Study On Relationship Between Skeletal Muscle Wasting And Apoptosis In Rats With Burn Injury And Sepsis

In clinical observation, we can find that there are obviously high protein catabolism in skeletal muscle in burn patients, and it can become more intense if combined with systemic inflammation, as result, the patients can fall into severe hypoproteinemia and negative nitrogen balance. As we all know, the skeletal muscle is the largest nitrogen resources, massive wasting of skeletal muscle could influence the prognosis of the burn patients gravely. Up to now, the precise molecular mechanism of skeletal atrophy and wasting remains unknown. Objective: In this study, we investigated the transcriptional changes in apoptosis-related proteins in different types of skeletal muscle in burned and sepsis rats. With the help of cell-free system, we analyzed the role the caspase-3 in skeletal muscle wasting and its relationship with ubiquitin-proteosome during skeletal muscle wasting. From the above results, we preliminarily investigated the relationship between skeletal muscle wasting and apoptosis, and the potential role of caspase-3 in skeletal muscle wasting. Also, the mechanism of skeletal muscle wasting and possible modulating mechanism were studied, which provided theoretical evidence for modulating skeletal muscle wasting and atropy in clinical settings.Materials and Methods: 288 male Wistar rats, weighted 60-70g, were randomly divided into 2 groups: (Dburn injury group (B group, n=120), a 30% TBSA (total body surface area) full-thickness scald was inflicted on rats back by 100℃ boiling water for 12s, the rats were resuscitated by administering 5% glucose-saline (40ml/kg) into the peritoneal cavity;(2)bura injury followed by endotoxin-challenge group(B+E group, n=96), the rats were subjected to 30% Ⅲ scald and 10mg/kg endotoxin challenge. All the rats were sacrificed at 6h, 1d, 4d, 7d and 14d (n=24, 12 animals served as normal controls in each time point) afterburn injury and/or endotoxin administration. Skeletal muscle samples were also collected to determine the proteolytic rate of skeletal muscle by in vitro muscle incubation system with sufficient oxygen supply. Transcriptal expression of Akt, Bcl-2 and caspase-3 were measured by RT-PCR method. With the help of skeletal muscle cell culture and cell-free system, we determined the skeletal muscle proteolytic rate and the expression of small molecular protein 14kDa-actin after given caspase-3 stimulator (stauroporine) or inhibitor (DEVD) or preoteasome inhibitor by amino acid analyzer and Western blots. Results:1. In burned rats, the Akt expression in EDL (Extensor Digital Longus) muscle decreased at different time points, the Bcl-2 expression increased slightly at the early stage, but decreased at late stage, caspase-3 expression increased significantly on days 7 and 14 postburn. But in SOL (Soleus) muscle, there were no significant changes in Akt expression at other time points except significant decrease on day 14. There were significant increase in Bcl-2 at 6h, Id and 4d, and then decrease gradually on days 7 and 14 postburn, no significant changes were found. There were no marked change in caspase-3 expression in SOL muscle in burned rats.2. In burn injury combined with sepsis rats, the Akt expression in EDL muscle decreased at 6 h, and kept low expression on days 1, 4, 7 and 14, and decreased significantly lower than normal controls. The Bcl-2 expression increased slightly at 6h, and no change on day 1, decreased on days 4, 7 and 14. The caspase-3 expression gradually increased, and peaked on day 14. In SOL muscle, the Akt and Bcl-2 expression decreased, but caspase-3 expression increased on days 7 and 14, and significantly higher on day 14.3. Caspase-3 could breakdown actomyosin complex, and exert 14kDa-actin small fragment.4. In cultured skeletal muscle cells, caspase-3 addition could enhance the cell proteolysis, while treatment with DEVD could inhibite proteolysis of skeletal muscle cells. After addition of degraded actomyosin by caspase-3 couldsignificantly stimulate total and myofibrilllar proteolysis of skeletal muscle cells.5. In cultured skeletal muscle cells in vitro, after treatment with staurosporine and Lactacystin, we could find high molelular weigh band actomyosin and high gray scale band 14kDa-actin. After treatment with staurosporine only, we could see lower gray scale band 14kDa-actin than before, however, after solely Lactacystin treatment, the band 14kDa-actin became higher than staurosporine only, but lower than both staurosporine and Lactacystin treatment. After DEVD treatment only, the band 14kDa-actin disappeared again.6. Caspase-3 inhibitor could down-regulate the expression of 14kDa-actin.7. IGF (Insulin-like Growth Factor)-1 and insulin could inhibit the expression of 14kDa-actin.Conclusion:1. In severely burned rats, apoptosis could commonly occur in fast-white skeletal muscle, and become markedly along with the prolongation of the disease course, with no obvious chagne in slow-Ted skeletal muscle. In rats subjected to burn combine with sepsis, apoptosis could occur not only in fast-white skeletal muscle but also in slow-red skeletal muscle. The apoptosis severity was more significant than simply burned rats.2. Caspase-3 located in the upstream of the sequence of skeletal muscle wasting reaction, and ahead of ubiquitin-proteosome pathway.3. IGF-1 and insulin could reverse the apoptosis of skeletal muscle.4. The expression 14kDa-actin could be used as a dependent index for skeletal muscle proteolysis.In summary, apoptosis plays an important role in skeletal muscle wasting and atrophy in burns and sepsis, and it locates in upstream upstream of the sequence of skeletal muscle wasting reaction. Especially, caspase-3 can trigger skeletal muscle wasting, which provides important theoretical evidence for modulating skeletal muscle wasting from the view of apoptosis.