| Background:The rapid growth of the domestic aerospace industry has led to extended durations of space travel for astronauts.In the weightless environment of space,astronauts experience muscle atrophy,characterized by a progressive decline in both muscle mass and strength,particularly affecting the"anti-gravity muscles."This phenomenon not only poses risks to the physical health and operational efficiency of astronauts during space missions but also impairs their functional capacity upon reentry to Earth.Consequently,elucidating the molecular mechanisms underlying microgravity-induced muscle atrophy and identifying effective interventions have become critical research objectives.Recent investigations have shown that time-restricted feeding（TRF）,a dietary approach involving a limited time window for daily food intake,exerts notable effects on various health parameters,including delayed aging,improved metabolism,and reduced cancer incidence.In the context of space-related physiology,previous studies conducted by our research group have demonstrated the potential of TRF in ameliorating simulated-induced cardiac dysfunction.However,the influence of TRF on simulated-induced muscle atrophy remains poorly understood.Therefore,the present study aims to investigate the impact of TRF as a potential protective measure against simulated-induced muscle atrophy,with the goal of shedding light on its underlying mechanisms and evaluating its therapeutic potential in the context of prolonged space travel.Purpose:To clarify whether TRF improves microgravity-induced muscle atrophy and the underlying mechanisms.Methods:1.Hindlimb unloading（HU）was used to establish a simulated microgravity mouse model by tail suspension.The TRF was implemented as follows:HU mice were allowed access to food between zeitgeber time（ZT）14（2 hour after lights off）and ZT22（2 hours before lights on）every day,while during the remaining period,they were restricted from eating but allowed ad libitum access to water.The control group of mice had unrestricted access to both food and water.2.Animal treadmill exhaustion experiment was used to analyze exercise capacity in mice.Hematoxylin-eosin（HE）and immunofluorescence staining techniques were utilized to analyze parameters such as muscle fiber diameter,cross-sectional area（CSA）,and microvascular density within the soleus muscle.Microvascular endothelial cells were isolated from mouse skeletal muscle and aging-related markers including p16,p53,IL-1α,IL-6,and theβ-galactosidase positive cells were measured.3.The GEO2R online analysis tool was employed to perform a comprehensive analysis of gene expression data.Specifically,we conducted a joint analysis of the soleus muscle gene chip sequencing dataset obtained from mice exposed to weightlessness,obtained from the GEO database,and the skeletal muscle transcriptome dataset derived from our lab on mice subjected to TRF intervention.Differential genes exhibiting opposite expression trends were identified and subsequently subjected to KEGG pathway enrichment analysis to elucidate their functional significance.To validate the findings,quantitative real-time polymerase chain reaction（q RT-PCR）was employed to measure the m RNA expression levels of a key differential gene,namely Serpine1.Moreover,western blotting,immunofluorescence,and immunohistochemistry staining techniques were utilized to evaluate the expression of plasminogen activator inhibitor-1（PAI-1）,a protein encoded by the Serpine1 gene,in the soleus muscle tissue of mice.4.Serum of mice in Control,HU and HU+TRF groups was collected,and the content of PAI-1 in serum was detected by ELISA.The serum of mice from different treatment groups was added to serum-free medium to culture HUVECs.The expression changes of aging related markers including p21,p53 and gamma-H2Ax in HUVECs were detected by Western Blotting and immunofluorescence.5.A co-culture system was established between human skeletal muscle cells（HSMCs）and human umbilical vein endothelial cells（HUVECs）by using a transwell chamber.The cells were subjected to simulated microgravity（SM）via a 2D clinostat.Cells starvation（Fasting）was administered by using serum-free medium to simulate the in vivo condition of cellular nutrient deficiency observed during TRF.6.AAV-PAI-1 and its negative control（AAV-NC）constructs were developed for the specific overexpression of PAI-1 in mouse skeletal muscle cells.These viral constructs were locally injected into the hind limbs of mice,and the effectiveness of viral infection was evaluated using a small animal imaging facility,along with Western Blotting and immunofluorescence staining.7.The experimental data were analyzed using SPSS 18.0 statistical software.Statistical analysis was performed using t-tests or one-way analysis of variance（ANOVA）as appropriate.The data are presented as mean±standard error.A significance level of P<0.05 was used to determine statistical significance.Results:1.Compared to the control group,4 weeks of HU significantly reduced exercise capacity of mice as evidenced by decreased exhaustion time and distance.Furthermore,HU led to a notable decrease in muscle mass,muscle fiber diameter,CSA,and microvascular density in the soleus muscle of mice,all of which were effectively ameliorated by TRF.Compared with the control group,mice subjected to HU exhibited an aging phenotype in the MMECs,as characterized by a significant increase in the number of SAβ-gal blue-stained positive cells,along with elevated expression of p16 and p53,IL-1αand IL-6.TRF mitigated the aging phenotype observed in MMECs induced by simulated microgravity.2.The integrated analysis of two datasets unveiled a compelling finding of 57distinct genes exhibiting divergent expression trends.q RT-PCR analysis demonstrated a close association between Serpine1 expression,microgravity-induced muscle atrophy,and TRF intervention.In the HU group,Serpine1 m RNA and PAI-1 protein levels were significantly elevated compared to the control group,which could be mitigated by TRF.Additionally,aged mice exhibited higher PAI-1 expression in the soleus muscle compared to young mice.3.Compared with the control group,the serum PAI-1 content in HU group was significantly increased.Compared with HU group,serum PAI-1 content in HU+TRF group was significantly decreased.Compared with the control group,the aging phenotype of HUVECs cultured with the serum of HU group mice was obvious,which showed that the expression of age-related molecules p21,P53 and gamma-H2Ax was significantly increased which could be improved by TRF.4.Compared to the control group,the SM group exhibited increased expression of Serpine1 and its protein PAI-1 in HSMCs,along with elevated secretion of PAI-1 into the culture medium.HUVECs co-cultured with HSMCs exposed to SM exhibited a significant aging phenotype,characterized by higher expression of p53 protein and an increased number of SAβ-gal-positive cells.Mimicking nutrient deficiency through cell starvation treatment improved p53 expression and reduced the number of SAβ-gal-positive cells in the HUVECs.5.Mice subjected to AAV-PAI-1 injection exhibited a significant increase in PAI-1expression within the soleus muscle compared to the group.Compared to HU mice infected with AAV-NC,TRF treatment demonstrated notable improvements in treadmill endurance time and distance,and significant increases in the soleus muscle mass,muscle fiber diameter,cross-sectional area（CSA）,and microvascular density in mice infected with AAV-NC.However,TRF group did not exhibit significant changes in the aforementioned indices in mice infected AAV-PAI-1 when compared to HU group.These findings have suggested that overexpression of PAI-1 in skeletal muscle cells impedes the protective effects of TRF against microgravity-induced muscle atrophy in mice.Conclusions:Elevation of PAI-1 promotes the senescence of microvascular endothelial cells,resulting in decresed microvascular density in skeletal muscle,and aggravates the development of microgravity-induced muscle atrophy.TRF demonstrates the ability to delay endothelial cell senescence and ameliorate muscle atrophy via inhibiting the production and secretion of PAI-1 in skeletal muscle.This highlights the potential of TRF as a promising strategy to mitigate the detrimental effects of weightlessness. |
