Effects Of Acupuncture On Mitochondrial Redox Homeostasis After Centrifugal Exercise Skeletal Muscle Injury Via TRP Channel

1 ObjectiveSkeletal muscle injuries,especially strains caused by eccentric contractions,are among the most common exercise injuries.Skeletal muscle injuries are prone to scar tissue formation and hinder regeneration when severe,and are associated with excessive mitochondrial oxidative stress caused by cytoplasmic Ca2+ overload.The transient receptor potential(TRP)channel,which regulates mitochondrial redox homeostasis,may be a key factor in the mechanism of the effect of acupuncture,but the mechanism is not clear.In this paper,we constructed an animal model of skeletal muscle mitochondrial redox-sensitive gene transfection and an animal model of eccentric exercise skeletal muscle injury by electrical stimulation,and analyzed the effect of acupuncture on the alteration of mitochondrial redox homeostasis(mitochondria,oxidative stress)after skeletal muscle injury by activating TRP channels in vivo(intravital)using two-photon laser confocal microscopy,aiming to reveal the mechanism of acupuncture intervention in skeletal muscle injury.2 MethodsEighty-four 8-week-old C57BL/6 male mice were randomly divided into:control group(C);exercise injury group(I);exercise injury injury+acupuncture intervention group(IA);exercise injury+acupuncture intervention+TRP channel blocker GdCl3 group(IAT);exercise injury+acupuncture intervention+mitochondrial Ca2+uptake blocker ruthenium red group(IAC);exercise injury+acupuncture intervention+ROS quencher(IAR)group;exercise injury+acupuncture intervention+mitochondrial division blocker(IAM)group;exercise injury+acupuncture intervention+microtubule blocker(IAN)group.Subsequently,100 μL of plasmid pMitoTimer was injected into the tibialis anterior muscle,and a mouse model of in vivo transfection of the mitochondrial redox-sensitive probe pMitoTime was constructed using the electroporation technique.14 d later,the mouse sciatic nerve was exposed,and two electric needles were placed behind the mouse sciatic nerve to start electrical stimulation of the fully activated inner tibialis anterior muscle(15 V,250 HZ,pulse number 38,10s interval between groups,150 electrical stimulations in total)to replicate the eccentric exercise skeletal muscle injury model.In the acupuncture intervention group,the tibialis anterior muscle was diagonally pricked 30 min after electrical stimulation(maintaining the same sample collection time as the blocker group).Immediately after electrical stimulation in the blocker group,a blocker was injected intraperitoneally,respectively,and 30 min after the pinprick intervention.Afterwards,samples were collected at 6h,48h,7d and 14d for the I and IA groups respectively;and at 6h for the blocker group.After intraperitoneal injection of sodium pentobarbital(75 mg/kg),the mice were fixed supine on a dissecting table after anesthesia to expose the tibialis anterior muscle,after which the tibialis anterior muscle was fixed in a plane using a cuvette as a scaffold and observed by two-photon laser confocal microscopy with green channels(488/518 nm)and red channels(543/572 nm),with solid red indicating mitochondrial autophagy.Image J software was used to analyse the changes in mitochondrial autophagy and oxidative stress.3 Results(1)Compared with group C,group I showed a significant increase in mitochondrial autophagy at the injury site at the 6h and 48h time phases(P<0.001),a highly significant increase in the red/green ratio and the yellow area of oxidative stress(P<0.001),increased mitochondrial division,changes in the Z-line of skeletal muscle and loss of mitochondrial network;at the 7d and 14d time phases,regenerating myotubes with a central nucleus appeared,covered with pure red The autophagic mitochondria were smaller in diameter than the myofibril diameter in group C(P<0.001).The mitochondrial network started to appear at 7d and oxidative stress was still observed.(2)Compared with group I,group IA increased the number of mitochondrial autophagy at 6h(P<0.01),and there was no significant difference in the level of oxidative stress;the area and degree of oxidative stress at 48h were lower than in group I(P<0.001),the degree of mitochondrial fragmentation was reduced,and the network homeostasis was overall better than in group I,but there was no significant difference in the level of mitochondrial autophagy;regenerated myotubes had appeared at 48h and A large number of regenerated myotubes appeared at 7d and 14d,and their diameters were much higher than those in group I.There was no oxidative stress,and the number of mitochondrial autophagy on myotubes was higher than that in group I(P<0.001).(3)After blocking TRP,the mitochondrial network in the IAT group was neater,the number of mitochondrial autophagy was significantly lower than that in the IA group(P<0.001),and the degree of mitochondrial oxidative stress was significantly higher than that in the IA group(P<0.05);after blocking mitochondrial Ca2+ uptake channels,the mitochondrial network in the IAC group was severely disordered,the number of mitochondrial autophagy was not significantly different from that in the IA group,and the area of oxidative stress was significantly lower than that in the I and IA groups(P<0.05).After quenching mitochondrial ROS,the mitochondrial network was neatly organized,the number of autophagy was significantly lower than that of the IA group(P<0.05),and the oxidative stress area and red/green ratio were significantly smaller than those of the IAT and IA groups(P<0.001);after blocking mitochondrial division,the mitochondrial network was disturbed,the mitochondrial volume was were larger,the number of autophagy was not significantly different from the IA group,and the level of oxidative stress was also not significantly different from the IA group,but the degree of mitochondrial oxidative stress was significantly higher than that of the I group(P<0.01);after blocking microtubule generation,the mitochondrial network was severely disrupted,the number of autophagy was significantly lower than that of the IA group(P<0.001),the red/green was lower than that of the IA group(P<0.01),and the area of oxidative stress was smaller than that of the I and IA groups(P<0.01).4 Conclusion(1)After eccentric exercise-induced skeletal muscle injury,mitochondrial network homeostasis was imbalanced,the degree of mitochondrial oxidative stress damage was increased,and mitochondrial autophagy was inhibited,which hinders the regenerative capacity of skeletal muscle after injury.(2)Acupuncture intervention promoted the recovery of mitochondrial network homeostasis,reduced the degree of mitochondrial oxidative stress damage,increased the level of mitochondrial autophagy,and enhanced the regenerative capacity of skeletal muscle after eccentric exercise-induced skeletal muscle injury,which is related to the activation of TRP channels and microtubule transport.