Effect Of Proteasome Inhibitor MG-132 On Myf-5 Protein Expression In Rat Denervated Skeletal Muscles

Objective The purpose of this paper is to investigate the influence of proteasome inhibitor MG-132 on expression of myogenic regulatory factor Myf-5 gene of denervated skeletal muscle in rats. Method A total of 54 Sprague-Duwley rats were selected. They were devided randomly into three groups, with 18 in each.group A:control group; group B: denervated group; group C:denervated and MG-132 group. Sham operation were made only for the rats in group A (sciatic nerve were not cut off) and sciatic nerve were cut off more than one centimeter at the mid-level of sciatic nerve on their right lower limb for the rats in group B and group C. Local multi-point injection concertration of 20μmol/L of proteasome inhibitor MG-132 100μL,1 time/d was did in group C. The rats were executed by vertebrae dislocation method at 2d,7d,28d after denervation. Dissect and dissociate the leg skeletal muscle of the right lower limb (tibialis anterior,soleus,gastrocnemius,plantar), whereafter muscle specimen were transected and placed quickly in -80℃refrigerator for reserve. Myf-5 mRNA level detected by RT-PCR:GAPDH,as a inner reference,were used to make semiquantitative detection for Myf-5mRNA.Took 50mg sample tissue to homogenate, and then extracted total RNA by extract reagent from hanhai sangon, next to had it inversely transcripted by RT-PCR reagent box from MBI according as 2μl sample RNA and 20μl system. Myf-5 primer were derived from primer 5.0 software design. the upper stream primer was5'-TGC CAT CCG CTA CAT TGA GAG-3', the downstream primer was 5'-CCG GGG TAG CAG GCT GTG AGT TG-3', As a inner reference,GAPDH primer came from Shanxi medical university molecule experiment centre and its piece length hd 308bp. the upper stream primer was 5'-TCC CTC AAG ATT GCT AGC AA-3',the downstream primer was5'-AGA TCC ACA ACG GAT ACA TT-3'. polymerase chain reaction went on by 50μl system and its reaction condition as followed:96℃,2', 1cycle。96℃,30"; Tm(Myf-5 53℃)/(GAPDH 50℃),30"; 72℃,2', 36 cycle. Myf-5 protein level detected by Westernblot. Result Compared with the control group,the expression of Myf-5 mRNA and protein were increased in the denerved and MG-132 intervention groups at 2d,7d,28d after operation(P<0.01). The increasing of Myf-5 mRNA and protein in the MG-132 intervention group was greater than that in the denerved group at 2d,7d,28d after operation (P<0.01).Conclusion In the early skeletal muscle atrophy, proteasome inhibitor MG-132 can inhibit the ubiquitin- proteasome pathway to increase the expression of Myf-5 gene in order to play the role of slowing skeletal muscle atrophy.