Study On The Mechanism Of Protocatechuic Acid Regulating Skeletal Muscle Fiber Type Transformation

Protocatechuic acid(PCA)is a naturally occurring phenolic acid.It has rich biological functions,including antibacterial,anti-inflammatory,antioxidant and antitumor.However,studies of PCA on myofiber types transformation have not been reported.The aim of this study was to investigate the effect and mechanism of PCA on myofiber type transformation.The main studies and results are as follows:Experiment 1.Effect of PCA on the transformation of skeletal muscle fiber types in miceIn this experiment,sixty 4-week-old healthy male Kunming mice with similar body weights were randomly divided into three groups with three treatments: control group(a basal diet),50 mg/kg PCA group(a basal diet + 50 mg/kg PCA)and 100 mg/kg PCA group(a basal diet + 100 mg/kg PCA),and fed for 28 days.The results showed that compared with the control group,the dietary addition of 50 and 100 mg/kg PCA significantly up-regulated the protein expression of slow myosin heavy chain(Slow My HC)and the m RNA expression of My HC I and My HC IIa in the gastrocnemius(GAS)muscle of mice,significantly increased the activities of succinate dehydrogenase(SDH)and malate dehydrogenase(MDH),significantly down-regulated the protein expression of Fast My HC and the m RNA expression of My HC IIa in the GAS muscle,and significantly decreased lactate dehydrogenase(LDH)activity.Immunofluorescence results showed that PCA significantly increased the number of slow muscle fibers and significantly decreased the number of fast muscle fibers.Furthermore,in terms of antioxidant capacity,dietary supplementation with 50 and 100mg/kg PCA significantly enhanced the activities of catalase(CAT),superoxide dismutase(T-SOD),total antioxidant capacity(T-AOC)and glutathione peroxidase(GSH-Px)and the content of reduced glutathione(GSH),and significantly decreased the content of malondialdehyde(MDA)in serum,liver and GAS muscle of mice compared to the control group.Dietary supplementation with 50 and 100 mg/kg PCA significantly upregulated the protein expression levels of quinone oxidoreductase 1(NQO1),heme oxygenase 1(HO-1)and nuclear transcription factor E2-related factor 2(Nrf2)in liver and GAS muscle,significantly upregulated the m RNA expression levels of antioxidant-related genes Nrf2,HO-1,CAT,SOD1,SOD2 and GSH-Px in liver and GAS muscle.In terms of mitochondrial function,dietary supplementation with 50 and 100 mg/kg PCA significantly promoted the m RNA expressions of cytochrome c(Cytc),nuclear respiratory factor 1(NRF1),transcription factor A,mitochondrial(TFAM),transcription factor B1,mitochondrial(TFB1M),citrate synthase(CS)and mitochondrial ATP synthase lipid-binding protein(ATP5G)in liver and GAS muscle compared with the control group,and significantly up-regulated the protein expression level of Cytc.Meanwhile,dietary supplementation with 50 and 100 mg/kg PCA significantly promoted the phosphorylation of AMP-activated protein kinase(AMPK)protein in GAS muscle and significantly upregulated the protein expression levels of silent message regulator 1(Sirt1)and peroxisome proliferator-activated receptor-γ coactivator-1α(PGC-1α).These results revealed that PCA significantly enhanced the antioxidant capacity and mitochondrial function of the mice and induced the conversion of mice skeletal muscle myofiber type from fast to slow muscle fibers,the effects of which may be related to the AMPK signaling pathway.Experiment 2.PCA-induced skeletal muscle fiber type transformation through AMPK signaling pathwayThis study was performed by adding different concentrations(0 μg/ml,12.5 μg/ml and25 μg/ml)of PCA to C2C12 myotubes to explore the effect of PCA on myofiber type conversion in vitro.Compound C,an AMPK inhibitor,was subsequently added to inhibit AMPK signaling and to explore the possible mechanism of PCA-induced myofiber type conversion.The results showed that PCA significantly upregulated the protein expression level of Slow My HC and the m RNA expression levels of My HC I and My HC IIa,and significantly decreased the protein expression level of Fast My HC and the m RNA expression level of My HC IIb in C2C12 myotubes.Meanwhile,PCA significantly enhanced the activities of SDH and MDH and decreased the activity of LDH.In terms of antioxidant capacity,PCA significantly upregulated the m RNA expression levels of Nrf2,CAT,SOD1,SOD2 and GSH-Px in C2C12 myotubes and significantly downregulated the gene expression level of Kelch-like ECH-associated protein 1(Keap1).In terms of mitochondrial function,PCA significantly upregulated the m RNA expression levels of Cytc,TFAM,TFB1 M,CS and ATP5 G.In addition,the addition of PCA significantly enhanced the phosphorylation of liver kinase B1(LKB1)and AMPK proteins in C2C12 myotubes,significantly increased the proteins expression of Sirt1 and PGC-1α,and significantly up-regulated the m RNA expression levels of AMPKα2 and PGC-1α.However,after adding AMPK inhibitor Compound C to inhibit AMPK signaling pathway,the effect of PCA on promoting the transformation of muscle fiber type from fast muscle to slow muscle was significantly inhibited.The above results suggest that the conversion of C2C12 myotubes muscle fiber type from fast to slow muscle induced by PCA is mediated through the AMPK signaling pathway.In conclusion,in vitro and in vivo,results showed that PCA induced the conversion of muscle fiber types from fast-twitch to slow-twitch in mice and C2C12 myotubes.Its mechanism was related to increased antioxidant capacity,enhanced mitochondrial function and activation of AMPK signaling pathway.This study will not only further enrich the nutritional physiological functions of PCA,but also accumulate information on the use of nutritional strategy to regulate the transformation of skeletal muscle fiber types.