Isoprenaline Retarding Denervated Skeletal Muscle Atrophy Through Inhibiting NF-kappaB/MuRF1 Pathway

Objective To study the influence of Isoprenaline on the expression pattern of nuclear factor of kappaB (NF-kappaB) and muscle atrophy F-box MAFbx and explore the preventive and therapeutic effect and mechanisms of Isoprenaline on denervated skeletal muscle atrophy.Method A total of 54 healthy male and 8w age Sprague-Duwley rats were selected. They have 195±5g weight and were devided randomly into nine group, A control group (innervations),experimental B group (denervated 2d),experimental C group (denervated 7d),experimental D group (denervated 14d), experimental E group (denervated 28d) Isoprenaline F group (denervated 2d),Isoprenaline G group (denervated 7d),Isoprenaline H group (denervated 14d), Isoprenaline J group (denervated 28d) six rats in every group. After the rats were anesthetized by pentobarbital sodium (40mg/kg) intraperitoneal injection in batch, The midpiece_sciatic nerve of their right lower limb posterior part were cut off for making animal model of denervated skeletal muscle. Sham operation was made only for the rats in normal group (sciatic nerve were not cut off) and sciatic nerve were cut off more than one centimeter for the rats in denervated group. The rats were executed by vertebrae dislocation method in batch. The rats in control group and experimental a group were executed in 48 hour after operation, experimental B and F group for seven day, experimental C and G group for twenty-eight day and so on. whereafter muscle specimen were quickly placed in -80℃refrigerator for reserve. MAFbx and NF-kappaB mRNA level detected by RT-PCR: GAPDH,as a inner reference, are used to make semiquantitative detection for Myf-5mRNA.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. MAFbx primer derives from primer5.0 software design. the upper stream primer is 5'cta cga tgt tgc agc caa ga 3', the downstream primer is 5'Ggc agt cga gaa gtc cag tc3', 167bp.P65:5'gac cag gaa gtc agc gag tc 3'. the downstream primer is:5'tcc aga ggg aca get ctt gt 3',176bp.As a inner reference,GAPDH primer come from Shanxi medical university molecule experiment centre and its piece length have 308bp. polymerase chain reaction goes on by 50μl system and its reaction condition as follow:96℃,2',1cycle。96℃-30″; Tm(Myf-5 53℃)/(GAPDH 50℃)-30″; 72℃,2′,36 cycle. MyoD protein level detected by Westernblot:β-actin,as a inner reference, are used to make semiquantitative detection for MAFbx and NF-kappaB 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,ImageJ software from NIH was used to get image data about mean grey value.then SPSS software-was used to statistics ordered data by one-way anova analysis. Sigmaplot software was used to statistics plotting.Result Expression of MAFbx,NF-κB mRNA and protein all are up-regulation at 2d,7d,14d,28d after denervation. The effect shows that only the expression of NF-κB mRNA and protein level in specimens of Isoprenaline group are down-regulated at 2d,7d,14d,28d after denervation compared with that in denervation Group (P<0.05).Conclusion:Expression of MAFbx,NF-κB were up-regulation at 2,7,14,28 days after denervation In the early stage of skeletal muscles.only NF-κB expression of gastrocnemius denervation model of rats can be down-regulated by Isoprenaline, which shows that Isoprenaline is a effective way of delaying denervated skeletal muscle atrophy through participate in NF-κB path.