APOPTOSIS IN SKELETAL MUSCLE FOLLOWING BURN INJURY

烧伤损伤，伴或不伴败血症，与许多功能和代谢失常有关。在骨骼肌中，重要的功能改变是肌肉无力，导致通气不足、依赖呼吸器和活动减少。与烧伤相关的神经肌肉功能障碍和肌肉无力延长住院时间，增加费用、发病率和死亡率。因此，这些研究的长期目标是确定肌肉无力的病因，并设计策略来改善人类肌肉产生张力的能力。基于令人信服的初步数据，在本提案中测试的假设是，细胞凋亡或程序性细胞死亡发生在骨骼肌中，发生在烧伤的远处和直接局部，伴随着促凋亡信号通路的激活。本研究的目的是：(1)确认和研究烧伤后骨骼肌局部和远处部位以及不同纤维类型（慢肌纤维和快肌纤维）中细胞凋亡的演变。(2)描述先前仅在体外系统中记录的可能导致细胞凋亡的信号通路。(3)有效抑制部分活化的促凋亡通路，减少肌肉萎缩和肌无力。凋亡将通过三种独立的方法-阶梯法、TUNEL法和ELISA法确认。细胞膜和下游信号分子调节凋亡（如TNFalpha， Fas, FasL，磷脂酰肌醇3-激酶，神经酰胺，BCL-2， BCL-X和caspases）将通过分子药理学或生化技术评估表达和/或活性。某些信号分子的重要性将通过敲除或转基因（TNF、lpr或Fas、神经酰胺、BC12和caspase）小鼠得到证实。一些凋亡信号分子的特异性外源性调节剂（如IGF-1和caspase抑制剂）将在体内用于减轻细胞凋亡引起的肌肉萎缩。这些“逆转细胞凋亡”的研究将与蛋白质转换和功能研究一起进行，以测试这些药物逆转烧伤神经肌肉功能障碍的功效。因此，拟议的研究将描述与烧伤相关的神经肌肉功能障碍，细胞凋亡的组成部分，以及通过药物治疗减少细胞凋亡和神经肌肉功能障碍的潜力。从大鼠研究中获得的信息将为预防和/或纠正人类烧伤或危重疾病的神经肌肉并发症的治疗方法提供科学依据和理论依据。

Burn injury, with and without sepsis, is associated with many functional and metabolic aberrations. In skeletal muscle, the important functional change is muscle weakness resulting in hypoventilation, dependence on respirators, and decreased mobilization. The neuromuscular dysfunction and muscle weakness associated with burns prolongs hospital stay, increases cost, morbidity and mortality. The long-term goals of these studies are, therefore, to characterize the etiology of muscle weakness and design strategies to improve tension-generating capacity of muscle in humans. Based on convincing preliminary data, the hypotheses tested in the present proposal is that apoptosis or programmed cell death occurs in skeletal muscle, at sites distant and immediately local to burn, with activation of pro-apoptotic signaling pathways. The goals of the present studies, using rats, are to: (1) Confirm and study the evolution of apoptosis in skeletal muscle at local and distant sites from burn, and in different fiber types (slow vs. fast twitch muscles). (2) Characterize the putative signaling pathways leading to apoptosis, previously documented only in in vitro systems. (3) Effectively inhibit some of the activated pro-apoptotic pathways and decrease muscle wasting and muscle weakness. Apoptosis will be confirmed by three independent methods- the ladder, TUNEL and ELISA assays. The cell membrane and downstream signaling molecules modulating apoptosis (e.g., TNFalpha, Fas, FasL, phosphatidylinositol 3-kinase, ceramide, BCL-2, BCL-X and caspases) will be assessed for expression and/or activity by using molecular pharmacological or biochemical techniques. The importance of certain signaling molecules will be confirmed by the use of knock out or transgenic (TNF, lpr or Fas, ceramide, BC12, and caspase) mice. Specific exogenous modulators (e.g., IGF-1 and caspase inhibitors) of some of the apoptotic signaling molecules will be used in vivo to attenuate muscle wasting due to apoptosis. These "reversal of apoptosis" studies will be performed in conjunction with protein turnover and functional studies to test the efficacy of these drugs to reverse the neuromuscular dysfunction of burns. The proposed studies will, therefore, delineate the neuromuscular dysfunction associated with burns, the component contributed to by apoptosis, and the potential for attenuation of both the apoptosis and the neuromuscular dysfunction by pharmacotherapy. Information obtained from the rat studies will provide a scientific basis and rationale for therapeutic maneuvers to prevent and/or rectify neuromuscular complications of burns or critical illness in humans.