Objective

2

Based on the PTEN-induced hypothetical kinase 1 (PINK1)/Parkin pathway

the effect of acupuncture pretreatment on the expression of mitochondrial autophagy-related proteins in gastrocnemius muscle tissue of rats with exercise-induced muscle damage (EIMD) was observed

and the underlying mechanism of acupuncture pretreatment for the prevention and treatment of EIMD was explored.

Methods

2

Of 88 SD male rats

aged 6 weeks

8 rats were randomly selected as a blank group

and the remaining 80 rats were randomized into a model group and an acupuncture pretreatment group

with 40 rats in each group. Either the model group or the acupuncture pretreatment group was subdivided randomly into 5 subgroups with 8 rats in each one according to the time points of sample collection

0 h

12 h

24 h

48 h and 72 h after modeling. An intermittent downhill running centrifugal exercise was carried out on an animal experimental treadmill to establish the EIMD model in the model group and the acupuncture pretreatment group. The rats in the acupuncture pretreatment group received acupuncture at "Guanyuan" (CV6) and bilateral "Zusanli" (ST36)

once a day for 20 min each time

for 7 consecutive days before EIMD model preparation. Transmission electron microscopy was used to observe the ultrastructure of gastrocnemius muscle tissue in each group. The contents of malondialdehyde (MDA)

superoxide dismutase (SOD) and catalase (CAT) in serum were detected by ELISA. Western blot was used to detect the protein expression of PINK1

Parkin

sequestosome 1 (p62) and microtubule-associated protein light chain 3B (LC3B) in rat gastrocnemius muscle tissue. Real-time PCR was adopted to detect the mRNA expression of PINK1

Parkin

p62 and LC3B in rat gastrocnemius muscle tissue.

Results

2

Compared with the blank group

the mitochondria of gastrocnemius muscles showed obvious swelling in the 0 h

12 h

24 h

and 48 h model subgroups

autophagosomes were formed in the 12 h and 24 h model subgroups

and the mitochondrial morphology returned to normal gradually in the 72 h model subgroup. The serum MDA contents of rats in 5 model subgroups increased (

P

<

0.01

P

<

0.05). The contents of SOD and CAT in the subgroups of 0 h

12 h

24 h and 48 h decreased (

P

<

0.05

P

<

0.01). The protein and mRNA expression levels of PINK1

Parkin and LC3B in gastrocn

emius muscle tissue of rats in 0 h

12 h and 24 h subgroups were elevated (

P

<

0.01); and the protein and mRNA expression levels of p62 in the 0 h

12 h

24 h and 48 h subgroups were reduced (

P

<

0.01

P

<

0.05). Compared with the model subgroup at the same time point

the myofibril damage and the degree of mitochondrial swelling were mild in each acupuncture pretreatment subgroup

and the numbers of autophagosomes were fewer. The contents of MDA in the acupuncture pretreatment subgroups decreased at 0 h

12 h

24 h

and 48 h (

P

<

0.05

P

<

0.01). The contents of SOD and CAT in the 12 h acupuncture pretreatment subgroup increased (

P

<

0.05

P

<

0.01). The protein and mRNA expression levels of PINK1 and Parkin in the 0 h

12 h

and 24 h acupuncture pretreatment subgroups decreased (

P

<

0.01

P

<

0.05). The protein and mRNA expression levels of LC3B in the 12 h acupuncture pretreatment subgroup decreased (

P

<

0.01)

and that of p62 in the 0 h and 24 h acupuncture pretreatment subgroups increased (

P

<

0.01

P

<

0.05).

Conclusion

2

The intermittent downhill running centrifugal exercise induces the excessive mitochondrial autophagy. Acupuncture pretreatment may attenuate EIMD

and the underlying mechanism is related to the regulation of PINK1/Parkin signaling pathway expression

reducing oxidative stress damage in skeletal muscle cells

and inhibiting mitochondrial autophagy overactivation.