New application of indole-3-carbinol or dimer 3, 3 '-diindolylmethane thereof in preparation of medicine for treating muscle degenerative diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a novel application of indole-3-methanol or dimer 3, 3 '-diindolylmethane thereof in preparation of medicines for treating muscle degenerative diseases. According to the indole-3-carbinol and the dimer 3, 3 '-diindolylmethane thereof, muscle function decline is obviously delayed, a myofibril structure and a mitochondrial form are protected, and amyloid protein deposition and accumulation are reduced, so that the indole-3-carbinol and the dimer 3, 3'-diindolylmethane thereof play a good role in treating muscle decline related diseases such as sarcopenia and inclusion body myositis. The compound is natural in source and high in safety, can be applied to prevention and treatment of the muscle diseases in forms of oral administration, dietary supplements or pharmaceutical preparations and the like, and has a good industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a new use of indole-3-carbinol or its dimer 3,3'-diindolylmethane in the preparation of a medicament for treating muscle degenerative diseases. Background Art

[0002] With the intensification of the aging trend of the population, the incidence of age-related muscle diseases (such as sarcopenia, inclusion body myositis) has been increasing year by year, and has become an important factor affecting the quality of life and independent living ability of the elderly. Among them, sarcopenia is a syndrome characterized by progressive decline in muscle mass, strength and function, which is common in the elderly population and is closely related to an increased risk of falls, fractures, disability and death; inclusion body myositis is a chronic, progressive inflammatory myopathy characterized by muscle fiber degeneration and amyloid deposition, and its pathogenesis is complex, and there is currently no effective treatment. Currently, the commonly used intervention measures include exercise rehabilitation, drug treatment and nutritional supplementation. Although exercise rehabilitation is widely recommended, its poor compliance and high implementation difficulty often limit its actual effect. Although drug treatment has a certain effect, it has more side effects and poor long-term safety. As a low-risk, highly acceptable and long-term sustainable method, nutritional intervention has shown the potential to significantly improve muscle mass and function in many studies. In view of this, there is an urgent need to develop natural functional factors with definite biological activity, safety and reliability, and suitable for use in the form of nutritional intervention to effectively prevent and treat muscle degenerative diseases.

[0003] Indole-3-carbinol (I3C) is a natural alkaloid widely present in cruciferous vegetables, which has good biological activity, especially has significant effects in aspects such as anti-inflammation, antioxidant, regulation of hormone balance and anti-tumor. I3C can spontaneously transform into a series of oligomers under acidic conditions of gastric acid, and among them, 3,3'-diindolylmethane (DIM) is the main metabolite, and also has biological activity similar to or stronger than that of the parent compound. In recent years, the research on I3C and its metabolite DIM in regulating immunity, improving mitochondrial function, delaying cell aging and other aspects has been continuously deepened. Existing research has preliminarily shown that I3C and DIM may have a potential protective effect on muscle function through mechanisms such as improving muscle energy metabolism, antioxidant damage and regulating inflammatory response. However, there is currently no clear medicinal report on their use in treating muscle diseases such as sarcopenia and inclusion body myositis. Therefore, developing a new medicinal use of I3C or its dimer in the above-mentioned muscle diseases has important theoretical value and application prospects. Summary of the Invention

[0004] The object of the present invention is to provide a compound of natural origin with good biological activity and safety for the treatment of sarcopenia, inclusion body myositis and other aging-related muscle diseases.

[0005] To achieve the above object, the present invention discloses a new use of I3C or its dimer DIM in the preparation of a medicament for treating muscle degenerative diseases.

[0006] The present invention is achieved by the following technical solutions:

[0007] A new use of indole-3-methanol or its dimer 3,3'-diindolylmethane in the preparation of a medicament for treating muscle degenerative diseases, said muscle degenerative diseases including sarcopenia, inclusion body myositis and their related muscle diseases.

[0008] Preferably, the indole-3-methanol or 3,3'-diindolylmethane exerts its effect by delaying the degeneration of muscle function and quality.

[0009] Preferably, the indole-3-methanol or 3,3'-diindolylmethane exerts its effect by protecting the myofibril structure and mitochondrial morphology.

[0010] Preferably, the indole-3-methanol or 3,3'-diindolylmethane exerts its effect by reducing the accumulation of amyloid deposits.

[0011] Preferably, the medicament is prepared by using I3C and DIM alone, or in combination with other active ingredients or excipients.

[0012] Preferably, the dosage form of the medicament is one of oral liquid, tablet, pill, infusion, capsule, injection, dropping pill, syrup, ointment.

[0013] Preferably, the medicament of the present invention can be administered orally, intravenously, nasally, rectally or by any other means capable of delivering an effective dose of the active substance.

[0014] Preferably, the medicament of the present invention can be administered through a pharmaceutical carrier or diluent, and can also be administered in combination with other reagents such as chemotherapy or immune activation drugs or therapeutic drugs. Suitable water-soluble organic carriers include but are not limited to cyclodextrin, corn oil, dimethyl sulfoxide (DMSO), capsules, etc.

[0015] The present invention has the following advantages compared with the prior art:

[0016] The indole-3-carbinol (I3C) and its dimer 3,3′-diindolylmethane (DIM) provided by the present invention can effectively delay the occurrence and development of sarcopenia and inclusion body myositis caused by aging. In a specific embodiment, Caenorhabditis elegans was used as a model system. After treatment with the above compounds, the muscle function of the model individuals was significantly improved, specifically manifested as the swimming speed, crawling speed, and digging speed being significantly higher than those of the control group. Further, fluorescence labeling and imaging techniques were used to analyze the myofibril structure and mitochondrial network morphology in the body wall muscles of C. elegans. The results showed that I3C and DIM could effectively maintain the integrity of myofibrils, reduce mitochondrial fragmentation, and maintain the stability of their network structure, thereby significantly improving the muscle cell mass. Secondly, I3C and DIM also delay the process of inclusion body myositis by reducing the aggregation of amyloid-β (Aβ) protein in muscle cells. In summary, I3C and its dimer DIM play a role in delaying senile muscle degeneration through multiple mechanisms, have the potential to improve muscle structure and function, and delay the onset of sarcopenia and inclusion body myositis, and are applicable to the preparation of drugs or functional dietary supplements for preventing or treating the above-related muscle diseases. Description of the Drawings

[0017] Figure 1 Shows the changes in the crawling speed of Caenorhabditis elegans in different treatment groups;

[0018] Figure 2 Shows the changes in the swimming speed of Caenorhabditis elegans in different treatment groups;

[0019] Figure 3 Shows the changes in the digging speed of Caenorhabditis elegans in different treatment groups;

[0020] Figure 4 Shows the changes in the myofibril morphology of the body wall muscles of Caenorhabditis elegans in different treatment groups;

[0021] Figure 5 Shows the changes in the mitochondrial morphology of the body wall muscles of Caenorhabditis elegans in different treatment groups;

[0022] Figure 6 Shows the aggregation of amyloid-β (Aβ) protein in the body wall muscle cells of Caenorhabditis elegans in different treatment groups. Detailed Description of the Invention

[0023] The present invention provides the use of indole-3-carbinol or its dimer 3,3′-diindolylmethane in the preparation of drugs for treating muscle degenerative diseases. The following examples are used to explain the specific details of this invention, but should not be construed as limiting the scope of the functions of this invention.

[0024] Application of I3C and DIM described in the present invention in the treatment of muscle diseases:

[0025] In the present invention, the structural formulas of I3C and DIM are as shown in Formulas (Ⅰ) and (Ⅱ).

[0026]

[0027] In the present invention, the biological model to which I3C and DIM are applied is preferably the Caenorhabditis elegans model. Caenorhabditis elegans has 60 - 80% homologous genes with humans, and at least 42% of its genes are related to human diseases. Moreover, the sarcomere, the basic functional unit of the nematode's striated muscle, is highly conserved in overall structure, composition, and function between nematodes and mammals. Functional body wall muscles are necessary for the nematode's sinusoidal movement on a semi-solid surface, its "C"-shaped swing in liquid, and its "W"-shaped movement in a gel medium. Therefore, the muscle function of nematodes is easily monitored. The optical transparency of nematodes allows for non-invasive localization of fluorescently labeled proteins or cationic dyes in mitochondria and sarcomeres, enabling visualization of these structures in vivo and prospective studies on muscle structure and function throughout the life process. Therefore, Caenorhabditis elegans is an ideal model for studying muscle diseases.

[0028] In the present invention, the treatment concentrations of I3C and DIM are the same, and both are effective doses that can significantly reduce the degree of muscle function and muscle structure degradation.

[0029] In the present invention, I3C and DIM play a good role in treating sarcopenia and inclusion body myositis-related muscle diseases by significantly improving age-related muscle function decline, protecting myofibril structure and mitochondrial morphology, and reducing amyloid deposition accumulation. The I3C and DIM of the present invention can increase the swimming speed, crawling speed, and digging speed of nematodes, effectively maintain the integrity of myofibrils, reduce mitochondrial fragmentation, and effectively reduce the aggregation of Aβ amyloid in muscle cells.

[0030] In the present invention, the drug is prepared by separately combining I3C and DIM with or without other active ingredients or excipients. The dosage forms of the drug of the present invention include oral liquid, tablets, pills, granules, capsules, injections, dripping pills, syrups, and ointments.

[0031] To fully understand the purpose, features, and effects of the present invention, the following will clearly and completely describe the present invention in combination with examples. However, they should not be construed as limiting the protection scope of the present invention. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0032] Example 1

[0033] 1. Experimental materials and experimental methods

[0034] 1.1 Experimental materials

[0035] Indole-3-carbinol I3C (purity ≥ 96%) and 3,3'-diindolylmethane DIM (purity ≥ 98%) were provided by Sigma-Aldrich. Uracil-deficient Escherichia coli (E. coli) OP50 and the Caenorhabditis elegans used were purchased from the Caenorhabditis elegans Genetics Center (CGC).

[0036] The eggs of nematodes of the same strain were randomly divided into 4 groups and placed on 4 different nematode growth media (NGM) for cultivation. They were cultivated at 20 °C until the 6th day of adulthood. Among them, the 4 media were the blank control group (OP50), the solvent control group (DMSO), the indole-3-carbinol group (I3C), and the 3,3'-diindolylmethane group (DIM). Preparation of NGM containing samples: The I3C and DIM samples were initially dissolved in dimethyl sulfoxide (DMSO) to form a stock solution, and then diluted to the required experimental concentration with E. coli OP50. At the same time, a 0.2% DMSO solution was prepared with E. coli OP50 as a solvent control. Finally, each drug working solution was spread on NGM, air-dried and sealed with a film, and stored at 4 °C for later use. To exclude the interference of offspring, the nematodes were first cultivated on normal NGM plates from eggs until the late L4 stage, and then 5-fluoro-2'-deoxyuridine (FUDR) was used to inhibit nematode egg-laying (a 0.05 g / ml FUDR stock solution was prepared with sterile water, and then the culture medium was prepared according to a 1:1000 system). The culture dish was changed every 1-2 days until the 6th day of adulthood.

[0037] 1.2 Measurement methods

[0038] 1.2.1 Crawling speed

[0039] Pick about 30 N2 wild-type nematodes and place them in a blank NGM for video recording. After the nematodes adapted for 5 min, use the WormLab nematode video acquisition system (MBF Bioscience, Williston, VT, USA) to record videos at an interval of 0.5 s / frame for 1 min, and then use the WormLab behavior analysis software (MBF Bioscience, Williston, VT, USA) to analyze the average movement speed of the nematodes according to the collected videos.

[0040] 1.2.2 Swimming speed

[0041] Pick N2 wild-type nematodes into a drop of M9 buffer on a microscope slide. After the nematodes adapted for 30 s, calculate the number of body bends within 30 s. The nematodes swim in a "C" shape. The front part swings from the left to the right and then back to the left is counted as one time. More than 15 nematodes were used under each condition, and the experiment was independently repeated 3 times.

[0042] 1.2.3 Excavation rate

[0043] At room temperature, 20 - 30 μL of M9 buffer solution was added to the center of the wells in a 12-well plate, and then approximately 30 - 35 N2 wild-type nematodes were immediately transferred into the droplets using a worm pick. Then 2 mL of Pluronic F-127 solution was added. After Pluronic F-127 was completely gelled (10 min), 2 μL of 10% methyl ethyl ketone (prepared with absolute ethanol) was spotted on the top surface, and then 20 μL of the concentrated OP50 bacterial culture was added to the dry methyl ethyl ketone. At this time, t = 0 min. The animals that burrowed into the surface layer were monitored under a microscope, and the proportion of animals on the gel surface was recorded every 15 min for 3 h. The percentage of animals on the top surface was defined as the number of animals on the top surface divided by the total sample size of the well. The experiment was independently repeated three times, and the results were expressed as the mean ± SEM of the three replicates. Two-way ANOVA analysis was used for statistical comparison of the excavation motility, and Tukey's multiple comparison test was performed.

[0044] 1.2.4 Morphology of myofibrils in body wall muscle

[0045] At least 15 nematodes of the RW1596 strain were anesthetized (1% NaN₃) and fixed on a glass slide. Images were taken using a 40× objective magnification of an upright fluorescence microscope (three biological replicates), and the fixed shooting position was the abdomen of the nematode. Normal myofibrils showed a typical parallel and dense arrangement. Myofibril damage was divided into 3 types: wavy muscle fibers, actin accumulation, and tearing or reduction of actin fibers. Damage less than 1 / 2 in the image was defined as mild damage, and more than 1 / 2 was severe damage.

[0046] 1.2.5 Morphology of mitochondria in body wall muscle cells

[0047] The changes in mitochondrial morphology were observed using the GFP-labeled fluorescence visible in the PD4251 strain of nematodes. The mitochondrial network was divided into 4 levels: normal (more than 90% of the mitochondria were in continuous parallel lines), mild (the network was disorganized with gaps but still continuous), moderate (the network was slightly fragmented, <50%), and severe (the network was severely fragmented, >50%). After the drug treatment of PD4251, at least 15 nematodes were anesthetized (1% NaN₃) and fixed on a glass slide. Images were taken using a 40× objective magnification of a fluorescence microscope, and the distribution of mitochondria with different morphologies was observed, statistically analyzed, and each experiment was repeated three times biologically.

[0048] 1.2.6 Aggregation of Aβ amyloid protein in body wall muscle

[0049] Nematodes of the CL4176 strain deposit Aβ in the body wall muscle under temperature induction 1-42, Thioflavin T binds specifically to Aβ, producing green fluorescence. CL4176 strain nematodes were cultured at 16 °C for 3 days (starting from eggs), and then subjected to a 36-hour induction at 25 °C high temperature before the staining experiment. The collected nematodes were washed 2-3 times with M9 buffer, then immediately frozen in liquid nitrogen, thawed with running water, and subjected to repeated freeze-thaw cycles 3 times (the last thawing was on ice). Subsequently, the washed nematodes were stained with 0.125% Thioflavin T in 50% ethanol (M9 buffer) for 10 minutes, and then washed with M9 buffer until the supernatant was colorless. Then, at least 10 nematodes were fixed on a glass slide for microscopic observation. The experiment was independently repeated three times.

[0050] 1.3 Data processing

[0051] All plotting and statistical analyses were performed using Prism 9.0 (GraphPad Software, San Diego, CA, USA). Unless otherwise specified, the results are expressed as the mean ± SD of three replicates, and significance was evaluated using one-way ANOVA, followed by Tukey's multiple comparison test. * indicates significant differences (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). Different letters between groups indicate statistical differences.

[0052] 2. Experimental results

[0053] 2.1 Multidimensional evaluation of I3C and DIM enhancing muscle function

[0054] The functional body wall muscles of Caenorhabditis elegans are responsible for generating the contractions and tensions necessary for movement and resistance to external forces. This muscle system generates the mechanical forces required for movement through coordinated contraction-relaxation cycles and maintains the tension balance against fluid resistance. To a certain extent, the faster the movement rate, the faster the mobilization of the nematode body wall muscles and the healthier the motor system. As Caenorhabditis elegans ages, its muscles gradually degenerate, manifested as a progressive loss of age-related muscle mass and function.

[0055] Caenorhabditis elegans has three known forms of locomotion, namely crawling on a flat plate, swimming in liquid, and burrowing in a Pluronic F-127 gel medium. Crawling is a spontaneous two-dimensional movement that relies on the coordination of muscle groups to move forward. By quantifying the crawling speed of the nematode, the state of muscle function, neural regulation, and energy metabolism can be indirectly reflected. Swimming requires intense contraction of the body wall muscles against fluid resistance and consumes more energy compared to crawling on a solid substrate. By quantifying the body swing speed of the nematode in a liquid environment, the endurance and functional integrity of the muscles can be evaluated. Pluronic F-127 gel provides higher mechanical resistance and requires additional muscle strength regulation and neuromuscular coordination. Animals with better muscle strength and neuromuscular performance will reach the gel surface faster, which is a more challenging method for evaluating locomotor endurance and neuromuscular coordination. The present invention explores these three forms respectively.

[0056] The research results show that compared with the solvent control group, supplementing I3C and DIM significantly improved the declining trend of age-related crawling ability and swimming ability of nematodes. Specifically, the crawling rates of N2 wild-type nematodes on the 6th day of adulthood increased by 14.6% (p < 0.05) and 23.5% (p < 0.001) after I3C and DIM treatments respectively ( Figure 1 ), and the swimming rates of the nematodes increased from 41.5 (bends / 30s) in the control group to 45.1 and 46.2 (bends / 30s) respectively after I3C and DIM treatments (p < 0.001) ( Figure 2 ). In addition, the present invention evaluated the effects of these two compounds on the burrowing ability of nematodes by comparing the proportion of nematodes reaching the surface in the I3C and DIM treatment groups with that in the solvent control group. The results show that the proportion of nematodes on the gel surface increased from 12.22% in the control group to 24.44% after I3C treatment, but this increase did not reach statistical significance ( Figure 3 , p > 0.05). The proportion of nematodes on the gel surface increased significantly to 28.89% after DIM treatment ( Figure 3 , p < 0.05). From the results, DIM has potential advantages in improving the burrowing ability of nematodes.

[0057] In summary, through multi-dimensional evaluation, the present invention systematically explored the effects of I3C and DIM on improving the muscle function of nematodes. The main conclusions are as follows: Both I3C and DIM can effectively improve the decline in locomotor ability related to aging, and DIM is more significant in enhancing crawling, swimming, and burrowing abilities.

[0058] 2.2 I3C and DIM significantly improve muscle mass

[0059] Myofibrils are the most basic contractile units in muscle cells. The orderly arrangement of myofibrils ensures uniform distribution and efficient conduction of force during contraction, maintaining intracellular energy transfer and signal synchronization. Disordered arrangement or breakage can lead to uneven local contraction force, thereby reducing overall muscle function. Through fluorescence image analysis of RW1596 nematodes in different treatment groups, I3C treatment significantly improved the myofibril integrity of RW1596 nematodes on the 6th day of adulthood, and the proportion of nematodes with intact myofibrils increased significantly from 37.67% in the control group to 77.08% ( Figure 4 , p<0.001). DIM treatment also showed a protective effect, increasing the proportion of nematodes with intact myofibrils to 64.69% ( Figure 4 , p<0.05). This shows that I3C and DIM can well protect the myofibril structure.

[0060] Mitochondria play a vital role in skeletal muscle cells. They not only provide the ATP required for muscle contraction, but also participate in regulating intracellular homeostasis, redox balance and apoptosis signaling pathways. Under normal circumstances, mitochondria present a highly dynamic network structure in muscle fibers, maintaining functional integrity through fusion and fission processes. However, aging leads to a decrease in mitochondrial fusion ability and enhanced mitochondrial fission, which ultimately causes mitochondria to be broken and fragmented. This morphological abnormality disrupts the exchange of substances and energy between mitochondria, affecting the energy metabolism and functional maintenance of muscle cells. Mitochondrial dysfunction is considered to be one of the important pathogenesis of a variety of muscle diseases, including sarcopenia and inclusion body myositis. The present invention found that I3C and DIM have a significant protective effect on the mitochondrial network structure of nematode body wall muscles. Specifically, after I3C treatment, the proportion of nematodes with normal mitochondrial structure increased significantly from 7.55% in the control group to 25.53% (p<0.05), while the proportion of nematodes with moderate mitochondrial damage decreased significantly from 56.73% to 21.93% (p<0.05), and the proportion of nematodes with severe mitochondrial damage decreased to 0 ( Figure 5 , p<0.05). DIM also showed the effect of improving the mitochondrial network structure, significantly increasing the proportion of nematodes with mild mitochondrial damage from 28.84% in the control group to 55.90% (p<0.05), reducing the proportion of nematodes with moderate mitochondrial damage to 23.94% (p<0.001), and reducing the proportion of nematodes with severe mitochondrial damage from 7.18% to 1.67% ( Figure 5 , p<0.05). These data suggest that I3C and DIM play a significant role in protecting the structural integrity of the mitochondrial network.

[0061] 2.3I3C and DIM significantly reduced Aβ amyloid deposition in muscle cells

[0062] In the muscle tissues of patients with inclusion body myositis, the abnormal deposition of Aβ amyloid protein is not only a typical pathological feature but also one of the key pathogenic factors inducing muscle injury and motor function decline. This protein causes the loss of muscle fiber function and then progressive movement disorders through the synergistic action of multiple mechanisms such as inducing mitochondrial dysfunction, protein metabolism disorders, and inflammatory stress. This study found that both I3C and DIM treatments could significantly inhibit the abnormal deposition of Aβ amyloid protein in the body wall muscle cells of nematodes, reducing its accumulation levels by 21.83% and 25.78% respectively( Figure 6 , p<0.05), suggesting their potential intervention effects in alleviating pathological deposition and improving muscle function.

[0063] Taken together, the above results indicate that I3C and DIM have great potential application value in delaying sarcopenia and inclusion body myositis-related muscle diseases by enhancing muscle function, improving muscle mass, and reducing the abnormal deposition of Aβ amyloid protein.

[0064] As mentioned above, the above is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent replacements or changes should be covered by the protection scope of the present invention.

Claims

1. A new use of indole-3-methanol or its dimer 3,3′-diindolylmethane in the preparation of a drug for treating muscle degenerative diseases, characterized in that, The muscle degenerative diseases include sarcopenia, inclusion body myositis and related muscle diseases.

2. The new use of indole-3-methanol or its dimer 3,3'-diindolylmethane according to claim 1 in the preparation of a medicament for treating muscle degenerative diseases, characterized in that, The indole-3-methanol or 3,3'-diindolylmethane exerts its function by delaying the degeneration of muscle function and mass.

3. A new use of indole-3-methanol or its dimer 3,3'-diindolylmethane in the preparation of a medicament for treating muscle degenerative diseases, characterized in that, The indole-3-methanol or 3,3'-diindolylmethane exerts its function by protecting the myofibril structure and mitochondrial morphology.

4. A new use of indole-3-methanol or its dimer 3,3′-diindolylmethane in the preparation of a medicament for treating muscle degenerative diseases, characterized in that, The indole-3-methanol or 3,3'-diindolylmethane exerts its function by reducing the accumulation of amyloid deposition.

5. A new use of indole-3-methanol or its dimer 3,3′-diindolylmethane in the preparation of a drug for treating muscle degenerative diseases, characterized in that, The drug is prepared by using I3C and DIM alone, or in combination with other active ingredients or excipients.

6. A new use of indole-3-methanol or its dimer 3,3'-diindolylmethane in the preparation of a drug for treating muscle degenerative diseases, characterized in that, The dosage form of the drug is one of oral liquid, tablet, pill, infusion, capsule, injection, dropping pill, syrup, and paste.

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