胰岛素强化治疗对烫伤脓毒症兔骨骼肌蛋白高降解的调节及其机制

目的探讨胰岛素强化治疗对烫伤脓毒症骨骼肌蛋白高降解的调节及机制。方法雄性日本大耳白兔30只，按随机数字表法将动物分为烫伤组（S组）、烫伤脓毒症组（SS组）、烫伤治疗组（SI组）、烫伤脓毒症治疗组（SSI组）以及对照组（C组），每组6只动物。S组使用沸水致背部30％总体表面积Ⅲ度烫伤；SS组同样条件致伤后，立即腹腔注射内毒素（2mg／kg）模拟烫伤脓毒症。SI组和SSI组从伤后2h开始经静脉泵入胰岛素，使血糖值始终波动在4．4～6．1mmol／L。通过高效液相-荧光法检测伸趾长肌和尿内三甲基组氨酸（3-MH）的含量；采用核糖核酸印迹法（Northern blot）检测伸趾长肌内泛素基因的表达变化。结果S组和SS组伸趾长肌和尿内3-MH含量较C组均显著升高（P均〈0．01）；SI组和SSI组伸趾长肌和尿内3-MH含量分别较S组和SS组显著降低（P均〈0．01）。S组和SS组伸趾长肌内编码泛素的基因转录水平则较C组显著增强（P均〈0．01）；SI组和SSI组伸趾长肌内编码泛素的基因转录水平分别较S组和SS组显著降低（P均〈0．01）。结论严重烫伤特别是合并内毒紊攻击后早期伸趾长肌细胞内泛素-蛋白酶体途径活性即显著增强，蛋白降解率显著增加。胰岛素强化治疗能通过基因水平抑制细胞内泛素-蛋白酶体途径的活性，有效降低烫伤脓毒症时骨骼肌蛋白高降解。     

蛋白酶体-C2亚基基因在烫伤脓毒症大鼠心肌内的表达及意义

目的　探讨烧伤脓毒症时动物心肌内蛋白酶体核心亚基C2亚基m RNA表达及蛋白降解的变化及意义。方法　雄性Wistar大鼠4 5只,随机分为烫伤组、脓毒症组及对照组。烫伤组大鼠使用沸水致背部30 %总体表面积 度烫伤;脓毒症组大鼠用同样方法烫伤后,立即腹腔注射内毒素(6 m g/ kg)制成烫伤脓毒症大鼠模型。通过高效液相荧光法检测心肌内三甲基组氨酸(3MH)的含量,用核糖核酸印迹杂交(Northern杂交)检测心肌内蛋白酶体C2亚基m RNA表达的变化。结果　脓毒症大鼠烫伤后2 h和6 h,单位心肌内3MH含量较对照组和烫伤组均显著升高(P均<0 .0 1) ;烫伤组大鼠伤后2 h与对照组比较差异无显著性(P>0 .0 5 ) ,而伤后6 h较对照组显著升高(P<0 .0 1)。脓毒症大鼠伤后2 h和6 h心肌内蛋白酶体C2亚基m RNA表达较对照组和烫伤组均显著升高(P均<0 .0 1) ,烫伤组大鼠伤后2 h和6 h较对照组也均显著升高(P均<0 .0 1)。结论　严重烫伤特别是合并内毒素攻击后,早期动物心肌细胞内泛素蛋白酶体途径活性呈持续增强现象,蛋白降解率显著增加。这可能是烧伤脓毒症时心功能异常的蛋白代谢机制。     

泛素-蛋白酶体途径对烫伤脓毒症大鼠肠道炎症反应与屏障功能的作用研究

目的研究泛素-蛋白酶体途径对烫伤脓毒症大鼠肠组织核转录因子-κB（NF-κB）活化、肿瘤坏死因子-α（TNF-α）表达以及血浆二胺氧化酶（DAO）活性的作用。方法采用30％总体表面积（TBSA）Ⅲ度烫伤合并内毒素攻击大鼠为模型模拟临床烫伤哝毒症。60只Wistar大鼠随机分为正常对照组、烫伤哝毒症模型组、泛素-蛋白酶体抑制剂N-乙酰亮氨酰亮氨酰正亮氨酸（ALLN）组、NF-κB抑制剂吡咯烷二硫基甲酸酯（PDTC）组。采用凝胶电泳迁移率改变分析法（EMSA）分析肠组织NF—κB活性；采用酶联免疫吸附法（ELISA）检测肠组织TNF-α含量；采用分光光度法检测血浆DAO活性。结果各组肠组织NF—κB活性于伤后1h均明显增强，并达到高峰（P均〈0．01）．之后呈现下降趋势；两种抑制剂均可显著降低伤后1h和2hNF-κB的活性。ALLN可明显降低伤后1h肠组织中TNF—α含量（P〈0.01）。两种抑制剂对伤后1h和2h血浆I）AO活性均无明显影响。结论早期使用泛素-蛋白酶体抑制剂可降低烫伤脓毒症大鼠肠组织NF—κB活性．降低肠组织中炎症反应．但对肠组织屏障功能无保护作用。     

蛋白酶体抑制剂在烫伤脓毒症大鼠肺脏炎症反应中的作用

目的研究干预泛素-蛋白酶体途径对烫伤脓毒症大鼠肺脏核因子-(?)B(NF-(?)B)活化、肿瘤坏死因子-α的产生以及髓过氧化物酶(MPO)活性的影响。方法采用30%TBSAⅢ度烫伤加内毒素攻击大鼠为模型模拟临床烫伤脓毒症,72只Wistar大鼠随机分为正常对照组、烫伤脓毒症组、烫伤脓毒症 蛋白酶体抑制剂N-Acetyl-leucinyl-leucinyl-norleucinal(ALLN)组、烫伤脓毒症 NF-(?)B抑制剂吡咯烷二硫基甲酸酯(Pyrrolidine Dithiocarbamate．PDTC)组,采用凝胶电泳迁移率改变分析法(EMSA)分析肺脏NF-(?)B活性,采用酶联免疫吸附试验检测肺脏TNF-α的变化,采用分光光度法检测肺组织髓过氧化物酶(MPO)的活性。结果肺组织NF-(?)B活性于伤后1h明显增强达到高峰(P＜0．01),伤后2h仍保持较高的活化水平,之后呈逐渐下降趋势。PDTC可明显降低其在伤后1h和2h的活性(P＜0．01),而ALLN可明显降低其在伤后1h的活性(P＜0．01)。PDTC两种抑制剂均可明显降低伤后2h肺组织TNF-α产生,明显降低伤后2h和6h肺组织MPO的活力(P＜0．01)。结论蛋白酶体抑制剂可降低烫伤脓毒症大鼠肺组织NF-(?)B的活性。降低肺组织的炎症反应。     

脓毒症大鼠肌肉恶病质发生机制的实验研究

目的探讨脓毒症大鼠肌肉恶病质的发生机制。方法20只SD大鼠随机分为正常组和脓毒症组,每组10只。以盲肠结扎穿孔法（CLP）建立脓毒症模型,喂养5d后分别检测血清肿瘤坏死因子-α（TNF-α）、白介素-1（IL-1）、白介素-6（IL-6）和皮质醇浓度,趾长伸肌组织3-MH浓度;电镜观察骨骼肌纤维超微结构;免疫组化测定泛素蛋白表达;实时荧光定量PCR技术（reaL-time qPCR）检测泛素mRNA表达。结果脓毒症组血清TNF-α、IL-1、IL-6、皮质醇浓度升高;趾长伸肌组织3-MH明显升高;脓毒症组骨骼肌泛素蛋白和泛素mRNA的表达明显增强。结论在大鼠脓毒症肌肉恶病质的发生发展过程中,泛素-蛋白酶体途径居于主导地位,而糖皮质激素和一些细胞因子也起着调节作用。     

烫伤脓毒症大鼠肾脏核转录因子-kB活化与肾损伤关系的研究

目的 研究烫伤脓毒症大鼠肾脏核转录因子-kB（NF-kB）活化与肾损伤的关系。方法采用30％总体表面积Ⅲ度烫伤加内毒素攻击制备烫伤脓毒症大鼠模型。54只Wistar大鼠随机分为正常对照组、烫伤脓毒症1、2、6、12和24h组，烫伤脓毒症1、2和6h＋NF-kB抑制剂吡咯烷二硫基甲酸酯（PDTC）组。采用凝胶电泳迁移率改变分析法（EMSA）检测肾组织NF-kB活性；采用酶联免疫吸附法检测血浆及肾组织中肿瘤坏死因子-α（TNF-α）含量的变化；采用自动生化分析仪检测血肌酐（SCr）和尿素氮（BUN）含量。结果肾组织NF-kB活性于烫伤脓毒症后1h明显增强并达到高峰（P〈0．01），PDTC可显著降低烫伤脓毒症后1h NF-kB的活性。烫伤脓毒症后1h和2h血浆及肾组织中TNF-α水平均明显增高（P均〈0．01），PDTC可显著降低伤后血浆TNF-α水平（P均〈0．01），对肾组织中TNF-α水平影响不明显。烫伤脓毒症后BUN及SCr含量均明显增高（P均〈0．01），PDTC对BUN和SCr含量均无显著影响。结论NF-kB抑制剂可降低烫伤脓毒症大鼠肾组织NF-kB活性，但对肾脏功能无明显保护作用。     

脓毒症大鼠肌肉恶病质发生机制的实验研究

目的探讨脓毒症大鼠肌肉恶病质的发生机制。方法 20只SD大鼠随机分为正常组和脓毒症组,每组10只。以盲肠结扎穿孔法(CLP)建立脓毒症模型,喂养5d后分别检测血清肿瘤坏死因子-α(TNF-α)、白介素-1(IL-1)、白介素-6(IL-6)和皮质醇浓度,趾长伸肌组织3-MH浓度;电镜观察骨骼肌纤维超微结构;免疫组化测定泛素蛋白表达;实时荧光定量PCR技术(reaL-time qPCR)检测泛素mRNA表达。结果脓毒症组血清TNF-αI、L-1、IL-6、皮质醇浓度升高;趾长伸肌组织3-MH明显升高;脓毒症组骨骼肌泛素蛋白和泛素mRNA的表达明显增强。结论在大鼠脓毒症肌肉恶病质的发生发展过程中,泛素-蛋白酶体途径居于主导地位,而糖皮质激素和一些细胞因子也起着调节作用。     

烧伤脓毒症早期膈肌内泛素基因和蛋白的表达变化及意义

目的 :探讨烧伤脓毒症时大鼠膈肌内蛋白降解信号分子——泛素的基因和蛋白表达变化及意义。方法 :大鼠背部 30 %总体表面积 度烫伤后 ,立即腹腔注射内毒素 (6 mg/ kg)制成烧伤脓毒症动物模型 ,随机分为脓毒症 2 h和 6 h组 ,另设对照组 ,每组 9只。通过 Northern杂交检测大鼠膈肌内泛素 m RNA表达的变化 ;免疫组织化学法测定膈肌内泛素蛋白水平的表达。结果 :烧伤脓毒症大鼠伤后 2和 6 h膈肌内泛素 m RNA2 .4 kb条带和 1.2 kb条带的表达较对照组均显著增强 ,且呈持续升高现象 ,其中 2 .4 kb条带的表达增强更明显 ,差异有显著性 (P均 <0 .0 1)。烧伤脓毒症大鼠伤后 2和 6 h膈肌内泛素蛋白的表达较对照组也显著增强 ,以伤后 6 h增强更明显。结论 :烧伤脓毒症早期膈肌内泛素基因和蛋白表达即显著增强 ,且呈持续升高现象 ,提示膈肌内泛素系统活性上调 ,蛋白降解增强 ,这对进一步从肌组织蛋白代谢角度研究烧伤脓毒症时膈肌功能变化及其对呼吸功能的影响机制有重要意义。     

血必净注射液对脓毒症大鼠蛋白C及肿瘤坏死因子基因表达的影响

目的探讨血必净注射液对脓毒症大鼠蛋白C（PC）及肿瘤坏死因子-α（TNF—α）基因表达的影响。方法将96只健康Wistar大鼠按随机数字表法分为正常对照组、假手术组、模型组和血必净治疗组，后两组又按处死时间分为术后2、8、24、48和72h亚组，每组8只。采用盲肠结扎穿孔术（CLP）制备脓毒症模型。取腹主动脉血进行血小板计数，并留取肝、肺组织分别检测各组动物组织PC和TNF—α的mRNA表达。结果CLP后8～72h模型组大鼠肝组织PC mRNA表达显著下调（P均〈0．01），血必净注射液可显著提高脓毒症大鼠PC mRNA表达水平（P均〈0．01）。CLP后2h模型组动物肝、肺组织TNF—α mRNA表达均迅速升高并持续至伤后24h（P〈0．05或P〈0．01）；血必净注射液治疗可显著降低肝、肺组织TNF—α mRNA水平（P〈0．05或P〈0．01），术后24h均恢复至伤前正常范围。模型组动物CLP后8～72h血小板计数不同程度减少，血必净治疗组较模型组能明显提高血小板计数（P〈0．05或P〈0．01）。结论血必净注射液可以从基因水平影响脓毒症动物组织PC和TNF—α的mRNA表达。     

早期肠内营养对重型颅脑损伤大鼠胃黏膜能量代谢的影响

[目的]研究早期肠内营养对重型颅脑损伤大鼠胃黏膜能量代谢的影响。[方法]建立大鼠重型颅脑损伤模型，随机分为早期肠内营养组（A组）、单纯损伤组（B组）和正常对照组（C组）。采用高效液相色谱法检测伤后6h、12h、24h、48h、72h胃黏膜组织及其线粒体内腺苷酸池含量的变化。[结果]B组各时相点胃黏膜组织及其线粒体内三磷酸腺苷（ATP）与总腺苷酸含量显著低于C组（P〈0．05或P〈0．01），胃黏膜组织能荷（EC）无明显变化，胃黏膜线粒体能荷水平在伤后6h、72h显著低于C组（P〈0．05或P〈0．01）。而A组胃黏膜组织及其线粒体内ATP与总腺苷酸含量较B组显著增加（P〈0.05或P〈0．01），胃黏膜线粒体能荷水平在伤后72h较B组显著增高（P〈0．05）。[结论]重型颅脑损伤可导致胃黏膜组织及其线粒体内能量代谢紊乱，早期肠内营养可能通过有效增加胃黏膜的能量贮备，来实现保护胃黏膜的作用，从而减轻急性胃黏膜损伤的程度。     

脑缺血再灌注损伤大鼠患侧骨骼肌早期形态及atrogin-1、MuRF-1 mRNA表达的变化

摘要 目的：观察大鼠脑缺血再灌注损伤后患侧骨骼肌早期形态学变化及肌萎缩素1（atrogin-1）和肌环指蛋白1（MuRF-1） mRNA表达水平的变化。 方法：60只雄性Wistar大鼠,10只为假手术对照组（A组）,其余50只采用Longa线栓法建立大鼠左侧大脑中动脉栓塞（MCAO）模型,其中30只造模成功大鼠随机分为B、C、D组,每组10只。B组为造模成功后1d组, C组为造模成功后4d组, D组为造模成功后7d组。应用Bederson评分评价大鼠的神经损伤后的恢复情况;分别获取A、B、C、D四组右侧肱二头肌,对各组通过HE染色检测肌纤维横截面积;通过荧光定量RT-PCR观察大鼠患侧骨骼肌中atrogin-1和MuRF-1 mRNA表达的变化。 结果：B、C、D组大鼠Bederson评分明显高于对照组A组（P<0.05）;B、C、D组大鼠间Bederson评分差异无显著性（P＞0.05）。患侧骨骼肌HE染色显示A、B、C组肌纤维横截面积两两比较,差异无显著性意义（P＞0.05）;D组大鼠患侧骨骼肌肌纤维横截面积分别与其余3组相比,差异均有显著性意义（P<0.05）。B、C、D组大鼠患侧atrogin-1和MuRF-1 mRNA的表达水平分别与对照组相比,差异均有显著性意义（P<0.05）;B组与C组相比较差异无显著（P＞0.05）;D组大鼠分别与B、C组相比,差异均有显著性意义（P<0.05）。 结论：脑缺血再灌注损伤大鼠早期患侧骨骼肌形态学即发生变化,其机制可能与Atrogin-1 和MuRF-1 mRNA在骨骼肌中高表达,泛素连接酶蛋白降解通路被激活有关。     

The study of respiratory muscles activation during respiratory muscle strength effort in adult females with chronic neck pain

[Purpose] This study aimed to compare maximal inspiratory pressure (MIP), maximal expiratory pressure (MEP) values and muscle activity during MIP and MEP between chronic neck pain and healthy participants. [Participants and Methods] Twenty chronic neck pain and 20 non-symptomatic females participated in this study. Maximal airway pressure (MIP and MEP) and surface electromyography (sEMG) for both sides of the upper trapezius, anterior scalene, pectoralis major and 6th intercostal muscles were recorded simultaneously. [Results] Significant differences of MIP and MEP values were found between the groups. The muscle activities of both sides of upper trapezius and 6th intercostal muscles during MEP were significantly higher in the chronic neck pain group than the healthy group except both sides of anterior scalene and pectoralis major muscles. During MIP, the activities of upper trapezius, 6th intercostal muscles and anterior scalene were significantly different between the two studied groups. Higher activity of left pectoralis major was found in the chronic neck pain group. [Conclusion] Decreasing values of MEP and MIP as well as muscles activities elevation in chronic neck pain participants were clearly demonstrated. Besides the musculoskeletal treatment, we suggest breathing exercise training to be considered in treatment programs.     

Sepsis is associated with increased mRNAs of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle.

Previous studies provided evidence that sepsis-induced muscle proteolysis in experimental animals is caused by increased ubiquitin-proteasome-dependent protein breakdown. It is not known if a similar mechanism accounts for muscle proteolysis in patients with sepsis. We determined mRNA levels for ubiquitin and the 20 S proteasome subunit HC3 by Northern blot analysis in muscle tissue from septic (n = 7) and non-septic (n = 11) patients. Plasma and muscle amino acid concentrations and concentrations in urine of 3-methylhistidine (3-MH), creatinine, and cortisol were measured at the time of surgery to assess the catabolic state of the patients. A three- to fourfold increase in mRNA levels for ubiquitin and HC3 was noted in muscle tissue from the septic patients concomitant with increased muscle levels of phenylalanine and 3-MH and reduced levels of glutamine. Total plasma amino acids were decreased by approximately 30% in the septic patients. The 3-MH/creatinine ratio in urine was almost doubled in septic patients. The cortisol levels in urine were higher in septic than in control patients but this difference did not reach statistical significance. The results suggest that sepsis is associated with increased mRNAs of the ubiquitin-proteasome pathway in human skeletal muscle.     

烧伤早期大鼠骨骼肌组织泛素转录表达的变化研究

目的：研究烧伤后大鼠骨骼肌组织泛素－蛋白毒体（ubiquitin-proteasome)蛋白降解途径组成成分中泛素表达的变化,探讨烧伤后骨骼肌蛋白降解的增强与泛素－蛋白酶体蛋白降解途径表达变化间的关系。方法：借助烫伤大鼠动物模型,利用骨骼肌离体孵育系统,应用氨基酸自动分析仪测定孵育液和骨骼肌组织中酪氨酸（Tyr)和3－甲基组氨酸（3－MH）量的变化,用RNA印迹技术（Northern blot)方法测定骨骼肌组织中泛素mRNA表达水平。结果：烧伤后大鼠骨骼肌释放Tyr和3－MH增多,以3－MH增加显著;烧伤后骨骼肌组织中泛素－2．4kb的mRNA表达上调明显。泛素mRNA表达增加量与Tyr和3－MH增加量间均呈显著正相关。结论：烧伤后大鼠骨骼肌蛋白降解明显增强,其机制与泛素－蛋白酶体蛋白降解途径在转录水平被激活密切相关。     

Energy-ubiquitin-dependent muscle proteolysis during sepsis in rats is regulated by glucocorticoids.

Recent studies suggest that sepsis-induced increase in muscle proteolysis mainly reflects energy-ubiquitin-dependent protein breakdown. We tested the hypothesis that glucocorticoids activate the energy-ubiquitin-dependent proteolytic pathway in skeletal muscle during sepsis. Rats underwent induction of sepsis by cecal ligation and puncture or were sham-operated and muscle protein breakdown rates were measured 16 h later. The glucocorticoid receptor antagonist RU 38486 or vehicle was administered to groups of septic and sham-operated rats. In other experiments, dexamethasone (2.5 or 10 mg/kg) was injected subcutaneously in normal rats. Total and myofibrillar proteolysis was determined in incubated extensor digitorum longus muscles as release of tyrosine and 3-methylhistidine, respectively. Energy-dependent proteolysis was determined in incubated muscles depleted of energy with 2-deoxyglucose and 2,4-dinitrophenol. Levels of muscle ubiquitin mRNA and free and conjugated ubiquitin were determined by Northern and Western blot, respectively. RU 38486 inhibited the sepsis-induced increase in total and myofibrillar energy-dependent protein breakdown rates and blunted the increase in ubiquitin mRNA levels and free ubiquitin. Some, but not all, sepsis-induced changes in ubiquitin protein conjugates were inhibited by RU 38486. Injection of dexamethasone in normal rats increased energy-dependent proteolysis and ubiquitin mRNA levels. The results suggest that glucocorticoids regulate the energy-ubiquitin-dependent proteolytic pathway in skeletal muscle during sepsis.     

Enterococcus faecalis exacerbates burn injury-induced host responses in rats

Pathophysiology of burn injury with complications of gram-positive infections is not well characterized. We have developed an in vivo rat model to study the effects of burn injury along with intra-abdominal inoculation of Enterococcus faecalis. We hypothesized that although burn injury or E. faecalis inoculation by itself may not induce significant pathophysiological responses, the combination of the two can lead to adverse pathophysiological consequences. Sprague-Dawley rats were divided into 4 groups: group 1(C), controls; group 2(B), burn injury on 30% total body surface area; group 3(EF), intra-abdominal implantation of bacterial pellet impregnated with E. faecalis; group 4(B+EF), burn injury plus bacterial pellet implantation. The mortality was 25% and 60% on day 1 and 2 in Group 4(B+EF), respectively; no significant mortality was observed in other groups. In group 4(B+EF), metabolic acidosis, respiratory alkalosis, and a hyperdynamic state developed on day 1, and metabolic and respiratory acidosis and a hypodynamic state on day 2. There were no significant alterations in metabolic or hemodynamic measurements in other groups. Intestinal microvascular permeability to albumin on day 1 and 2 was increased in group 4(B+EF). In group 2(B), microvascular permeability was not increased significantly. Although the permeability was increased on day 1 in group 3(EF), it declined on day 2. The metabolic and hemodynamic alterations were correlated with increased intestinal microvascular permeability to albumin. E. faecalis appeared to be involved in initiating a vicious cycle of burn injury-mediated disruption of intestinal integrity along with metabolic and hemodynamic derangements.     

Proteasome proteolytic activity in skeletal muscle is increased in patients with sepsis

Patients with sepsis in the ICU (intensive care unit) are characterized by skeletal muscle wasting. This leads to muscle dysfunction that also influences the respiratory capacity, resulting in prolonged mechanical ventilation. Catabolic conditions are associated with a general activation of the ubiquitin-proteasome pathway in skeletal muscle. The aim of the present study was to measure the proteasome proteolytic activity in both respiratory and leg muscles from ICU patients with sepsis and, in addition, to assess the variation of proteasome activity between individuals and between duplicate leg muscle biopsy specimens. When compared with a control group (n=10), patients with sepsis (n=10) had a 30% (P<0.05) and 45% (P<0.05) higher proteasome activity in the respiratory and leg muscles respectively. In a second experiment, ICU patients with sepsis (n=17) had a 55% (P<0.01) higher proteasome activity in the leg muscle compared with a control group (n=10). The inter-individual scatter of proteasome activity was larger between the patients with sepsis than the controls. We also observed a substantial intra-individual difference in activity between duplicate biopsies in several of the subjects. In conclusion, the proteolytic activity of the proteasome was higher in skeletal muscle from patients with sepsis and multiple organ failure compared with healthy controls. It was shown for the first time that respiratory and leg muscles were affected similarly. Furthermore, the variation in proteasome activity between individuals was more pronounced in the ICU patients for both muscle types, whereas the intra-individual variation between biopsies was similar for ICU patients and controls.