The Influences Of Proteasome Inhibitor MG-132 On The Gene Expression Of MyoD Of Skeletal Muscle Of Rats

Objective The purpose of this article is to investigate the influences of proteasome inhibitor MG-132 on the gene expression of MyoD of skeletal muscle of rats.Method A total of 72 healthy male and 8w age Sprague-Duwley rats were selected. They have 190±5g weight and were devided randomly into three group, with 24 in each, group A: control group; group B: denervated group ; group C: 100μL MG-132 group. Standard model of denervated gastrocnemius muscle was established in right limb of each rat in group B and C . 100μL MG-132 (concentration 20μmol /L) were respectively given by intramuscular injection once per day to right gastrocnemius muscle of each rat of group C. The rats were executed by vertebrae dislocation method at 2d,7d,14d,28d after denervation .Dissect and dissociatethe leg skeletal muscle of the right lower limb gastrocnemius,whereafter muscle specimen were transected and placed quickly in -80℃refrigerator for reserve . MyoDmRNA level detected by RT-PCR: GAPDH ,as a inner reference , are used to make semiquantitative detection for MyoD mRNA .Take 50mg sample tissue to homogenate , and then extract total RNA by extract reagent from shanhai sangon , next to have it inversely transcripted by RT-PCR reagent box from MBI according as 2μl sample RNA and 20μl system . MyoD primer derive from primer5.0 software design.its piece length is 137bp.As a inner reference ,GAPDH primer come from shanxi medical university molecule experiment centre and its piece length have 307bp. polymerase chain reaction goes on by 50μl system and its reaction condition as follow : 94℃,2′,1cycle。94℃- 30″;Tm(MyoD 53℃)/(GAPDH 50℃)- 30″;72℃,2′,36 cycle. Then take agarose gel horizontal strip electrophoresis with 160mv voltage and get image through gel formatter in the end . MyoD protein level detected by Westernblot:β-actin ,as a inner reference ,are used to make semiquantitative detection for MyoD protein . Take 100mg sample tissue to homogenate , and then extract total protein by protein extract reagent box from Applygen Technologies Inc. after boiled for 5min and centrifugated in sample buffer for 10 min ,RNA samples were spotted into pore of vertical electrophoresis bath and were separated in 12% SDS-PAGE for about three hour and transferred onto nitrocellulose membrane (Bio-Rad) for 1 hour and thirty minutes . Nonspecific reactivity was blocked by incubation 4h at 20℃in buffer(10mM Tris-HCl, pH7.5,150mM NaCl, 2%Tween-20, 4% bovine sreum albumin). After floating in TBST buffer, The membrane was then incubated with primary antibody overnight at 4℃. After floating in TBST buffer, The secondary antibody was used to incubated with the membrane for 4h at 4℃. After floating in TBST buffer and TBS buffer, The membrane was then placed in dark room and reactive protein was detected by ECL chemiluminescence system(santa cruz). the membrane was immediately placed into camera obscure for exposition about 1 min ,and was taken out to immerse into developer until developing was optimization . then the membrane was immediately got out of the developer , placed into fixerfor about fifteen minutes , taken out for open-air drying , photographed by figure camera for store .Finally ,Photoshop software from was used to get image data about mean grey value .then SPSS software was used to statistics ordered data by one-way anova analysis .Result (1)The effects shows that the expression of MyoD mRNA and protein indenerved skeletal muscle all are up-regulation at 2d,7d,14d,28d after denervation compared with that in A group (P<0.05); (2)There were also statistic differences between group B and C (P < 0.05) ;(3) There were two-way and two-way interactions among time and the treatment.The factors interacted to affect mRNA and protein expression of MyoD. (P<0.05)Conclusion (1)The expression of MyoD mRNA and protein indenerved skeletal muscle all are up-regulation at 2d,7d,14d,28d ; (2)Two factors interacted to affect mRNA and protein expression of MyoD; (3)After denervation in SD rat, proteasome inhibitor MG-132 can up-regulatie the expression of MyoD mRNA and protein by preventing ubiquitin-proteasome pathway. Proteasome inhibitor MG-132 retard the denervation induced muscular atrophy by upregulating the expression of MyoD.