The Association Of Skeletal Muscle Growth With NFκB Inhibition By N-3PUFA

The aim of the study was to investigated that the effect of duration of feeding linseed diet on the skeletal muslce growth of the growing-finishing pigs,and the molecular mechanism of n-3PUFA regulating skeletal muscle growth of growing-finishing pigs.The contents of the research were mainly composed of two parts:the first part was the animal feeding experiments,which was to investigated that the effect of duration of feeding linseed diet on the skeletal muslce growth of the growing-finishing pigs and explore the relationship between gene expression and muslce mass;the second part was in vitro experiment,which investigated the molecular mechanism of n-3PUFA regulating skeletal muscle growth of growing-finishing pigs.The fisrt part:the animal feeding experiments.Two isoenergetic and isonitrogenous diets were formulated,and one of which was the basal diet and another one was the linseed diet including linseed at the level of 10%.Twenty-four Landrace×Yorkshire barrows weighing 35±3.7 kg were randomly assigned to four treatments with six individuals per treatment.Pigs in treatment 1(T1) fed the control diet throughout the experimental period,while pigs in T2,T3 and T4 fed the control diet except for 30,60,and 90 d prior to slaughter when the linseed diet were fed.The experiment was conducted for 90 days.Carcass quality and meat quality data were collected and analyzed.The longissimus dorsi muscle mass,posas minor muscle mass and each muscle mass in the hind leg were weighted.Additionally,fatty acid composition(%) of the diet,the longissimus dorsi muscle and the backfat were analyzed by gas chromatography method.PPARγand TNFαmRNA expression levels in muscle,spleen and adipose tissue,and plasma concentrations of TNFαdata were measured and analyzed.1.Duration of dietary linseed feeding affects the intramuscular fat,muscle mass and fatty acid composition in pig muscle.No significant difference(P＞0.05) was observed for average backfat thickness,lean meat percentage,loin muscle area, whereas the intramuscular fat content increased linearly(P＜0.01) as prolonged the time of feeding linseed diet.As prolonged the time of feeding linseed diet,the longissimus dorsi muscle mass,quadriceps femoris muscle mass and semitendinosus muscle mass increased linearly(P＜0.01).Duration of feeding linseed diet linearly increased(P＜0.01) the LNA,EPA and C22:5n-3 concentrations in the longissimus dorsi muscle and backfat.There was significant quadratic relation between the intramuscular fat content and the n-3 polyunsaturated fatty acids(PUFA) enrichment (R~2=0.87,P＜0.01),or LNA enrichment(R~2=0.91,P＜0.01) in the longissimus dorsi muscle.Likewise,the longissimus dorsi muscle mass was also quadratically related to the n-3PUFA enrichment(R~2=0.89,P＜0.01),or LNA enrichment(R~2=0.86,P＜0.01) in the longissimus dorsi muscle.These results revealed that duration of feeding linseed diet may stimulate intramuscular fat accumulation,and promote the hypertrophy of the longissimus dorsi muscle,quadriceps femoris muscle and semitendinosus muscle mass by increasing the n-3PUFA enrichment,especially LNA enrichment in the longissimus dorsi muscle.2.Duration of feeding linseed diet influences peroxisome proliferator-activated receptorγand tumor necrosis factor gene expression,and muscle mass of growing-finishing barrows.The expression of PPARγin longissimus muscle and spleen increased(P＜0.01) linearly as prolonged the time of feeding linseed diet,while the expression of PPARγin adipose tissue were not affected(P=0.095).Duration of linseed addition linearly decreased(P＜0.01) TNFαgene expression levels in the longissimus dorsi muscle,adipose and spleen,and serum concentration of TNFαas well.The expression levels of PPARγnegatively correlated with the expression of TNFαin muscle(R~2=0.70,P＜0.001) and spleen(R~2=0.77,P＜0.001) respectively. Likewise,PPARγexpression level in spleen(R~2=0.59,P＜0.01) or muscle(R~2=0.52, P＜0.05) negative correlated with serum TNFαconcentration.There were significant quadratic relation between muscular PPARγ(R~2=0.80,P＜0.01) or muscular TNFα(R~2=0.87,P＜0.01) expression and the longissimus dorsi muscle mass.These results demonstrated that duration of feeding linseed diet lead to a linear decrease of TNFαgene expression,which may increase the muscle mass in growing-finishing barrows, at least in part,through a PPARγ-dependent mechanism.The second part:in vitro experiment.In order to investigate the molecular mechanism of n-3PUFA regulating skeletal muscle growth of growing-finishing pigs. C2C12 myotube were incubated with BSA(the postive control),PTA(the negative control),ALA an EPA,respectivley.After treatment with 600μM BSA,LNA,EPA or PTA for 24 hours PPARγ,TNFαand MuRF1 mRNA expression levels were measured by Real-time quantitative PCR method.After incubation of C2C12 myotubes with 300μM,600μM BSA,LNA,EPA or PTA for 24 hours,respectively,the abundance of IκBαwas measured by Western blot method.Additionally,C2C12 myotubes were incubated with 600μM LNA,600μM EPA,600μM PTA or for 24 h.Total nuclear protein was subsequently isolated and analyzed by EMSA for NF-κB DNA binding activity.The effect of n-3PUFA on NF-κB activation and MuRF1 gene expression in C2C12 skeletal muscle Cells.C2C12 myotubes incubated in the presence of 600μM EPA for 24 hours resulted in a 2.3-fold induction of the PPARγepression(P＜0.01).A 1.08-fold induction(P＜0.01) was observed in the presence of 600μM LNA for 24 hours.However,24-h incubation period with 600μM palmitate decreased PPARγexpression(P＜0.01).Treatment with 600μM LNA or EPA for 24 hours caused a 1.06-fold and 2.93-fold reduction in the mRNA levels of TNFαin C2C12 myotubes (P＜0.001),respectively.Whereas 24-h incubation period with 600μM palmitate increased TNFαexpression(P＜0.01).Incubation of C2C12 myotubes with 300μM LNA,EPA or palmitate for 24 hours did not affect the abundance of IκBα.However, 600μM EPA addition to cells caused approximately a 86%(P＜0.01) increase in the abundance of IκBα,and 600μM palmitate addition to cells caused approximately a 39%(P＜0.01) decrease in the abundance of IκBα.Incubation of C2C12 myotubes with 600μM EPA for 24 hours decreased the NF-κB DNA binding activity.However, Treatment with 600μM LNA or EPA for 24 hours increased the NF-κB DNA binding activity.C2C12 myotubes incubated in the presence of 600μM EPA for 24 hours caused a 3.38-fold induction in the levels of MuRF1 mRNA(P＜0.01).Whereas 24-h incubation period with 600μM palmitate increased MuRF1 expression(P＜0.01). These results revealed EPA treatment of skeletal muscle cells represses MuRF1 expression through mechanisms involving the activation of the axis PPARγ/TNFα/IκBα/NF-κB,decreases skeletal muscle protein degradation,and promotes the skeletal muscle growth.