Rotating magnetic field delays human umbilical vein endothelial cell aging and prolongs the lifespan of Caenorhabditis elegans

RMF characterization

The entire experimental equipment consisted mainly of natural magnets (Figure 1A). The experimental material was placed 6 cm (0.2 T) from the motor-driven rotating magnet, which produced a 4 Hz magnetic field change frequency. The frequency of the magnet rotation and the timing of the changes in the magnetic field direction were constant and the entire experimental setup provided an environment for culturing the cells (Figure 1B, 1C).

Figure 1. Characterization of rotating magnetic field and experimental setup. (A) Experimental setup for treatment of Caenorhabditis elegans and cells with a rotating magnetic field (RMF). C. elegans and cells were positioned above the RMF generator and exposed to RMFs of different amplitudes consisting of two overlaying components: translational (with varying inversion time) and rotational (with varying rotational frequencies). (B, C) B(t, ω) represents the magnetic field induction as a function of time; B0 represents the amplitude; Fτ represents the contribution of the translational movement of different inversion time; and ωt represents the contributing rotation frequency.

RMF delayed HUVEC aging

In the logarithmic growth phase of cells, HUVECs were cultured in high-glucose (40 mM) to construct a cell senescence model. As a classic anti-aging drug, metformin (20 μM) was used as a positive control. HUVECs were exposed to RMF for 2 h/d and 4 h/d and cell senescence was measured after 3 d. The number of SA-β-Gal-positive cells decreased with increasing RMF exposure time (Figure 2A, 2B. P<0.01), and flow cytometric analysis revealed that the number of apoptotic cells was decreased (Figure 2C, 2D. P<0.01). Western blot analysis showed that RMF exposure increased AMPK protein expression, and decreased expression of P21, P53 and mTOR proteins in a time-dependent manner (Figure 2E). The results indicated that RMF delays the senescence of HUVECs by upregulating signaling via the AMPK pathway and downregulating the mTOR pathway.

Figure 2. Rotating magnetic field exposure delays HUVEC senescence. (A, B) HUVECs were exposed to RMF daily for 0 h, 2 h, and 4 h; 20 μM metformin was used as a positive control. SA-β-Gal staining was performed and the number of β-Gal-positive (blue) cells was calculated as a percentage of the total cell number using Image J software. (C, D) Flow cytometry was used to detect the apoptosis of HUVEC after RMF treatment. (E) Western blot analysis showed that RMF exposure increased in AMPK protein expression and decreased P21, P53 and mTOR protein expression.

HUVEC genome sequencing

To broadly identify the mechanisms underlying RMF-induced longevity, we performed RNA sequencing analysis of HUVECs with or without exposure to RMF for 4 h. Transcriptome volcano map analysis showed that, after 4 h of RMF treatment, 2,340 gene were upregulated genes and 656 downregulated genes (Figure 3A). GO and KEGG analysis indicated that the downregulated genes were mainly involved in centrosomes, cell projection assembly, and ATP activity, suggesting that RMF adjusted the energy metabolism of HUVECs (Figure 3B, 3C). In addition, we speculated on the possible protein pathways and interactions (Figure 3D, 3E). The results showed that RMF influences the senescence of HUVECs by affecting the activity of ATPase activation, centrosomes, and cell projection assembly.

Figure 3. RMF extends the life of HUVECs in a multi-target manner. (A) Volcano map showing the RNA-seq expression pattern in HUVECs exposed to RMF for 4 h compared to that of untreated control cells. Red indicates a higher expression level and green indicates a lower expression level. The distance of the dot from the x-axis reflects the size of the P-value. (B, C) Gene enrichment map in HUVECs exposed to a RMF for 4 h compared to untreated control cells. (D) Signal transduction pathways affected by RMF. (E) Predicted protein interactions.

RMF improved Ca²⁺ channel permeability in the endoplasmic reticulum and decreases ATP production

To further explore the mechanism of RMF resistance to endothelial cell senescence, we examined the levels of Ca²⁺ and ATP. The results showed that the concentration of Ca²⁺ was obviously increased in glucose-induced senescent HUVECs after RMF treatment. The intracellular Ca²⁺ content changed very fast due to magnetic field interference, and the increase in Ca²⁺ concentration slowed as the RMF treatment time increased (Figure 4A. P<0.01). There was no significant difference between the effects of exposure to RMF for 4 min and 2 min, indicating that the RMF-induced increase in intracellular Ca²⁺ concentration in HUVECs reached saturation at a rapid rate within 4 min. In this study, we used the endoplasmic reticulum calcium channel-specific inhibitor, BAPTA (25 μM). There was no significant increase in Ca²⁺ content in the inhibitor-treated group compared to that in the control group, indicating that the calcium channel in the endoplasmic reticulum is a magnetic field target. Subsequently, changes in Ca²⁺ concentration in the same batch of HUVECs were detected by continuously turning the magnetic field on and off at intervals of 4 min (Figure 4B. P>0.01). When the RMF was turned on, the intracellular Ca²⁺ concentration increased, whereas when the RMF was turned off, the intracellular Ca²⁺ concentration slowly decreased, which provides evidence that the calcium ion channel in the endoplasmic reticulum is a sensitive target of the magnetic field. Thereafter, we examined changes in the MMP by flow cytometric analysis of JC1-stained HUVECs. The results showed that as the intracellular Ca²⁺ concentration increased (Figure 4D, E. P<0.01), the MMP difference and intracellular ATP production decreased (Figure 4C. P<0.01). These findings indicated that RMF reduces mitochondrial activity by increasing the intracellular Ca²⁺ concentration.

Figure 4. RMF induced Ca²⁺ outflow from the endoplasmic reticulum to reduce intracellular MMP and ATP levels. (A) HUVECs were treated with RMF for 0 min, 2 min, 4 min and 4 min with a calcium channel inhibitor (BAPTA). After the addition of the Ca²⁺ probe Fluo-4-AM, the intracellular Ca²⁺ concentration was detected by flow cytometry. (B) The intracellular Ca²⁺ concentration in the same cells was detected following RMF treatment at 4-min intervals. (C) HUVECs were treated with RMF for 0 h, 2 h, 4 h, and 8 h. The intracellular ATP content was then detected. (D, E) After RMF treatment of HUVECs at 0 min, and 4 min in the presence and absence of the endoplasmic reticulum calcium channel inhibitor (BAPTA), changes in MMP levels were detected by flow cytometric analysis of the MMP dye, JC-1.

RMF increased the lifespan of C. elegans and reduced damage under stress conditions

We found that the levels of Ca2+ and ATP in the C. elegans exposed to RMF were consistent with HUVECs (Figure 5A, 5B). Lifespan is the most intuitive indicator to study the effects of magnetic fields on Aging evaluative systems. Because of its short growth cycle, ease to observation and high degree of gene homology with humans, C. elegans is an ideal model organism to study the effect of RMF on lifespan. In this study, exposure of C. elegans to RMF at 1 h/d, 2 h/d and 4 h/d revealed that RMF significantly increased the average lifespan by 0.6%, 13.99% and 8.39%, respectively, compared with the control (Table 1), with the most significant effect observed following 2 h/d RMF exposure (Figure 5C, Table 1. P<0.01); therefore, 2 h/d RMF was used for subsequent experiments.

Figure 5. RMF increased the lifespan of C. elegans and reduced damage under stress conditions. (A, B) Changes of Ca2+ and ATP levels in C. elegans after RMF treatment. (C) The lifespan of 240 N2 C. elegans eggs following RMF treatment for 0 h/d (control), 1 h/d, 2 h/d and 4 h/d was recorded. (D) The lifespan of 240 N2 C. elegans eggs following RMF treatment for 0 h/d (control) and 2 h/d in the presence and absence of glucose was recorded. (E) Worms were treated with RMF for 0 h/d (control) and 2 h/d at 20°C for 4 days, and then exposed to thermal shock at 37°C. (F) Curves showing the non-paralyzed fraction nematodes in each group following treatment of C. elegans eggs with RMF for 0 h/d (control) and 2 h/d. CL4176 C. elegans were raised from the egg stage to the L3 stage at 15°C and then transferred to an environment at 25°C.

Glucose causes developmental retardation and shortens the lifespan of C. elegans; therefore, we investigated the effect of RMF on the longevity of C. elegans exposed to toxic glucose stimuli. We observed that RMF attenuated the toxic effects of glucose on C. elegans, with the average life increased by 14.63% (Figure 5D, Table 1. P<0.01) compared with that of the control.

The resistance of C. elegans to external stimuli decreases dramatically with age. To determine the ability of RMF to increase the resistance of C. elegans to heat stress, we analyzed the survival of day 7 C. elegans treated with 2 h/d RMF under heat stress conditions at 37°C. We found that RMF preconditioning had a significant protective effect against this acute stress (Figure 5E. P<0.01), indicating that RMF increased the resistance of C. elegans to heat stress. Increased longevity has been reported to be closely correlated with the prevention of age-related diseases. Extensive experimental studies have shown that accumulation of β-amyloid (Aβ) in Alzheimer’s disease is a core event that triggers neuronal degeneration. In CL4176 C. elegans, the smg-1 system is inactivated by raising the temperature, resulting in high Aβ 1–42 expression in muscle tissue and formation of a polymer. The mutant CL4176 cultured under RMF showed a significant anti-disease effect compared to that observed in the control group. The average lifespan of CL4176 was extended by 19.32% (Figure 5F, Table 2. P<0.05). These results showed that RMF increases the lifespan of C. elegans and reduces the damage under stress conditions.

RMF improved C. elegans healthspan

Healthspan is a new term for the period during which adult activity and function remain intact prior to age-related decline. As the incidence of age-related diseases increases, the impact of current life-extending methods on health is unclear. To determine whether RMF simply extends life or merely youth and health, we assessed the effects of RMF on aging-related physiological functions, including head-swinging, body-bending, food-intake, and distance moved. During aging, muscle cells gradually lose their vitality, leading to decreased exercise ability and pharyngeal pump function as well as changes in other phenotypes. We measured pharyngeal pump volume and body curvature in RMF-treated animals on days 6, 10, and 14, respectively, as indicators of physiological changes. RMF significantly increased the flexibility of body-bending and the frequency of pharyngeal muscle contraction (Figure 6A–6D. P<0.01). Furthermore, RMF had a slight but not significant increase in N2 C. elegans food-intake measured in day 10, suggesting that its effect on longevity may not be dependent on the dietary restriction (DR) pathway (Figure 7A. P>0.05). Interestingly, food-intake increased significantly in RMF-treated C. elegans under high-glucose conditions, indicating the involvement of other mechanisms that remain to be explored. Previous studies have shown that gonadal function declines with age. Prolonging the lifespan of C. elegans may have reproductive side-effects. RMF treatment had no effect on brood size, suggesting that RMF-induced longevity was not dependent on reproductive signaling pathways (Figure 7B. P>0.05). These results showed that RMF exposure significantly prolonged the youthful vigor and healthy life of C. elegans. Subsequently, we showed that the decrease in ATP levels observed in RMF-treated C. elegans reflected that in HUVEC (Figure 7C. P<0.01).

Figure 6. RMF improved lifespan in C. elegans. (A) Head-swing frequency. (B) Body-bend frequency. (C) The four levels of locomotivity. Motor ability assays were performed on day 6, 10, and 14. (D) Pharyngeal-pumping frequency (20 s). Different letters indicate a significant difference among groups (P < 0.05). All analyses were based on the data from three independent experiments.

Figure 7. The effects of RMF on ROS and ATP levels and C. elegans physiology. (A) C. elegans food-intake. (B) C. elegans brood size. (C) Total ATP content in C. elegans was detected using a microplate reader. (D, E) The ROS levels in C. elegans were detected with the H2DCF–DA probe by fluorescence microscope and microplate reader.

RMF reduced ROS levels in C. elegans via a mev-1-dependent pathway

In addition to the ROS produced during metabolism, the endogenous ROS levels are increased by adverse external stimuli accelerate the aging process. Furthermore, studies have shown that C. elegans produces large amounts of ROS in high-glucose environments [55]. To investigate the ability of RMF to promote antioxidant mechanisms, including clearance of ROS, we used H2DCF-DA as a free radical probe to compare ROS levels in C. elegans exposed to RMF for 10 days and those in the control group. The results showed that RMF pretreatment had a significant protective effect against the increased ROS production induced by a high-glucose stress environment, indicating that the increased lifespan induced by RMF treatment was related to reduced ROS accumulation and enhanced resistance of C. elegans to oxidation (Figure 7D, 7E, P<0.01). Studies have shown that the mev-1 gene affects the expression of antioxidant enzymes. The mev-1 mutant C. elegans carries a defective complex II subunit in the electron transport chain (ETC), which significantly shortens life at high oxygen concentrations. Thus, we investigated the possible antioxidant effects of RMF on TK22 C. elegans, which carries a mutation in the mitochondrial complex II cytochrome B large subunit. There was no significant difference in ROS levels between the two mev-1-mutation carrying forms of C. elegans, suggesting that the mev-1 gene is critically involved in the mechanism by which RMF extends life.

RMF reduced the accumulation of fat and age-related pigments in C. elegans

Age-related pigments are a biomarker of aging of C. elegans and humans. In this study, we employed a fluoroenzyme marker to show that the fluorescence intensity of age-related pigments was significantly reduced in C. elegans on day 10 and 14 after RMF treatment (Figure 8A, 8B. P<0.01).

Figure 8. RMF reduced the accumulation of fat and age-related pigments in C. elegans. (A) Representative image of age-related pigments in N2 C. elegans; scale bar = 100 μm. (B) The fluorescence of the fat normalized to tryptophan fluorescence was measured using a microplate reader. (C) The fluorescence of the age-related pigment normalized to tryptophan fluorescence was measured using a microplate reader. (D) Representative image of fat in N2 C. elegans; scale bar = 100 μm. Both experiments were performed on four independent four occasions (days 6, 10 and 14 after the start of RMF treatment). Different letters indicate a significant difference among groups (P < 0.05).

In C. elegans, an increase in ROS production leads to a marked fat accumulation. Considering our previous observation that RMF effectively reduced ROS accumulation, we next investigated the effects of RMF and glucose pretreatment on fat accumulation in N2 C. elegans. At day 6, 10, and 14 after RMF treatment, by oil red O staining showed that fat accumulation was reduced by 2 h/d RMF compared with the control (Figure 8C, 8D. P<0.01).

RMF improved autophagy

Autophagy is the phagocytosis of cytoplasmic proteins or organelles in a process that occurs in vesicles, which fuse with lysosomes to form autophagosomes. The contents of these organelles are then degraded, thereby realizing the metabolic needs of cells and the renewal of some organelles. Numerous studies have demonstrated that abnormalities in autophagy function are closely related to the development of age-related diseases such as Alzheimer’s disease. Bec-1 is a key positive regulator of the level of autophagy. LGG, which is the homolog of mammalian LC3, is one of the well- described and widely available of the autophagy matrices and is used as a marker for autophagy measurement because of its involvement in the formation of autophagic vacuoles. In this study, we found that bec-1 mRNA levels were increased 2-fold compared with those in the control group after 10 days of 2 h/d RMF treatment, (Figure 9A), and LGG mRNA levels were increased 2.5-fold compared with those in the control group (Figure 9B, P<0.01). The effect of RMF on autophagy activity was assessed in the DLM1 strain, which expresses the LGG-1-GFP fusion protein in vivo. The results showed that both RMF and glucose treatment increased autophagy in C. elegans (Figure 9C, 9D, P<0.01). The relationship between autophagy and aging is controversial; however, our studies showed that RMF induces an appropriate autophagy pathway to prolong life and reduce Aβ-induced toxic effects in C. elegans.

Figure 9. Autophagy was involved in lifespan extension and protection against Aβ-induced toxicity by RMF. (A, B) The lgg-1 and bec-1 gene transcript levels in CL4176 C. elegans. (C, D) dFP::LGG-1 was detected by laser confocal microscope (Leica-sp5ii, TCS SP5II, Shenzhen).

RMF prolonged the lifespan of C. elegans via the insulin/IGF-1 signaling pathway

Many studies have shown that insulin/igf-1 signaling pathway (IIS) is involved in regulation of biological process such as the life cycle in C. elegans. After insulin-like peptide ligands bind to the insulin/igf-1 transmembrane receptor (IGFR)-derived daf-2, daf-2 ultimately regulates FOXO transcription by controlling the phosphoinositide 3-kinase (PI3K)/Akt kinase cascade. Factor daf-16, then controls the life cycle and neurodegeneration process. To determine the role of the IIS pathway in the mechanism by which RMF increases longevity, we investigated the ability of RMF to promote daf-16 transcription and translation. Under high-glucose conditions, 2 h/d RMF significantly increased daf-16 protein levels in C. elegans. In addition, real-time quantitative PCR analysis showed that RMF increased daf-16 mRNA levels in C. elegans (Figure 10A–10C. P<0.01). Considering the antioxidant effect of RMF on C. elegans, we subsequently investigated the influence of RMF on SOD-3 (a downstream antioxidant gene in the IIS pathway). Quantitative real-time PCR analysis showed that SOD-3 mRNA levels were significantly increased in C. elegans following RMF exposure. Furthermore, evaluation of SOD-3 antioxidant enzyme protein levels in transgenic SOD-3::GFP (CF1553) revealed that SOD-3::GFP were increased following RMF treatment compared with the levels in the control group (Figure 10D–10F. P<0.01). Subsequently, we further investigated the involvement of other age-related genes (age-1, ctl-1, sir-2.1, hsp-16.2, hsp-16.1, skn-1, gst-4, and gcs-1) in the mechanism by which RMF increases longevity. We found that mRNA levels of sir2.1, ctl-1, hsp-16.1, hsp-16.2 and gcs-1 were significantly increased after RMF treatment. These results indicated that the effect of RMF on longer life span and increased stress resistance depends in part on the promotion of related stress-inducing genes (Figure 11A). Hsf-1 is a heat shock transcription factor known to play an important role in regulation of the insulin/igf-1 signaling pathway (IIS). According to previous reports, hsf-1 can downregulate the IIS pathway to further extend lifespan. The eat-2 gene plays a role in regulating the post-synaptic pumping rate of the pharyngeal muscle and is closely related to the DR? pathway. In this study, we evaluated the influence of RMF on the lifespan of daf-2 (CB1370), daf-16 (GR1307), hsf-1 RMF-1 (AGD794) and eat-2 (DA1116) mutants. The results show that hsf-1 is not required for the function of RMF and the DR pathway is not involved (P>0.05). This is consistent with the results of the food-intake experiment, indicating that RMF extends lifespan via the IIS pathway (Figure 11B, Table 3). Finally, we summarize the molecular mechanisms by which RMF interacts with HUVEC and infer the RMF action site and the magnetic induction pathway (Figure 11C).

Figure 10. RMF promoted the expression of daf-16 and SOD-3. (A) The daf-16 transcript level. (B) Expression of daf-16::GFP observed under a fluorescence microscope (C) The relative fluorescence intensity of the daf-16::GFP was quantified using ImageJ software. (D) The SOD-3 transcript level. (E) Expression of SOD-3::GFP expression observed under a fluorescence microscope. (F) The relative fluorescence intensity of the SOD-3::GFP was quantified using ImageJ software.

Figure 11. The molecular mechanism underlying the anti-aging effect of RMF. (A) The mRNA levels of hsp-16.1, hsp-16.2, age-1, sir-2.1, ctl-1, skn-1, gst-4 and gcs-1 were determined by quantitative real-time RT-PCR and normalized to the expression of act-1. At least 3000 C. elegans were used in each group and the experiments were performed on three independent occasions. Means with different letters were significantly different at P < 0.05 for each gene. (B) RMF extended lifespan of the hsf-1 (AGD794) and eat-2 (DA1116) mutants. RMF did not extend lifespan of the daf-2 (CB1370) and daf-16 (CR1307) deletion mutants. The lifespan analysis of each mutant was performed on three independent occasions. (C) A possible mode of action of the longevity extension mediated by RMF.

RMF system

RMFs were generated by the Rotating Magnetic Bed System (RMBS; designed by Prof. Xiaoyun Zhang University of Shenzhen, China), equipped with a permanent magnet capable of generating up to 0.2 T RMF. The RMBS was equipped with a closed glass chamber, which included a heating element and a gas-holding device to maintain a defined environment for C. elegans and cells. C. elegans were maintained according to standard protocols.

C. elegans

The following strains were used in this study: the wild-type N2, Bristol (wild-type); GR1307, daf-16 (mgDf50); CB1370, daf-2 (e1368); TK22, mev-1 (kn1); AGD794, hsf-1; DA1116, eat-2; DLM1, dFP:: LGG-1; TJ356 [zIs356IV(daf16p::daf16a/b::GFP+rol6 (su1006))]; CF1553 [(pAD76)SOD-3::GFP+rol6 (su1006)]; and CL4176 [(pAF29) myo-3p::Aβ1-42+(pRF4)rol6(su1006)].

Cell culture

HUVECs from neonatal umbilical cord were washed three times with phosphate-buffered saline (PBS) and digested with 0.2% type II collagenase at room temperature for 15 min. After centrifugation for at 1,200 r/min for 5 min, cells were resuspended in complete medium in the process of the temperature after abandon supernatant (800 mL/L M199, 200 mL/L FBS, 20 μg/mL ECGS, 1 mmol/L glutamine) heavy suspension cells, beat after blending in cultivation in the bottle. Passage 1–2 cells were used for follow-up experiments.

Lifespan measurement

Lifespan was measured in 3.5 cm tissue culture plates containing 10 μM 5-fluoro-2′-deoxyuridine (FUDR). C. elegans at the young adult stage were cleaned and collected for disruption with 5% sodium hypochlorite solution (composed of 0.4g NaOH, 4mL H2O, 2mL 10% bleach), and cleaned with M9 (1LH2O, 3gKH2PO4, 6gNa2HPO4, 5gNaCl and 1ml 1molLMgSO4) solution three times. The eggs and M9 were then transferred to the 3.5 cm tissue culture plates for synchronization. The synchronized C. elegans were grown to the adult stage on the NGM (2.5g peptone, 3.0g NaCl, 17g agar powder and 975ml water were sterilized by high temperature and cooled to 55 degrees Celsius. Stirred CaCl2, MgSO4, cholesterol solution and PBS were shaken to the plate) plate inoculated with Escherichia coli OP50, and were then were transferred to plates containing E. coli OP50 alone, and maintained at 20°C as described previously [62]. Subsequently, C. elegans were transferred to a new petri dish every 2–3 days until no response to mechanical stimulation was observed, when the nematodes were presumed dead. The significance of differences in the survival curves were evaluated by log-rank tests. There were more than 100 C. elegans in each group, and each experiment was repeated on at least three independent occasions. Lifespan tests were carried out 20°C, and heat shock experiments were carried out at 37°C.

Healthspan evaluation

The responses of nematodes to radiation and physical stimuli were evaluated every two days and death was determined by the lack of response. To measure head-swing and body-bending frequencies as well as locomotivity, C. elegans maintained on NGM plates without food were treated with or without RMF 2 h/d for 6, 10, and 14 d. This assay was repeated on two independent occasions. Each group included at least 30 C. elegans [63, 64].

Brood size

The incubation size of an adult worm was measured. Starting from the L1 stage, C. elegans were first treated with RMF for 2 h/d, and then maintained on NGM plates with E. coli OP50 at 20°C for 72 h to allows each worm to lay eggs. The eggs were then allowed to hatch and counted. The experiment was repeated on three independent occasions.

Measurement of ROS accumulation

Endogenous ROS levels were measured using 2′,7′-dichlorofluorescein diacetate (H2DCF–DA). On day 10 after RMF2 h / d treatment, one hundred C. elegans were incubated with 10 μM H2DCF-DA for 30 μ min at 37 °C. Fluorescence intensity (excitation and emission wavelengths of 485 and 535 nm, respectively) was measured. The assay was performed on three independent occasions.

Measurement of fat accumulation

To investigate the effect of 2 h / d RMF treatment on fat accumulation, RMF was treated for 7 days, 10 days, and 14 days, followed by oil red O staining. The C. elegans were then immobilized under a fluorescence microscope and visualized with paraformaldehyde. The fat accumulation test was repeated three times. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the images and calculate the total number of C. elegans (at least 60 in each group).

Analysis of cytosolic Ca²⁺

HUVECs were seeded in culture dishes (35 mm thin-bottomed and 7 mm low wall; Ibidi, #80136, Germany) and cultured for three generations. HUVECs were loaded with 2 mM Fluo-4-AM (Solarbio, F8500, China), cell clear fluorescent Ca²⁺ probe and 0.04% pluronic F-127 in HEPES-buffered Tyrode solution (HEPES 10 mM, KCl 2.8 mM, NaCl 129 mM, MgCl₂ 2 0.8 mM, NaHCO 3 8.9). M1, KH₂PO₄ 0.8 mM, glucose 5.6 mM, CaCl₂ 1.2 mM and 0.1% BSA, pH 7.4 were incubated at 37°C as described previously [65]. Then, the myotubes were washed with buffer and incubated for an additional 40 min at room temperature. The acetoxymethyl ester (AM) group was cleaved by intracellular esterase. The endoplasmic reticulum calcium channel inhibitor BAPTA (MCE, HY-100168, China) was added during the AM cutting phase. HUVECs were then analyzed at 37°C. Cells were placed 6 cm above the magnetic coil and exposed to RMF using a 20 min kinetic cycle. Three RMFs were applied during this cycle: 0–4 min (interval 1), 8–12 min (interval 2) and 16–20 min (interval 3); these three intervals were marked as “ON”. In between, the RMF was switched off for 4 min; this interval was marked as “OFF”. Changes in cytosolic Ca²⁺ were monitored using an automated inverse fluorescence microscope (Ti-E, Nikon, Japan) and flow cytometry (BD C6, USA) at an excitation wavelength (Ex) of 490 nm and an emission wavelength (Em) of 515 nm.

Mitochondrial membrane potential detection

Changes in MMP were evaluated using a MMP detection kit (JC-1) (Solarbio, M8650). HUVECs were exposed to RMF for 0 h, 4 h, 4 h plus BAPTA. After treatment, the medium was removed and the cells were washed once with PBS before adding fresh medium and JC-1. Cells were incubated at 37°C for 20 min before the JC-1 staining buffer was removed by washing twice. The cells were digested and analyzed by flow cytometry.

Total ATP detection

The ATP content of C. elegans and HUVECs was analyzed using an ATP Analysis System Bioluminescence Detection Kit (Promega, FF2000). HUVECs (20,000/well) or nematodes (20/well) were added to 96-well plates. The ATP kit test solution was added and plates were incubated for 1 h at room temperature. The ATP content was detected by chemiluminescence.

RNA sequencing

Gene expression in HUVECs treated with RMF for 4 h or the vehicle control was performed on day 10 with the assistance of Novo Gene Corporation (Beijing, China). Up- or down-regulated genes were identified by filtering the RNA-seq data with the following cut-off: twofold change in expression level and a false discovery rate analogue of q value less than 0.05.

Quantitative real-time PCR (RT-PCR)

Approximately 500 synchronized C. elegans were cultured at 20°C with or without RMF 2 h/d. Total RNA was extracted using TRIzol A+ (Tiangen, China). RNA (3 μg) was reverse transcribed using a GoScript Reverse Transcription System (Promega, A5004) as described previously. The expressed genes were amplified in triplicate and quantified in a SYBR Green PCR Mix (Applied Biosystems) with an ABI 7500 DNA Analyzer (Applied Biosystems) as described previously [66]. Each experiment was performed in triplicate and the fold change was calculated by a relative quantification method (2-∆CT). The primer sequence of the gene to be detected is in Supplementary Table 1.

Western blot analysis

After treatment, the culture medium was discarded, and cells were washed twice with precooled PBS. Appropriate amounts of cell lysate were added, and cell protein was scraped with a cell blade after the ice was fully cleated. The supernatant was a centrifuged at 12,000r/min for 10 min at 4°C for, and the protein content was determined using the BCA method. Proteins were boiled for 5–10 min and separated by SDS-PAGE before transfer to a PVDF membrane. Membranes were then blocked with 5% skimmed milk powder solution at room temperature for 1 h, and then incubated overnight at 4°C with primary antibodies for the detection of P21, P53, AMPK (Invitrogen, MA5-12557, MA5-15815, all 1:1,000) and mTOR (CST, #2983, 1:1,000). After washing three times with 1× TBST for 5 min, membranes were incubated with the appropriate secondary detection antibody (Invitrogen, G-21040, G-21234, all 1:10,000) at room temperature for 1 h. After washing three times with 1× TBST for 5 min, the antibody-reactive protein bands were detected by chemiluminescence.

Statistical analysis

All statistical analyses were conducted with GraphPad Prism 8.0. Lifespan and paralysis assay data were analyzed using the Kaplan–Meier survival method and P-values were calculated using the log-rank test. Student’s t-test was used to analyze differences between two groups. One-way analysis of variance (ANOVA) with Duncan’s test was used to compare multiple groups. P < 0.05 was considered to indicate statistical significance.