| Propolis is an important functional food with a variety of biological properties such as antioxidant,antiinflammatory and immunomodulatory.In China,many functional foods containing propolis are marketed and widely consumed to promote health and prevent diseases.The flavonoids and polyphenols rich in propolis help prevent muscle atrophy caused by oxidative stress.However,it is still unclear about the efficacy and mechanism of action of propolis on senile muscular atrophy.In this study,the effects of propolis ethanol extract(PEE)on grip strength,gastrocnemius index and body weight of mice were determined using a D-gal-induced senescence mouse model;the effects of PEE on the activities of antioxidant enzymes(SOD,GSH-PX,CAT and MDA)in muscle and serum of senescent mice were also determined;the effects of PEE on muscle fibers of senescent mice were investigated by observing the changes in The effects of PEE on muscle fibers in aging mice were investigated by observing the changes in muscle fiber morphology and measuring the cross-sectional area and diameter of muscle fibers;finally,the effects of PEE on D-gal-induced skeletal muscle atrophy in mice were investigated by analyzing the Nrf-2/HO-1 signaling pathway,apoptosis and muscle E3 ubiquitin ligase protein expression.We used broad-target metabolomics techniques to explore the effects of PEE on the metabolic profile of D-gal-induced senescent mouse gastrocnemius muscle to reveal essential biomarkers and metabolic pathways associated with PEE activity.This study also determined the effects of PEE on D-gal-induced senescence,proliferation,oxidative stress,and differentiation of C2C12 cells using D-gal-induced C2C12 cells as a model.The effects of PEE on the m RNA and protein expression of Nrf2/HO-1 and P38/P53 signaling pathways in D-gal-induced C2C12 cells were also investigated.Finally,we investigated the core components of the muscle atrophy-relieving effect of PEE and their main targets of action with the help of network pharmacology and molecular docking,and validated them by in vitro experiments.The main results of the study are as follows:(1)PEE can effectively alleviate D-gal-induced weight loss and reduced gastrocnemius index in mice.The results of limb draping test revealed that PEE could reverse the loss of muscle strength in aging mice induced by D-gal.PEE increased SOD and GSH-Px levels and decreased MDA levels in muscle homogenate and serum of aging mice induced by D-gal.PEE significantly increased the expression of Nrf2 and HO-1 proteins in the gastrocnemius muscle and increased the expression of Nrf2 protein in the nucleus of gastrocnemius cells,and PEE promoted the nuclear translocation of Nrf2 in the gastrocnemius muscle.PEE significantly decreased the expression of myo-specific E3 ubiquitin ligases MAFbx and Mu RF1 in the gastrocnemius muscle of aging mice.(2)A total of 835 substances were detected in the gastrocnemius muscle of D-gal-induced senescent mice by a broad-target metabolomics approach.the OPLS-DA score maps clearly distinguished the control group from the senescence model group and the senescence model group from the PEE group,and these results indicated that the muscle metabolic profiles of senescent mice were different between the control and senescence model groups and the senescence model group and the PEE group.A total of 49 differential metabolites were found between the control and model group groups.Compared to the control group,23 metabolites were upregulated and 26 metabolites were down-regulated in the model group.There were a total of 49 differential metabolites between the control and model group groups.There were 23 up-regulated metabolites and 26 downregulated metabolites in the model group compared to the control group.The total number of differential metabolites between these three groups was 17,and among these 17 differential metabolites,9 differential metabolite levels were restored to the same level as the control group after PEE treatment.Among the nine metabolites,the expression of two metabolites,Ala-Ile and Leu-Pro-Tyr,was down-regulated and the expression of seven metabolites was up-regulated in the model group compared with the control group,namely 15-ketoprostaglandin E1,15(R)-prostaglandin E1,8iso-prostaglandin E2,estriol,isoprenaline,9(S),12(S),13(S)-Tri HOME,and pyridine-3,4dicarboxylicacid.(3)PEE treatment at 1μg/m L increased the survival rate of D-gal-induced C2C12 cells.PEE treatment effectively reduced the number of SA-β-GAL positive cells in D-gal-induced C2C12 cells.PEE treatment increased the number and size of fused myotubes in D-gal-induced C2C12 cells.PEE significantly increased the expression of myogenic markers Myo D,Myogenin and My HC in C2C12 cells.PEE treatment significantly increased the expression of CAT,GSH-PX,T-AOC and Myogenin in D-gal-induced C2C12 cells.cells,and PEE significantly increased the expression of myogenic markers Myo D,Myogenin and My HC in C2C12 cells.PEE treatment significantly increased the levels of CAT,GSH-PX,T-AOC and SOD in D-gal-induced C2C12 cells and significantly decreased the levels of ROS in C2C12 cells.PEE significantly upregulated the levels of Nrf2 and PEE significantly up-regulated the m RNA levels of Nrf2 and HO-1 in D-gal-induced C2C12 cells and significantly up-regulated the levels of Nrf2 in the nucleus of C2C12 cells,but had no effect on the expression of Nrf2 in the cytoplasm.The above effects were reversed by the addition of ML385,which also abolished the improvement of PEE on D-gal-induced senescence and on the pro-differentiation ability of C2C12 cells.pee treatment significantly decreased the expression of P38,P53 and Bax m RNA and increased the expression of Bcl-2m RNA in D-gal-induced C2C12 cells.pee treatment significantly decreased the expression of D-galinduced phosphorylation of P38,expression of P53 and Bax and increased expression of Bcl-2 protein in C2C12 cells.(4)A total of 12 intersectional targets between PEE and sarcopenia were identified by network pharmacology prediction.The "component-target-pathway" network established by Cytoscape 3.9.1 software identified Apigenin(Ap)as the core component of PEE to alleviate sarcopenia,and TNFα and IL-6 as the main core targets.6 tightly.In vitro experiments showed that 2.5 μM Ap treatment significantly increased the viability of D-gal-induced C2C12 cells and significantly increased the expression of differentiation marker proteins Myo D1 and Myogenin and the number of fused myotubes.Ap also significantly inhibited the expression of EGFR,IFNγ,TNFα and IL6 in D-gal-induced C2C12 cells.Ap was able to inhibit D-gal-induced phosphorylation of JAK2 and STAT3 in C2C12 cells.In this study,the effect and mechanism of action of PEE on senile muscle atrophy were demonstrated in vivo and in vitro with the help of metabolomics and network pharmacology,respectively,and the core components of its action were predicted and validated.The results of this study will provide an important theoretical basis for the development of PEE for the alleviation of senile muscle atrophy. |
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