NOUXS Age-Reversing Factor product labreport

—— Experiment to extend lifespan and delay aging by activating the Cisd2 gene in naturally aged mice

Abstract

Background

The CISD2 gene is located in the longevity-related region of human chromosome 4q and plays a core role in aging regulation. With age, body functions gradually decline, cell activity decreases, and tissues and organs are damaged, which ultimately affects lifespan. Studies have shown that the expression level of the Cisd2 gene decreases during mouse aging, while maintaining its high expression can significantly prolong lifespan and improve health status.

This study aimed to evaluate the anti-aging potential of Cisd2 gene activators and verify their effectiveness in delaying aging and improving physiological functions.

Methods

This experiment used a natural aging mouse model and randomly divided them into an experimental group and a control group:

• Experimental group: received anti-aging product intervention.
• Control group: given an equal amount of placebo.

The experiment lasted for 24 months, and the mice's Cisd2 gene expression levels, physiological functions (metabolism, motor ability, etc.) and aging-related biomarkers were regularly tested.

After screening the herbal compound library, Agingin was identified as a potential Cisd2 activator. In vitro and in vivo experiments showed that the product had no detectable toxicity. The study further evaluated its effects in naturally aged mice, including the extension of lifespan and the improvement of age-related structural and functional defects. In addition, the Cisd2 gene knockout model was analyzed through tissue experiments to study the Cisd2-induced antioxidant effect of Agingin, and RNA sequencing was used to explore its anti-aging biological mechanism.

Results

The experimental results revealed three key findings:

1. Anti-aging factors can significantly activate the Cisd2 gene, increase its expression level in aged mice, and effectively prolong healthy lifespan.
2. Its anti-aging effect mainly depends on the Cisd2 gene, improving age-related lesions, changes in body composition, cardiovascular and organ aging and other problems.
3. Mice that received anti-aging hormone intervention in the elderly stage had a physiological state close to that of young individuals and showed stronger tissue repair ability.

The expression of Cisd2 gene in the experimental group mice was significantly increased, the metabolic level was higher, the athletic ability was enhanced, the skin and internal organs aging markers were reduced, and the average lifespan was longer than that of the control group.

Conclusions

The results of the study showed that anti-aging factor, as a Cisd2 activator, can effectively delay aging, improve physiological functions and prolong healthy life span. This discovery not only provides a scientific basis for its anti-aging effect, but also opens up a new direction for future intervention strategies for healthy aging.

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Background

Aging has become a major global challenge. With the increase in life expectancy, the global population is facing an increasingly severe aging problem. Aging is caused by the accumulation of cellular and molecular damage, which leads to the gradual decline of the functions of multiple organ systems, eventually causing a variety of age-related complications and increasing the risk of morbidity and mortality. Although the scientific community has made progress in aging research, there is still a lack of treatment options that can effectively delay aging and increase healthy life expectancy.

Mitochondrial dysfunction is one of the prominent features of aging. Many cellular and molecular features associated with aging have been identified, including DNA damage, telomere shortening, epigenetic changes, loss of protein homeostasis, dysregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and changes in intercellular communication. There may be extensive interconnections between these aging features. Therefore, improving one of these features may affect other features, thereby producing a comprehensive intervention effect on the aging process.

Although the molecular mechanisms that lead to aging have not yet been fully elucidated, studies have shown that the decline of mitochondrial function plays a vital role in aging and related pathophysiology. Impairment of mitochondrial function not only reduces the energy metabolism capacity of cells, but also leads to increased oxidative stress and imbalance of intracellular calcium ion homeostasis, thereby exacerbating the aging process. Existing studies have shown that maintaining mitochondrial function is essential for delaying aging and maintaining the health of multiple organ systems.

According to the database collected by the existing human aging genome resources, there are currently 8 genes that can regulate aging (Bub1b, Cisd2, Klotho, Pawr, Pparg, Pten, Sirt1, Sirt6), which have been experimentally proven to effectively reduce and increase lifespan in mice through gene knockout and overexpression. However, only two genes, Cisd2 and hMTH1, have evidence to show that they are associated with delayed aging. The Cisd2 (CDGSH iron-sulfur domain 2) gene is located in the human 4q22-24 chromosome region and is one of the genes associated with longevity. Mouse studies have shown that the expression level of Cisd2 decreases significantly during natural aging, while maintaining its high level of expression can significantly extend the lifespan of mice and improve aging-related physiological functions. Specifically, the Cisd2 gene protects cells from oxidative stress and maintains cell function by regulating mitochondrial function and intracellular calcium ion homeostasis.

In addition, the synergistic effect of Cisd2 between the endoplasmic reticulum and mitochondria is considered to be one of the mechanisms by which it plays a key role in the aging process. Studies have found that Cisd2 can maintain intracellular calcium ion homeostasis and optimize mitochondrial-endoplasmic reticulum communication, thereby improving cellular metabolism and reducing oxidative stress levels. The absence of Cisd2 can lead to impaired cardiac function, while its high expression can delay cardiovascular decline and improve age-related cardiac dysfunction. In addition, ongoing studies have shown that increased levels of Cisd2 can delay respiratory aging, slow the progression of neurodegenerative diseases, and reduce Alzheimer's-related neuronal loss, while promoting neural regeneration and repair, which helps improve cognitive function.

Based on these studies, Cisd2 has been established as an important target for delaying aging. This study aims to evaluate the anti-aging effect of the Cisd2 activator, anti-aging hormone, and explore its potential in delaying aging and improving physiological functions. By activating the Cisd2 gene, anti-aging hormone may slow down the natural aging process, restore the vitality of aging tissues, and thus prolong the healthy life span, providing a scientific basis and new treatment strategies for aging intervention.

Methods

Construction of HEK293-CISD2 reporter cell line

In this study, a 102 kb human bacterial artificial chromosome (BAC) clone (CTD-2303J4, Invitrogen, CA, USA, #96012) was used to construct a CISD2 BAC reporter gene clone. This BAC clone contains the complete CISD2 gene and its flanking sequences in its natural chromosomal context, including 23.8 kb of the coding region, 31.3 kb of the upstream regulatory region, and 46.9 kb of the downstream region (Additional file : Figure S1A).

To construct the CISD2 BAC reporter clone, we used the central recombineering technique to insert the IRES-Luc-pA element (i.e., internal ribosome entry site (IRES), luciferase (Luc), and polyA signal (pA)) into exon 2 of the CISD2 gene (Additional file : Figure S1B). This was done in the E. coli system.

Subsequently, to establish the HEK293-CISD2 reporter cell line, we co-transfected HEK293 cells with the linearized CISD2 BAC reporter construct and the pCI-neo plasmid (Promega, Mannheim, Germany, #E1841). Electroporation parameters were set at 250 V, 500 μF. After transfection, cells were selected in growth medium (DMEM; Gibco, Carlsbad, CA, USA, #11965) containing 1 μg/mL puromycin (Invitrogen, #A11138-03) for 12–14 days. The medium was supplemented with 10% fetal bovine serum, 1% glutamine/penicillin/streptomycin, 1% non-essential amino acids, and 1 mM sodium pyruvate to support cell growth.

Finally, the correct cloning of the HEK293-CISD2 reporter cell line was verified by PCR and luciferase reporter assays.

Luciferase reporter gene assay

HEK293-CISD2 BAC reporter cells were seeded at a density of 2 × 10⁴ cells/well in 96-well plates and cultured for 24 hours. Subsequently, the cells were treated with different doses of anti-aging factors and other cosmetic ingredients for 24 hours.

After treatment, luciferase activity was measured using the ONE-Glo™ Luciferase Assay System Kit (Promega, #E6120) according to the manufacturer’s instructions. Luminescence intensity was monitored using an Infinite 200 microplate reader (Tecan Group Ltd., Mannedorf, Switzerland).

Herbal Compound Library

This study used a herbal ingredient library, which originated from the "Shennong Bencao Jing Baizhonglu", a pharmaceutical work compiled by Xu Dachun in the Qing Dynasty. The book was completed in 1736 and contains 100 major drugs in the "Shennong Bencao Jing" with brief annotations based on clinical experience. The library contains 100 extracts and prescriptions, and the purpose of establishing this library is to systematically screen potential CISD2 activators.

The extraction process is as follows:
First, 50 g of carrot herb was crushed and extracted with 200 mL of 60% ethanol at 25 °C by shaking for three times. Subsequently, partition extraction was performed using 600 mL of dichloromethane (CH₂Cl₂), and the extract was dried by a rotary evaporator.

Then, a silica-based high performance liquid chromatography (HPLC) column and a C18 HPLC column were used for chromatographic separation, respectively. The CH₂Cl₂ layer was eluted with 100 mL of 100% CH₂Cl₂, CH₂Cl₂/methanol (95/5), CH₂Cl₂/methanol (9/1), CH₂Cl₂/methanol (8/2), and 100% methanol in a gradient manner to obtain C1–C5 fractions; the C18 column was eluted with 200 mL of ddH₂O, 30% methanol (aqueous solution), 60% methanol (aqueous solution), 90% methanol (aqueous solution), and 100% methanol to obtain M1–M5 fractions.

Finally, 13 fractions were obtained in this study, including crude extract, CH₂Cl₂ layer, 60% ethanol layer, C1–C5 fractions, and M1–M5 fractions. All fractions were dried by rotary evaporator and stored at −20°C for subsequent experiments.

Identification of CISD2 activators

After multiple screening and comparison, this experiment finally selected 36 plants such as Citrus aurantium, blueberry, and sunflower, extracted flavonoids, polyphenols, kaempferol, luteolin and other compounds with strong antioxidant effects, and synthesized them through scientific proportions of multiple natural ingredients. The ability of these compounds to enhance CISD2 expression was detected using HEK293-CISD2 reporter cell line analysis (Additional file: Figure S1C, D).

Finally, a multi-herbal compound (anti-aging agent) with a purity of >98% was born and identified as a promising CISD2 activator that could significantly enhance the expression of CISD2 inside and outside cells (Additional file : Figure S1E-G).

Analysis of anti-aging factors and their combined auxiliary substances

Levels of nervosa, nervosa-7-O-β-d-alduronate (N7G), and nervosa-7-O-sulfate (N7S) in mouse serum and tissues were quantified by LC-MS/MS. In the experimental model, mice were fed a nervosa-supplemented diet ad libitum for 4 months until euthanasia. To synchronize food intake, the experimental mice were fasted for 6 hours (2 pm to 8 pm).

Sample preparation for LC-MS/MS was as follows: 50 µL of mouse plasma or homogenized tissue fluid (cardiac, liver, or neonatal muscle tissue) was mixed with 50 µL of 250 ng/mL sirenolide-d3 (from Toronto Research Chemicals, No. H289502). Sirenolide-d3 was used as an internal standard to label sirenolide with three hydrogens replaced. The mixture was vortexed and centrifuged at 15,000 × g for 20 minutes in a Beckman Coulter Microfuge 22R centrifuge. The supernatant was transferred to a clean tube and 15 µL of the supernatant was used for LC-MS/MS analysis.

LC-MS/MS analysis

LC-MS/MS analysis was performed using an Agilent 1200 Series LC system equipped with an Agilent ZORBAX Eclipse XDB-C8 column (5 μm, 3.0 × 150 mm) connected to an MDS Sciex API 4000 mass spectrometer. The MS/MS ion transitions of anti-aging factor, N7G, N7S and anti-aging factor-d3 are m/z 300.9 → 163.9 (anti-aging factor), 477.0 → 301.0 (N7G), 380.9 → 301.0 (N7S) and 303.9 → 163.8 (anti-aging factor-d3), respectively.

The samples were separated by high performance liquid chromatography (HPLC) with mobile phase A consisting of 10 mM ammonium formate aqueous solution (containing 0.1% formic acid) and mobile phase B consisting of acetonitrile. The gradient elution program was as follows (time/min – %B):

• 0.0–0.5 min：10%
• 0.5–1.2 min：60%
• 1.2–3.4 min：80%
• 3.5–5.0 min：10%

The flow rate was set to 1.5 mL/min, with part of the sample entering the mass spectrometer and the rest being diverted to waste. The retention times of nivolumab, N7G, N7S, and nivolumab-d3 were 2.46 min, 2.07 min, 2.16 min, and 2.44 min, respectively.

Mouse model and anti-aging hormone treatment

The construction method of CISD2 reporter transgenic (TG) mice is as follows. Briefly, the linearized CISD2 BAC reporter construct (which contains the luciferase gene driven by the human CISD2 promoter) was microinjected into the pronuclei of C57BL/6 mouse fertilized eggs to obtain CISD2 reporter TG mice.

Cisd2 Muscle-specific knockout mice (mcKO) were constructed. The brief steps are as follows:

• Mice carrying the Cisd2 floxed allele (Cisd2 f/f) were used as a base.
• They were crossed with MCK-Cre transgenic mice (JAX006475) for two generations, and finally Cisd2 mcKO (Cisd2 f/f; MCK-Cre) mice were obtained, in which the Cisd2 gene was specifically deleted in skeletal muscle.

All experimental mice used in this study were of purebred C57BL/6 background and housed in a specific pathogen free (SPF) environment. The experimental conditions were as follows:

• Photoperiod: 12 hours light / 12 hours dark

• Temperature control: 20-22°C

Age-Reversing Factor diet therapy

Wild-type (WT) mice aged 19.5 to 23.5 months were fed an anti-aging diet with an AIN-93G growing diet (TestDiet, St. Louis, MO, USA, Additional file : Table S2). The experimental groups were as follows:

• Control group (Veh): The diet contained 3.04% propylene glycol (w/w) as a carrier (Sigma-Aldrich, Munich, Germany, #16033).

• Aging-reversing group: 0.07% Aging-reversing (w/w) (Sigma-Aldrich, #H4125, HPLC purity > 95%) was added to the feed at a dose of 100 mg/kg/day for 3 to 6 months.

After the experiment, the mice were euthanized by CO₂. All animal experimental protocols in this study strictly followed the 3R principle (replacement, reduction, improvement) of the Animal Protection Act for experimental design.

In Vivo Imaging System (IVIS)

To evaluate the effects of dietary anti-aging treatment (100 mg/kg/day) on CISD2 reporter transgenic (TG) mice, luciferase activity was detected using an in vivo bioluminescence imaging system (IVIS 50, Xenogen Corp., Alameda, CA, USA). The experimental procedure was as follows:

1. Substrate injection: Mice were intraperitoneally injected with d-luciferin (150 mg/kg, prepared in PBS).

2. Imaging acquisition: Anesthesia was performed using 2.5% isoflurane, and images were acquired in an IVIS 50 system.

3. Data analysis: The bioluminescent signals of the ventral views of the mice were analyzed using Living Image 3.2 software (IVIS 50 Imaging System, Xenogen Corp.) and the units of signals are average photons/sec/cm²/sr.

Blood biochemistry analysis

Biochemical parameters of blood samples were tested using a Fuji Dri-Chem 4000i automatic biochemical analyzer (Fuji Film, Tokyo, Japan) and included:

• Liver function indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST)

• Renal function and metabolic indicators: urea nitrogen (BUN), creatinine (Cr), total cholesterol (TC), triglyceride (TG)

• Electrolyte levels: Ca²⁺, Mg²⁺, Na⁺, K⁺, Cl⁻

Hematological analysis Complete blood counts (CBC) were performed using a ProCyte Dx hematology analyzer (IDEXX, Columbus, OH, USA). Whole blood samples were immediately anticoagulated by the addition of EDTA (final concentration 5 mM).

Systemic Composition Analysis

Human lean and fat volumes were measured using a micro-CT scanner (SkyScan 1076, Bruker, Kontich, Belgium). The numerical results of whole body lean, fat, and visceral fat in mice were analyzed using the three-dimensional structures obtained by micro-CT and the software SkyScan 1076 (Bruker).

Whole body metabolic rate measurement

The oxygen consumption rate (VO₂), carbon dioxide production rate (VCO₂), and energy expenditure (EE) of mice were measured using the TSE LabMaster system (TSE Systems GmbH, Homburg, Germany) as follows:

1. Acceleration period: Each mouse acclimatized for 72 hours before the experiment.

2. Measurement cycle: A 12:12 h light-dark cycle (lights on at 8:00 AM) was used, followed by 48 h of continuous monitoring.

3. Data analysis: The whole body metabolic rate of mice was calculated based on reference calorimetry and corrected for lean body mass (LBM) using the following formula:

   $$\text{Lean body mass}=\text{Lean body volume}\times 1.06{\text{ g/cm}}^3$$ $$瘦体重 = 瘦体体积 \times 1.06 \text{ g/cm}^3$$

Ischemic tolerance test and insulin tolerance test

1. Ischemia Tolerance Test

• Fast for 6 hours (9:00 a.m. to 3:00 p.m.).

• Anemic saline (1.5 mg/g) was injected, and blood samples were collected at designated time points.

• Blood glucose determination: Measurements were performed using OneTouch Ultra blood glucose test strips and a SureStep Brand blood glucose meter (LifeScan, Milpitas, CA, USA).

2. Insulin Tolerance Test (ITT)

• Fast for 2 hours (9:00 am to 11:00 am).

• Insulin injection: 0.75 U/kg human insulin (Actrapid, Novo Nordisk, Bagsværd, Denmark) was administered intraperitoneally.

• Insulin concentration determination: The insulin ELISA kit (Mercodia, Uppsala, Sweden, No. 10-1249-01) was used for detection.

Rotarod Test

The rotarod test is used to evaluate the motor coordination, balance ability and fatigue resistance of mice. The experiment is conducted using the RT-01 rotarod apparatus. The specific process is as follows:

1. Pre-training phase

• Each mouse underwent three adaptation training sessions (5 rpm, 5 min) before the formal test to ensure that it was familiar with the experimental equipment.

2. Testing Phase

• The rotation speed was set to 10 rpm, 20 rpm, and 30 rpm with an acceleration rate of 1 rpm/s.
• Experiment duration: 5 minutes per test.
• Data recording: When the mouse falls from the rotating rod, the infrared sensor at the bottom automatically records the falling time.

Transthoracic echocardiography

Mouse cardiac function was assessed using the VisualSonics VeVo 2100 imaging system (VisualSonics, Toronto, Ontario, Canada). The specific procedure is as follows:

1. Anesthesia: Mice were anesthetized using 1% isoflurane in 95% O .

2. Temperature maintenance: The body temperature of mice was maintained at 36°C to 37°C using a heating pad (TC-1000, CWE Inc., Ardmore, PA, USA) to ensure stability during the experiment.

3. Ultrasound examination: Cardiac function was assessed using a high-frequency 30-50 MHz probe, as described above.

4. Data Analysis: Ultrasound data were analyzed using VisualSonics software.

5. Blind design: Data collectors were kept blind to the grouping of mice to eliminate bias.

Electrocardiogram (ECG)

The cardiac function of mice was tested by electrocardiogram (ECG). The experimental design was as follows:

1. Experimental cycle: Mice were subjected to a 12:12 h dark-light cycle with light on at 6:00 AM.

2. Anesthesia: Mice were first placed in an environment filled with 3% isohelane to induce anesthesia for 3-5 minutes. Mice were then placed on a heating pad (ALA Scientific, New York, USA) to ensure stable body temperature.

3. ECG recordings: Mice were anesthetized and ECG recordings were performed using subcutaneous electrodes attached to the limbs for 5 min, and data were recorded by a PowerLab data acquisition system (ML866, ADInstruments, Colorado Springs, CO, USA) and an animal bioamplifier (ML136, ADInstruments).

4. Data analysis: The ECG analysis was completed using LabChart 7 Pro (version 7.3.1, ADInstruments), analyzing 1500 heart beats, mainly detecting myocardial electrophysiological parameters such as the QTc interval, QRS interval and Tpeak-Tend interval.

5. Statistical analysis: The obtained data were statistically compared using the Mann-Whitney U test, and p < 0.05 was considered significant.

Western Blotting

1. Tissue sample processing: Femoris, gastrocnemius, and myocardium were homogenized using RIPA buffer (containing Tris, NaCl, EDTA, Triton X-100, sodium deoxycholate, SDS), followed by denaturation at 100°C for 15 min using SDS sample buffer (containing Tris, dithiothreitol, SDS, glycerol).

2. Separation and transfer: Separate by SDS-PAGE, transfer proteins to polyvinylidene fluoride membrane, and block with TBST buffer for 1 hour.

3. Antibody incubation: The membrane was incubated with specific antibodies (such as Cisd2 and Gapdh), washed three times, and then incubated with appropriate secondary antibodies for 1 hour.

4. Signal detection: Protein signals were detected using the ECL detection method (Thermo Fischer Scientific).

Histopathology and electron microscopy (TEM)

1. Tissue fixation: Fix femoris and gastrocnemius muscles and diabetic tissues in 10% formalin for 14-16 hours.

2. Paraffin embedding and sectioning: Samples were processed by a tissue processor (STP120, MICROM, Walldorf, Germany) and embedded in paraffin.

3. Staining: Sections were stained with H&E, Masson's trichrome, and Sirius red.

4. TEM analysis: Fixation with 1.5% glutaraldehyde and 1.5% paraformaldehyde and 1% OsO4 followed by sectioning and TEM analysis.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) detection

ROS and RNS levels in dental tissues were quantified using an external ROS/RNS detection kit, and DCF fluorescence intensity was monitored using an Infinite 200 microplate reader (Tecan Group Ltd.).

RNA analysis and sequencing

1. RNA Isolation: Total RNA was isolated from tibialis (gastrocnemius) muscle, cardiac muscle, and liver tissues using TRI Reagent.

2. RNA sequencing: RNA-seq was performed by the Genomics Panel, with cross-single-end sequencing generating datasets with a read depth of at least 2000.

3. Gene expression analysis: Gene expression was calculated by RPKM (reads per kilobase of exon model per million). Comparative analyses included 26 month WT-Veh vs. 3 month WT, 26 month WT-Veh vs. 26 month WT-Veh, and gene ontology functional characterization using PANTHER and MGI GO term finder were performed.

Data Analysis and Visualization

1. PCA analysis: Principal component analysis (PCA) was performed using EZinfo 3.0.3 software.

2. Heat map drawing: Gene expression heat maps were generated using Multi Experiment Viewer 4.9 software to further visualize the data.

Statistical analysis

Data are expressed as mean ± standard deviation (SD) or mean ± standard error (SEM) as indicated in the figures.

1. Between-group comparisons: Comparisons between the two groups were performed using the two-tailed Student's t-test.

2. Multiple Group Comparisons: For comparisons between more groups, one-way or multi-way analysis of variance (ANOVA) was used with a Bonferroni correction for multiple comparisons adjustment.

3. Survival Analysis: Survival analysis was performed using the Mantel-Cox log-rank test.

4. Software and Statistical Methods: All statistical analyses were performed using SPSS Statistics 26.0 (IBM Corp., New York, USA) and GraphPad Prism 6.0 (GraphPad Software, CA, USA).

5. Power analysis: The statistical power was 0.9608, and the sample size was 47 animals (including the untreated group, the Veh group, and the anti-aging hormone group).

6. Significance: p < 0.05 was considered statistically significant.

Result

Anti-aging factor is a promising Cisd2 activator with no detectable toxicity

The key role of Cisd2 in anti-aging prompted us to explore its potential pharmaceutical applications. To this end, we utilized the BAC (embryonic artificial chromosome) clone CTD-2303J4 containing the human CISD2 gene, constructed the HEK293-CISD2 reporter cell line and the CISD2-TG (transgenic) mouse model to systematically screen for active compounds that can upregulate CISD2 expression.

In the active ingredient library of 100 traditional precious herbs, based on the records of the Chinese medicine classic Shennong's Herbal Classics 100 Records, combined with chemical separation, structure identification and biological activity screening, we finally identified Anti-aging factor (purity > 98%), which can significantly increase the expression level of Cisd2 (Additional file : Figure S1C-G).

Notably, sildenafil showed no detectable toxicity within the applicable dose range in HEK293 cells (10-30 μM) and WT young and old mice. We supplemented the diet of old mice with sildenafil (100 mg/kg/day) to assess its long-term safety during natural aging. This dose corresponds to a human dose of approximately 491 mg/60 kg/day based on cross-species dose conversion.

In 20-26 month old mice, serum biochemical analysis after 6 months of sildenafil treatment showed that sildenafil did not cause detectable toxicity compared with the age- and sex-matched Veh group. The serum parameters tested included:

1. Biochemical indicators related to heart, liver and kidney function

2. Metabolic indicators such as plasma insulin, total cholesterol, triglycerides (TG)

3. Electrolyte levels such as calcium (Ca²⁺), magnesium (Mg²⁺), sodium (Na⁺), potassium (K⁺), and chloride (Cl⁻)

In addition, ANTI-AGE can reduce aspartate aminotransferase (AST), a marker of liver damage (Additional file : Figure S2). Blood cell count (CBC) analysis showed that after 7 months of long-term treatment, ANTI-AGE had no significant effect on hematological parameters (Additional file : Figure S3). In summary, serum biochemistry and CBC results confirmed the safety of ANTI-AGE in long-term application.

Further analysis showed that N7G and its major metabolites (N7S) could be detected in the liver, bone marrow, heart, and blood (Additional file : Figure S4A-D). In HEK293-CISD2 cells, both N7G (10 μM) and its metabolite N7S (30 μM) activated the CISD2 reporter gene (Additional file : Figure S4E). Therefore, N7G's anti-aging effect may originate from the synergistic effect of N7G and its metabolites (such as N7S).

Anti-aging factor delays natural aging and extends lifespan in mice

To investigate whether antiagelin can slow aging and extend healthy lifespan, we administered dietary antiagelin to naturally aged mice starting at 21 months of age and monitored their survival.

The results showed that anti-aging factor significantly extended the lifespan of mice (Figure 1A):

• Median lifespan: increased from 25.95 months to 28.2 months (+2.25 months, +8.7%, p = 0.04)

• Maximum lifespan: From 29.5 months to 33.6 months (+4.1 months, +13.9%)

Furthermore, in 26-month-old aged mice, Cisd2 expression levels were significantly decreased in skeletal muscle (femur and gastrocnemius) and cardiac muscle. However, dietary anti-aging restored Cisd2 levels in these tissues to those of 3-month-old young mice (Figure 1B-D).

The combined results suggest that Cisd2 could be a drug target and activated by anti-aging factors in late life. Increased levels of Cisd2 could help slow natural aging and extend lifespan.

Anti-aging factor increases Cisd2 levels in aged wild-type mice

We monitored the survival of mice weekly in untreated wild-type mice (n = 20), vehicle control group (Veh, n = 8), and NLS-treated groups (NLS, n = 19).

Experimental design: Wild-type mice aged 21 months were treated with either 100 mg/kg/day of vehicular regeneration factor or Veh control, and statistical analysis was performed using the Mantel-Cox survival curve test. The results are as follows:

• Veh vs. Age-Reversing Factor: p = 0.04

• Untreated WT vs. Anti-Aging Agent: p = 0.029

• Untreated WT vs. Veh: p = 0.5 (no significant difference)

Further analysis of Cisd2 expression in aging tissues (Figure 1B-D) revealed that:

• In 26-month-old mice, Agingin significantly increased the Cisd2 protein expression level in femur (B), gastrocnemius (C), and myocardium (D).

• After 5 months of anti-aging treatment (from 21 to 26 months of age), Cisd2 levels returned to the level of 3-month-old mice.

• Data are expressed as mean ± SD, *p < 0.05, **p < 0.005, and statistical analysis was performed using one-way analysis of variance (ANOVA) with Bonferroni post hoc test.

These results suggest that dietary anti-aging factors can effectively reverse the decline of Cisd2 in aging tissues and may help delay the age-related aging process.

Anti-aging factor alleviates systemic metabolic decline in aged mice

Aging is closely associated with metabolic changes, which are often manifested by a decrease in whole-body metabolic rate, increased fat accumulation, loss of muscle mass, and impaired glucose homeostasis. To evaluate the anti-aging effects of anti-aging agents and their effects on age-related metabolic decline, we analyzed whole-body energy metabolism and body composition in aged mice.

The experimental results show (Figure 2A-C):

• Compared with young (3 months old) WT mice, Veh-treated old (28 months old) WT mice had significantly reduced VO₂ (oxygen consumption), VCO₂ (carbon dioxide production), and EE (energy expenditure).

• After 6 months of anti-aging treatment, these metabolic indices were significantly improved, especially in the dark period (active period), indicating that anti-aging can improve the whole-body metabolism of aged mice.

• Further comparison of mice treated with different durations of treatment (Additional file : Figure S5) revealed that the metabolic levels of mice aged 28 months (treated with 6 months of anti-aging hormone) were significantly higher than those of mice aged 23.5 months (treated with 1.5 months of anti-aging hormone).

To evaluate the effects of NLS on metabolic function in aged mice, we performed metabolic monitoring on 3-month-old WT mice, 28-month-old Veh-treated WT mice, and 28-month-old NLS-treated WT mice, including:

• Whole body oxygen consumption (VO₂) (Figure 2A)

• Carbon dioxide production (VCO₂) (Figure 2B)

• Energy expenditure (EE) (Figure 2C)

Experimental design:

• Old mice (22 months of age) were treated with dietary anti-aging agents (100 mg/kg/day) or Veh control for 6 months, and metabolic monitoring was performed at 28 months of age.

• The metabolic rate of individual mice was recorded by 48 h continuous monitoring, and the area under the curve (AUC) during the dark period (20:00–02:00) was calculated.

• The metabolic indicators of each mouse were taken from two 24-hour measurement cycles to calculate the AUC value and normalized according to lean mass.

Data Analysis:

• Hourly metabolic monitoring data are expressed as mean ± SEM.

• AUC quantification data are expressed as mean ± SD.

• Statistical analysis was performed using one-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons.

• *p < 0.05; **p < 0.005, UT stands for untreated group.

The experimental results show that: Anti-aging factor can significantly improve the energy metabolism of elderly mice, enhance VO₂ and VCO₂, increase EE, and promote the recovery of energy metabolism, thereby alleviating the metabolic decline caused by aging and delaying the aging process.

Anti-aging factor reduces fat accumulation and improves glucose homeostasis in aged mice

Body composition analysis (including total body fat and muscle mass) can be used to assess an individual's health status.

The experimental results show (Figure 3A-D):

• Compared with young (3-month-old) WT mice, 28-month-old Veh-treated WT mice had significantly increased total body fat and visceral fat.

• Six months of dietary anti-aging hormone treatment can significantly reduce the accumulation of whole body fat and visceral fat in aged mice (Figure 3A, B).

• Agingin treatment also attenuated age-related muscle loss and increased the body lean percentage of aged mice (Figure 3C, D).

• There was no significant difference in body weight change between the Veh group and the anti-aging group (Figure 3E).

Fat Reduction and Muscle Protection We observed significant metabolic improvements after 6 months of Veh treatment in mice aged 22 to 28 months. Micro-CT analysis showed that compared with young (3 months) WT mice, old (28 months) WT mice treated with Veh had significantly increased total body fat and visceral fat volume, and Veh treatment effectively reduced this age-related fat accumulation (Figure 3A, B). In addition, Veh also improved age-related muscle loss and increased lean meat percentage (Figure 3C, D), but there was no significant change in overall body weight (Figure 3E).

Improvement of blood glucose homeostasis Age-related impaired blood glucose homeostasis and insulin resistance are closely related to muscle mass loss. To evaluate the effects of anti-aging on blood glucose homeostasis, we performed glucose tolerance test (GTT) and insulin tolerance test (ITT). Although there were no significant differences in GTT and ITT between the two groups of mice (Figure S6), the anti-aging group showed a better trend. Importantly, 6 months of anti-aging treatment significantly reduced fasting blood glucose levels (6 hours) and blood glucose levels at 120 minutes of GTT (Figure 3F, G).

Liver transcriptome analysis To explore the molecular mechanism by which Veh improves glucose homeostasis, we performed RNA sequencing analysis. The results showed that the expression of insulin signaling-related genes in the liver of aged mice treated with Veh changed significantly, affecting insulin signaling, glucose metabolism, lipid metabolism, and cell proliferation/differentiation (Figure S7A). However, Veh treatment reversed these changes, causing gene expression levels to tend toward those of young mice (Figures S7B-D). In addition, Veh reduced the expression of IKKβ, a gene that inhibits insulin signaling and leads to insulin resistance (Figure S7B).

Glycogen metabolism regulation In terms of glycogen metabolism, key enzymes related to hepatic glycogenolysis (Agl, Pygl, PKAα, and PhKδ) were significantly upregulated in aged Veh-treated mice, while anti-aging treatment inhibited the expression of these enzymes (Figure 3H, I), thereby reducing hepatic glycogenolysis and improving blood glucose homeostasis. Similarly, enzymes related to glycogen synthesis (Gys2, Gbe1, Gsk3β, and PP1) were significantly upregulated in aged Veh-treated mice, while anti-aging treatment restored them to the levels of young mice (Figure 3I, Figure S7E, F).

In summary, Agerin improves body composition, reduces fat accumulation, and preserves lean body mass in aged mice, which may help maintain healthy aging.

Anti-aging factor inhibits muscle aging in old mice

Sarcopenia and Mitochondrial Function Sarcopenia is characterized by a decrease in muscle mass and strength, accompanied by impaired mitochondrial function. To investigate the anti-muscle aging effects of sarcosin, we evaluated the effects of sarcosin treatment on skeletal muscle function and structure in aged mice. The results of the rotarod test showed that sarcosin treatment significantly improved the motor capacity of 26-month-old WT mice (Figure 4A).

Histological analysis Masson trichrome staining analysis showed that the muscle fibers of 26-month-old Veh-treated mice were significantly atrophied and accompanied by fibrosis, and anti-aging treatment significantly alleviated these aging-related muscle pathological changes (Figure 4B). In addition, the muscle fiber area in the quadriceps and gastrocnemius muscles of the anti-aging group was significantly increased, and the fiber degenerative changes were reduced (Figure 4C-E), indicating that anti-aging can effectively improve muscle health.

Aged wild-type mice (n=4-7) were given diets supplemented with anti-aging agents (100 mg/kg/day) or control food for 3 and 6 months (starting at 20 months of age), respectively, and subsequently subjected to the Rotarod motor capacity test (Figure 4A).

To evaluate changes in muscle tissue structure, we analyzed the femur and gastrocnemius using Masson trichrome staining (Figure 4B). After 21-month-old wild-type mice were given anti-aging drugs in their diet, we observed improved myofiber integrity in the femur and gastrocnemius (Figure 4C). In addition, quantitative analysis of myofiber integrity and injury morphology further confirmed this (Figure 4D, E). All data are presented as mean ± standard deviation (SD), and statistical tests were performed using Bonferroni one-way analysis of variance (ANOVA, Figure 4D, E) or Student's t test (Figure 4A); * p < 0.05, ** p < 0.005.

Transmission electron microscopy (TEM) analysis further revealed the effects of Veh on the ultrastructure of gastrocnemius muscle (Fig. 4F-H). The 3-month-old young mice (Fig. 4F) showed intact myofibril structure, while the 24-month-old control mice (treated with Veh) exhibited obvious mitochondrial degeneration (MD) and severe fibrosis (*), which may be caused by myofibril degeneration (Z-line misalignment, ZLb) (Fig. 4G).

Surprisingly, mice treated with 3 months of silencing showed ultrastructural recovery, including intact Z-lines (ZL) and multiple normal-sized triplets (Figure 4H). This suggests that silencing treatment can effectively alleviate aging-related muscle degeneration and even reverse its progression to some extent. The intact Z-lines and normal triplets shown in Figure 4H further support the hypothesis that silencing promotes muscle rejuvenation. Labels: M (mitochondria), SR (sarcoplasmic reticulum), TC (SR terminal cisterna), scale bar 500 nm.

In summary, anti-aging hormone treatment can significantly delay metabolic decline and muscle aging in elderly mice by improving fat accumulation, enhancing muscle mass, restoring blood sugar homeostasis and regulating glycogen metabolism, and has potential anti-aging application value.

Anti-aging factor interrupts heart aging in old mice

As people age, the incidence of cardiovascular disease increases significantly, but there is currently no effective treatment. The main characteristics of human heart aging include decreased ejection fraction (EF), increased cardiac function index (HFI), increased incidence of arrhythmias, and increased perivascular fibrosis.

In this study, we systematically evaluated cardiac aging phenotypes in wild-type mice treated with Veh at 24 months of age using echocardiography (to assess mechanical function), electrocardiography (ECG, to assess electrical function), and histopathological analysis ( Fig. 5 ).

The experimental results showed that anti-aging factors can improve the mechanical function of the elderly heart, significantly increase the systolic ejection fraction (EF) and reduce the diastolic myocardial function index (Figure 5A, B).

To further explore the effects of anti-aging on age-related arrhythmias and cardiac electrophysiology, we recorded ECG signals for 5 minutes. Surprisingly, anti-aging significantly reduced the incidence of arrhythmias in aged mice (Figure 5D) and corrected the abnormal QT interval (Figure 5E) and Tpeak-Tend interval (Figure 5F) observed in naturally aged mutant mice.

In addition, to evaluate the structural changes in cardiac tissue, we used Sirius Red/Fast Green staining to detect collagen deposition. The results showed that anti-aging treatment significantly reduced perivascular fibrosis in the hearts of aged mice (Figure 5G).

The experimental results showed that Veh can effectively slow down the cardiac aging process in old wild-type mice. Echocardiographic analysis (Figures B and C) showed that the ejection fraction (EF) and left ventricular function index of the Veh treatment group were improved compared with the control group. In addition, electrocardiographic analysis (Figure D) revealed that untreated old mice or control group (Veh) mice showed obvious arrhythmias, including premature contractions (VPC), atrioventricular block (AV block), irregular PR interval, and prolonged QT interval, while Veh treatment significantly alleviated these abnormalities (Figures E and F).

To further evaluate the protective effect of anti-aging on cardiac structure, we used Sirius red/fast green staining (Figure G) to detect fibrosis around cardiac vessels. The results showed that collagen deposition in the anti-aging group was significantly reduced compared with the control group, suggesting that it may have anti-fibrotic effects. Transmission electron microscopy (TEM) analysis (Figures H-K) further revealed that untreated 24-month-old mice (Figures I, J) showed significant mitochondrial degeneration, myofibril rupture, and abnormal intercellular junction (ICD) structure, while after 3 months of anti-aging treatment (Figure K), these ultrastructural damages were significantly repaired, similar to the myocardial ultrastructure of 3-month-old young mice (Figure H).

The experiment used a 21-month-old WT mouse model, which was treated with 100 mg/kg/day of Veh or control (Veh) for 3 months and analyzed at 24 months of age. All quantitative data are expressed as mean ± standard deviation (SD) and statistically tested using one-way analysis of variance (ANOVA) with Bonferroni correction. The significance level was set at *p < 0.05; **p < 0.005.

In summary, this study shows that anti-aging factors can not only slow down muscle aging in elderly mice, but also improve their cardiac function, reduce age-related abnormal heart rhythms and cardiac fibrosis, and have potential anti-aging therapeutic value.

Anti-aging factor improves myocardial ultrastructure

At the ultrastructural level, anti-aging can effectively improve aging-related damage to the intercardiomyocyte disc (ICD), mitochondria, and sarcomeres. The ICD is responsible for maintaining mechanical coupling and electrical signal transmission between cardiomyocytes, and its structural integrity is essential for cardiac function. Transmission electron microscopy (TEM) observations showed that the ICD structure of 3-month-old WT mice was intact (Figure 5H), while the 24-month-old control mice (Figure 5I, J) showed severe ultrastructural abnormalities, including focal adhesion (FA) destruction, gap junction (GJ) rupture, partial tearing of desmosomes (DS), and widening of the membrane spacing on both sides of the ICD. In addition, significant mitochondrial swelling, rupture, and myofibril atrophy were observed in the cardiomyocytes of aged mice. However, after 3 months of anti-aging treatment (Figure 5K), these structural abnormalities were significantly improved, the ICD structure was restored to its integrity, and the mitochondrial morphology and myofibril arrangement were close to the levels of 3-month-old mice.

Taken together, these results suggest that anti-aging factors can effectively improve the electrophysiological function of the heart and delay the aging process of the heart in naturally aged mice.

Anti-aging effects of Anti-aging factor are mediated through a Cisd2-dependent pathway

This study found that the improvement of anti-aging and related phenotypes by anti-aging factors is largely dependent on Cisd2. To verify this hypothesis, we used Cisd2 mcKO mice (MCK-Cre; Cisd2^f/f), a model that specifically knocks out Cisd2, affecting its skeletal and cardiac muscle function. The results showed that Cisd2 mcKO mice showed obvious premature aging characteristics at 3 months of age and had a shortened lifespan, and their aging phenotype was comparable to that of 26-month-old WT mice.

To further evaluate the importance of Cisd2 in the anti-aging effects of velin, we treated Cisd2 mcKO and WT mice with velin or control (Veh) for 4 months starting from 3 months of age and analyzed them at 7 months of age (Additional file : Figure S8A). The results showed that there was no significant difference in the concentration of velin in the serum and various tissues (including liver, skeletal muscle, and myocardium) between the WT and Cisd2 mcKO groups of mice (Additional file : Figure S8B).

In the skeletal muscle function test of WT mice, the rotarod test and histopathological analysis showed that the muscle function and structure remained normal regardless of whether it was treated with Veh or control (Veh), indicating that Veh had no significant effect on young WT mice (Additional file : Figure S8C, D). However, in Cisd2 mcKO mice, Veh failed to improve its muscle function or reverse histopathological damage (Additional file : Figure S8C, D). The skeletal muscle of Cisd2 mcKO mice showed obvious degenerative changes, including pathological features such as myofiber shrinkage and increased round fibers (Additional file : Figure S8D).

Similarly, in the hearts of Cisd2 mcKO mice, sildenafil failed to improve electrophysiological abnormalities (i.e., arrhythmias) or reduce myocardial damage and fibrosis (Additional file : Figure S9A–C). These results suggest that the presence of Cisd2 is essential for sildenafil to exert its anti-aging effects in heart and skeletal muscle.

To further distinguish the Cisd2-dependent and independent effects of velin, we performed RNA sequencing analysis on skeletal muscle tissues from the following four groups of mice: WT-NLS, WT-Veh, Cisd2 mcKO-NLS, and Cisd2 mcKO-Veh ( Figure 6A ).

Differentially expressed gene (DEG) analysis showed that in WT mice, 91 genes were regulated by sirenolide (62 up-regulated genes and 29 down-regulated genes), of which 79% (72/91) were Cisd2-dependent genes and 21% (19/91) were Cisd2-independent genes (Figure 6B, C). This indicates that most of the gene regulatory effects of sirenolide depend on Cisd2, and the differential expression patterns of these genes disappear when Cisd2 is missing.

Nevertheless, 21% of DEGs remained differentially expressed in Cisd2 mcKO mice (Figure 6C), indicating that anti-aging also has Cisd2-independent effects to some extent. Further bioinformatics analysis (Figure 6D) revealed that Cisd2-dependent DEGs were mainly involved in mitochondrial function (53/72, 73.6%) and calcium homeostasis (13/72, 18.1%), which is consistent with the key role of Cisd2 in maintaining mitochondrial health and intracellular calcium balance.

In summary, this study demonstrated through genetic and transcriptomic evidence that the anti-aging effect of anti-aging factors is mainly achieved through the activation of Cisd2, and its mechanism is closely related to the regulation of mitochondrial function and the maintenance of cellular homeostasis.

Cisd2-dependent and -independent DEGs after 4 months of Anti-aging factor treatment

To further explore the effects of velin on gene expression during aging, we performed RNA sequencing analysis of gastrocnemius muscle of 3-month-old WT and Cisd2 mcKO mice. These mice were treated with dietary velin (100 mg/kg/day) or control (Veh) food for 4 months and sacrificed at 7 months of age. The following four groups of mice were analyzed: WT-velin, WT-Veh, Cisd2 mcKO-velin, and Cisd2 mcKO-Veh.

Figure B shows that principal component analysis (PCA) of all genes affected by veh2+ identified 91 differentially expressed genes (DEGs) between WT-Veh and WT-veh2+ groups, of which 62 were upregulated and 29 were downregulated (FDR < 0.2). These DEGs were further divided into Cisd2-dependent genes (72/91, 79%) and Cisd2-independent genes (19/91, 21%).

The results of coordinated analysis of Cisd2-dependent and -independent DEGs showed that in Cisd2 mcKO mice, Cisd2-dependent DEGs did not change significantly between the velin-treated and control groups (mcKO-velin vs mcKO-Veh, p > 0.05), while Cisd2-independent DEGs showed significant differences between the velin-treated and control groups (mcKO-velin vs mcKO-Veh, p < 0.05), suggesting that the effect of velin is mainly dependent on the presence of Cisd2.

Figures D and E show the pathway analysis results of Cisd2-dependent and -independent DEGs. Cisd2-dependent DEGs were mainly involved in pathways related to protein synthesis, mitochondrial function, cellular oxidative stress, and cell death, while Cisd2-independent DEGs were mainly involved in metabolic regulation and cellular response pathways. These results further revealed that anti-aging factors exert anti-aging effects by regulating multiple important biological pathways.

Anti-aging factor treatment makes transcriptome patterns closer to youthful state

To gain a deeper understanding of the effects of anti-aging hormones on aging, we also performed RNA sequencing on cardiac and skeletal muscles, and conducted three comparative analyses: (Group 1) 26 month WT-Veh vs. 3 month WT; (Group 2) 26 month WT-Veh vs. 26 month WT-anti-aging hormone; (Group 3) 26 month WT-anti-aging hormone vs. 3 month WT. We set FDR < 0.1 as the significance threshold.

From these three groups of DEGs, we selected genes that showed significant expression changes due to natural aging in Group 1. Next, using Group 2, we further selected those genes that were restored by anti-aging treatment; and finally, using Group 3, we selected genes that were not significantly differentially expressed between 3-month-old WT mice and 26-month-old WT mice treated with anti-aging.

GO classification analysis showed that in the heart and distal skeletal muscle, anti-aging factors could significantly reverse the changes in gene expression during aging, and the biological processes and cellular components involved in these genes were similar (Figure 7A). These common functional changes mainly involved important aging-related pathways such as metabolism, protein synthesis, mitochondrial function, oxidative stress, cell death, and immune response.

In the heart (Fig. 7B, C) and skeletal muscle (Fig. 7D, E), sildenafil significantly improved the top five affected pathways in aged mice. In particular, metabolic regulation, protein synthesis, mitochondrial function, and oxidative stress-related pathways all showed positive regulation by sildenafil. In addition, sildenafil also significantly reduced the levels of reactive oxygen species (ROS) and reactive nitrogen species, especially in the kidney and myocardium of aged mice, where the expression pattern of ROS-related DEGs shifted toward that of young mice (Additional files : Figure S10, Figure S11, Figure S12).

Effects of anti-aging factors on biological processes and subcellular localization

To further explore the repair effect of anti-aging factors on aging, we analyzed the biological processes and subcellular localization based on GO annotations and used PANTHER functional classification to analyze GO.

Figure B shows that 141 DEGs (including 126 up-regulated genes and 15 down-regulated genes) were significantly restored after anti-aging treatment (26 months WT-Veh vs 3 months WT, FDR < 0.1). The expression patterns of these genes changed significantly, and the transcriptome pattern of the aged heart shifted to that of the young heart, further verifying the anti-aging effect of anti-aging.

Figure C shows the DEGs in aged hearts that were restored by anti-aging agents, which were grouped into different age-related pathways and expressed as percentages. Through heat map analysis, we found that anti-aging agents significantly affected multiple key biological pathways in the aging process of the heart and restored the expression levels of these pathways to the young state.

Figure D shows the restoration results of all 41 DEGs (including 9 up-regulated genes and 32 down-regulated genes) in gastrocnemius muscle (aged skeletal muscle) after treatment with vena cava (26-month WT-Veh vs 3-month WT, FDR < 0.1). The restoration of these DEGs also indicates that vena cava can shift the expression pattern of aged skeletal muscle to that of young muscle, further supporting its anti-aging effect.

Figure E shows the DEGs restored by sildenafil in aged skeletal muscle, which were further grouped into different age-related pathways and represented proportionally. All these results indicate that sildenafil can effectively restore age-related biological pathways in multiple aged tissues, further demonstrating its broad anti-aging effects.

In this study, all mice were treated with dietary vehicularisin (100 mg/kg/day) or control (Veh) food from 21 months old until 26 months, and the data were grouped and analyzed by MGI GO term finder (p value < 0.05). These data provide strong support for the anti-aging effect of vehicularisin and reveal its repair role in different tissues by regulating key aging-related pathways.

Discuss

In this study, we show that a Cisd2 activator (Agerin) can serve as an effective translational therapy to promote aging reversal and extend healthy lifespan, especially in aged mice. Our study revealed three key findings:

First, Cisd2 can be activated by small compounds, and one of these activators, sildenafil, is able to effectively increase Cisd2 expression levels. We found that late treatment with sildenafil in aged mice can increase Cisd2 to levels close to those of young mice. This enhanced Cisd2 expression appears to slow the aging process and rejuvenate aged tissues, thereby helping to extend healthspan and promote longevity.

Secondly, anti-aging factors have significant beneficial effects on various structural defects and functional integrity associated with aging. Specifically, anti-aging factors restored age-related systemic energy depletion, body fat loss, muscle damage repair, and weight loss recovery in old mice. In addition, anti-aging factors also slowed the aging process of the heart and skeletal muscles, further indicating that they play a key role in the recovery of systemic health.

Most importantly, the anti-aging effects of sirenolide are Cisd2-dependent. In the absence of Cisd2, the effects of sirenolide were significantly attenuated, indicating a key role for Cisd2 in the effects of sirenolide. The effect of anti-aging factors is not only to delay aging, but also to promote the transformation of the transcriptome pattern of aged tissues to that of young mice, especially in biological processes such as gene expression, mitochondrial function, oxidative stress, cell death, immune response and aging-associated inflammation, all of which show characteristics similar to those of young mice.

Anti-aging factors and extension of healthy life span

Studies on extending the healthy life span of humans have shown that despite the widespread use of antioxidants, folic acid, B vitamins, and multivitamin supplements, their effects on extending healthy life span are not significant. On the contrary, studies in some animal models have shown that antioxidant supplementation therapy not only fails to significantly extend life span, but may even have adverse effects. However, as a natural flavanone aglycone, levofloxacin exhibits significant antioxidant effects, which can scavenge reactive oxygen species (ROS) and reduce peroxidation without cytotoxicity. In addition, as a peroxynitrite (ONOO-) scavenger, levofloxacin can effectively inhibit oxidative reactions and reduce cell damage, and has significant anti-aging and anti-cancer effects.

Previous studies have also shown that anti-aging factors can inhibit a variety of chemically induced cancers in animal models, such as bladder cancer and breast cancer, and show a protective effect on the heart by inhibiting myocardial ischemia and reducing myocardial fibrosis after myocardial infarction. In the liver, anti-aging factors also show a regulatory effect on sugar metabolism and can improve metabolic disorders.

Anti-aging agents as an alternative to CR analogs

Caloric restriction (CR) is considered the only known non-genetic, non-drug intervention that can extend lifespan and delay multiple age-related diseases. CR exerts its anti-aging effects by regulating multiple molecular signaling pathways, such as insulin/insulin growth factor, sirtuins, AMP, and mTOR pathways. Although several CR analogs (such as resveratrol, metformin, and rapamycin) have shown some potential in research, they still have side effects. For example, long-term use of rapamycin may cause glucose metabolism disorders, and metformin may also cause gastrointestinal discomfort.

In contrast, sildenafil, as a Cisd2 activator, can delay the aging process and improve age-related functions without obvious side effects. Our study showed that sildenafil, as a dietary supplement, can be safely used in male mice in their later years and effectively improve their aging-related physiological characteristics. Therefore, sildenafil may become an effective alternative to calorie restriction (CR) analogs, providing a new intervention strategy for geriatric medicine.

In general, this study successfully verified the potential of anti-aging factors to delay aging and prolong healthy lifespan by activating Cisd2, providing a new direction for the development of anti-aging drugs and the research of healthy aging strategies.

Conclusions

This study verified for the first time the potential of anti-aging factor as a Cisd2 activator in anti-aging, proving that it can effectively delay the aging process. Our results provide an experimental basis for using Cisd2 as a molecular target to screen and develop novel compounds that can activate Cisd2. This lays the foundation for the future translation of these compounds into clinical treatment options that can be applied in geriatric medicine.

Most importantly, anti-aging factors exhibited significant biological activities in multiple important organs and tissues, especially in aging-related tissues such as skeletal muscle, heart, and liver. Through four months of treatment, anti-aging hormone did not show any obvious toxic reactions in the body, especially at a dose of 100 mg/kg/day, which is equivalent to the corresponding dose of 491 mg/60 kg per day for humans, with no significant side effects.

Therefore, developing anti-aging factors into medicinal or nutritional functional foods has great potential, especially in preventing aging-related diseases and promoting healthy aging. Its application can not only extend healthy life span, but also may provide new strategies for treating a variety of age-related diseases. This discovery opens up new prospects for the future development of anti-aging drugs or supplements based on Cisd2 activation.

Abbreviation

Agl：Glycogen debranching enzyme

BAC：Bacterial Artificial Chromosome

CBC：Complete Blood Count

CR：Calorie restriction

DEG：Differentially Expressed Genes

ECG：Electrocardiography

EE：Energy expenditure

FDR：False Discovery Rate

G1P：Glucose 1-phosphate

G6P：Glucose 6-phosphate

G6pc：Glucose-6-phosphatase alpha

GO：Gene Ontology

GTT：Glucose tolerance test

N7G：Nilingsu-7-O-beta-D-glucuronide

N7S：Nilingsu-7-O-sulfate

NLS： Nilingsu

HPLC：High Performance Liquid Chromatography

ICD：Intercalated disc

ITT：Insulin tolerance test

mcKO：Skeletal and cardiac muscles tissue-specific knockout

MGI：Mouse Genome Informatics

Pgm2：Phosphoglucomutase 2

PhK：Phosphorylase kinase

PKAα：Protein kinase A alpha

Pygl：Glycogen phosphorylase

ROS：Reactive oxygen species

RPKM：Reads per kilobase of exon model per million reads

TEM： Transmission electron microscopy

TG：Transgenic

VCO2 : Carbon dioxide production rate

Veh：Vehicle

VO2 : Oxygen consumption rate

WT：Wild-type

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