Effects Of Interleukin-15 On Skeletal Muscle Hyperproteolysis In Sepsis Rats

BackgroundSepsis is a serious common complication after major surgery, trauma, burn injury, and shock.under severe risks of illness evolve rapidly, often complicated by multiple organ dysfunction syndrome (MODS), there is a very high mortality rate;.negative nitrogen balance is a very typical characteristics of metabolism in septic states, accounting for 50%of the dry weight of the body cells, skeletal muscle protein catabolism is one major reason for that.Sepsis, a high state of skeletal muscle protein catabolism involves a complex metabolic regulatory networks. Ubiquitin system is a newly discovered protein degradation pathway in recent years, plays an important role in metabolism regulation. Studies suggest that,in sepsis, skeletal muscle protein catabolism and the ubiquitin-proteasome system are closely related. In this state, the body to release a variety of factors (eg, growth factor, interleukin family factors, tumor necrosis factor, etc.), can induce ubiquitin system activation directly or indirectly, affecting the rate of skeletal muscle protein degradation. Interleukin-15 (IL-15) has a wide range of biological activity, which is highly expressed in skeletal muscle. Studies have shown that IL-15 could significantly reverse the skeletal muscle weight of a downward trend in cancer cachexia mouse model, and could reduce the activity of ubiquitin (Ub). At present, the study about the impact of IL-15 in the role of skeletal muscle protein degradation on sepsis state is rare, and the mechanism is unclear. Studies suggest that TNF-a can significantly enhance the expression and activity of ubiquitin system, promoting skeletal muscle protein degradation. Related studies also indicate that there is interaction between IL-15 and TNF-a,It is possible that IL-15 affect the Ubiquitin-proteasome system pathway mediated by TNF-a, but also may be to act on ubiquitin-Protease way directly, what the specific mechanism remains to be further clarified.AimBased on the above findings, the subjects attempted to Further describe the skeletal muscle protein degradation, the gene-protein expression changes and the Interaction mechanism of IL-15 and ubiquitin system in sepsis through animal experiments. Exploration the best time point and dose about exogenous IL-15 to intervention in septic rats. Ask a hypothetical:the skeletal muscle protein degradation rate of sepsis body can be reduced by the intervention of Exogenous IL-15.The use of exogenous IL-15 in septic cases Can down-regulate the body skeletal muscle protein degradation rate, to explore new ways of prevention and treatment. For the High metabolic cachexia in sepsis.Methods1. By cecal ligation and puncture (CLP) to establish a stable rat model of sepsis: Observe the general observation of vital signs parameters in rats, the degree of organ injury, and the stability, of model.2. Healthy SD rats were randomly divided into CLP group (A group), Sham-operated control group (B group). At the observation time points (2h,4h,8h,12h, 24h),kill the rats to take specimens of living. The extensor digitorum longus were incubated in vitro.high performance liquid mass spectrometry (HPLC/MS) determination of total protein (Tyr), and muscle fiber protein (3-MH) degradation rate. Take blood, skeletal muscle and organ, using enzyme-linked immunosorbent assay (ELISA), tissue microarray immunohistochemical staining (IH) and real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) to analyze the gene and protein expression of Ub and IL-15.3. Exploration of the best time point and dose of exogenous IL-15 use.In accordance with previous findings, the 8 hours after CLP was chosen as the Effective point. The exogenous high-purity IL-15 (100μg/Kg) were Injected through the tail vein at the intervention time points,CLP after 6h (C group),7.5h (D group),8h (E group),the remaining time points and non-intervention group (F group) injected the same dose of normal saline. Rats were killed at 12h after CLP to take samples, analyzes the change of indicators. To select the best time point (T0).for IL-15 to intervene.4. Exogenous IL-15 intervention:sepsis rats (CLP surgery) were divided into two groups, Leave control group (G group) of tail vein injection of saline at T0 (7h after CLP); intervention group (H group) were Injected of high-purity recombinant IL-15, dose of M0, Analysis the differences of indicators5. TNFαmonoclonal antibody intervention:control group (I group) to give a simple exogenous IL-15 intervention, in the 2h and 8h after CLP to be saline injection. Exogenous IL-15+TNFαantibody intervention groups (J group):In the CLP tail vein injection of recombinant 7h after the IL-15 at the same time, at 2h after CLP, and 8h, in two tail vein injection of TNFαmonoclonal antibody (3mg/kg). Analysis of indicators of difference.results and discussion:1. The establishment of a stable rat model of sepsis. With time change, the General condition of rats were deteriorated, the plasma levels of endotoxin increased. Parameter stability in the control group. Significant difference between the two groups (P<0.01). Pathology show that the tissues and cells of rats have been serious injuryafter CLP2.The gene-protein level of IL-15 and ubiquitination in group A are higher than group B in all (P<0.01), show The initial increase and late decline. Compared with group B, rats Serum IL-15 protein of group A peaked at 8h after CLP and then decreased; IL-15 and Ub m RNA levels of skeletal muscle increased at 4h, and 12h to the peak; skeletal muscle protein metabolic rate increased with time (P<0.01), reached the highest at 24h. We conclude that 8h after CLP as a valid compensatory time windows.3. Exploration of the best time point and dose of exogenous IL-15 compared with group F, rat skeletal muscle metabolic rate decreased in group C, group D, group E,the serum concentr of IL-15 increased and the Ub declined (P<0.01), we select 7h after CLP. (T0) as the best time point for IL-15 to intervene. Metabolic rate of skeletal muscle fiber protein was negatively correlated with serum IL-15 concentration (r=-0.41374, P<0.05), and positively correlated with Ub(r= 0.76247, P<0.01).4. Exogenous recombinant IL-15 cytokine intervention experiments, compared with group G, protein metabolism was significantly lower in group H (P<0.01), IL-15 protein expression in plasma and tissue was elevated in group H (P<0.05). Description IL-15 can effectively antagonize degradation effects of ubiquitin system in skeletal muscle protein.5. the levels of IL-15 and Ub express in plasma tissue were significantly difference between Two groups (P<0.01), the skeletal muscle fiber protein metabolic rate of groupⅠis lower than group J (P<0.01), but the total protein metabolic rate was no Statistical difference. shows that IL-15 affect the Ubiquitin-proteasome system pathway mediated by TNF-α, but not the unique pathway. Prompted the surface of skeletal muscle cells have the specific receptor for IL-15.SummaryThis study established a stable rat model of sepsis①; a aerobic incubation system of skeletal muscle in vitro②; a detection method about 3-methyl histidine and tyrosine by High Performance Liquid Mass Spectrometry③; the methods and conditions to analyze the IL-15 and ubiquitin gene and protein expression systems④.The experimental results described the protein and gene expression levels of IL-15 as well as the ubiquitin on the pathological state in sepsis, observed that IL-15 and Ub expression have relations with the metabolic rate of skeletal muscle, il-15 is activated in the pathological state. By exogenous recombinant IL-15 factor intervention trial, Our preliminary exploration of the IL-15 intervention point (7h after CLP) and dose (100μg/Kg). Experiments showed that IL-15 intervention can reduce the rate of skeletal muscle protein metabolism in sepsis, suggesting 7h after CLP as the best intervention point. By TNF-a Blocker intervention, shows that IL-15 affect the Ubiquitin-proteasome system pathway mediated by TNF-α, but not the unique pathway. For the Patients in critical condition, IL-15 Synthesized by the endogenous is far from meeting the needs of the body, Stimulate the synthesis and exogenous intervention is necessary.