小鼠前脑线粒体蛋白的增龄性变化

目的　探讨小鼠前脑不同发育阶段线粒体蛋白的表达变化。 方法　分别取妊娠12.5 d（E12.5）及17.5 d（E17.5）的胎鼠、出生2 d的新生鼠、出生3周的幼鼠和2月龄成年鼠的前脑组织,利用QRT-PCR和Western blotting检测小鼠前脑发育不同阶段中COX IV、HSP60、OGDH、PDHE1α的mRNA和蛋白表达水平。 结果　在E12.5 ~2 d的前脑组织中COX IV mRNA呈低水平表达,3周时表达量明显升高并达到顶峰;HSP60和PDHE1α mRNA在E17.5表达明显增高,OGDH mRNA则在2 d显著上升,三者表达水平均于2月达到顶峰。COX IV、HSP60、OGDH的蛋白水平从E12.5 ~3周无明显差异,但在2月时表达显著上升;PDHE1α蛋白随前脑发育其表达水平不断提高,并于2月达到高峰。此外,COX IV、HSP60、OGDH、PDHE1α mRNA与蛋白表达水平呈正相关。 结论　COX IV、HSP60、OGDH和PDHE1α可能参与了小鼠前脑发育过程且与年龄密切相关。     

维生素D通过抑制Pin1介导线粒体氧化应激拮抗高糖诱导的人脐静脉内皮细胞凋亡

目的:观察维生素D对高糖诱导的人脐静脉内皮细胞(HUVECs)氧化损伤的作用,对脯氨酰异构酶1(Pin1)蛋白表达和活性、p66Shc线粒体转位及活性氧簇(ROS)生成的影响,以及维生素D受体(VDR)在这一过程的作用。方法:以33 mmol/L葡萄糖干预建立高糖内皮细胞损伤模型,分别接受维生素D(10~(-8)~10~(-6)mol/L)和Pin1抑制剂胡桃醌(10~(-7)mol/L)共孵育;流式细胞术和TUNEL染色法检测HUVECs的凋亡率;流式细胞术和荧光显微术检测细胞内的ROS水平;Western blot法检测HUVECs中Pin1、p66Shc、p-p66Shc、p66Shc胞浆/线粒体比值及caspase-3的蛋白水平;多肽酶解法测定HUVECs裂解液中Pin1的活性;通过转染siRNA沉默VDR的表达,观察VDR是否介导Pin1蛋白及活性改变。结果:维生素D(10~(-8)~10~(-6)mol/L)抑制高糖诱导的HUVECs凋亡,并抑制高糖诱导的HUVECs中Pin1蛋白表达和活性增加。维生素D抑制高糖诱导的HUVECs中p66Shc磷酸化、p66Shc线粒体转位、ROS生成及caspase-3蛋白表达(P0.05)。通过转染siRNA沉默VDR表达,可削弱维生素D对高糖诱导的HUVECs中Pin1蛋白表达和活性增加的抑制作用。结论:维生素D通过激动VDR,抑制高糖环境下Pin1蛋白表达和活性增加,抑制p66Shc的线粒体转位,减少ROS生成,从而减轻血管内皮细胞凋亡。     

Tigecycline-induced inhibition of mitochondrial DNA translation may cause lethal mitochondrial dysfunction in humans

BackgroundA 65-year-old patient developed an unexplained and ultimately lethal metabolic acidosis under prolonged treatment with tigecycline. Tigecycline is known to have a selective inhibitory effect on eukaryotic mitochondrial translation. The underlying molecular mechanisms of the metabolic acidosis in this patient were explored.MethodsOxidative phosphorylation system (OXPHOS) analysis, blue native polyacrylamide gel electrophoresis followed by in-gel activity staining in mitochondria, molecular analysis of mitochondrial DNA (mtDNA) for genomic rearrangements and sequencing of the rRNA genes was performed on the subject's skeletal muscle.ResultsOXPHOS analysis revealed a combined deficiency of the complexes I, III, IV and V, with a preserved function of complex II (encoded by nuclear DNA), thus demonstrating a defective mtDNA translation. There were no known underlying mitochondrial genetic defects. The patient had a (m.1391T>A) variant within the 12SrRNA gene in heteroplasmy (50–60%).ConclusionsThis patient developed an ultimately lethal mitochondrial toxicity while receiving prolonged treatment with tigecycline, which was caused by a defective translation of the mtDNA. Tigecycline is known to suppress eukaryotic mitochondrial DNA translation, but until now this effect has been considered to be clinically insignificant. The observations in this patient suggest a clinically significant mitochondrial toxicity of tigecycline in this patient, and warrant further investigation.     

Sirtuins in mitochondrial stress: Indispensable helpers behind the scenes

Mitochondria play an essential part in guaranteeing normal cellular physiological functions through providing ATP and participating in diverse processes and signaling pathways. Recently, more and more studies have revealed the vital roles of mitochondria in coping with stressors in the aging process, metabolic disturbances and neurological disorders. Mitochondrial stress responses, including the mitochondrial unfolded protein response (UPR^mt), antioxidant defense, mitochondrial fission, mitochondrial fusion and mitophagy, are induced to maintain cellular integrity in response to stress. The sirtuin family, a group of NAD⁺-dependent deacetylases, has been the focus of much attention in recent years for their multiple regulatory functions, especially in aging and metabolism. Recent reports validated the significant link between mitochondrial stress responses and the sirtuin family, which may help to elucidate the pathogenesis and therapies for diseases such as Alzheimer’s disease or Parkinson’s disease. This review will summarize recent related studies and illuminate the interplay between sirtuins and mitochondrial stress.     

环孢素A对积水肾脏急性压力灌注损伤的保护

目的 在不同程度肾脏积水的前提下,观察线粒体渗透性转换孔(MPTP)抑制剂环孢素A(CsA)对于急性压力灌注所致损伤的保护作用.方法 健康新西兰大白兔20只,随机分为轻度积水组(M组,n=10)和重度积水组(S组,n=10),肾脏轻度积水及重度积水模型采用输尿管套扎法构建.M组及S组分为M1、M2、S1、S24个亚组,分别予以60、100、20、60 mmHg(1mmHg=0.133 kPa)压力灌注.灌注前M组及S组均给予CsA灌胃1周.灌注后48 h取标本,检测标本中超氧化物歧化酶(SOD)及丙二醛(MDA)的含量,JC-1染色检测线粒体膜电位(MMP)的变化.结果 M1、M2、S1、S2各组SOD活力分别为(30.11±2.23)、(28.41 ±2.26)、(44.82±6.61)、(40.31±5.21) U/mg,M1与M2之间SOD活力差异无统计学意义(P＞0.05),S1与S2之间SOD活力差异无统计学意义(P＞0.05);MDA的含量分别为(0.61 ±0.17)、(0.60±0.15)、(0.95 ±0.26)、(0.80±0.32) nmol/mg,M1与M2之间MDA含量差异无统计学意义(P＞0.05),S1与S2之间MDA含量差异无统计学意义(P＞0.05).JC-1染色橙红色阳性细胞百分比分别为(67.16±9.47)％、(60.75±11.89)％、(41.08 ±4.50)％、(42.36 ±3.44)％.M1与M2比较,S1与S2比较,阳性细胞百分比差异均无统计学意义(P＞0.05).结论 CsA对于积水肾脏因急性压力灌注所致的损伤有一定的保护作用.在经皮肾镜及输尿管镜治疗结石前给予MPTP抑制剂可有效减轻压力灌注所致损伤.     

The cGAS-STING pathway connects mitochondrial damage to inflammation in burn-induced acute lung injury in rat

ObjectiveDamage associated molecular patterns (DAMPs) are pathological mediators linking local tissue damage to systemic inflammation in various diseases. Some DAMPs, such as mitochondrial DNA (mtDNA), can be recognized by the cytoplasmic cGAS protein to trigger the activation of the stimulator of interferon genes (STING)-dependent innate immune pathway responsible for infection or sterile inflammation. The objective of our study was to evaluate the association between circulating mtDNA and cGAS-STING pathway activation in mediating inflammation following burn injury.Methods48 adult Sprague-Dawley male rats were divided into eight groups (Sham, 2, 4, 8, 12, 24, 48, 72 h after burn injury). The animals underwent 40% total body surface area scald injury to produce a full-thickness burn. Plasma samples were collected via cardiac puncture under deep anesthesia. Tissues were harvested and placed in formalin, followed by paraffin embedment. Total plasma DNA was isolated followed by measurement of mtDNA using quantitative polymerase chain reaction. Haemotoxylin-Eosin stain and Western blot was used for lung histology and protein assays, respectively. Statistical analyses were performed using ANOVA and student’s t-test and represented as mean ± s.d.ResultsPlasma mtDNA trended upward at early time-points following burn injury with peak levels at 8 h after burn when compared to the control group (345 ± 83.4 copies/μl vs. 239 ± 43.1 copies/μl, p = 0.07) and followed a bell-shaped distribution. Lung slices from burned rats showed acute injury marked by increased inflammatory infiltrate, with the maximum changes seen at 24 h, accompanied with significant upregulation of neutrophil elastase (p = 0.04). Compared with sham animals, cGAS and STING protein levels in lung tissue were up-regulated at 4 and 8 h after burn (p = 0.03 and p < 0.001, respectively).ConclusionActivation of the cGAS-STING pathway by increased plasma mtDNA is an important pathway driving neutrophil infiltration in burn-induced acute lung injury in rats. A further understanding of the STING-mediated immunopathology in lung and other susceptible organs may be important for the development of novel therapies for burn injury.     

Combination of sodium butyrate and probiotics ameliorates severe burn-induced intestinal injury by inhibiting oxidative stress and inflammatory response

Burns are a common traumatic injuries with considerable morbidity and mortality rates. Post-burn intestinal injuries are closely related to oxidative stress and inflammatory response. The aim of the current study was to investigate the combined effect of sodium butyrate (NaB) and probiotics (PROB) on severe burn-induced oxidative stress and inflammatory response and the underlying mechanism of action. Sprague-Dawley rats with severe burns were treated with NaB with or without PROB. Pathomorphology of skin and small intestine tissue was observed using hematoxylin and eosin staining and severe burn-induced apoptosis in small intestine tissue was examined via terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. The release of factors related to inflammation was quantified using ELISA kits and qRT-PCR and levels of oxidative stress markers were evaluated using biochemical assays. Furthermore, mitochondrial morphological changes in small intestinal epithelial cells were observed using transmission electron microscopy. In addition, the underlying mechanism associated with the combined effect of NaB and PROB on severe burn-induced oxidative stress and inflammatory response was investigated using western blotting. The combination of NaB and PROB exerted protective effects against severe burn-induced intestinal barrier injury by reducing the levels of diamine oxidase and intestinal fatty acid binding protein. Combined NaB and PROB treatment inhibited severe burn-induced oxidative stress by increasing superoxide dismutase levels and decreasing those of malondialdehyde and myeloperoxidase levels. Severe burn-induced inflammation was suppressed by combined NaB and PROB administration, as demonstrated by the decreased mRNA expression of tumor necrosis factor-α, interleukin-6, interleukin-1β, and high mobility group box-1 in the small intestine. In addition, this study showed that combined NaB and PROB administration increased nuclear factor-erythroid 2-related factor 2 (Nrf2) protein expression and decreased the phosphorylation of nuclear factor (NF)-κB and extracellular signal-regulated kinase 1/2 (ERK 1/2). In conclusion, our findings indicate that combined NaB and PROB treatment may inhibit severe burn-induced inflammation and oxidative stress in the small intestine by regulating HMGB1/NF-κB and ERK1/2/Nrf2 signaling, thereby providing a new therapeutic strategy for intestinal injury induced by severe burn.     

Mitochondrial transplantation ameliorates acute limb ischemia

ObjectiveAcute limb ischemia (ALI), the most challenging form of ischemia-reperfusion injury (IRI) in skeletal muscle tissue, leads to decreased skeletal muscle viability and limb function. Mitochondrial injury has been shown to play a key role in skeletal muscle IRI. In previous studies, we have demonstrated that mitochondrial transplantation (MT) is an efficacious therapeutic strategy to replace or to augment mitochondria damaged by IRI, allowing enhanced muscle viability and function in cardiac tissue. In this study, we investigated the efficacy of MT in a murine ALI model.MethodsC57BL/6J mice (male, 10-12 weeks) were used in a model of ALI. Ischemia was induced by applying a tourniquet on the left hindlimb. After 2 hours of ischemia, the tourniquet was released, and reperfusion of the hindlimb was re-established; either vehicle alone (n = 15) or vehicle containing mitochondria (n = 33) was injected directly into all the muscles of the hindlimb. Mitochondria were delivered at concentrations of 1 × 10⁶ to 1 × 10⁹ per gram wet weight to each muscle, and the animals were allowed to recover. Sham mice received no ischemia or injections but were anesthetized for 2 hours and allowed to recover. After 24 hours of recovery, limb function was assessed by DigiGait (Mouse Specifics Inc, Boston, Mass), and animals were euthanized; the gastrocnemius, soleus, and vastus medialis muscles were collected for analysis.ResultsAfter 24 hours of hindlimb reperfusion, infarct size (percentage of total mass) and apoptosis were significantly decreased (P < .001, each) in the gastrocnemius, soleus, and vastus medialis muscles in MT mice compared with vehicle mice for all mitochondrial concentrations (1 × 10⁶ to 1 × 10⁹ per gram wet weight). DigiGait analysis at 24 hours of reperfusion showed that percentage shared stance time was significantly increased (P < .001) and stance factor was significantly decreased (P = .001) in vehicle compared with MT and sham mice. No significant differences in percentage shared stance time (P = .160) or stance factor (P = .545) were observed between MT and sham mice.ConclusionsMT ameliorates skeletal muscle injury and enhances hindlimb function in the murine model of ALI.     

Src羧基端激酶结合蛋白影响线粒体分裂及NK细胞杀伤肺癌细胞活性的分子机制探讨

目的:探究Src羧基端激酶结合蛋白（csk-binding protein,CBP）在人肺癌组织中的表达水平,及其对线粒体分裂和自然杀伤（natural killer,NK)细胞杀伤肺癌细胞活性的影响。方法:免疫组织化学染色和实时荧光定量PCR（qRT-PCR）检测肺癌组织及癌旁组织中CBP的表达;含CBP慢病毒感染人肺癌细胞株A549,通过荧光倒置显微镜、qRT-PCR 和Western blot检测细胞感染效果;CCK-8法检测各组A549细胞活性,Mito-Tracker染色观察各组A549细胞内线粒体形态和长度变化,Western blot检测各组A549细胞线粒体动态相关蛋白Mfn1、Mfn2、Drp1的表达,免疫荧光染色检测各组A549细胞内细胞色素C（Cyt C）的表达,流式细胞术检测各组A549细胞凋亡情况;从人外周血单个核细胞（peripheral blood mononuclear cell,PBMC）中分离NK细胞并进行鉴定,乳酸脱氢酶释放实验检测NK细胞对各组A549细胞的杀伤率。结果:肺癌组织中CBP的表达水平较癌旁组织中明显下降（P＜0.01）;感染CBP过表达慢病毒的A549细胞中CBP mRNA和蛋白的相对表达量均升高,培养48 h、72 h、96 h后细胞增殖活性下降,线粒体呈椭圆状或短杆状,长度明显缩短,线粒体分裂相关蛋白Mfn1、Mfn2蛋白表达减少,Drp1蛋白表达增加,有大量Cyt C从线粒体释放,细胞凋亡率升高,同时NK细胞对A549细胞的杀伤率明显提高,差异均具有统计学意义（P＜0.01）;而在使用线粒体分裂蛋白抑制剂Mdivi-1预先处理A549细胞后再感染CBP过表达慢病毒,线粒体分裂受到抑制,Mfn1、Mfn2蛋白表达增加,Drp1蛋白表达减少,Cyt C释放减少,细胞凋亡率降低,NK细胞对A549细胞的杀伤率也受到抑制,差异均具有统计学意义（P＜0.01）。结论:CBP在人肺癌组织中低表达,在A549细胞中过表达CBP能够促进线粒体分裂与细胞凋亡,并提高NK细胞对A549细胞的杀伤率。