| Objective In this study,based on the research status and hotspot of skeletal muscle fatigue and mitochondrial function,we explored the anti-skeletal muscle fatigue effect of epimedin C,the main extract component of Epimedium wushanense,and its effect on mitochondrial energy supply.The effects of epimedin C on mitochondrial energy supply were studied in the rodent animal model,isolated muscle tissue,cell and molecular levels to find targets regulated by epimedin C and related anti-fatigue mechanisms.In order to provide a new research direction for the anti-fatigue effect of Epimedium wushanensis and its main extract epimedin C,and provide a theoretical basis for new drug research and clinical application.Methods Part Ⅰ Four weeks old healthy C57BL/6 male mice were divided into model group and experimental group.Daily running training and swimming pool training,10 minutes a day,for 10 consecutive days.Mice with poor motor ability were removed after 10 days.Mice in the experimental group were fed with epimedin C in a dose of 10ug/kg.Epimedin C was fed daily and exercise training was continued during the feeding period.14 days later,the exercise ability of explosive power（swimming pool test）and endurance（running platform test）was tested.The leg gastrocnemius muscle of the feeding group and the experimental group were isolated,the tendons at both ends were resected,and the protein was extracted and sent for Mass Spectrum analysis.Fresh leg slow muscle was used for electrophysiological experiment to measure the energy output waveform of slow muscle.Fresh skeletal muscle tissue of C57BL/6 mice was used to isolate and culture skeletal muscle primary cells for cell experiment and mitochondrial enzyme staining.Part Ⅱ Total protein was extracted from skeletal muscle tissue of experimental group and control group respectively.Protein secondary spectrum detection was performed,and KEGG and NCBI online comparison systems were used to compare the complete domain to be measured with the corresponding protein sequences of mice,rats and zebrafish to generate an Alignment table and draw an evolutionary tree.The interaction between proteins was acquired by the Pull down-screening experiment.MIC25 was used as bait to screen for the candidate interaction proteins,and the obtained candidate interaction proteins were confirmed by secondary spectrum.The related interacting proteins were verified by yeast two-hybrid assay and worm luciferase complementation assay.Part Ⅲ In this stage,a two-factor intervention experiment was conducted to explore the regulation process of MIC25 at the protein level,using Epimedin C as the first intervention factor and RNAi/CRISPR or overexpression related protein as the second intervention factor.Co-ip protein interaction experiment was conducted to test the ubiquitination level of MIC25 in this intervention process,to verify the effect of UBC on MIC25 ubiquitination under the action of Epimedin C,and explore the basic model of Epimedin C on this regulatory pathway.At the same time,the mitochondrial staining of skeletal muscle and the staining of key enzymes related to ATP synthesis were used as indicators to verify the possibility of this model and further explain the mechanism of Epimedin C.Statistical method SPSS21.0 software was used for statistical processing and analysis of experimental data,and measurement data were expressed as mean ± standard deviation（± s）.If the obtained data were empirically consistent with normal distribution,one-way ANOVA was performed,LSD method was used for homogeneity of variances,and Games-Howell method was used for heterogeneity of variances.If it does not conform to normal distribution,nonparametric test is used.P < 0.05 indicated statistical difference.Result Part Ⅰ The treadmill and swimming pool experiments showed that the exercise ability of the experimental group mice fed with Epimedin C was improved,mainly as the running time of the experimental group mice was significantly longer than that of the control group mice,and the number of sprints in the swimming pool was significantly more than that of the control group mice（P < 0.05）.After sampling the living skeletal muscle tissue of the experimental group and the control group,the output work of the slow muscle in the experimental group increased by 5.8% on average compared with that in the control group（P < 0.05）under the same electric stimulation.The staining experiment of ATP5 MF,a key enzyme affecting mitochondrial ATP synthesis in tissue cells,showed that the positive signal of epimedin C treated muscle cells was significantly higher than that of the control group（P < 0.05）.Part Ⅱ 1.The results of muscle protein spectrum test showed that compared with the control group,the difference proteins in the muscle tissue of mice in the experimental group were mainly mitochondria-related proteins.GO diagram analysis showed that the functions related to the formation of mitochondrial crest had a high degree of fit.Using the Cluster Profiler Online tool of KEGG online system,MIC25 in the MIC protein family was found to be highly correlated with Epimedin C processing and mitochondrial crest formation based on functional cluster analysis and raw value peptide abundance analysis.2.Comparison of MIC25 transcriptome showed that MIC25 in mouse skeletal muscle cells and human skeletal muscle cells had similar transcriptional intensity trends after epimedin C treatment.3.Amino acid sequence alignment results showed that after protein spectrum data analysis,the comparison results of mouse and human proteins were obtained.The material background was set as "muscle",and the functional background was set as "exercise and energy supply related",and the suspected MIC protein family information was obtained,which were Q9BRQ6 and Q9IVN4,respectively.It was found that it was the most homologous with MIC25 protein.It was compared with the same protein from other species using MEGA 4.0 and an evolutionary tree was drawn,which showed that the human MIC25 protein was most similar to mouse and rat MIC25.4.Protein-protein interaction test results showed that the five proteins CHCH3,APOO,IMMT,PPPICA and UBC had different degrees of positive signals with MIC25 protein as bait protein in yeast two-hybrid experiment compared with positive control.In the luciferase complementation experiment,the five proteins also had different degree of binding signal with MIC25.Part Ⅲ 1.The results of mitochondrial and related enzyme staining in skeletal muscle tissues of mice showed that Epimedin C treated the mitochondrial staining signal was stronger than the control group,and the MIC25-Rnai signal was significantly weakened,while the MIC25-bound UBC was enhanced after RNAi operation.In the same treatment group,mitochondrial complex 1 and mitochondrial respiration related complex ⅴ also showed a similar trend.2.Energy-related enzyme staining results after Epimedin C treatment showed that the ATP5 MF enzyme in normal muscle cells could be upregulated by Epimedin C and the addition of MIC25,and both had additive effects.Epimedin C failed to restore ATP5 MF in myocytes with mic25 decreased expression profile.The addition of exogenous MIC25 did not significantly restore ATP5 MF abundance,but the abundance of ATP5 MF rapidly increased under the combined action of external MIC25 and Epimedin C.3.The protein interaction experiment results showed that the binding of MIC25 with CHCH3（MIC19）,APOO（MIC26）,IMMT（MIC60）and PPP1 CA was enhanced after Epimedin C treatment（P < 0.05）.The binding ability to UBC was decreased（P < 0.05）.4.The experimental results of interaction between MIC25 and respiratory chain proteins showed that when UBC was overexpressed,CHCHD3,APOO,IMMT and Mic25 had significant interaction（P < 0.05）,while PPPICA and Mic25 had no significant change（P > 0.05）.The results showed that Epimedin C could alleviate the UBC induced weakening of the interaction between MIC25 and CHCHD3,APOO,IMMT to some extent.5.UBC regulation of MIC25 ubiquitination on the expression of proteins related to energy metabolism in skeletal muscle mitochondria showed that the increase of MIC25 directly increased the abundance of Mito Complex ⅰ and ⅴ enzymes,and energy metabolism was enhanced（P < 0.05）.The presence of UBC can inhibit the effect of MIC25 to some extent and weaken the Mito Complex signal（P < 0.05）.6.Epimedin C influences the expression of mitochondrial energy metabolism proteins by regulating UBC and MIC25.Epimedin C helps to maintain the abundance of Mito Complex ⅰ and ⅴ enzymes.Epimedin C could inhibit the weakening of Mito Complex signal under UBC to a certain extent（P < 0.05）.After CRISPR system editing down-regulated UBC,MIC25 expression was significantly increased（P < 0.05）.Conclusion 1.Epimedin C has a significant phenotype in both animal tissues and cells,suggesting that Epimedin C can improve motor function by enhancing ATP synthesis.2.Epimedin C significantly increases the upregulation of MIC25,through which Epimedin C affects the activity of enzymes related to energy metabolism.3.The interaction between MIC25 and UBC protein is weakened under the action of Epimedin C,which results in MIC25 being weakened by UBC’s ubiquitination and thus prevented from entering the degradation pathway,thus increasing the abundance of MIC25 and enhancing mitochondrial energy metabolism. |
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