNA-based PEI complex (200 μl) via the external jugular vein. After three weeks of PLCA treatment, we performed histological analysis of the left carotid artery. Figure 6 i). The results showed that after treatment with MT-CO2 siRNA, the levels of MT-CO2 in serum and arteries were significantly reduced (i). Figure 6 j). Notably, knocking down MT-CO2 effectively inhibits the formation of atherosclerotic plaques (j). Figure 6 l). Typical results of H&E-stained vascular sections and vascular ultrasound images are shown in [reference needed]. Figure 6k and Figure 6 m.

[0071] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

Claims

1. Use of an inhibitor of the MT-C02 gene for the preparation of a medicament for organ damage diseases, characterized in that, The sequence of the MT-CO2 gene is shown as SEQ ID NO.

1.

2. The use of the inhibitor of the MT-CO2 gene according to claim 1 in the preparation of a medicament for organ injury disease, characterized in that, The organ injury disease includes acute myocardial infarction, sepsis and atherosclerosis.

3. The use of the inhibitor of the MT-CO2 gene according to claim 1 in the preparation of a medicament for organ injury disease, characterized in that, The MT-CO2 gene inhibitor is selected from small interfering RNA, MT-CO2 gene knockout reagent.

4. The use of the inhibitor of the MT-CO2 gene according to claim 1 in the preparation of a medicament for organ injury disease, characterized in that, The medicine also includes pharmaceutically acceptable adjuvant.

5. The use of the MT-CO2 gene inhibitor in claim 1 for reducing apoptosis in vitro.

6. The use of the inhibitor of the MT-CO2 gene according to claim 5 for reducing apoptosis in vitro, characterized in that, The cell is myocardial cell or vascular endothelial cell.

7. The use of the inhibitor of the MT-CO2 gene according to claim 6 for reducing apoptosis in vitro, characterized in that, The apoptosis is caused by hypoxia / reoxygenation, serum starvation or H2O2 treatment.

8. The use of the MT-CO2 gene inhibitor in claim 1 for inhibiting the transcription of downstream gene DYNC2H1 in myocardial cell in vitro.

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