【作者】 邓虎平；

【导师】 柴家科；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2010， 博士

【摘要】 目的：严重烧伤后骨骼肌蛋白代谢以高分解代谢和负氮平衡为特点,我所以往研究发现泛素蛋白酶体途径是骨骼肌蛋白分解的主要机制,其中肌肉萎缩盒F蛋白(MAFbx)和肌肉特异性环指蛋白1(MuRF1)是骨骼肌蛋白分解的关键酶,胰岛素能够抑制骨骼肌蛋白分解。但目前对于烧伤后骨骼肌MAFbx和MuRF1表达变化及其调节机制的研究尚不完善。已知泛素蛋白酶体参与的蛋白分解过程需要ATP提供能量,骨骼肌的能量状态可能影响着蛋白分解代谢。单磷酸腺苷激活的蛋白激酶(AMPK)是调节三磷酸腺苷(ATP)生成的关键分子,近年来又发现AMPK的活化抑制蛋白合成,然而,迄今为止对于骨骼肌能量状态及AMPK对骨骼肌蛋白分解代谢影响和机制的报道甚少。为进一步阐明烧伤后骨骼肌蛋白分解代谢的机制,指导临床治疗,本课题试图通过动物实验和细胞培养实验探讨：(1)烫伤大鼠骨骼肌蛋白分解、能荷和AMPK的变化；(2)烫伤大鼠血清对L6肌管蛋白分解、能荷和AMPK的影响；(3)L6肌管AMPK对蛋白分解的调节作用,及其与胰岛素信号通路相互作用的机制。方法：1.动物实验：雄性Wistar大鼠100只(180-220g),随机分为两组：(1)对照组(n=50)；(2)烫伤组(n=50),背部置于94℃热水12s致30%总体表面积(TBSA)Ⅲ度烫伤。分别于伤后6h、1d、3d、5d和7d(n=10)麻醉处死大鼠,解剖分离双侧胫骨前肌、趾长伸肌(EDL)和比目鱼肌。实验过程中每日称量体重,实验结束时称量肌肉重量。Western blotting检测胫骨前肌AMPK表达和活性,实时定量PCR(Q-PCR)检测胫骨前肌AMPKα、丝氨酸／苏氨酸激酶11(LKB1)、MAFbx及MuRF1的mRNA表达水平,高效液相色谱法(HPLC)测定胫骨前肌核苷酸水平,计算AMP(单磷酸腺苷)／ATP比值和细胞能荷(EC)。2.细胞培养实验A：采用上述烫伤动物模型,雄性Wistar大鼠40只(200-250g),随机分为两组：对照组(n=20)和烫伤组(n=20),伤后12h无菌抽取大鼠腹主动脉血液,制备血清。10%胎牛血清(FBS)预处理低血清分化的L6肌管12h后,分别采用含10%假伤大鼠血清的DMEM(10%SS组)、或含10%烫伤大鼠血清的DMEM(10%BS组)孵育肌管不同时间(0、10min、1h、6h、24h)。L6肌管蛋白、mRNA和核苷酸水平的测定同方法1。3.细胞培养实验B：低血清分化的L6肌管于无血清DMEM中预处理12h后,分别于不同浓度或不同时间条件下,用含AMPK活化剂(AICAR,5-咪唑-4-酰胺-1-β-D-呋喃糖核苷)或含胰岛素的DMEM处理L6肌管,部分实验中采用PI3K抑制剂(wortmannin)、Akt抑制剂(Akt InhibitorⅣ)或AMPK抑制剂(Compound C)预处理肌管1h。Western blotting检测L6肌管AMPK、Akt、糖原合成酶激酶(GSK)3-β和乙酰辅酶A羧化酶(ACC)的表达及其活性,mRNA和核苷酸水平的检测同方法1。结果：1.(1)大鼠烫伤后体重较对照组下降,于伤后3d最为显著(p<0.05)。大鼠EDL重量和EDL重量／体重比值于伤后1～3d显著低于对照组(p<0.01)。(2)与对照组比较,烫伤后6h胫骨前肌MAFbx mRNA表达明显上调(p<0.01),烫伤后6h和1d,胫骨前肌MuRF1 mRNA表达明显上调(p<0.01),于5d和7d明显下降(p<0.01或p<0.05)。(3)烫伤后6h,大鼠胫骨前肌AMPKα(Thr172)磷酸化水平高于对照组(p<0.05),其后逐渐下降,于3d显著低于对照组(p<0.01),以后又升高。烫伤对胫骨前肌AMPKα蛋白表达无影响(p<0.05)。(4)烫伤后1d,AMPKα2mRNA表达明显低于对照组(p<0.01)。(5)烫伤后6h,LKB1 mRNA水平较对照组上调(p<0.01),其后下降,于3d和5d明显低于对照组(p<0.01),7d有所回升。(6)烫伤后6h、1d、5d和7d,AMP／ATP比值较对照组升高(p<0.01),而EC较对照组降低(p<0.01或p<0.05)。2.(1)烫伤大鼠血清孵育L6肌管24h显著上调MAFbx和MuRF1的mRNA表达(p<0.05)。(2)与10%SSs组比较,10%BS组L6肌管AMPKα蛋白表达显著下降(30min、1h、6h,p<0.01),而AMPKα磷酸化水平升高(1h、6h,p<0.05；24h,p<0.01)。(3)10%BS组L6肌管AMPKα1 mRNA表达于24h较10%SS组降低(p<0.01),AMPKα2 mRNA表达于1h、6h和24h均较10%SS组降低(p<0.05)。(4)10%BS组L6肌管LKB1 mRNA表达于24h较10%SS组升高(p<0.01),然而,烫伤大鼠血清对L6肌管AMP／ATP比值和EC无影响(p>0.05)。3.(1)AMPK活化剂AICAR以剂量依赖方式抑制MAFbx和MuRF1 mRNA表达,Compound C预处理可抑制AICAR对MuRF1基因表达的上调作用(p<0.01)。(2) AICAR以剂量依赖方式上调Akt (Ser473)和GSK3-β(Ser9)的磷酸化水平,与激活PI3K有关。(3)胰岛素以剂量依赖的方式下调MAFbx和MuRF1 mRNA表达,AICAR能够逆转胰岛素对MAFbx和MuRF1 mRNA表达的抑制作用(p<0.01)。(4)胰岛素以剂量依赖方式去磷酸化抑制AMPK,其机制为通过激活Akt实现。(5)胰岛素以剂量依赖方式抑制肌管LKB1 mRNA表达,而对AMP/ATP和EC无显著影响(p>0.05)。结论：烫伤大鼠骨骼肌能量状态的下降激活AMPK, AMPK的活化上调骨骼肌泛素E3连接酶的基因表达,蛋白分解增强。AMPK与蛋白代谢密切相关的胰岛素信号通路相互作用,AMPK激活Akt,而胰岛素抑制AMPK,胰岛素下调蛋白分解代谢的可能机制之一为抑制AMPK。因此,烫伤后骨骼肌AMPK的活化可促进蛋白分解代谢,针对AMPK的治疗措施可能是抑制烧伤后蛋白高分解的新靶点。更多还原

【Abstract】 Objective:The protein metabolism in skeletal muscle post severe burn is characterized by hyperproteolysis and negative nitrogen balance. Our previous studies have deduced that ubiquitin-proteasome played a key role in skeletal muscle proteolysis. Especially, muscle atrophy F-box (MAFbx) and muscle-specific RING finger protein 1 (MuRF1) are key enzymes in regulating muscle proteolysis. Furthermore, the enhanced proteolysis can be inhibited by administration of intensive insulin therapy. However, the changes and molecular mechanisms in expression of skeletal muscle MAFbx and MuRF1 post-burn are still obscure.Adenosine triphosphate (ATP) supplies energy to ubiquitin-proteasome pathway of proteolysis. Energy status may affect proteolysis in skeletal muscles. It is well known that AMP-activated protein kinase (AMPK), the’Energy Gauge’, regulates production of ATP. Recently, several studies revealed the inhibitory effects of AMPK activation on protein synthesis. However, little has been studied on the effects of energy status and AMPK activity on skeletal muscle proteolysis and its mechanisms. In order to further elucidate the mechanisms of proteolysis in skeletal muscles post-burn, and to guide clinical treatment, this project was aimed to explore:(1) changes of energy status, AMPK and proteolysis in skeletal muscles of scalded rats, (2) changes of energy status, AMPK and proteolysis in L6 myotubes treated with serum from scalded rats, (3) molecular mechanisms of interaction among AMPK, proteolysis and insulin signaling pathway in L6 myotubes.Methods:1. Animal experiments:One hundred male Wistar rats (180-220g) were randomly divided into two groups:(1) control group (n=50), (2) scald group (n=50). In scald group, rats were inflicted on with 94℃water for 12s to create full-thickness burns, 30% total body surface area (TBSA). The rats were anesthetized 6h, Id,3d,5d, or 7d after injury and sacrificed for collecting muscles:tibialis anterior, extensor digitorum longus (EDL), and soleus. Rats and muscles were weighed. Then, protein levels and activity of AMPK were determined by Western blotting. Quantitative real time PCR (Q-PCR) was used to measure mRNA levels of AMPKa, serine/threonine kinase 11 (LKB1), MAFbx and MuRF1. Furthermore, adenosine monophosphate (AMP)/ATP ratio and energy charge (EC) were measured by high performance liquid chromatography (HPLC).2. Cell culture experiment A:Forty male Wistar rats were randomly divided into sham scald group (n=20) and scald group (n=20). Scald animal model was established according to aforementioned method 1. Rats were sacrificed to harvest serum 12h post-injury. Furthermore, after pre-incubation with DMEM plus 10% FBS for 12h, the differentiated L6 myotubes were cultured with DMEM containing 10% sham scald rat serum (10%SS) or 10% scald rat serum (10% BS) for different durations of time (0, 10min, 1h,6h,24h). The levels of protein, mRNA and nucleotides were measured according to method 1.3. Cell culture experiment B:The L6 myotubes were pre-incubated with DMEM for 12h, followed by replacement with DMEM plus different doses of AMPK activator (AICAR,5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside) or insulin for various time durations. In some groups, PI3K inhibitor (wortmannin), protein kinases B (PKB, Akt) inhibitor (Akt Inhibitor IV) or AMPK inhibitor (Compound C) was applied as pretreatment for 1h. Protein levels and activity of AMPK, acetyl-CoA carboxylase (ACC), Akt, and glycogen synthase kinase (GSK) 3-βwere determined by Western blotting, mRNA levels of AMPKa, LKB1, MAFbx and MuRF1 by Q-PCR, nucleotides levels by HPLC.Results:1. Scald reduced body weight of rats, especially on 3d post-injury (p<0.05). Meanwhile, muscle mass of EDL, and EDL to body weight ratio were decreased from Id through 3d in scald group compared with that in control group (p<0.01). Further, compared with control group, the mRNA level of MAFbx in tibialis anterior was-increased 6h post-injury (p<0.01), the mRNA level of MuRF1 increased both at 6h and on 1d post-injury (p<0.01), followed by a decrease from 5d to 7d in scald group (p<0.01 or p<0.05). Compared with control group, phosphorylation level of tibialis anterior AMPKa (Thr172) in scald group was increased at 6h (p<0.05), then was decreased to the lowest level on 3d (p<0.01), and finally was increased on 7d (p<0.05). But there were no differences in protein levels of AMPKa between the two groups (p>0.05). The mRNA level of AMPKa2 subtype decreased on 1d in scald group (p<0.01). The gene expression of LKB1 in scald group was upregulated at 6h (p<0.01), then reduced significantly 3d and 5d (p<0.01), followed by an increase on 7d. Interestingly, tibialis anterior AMP/ATP ratio was higher in scald group, except on 3d, than that in control group (p<0.01), while changes of EC was contrary to changes of AMP/ATP ratio (p<0.01 or p<0.05).2. Scald rat serum caused upregulation of MAFbx and MuRF1 mRNA expression in L6 myotubes at 24h (p<0.05). Compared with 10%SS group, the protein level of AMPKa was decreased in 10%BS group (30min, 1h and 6h, p<0.01), while pAMPKa level was increased in 10%BS group (1h and 6h,p<0.05; 24h,p<0.01). In 10%BS group, the mRNA level of AMPKα1 were reduced at 24h (p<0.01), and the mRNA level of AMPKa2 were reduced at 1h,6h and 24h (p<0.05), and the mRNA level of LKB1 was increased at 24h (p<0.01), as compared to that in 10%SS group. But the AMP/ATP ratio and EC showed no significant differences between the two groups (p>0.05).3. In L6 myotubes, AMPK activator, AICAR, downregulated mRNA expression of MAFbx and MuRF1 in a dose dependent pattern. Howerver, pretreatment with Compound C inhibited the upregulation of MuRF1 level caused by AICAR (p<0.01). Furthermore, AICAR enhanced both pAkt (Ser473) and pGSK3-β(Ser9) levels, relating to activating PI3K. On the other hand, insulin reduced both MAFbx and MuRF1 mRNA levels dose-dependently. This inhibitory effect could be blocked by administration of AICAR (p<0.01). Concomitantly, insulin inhibited AMPKa through activation of Akt, and downregulated mRNA level of LKB1 dose-dependently. Moreover, insulin had no effects on AMP/ATP ratio and EC (p>0.05).Conclusion:A lower energy status activates AMPK, which then enhancing expression of ubiquitin E3 ligases and skeletal muscle proteolysis in scalded rats. There are tight connections between AMPK and insulin signaling pathway. AMPK phosphorylates Akt, while insulin inhibits AMPK, which might be one of signaling pathways counting for downregulating proteolysis. Thus, enhanced proteolysis in skeletal muscle could be caused by activated AMPK after burns. It could be implied that targeting AMPK would be a new strategy to inhibit skeletal muscle proteolysis after burns.更多还原