Use of tetrahydrocurcumin in preparation of drugs for improving cardiovascular and skeletal muscle aging

Abstract

The present application relates to the technical field of biological medicine, in order to solve the problem that there is no medicine to effectively improve the cardiovascular and skeletal muscle aging damage caused by simulated weightlessness and natural aging at present, the application of tetrahydrocurcumin in the preparation of medicine for preventing and / or improving cardiovascular and skeletal muscle aging is proposed. The present application carries out in vivo verification experiment by constructing simulated weightlessness tail suspension model and natural aging mouse model, and confirms that tetrahydrocurcumin can effectively improve the cardiac systolic function damage induced by aging and weightlessness, repair vascular structure damage, improve vascular elasticity and inhibit vascular cell aging, and at the same time significantly alleviate the loss of skeletal muscle mass, improve the skeletal muscle movement function and tissue structure damage. Molecular mechanism research shows that tetrahydrocurcumin can activate the SIRT5 protective pathway, down-regulate the expression of aging, inflammation and atrophy related genes and proteins, and inhibit the degenerative inflammation and atrophy process of the body.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of tetrahydrocurcumin in the preparation of drugs that improve cardiovascular and skeletal muscle aging. Background Technology

[0002] The unique environment of weightlessness in space and the natural aging process can both induce degenerative aging damage in multiple systems of the body, with cardiovascular function decline and skeletal muscle atrophy being the most typical. During prolonged weightlessness in space, the human body is continuously under weightlessness stress, which can lead to a series of cardiovascular pathological changes, such as decreased cardiac contractility, and disuse atrophy of skeletal muscles, significantly hindering the implementation of long-term space missions. Simultaneously, with natural aging, the body's metabolic capacity continues to decline, and the body spontaneously develops systemic degenerative diseases highly similar to those caused by weightlessness. The two types of damage have highly overlapping pathological manifestations, making it possible to simultaneously apply the same intervention target to both space weightlessness damage protection and natural aging intervention.

[0003] Currently, intervention methods for cardiovascular and skeletal muscle degenerative diseases caused by weightlessness and natural aging are extremely limited. Existing protective measures mainly rely on physical exercise and nutritional support, with limited intervention effects and poor adherence. Clinically and in existing research, there is a lack of highly effective intervention drugs that can simultaneously target and improve cardiovascular function decline, vascular aging and hardening, and skeletal muscle atrophy and aging. Most drugs can only improve cardiac function or muscle damage individually, failing to achieve synergistic protection of both the cardiovascular and skeletal muscle systems, and are difficult to adapt to the intervention needs of both the special scenarios of spaceflight weightlessness and natural aging.

[0004] Tetrahydrocurcumin (THC) is the main active metabolite of curcumin in vivo, possessing various biological activities such as antioxidant, anti-inflammatory, and anti-tumor effects. Compared to curcumin, THC exhibits higher chemical stability and bioavailability. However, whether THC can simultaneously improve cardiovascular and skeletal muscle aging-related damage caused by simulated weightlessness and natural aging has not yet been systematically reported. Summary of the Invention

[0005] To comprehensively address the above-mentioned problems, this invention aims to provide the application of tetrahydrocurcumin in the preparation of drugs that improve cardiovascular and skeletal muscle aging. It can effectively improve degenerative damage to the heart, blood vessels, and skeletal muscles caused by weightlessness stress and natural aging, providing a novel candidate drug for the protection against space weightlessness injury and the prevention and treatment of degenerative diseases caused by natural aging.

[0006] The first aspect of this invention provides the use of tetrahydrocurcumin in the preparation of medicaments for preventing and / or improving cardiovascular and skeletal muscle aging.

[0007] Furthermore, the cardiovascular aging is caused by weightlessness and / or natural aging.

[0008] Furthermore, the skeletal muscle aging is caused by weightlessness and / or natural aging.

[0009] A second aspect of the present invention provides a pharmaceutical formulation for preventing and / or improving cardiovascular and skeletal muscle aging, the pharmaceutical formulation comprising an active ingredient and pharmaceutically acceptable excipients.

[0010] Preferably, the active ingredient is tetrahydrocurcumin or at least one of its pharmaceutically acceptable salts, esters, or solvates.

[0011] Preferably, the pharmaceutically acceptable excipients include one or more of the following: excipients, binders, disintegrants, lubricants, solvents, solubilizers, flavoring agents, colorants, pH adjusters, isotonic agents, suspending agents, thickeners, preservatives, stabilizers, antioxidants, wetting agents, surfactants, suspending agents, propellants, absorption enhancers, absorption delaying agents, or coating materials.

[0012] Preferably, the dosage form of the pharmaceutical preparation includes one or more of oral dosage forms, injectable dosage forms, inhaled dosage forms, and transdermal dosage forms.

[0013] Preferably, the oral formulation is selected from any one of tablets, capsules, granules or oral liquids.

[0014] Compared with the prior art, the advantages of this invention are as follows: This invention discloses for the first time the application of the natural active ingredient tetrahydrocurcumin in the preparation of drugs to improve cardiovascular and skeletal muscle aging induced by weightlessness and natural aging. Research in this invention shows that tetrahydrocurcumin intervention can significantly improve cardiac function damage, vascular elasticity decline, and pathological vascular tissue damage induced by weightlessness stress and natural aging in mice, restore cardiovascular physiological function, and significantly inhibit the accumulation of aging in vascular cells. Simultaneously, it can effectively improve the skeletal muscle atrophy phenotype, alleviate skeletal muscle mass loss, muscle strength decline, and muscle fiber structure damage, and downregulate the abnormal expression of aging, inflammation, and muscle atrophy-related factors. Molecular-level studies show that THC can significantly downregulate the mRNA and protein expression levels of aging markers P16 and P53, the pro-inflammatory factor IL-6, and muscle atrophy-related genes MuRF1 (Trim63) and Fbxo32 in muscle tissue; while simultaneously upregulating the expression of anti-aging-related silencing information regulator 5 (SIRT5). This application is the first to demonstrate that tetrahydrocurcumin can synergistically improve cardiovascular function and skeletal muscle mass by inhibiting inflammation, downregulating aging and atrophy-related pathways, and activating SIRT5 anti-aging signaling, providing a novel natural source drug for weightlessness medical protection and chemoprevention of age-related cardiovascular degeneration and sarcopenia. Attached Figure Description

[0015] Figure 1 Schematic diagram of the effect of tetrahydrocurcumin on cardiac function (echocardiography) in a tail-suspended mouse model. Ctrl, control mice; HU4w, tail-suspended simulated weightlessness mice; mice subjected to weightlessness under treatment with tetrahydrocurcumin at a concentration of HU4w+90THC, 90 mg / kg / d; mice subjected to weightlessness under treatment with tetrahydrocurcumin at a concentration of HU4w+120THC, 120 mg / kg / d. A: Comparison of mouse echocardiograms; B: Statistical graph of left ventricular ejection fraction (EF) measured in mouse echocardiography, n=8; C: Statistical graph of short axis shortening (FS) measured in mouse echocardiography, n=8. ***P<0.001 vs. Con(mean±SEM).

[0016] Figure 2 Schematic diagram of the effect of tetrahydrocurcumin on cardiac function (echocardiography) in naturally aging mice. Ctrl, 8-week-old control mice; Old, 24-month-old naturally aging mice; Old+THC, naturally aging mice treated with tetrahydrocurcumin. A: Comparison of mouse echocardiograms; B: Statistical graph of left ventricular ejection fraction (EF) measured by echocardiography in mice, n=6; C: Statistical graph of short axis shortening (FS) measured by echocardiography in mice, n=6. ***P<0.001 vs. Con(mean±SEM).

[0017] Figure 3 Schematic diagram of the effect of tetrahydrocurcumin on pulse wave velocity (PWV) in a tail-suspended mouse model. Ctrl, control mice; HU4w, tail-suspended simulated weightlessness mice; mice under treatment with tetrahydrocurcumin at a concentration of 90 mg / kg / d (HU4w + 90 THC); mice under treatment with tetrahydrocurcumin at a concentration of 120 mg / kg / d (HU4w + 120 THC). A: Comparison of aortic pulse wave velocity (PWV) in mice; B: Statistical graph of aortic pulse wave velocity (PWV), n=8; C: Statistical graph of left common carotid artery pulse wave velocity (PWV), n=8. *P<0.05, **P<0.01, ***P<0.001 vs. Con (mean±SEM).

[0018] Figure 4 Yes; Schematic diagram of the effect of tetrahydrocurcumin on pulse wave velocity (PWV) in naturally aging mice. Ctrl, 8-week-old control mice; Old, 24-month-old naturally aging mice; Old+THC, naturally aging mice treated with tetrahydrocurcumin. A: Comparison of pulse wave velocity (PWV) in the aorta of mice; B: Statistical graph of pulse wave velocity (PWV) in the aorta, n=6. **P<0.01 vs. Con(mean±SEM).

[0019] Figure 5 Yes; Schematic diagram of the effect of tetrahydrocurcumin on the morphology (HE staining) of aortic tissue in a suspended tail model mouse. Ctrl, control mice; HU4w, suspended tail simulated weightlessness model mice; mice under treatment with tetrahydrocurcumin at a concentration of HU4w+90THC, 90 mg / kg / d; mice under treatment with tetrahydrocurcumin at a concentration of HU4w+120THC, 120 mg / kg / d. A: Comparison of HE staining of mouse aorta; B: Statistical graph of HE staining of mouse aorta, n=6. *P<0.05, **P<0.01, ***P<0.001 vs. Con (mean±SEM).

[0020] Figure 6 Yes; Schematic diagram of the effect of tetrahydrocurcumin on the morphology (HE staining) of aortic tissue in naturally aging mice. Ctrl, 8-week-old control mice; Old, 24-month-old naturally aging mice; Old+THC, naturally aging mice treated with tetrahydrocurcumin. A: Comparison of HE staining in mouse aorta; B: Statistical graph of HE staining in mouse aorta, n=6. *P<0.05, **P<0.01, ***P<0.001 vs. Con (mean±SEM).

[0021] Figure 7 Yes; Schematic diagram of the effect of tetrahydrocurcumin on the activity of senescence-related β-galactosidase in the aortic tissue of a suspended tail model mice. Ctrl, control mice; HU4w, mice in a suspended tail simulated weightlessness model; mice in weightlessness treated with tetrahydrocurcumin at a concentration of HU4w+90THC, 90 mg / kg / d; mice in weightlessness treated with tetrahydrocurcumin at a concentration of HU4w+120THC, 120 mg / kg / d.

[0022] Figure 8 Yes; Schematic diagram of the effect of tetrahydrocurcumin on skeletal muscle mass in tail-suspended mouse model. Ctrl, control group mice; HU4w, tail-suspended simulated weightlessness mice; mice subjected to weightlessness under treatment with tetrahydrocurcumin at a concentration of HU4w+90THC, 90 mg / kg / d; mice subjected to weightlessness under treatment with tetrahydrocurcumin at a concentration of HU4w+120THC, 120 mg / kg / d. A: Comparison of mouse soleus muscle mass; B: Statistical graph of mouse gastrocnemius muscle mass, n=10; C: Statistical graph of the ratio of mouse gastrocnemius muscle mass to tibia length, n=8; D: Statistical graph of mouse soleus muscle mass, n=10; E: Statistical graph of the ratio of mouse soleus muscle mass to tibia length, n=8. **P<0.01, ***P<0.001 vs. Con(mean±SEM).

[0023] Figure 9Yes; Schematic diagram of the effect of tetrahydrocurcumin on skeletal muscle mass in naturally aging mice. Ctrl, 8-week-old control mice; Old, 24-month-old naturally aging mice; Old+THC, naturally aging mice treated with tetrahydrocurcumin. A: Statistical graph of gastrocnemius muscle mass in mice, n=6; B: Statistical graph of the ratio of gastrocnemius muscle mass to tibia length in mice, n=6; C: Statistical graph of soleus muscle mass in mice, n=6; D: Statistical graph of the ratio of soleus muscle mass to tibia length in mice, n=6; E: Grasping force value in mouse gripping test, n=6; F: Grasping force-to-gravity ratio in mouse gripping test, n=6. *P<0.05, **P<0.01, ***P<0.001 vs. Con(mean±SEM).

[0024] Figure 10 Yes; Schematic diagram of the effect of tetrahydrocurcumin on the morphology (HE staining) of gastrocnemius muscle tissue in a tail-suspended mouse model. Ctrl, control mice; HU4w, tail-suspended simulated weightlessness mice; mice under treatment with tetrahydrocurcumin at a concentration of HU4w+90THC, 90 mg / kg / d; mice under treatment with tetrahydrocurcumin at a concentration of HU4w+120THC, 120 mg / kg / d. A: Comparison of HE staining of mouse gastrocnemius muscle; B: Statistical graph of average muscle area of ​​mouse gastrocnemius muscle stained with HE, n=6. *P<0.05, **P<0.01, ***P<0.001 vs. Con (mean±SEM).

[0025] Figure 11 Yes; Schematic diagram of the effect of tetrahydrocurcumin on the morphology (HE staining) of gastrocnemius muscle tissue in naturally aging mice. Ctrl, 8-week-old control mice; Old, 24-month-old naturally aging mice; Old+THC, naturally aging mice treated with tetrahydrocurcumin. A: Comparison of HE staining of mouse gastrocnemius muscle; B: Statistical graph of average muscle area of ​​mouse gastrocnemius muscle stained with HE, n=6. *P<0.05, **P<0.01, ***P<0.001 vs. Con (mean±SEM).

[0026] Figure 12Yes; Schematic diagram of the effect of tetrahydrocurcumin on the expression of aging and atrophy-related genes (PCR) in the muscles of tail-suspended mice. Ctrl, control mice; HU4w, tail-suspended simulated weightlessness mice; mice subjected to weightlessness under treatment with tetrahydrocurcumin at a concentration of HU4w+90THC, 90 mg / kg / d; mice subjected to weightlessness under treatment with tetrahydrocurcumin at a concentration of HU4w+120THC, 120 mg / kg / d. AF: RNA levels of pro-inflammatory factors IL-6 and IL-8, aging markers P16 and P53, silencing information regulator 5SIRT5, and muscle atrophy marker MuRF1 in mouse gastrocnemius muscle, n=4. *P<0.05, **P<0.01, ***P<0.001 vs. Con (mean±SEM).

[0027] Figure 13 Yes; Schematic diagram of the effect of tetrahydrocurcumin on the expression of aging and atrophy-related proteins in the muscles of tail-suspended mice (Western Blot). Ctrl, control mice; HU4w, tail-suspended simulated weightlessness mice; mice under treatment with tetrahydrocurcumin at a concentration of HU4w+90THC, 90 mg / kg / d; mice under treatment with tetrahydrocurcumin at a concentration of HU4w+120THC, 120 mg / kg / d. A: Western blot results of mouse gastrocnemius muscle, n=4; BF: aging markers P53 and P16, muscle atrophy molecules MuRF1 (Trim63) and Fbxo32, and silencing information regulator 5SIRT5 protein levels in mouse gastrocnemius muscle, n=4. *P<0.05, **P<0.01, ***P<0.001 vs. Con (mean±SEM). Detailed Implementation

[0028] The following is in conjunction with embodiments of the present invention. Figures 1-13 It should be understood that the preferred embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0029] Example 1: Application of tetrahydrocurcumin in the preparation of drugs for preventing and / or improving cardiovascular and skeletal muscle aging. Wherein, cardiovascular aging is caused by weight loss and / or natural aging; skeletal muscle aging is caused by weight loss and / or natural aging.

[0030] Example 2: A pharmaceutical formulation for preventing and / or improving cardiovascular and skeletal muscle aging, said pharmaceutical formulation comprising an active ingredient and pharmaceutically acceptable excipients.

[0031] The active ingredient is tetrahydrocurcumin or at least one of its pharmaceutically acceptable salts, esters, or solvates.

[0032] The pharmaceutically acceptable excipients include one or more of the following: excipients, binders, disintegrants, lubricants, solvents, solubilizers, flavoring agents, colorants, pH adjusters, isotonic agents, suspending agents, thickeners, preservatives, stabilizers, antioxidants, wetting agents, surfactants, suspending agents, propellants, absorption enhancers, absorption delayers, or coating materials.

[0033] The dosage form of the pharmaceutical preparation includes one or more of the following: oral dosage form, injection dosage form, inhalation dosage form, and transdermal dosage form.

[0034] The oral preparation is selected from any one of tablets, capsules, granules or oral liquids.

[0035] The daily dose of the oral formulation is 50 mg to 150 mg per kilogram of body weight, preferably 90 mg / kg / day or 120 mg / kg / day, and can be flexibly adjusted according to the intervention scenario, degree of aging and dosing cycle.

[0036] Animal experiments: Unless otherwise specified, the experimental methods described in the examples of animal experiments herein are conventional methods, and the reagents and biological materials mentioned herein are commercially available unless otherwise specified.

[0037] 1. Materials and Methods 1.1 Laboratory animals and their housing conditions This experiment used 50 healthy male C57BL / 6J mice (purchased from the Animal Experiment Center of Air Force Medical University) and housed them in standard animal housing environments, maintaining a 12h light / 12h dark circadian rhythm, an ambient temperature of 22±2℃, and a humidity of 50%±10%. The mice had free access to food and water. After one week of acclimatization, the modeling and drug administration experiments were carried out.

[0038] 1.2 Animal Model Construction and Dosing Regimen This invention sets up two animal models: a simulated weightlessness tail suspension model and a natural aging model, which correspond to the space weightlessness injury scenario and the natural aging scenario of human beings, respectively.

[0039] 1.2.1 Simulation of Weightlessness and Suspended Tail Model: Healthy 14-week-old SPF-grade male C57BL / 6J mice were randomly divided into four groups (n=8 per group) after one week of acclimatization: control group (Ctrl), tail suspension model group (HU4w), tail suspension + low-dose THC group (HU4w+90THC), and tail suspension + high-dose THC group (HU4w+120THC). Except for the control group (Ctrl), all other groups were suspended by tail suspension to simulate hind limb stress injury caused by weightlessness in space. From day 1 of modeling, the tail suspension + low-dose THC group (HU4w+90THC) and the tail suspension + high-dose THC group (HU4w+120THC) were administered tetrahydrocurcumin solution by gavage at fixed times daily, at doses of 90 mg / kg / day and 120 mg / kg / day, respectively. The control group (Ctrl) and the tail suspension model group (HU4w) were administered an equal volume of 0.5% physiological saline by gavage. This intervention lasted for four weeks.

[0040] 1.2.2 Natural Aging Model: Eight-week-old young C57BL / 6J mice were used as the control group (Ctrl), and 24-month-old aged C57BL / 6J mice were used as a natural aging model. The aged mice were randomly divided into a natural aging group (Old) and an aging + THC treatment group (Old+THC), with six mice in each group. The aging + THC treatment group (Old+THC) was administered 120 mg / kg / day of tetrahydrocurcumin solution by gavage at a fixed time every day, while the control group (Ctrl) and the natural aging group (Old) were administered an equal volume of 0.5% physiological saline by gavage. The intervention was carried out for 8 consecutive weeks.

[0041] 2. Detection Method 2.1 Ultrasound detection of cardiac function in mice Mouse cardiac function data were acquired using a Vevo 3100 high-resolution imaging system (purchased from VisualSonics, Canada). Mice were shaved of hair from the neck and chest and then fixed in a supine position on a heated plate (maintaining body temperature of 37±0.5℃). They were anesthetized with isoflurane inhalation (induction 4-5%, maintenance 1.5-2%). After applying ultrasound gel, the depth and gain were adjusted to ensure clear and sharp endocardial boundaries. A stable heart rate was maintained (400-600 beats / min for normal mice). M-mode images of the left ventricle's long and short axes were acquired using the probe. Left ventricular ejection fraction (EF%) and fractional shortening (FS%) were measured. Each parameter was measured continuously for three cardiac cycles, and the average value was taken. The results are shown below. Figure 1 , Figure 2 As shown.

[0042] 2.2 Detection of vascular pulse wave propagation velocity Pulse wave velocity (PWV) was acquired using a Vevo 3100 high-resolution imaging system (purchased from VisualSonics, Canada). Mice were shaved and fixed in a supine position with their necks fully exposed. A constant-temperature heating plate was used to maintain the mice's normal body temperature. Mice were anesthetized with isoflurane inhalation (induction 4-5%, maintenance 1.5-2%). Ultrasonic gel was applied to the neck, the probe position was fixed, and the pulse wave conduction time and distance in the aorta and carotid arteries were measured. The pulse wave velocity was calculated using the formula PWV = ΔL / Δt, where ΔL is the arterial distance between the two measurement points, and Δt is the pulse wave conduction time. The results are shown below. Figure 3 , Figure 4 As shown.

[0043] 2.3 HE staining of vascular tissue Mouse aortic and carotid artery tissues were isolated, fixed in 4% paraformaldehyde, dehydrated in a gradient manner, cleared, impregnated with paraffin, and embedded in paraffin to prepare serial tissue sections. The sections were dewaxed to water, stained with hematoxylin and eosin, dehydrated in a gradient manner, cleared with xylene, and mounted with neutral resin. Changes in vascular tissue structure and medial thickness were observed under a microscope to evaluate vascular structural damage and repair. The results are shown below. Figure 5 , Figure 6 As shown.

[0044] 2.4 SA-β-gal aging staining detection in vascular tissue After anesthetizing and fixing the mice, the chest was quickly opened to fully expose the heart and aortic arch. The aorta and bilateral common carotid arteries were completely dissected, and the fat and connective tissue attached to the perivascular area were carefully removed. The dissected vascular tissue was rinsed in pre-cooled PBS buffer to remove residual blood, trimmed to an appropriate length, and then placed in cryovials for storage at -80°C for later use.

[0045] OCT embedding was performed on the vascular tissue to be tested. The vascular tissue was placed vertically in the embedding medium, ensuring that the vascular ring structure was intact and facing the section direction. After replenishing the embedding medium to completely cover the tissue, it was quick-frozen at -80℃ for shaping. The cryostat was set to a working temperature of -20℃. The quick-frozen tissue block was trimmed, and after the intact vascular tissue was exposed, continuous frozen sections with a thickness of 8-10 μm were prepared. The intact sections were collected using pre-cooled glass slides for later use.

[0046] After preparing the frozen sections of blood vessels, thaw them at room temperature for 10 min. Use a histochemical pen to draw circles along the tissue edges to define the staining area. Add an appropriate amount of β-galactosidase staining fixative to the tissue surface, ensuring complete coverage, and fix at room temperature for 20 min. After fixation, wash the sections three times with PBS buffer, 5 min each time. After washing, place the sections in a light-protected humidified chamber, add sufficient β-galactosidase staining working solution to completely cover the tissue, and incubate at a constant temperature of 37°C in the dark until senescent vascular cells show specific staining.

[0047] After staining, the staining working solution was discarded, and the tissue was thoroughly washed with PBS buffer, followed by gradient dehydration with anhydrous ethanol, clearing with xylene, and finally mounting with neutral resin. The vascular tissues of each group of mice were observed using an optical microscope to analyze the distribution and staining of senescent positive cells, thereby evaluating the degree of cellular senescence in the vascular tissues. The results are as follows: Figure 7 As shown.

[0048] 2.5 Skeletal muscle mass and strength testing The skin and subcutaneous connective tissue of the mouse hind limbs were dissected layer by layer to fully expose the anatomical structures of the gastrocnemius and soleus muscles. The gastrocnemius and soleus muscles were completely dissected according to their origin and insertion points, and surface fat and adhering connective tissue were removed. The absolute wet weight (mg) of the gastrocnemius and soleus muscles was measured using an analytical balance. Combined with the mouse tibia length, the relative wet weight (mg) of the gastrocnemius / tibia length ratio (mg / mm) and soleus / tibia length ratio (mg / mm) were calculated to obtain two groups of relative wet weights (mg) to evaluate the degree of skeletal muscle atrophy and loss. Simultaneously, a small animal muscle strength testing system was used to perform limb grip strength tests on mice, obtaining the maximum grip strength and grip strength-to-gravity ratio to comprehensively evaluate the overall functional status of skeletal muscles. The test results for each group are shown below. Figure 8 , Figure 9 As shown.

[0049] 2.6 HE staining of skeletal muscle tissue After the isolated soleus and gastrocnemius muscle tissues were fixed in muscle fixative for 24 hours, they underwent sequential dehydration, tissue clearing, and paraffin embedding to prepare serial paraffin tissue sections. The sections were then routinely dewaxed to water, stained with hematoxylin and iodine for nuclear staining, bluing with tap water, and stained with eosin for cytoplasmic staining. They were then dehydrated using a series of ethanol solutions, cleared with xylene, and finally mounted with neutral resin to prepare permanent sections. Microscopic observation was performed on the regularity of muscle fiber arrangement, muscle fiber integrity, and tissue structure morphology. The average cross-sectional area of ​​muscle fibers was statistically analyzed to systematically assess skeletal muscle atrophy, damage, and repair. The results are as follows: Figure 10 , Figure 11 As shown.

[0050] 2.7 Detection of gene expression related to skeletal muscle aging, inflammation and atrophy 2.7.1 Sample Pretreatment and Total RNA Extraction 30–50 mg of mouse gastrocnemius muscle tissue was collected and processed entirely on ice. The tissue was minced into small pieces of 1–2 mm³ using ophthalmic scissors. RNA was extracted from the samples using the RNAsimple kit (purchased from Beijing Tiangen Biotech Co., Ltd.). 1 mL of pre-chilled RZ lysis buffer was added to each tissue piece, and the tissue was thoroughly homogenized using a homogenizer (purchased from Shanghai Jingxin Industrial Development Co., Ltd.). The homogenate was incubated at room temperature for 5 min to allow for complete dissociation of the nucleic acid-protein complex. Then, 200 μL of chloroform was added, and the mixture was vigorously vortexed for 15 s, incubated at room temperature for 3 min, and then centrifuged at 4°C and 12000 rpm for 10 min. After centrifugation, the sample separated into layers, with RNA concentrated in the upper colorless aqueous phase. The upper aqueous phase was carefully transferred to a brand new enzyme-free centrifuge tube, strictly avoiding contact with the middle layer to prevent DNA contamination. Add 0.5 times the volume of anhydrous ethanol to the aqueous phase solution and mix thoroughly. Transfer the mixture and the precipitated white precipitate into a CR3 adsorption column and centrifuge at 12000 rpm for 30 seconds at 4°C to ensure complete RNA adsorption onto the column membrane. Wash the adsorption column repeatedly with protein removal buffer RD and wash buffer RW to effectively remove tissue proteins, salts, and residual impurities. Finally, centrifuge at high speed for 2 minutes to thoroughly remove residual wash buffer. Add [RNA] dropwise to the center of the adsorption column membrane. After standing at room temperature for 2 minutes, the tissue was centrifuged and eluted to obtain total RNA from skeletal muscle tissue. The concentration was measured and the tissue was stored at -80℃.

[0051] 2.7.2 Genomic DNA Removal and Reverse Transcription to Synthesize cDNA RNA reverse transcription was performed using the EvoM-MLV reverse transcription premix kit (purchased from Hunan Aikerui Biotechnology Co., Ltd.). A 10 μl system was selected based on RNA concentration. 2 μg of DNACleanReactionMixVer.2 was added, along with RNA and RNase-free reagents. The mixture was incubated at 42℃ for 2 min, followed by 5 cycles at 4℃. Subsequently, 4 μl of 5×EvoM-MLVRTReactionMixVer.2 and 6 μL of RNase-free reagents were added to the reaction solution from the previous step and mixed thoroughly. The mixture was incubated at 37℃ for 15 min, then at 85℃ for 5 s, followed by 4 cycles at 4℃ for reverse transcription. Rapid synthesis and stable termination of the reaction yielded a highly pure cDNA template, which could be directly used for subsequent quantitative PCR amplification experiments.

[0052] 2.7.3 Real-time quantitative PCR detection and data analysis The SYBR Green ProTaq HS premixed qPCR kit (purchased from Hunan Aike Rui Biotechnology Co., Ltd.) was used to conduct real-time quantitative PCR experiments using the prepared cDNA as amplification template. The relative mRNA expression levels of aging marker genes P16 and P53, inflammatory cytokine genes IL-6, IL-1β, and IL-8, key muscle atrophy genes MuRF1 (Trim63) and Fbxo32, and protective regulatory gene SIRT5 in mouse skeletal muscle tissue of each group were detected. Stable-expressing internal reference genes were selected to calibrate the sample loading error of each group. Data analysis was performed using a relative quantification method (2^-ΔΔCt method). The calculation formula was: ΔCt = Ct(target gene) - Ct(internal reference gene), ΔΔCt = ΔCt(experimental group) - ΔCt(control group), and relative expression level = 2^(-ΔΔCt). The relative expression level of each target gene was finally calculated. By comparing and analyzing the differences in gene transcription levels among different groups, this study elucidates the molecular mechanism by which tetrahydrocurcumin regulates skeletal muscle aging, inflammatory infiltration, and atrophy. Specific experimental results are as follows: Figure 12 As shown.

[0053] 2.8 Detection of protein expression related to skeletal muscle aging and atrophy 2.8.1 Extraction of total protein from tissues Approximately 30 mg of mouse muscle tissue was collected and processed entirely on ice. The tissue was minced into small pieces of 1–2 mm³ using ophthalmic scissors. Pre-chilled RIPA lysis buffer containing protease inhibitors was prepared in advance at a ratio of 1:100. 500 μL of this lysis buffer was added to the tissue pieces. Low-temperature homogenization was performed using a tissue homogenizer, with homogenization parameters set at 4°C for 45 seconds, followed by a 10-second interval, repeated 8 times to ensure complete tissue lysis. After homogenization, the sample was placed on ice for 30 minutes to allow for the release of total tissue protein. Subsequently, the sample was centrifuged at 4°C and 12,000 rpm for 15 minutes. The clear supernatant was carefully transferred to a new centrifuge tube, avoiding sedimentation at the bottom. The resulting protein sample was stored at -80°C for later use.

[0054] 2.8.2 BCA method for protein concentration quantification Protein concentrations in each group of samples were determined using the BCA protein quantification method. 5 μL of the protein sample was diluted 10-fold with RIPA lysis buffer containing a protease inhibitor. Freshly prepared BCA working solution (A:B = 50:1) was added to each well of a 96-well plate along with 20 μL of standard and diluted sample. 200 μL of BCA working solution was then added to each well. The plates were incubated at 37°C for 30 minutes in the dark. After incubation, the absorbance at 562 nm was measured using a microplate reader. A standard curve was plotted based on the standard concentrations and corresponding absorbance values ​​to calculate the actual protein concentrations in each group, providing a basis for consistent protein loading ratios in subsequent tests.

[0055] 2.8.3 Gel electrophoresis separation Prepare gels of appropriate concentrations according to the molecular weight of the target protein, assemble the electrophoresis tank, and add sufficient electrophoresis buffer. Load samples sequentially, adding 3 μL of protein marker and 5 μL of denatured protein sample to each well. Electrophoresis is performed in a segmented constant-voltage mode. The stacking gel stage is set to 80V. Once the sample bands are compressed and aligned and enter the separating gel, the voltage is adjusted to 120V until the protein bands migrate to the bottom of the gel, at which point electrophoresis is terminated.

[0056] 2.8.4 Transfer Treatment After activating the PVDF membrane with methanol for 15 seconds, equilibrate it in transfer buffer for 5 minutes. After electrophoresis, remove the gel and equilibrate it in transfer buffer for 10 minutes. Completely immerse the transfer clip, filter paper, and sponge in pre-cooled transfer buffer. Assemble the transfer "sandwich" structure in the order of sponge-filter paper-gel-PVDF membrane-filter paper-sponge, ensuring all air bubbles are removed between layers to avoid affecting protein transfer. Place the assembled transfer clip in the transfer tank and use a 300mA constant current mode. Adjust the ice bath transfer time according to the target protein molecular weight to complete the protein transfer from the gel to the PVDF membrane.

[0057] 2.8.5 Blocking and Antibody Incubation After transfer, remove the PVDF membrane and place it in 5% skim milk blocking buffer. Block on a shaker at low speed for 2 hours at room temperature to block non-specific binding sites on the membrane. After blocking, discard the blocking buffer and wash the membrane three times with TBST buffer for 10 minutes each time to thoroughly remove residual blocking buffer. Dilute the primary antibody according to the antibody instructions and immerse the PVDF membrane in the diluted primary antibody working solution. Incubate overnight at 4°C with low speed on a shaker. After primary antibody incubation, wash the membrane thoroughly three times with TBST buffer for 10 minutes each time. Select an HRP-labeled secondary antibody according to the species matching of the primary antibody and dilute it at a ratio of 1:2000 to 1:5000 to prepare the secondary antibody working solution. Incubate on a shaker at room temperature for 1 hour. After incubation, wash three more times with TBST buffer for 10 minutes each time.

[0058] 2.8.6 Chemiluminescence Imaging Detection Mix ECL chemiluminescence solution A and solution B in a 1:1 volume ratio, and use immediately. Place the washed PVDF membrane face up on an imaging plate, and evenly add sufficient chemiluminescence solution to completely cover the membrane surface. Incubate at room temperature in the dark for 1–2 minutes. Acquire protein band images using a chemiluminescence imaging system. Detect the grayscale value of each target protein band using image analysis software, calibrate with the internal control protein grayscale value, and calculate the relative expression level of each target protein. Analyze the regulatory effect of tetrahydrocurcumin on the expression of proteins related to weightlessness-induced skeletal muscle injury. Experimental results are as follows: Figure 13 As shown.

[0059] 2.9 Statistical Analysis All data in this study are expressed as mean ± standard error (mean ± SEM) and were analyzed using one-way ANOVA with GraphPad Prism 10.0 software. Differences between groups were considered statistically significant as *P < 0.05.

[0060] 3. Experimental Results 3.1 Tetrahydrocurcumin significantly improves cardiac function damage caused by weight loss and natural aging. To investigate the therapeutic effects of tetrahydrocurcumin on cardiac function damage caused by weightlessness and natural aging, this invention conducted in vivo intervention experiments using a simulated weightlessness tail suspension model and a naturally aging mouse model.

[0061] For the simulated weightlessness tail model: such as Figure 1The AC results showed that, compared with the blank control group (Ctrl), the left ventricular ejection fraction (EF%) and left ventricular fractional shortening (FS%) of mice in the tail suspension model group (HU4w) were significantly reduced, indicating that weightlessness stress can lead to a decline in cardiac contractile function in mice and induce degenerative damage to cardiac function. After intervention with different doses of tetrahydrocurcumin, the EF% and FS% of mice in the tail suspension + low-dose THC group (HU4w + 90THC) and the tail suspension + high-dose THC group (HU4w + 120THC) were significantly improved compared with those in the tail suspension model group (HU4w), effectively improving the cardiac contractile function damage caused by weightlessness stress.

[0062] For natural aging models: such as Figure 2 The AC results showed that, compared with the control group (Ctrl), the cardiac EF% and FS% of naturally aging mice (Old) were significantly reduced, confirming that the natural aging process can be accompanied by a significant decline in cardiac pumping function and a decrease in cardiac contractility. After treatment with tetrahydrocurcumin, cardiac function-related indicators of mice in the aging + THC treatment group (Old + THC) were significantly improved, effectively alleviating the degenerative damage to cardiac function caused by natural aging.

[0063] The above results indicate that tetrahydrocurcumin can effectively reverse both weightlessness stress and age-induced cardiac function decline, and has a significant protective and improving effect on both weightlessness simulation and age-related cardiovascular function damage.

[0064] 3.2 Tetrahydrocurcumin improves vascular elasticity, repairs vascular tissue structure, and inhibits vascular cell aging. To further investigate the intervention effect of tetrahydrocurcumin on vascular aging damage caused by weightlessness and natural aging, this invention tested the vascular elasticity function, vascular tissue structure and morphology, and vascular cell aging level in mice.

[0065] For the simulated weightlessness tail model: by Figure 3 The AC results showed that, compared with the control group (Ctrl), the vascular pulse wave conduction velocity of mice in the tail suspension model group (HU4w) was significantly increased, exhibiting typical pathological characteristics of decreased vascular elasticity and vascular sclerosis and aging; HE staining results of vascular tissue showed (see...) Figure 5In the AB group (tail suspension model group, HU4w), the blood vessels of mice showed significant damage including tunica media thickening, tissue structure disorder, and pathological remodeling. After intervention with tetrahydrocurcumin, the pulse wave velocity of blood vessels in mice in the tail suspension + low-dose THC group (HU4w + 90THC) and the tail suspension + high-dose THC group (HU4w + 120THC) significantly decreased, effectively improving vascular elasticity decline and arteriosclerosis. Simultaneously, it significantly alleviated tunica media thickening and structural disorder, repaired pathological damage to vascular tissue structure caused by weightlessness stress, and significantly reduced the number of SA-β-gal senescent positive cells in blood vessels, inhibiting abnormal senescence of vascular cells (see [link to article]). Figure 7 ).

[0066] For natural aging models: by Figure 4 AB in Figure 6 The A / B results showed that, compared with the control group (Ctrl), the naturally aging group (Old) mice exhibited significantly increased pulse wave velocity, significantly decreased vascular elasticity, and obvious signs of vascular sclerosis and aging. The vascular tissue morphology and structure showed aging damage characteristics such as tunica media thickening, disordered arrangement, and pathological remodeling. After tetrahydrocurcumin intervention, the aging + THC treatment group (Old + THC) mice showed significantly reduced vascular pulse wave velocity, effective recovery of vascular elasticity, significant improvement in pathological damage to vascular tissue structure, and effective repair of vascular tissue morphology, effectively inhibiting the aging process of vascular cells induced by natural aging.

[0067] The above results indicate that tetrahydrocurcumin can improve vascular degenerative damage induced by weightlessness stress and natural aging from multiple levels, including improving vascular function, repairing tissue structure, and inhibiting cell aging, and has a significant protective and repairing effect on vascular aging.

[0068] 3.3 Tetrahydrocurcumin alleviates skeletal muscle mass loss caused by weight loss and natural aging, and improves skeletal muscle motor function. Weight loss and natural aging can both induce skeletal muscle degenerative atrophy. To verify the protective effect of tetrahydrocurcumin on skeletal muscle phenotype and function, this invention tested the wet weight of mouse skeletal muscle, relative muscle ratio, and limb muscle strength indices.

[0069] For the simulated weightlessness tail model: by Figure 8The results of the AE study showed that, compared with the control group (Ctrl), the wet weight and relative muscle ratio of the gastrocnemius and soleus muscles in the tail suspension model group (HU4w) were significantly reduced, indicating that weightlessness stress can induce significant skeletal muscle disuse atrophy, resulting in muscle loss and limb motor function decline. After intervention with different doses of tetrahydrocurcumin, the wet weight and relative muscle ratio of skeletal muscle in the tail suspension + low-dose THC group (HU4w + 90THC) and the tail suspension + high-dose THC group (HU4w + 120THC) significantly recovered, and limb muscle strength was effectively improved, which can significantly antagonize the weightlessness stress-induced skeletal muscle disuse atrophy and motor function impairment.

[0070] For natural aging models: by Figure 9 The AF results showed that, compared with the control group (Ctrl), the naturally aged group (Old) mice exhibited significant weight loss in the gastrocnemius and soleus muscles, with significantly reduced muscle wet weight and relative muscle ratio, and severe limb muscle weakness, confirming that natural aging induces typical age-related skeletal muscle atrophy and degenerative decline in motor function. After tetrahydrocurcumin intervention, the skeletal muscle mass of the aged + THC treatment group (Old + THC) mice was effectively restored, the relative muscle ratio was significantly increased, and limb motor function was significantly improved, effectively alleviating skeletal muscle atrophy and degeneration caused by natural aging.

[0071] The above results indicate that tetrahydrocurcumin can simultaneously improve the loss of skeletal muscle mass and decreased muscle strength caused by weightlessness and natural aging, effectively protect against skeletal muscle atrophy and motor function damage caused by weightlessness and natural aging, and maintain normal skeletal muscle morphology and motor function.

[0072] 3.4 Tetrahydrocurcumin improves skeletal muscle tissue structural damage caused by weight loss and natural aging. To further verify the effect of tetrahydrocurcumin on improving skeletal muscle aging and atrophy from the perspective of tissue morphology, this invention observed the changes in skeletal muscle fiber tissue structure in mice of each group by HE staining.

[0073] For the simulated weightlessness tail model: by Figure 10 The A / B results showed that, compared with the control group (Ctrl), the skeletal muscle tissue of mice in the tail suspension model group (HU4w) exhibited significant pathological damage, mainly manifested as loose and disordered muscle fiber arrangement, increased gaps, and a significant reduction in muscle fiber cross-sectional area, presenting typical characteristics of disuse-induced muscle fiber atrophy. After intervention with different doses of tetrahydrocurcumin, the skeletal muscle fibers of mice in the tail suspension + low-dose THC group (HU4w + 90THC) and the tail suspension + high-dose THC group (HU4w + 120THC) were more regularly and tightly arranged, with reduced gaps between muscle fibers, and the muscle fiber atrophy was significantly alleviated, effectively repairing the structural damage to skeletal muscle tissue induced by weightlessness stress.

[0074] For natural aging models: by Figure 11 The A / B results showed that, compared with the control group (Ctrl), the naturally aging group (Old) mice exhibited significant skeletal muscle tissue damage, with irregular muscle fiber morphology, disordered arrangement, significantly widened interfiber spaces, and a substantial decrease in muscle fiber cross-sectional area, confirming that natural aging can induce severe degenerative changes in skeletal muscle tissue structure. After treatment with tetrahydrocurcumin, the skeletal muscle fiber structure integrity of the aging + THC treatment group (Old + THC) mice was significantly improved, with muscle fiber arrangement becoming more orderly and dense, effectively reversing the morphological damage and muscle fiber atrophy caused by natural aging.

[0075] The above results indicate that tetrahydrocurcumin can significantly improve the structural disorder and muscle fiber atrophy damage of skeletal muscle tissue induced by weight loss and natural aging, effectively maintain the integrity of skeletal muscle tissue morphology and structure, and has a good repair and protection effect on degenerative structural damage of skeletal muscle.

[0076] 3.5 Tetrahydrocurcumin regulates the transcriptional expression of genes related to skeletal muscle aging, inflammation, and atrophy. To elucidate the molecular mechanism by which tetrahydrocurcumin improves skeletal muscle aging and atrophy, this invention uses qPCR technology to detect the mRNA expression levels of genes involved in skeletal muscle tissue aging, inflammation, atrophy, and protection.

[0077] Depend on Figure 12 The AF results showed that, compared with the control group (Ctrl), the skeletal muscle tissue of mice in the tail suspension model group (HU4w) showed significantly high expression of aging genes P16 and P53, inflammatory factors IL-6 and IL-8, and atrophy-related gene MuRF1, while the expression level of the protective gene SIRT5 was significantly downregulated. This indicates that weightlessness stress can abnormally activate skeletal muscle aging, inflammation, and atrophy pathways, inhibit the expression of muscle protective pathways, and induce skeletal muscle transcriptional disorders and degenerative damage. After intervention with different doses of tetrahydrocurcumin, the abnormal activation of skeletal muscle aging, inflammation, and atrophy-related genes in mice in the tail suspension + low-dose THC group (HU4w + 90THC) and the tail suspension + high-dose THC group (HU4w + 120THC) was significantly inhibited, while the expression of the protective gene SIRT5 was significantly upregulated, effectively blocking the weightlessness stress-induced skeletal muscle aging, inflammatory response, and atrophy process at the transcriptional level.

[0078] 3.6 Tetrahydrocurcumin regulates the expression levels of proteins related to skeletal muscle aging and atrophy. To further validate the regulatory mechanism of tetrahydrocurcumin at the protein level, this invention uses Western blotting to detect differences in the expression of skeletal muscle aging, atrophy, and protective proteins.

[0079] Depend on Figure 13The AF results showed that, compared with the control group (Ctrl), the skeletal muscle aging and atrophy-related proteins MuRF1 (Trim63) and Fbxo32 were abnormally highly expressed in the tail suspension model group (HU4w), while the expression of SIRT5 protective protein was significantly inhibited, suggesting that weightlessness stress can accelerate the process of skeletal muscle degenerative damage by regulating protein pathways. After intervention with different doses of tetrahydrocurcumin, the abnormal expression of skeletal muscle aging and atrophy-related proteins in the tail suspension + low-dose THC group (HU4w + 90THC) and the tail suspension + high-dose THC group (HU4w + 120THC) was significantly downregulated, while the expression level of SIRT5 protective protein was significantly upregulated, effectively inhibiting the weightlessness-induced skeletal muscle aging and atrophy process at the protein level.

[0080] 4. Conclusion In summary, this invention, through the construction of a simulated weightlessness tail suspension model and a naturally aging mouse model, demonstrates from multiple dimensions—functional, tissue morphology, gene, and protein—that tetrahydrocurcumin can simultaneously target both the cardiovascular and skeletal muscle systems. It effectively improves cardiac function damage, vascular elasticity decline, and vascular cell aging caused by weightlessness and natural aging, while significantly alleviating skeletal muscle mass loss, muscle fiber atrophy, and aging-related inflammatory responses. Tetrahydrocurcumin can exert anti-aging protective effects through multi-target and multi-pathway synergistic action, significantly improving cardiovascular and skeletal muscle degenerative damage mediated by weightlessness stress and natural aging. It possesses significant research value in the preparation of drugs for preventing and improving cardiovascular and skeletal muscle aging caused by weightlessness and natural aging.

[0081] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Application of tetrahydrocurcumin in the preparation of drugs for the prevention and / or improvement of cardiovascular and skeletal muscle aging.

2. The application according to claim 1, characterized in that, The cardiovascular aging mentioned above is caused by weightlessness and / or natural aging.

3. The application according to claim 1, characterized in that, The skeletal muscle aging mentioned above is caused by weightlessness and / or natural aging.

4. A pharmaceutical formulation for preventing and / or improving cardiovascular and skeletal muscle aging, characterized in that, The pharmaceutical preparation includes an active ingredient and pharmaceutically acceptable excipients.

5. The pharmaceutical preparation according to claim 4, characterized in that, The active ingredient is tetrahydrocurcumin or at least one of its pharmaceutically acceptable salts, esters, or solvates.

6. The pharmaceutical preparation according to claim 5, characterized in that, The pharmaceutically acceptable excipients include one or more of the following: excipients, binders, disintegrants, lubricants, solvents, solubilizers, flavoring agents, colorants, pH adjusters, isotonic agents, suspending agents, thickeners, preservatives, stabilizers, antioxidants, wetting agents, surfactants, suspending agents, propellants, absorption enhancers, absorption delayers, or coating materials.

7. The pharmaceutical preparation according to claim 6, characterized in that, The dosage form of the pharmaceutical preparation includes one or more of the following: oral dosage form, injection dosage form, inhalation dosage form, and transdermal dosage form.

8. The pharmaceutical preparation according to claim 7, characterized in that, The oral preparation is selected from any one of tablets, capsules, granules or oral liquids.

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