An energy drug for treating mitochondrial damage under hypoxic conditions

Abstract

This invention relates to an energy drug for treating mitochondrial damage under hypoxic conditions. The energy drug is the application of racemic scopolamine (Rac) in remodeling mitochondrial function. Hypoxic conditions include physiologically or pathologically induced hypoxia such as high altitude or vascular occlusion. This invention uses racemic scopolamine to treat hypoxic rat embryonic cardiomyocyte H9C2 and BALB / c mouse models, demonstrating that the compound has the characteristic of remodeling mitochondrial function under hypoxic conditions. It is a highly effective energy drug with promising clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of racemic scopolamine in mitochondrial functional remodeling under hypoxic conditions. Background Technology

[0002] Scopolamine (Rac) is derived from anisodamine, a plant native to my country belonging to the Solanaceae family. Anisodus tanguticus Rac is an alkaloid extracted from [a specific plant / organism], and is a non-selective cholinergic antagonist. Previous studies have shown that Rac is an antioxidant that can prevent cell damage caused by free radicals. In addition, Rac can activate the Nrf2 / ARE signaling pathway, enhance cellular antioxidant capacity, and inhibit cellular senescence.

[0003] Normally, the number of mitochondria within a cell depends on the cell's metabolic level; the more vigorous the cell's metabolism, the more mitochondria it contains. Therefore, cardiomyocytes are an excellent model for studying mitochondrial function regulation. When cardiomyocytes are under physiological or pathological hypoxia, such as hypoxia induced by high altitude or vascular occlusion, energy metabolism becomes disordered, mitochondrial function becomes abnormal, and energy supply cannot meet the metabolic needs of cardiomyocytes, causing myocardial hypoxia damage. The accumulation of reactive oxygen species (ROS) induces cardiomyocyte apoptosis, thereby affecting myocardial function. Clinical studies show that mitochondrial dysfunction is one of the most important mechanisms inducing cardiovascular disease, but currently, there are no effective treatments or drugs. Therefore, developing new drugs for mitochondrial diseases is an urgent need for patients, and the application of Rac in mitochondrial functional remodeling under hypoxic conditions has significant clinical implications. Summary of the Invention

[0004] The purpose of this invention is to provide an application of Rac for mitochondrial functional remodeling under hypoxic conditions.

[0005] This invention uses CoCl2 to induce cellular hypoxia, establishes a rat cardiomyocyte H9C2 hypoxic injury cell model, and verifies its effectiveness. Then, α-lipoic acid is used as a positive control to detect mitochondrial function-related indicators. The optimal conditions for CoCl2-induced H9C2 cell hypoxia were determined using the CCK-8 assay to be 700 μM treatment for 24 h. Figure 1 Furthermore, qPCR and Western blotting revealed that CoCl2 significantly upregulated the mRNA and protein expression levels of HIF-1α in H9C2 cells, validating the hypoxic response observed in the cell model. Figure 2Furthermore, flow cytometry and qPCR were used to detect mitochondrial-related functional indicators in hypoxia-induced H9C2 cells, including mitochondrial membrane potential, mitochondrial dynamics-related genes, relative mitochondrial copy number, intracellular calcium ions, ROS levels, and ATP content, verifying that mitochondrial dysfunction occurred in the cell model. Figure 3 — Figure 7 Next, using the well-known mitochondrial protectant α-lipoic acid as a positive control, we found that the aforementioned mitochondrial functional indicators damaged by CoCl2 hypoxia could be reversed by racemic Rac, specifically manifested as an increase in mitochondrial membrane potential (Figure 8) and the presence of mitochondrial fusion-related genes. Mfn1 , Mfn2 , OPA1 Significantly upregulated genes related to mitochondrial division Drp1 , FIS1 , Mff Significant downregulation resulted in an increase in the relative copy number of mitochondria (Figure 9), a decrease in free calcium ion concentration, a decrease in ROS levels, an increase in ATP content, and a decrease in the number of apoptotic cells (Figure 10). These results indicate that Rac can also serve as a mitochondrial protectant with the potential to combat hypoxia-induced damage. Furthermore, 10 μM Rac and 100 μM α-lipoic acid showed comparable efficacy in protecting H9C2 cells from hypoxia-induced mitochondrial dysfunction, suggesting that Rac may achieve similar efficacy with a lower dosage than other drugs, indicating its potential application prospects. Figure 11 and Figure 12 ).

[0006] A mouse hypoxia model was constructed to investigate the restorative effect of Rac on mitochondrial dysfunction in vivo. Experimental results showed that CoCl2 hypoxia treatment upregulated mitochondrial function in mice. HIF-1α mRNA ( Figure 13 This indicates that the in vivo hypoxia model was successfully established, and mitochondrial fusion-related genes were also detected. Mfn1 , OPA1 Significantly upregulated genes related to mitochondrial division Drp1 , Mff Significantly downregulated genes related to mitochondrial production PGC-1α Downregulation indicates mitochondrial dysfunction, and Rac can reverse this phenomenon. Figure 14 Immunohistochemical results further confirmed that Rac could restore the expression levels of HIF-1α, Drp1, Mfn2, and mtTFA proteins. Figure 15 ), remodeling mitochondrial function. Furthermore, through H&E staining and Masson staining ( Figure 16 Rac was found to improve myocardial fibrosis and prevent excessive collagen deposition. Serum tests in mice showed that, compared to hypoxic mice, Rac was able to restore serum levels of the enzyme-related marker LDH (…). Figure 17 ).

[0007] The beneficial effects of this invention are as follows:

[0008] (1) Rac can be a potential drug to alleviate mitochondrial dysfunction by improving mitochondrial dynamics-related genes and enhancing mitochondrial function in H9C2 cells.

[0009] (2) A mouse hypoxia injury model was successfully constructed using CoCl2. The results of the study on cardiac tissue and mitochondrial functional indicators were consistent with the results of the cellular level study. Attached Figure Description

[0010] Figure 1 shows the effect of CoCl2 treatment on the viability of H9C2 rat cardiomyocytes after 24 h, 48 h and 72 h.

[0011] Figure 2 shows CoCl2-induced intracellular hypoxia in H9C2 cells.

[0012] Figure 3 shows the effect of CoCl2 on mitochondrial membrane permeability and membrane potential in H9C2 cells.

[0013] Figure 4 shows the effect of CoCl2 on mitochondrial fusion and division genes in H9C2 cells.

[0014] Figure 5 shows the effect of CoCl2 on intracellular calcium ions in H9C2 cells.

[0015] Figure 6 shows the effect of CoCl2 on the intracellular ROS level in H9C2 cells.

[0016] Figure 7 shows the effect of CoCl2 on the intracellular ATP content of H9C2 cells.

[0017] Figure 8 shows the effects of Rac and α-lipoic acid on the recovery of damaged mitochondrial membrane potential in CoCl2-induced H9C2 cells.

[0018] Figure 9 shows the effects of Rac and α-lipoic acid on the expression of genes related to mitochondrial dynamics and mitochondrial copy number induced by CoCl2 in H9C2 cells.

[0019] Figure 10 shows the therapeutic effects of Rac and α-lipoic acid on CoCl2-damaged H9C2 cells in terms of calcium ion concentration, ROS, ATP content, and apoptosis.

[0020] Figure 11 shows the effect of different concentrations of Rac treatment for 24 h on the viability of H9C2 cells induced by CoCl2 hypoxia.

[0021] Figure 12 shows the effect of different concentrations of α-lipoic acid on the viability of H9C2 cells induced by CoCl2 hypoxia after 24 h of treatment.

[0022] Figure 13 Mouse heart HIF-1α The relative content of mRNA.

[0023] Figure 14. Relative content of mRNA related to mitochondrial fusion, division, and generation in mouse heart.

[0024] Figure 15 Immunohistochemical results of related proteins in mouse heart tissue during hypoxia, fusion, division, and generation.

[0025] Figure 16 HE staining and Masson staining of mouse heart tissue.

[0026] Figure 17. Measurement of LDH content in mouse serum. Detailed Implementation Plan

[0027] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0028] Unless otherwise specified, the experimental methods used in the following examples are commercially available.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] Example 1

[0031] Cell viability assay

[0032] 1.1 Cell Culture and Plating

[0033] (1) The complete culture medium used for rat embryonic cardiomyocytes H9C2 was DMEM, and the culture conditions were 37℃ and 5% CO2.

[0034] (2) Cell passage. When the cell density reaches 80%, the cells are passaged and then placed back into a 37°C, 5% CO2 cell culture incubator.

[0035] 1.2 Cell viability assay

[0036] (1) Inoculate well-grown H9C2 cells into 96-well plates, with at least 3 replicates per group.

[0037] (2) Cell viability was detected using the CCK-8 kit.

[0038] The effects of treating H9C2 cells with 125, 250, 500, 1000, and 2000 μM CoCl2 for 24 h, 48 h, and 72 h on rat cardiomyocyte viability were investigated. Results are attached. Figure 1 As shown.

[0039] The effect of CoCl2 on H9C2 cell viability was concentration- and time-dependent. At 24 h and 48 h of treatment, cell viability was impaired starting at 500 μM (p < 0.01). At 72 h of treatment, only 125 μM significantly impaired cell viability (p < 0.05). 700 μM CoCl2 treatment for 24 h reached the half-lethal concentration.

[0040] 2. Detection of hypoxia-inducible factor mRNA levels

[0041] Total RNA was extracted, and the transcriptional levels of relevant genes were detected using RT-PCR. Genes detected included... HIF-1α The results are attached. Figure 2 As shown in Figure A.

[0042] Hypoxia-inducible factor under CoCl2 treatment HIF-1α The mRNA level was upregulated in a dose-dependent manner.

[0043] 3. Protein level detection of hypoxia-inducible factor and genes related to mitochondrial dynamics

[0044] Protein was extracted using protein lysis buffer, and HIF-1α expression was detected by Western blotting under 350 μM and 700 μM CoCl2 treatment to determine whether the hypoxia model was successfully constructed. Results are attached. Figure 2 As shown in B and 2C.

[0045] Treatment with 350 μM and 700 μM CoCl2 both increased the expression of hypoxia-inducible factor HIF-1α (p<0.001), indicating that the hypoxia model was successfully constructed.

[0046] 4. Mitochondrial membrane permeability detection

[0047] Mitochondrial membrane permeability was assessed by staining with Mito Tracker Green and then detecting the fluorescence values ​​of the Control group, the 350 μM CoCl2 treatment group, and the 700 μM CoCl2 treatment group by flow cytometry. Results are attached. Figure 3 As shown in Figure A.

[0048] 350 μM CoCl2 was insufficient to damage the structural integrity of mitochondria and did not affect mitochondrial membrane permeability, while 700 μM CoCl2 significantly disrupted mitochondrial membrane permeability, resulting in a higher fluorescence intensity than the control group (p<0.005).

[0049] 5. Flow cytometry detection of mitochondrial membrane potential

[0050] JC-1 was used to detect the intracellular membrane potential levels in each group. In normal cells, the mitochondrial membrane potential is high, and JC-1 exists in the mitochondrial matrix in polymeric form, producing red fluorescence. When the mitochondrial structure is damaged, the mitochondrial membrane potential decreases, and JC-1 exists in the mitochondrial matrix in monomeric form, producing green fluorescence. The results are attached. Figure 3 As shown in B and 3C.

[0051] Upon addition of CoCl2, the red fluorescence emitted by the multimeric form of JC-1 decreased, while the green fluorescence emitted by the JC-1 monomer, representing mitochondrial structural damage and entry into the mitochondrial matrix, increased. The 700 μM CoCl2 group showed stronger green fluorescence and weaker red fluorescence compared to the 350 μM CoCl2 group.

[0052] 6. Detection of mRNA levels of genes related to mitochondrial dynamics

[0053] Total RNA was extracted, and the transcriptional levels of relevant genes were detected using RT-PCR. Genes detected included... Mfn1、 Mfn2, OPA1, Drp1, FIS1, Mff The results are attached. Figure 4 As shown.

[0054] Mitochondrial fusion-related genes under the action of CoCl2 Mfn1, Mfn2, OPA1 Downregulation of expression levels in genes related to mitochondrial division OPA1, Drp1, FIS1, Mff The expression level was upregulated.

[0055] 7. Flow cytometry detection of cellular calcium

[0056] Intracellular calcium ion levels were detected using the Fluo-3, AM probe. Results are attached. Figure 5 .

[0057] The intracellular calcium ion content remained unchanged under the treatment of 350 μM CoCl2. However, the intracellular calcium ion content increased under the treatment of 700 μM CoCl2.

[0058] 8. Flow cytometry detection of ROS

[0059] ROS content was detected using a DCFH-DA probe. Stronger fluorescence intensity indicates higher ROS content. Results are attached. Figure 6 As shown.

[0060] After CoCl2 treatment, the ROS level increased relative to the control group (p<0.05).

[0061] 9. ATP content determination

[0062] ATP levels were measured using an ATP assay kit. Mitochondria are the primary site of ATP production and their levels reflect the normality of mitochondrial function. High ATP levels indicate good functioning of mitochondrial oxidative phosphorylation-related enzymes. Results are attached. Figure 7 As shown.

[0063] After hypoxia induced by 700 μM CoCl2, ATP content decreased to 30% of that in the control group (p<0.001).

[0064] Example 2

[0065] 1. Flow cytometry detection of mitochondrial membrane potential.

[0066] The intracellular membrane potential levels in each group were detected using JC-1. Results are attached. Figure 8 As shown.

[0067] Under hypoxia, the ratio of red fluorescence to green fluorescence decreases, and the ratio increases after the addition of Rac.

[0068] 2. mRNA expression levels of genes related to mitochondrial dynamics

[0069] Gene detection includes Mfn1, Mfn2, OPA1, Drp1, FIS1, Mff The level of expression, and the results are attached. Figure 9 As shown in Figure A.

[0070] Compared to the control group, the hypoxia group Mfn1, Mfn2, OPA1 The expression levels of these three genes were downregulated, but were restored in the hypoxia+Rac group. Drp1, FIS1, Mff Rac reversed this effect, as it was upregulated during periods of hypoxia.

[0071] 3. Relative quantification of mitochondrial DNA copy number

[0072] Total genomic DNA was extracted from cells according to the instructions of the genomic DNA extraction kit. Then, the mitochondrial cytochrome b gene was measured using qPCR. Ctyb ) and actin gene ( β-actin The relative abundance of ) is shown in the attached figure. Figure 9 As shown in B.

[0073] The mtDNA copy number in the Hypoxia group was much lower than that in the Control group (p<0.001), while the mtDNA copy number in the Hypoxia+Rac group was higher than that in the Hypoxia group (p<0.05). The positive control group, Hypoxia+α-LA, had a more significant restorative effect on mtDNA copy number (p<0.001).

[0074] 4. Flow cytometry detection of cellular calcium

[0075] Intracellular calcium ion levels were detected using the Fluo-3, AM probe. Results are attached. Figure 10 As shown in Figure A.

[0076] The increased fluorescence in the Hypoxia group compared to the control group indicates an increase in intracellular calcium ion content. The decreased fluorescence intensity in the Hypoxia+Rac group suggests that the intracellular calcium ion balance has been restored under the action of Rac.

[0077] 5. Flow cytometry detection of ROS

[0078] ROS content was detected using a DCFH-DA probe. Results are attached. Figure 10 As shown in B, Rac treatment reduces ROS levels.

[0079] 6. ATP content determination

[0080] ATP levels were measured using an ATP assay kit. Mitochondria are the primary site of ATP production and their levels reflect the normality of mitochondrial function. High ATP levels indicate good functioning of mitochondrial oxidative phosphorylation-related enzymes. Results are attached. Figure 10 As shown in C.

[0081] After the addition of Rac, ATP levels recovered to 40% of the control group, which was higher than that of the hypoxia group (p<0.05). These results indicate that Rac can restore ATP levels compared to the CoCl2-induced hypoxia group.

[0082] 7. Detection of apoptosis using fluorescence microscopy and flow cytometry

[0083] Annexin V-FITC / PI staining was performed, and the FITC and PE channels were detected by flow cytometry. Results are attached. Figure 10 As shown in D.

[0084] Under hypoxic conditions, the rate of apoptosis was higher than in the control group. Rac treatment can alleviate apoptosis under hypoxic conditions.

[0085] 8. Cell viability assay

[0086] H9C2 cells were treated with 1, 10, 100, or 1000 μM Rac or α-LA for 24 h; cells were first treated with CoCl2 for 24 h, followed by treatment with Rac or α-LA for 24 h. Results are attached. Figure 11 and Figure 12 As shown.

[0087] Rac or α-LA at concentrations up to 100 μM did not damage cell viability, while 10 μM Rac showed a significant therapeutic effect on CoCl2-induced cell viability damage (p<0.001). 100 μM α-LA could also treat CoCl2-induced cell viability damage (p<0.05).

[0088] Example 3

[0089] 1. Hypoxia-inducible factor in mouse heart tissue HIF-1α mRNA level measurement

[0090] Mouse hearts were collected, lysed, and thoroughly ground. RNA was then extracted, reverse transcribed, and analyzed using qPCR. Results are attached. Figure 13 .

[0091] Hypoxia-inducible factor in the hearts of mice in the hypoxia group HIF-1α Significantly elevated expression levels (p<0.005) indicate successful establishment of the hypoxic mouse model. Hypoxia+Rac group mice. HIF-1α The expression level was significantly lower than that in the hypoxia group (p<0.005).

[0092] 2. Detection of mRNA levels of genes related to mitochondrial dynamics in mouse heart tissue

[0093] Mouse hearts were collected, lysed, and thoroughly ground. RNA was then extracted and reverse transcribed, and measured using qPCR. Mfn1, OPA1, Drp1, Mff, PGC-1α The results are attached. Figure 14 .

[0094] Consistent with in vitro experimental results, mitochondrial fusion-related genes Mfn1, OPA1 and genes related to mitochondrial formation PGC-1α The expression levels of these genes decreased in mice under hypoxic conditions. After Rac treatment, the expression levels of mitochondrial fusion-related and gene generation-related genes were upregulated relative to the hypoxic group. (Mitochondrial division-related genes) Drp1, Mff It showed an upregulation in the hypoxia group and a reversal in the Hypoxia+Rac group.

[0095] 3. Immunohistochemistry

[0096] After dewaxing and hydration, antigen retrieval was performed, followed by enzyme inactivation and blocking, antibody incubation, DAB staining, hematoxylin staining, and finally microscopic examination. The expression of various proteins in the tissue samples was observed. Results are attached. Figure 15 As shown.

[0097] Compared to the control, in the hypoxic mouse Hypoxia group, the expression levels of hypoxia-inducible factor HIF-1α protein were increased, the expression levels of mitochondrial fission-related protein Drp1 were increased, the expression levels of fusion-related protein Mfn2 were decreased, and the expression level of synthesis-related protein mtTFA was increased. In the Hypoxia+Rac group, compared with Hypoxia, the expression levels of HIF-1α were decreased, the expression levels of Drp1 and mtTFA were decreased, and the expression level of Mfn2 was increased. The expression levels of each protein in the Rac group were almost the same as those in the control group.

[0098] 4. HE staining

[0099] Tissue was fixed, sectioned, dewaxed, and hydrated. Then, it was stained with hematoxylin, differentiated, and subjected to blue-reverse staining, followed by eosin staining and dehydration. Finally, the slides were mounted, examined under a microscope, and photographed. Results are attached. Figure 16 As shown.

[0100] Rac alleviates symptoms such as cell lysis and rupture in myocardial tissue and tissue fibrosis.

[0101] 5. Masson staining

[0102] Tissue was fixed, sectioned, dewaxed, and hydrated, then mordanted, followed by nucleus staining, muscle fiber staining, and collagen fiber staining. After dehydration and mounting, the sections were examined under a microscope. Results are attached. Figure 16 As shown.

[0103] Compared to the control group, the Hypoxia group showed collagen deposition and myocardial tissue damage, while the Hypoxia+Rac group essentially recovered to the level of the control group. The cell morphology and tissue state of the Rac group were almost identical to those of the control group.

[0104] 6. Serum LDH level detection

[0105] According to the LDH assay kit instructions, coenzyme I was prepared into a working solution, and a 0.4 mol / L NaOH solution was prepared. The assay was performed according to the operating procedure. The results are attached. Figure 17 As shown.

[0106] Higher serum LDH levels indicate greater myocardial damage. Compared to the control group, the LDH level in the Hypoxia group was significantly higher (p<0.05), indicating greater myocardial tissue damage. In contrast, the LDH level in the Hypoxia+Rac group was significantly lower than that in the Hypoxia group (p<0.05). The LDH levels in the Rac group were almost identical to those in the control group.

Claims

1. An energy drug for treating mitochondrial damage under hypoxic conditions, characterized in that: The energy drug is the application of racemic scopolamine (Rac) in remodeling mitochondrial function, and the hypoxic conditions include physiological or pathological hypoxic stress induced by high altitude or vascular occlusion diseases.

2. The compound according to claim 1, characterized in that: (i) Rac remodels mitochondrial function and calcium uptake under hypoxic conditions; (ii) Rac protects the integrity of mitochondrial structure and enhances antioxidant capacity; (iii) Rac restores mitochondrial dynamics under hypoxic conditions.

3. The compound according to claims 1 to 2, characterized in that, Protecting mitochondrial membrane integrity, enhancing mitochondrial membrane potential, and restoring mitochondrial calcium uptake capacity under hypoxic conditions refer to maintaining intracellular calcium balance by remodeling mitochondrial function.

4. The compound according to claims 1 to 3, characterized in that, The Rac reduces free radical production, enhances antioxidant capacity, resists hypoxia-induced apoptosis, improves cell survival rate, and repairs myocardial damage by remodeling mitochondrial function.

5. The compound according to claims 1 to 4, characterized in that, The restoration of mitochondrial dynamics includes reversing the upregulation of mitochondrial division-related genes and proteins and the downregulation of mitochondrial fusion-related genes and proteins caused by hypoxia. Rac can inhibit excessive mitochondrial division, promote fusion, and restore the ability of mitochondria to produce ATP.

6. The use of the compound of claim 1 in the preparation of a pharmaceutical or nutritional product for improving mitochondrial function.

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