Study On Proteolytic Mechanism Of Different Types Of Skeletal Muscle In Rats During Burn Injury And Sepsis

Patients with severe burn injury are always associated with hypermetabolism and protein catabolism If sepsis followed by, this kind of catabolic response will be accelerated, and severe negative protein balance and hypoproteinemia will occur Skeletal muscle, as the largest mtrogen resource, protein will endure a great loss in this process, which will exert a detericus influnce on the prognosis of patients Until recently, it is unclear about the molecular mechanism of skeletal muscle proteolysis dunng burn injury and sepsisObjectives:Through the observation of the changes in proteclytic rate of different kinds of skeletal muscle and changing orderliness in transcriptional and translational level of main components of ubiquitin-prcteascme pathway which plays important roles in proteolysis, to investigate preliminarily the molecular mechanism and possible modulating mechanism of skeletal muscle proteolysis enhancement, and to provide evidence for its clinical interferenceMethods:288 wlstar rats(weight 40-60g) were randomly divided into three groups (1)burn injury group(B group, n=96), a 30% TBSA (total body surface area) fl.ill-thickness scald model was created on rats back after immersed in 100 0C boiling water for 12s, the rats were resuscitated immediately by peritoneal cavity injection of 5% glucose-sahne(40m1/kg); (2)endotoxin insult group(E group, n=96), Bactenum Ccli endotoxm(055B5, 10mg/kg) was administered into the perztoneal cavity of rats under unanaesthesia condition; (3) burn injury and endotoxin-insult group(B+E group,n=96), the rats were subjected to 30% lIP scald and 10mg/kg endotoxin attack The rats were killed at 2h~ 6h~ 12h~ 24h(n=12,each time point containing normal control 12 rats) time points after burn injury and/cr endotoxin administration The plasma samples were collected to determine the concentration of free amino acids with amino acid automatic analyzer, TNF- a IL-6 with enzyme-linked immunosorbent assay(IELISA),glucocorticoid with radiant-immunological assay(RIA), as well as hppolysacchande with chromogemc Limulus Amebocyte Lysate(LAL) which was modified by perchloric acid(PCA) pretreatment of blood samples Skeletal muscle samples were also collected to examine the proteolytic rate in vitro muscle incubation system with sufficient oxygen supply Transcriptional expression of ubiquttin, E2-14K and subunit RC2 of 20s proteasome were measured with Northern Blot method, the translational expression of ubiquitin and ubiquitinated protein were measured with Western Blot method Also , the distribution and content of ubiquitin and ubiquitinated protein was analyzed with immunohistochemical method in rats skeletal muscleResults:1 There was remarkable decrease in plasma total amino acid content at 24h in B+E group, no obvious change was found in B and E group Phe and 3-MEL contents increased significantly, while Arg and Gin contents decreased prominently The increase of 3-MEL in B+E group was the greatest among 3 groups2 There was a significant increase in plasma concentration of cortisol, lipopolysaccharide, TNF- a and ]IL-6 in rats during different time points in B, E and B+E group, but the level in B+E group were significantly higher than that in other two groups3 The release of Tyr and 3-MEL in rats EDL muscle increase markedly in B , E and B+E group The increase of 3-Mu level is greater than that of Tyr Only in B+E group did the proteolytic rate in rat SOL muscle increase markedly4 The transcriptional expression ofubiquitin~E2-14K.2Os and RC2 increased significantly in rat EDL muscle in B, E and B+E group No significant changed were observed in rats SOL muscle in B and E group, except for 6h time point in B+E group5 The translational expression of ubiquitin and ubiquitinated protein increased evidently in rat EDL muscle in B, E and B+E group No significant changes were observed in rat SOL muscle in B and E group, except at 6h time point in B+E group6 There was a significant positive correlation between respective level of 3-MH, ubiquitm-mRNA, ubi...