Hyperoxia but not ambient pressure decreases tetrahydrobiopterin level without affecting the enzymatic capability of nitric oxide synthase in human endothelial cells

Nitric oxide (NO) seems to be related to bubble formation and endothelial dysfunction resulting in decompression sickness. Bubble formation can be affected by aerobic exercise or manipulating NO. A prior heat stress (HS) has been shown to confer protection against decompression sickness in rats. An important question was if the oxidative environment experienced during diving limits the availability of the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin (BH4). Human endothelial cells were used to investigate how HS and simulated diving affected NO synthesis and defense systems such as heat shock protein 70 (HSP70) and glutathione (GSH). BH4 was measured using a novel LC–MS/MS method and NOS by monitoring the conversion of radiolabeled l-arginine to l-citrulline. Increased pO₂ reduced BH4 levels in cells in a dose-dependent manner independently of high pressure. This effect may result in decreased generation of NO by NOS. The BH4 decrease seemed to be abolished when cells were exposed to HS prior to hyperoxia. NOS enzyme was unaffected by increased pO₂ but substantially reduced after HS. The BH4 level seemed to a minor extent to be dependent upon GSH and probably to a higher degree dependent on other antioxidants such as ascorbic acid. A simulated dive at 60 kPa O₂ had a potentiating effect on the heat-induced HSP70 expression, whereas GSH levels were unaffected by hyperoxic exposure. HS, hyperoxia, and dive affected several biochemical parameters that may play important roles in the mechanisms protecting against the adverse effects of saturation diving.     

Exercise and nitric oxide prevent bubble formation: a novel approach to the prevention of decompression sickness?

Nitrogen dissolves in the blood during dives, but comes out of solution if divers return to normal pressure too rapidly. Nitrogen bubbles cause a range of effects from skin rashes to seizures, coma and death. It is believed that these bubbles form from bubble precursors (gas nuclei). Recently we have shown that a single bout of exercise 20 h, but not 48 h, before a simulated dive prevents bubble formation and protects rats from severe decompression sickness (DCS) and death. Furthermore, we demonstrated that administration of N ^ω-nitro- l -arginine methyl ester, a non-selective inhibitor of NO synthase (NOS), turns a dive from safe to unsafe in sedentary but not exercised rats. Therefore based upon previous data an attractive hypothesis is that it may be possible to use either exercise or NO-releasing agents before a dive to inhibit bubble formation and thus protect against DCS. Consequently, the aims of the present study were to determine whether protection against bubble formation in 'diving' rats was provided by (1) chronic and acute administration of a NO-releasing agent and (2) exercise less than 20 h prior to the dive. NO given for 5 days and then 20h prior to a dive to 700 kPa lasting 45 min breathing air significantly reduced bubble formation and prevented death. The same effect was seen if NO was given only 30 min before the dive. Exercise 20h before a dive surpressed bubble formation and prevented death, with no effect at any other time (48, 10, 5 and 0.5h prior to the dive). Pre-dive activities have not been considered to influence the growth of bubbles and thus the risk of serious DCS. The present novel findings of a protective effect against bubble formation and death by appropriately timed exercise and an NO-releasing agent may form the basis of a new approach to preventing serious decompression sickness.     

Exercise before and after SCUBA diving and the role of cellular microparticles in decompression stress

Risk in SCUBA diving is often associated with the presence of gas bubbles in the venous circulation formed during decompression. Although it has been demonstrated time-after-time that, while venous gas emboli (VGE) often accompany decompression sickness (DCS), they are also frequently observed in high quantities in asymptomatic divers following even mild recreational dive profiles. Despite this VGE are commonly utilized as a quantifiable marker of the potential for an individual to develop DCS. Certain interventions such as exercise, antioxidant supplements, vibration, and hydration appear to impact VGE production and the decompression process. However promising these procedures may seem, the data are not yet conclusive enough to warrant changes in decompression procedure, possibly suggesting a component of individual response. We hypothesize that the impact of exercise varies widely in individuals and once tested, recommendations can be made that will reduce individual decompression stress and possibly the incidence of DCS. The understanding of physiological adaptations to diving stress can be applied in different diseases that include endothelial dysfunction and microparticle (MP) production.Exercise before diving is viewed by some as a protective form of preconditioning because some studies have shown that it reduces VGE quantity. We propose that MP production and clearance might be a part of this mechanism. Exercise after diving appears to impact the risk of adverse events as well. Research suggests that the arterialization of VGE presents a greater risk for DCS than when emboli are eliminated by the pulmonary circuit before they have a chance to crossover. Laboratory studies have demonstrated that exercise increases the incidence of crossover likely through extra-cardiac mechanisms such as intrapulmonary arterial-venous anastomoses (IPAVAs). This effect of exercise has been repeated in the field with divers demonstrating a direct relationship between exercise and increased incidence of arterialization.     

High intensity cycling before SCUBA diving reduces post-decompression microparticle production and neutrophil activation

Background

Venous gas emboli (VGE) have traditionally served as a marker for decompression stress after SCUBA diving and a reduction in bubble loads is a target for precondition procedures. However, VGE can be observed in large quantities with no negative clinical consequences. The effect of exercise before diving on VGE has been evaluated with mixed results. Microparticle (MP) counts and sub-type expression serve as indicators of vascular inflammation and DCS in mice. The goal of the present study is to evaluate the effect of anaerobic cycling (AC) on VGE and MP following SCUBA diving.

Methods

Ten male divers performed two dives to 18 m for 41 min, one dive (AC) was preceded by a repeated-Wingate cycling protocol; a control dive (CON) was completed without exercise. VGE were analyzed at 15, 40, 80, and 120 min post-diving. Blood for MP analysis was collected before exercise (AC only), before diving, 15 and 120 min after surfacing.

Results

VGE were significantly lower 15 min post-diving in the AC group, with no difference in the remaining measurements. MPs were elevated by exercise and diving, however, post-diving elevations were attenuated in the AC dive. Some markers of neutrophil elevation (CD18, CD41) were increased in the CON compared to the AC dive.

Conclusions

The repeated-Wingate protocol resulted in an attenuation of MP counts and sub-types that have been related to vascular injury and DCS-like symptoms in mice. Further studies are needed to determine if MPs represent a risk factor or marker for DCS in humans.     

Expression of heat shock protein 70 and endothelial nitric oxide synthase in placental tissue of preeclamptic and intrauterine growth-restricted pregnancies

Ischemia, hypoxia, and elevated vascular resistance disturb placental functions by increasing oxidative stress. Heat shock protein 70 (HSP70) is an oxidative stress marker. Endothelial nitric oxide synthase (eNOS) is a nitric oxide enzyme with a key role in pathologic and physiologic angiogenesis and vasculogenesis. This study was performed to investigate the role of oxidative stress in the pathogenesis of preeclampsia and intrauterine growth-restricted (IUGR) pregnancies by comparing the levels of HSP70 and eNOS in placentas from women with these diseases and those with healthy pregnancies.     

Hyperbaric oxygen pretreatment reduces the incidence of decompression sickness in rats

We have previously hypothesised that the number of bubbles evolving during decompression from a dive, and therefore the incidence of decompression sickness (DCS), might be reduced by pretreatment with hyperbaric oxygen (HBO). The inert gas in the gas micronuclei would be replaced by oxygen, which would subsequently be consumed by the mitochondria. This has been demonstrated in the transparent prawn. To investigate whether our hypothesis holds for mammals, we pretreated rats with HBO at 304, 405, or 507 kPa for 20 min, after which they were exposed to air at 1,013 kPa for 33 min and decompressed at 202 kPa/min. Twenty control rats were exposed to air at 1,013 kPa for 32 min, without HBO pretreatment. On reaching the surface, the rat was immediately placed in a rotating cage for 30 min. The animal’s behaviour enabled us to make an early diagnosis of DCS according to accepted symptoms. Rats were examined again after 2 and 24 h. After 2 h, 65% of the control rats had suffered DCS (45% were dead), whereas 35% had no DCS. HBO pretreatment at 304, 405 and 507 kPa significantly reduced the incidence of DCS at 2 h to 40, 40 and 35%, respectively. Compared with the 45% mortality rate in the control group after 24 h, in all of the pretreated groups this was 15%. HBO pretreatment is equally effective at 304, 405 or 507 kPa, bringing about a significant reduction in the incidence of DCS in rats decompressed from 1,013 kPa.     

Distribution of 70kDa heat shock protein in rabbit brains after heat stress and heat stroke

OBJECTIVE: Evaluation of the relationship between the induction of 70kDa heat shock protein in rabbit brains and heat stress. METHODS: HSP70 was detected using monoclonal antibody by ABC method in rabbit hypothalamus, hippocampus and cerberal cortex. RESULTS: Intense HSP70 staining was displayed in rabbit brains of the heat stroke group (rectal temperature 43 degrees C to death). Positive cells were distributed mainly in the CA1, CA2 regions of the hippocampus; granular cell layer I and pyramidal layer (II) of the cerebral cortex; and the periventricular area of hypothalamus. HSP70-psoitive substances were localized in the cytoplasm and neuronal processes, a few neurons exhibited dark staining nucle. Hosever, the rabbit brains of the general heat stress group (rectal temperature 42.0 degrees C, 30 minutes) had much weaker staining. CONCLUSION: Hyperthermia causes neuronal expression of HSP70, particularly under strong heat stress, and may be sustained till death.     

Oxygen pretreatment as protection against decompression sickness in rats: pressure and time necessary for hypothesized denucleation and renucleation

Pretreatment with HBO at 300–500 kPa for 20 min reduced the incidence of decompression sickness (DCS) in a rat model. We investigated whether this procedure would be effective with lower oxygen pressures and shorter exposure, and tried to determine how long the pretreatment would remain effective. Rats were pretreated with oxygen at 101 or 203 kPa for 20 min and 304 kPa for 5 or 10 min. After pretreatment, the animals were exposed to air at 1,013 kPa for 33 min followed by fast decompression. Pretreatment at 101 or 203 kPa for 20 min and 304 kPa for 10 min significantly reduced the number of rats with DCS to 45%, compared with 65% in the control group. However, after pretreatment at 304 kPa for 5 min, 65% of rats suffered DCS. When pretreatment at 304 kPa for 20 min was followed by 2 h in normobaric air before compression and decompression, the outcome was worse, with 70–90% of the animals suffering DCS. This is probably due to the activation of “dormant” micronuclei. The risk of DCS remained lower (43%) when pretreatment with 100% O₂ at normobaric pressure for 20 min was followed by a 2 h interval in normobaric air (but not 6 or 24 h) before the hyperbaric exposure. The loss of effectiveness after a 6 or 24 h interval in normobaric air is related to micronuclei rejuvenation. Although pretreatment with hyperbaric O₂ may have an advantage over normobaric hyperoxia, decompression should not intervene between pretreatment and the dive.     

No changes in lung function after a saturation dive to 2.5 MPa with intermittent reduction in P_{{{{\rm O}}_{{{\rm 2}}} }} during decompression

Decompression stress and exposure to hyperoxia may cause a reduction in transfer factor of the lung for carbon monoxide and in maximal aerobic capacity after deep saturation dives. In this study lung function and exercise capacity were assessed before and after a helium–oxygen saturation dive to a pressure of 2.5 MPa where the decompression rate was reduced compared with previous deep dives, and the hyperoxic exposure was reduced by administering oxygen intermittently at pressures of 50 and 30 kPa during decompression. Eight experienced divers of median age 41 years (range 29–48) participated in the dive. The incidence of venous gas microemboli was low compared with previous deep dives. Except for one subject having treatment for decompression sickness, no changes in lung function or angiotensin converting enzyme, a marker of pulmonary endothelial cell damage, were demonstrated. The modified diving procedures with respect to decompression rate and hyperoxic exposure may have contributed to the lack of changes in lung function in this dive compared with previous deep saturation dives.     

Exercise-induced myofibrillar disruption with sarcolemmal integrity prior to simulated diving has no effect on vascular bubble formation in rats

Decompression sickness is initiated by gas bubbles formed during decompression, and it has been generally accepted that exercise before decompression causes increased bubble formation. There are indications that exercise-induced muscle injury seems to be involved. Trauma-induced skeletal muscle injury and vigorous exercise that could theoretically injure muscle tissues before decompression have each been shown to result in profuse bubble formation. Based on these findings, we hypothesized that exercise-induced skeletal muscle injury prior to decompression from diving would cause increase of vascular bubbles and lower survival rates after decompression. In this study, we examined muscle injury caused by eccentric exercise in rats prior to simulated diving and we observed the resulting bubble formation. Female Sprague–Dawley rats (n = 42) ran downhill (?16º) for 100 min on a treadmill followed by 90 min rest before a 50-min simulated saturation dive (709 kPa) in a pressure chamber. Muscle injury was evaluated by immunohistochemistry and qPCR, and vascular bubbles after diving were detected by ultrasonic imaging. The exercise protocol resulted in increased mRNA expression of markers of muscle injury; αB-crystallin, NF-κB, and TNF-α, and myofibrillar disruption with preserved sarcolemmal integrity. Despite evident myofibrillar disruption after eccentric exercise, no differences in bubble amounts or survival rates were observed in the exercised animals as compared to non-exercised animals after diving, a novel finding that may be applicable to humans.     

HSP70对大鼠肝脏局部缺血再灌注后炎症反应的影响

目的：通过热应激预处理诱导HSP70表达，探讨其对肝脏缺血再灌注损伤后炎症反应的影响。方法:采用大鼠局部缺血再灌注模型（IR组），并在热应激预处理（H+IR组）及槲皮素＋热应激预处理（Q+H+IR组）条件下观察肝脏缺血再灌注后HSP70、ICAM-1的表达及MPO的活性；测定血清ALT和AST的活性；电镜观察肝细胞结构的改变。结果:在H+IR组检测的各时点HSP70表达均明显高于其它两组、对肝脏进行缺血再灌注后，肝细胞损伤较轻，血清ALT、AST升高不明显(P<0.01)；肝组织中ICAM-1表达增加，以再灌注后6 h最显著，MPO活性升高以12 h最为显著，但两者变化均低于IR组和Q+H+IR组(P<0.01)。结论：〖HTSS〗热应激预处理诱导产生HSP70蛋白能够降低大鼠肝脏缺血再灌注损伤过程ICAM-1的表达和MPO活性的改变，进而抑制炎症反应引起的肝脏损伤。     

An important role of the heat shock response in infected cells for replication of baculoviruses

Baculoviruses serve as a stress factor that can activate both death-inducing and cytoprotective pathways in infected cells. In this report, induction of heat shock proteins (HSPs) of the 70-kDa family (HSP/HSC70) in Sf-9 cells after infection with AcMNPV was monitored by Western blot analysis. Two-dimensional electrophoresis in polyacrylamide gel revealed changes in the cellular pattern of HSP/HSC70s and synthesis of a new member of the HSP/HSC70 family in the infected cells. Although infection with AcMNPV moderately increased the HSP/HSC70 content in cells under standard conditions, the infection potentiated the response to heat shock boosting the HSP/HSC70s content in infected cells several-fold in comparison with uninfected cells. Addition of KNK437, a known inhibitor of inducible HSPs, decreased the rate of viral DNA synthesis in infected cells more than one order of magnitude and markedly suppressed the release of budded viruses indicating the importance of the heat shock response for baculovirus replication.     

Hg2+ and Ni2+ alter induction of heat shock protein-72 in THP-1 human monocytes

The biological liabilities that result from the release of metal ions from biomedical alloys, particularly Ni(2+) and Hg(2+), continue to be a concern. Heat-shock proteins (HSP) are a class of molecular chaperones that may be induced under conditions of cellular stress, including oxidative stress. Our hypothesis was that because Hg(2+) and Ni(2+) alter other cellular stress responses such as glutathione levels and cytokine secretion, these metal ions may alter HSP induction in monocytes, which are key cells in the response of tissues to biomedical alloys. THP-1 monocytes were exposed to sublethal concentrations of Hg(2+) or Ni(2+) for 1 h with or without heat stress (43 degrees C), then allowed to recover at 37 degrees C for 2-6 h. HSP72 was measured using immunoblotting with phosphorimage quantification. Hg(2+) exposures of 2-10 micromol/L induced HSP72 without heat stress. With heat stress, HSP72 levels were altered by Hg(2+) versus heat stress alone. The response depended on the concentration of Hg(2+) and the recovery time. Hg(2+) at 10 micromol/L caused uniformly lower HSP72 levels. Ni(2+) exposures of 20-100 micromol/L did not induce HSP72 without heat stress, but significantly altered heat-induced HSP72 expression, with a significant increase in expression over heat alone at 40 and 100 micromol/L. Results from the current study support the hypothesis that these metal ions can, at concentrations relevant to those released from biomedical alloys, modulate HSP expression in human monocytes. The modulation of HSP expression indicates an early sign of cellular stress that may be important to the overall biological response to biomedical alloys containing and releasing these metal ions.     

Roles of compatible osmolytes and heat shock protein 70 in the induction of tolerance to stresses in porcine endothelial cells

Studies of the responses of porcine pulmonary endothelial cells to acute hypertonic stress have been extended by examining the induction and underlying mechanisms of cell tolerance to both osmotic and heat stresses. Preliminary adaptation of these cells to 0.4osmol (kg H₂O)⁻¹ rendered them tolerant either to subsequent severe osmotic stress (0.7osmol (kg H₂O)⁻¹) or to subsequent severe heat shock (50 min at 49°C). In contrast, preliminary exposure of the cells to mild heat shock (44°C for 30 min) induced tolerance only to severe heat shock, not to hyperosmotic stress. Induction of tolerance to heat shock by either procedure correlated with the induced expression of heat shock protein 70 (HSP70). Induction of tolerance to hyperosmotic stress, on the other hand, was associated with the cellular accumulation of osmolytes, such as amino acids, betaine and myo -inositol, and did not correlate with the induced expression of HSP70. It also required a reduction in the final change of osmotic pressure applied to the cells, such that maximum cell shrinkage would not be much more than 40%. In general, therefore, HSP70 and compatible osmolytes have distinct roles in cellular adaptation to these stresses.     

Effect of endotoxin-induced cell injury on 70-kD heat shock proteins in bovine lung endothelial cells

Heat shock proteins (HSPs) have been remarkably conserved throughout evolution. It has been assumed that induction of HSPs remains a stereotypic response to injury, important for survival of eukaryotic cells during euthermic injury. However, there are few studies of this phenomenon in endothelial cells, and none in pulmonary endothelial cells. We studied the induction of synthesis of 70-kD proteins in bovine pulmonary artery endothelial cells (BPAECs) in response to heat shock and to euthermic injury induced by bacterial endotoxin. First, in response to heat, BPAECs showed rapid and reversible heat-induced synthesis of 70-kD proteins, readily detectable by one-dimensional SDS-PAGE of [35S]methionine-labeled BPAECs. Heat shock at 42 degrees C for 3 h or 43 degrees C for 2 h suppressed total protein synthesis by 30% (P less than 0.001) but an increased rate of synthesis of 70-kD protein continued, representing an increasing fraction of total protein synthesis. Heat-induced synthesis of 70-kD protein returned to baseline levels 8 h after heat shock. Northern analysis showed that mRNA for a protein homologous to a conserved amino acid sequence in the family of species-homologous 70-kD heat shock proteins (HSP 70) was induced by a 15-min incubation at 42 degrees C and remained detectably increased for 6 h. We next assessed whether euthermic injury by bacterial endotoxin (LPS) generated a similar response. LPS was cytotoxic by BPAECs as assessed morphologically, by release of 51Cr from prelabeled cells, and by a significant suppression of total protein synthesis (range, 35 to 70%; P less than 0.001). Despite cytotoxicity, LPS did not induce 70-kD protein at a level that could be detected by SDS-PAGE, and no increase in mRNA for HSP 70 was detected by Northern analysis. LPS-injured BPAECs remained "competent" to induce both 70-kD proteins and mRNA for HSP 70 in response to heat shock. We conclude that at least quantitatively, induction of HSP 70 by BPAECs is not a stereotypic response to injury but rather is at least relatively injury-specific. However, competence to induce HSP 70 appears to be extremely resilient: it is retained in dysfunctional BPAECs in the face of profound inhibition of global protein synthesis, suggesting an important homeostatic role.     

热应激蛋白70的研究进展

热应激蛋白70(HSP70)是由细胞核内高度保守的热应激基因编码的产物之一,作为对外界应激的一种保护性反应,它有助于机体维持正常的新陈代谢和细胞结构的完整性。当前,HSP70的研究已成为当今世界生命科学的热点和富有希望的研究领域。本文就其结构与功能、基因表达的调控以及应用前景作一综述。     

Venous gas embolism as a predictive tool for improving CNS decompression safety

A key process in the pathophysiological steps leading to decompression sickness (DCS) is the formation of inert gas bubbles. The adverse effects of decompression are still not fully understood, but it seems reasonable to suggest that the formation of venous gas emboli (VGE) and their effects on the endothelium may be the central mechanism leading to central nervous system (CNS) damage. Hence, VGE might also have impact on the long-term health effects of diving. In the present review, we highlight the findings from our laboratory related to the hypothesis that VGE formation is the main mechanism behind serious decompression injuries. In recent studies, we have determined the impact of VGE on endothelial function in both laboratory animals and in humans. We observed that the damage to the endothelium due to VGE was dose dependent, and that the amount of VGE can be affected both by aerobic exercise and exogenous nitric oxide (NO) intervention prior to a dive. We observed that NO reduced VGE during decompression, and pharmacological blocking of NO production increased VGE formation following a dive. The importance of micro-nuclei for the formation of VGE and how it can be possible to manipulate the formation of VGE are discussed together with the effects of VGE on the organism. In the last part of the review we introduce our thoughts for the future, and how the enigma of DCS should be approached.     

Effect of pre-breathing oxygen at different depth on oxidative status and calcium concentration in lymphocytes of scuba divers

Aim: In‐water pre‐breathing oxygen at various depths reduces decompression‐induced bubble formation and platelet activation, but it could induce side effects such as oxidative stress. The aim of this study was to investigate the effect of in‐water pre‐breathing oxygen, at different depths, on the oxidative status and intracellular calcium ([Ca²⁺]i) of peripheral blood lymphocytes isolated from six divers. They participated in a 4‐diving protocol. Two week recovery time was allowed between successive dives. Before diving, all divers, for 20 min, breathed normally at sea level (dive 1), 100% oxygen at sea level (dive 2), 100% oxygen at 6 msw (dive 3), 100% oxygen at 12 msw (dive 4). Then they dived to 30 msw for 20 min with air tank. Methods: Blood samples were collected before and after each dive. Hydrogen peroxide (H₂O₂) levels, catalase (CAT) activity, mRNA expression of CAT, glutathione peroxidase (GPx) and superoxide dismutase (SOD), and the [Ca²⁺]i in lymphocytes were measured. Results: The dives slightly decreased lymphocyte number and significantly reduced lymphocyte H₂O₂ levels. CAT activity was higher after scuba diving and, dive 3 enhanced mRNA gene expression of CAT, GPx and SOD. The [Ca²⁺]i was higher after dive 1 and 2 than pre‐diving, while was maintained at pre‐diving value after dive 3 and 4. Conclusion: Our results suggest that pre‐breathing oxygen, in particular at 12 msw, may enhance lymphocyte antioxidant activity and reduce reactive oxygen species levels. Pre‐breathing oxygen in water may also preserve calcium homeostasis, suggesting a protective role in the physiological lymphocyte cell functions.     

Human sympathetic nerve activity to glabrous skin does not increase during simulated diving

In humans, cardiovascular adjustment to simulated diving causes a marked increase in sympathetic outflow to intramuscular vessels and muscle vasoconstriction. Skin vasoconstriction in the hand also occurs during diving in humans. Skin nerve sympathetic activity (SSA), containing vasoconstrictor signals to glabrous skin, unexpectedly was reduced during diving in a previous study of SSA recorded in the peroneal nerve. SSA was recorded by microneurography in the median nerve in 13 healthy volunteers during simulated diving. Skin blood flow in the hand and one finger was monitored. The typical SSA response, irrespective of duration of diving and water temperature, was an increase during the control period immediately prior to immersion of the face and a sudden reduction of SSA when the face was immersed. The increase in SSA preceding the dive was accompanied by vasoconstriction, which continued during the dive, but re-dilation regularly occurred before the end of the dive. Inhibition of SSA was not total. Mental arithmetic during diving evoked strong bursts of SSA, similar to those seen normally during mental stress. It is concluded that the true response of SSA to simulated diving is an inhibition of the immediately preceding outflow, in agreement with observations of cutaneous blood flow in animals. The skin vasoconstriction recorded during simulated diving is a consequence of an SSA increase before the procedure, suggested to be a stress response before the forthcoming manoeuvre. The SSA response during simulated diving is the opposite to that of sympathetic outflow to muscle, which emphasizes the diversity of sympathetic regulation of different organ systems.     

热休克蛋白40和热休克蛋白70抑制N2a细胞内突变亨廷顿蛋白聚积和毒性

目的 研究热休克蛋白40(HSP40)和热休克蛋白70(HSP70)对N2a细胞内突变亨廷顿蛋白(htt) 聚集物形成和毒性的影响. 方法 应用荧光显微镜术和免疫印迹技术检测单独或共同过表达HSP40和HSP70对N2a细胞内转染的突变htt(含有150个谷氨酰胺重复数,150Q)聚集物形成的影响;应用四甲基偶氮唑盐(MTT)法分析细胞活力和比色法检测细胞内活性氧(ROS)含量. 结果 单独过表达HSP40或HSP70,尤其是共同过表达HSP40和HSP70显著减少150Q htt在N2a细胞内的聚积,各组含有聚集物的细胞为(n=1 000):仅表达150Q htt 组约50%,过表达HSP40组约12%,过表达HSP70组约15%,共同过表达HSP40和HSP70组约5%.MTT分析结果显示,单独尤其是共同过表达HSP40和HSP70能显著增加150Q细胞的活力( P<0.01, n =3),同时减少ROS产生( P<0.01, n =3). 结论 HSP40和HSP70通过抑制细胞内突变htt聚积以及减少氧化应激而增加150Q N2a细胞活力.