Acupuncture Intervention On The Early Phases Of Rat Skeletal Muscle Caused By Eccentric Exercise Ca²⁺, Na⁺, H₂O₂ Study On Changes In Flow Velocity

1 Objective:Skeletal muscle injury,especially contusion and strain,are one of the most common sports injuries.It is generally believed that fibrosis during skeletal muscle injury/repair is related to the inflammatory response caused by Ca²⁺overload in the early stage of injury.Acupuncture has a good curative effect on skeletal muscle injury.According to the recent research situation,the mechanism is related to the early recovery of plasma Ca²⁺homeostasis through the reactive oxygen?ROS?signaling pathway mediated by TRP channel.In this paper,the Non-invasive Micro-test Technique was used to detect the dynamic changes of Ca²⁺,Na⁺and H₂O₂ during the early phase of skeletal muscle injury after acupuncture intervention in order to explore the role of interaction between TRP channel and NADPH oxidase 2?NOX2?in the acupuncture mechanism.2 Methods:324 healthy male Wistar rats were randomly divided into 6 groups:blank control group?C?,electrical stimulation group?E?,electrical stimulation plus acupuncture group?EA?electrical stimulation plus acupuncture TRP channel blocking group?EAT?,electrical stimulation plus acupuncture NOX2 blocking group?EAN?,placebo group?EAP?.Except for group C,the animal model of skeletal muscle injury caused by eccentric contraction was established by electrostimulation of living gastrocnemius muscle.Immediately after electrical stimulation,blocking group and placebo group respectively accepted GdCl₃,apocynin and PBS buffer by tail vein injection,after 30min,Except for group E,the rest of the group were needled with a acupuncture needle?diameter of 0.13 mm?at the belly of gastrocnemius?oblique needling?.Afterwards,a special polypropylene ring-shaped perforated vessel wall was sutured to the exposed gastrocnemius muscle.Using non-invasive micro-test technique,EA,EAT,EAN and EAP group was detected after the phase retaining needle,needle drawing immediately,3h,6h and 24h.The dynamic flow rates of Ca²⁺,Na⁺,and H₂O₂ were changed during the10 minutes testing time.The phase and time of detection in the C and E groups were consistented.3 Results:3.1 When the gastrocnemius muscle was in a resting state,Ca²⁺and Na⁺were inflow in small amounts,and H₂O₂ had a small outflow.3.2 Effect of eccentric Exercise and acupuncture on Ca²⁺dynamic velocity changes of skeletal muscle at different phases:?1?In group E,a small amount of outflow occurred at each phase,and the outflow suddenly increased significantly at 6 h?p<0.05?,followed by a small outflow;?2?In the EA group,a small amount of outflow occurred during and after the needle acupuncture,and a small amount of inflow was observed at 3h.The outflow velocity increased suddenly at 6h and 24h,and the flow rate peaked at 24 h,which was significantly different from the E group at 24 h.?p<0.05?;?3?After blocking the TRP channel,the EAT group showed a significant influx trend.Specifically,except a small outflow in the retention period and 3h phase,significant inflow occurred immediately after the needle pulling?6h and 24h,compared with group C and group EA,there was no statistically significant difference in net flow velocity,but Ca²⁺oscillation amplitude?influx and outflow fluctuation amplitude?in EAT group was significantly increased?p<0.001,p<0.01,respectively?;?4?After blocking NOX2,the overall outflow of EAN group was dominant.Specifically,there was significant inflow at the time of retention and immediately after needle pulling,and suddenly significant outflow was observed at 3h and 6h.The 6h phase was significant difference than that of E group.?p<0.05?,afterwards,the outflow velocity was significantly decreased at 24h,and was significantly different from 6h?p<0.05?;?5?The EAP group was effluxed at all phases,and the overall trend was similar to the E group.The outflow's velocity peaked at 10min phase,which was significantly different from that of the concurrent phase E and EA groups?p<0.05?,and the outflow rate was significantly decreased at 24h.3.3 Effect of eccentric exercise and acupuncture on Na⁺dynamic velocity changes of skeletal muscle at different phases:?1?In group E,the outflow occurred at 0min and10min,and the inflow occurred suddenly at 3h and 6h.There was a significant difference between group 6h and group C?p<0.001?,and the outflow again occurred at24h;?2?In the EA group,the flow occurred immediately after the needling and after the pulling phase,while it was outflowed at the 3h,6h,and 24h;?3?After blocking the TRP channel,the EAT group flowed out at all phases,which was in line with the trend of changes in the E group.?4?After blocking NOX2,the EAN group only outflowed at 6h phase?p<0.05?,while the rest of the phases flowed in,the influx peaked at 3h,which was significantly different from that at 10min?p<0.05?.The inflow amplitude decreased at24h,showing a significant difference from 3h?p<0.01?.?5?After placebo injection,the EAP group only flowed inwards at 3h,and the rest of the phases were flowed out.3.4 Effect of eccentric Exercise and acupuncture on H₂O₂ dynamic velocity changes of skeletal muscle at different phases:?1?In group E,the inflow occurred only at 10 min phase,and the rest of the phases were flowed out with an increasing trend.The peak value were reached at 24h,showing a significant difference with C group and 0min?p<0.01?,and extremely significant with 3h?p<0.001?and 6h phase?p<0.05?;?2?EA group only inflowed during acupuncture,all other phases were outflowed and peaked at24h,but the outflow rate was less than that of E group and there was a significant difference at 6h phase with E group?p<0.05?;?3?After blocking the TRP channel,the EAT group flowed out at all phases and reached the peak at 3h,showing a significant difference compared with the E and EA groups at the same phases?p<0.001?;?4?After blocking NOX2,the EAN group effluxed at each time phase and peaking after needle pulling,but the flow velocity was higher than that of the EAT group.There was an extremely significant difference compared with EA group at the peaking phase?P<0.001?and a significant difference with EAT group at the same phase?p<0.05?;?5?After injection of placebo,all phases remained outflow,but the flow rate was less than the EAT and EAN groups.4 conclusion:4.1 In the resting state,Ca²⁺and Na⁺of skeletal muscle were all slightly inflowed,while H₂O₂ was slightly flowed out.4.2 In the early subsequent phase after skeletal muscle injury,Ca²⁺efflux velocity decreased,while Na⁺inflow rate increased,accompanied by increased H₂O₂ efflux velocity.4.3 Acupuncture intervention reduced Ca²⁺efflux velocity in the early subsequent phase of skeletal muscle injury and advanced the Na⁺influx phase,with the decrease of H₂O₂ efflux velocity,and the effect was related to the interaction of TRP channels synergize with NOX2 Activity.