Preliminary Study On Toxicity And Toxic Mechanism Of Maduramicin In Skeletal Muscle Of Mice And Broilers

Maduramicin,a polyether ionophore anticoccidial agent,is used as a feed additive in poultry.The safety range of maduramicin is narrow in livestock and poultry,which leads to death if improperly used,resulting in huge economic losses.In addition,due to eaten by accident or left in animal derived food,maduramicin poisoned or even killed people,leading to a serious threat to human health.The main toxic effects of maduramicin are myocardial and skeletal muscle damages.In our previous study,maduramicin increased the level of ROS,which led to inhibit PP5 and activate JNK,and finally induced apoptosis in skeletal muscle cells.Maduramicin also inhibited Akt and blocked autophagic flux,leading to autophagosome accumulation and consequently apoptosis in skeletal myoblasts.In order to further explore the toxicity and toxic mechanism of maduramicin in skeletal muscle,we used C2C12 and L6 skeletal myoblasts,ICR mice and yellow-feathered broilers as in in vitro and in vivo model,treated cells and animals with maduramicin and/or digoxin,and detected cell viability,intracellular ATP content,pH value,Ca2+concentration,mitochondrial membrane potential,AMPK,Na+/K+-ATPase and endoplasmic reticulum stress and autophagy related proteins expression,to study the mechanism of maduramicin-inhibited skeletal myoblasts growth,and to understand the toxic mechanism of maduramicin in skeletal muscle of mice and broilers,thereby provide theoretical basis for prevention and treatment of maduramicin-induced poisoning.In experiments of skeletal muscle myoblast cells in in vitro,C2C12 and L6 cells were treated with 0.2?1.0 ?M for 24?72 h,and then detected cell viability using MTT,determined intracellular ATP content by kit,detected intracellular Ca2+concentration,pH value and mitochondrial membrane potential through flow cytometry following fluorescence probe labeling,detected expressions of AMPK,Na+/K+-ATPase,endoplasmic reticulum stress and autophagy related proteins via Western blotting.In addition,in the experiments of maduramicin combined with digoxin treatment,the cells were divided into the control group,10.0 ?M digoxin treatment group,1.0 ?M maduramicin treatment group and 10.0 ?M digoxin+1.0 ?M maduramicin co-treatment group,cell viability was determined using MTT,expressions of Na+/K+-ATPase and autophagy related proteins were detected by Western blotting.The results showed that 0.2?1.0 ?M maduramicin inhibited cell viability,increased intracellular ATP,Ca2+ concentration,pH value,decreased mitochondrial membrane potential,and upregulated expressions of endoplasmic reticulum stress and autophagy related proteins in C2C12 and L6 cells.Cell viability and expression levels of Na+/K+-ATPase,LC3B-? and p62 in digoxin and maduramicin co-treatment group were lower than that in maduramicin treated group.In in vivo experiments in mice,20?25 g male ICR mice were divided into the blank control group,1.75 mg/kg b.w.maduramicin group and 3.50 mg/kg b.w.maduramicin group,mice were intragastrically administered with maduramicin once a day for 7 days.Rectus femoris were taken and expressions of AMPK,Na+/K+-ATPase,endoplasmic reticulum stress and autophagy related proteins were detected by Western blotting.The findings showed that expressions of p-AMPK(Thr183/Thr172),p-eIF2?(Ser51),ATF4,CHOP,Na+/K+-ATPase,LC3B-? and p62 were increased in maduramicin treated group.1.75 mg/kg b.w.maduramicin group were 1.44,1.61,1.35,2.58,1.41,3.79,2.10 fold as control group;3.5 mg/kg b.w.maduramicin group were 1.71,2.99,1.61,3.34,4.01,7.05,9.18 fold as control group.In in vivo experiments in broilers,28 day old male yellow-feathered broilers were divided into the blank control group,0.1 mg maduramicin group,0.2 mg maduramicin group and 0.5 mg maduramicin group,broilers were intragastrically administered with maduramicin once a day for 14 days.Rectus femoris were taken and expressions of p-AMPK(Thr183/Thr172),p-eIF2?(Ser51),LC3B,p62 and Na+/K+-ATPase were detected via Western blotting.The data showed that expressions of p-AMPK(Thr183/Thr172),p-eIF2?(Ser51),Na+/K+-ATPase,LC3B-? and p62 in maduramicin treated group were higher than that in control group.0.1 mg maduramicin group were 1.47,1.25,1.29,1.52,2.03 fold as control group;0.2 mg maduramicin group were 1.97,1.83,1.83,2.00,3.50 fold as control group;0.5 mg maduramicin group were 3.02,2.37,3.08,3.54,12.59 fold as control group.Taken together,maduramicin inhibits cell viability,disturbs the energy metabolism and ion balance,and destroys the homeostasis of in skeletal myoblast cells.Maduramicin activates AMPK and induces endoplasmic reticulum stress in skeletal muscle cells.Na+/K+-ATPase mediates the inhibitory effect of maduramicin on autophagic flux in skeletal muscle cells.