Effect Of Genistein On The Transformation Of Mouse Skeletal Muscle Fiber Types And Its Mechanism

The composition of skeletal muscle fiber types directly affects the meat yield and quality of livestock and poultry,and is related to muscle endurance and some metabolic diseases.Genistein,as a kind of phytoestrogens,has been proved to regulate the fatty acid oxidation and insulin sensitivity of skeletal muscle.miRNAs are small non-coding RNAs of about 22 nt in length and miRNA generally facilitates m RNA translation by binding to the 3’UTR of the m RNA.Our previous studies have shown that Genistein can inhibit the fusion of myotubes by modulating miRNA expression,but it is unknown whether Genistein can modulate skeletal muscle fiber types by this mechanism.In this study,Genistein was added into the diet of mice and the culture medium of C2C12 cells.And q PCR,immunofluorescence and other experimental methods were used to explore the effects of Genistein on myofiber type transformation and muscle metabolism and its epigenetic regulation mechanism.The main findings are as follows:(1)Adding genistein to the diet at the rate of 10 mg / kg body weight per day had no significant effect on the organ index and blood physiological and biochemical indexes in female mice(p>0.05).(2)The addition of Genistein in diet significantly increased the proportion of muscle(p<0.05)and the expression of Myo D,Myo G and Myf5 genes in muscle(p<0.05).Moreover,the diameter of muscle fiber increased obviously.(3)Adding genistein to the diet significantly improved the endurance of mice(p<0.05),changed the color of the leg muscle of the mice,showed a significantly increment in the ‘a’ value of leg muscles and a significantly decrease the ‘L’ value(p<0.05).Moreover,the proportion of slow muscle fibers and expression of MYHC I,MYHC IIa,MYHC IIx,Tnnc1 and Tnni1 in mouse Tibialis anterior muscle was significantly increased(p<0.05).(4)Genistein at a level of 10 μM/L could promote the differentiation of C2C12 cells and the formation of slow muscle fibers,significantly increase the expression of MYHC I,Tnnc1 and Tnnc2(p<0.05)and extremely significantly increase the expression of MYHC IIa,MYHC IIx and Tnni1(p<0.01).Fluorescent staining and RT-PCR showed that Genistein could also promote the Mitochondria biogenesis and the expression of metabolism-related genes(ATP6,ATP8,Sirt1,PGC1 and t FAM)in C2C12 cells(p<0.05).(5)Genistein significantly inhibited the expression of miR-222 in vitro and in vivo(p<0.05).Furthermore,in C2C12 cells,miR-222 significantly inhibited the expression of MYHC I,MYHC IIa,MYHC IIx,Tnnc1 and Tnni1,significantly increased the expression of MYHC IIB and Tnnc2(p<0.05).And MYHC I immunofluorescence staining showed that miR-222 significantly inhibited slow muscle fiber formation(p<0.05).Mitochondrial staining and RT-PCR showed that miR-222 significantly significantly inhibits mitochondria biogenesis and expressions of genes related to mitochondria biogenesis and metabolism(ATP6,ATP8,Sirt1,PGC1 and t FAM)in C2C12 cells(p<0.05).(6)There was a significant negative correlation between miR-222 and PGC1αm RNA expression in mouse muscle(p<0.01),and RT-PCR and Western Blotting showed miR-222 significantly inhibited PGC1α m RNA and protein expression(p<0.05).Double luciferase reporter system showed miR-222 can directly target PGC1α.In conclusion,Genistein may affect the composition and metabolism of skeletal muscle fibers by regulating the expression of miR-222 and its target gene.Genistein can be used as a natural antagonist to inhibit the expression of miR-222.Therefore,Genistein can be used as a potential additive to improve the yield and quality of animal meat.