| Objective: NADPH oxidase(Nox)in skeletal muscle can promote the production of reactive oxygen species(ROS).Studies in recent ten years have shown that Nox is the main way to increase ROS in skeletal muscle induced by exercise.However,it is not clear that which subtype of Nox facilitate the mechanism of exercise-induced ROS generation,and the upstream of Nox signaling pathway is still under definition.Hence,in the present study we generated a single bout of exhaustive treadmill running exercise model in SD rats to detect the effect of Nox2 and Nox4 expression induced by exercise in skeletal muscle.We also examined the upstream signal of Nox,PKC pathway during exercise-induced ROS generation by utilizing PKC inhibitor,Chelerythrine.Methods: Thirty male SD rats,weight about 240-260 g,were randomly divided into three groups,control group(group C),exhaustive exercise group(group E),and PKC inhibitor injection combined with exhaustive exercise group(group EC),10 rats in each group.Exhaustive exercise was executed on a treadmill and the speed was set initially at 5 m/min and then increased to final 25 m/min by 5 m/min every 5 min until the subjective rats could not sustain running.For rats in EC group,a PKC inhibitor,Chelerythrine(5mg/kg),was intraperitoneally injected respectively 1 day and 1 hour before exercise.All the subjective animals were sacrificed immediately after exercise,and both sides of plantaris muscle was harvested for further examination.ROS generation in skeletal muscle was detected by a DCF fluorescent probe,PKC,Nox2,and Nox4 protein expression was measured by western blot,and PKC-Nox4 interaction was examined by immunoprecipitation.Results: 1)The data of exhaustive running showed there was no differences in the total running duration between E(89±9.30 min)and EC(76.5±8.12 min)group(P>0.05).2)DCF fluorescence probe detection showed that compared to C,the ROS content in skeletal muscle of E group was significantly increased(P<0.05),while compared to E,the value of ROS content was significantly reduced(P<0.05).3)Compared to C,PKC protein expression in skeletal muscle in group E was obviously induced(P<0.01),while compared to E,the PKC protein expression was apparently decreased(P<0.01).4)Compared to C,the protein expressions of Nox2 and Nox4 in skeletal muscle of group E were significantly elevated(P<0.01 and P<0.05).Compared to E,the value of Nox2 protein expression in EC group was significantly decreased(P<0.05),while the Nox4 expression had no changes(P>0.05).5)The result of immunoprecipitation showed that PKC-Nox4 interaction.Compared to C,there was a significantly increase of PKC-Nox complex generation(P<0.05);while compared to E,the PKC-Nox complex content was significantly reduced in EC group(P<0.05).Conclusion: 1)There is no effect of PKC inhibition on exhaustive exercise performance.2)A single bout of exhaustive exercise immediately induces ROS generation in skeletal muscle and this effect was blocked by PKC inhibition probably by changing the activity of its downstream signal molecule,Nox.3)A single bout of exhaustive exercise promotes PKC protein expression in skeletal muscle and PKC inhibitor is sufficient to blunt this effect.4)A single bout of exhaustive exercise induces Nox2,and Nox4 protein expression in skeletal muscle,while PKC inhibition is only sufficient to block exercise-induced Nox2 protein expression and has no effects on the Nox4 protein expression.5)PKC-Nox4 functions as a complex in skeletal muscle.A single bout of exhaustive exercise induces PKC-Nox4 complex generation probably facilitating exercise-induced ROS generation,and PKC protein activity plays a critical role during this process. |
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