Apoptosis and apoptosis-related proteins in thyroid myopathies

DNA fragmentation and apoptosis-related proteins have been investigated in thyroid cells and there is evidence that Fas-mediated apoptosis is inhibited by thyroid stimulating hormone (TSH). We investigated DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and Bcl-2 and Fas antigen expression by immunocytochemistry in skeletal muscles from 12 patients with hypothyroid myopathy and 5 patients with hyperthyroid myopathy. The finding of very few TUNEL-positive muscle fibers in both conditions suggests that apoptosis does not play a role in the pathogenesis of thyroid myopathies. Bcl-2 expression increased significantly in hypothyroid myopathy, correlating with high serum TSH levels, and not with either triiodothyronine (T3) or thyroxine (T4) serum levels. By contrast, Fas antigen was overexpressed in hyperthyroid myopathy, correlating with low TSH levels. These findings suggest an anti-apoptotic role for TSH itself in skeletal muscle.     

缺血缺氧对体外培养星形胶质细胞细胞周期和周期相关蛋白的影响

目的 观察缺血缺氧损伤对星形胶质细胞细胞周期及细胞周期相关蛋白的影响。方法 用流式细胞仪及Brdu掺入法检测缺血缺氧后不同时间点星形胶质细胞细胞周期变化和细胞的增殖活力；用荧光免疫细胞化学技术测定增殖细胞核抗原(PCNA)及细胞周期蛋白cyclin D1的表达水平。结果 体外缺血缺氧损伤后S期星形胶质细胞较正常组明显增加，6h达高峰，Brdu掺入法显示损伤后6h星形胶质细胞的增殖活力最高，而随后S期细胞数目及细胞增殖活力都呈下降趋势。PCNA阳性反应损伤后表达增加，6h表达最高，而cyclin D1的表达在损伤后逐渐增加，在24h时达高峰。结论 缺血缺氧损伤激活星形胶质细胞，使其进入新的细胞周期，出现细胞的增殖反应；PCNA及cyclin D1参与了损伤后星形胶质细胞的修复和增殖；细胞周期事件与星形胶质细胞的增殖活化密切相关。     

DNA-fragmentation and apoptosis-related proteins of muscle cells in motor neuron disorders

Apoptosis has been decribed as one of the mechanisms of muscle fiber loss in infantile spinal muscular atrophy. In order to investigate if muscle fiber-apoptosis plays a role in other denervating disorders as well, we studied DNA-fragmentation, a hallmark of apoptosis, by the TUNEL-method and, moreover, the expression patterns of apoptosis-related proteins in 2 patients suffering from ALS and in 6 patients with polyneuropathy. We identified DNA-cleavage in muscle fibers of all these patients. Furthermore, we found strong expression of bax and ICE promoting apoptosis in muscle fibers. However, also strong expression of the anti-apoptotic factor bcl-2 was found. Our findings indicate that defective innervation may prompt muscle fibers to activate an intrinsic "suicide" programme which is promoted by the pro-apoptotic factors bax and ICE, which seems to induce formation of apoptotic bodies by cleavage of actin. Nevertheless, there are also anti-apoptotic strategies in muscle fibers manifested by expression of the bax-antagonist bcl-2 which is able to neutralize high bax levels.     

Neurosteroid modulation of embryonic neuronal survival in vitro following anoxia

Neurosteroids are synthesized de novo in the brain from cholesterol or peripheral steroid precursors and modulate inhibitory γ-aminobutyric acid (GABA_A) and excitatory N-methyl-d-aspartate (NMDA) receptors. Evidence indicates that neurosteroids are neuroprotective and important during neurodevelopment. We tested the hypothesis that neurosteroids increase embryonic neuronal survival following anoxia in rat embryonic day 18 cerebral cortical cultures to examine potential neurosteroid modulation of this insult during early development. Twenty-four hours after plating in serum-free medium, cultures were exposed to DHEA, DHEAS, or allopregnanolone (10⁻¹⁰, 10⁻⁸, or 10⁻⁶ M), or vehicle, for 24 h (n=9 per treatment condition). Cultures were then subjected to anoxia for 2 h and subsequently reincubated for 24 h prior to neuron immunostaining with microtubule-associated protein 2 (MAP-2) antibody. Supernatant from DHEA and DHEAS-exposed cultures was tested for 17β-estradiol metabolite formation by radioimmunoassay. DHEA 10⁻⁶ and 10⁻⁸ M significantly increased neuron survival by 85–87% following anoxia. DHEAS 10⁻⁶ M significantly increased neuron survival by 74% following anoxia, but DHEAS 10⁻¹⁰ M decreased neuron survival after this insult. Allopregnanolone had modest effects on neuron survival that did not attain statistical significance. 17β-Estradiol concentrations were below the limit of detection in all specimens tested (sensitivity 4.7 nM). Our data indicate that pretreatment with DHEA and DHEAS at physiologically relevant concentrations promotes neuronal survival following anoxia in embryonic rat cerebral cortical cultures, and that these effects are not secondary to 17β-estradiol metabolite formation. DHEA and DHEAS modulation of anoxia in embryonic neurons may be relevant to disorders of neurodevelopment involving this insult.     

Adaptation of adult brain tissue to anoxia and hypoxia in vitro

The rat hippocampal slice preparation was used in the present study to demonstrate the ability of adult brain tissue to adapt to anoxic and hypoxic conditions. Adaptation was induced by pre-exposure of hippocampal slices to a short (5 min) anoxic episode. The evoked electrical activity of pre-exposed slices recovered from a subsequent, longer anoxic insult, while that of controls (without pre-exposure), receiving the same insult, did not. The adaptation process is time-dependent; an interval of 0.5 h between the pre-exposure and the subsequent anoxic insult allowed slices to resist anoxic periods of 13 +/- 2 min while after an interval of 2 h an anoxic period of 16 +/- 2 min could be tolerated. Evoked electrical activity persisted in adapted slices during exposure to hypoxia while their non-adapted controls exhibited synaptic silence under hypoxic conditions.     

帕金森病小鼠模型脑黑质区热休克蛋白的表达

目的应用1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)制备帕金森病小鼠模型,观察中脑黑质部位热休克蛋白(hsps)的表达。方法175只C57BL小鼠分为4组:MPTP组,56只小鼠连续5d腹腔注射MPTP眼30mg/(kg·d)演,制备帕金森病小鼠模型;生理盐水组,56只小鼠连续5d腹腔注射生理盐水0.3ml/d(容积与MPTP组同);假手术组,56只小鼠仅用注射器刺穿腹部皮肤;另7只小鼠作为正常对照组。分别于最后1次注射后4h(视为0d)及第2、4、7、10、14、21、28天处死小鼠,应用免疫印迹法(West鄄ernblot)和免疫组化检测方法观察热休克蛋白的动态变化以及在中脑的定位;经原位杂交法检测MPTP对hsp40和hsp70mRNA表达的影响。结果免疫印迹法检测显示,MPTP组小鼠于药物注射后4h即出现hsp70表达升高,两周内与正常对照组、生理盐水组和假手术组相比差异均有显著性意义(P<0.01),其中以第2,4天为表达高峰,之后缓慢下降,至第21天降至正常水平。MPTP组小鼠在注射后2～14d,hsp40/HDJ鄄1表达水平高于其余各组(P<0.01),第4天为表达高峰。HDJ-2表达不如HDJ鄄1明显,仅第4天与另3组相比差异有显著性意义(P<0.01)。MPTP组小鼠在注射后第2～4天,hsp90表达水平高于正常对照组、生理盐水组和假手术组(P<0.01);生理盐水组和假手术组小鼠hsp90的表达与正常对照组差异无     

米诺环素对实验性自身免疫性脑脊髓炎大鼠凋亡相关蛋白表达的影响

目的 探讨米诺环素对实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)大鼠CNS内Fas和Bcl-2蛋白表达的影响.方法 将大鼠随机分为3组,并以豚鼠全脊髓匀浆(guinea pig spinal cord homogenate,GPSCH)为抗原免疫建立EAE模型.治疗组给予米诺环素,而对照组和模型组给予生理盐水灌胃;免疫组化检测Fas和Bcl-2的表达.结果 Fas的表达各组平均光密度和阳性细胞数模型组高于治疗组(P<0.05);Bcl-2的表达除脑干阳性细胞数外,平均光密度及细胞数治疗组高于模型组,对照组低于模型组(P<0.05).结论 米诺环素可能通过介导Fas和Bcl-2的表达,干预凋亡调节免疫反应对EAE起治疗作用.     

Effects of Rubus parvifolius L. on neuronal apoptosis and expression of apoptosis-related proteins in a rat model of focal cerebral ischemic-reperfusion injury

BACKGROUND:Studies have shown that the Ruhus parvifolius L.(RP)plant extract exhibits protective effects on cerebral ischemia.This effect is reflected in altered ischemic neuronal apoptosis and associated protein expression.OBJECTIVE:To explore the neuroprotective mechanism of RP after cerebral ischemia jniury.DESIGN,TIME AND SETTING:Randomized control experiment of cellular,molecular.and protein levels.The experiment was completed at Chongqing Medical University at the School of Pharmacy Laboratories and Basic Medical Institute from October 2005 to January 2006.MATERIALS:Twenty-four adult,male,Wistar rats,weighing(28±20)g.RP extract,which was a product of ethanol extraction.was provided by the LabOratory of Pharmaceutical Analysis,Chongqing Medical University.RP was dissolved in distilled water to a concentration of 10 mg/mL.All rats were randomly assigned into four groups:5 g/kg RP,10 g/kg RP,model,and sham-surgery,with 6 rats in each group.METHODS:In the 5 and 10 g/kg RP groups,as well as the model group,the middle cerebral artery was occluded(MCAO)for 60 minutes,resulting in focal cerebral ischemia,followed by reperfusion for 24 hours.Rats in the 5 and 10 g/kg RP groups received 5 and 10 g/kg RP,respectively.The RP treatment group received RP intragastrically(once a day)for 3 days.One hour after the last dose,rats were subjected to MCAO.The same surgical procedure was performed in the sham-surgery group,except the suture was introduced into the extemal carotid artery,but not advanced.Rats in the model group were subjected to MCAO.The sham-surgery and model groups received intragastrically administered normal saline once per day for 3 days.One hour after the last dose,the rats were subjected to surgery.MAIN OUTCOME MEASURES:TUNEL labeling and immunohistochemical methods were used to investigate changes in neuronal apoptosis and expression of the apoptosis-related proteins.Bax and Bcl-2.on the ischemic hemisphere,as well as the contralateral hemisphere,after administration of RP or normal saline.RESULTS:All 24 Wistar rats were included in the final analysis.without any loss.Compared with the sham-surgery group,the number of the TUNEL-positive cells in the model group,as well as the 5 and 10 g/kg RP treatment groups,were significantly increased in the ischemic hemisphere(P<0.05).Compared with the sham-surgery group,the number of Bcl-2-positive and Bax-positive cells increased significantly in the model group(P<0.01).The number of Bcl-2-positive cells increased,and the number of Bax-positive cells decreased in the model group,compared to the 5 and 10 g/kg RP treatment groups(P<0.05-0.01).CONCLUSION:RP can prevent neuronal apoptosis following cerebral ischemic-reperfusion of rats.The mechanism underlying the RP-induced neuroprotection in the cerebral ischemia iniury may be related to increased Bcl-2 expression and decreased Bax expression.     

老年与成年大鼠纹状体多巴胺含量及黑质部凋亡相关蛋白表达的研究

目的 研究老年与成年SD大鼠纹状体多巴胺含量及黑质部凋亡相关蛋白Bad、Bcl-2的表达情况,并探讨两者之间的关系.方法 取老年SD大鼠6只作为老年组,成年SD大鼠6只作为成年组;采用高效液相色谱-质谱联用法测定纹状体内多巴胺含量;免疫印迹法检测黑质Bad、Bcl-2的表达.结果 老年组纹状体DA含量(4.13±0.209 ng/mg)低于成年组(5.65±0.188ng/mg)(P＜0.05);老年组黑质部抗凋亡蛋白Bcl-2的表达(1.08±0.047)低于成年组(1.36±0.085)(P＜0.05);促凋亡蛋白Bad的表达(0.79±0.051)高于成年组(0.57±0.071)(P＜0.05).结论 老年SD大鼠纹状体DA含量降低,抗凋亡蛋白Bcl-2表达降低,促凋亡Bad表达增高.     

Effects of Rubus parvifolius L. on neuronal apoptosis and expression of apoptosis-related proteins in a rat model of focal cerebral ischemic-reperfusion injury*★

BACKGROUND： Studies have shown that the Rubus parvifolius L. （RP） plant extract exhibits protective effects on cerebral ischemia. This effect is reflected in altered ischemic neuronal apoptosis and associated protein expression.
OBJECTIVE： To explore the neuroprotective mechanism of RP after cerebral ischemia injury.
DESIGN, TIME AND SETTING： Randomized control experiment of cellular, molecular, and protein levels. The experiment was completed at Chongqing Medical University at the School of Pharmacy Laboratories and Basic Medical Institute from October 2005 to January 2006.
MATERIALS： Twenty-four adult, male, Wistar rats, weighing （28 ± 20） g. RP extract, which was a product of ethanol extraction, was provided by the Laboratory of Pharmaceutical Analysis, Chongqing Medical University. RP was dissolved in distilled water to a concentration of 10 mg/mL. All rats were randomly assigned into four groups： 5 g/kg RP, 10 g/kg RP, model, and sham-surgery, with 6 rats in each group.
METHODS： In the 5 and 10 g/kg RP groups, as well as the model group, the middle cerebral artery was occluded （MCAO） for 60 minutes, resulting in focal cerebral ischemia, followed by reperfusion for 24 hours. Rats in the 5 and 10 g/kg RP groups received 5 and 10 g/kg RP, respectively. The RP treatment group received RP intragastrically （once a day） for 3 days. One hour after the last dose, rats were subjected to MCAO. The same surgical procedure was performed in the sham-surgery group, except the suture was introduced into the external carotid artery, but not advanced. Rats in the model group were subjected to MCAO. The sham-surgery and model groups received intragastrically administered normal saline once per day for 3 days. One hour after the last dose, the rats were subjected to surgery.
MAIN OUTCOME MEASURES： TUNEL labeling and immunohistochemical methods were used to investigate changes in neuronal apoptosis and expression of the apoptosis-related proteins, Bax and Bcl-2, on the ischemic hemisphere     

Expression of axotomy-inducible and apoptosis-related genes in sensory nerves of rats with experimental diabetes

In diabetes, peripheral nerves suffer deficient neurotrophic support-a situation which resembles axotomy. This raises the question: does inappropriate establishment of an axotomised neuronal phenotype contribute to diabetic neuropathy, and in extremis, does this provoke apoptosis? We hybridized reverse-transcribed RNA, from the dorsal root ganglia (DRG) of 8-week streptozotocin (STZ)-induced diabetic rats, to Affymetrix Rat Genome U34A chips and scanned the array for expression of (a) genes that are upregulated by axotomy, (b) proapoptotic and (c) anti-apoptotic genes. Expression of the axotomy-responsive genes coding for growth-associated protein 43 (GAP-43), galanin, neuropeptide Y (NPY), pre-pro-vasoactive intestinal polypeptide (pre-pro-VIP), neuronal nitric oxide synthase (nNOS), protease nexin 1, heat-shock protein 27 (HSP 27) and myosin light chain kinase II (MLCK II) was unaffected in ganglia from diabetic rats compared to controls; thus, no axotomised phenotype was established. The expression of the majority of proapoptotic genes in the DRG was also unaltered (bax, bad, bid, bok, c-Jun, p38, TNFR1, caspase 3 and NOS2). Similarly there was no change in expression of the majority of antiapoptotic genes (bcl2, bcl-xL, bcl-w, NfkappaB). These alterations in gene expression make it clear that neither axotomy nor apoptotic phenotypes are established in neurones in this model of diabetes.     

Acrylamide intoxication modifies in vitro responses of peripheral nerve axons to anoxia

Decreased axolemmal Na+/K(+)-ATPase activity has been considered as a possible mechanism for peripheral nerve axon damage induced by acrylamide (ACR) or 2,5-hexanedione (HD). Reduced activity of this enzyme is also presumed to be the basis of peripheral nerve resistance to ischemia or hypoxia associated with other neuropathies (e.g., diabetes). In the present study, we tested the hypothesis that peripheral nerve of ACR (50 mg/kg/d x 10 d) or HD (400 mg/kg/d x 20 d) exposed rats are resistant to oxygen-limiting conditions as a result of reduced axonal Na+/K(+)-ATPase activity. As an index of resistance, effects of in vitro anoxia on subaxonal concentrations of Na, K and Ca were assessed in isolated segments of tibial nerve from control and neurotoxicant-treated animals. Results show axons from HD rats were not resistant to anoxic challenge; i.e., axons exhibited disrupted elemental composition comparable to anoxic control changes. In contrast, ACR-exposed axons displayed anoxic resistance. Ouabain-exposed tibial axons subjected to anoxic conditions were also resistant, but the corresponding elemental pattern did not resemble that associated with ACR axons. Moreover, ACR axons were capable of maintaining elemental gradients during normoxic exposure which should not be possible if Na+ pump activity is depressed. Considered together, these data are not consistent with a role for diminished Na+/K(+)-ATPase activity in neurotoxicant-induced peripheral axonopathy. We also assessed the ability of ACR- and HD-exposed tibial nerve axons to recover from anoxia. Unlike control fibers which can fully restore normal elemental composition, neurotoxicant-exposed axons were incapable of such restoration. These data suggest the axonal machinery responsible for post-anoxia recovery (e.g., energy metabolism, ion translocation, Ca2+ and free radical buffering) is compromised by ACR or HD intoxication.     

Aberrant expression of the apoptosis-related proteins BAK and MCL1 in T cells in multiple sclerosis

Pathogenic T cells of multiple sclerosis (MS) patients have been suggested to be endowed with an increased resistance to apoptosis, contributing to their increased survival. We report herein increased levels of the anti-apoptotic MCL1 protein and its half-life in activated lymphocytes of MS patients, which were not associated with differences in MCL1 RNA levels or with alterations in the expression levels of the known E3 ligases of MCL1-β-TrCP and HUWE1. Concomitantly, the expression levels of the pro-apoptotic protein BAK were decreased in MS patients at relapse. These findings suggest the dysregulation of the apoptosis-related proteins MCL1 and BAK in MS.