空降兵跳伞后血浆的生化变化：初步调查与分析
作者：邬堂春 熊一力 陈胜 冷顺堂 海涛 罗伯特.汤格
单位：邬堂春.同济医科大学职业医学研究所，武汉 430030
关键词：空降兵；跳伞；应激(生理)；激素；热应激蛋白70；丙二醛
航天医学与医学工程990401Biochemical Changes of Plasma in Paratroops after
Parachuting:A Preliminary Investigation*
WU Tang-chun1, XIONG Yi-li2,CHEN Sheng1,LENG Shun-tang2,HAI Tao,Robert M.Tanguay3
(1.Institute of Occupational Medicine, Tongji Medical University, Wuhan 430030；2. Hankou Airplane Hospital (No.457), Wuhan 430012；3.Lab of Cellular and Developmental Genetics, Life and Health Sciences Research Center, Laval University, Quebec, Canada G1K 7P4)
Abstract: Objective To study whether physiological and psychological stresses during parachuting jumps may result in biochemical changes of plasma in parachutists. Method Differences in the levels of hormones (cortisol, growth hormone, insulin, pancreatic glucagon, endothelin, angiotonin I and II, aldosterone), activities of enzymes (superoxide dismutase, glutathione peroxidase, glutathione S transferase), levels of the free radical damage indicator malondialdehyde (MDA), tumor necrosis factor alpha (TNF-α), and the main heat stress protein, HSP70,in the plasma and serum were investigated in control (non-parachuting) and parachuting paratroops. Result Significantly higher levels of serum hormones such as growth hormone, insulin, angiotonin I, renin activities, as well as MDA and plasma TNF-α and HSP70 were observed in the parachuting group. Conclusion Whether these changes can potentially serve as useful biomarkers to assess possible abnormal stress in parachutists and to evaluate the health condition and to select parachutists remains to be further studied.
Key words: parachutists; parachuting; stress(physiology); hormones;HSP70;malondialdehyde
摘要：目的 探讨跳伞期间空降兵经历的生理和心理应激是否会改变空降兵血浆的生化指标。方法 调查了对照组(未跳伞组)和跳伞组空降兵血浆和血清中激素水平(皮质醇、生长激素、胰岛素、胰高血糖素、类皮素、血管紧张素I和 II、醛固酮)、酶活性(超氧化物岐化酶、谷胱甘肽过氧化氢酶和谷胱甘肽硫转移酶)、自由基损伤指标水平(丙二醛，)、肿瘤坏死因子α(TNF-α)和主要热应激蛋白(HSP70)的差异性。结果 跳伞组血清中生长激素、胰岛素、血管紧张素I、肾素活性和血浆中丙二醛、TNF-α、HSP70等的水平比对照值显著高。结论 这些变化有可能作为评估空降兵是否经历异常应激、评价他们的健康状况和选择空降兵的有用生物标志物，但需进一步研究。
中图分类号：R851.3 文献标识码：A 文章编号：1002-0837(1999)04-0235-05
Parachuting has long been a subject of military interest,and is used to insert troops rapidly into a drop zone by air[1].During parachuting,paratroops may experience both physiological and psychological stresses caused by such environmental factors as high ambient temperature,altitude hypoxia,noise,vibration,and by such inherent psychogenic and emotional states as anxiety,fear,feeling of insecurity,and self-imposed stressors[2,3].These complex stressors can result in the stress response,but the biochemical changes in plasma of paratroops caused by these combined stresses and their possible significance in evaluating the health condition and selection of parachutists are unknown.It is known that organisms ranging from bacteria to human respond to high temperatures by inducing the synthesis of a group of proteins known as heat shock or heat stress protein (HSPs) and by reducing the rate of synthesis of most other proteins[4～6].The synthesis of HSPs is not only induced by heat but also by a variety of other physiological stresses such as ischemia and low pH,and by environmental stressors such as ethanol,carbon monoxide,nicotine,H2O2,benzene,mutagens,carcinogens,teratogens,dusts,drugs,ultraviolet light,amino acid analogs,heavy metals,free radicals,and others[4～7].HSP70 is the main heat stress protein and has been shown to have many important biological functions[8].For example,HSP70 may confer to organisms the ability to recover from stress,and it also protects organisms against the damage from heat or other harmful factors by raising the cellular resistance to these harmful factors[8～10].Moreover,HSP70 functions as a molecular chaperone,facilitating the synthesis,folding,assembly and intracellular transport of many proteins; this ability has been linked to thermotolerance and resistance to toxicants[11,12].To examine the stress response of paratroops,we investigated the levels of a number of factors.The levels of hormones (cortisol,growth hormone,insulin,pancreatic glucagon,endothelin,angiotonin I and II,aldosterone),activities of antioxidant enzymes [superoxide dismutase (SOD),glutathione peroxidase (GST-Px),glutathione S transferase (GST)],oxidative damage [malondialdehyde (MDA)],tumor necrosis factor alpha (TNF-α),and levels of the main heat stress protein,HSP70,were determined in serum and plasma of paratroops after parachuting and the results were compared with those in paratroops that had not parachuted within the last 6 months.The potential biomedical significance of the observed changes was discussed.
Method
Subjects 118 healthy male paratroopers aged 18～23 years (mean: 20 years)were selected as the subjects.All subjects were in excellent physical condition.Fifty of them,who had not jumped within the last 6 months,served as control.The other 68,were subjected to three parachute jump exercises (once a day for three consecutive days),and served as the test group.
Determination of Environmental Conditions Dry wet and black bulb temperatures were determined.The wind velocity was determined using an anemometer.The temperature and wind velocity were measured six times between 9:00～11:00 a.m.on three consecutive days.
Collection of blood samples Venous blood samples(5 ml) were obtained from both parachuting and control subjects at the same time on the last day of the experiment.Half of each sample was used for collecting sera (not heparinised),while the other half was heparinised for collecting plasma.
Determination of serum hormone levels The concentrations of serum cortisol,growth hormone,insulin,pancreatic glucagon,endothelin,angiotonin I and II ,and aldosterone were determined using the corresponding radioimmune reagent kit (all from the Institute of Northern Biotechnique,Beijing,except for the endothelin reagent kit which was from the Institute of Eastern-Asia Immunotechnique,Beijing).
Activities of SOD,GST-Px,GST and level of MDA The activities of SOD,GST-Px,GST,and the levels of MDA were determined with the corresponding reagent kits (Jiancheng Bioengineering Company,Nanjing).
Levels of TNF-α and HSP70 The concentration of TNF-α was determined using an ELISA Kit (Bangding Biomedical Company,Beijing).Plasma HSP70 was detected using an immunodot blot assay[13,14].Recombinant human HSP70,purified through the expression of the corresponding cDNA in E.coli BL2(DE3) cells using a pET vector as described earlier[15],was used as a standard reference.In this assay,antibodies against human HSP70 which were produced by the immunization of a rabbit with human HSP70 fusion protein in the pET-3c expression vector were used at a dilution of 1:5000[16].Immunoblot analysis was performed with a horseradish peroxidase conjugated goat anti-rabbit immunoglobin G (Sigma) and DAB (3,3-diaminobenzidine tetrahydrochloride).The density of each spot was determined by the absorbance read at 460 nm using a CS-930 Image Analyser (Japan).
Statistical Analysis Statistical analyses were performed using the Statistical Analysis Software (SAS) package[17].
Result
Environmental conditions Temperature and wind velocity data for three consecutive days were collected and are shown in Table 1.No significant differences (P>0.05) were seen between the environmental conditions of the two groups.
Table 1 Environmental conditions of control and parachuting
paratroops(
±s) groupsdry bulb temp.(℃)
wet bulb temp.(℃)
black bulb temp.(℃)
wind velocity(m/s)
control
30.8±1.2
22.8±0.9
34.5±2.5
0～3±1.1
parachuting
31.5±1.4
23.1±1.1
34.7±2.6
0～3±1.2
Differences in serum hormone levels The concentrations of cortisol,growth hormone,insulin,pancreatic glucagon,endothelin,angiotonin I and II,renin and aldosterone from sera of the two groups are listed in Table 2.When compared to the sera of control subjects,the levels of cortisol,pancreatic glucagon,endothelin,angiotonin II,and aldosterone in the sera of parachuting subjects were not statistically different,while the serum concentrations of growth hormone,insulin,angiotonin I and renin activities were significantly higher in the parachuting group.Table 2 Differences in hormone levels in sera of control and
parachuting paratroops(
±s) parameterscontrol
parachuting
cortisol (ng/ml)
104.82±40.84
41.29±31.60
growth hormone (ng/ml)
3.71±2.05
11.09±5.67**
insulin (μIU/ml)
5.73±2.53
4.44±1.94*
pancreatic glucagon (pg/ml)
23.54±4.64
25.65±5.30
endothelin (pg/ml)
19.60±11.54
27.24±15.11
angiotoninⅠ(incubate at 37℃for 1h,ng/ml)
12.01±3.07
15.33±2.65
angiotoninⅠ(ng/ml)
9.00±3.10
6.82±1.63**
renin activities (ng/ml.h)
3.01±2.98
8.51±2.81**
angiotoninⅡ(pg/ml)
239.31±67.29
219.60±50.84
aldosterone (pg/ml)
122.45±33.75
113.11±35.44
Note:*P<0.05,**P<0.01,as compared with the control
Activities of SOD,GST-Px,GST,and levels of MDA The activities of the antioxidant enzymes SOD,GST-Px,GST,and the concentration of MDA in plasma from the two groups are shown in Table 3.No significant difference in the enzyme activities was found between the control and parachuting group.However,significantly higher levels of MDA were found in the sera of parachuting subjects (P<0.05).
Table 3 Activities of SOD,GST-PX and GST,and levels of MDA in
plasma in two groups(
±s) GroupsSOD(NU/ml)
GST-PX(U/ml)
GST(U/ml)
MDA(nM/ml)
control
114.27±38.63
173.73±155.52
784.01±131.39
5.72±0.89
parachuting
125.34±32.93
176.83±153.57
803.34±116.55
8.83±1.27*
Note:*P<0.05,as compared with the control
TNF-α and HSP70 in plasma of parachutists Table 4 summarizes the frequency and differences in the concentrations of TNF-α and HSP70 in the plasma of subjects from the two groups.Plasma HSP70 was detectable in all subjects.Compared to control subjects,there was significantly more HSP70 in the plasma of parachuting subjects (P<0.05).A signi-ficantly greater number of subjects had detectable TNF-α in the parachuting group,and the levels of TNF-α were generally higher in this group (P<0.05).
Table 4 Occurrence and levels of HSP70 and TNF-α in plasma of control and parac
huting paratroops(
±s) GroupsHSP70
TNF-α
N
occurrence(%)
ug/ml
N
occurrence(%)
pg/ml
Control
37
100
5.30±0.80
50
56
67.2±58.3
Parachuting
46
100
6.20±1.70*
68
73.5*
88.4±62.3*
Note:sample number of determinations,*P<0.05,as compared with the control
Discussion
Living cells and organisms from bacteria to humans display a rapid molecular response when they are exposed to adverse environmental conditions.This ubiquitous phenomenon is commonly known as the stress response.The most striking features of this evolutionarily conserved response is the induced synthesis of a set of proteins,the HSPs[18].Several HSP families can be distinguished according to their average apparent molecular mass: HSP100,HSP90,HSP70,HSP60 and the small HSPs[4～6,19].The human stress response also includes the hormone and other biochemical changes.
During parachuting,paratroops must face both physiological and psychological stresses caused by such environmental factors as high ambient temperature,altitude hypoxia,noise,vibration,and by such inherent psychogenic and emotional states as anxiety,fear,feeling of insecurity,and self-imposed stressors[1～3].These conditions can induce the stress response,but the biochemical changes in plasma of parachutists and their possible significance in evaluating their health and selection are still unclear.Therefore,we investigated a number of factors in the plasma and sera of paratroops in an attempt to understand their response to these stresses.
Our results show significant differences in the levels of a number of hormones between parachuting paratroops and a control group of paratroops that have not parachuted for at least six months (Table 2).Although serum levels of cortisol,pancreatic glucagon,endothelin,angiotonin II,and aldosterone were not different between the two groups,the levels of growth hormone,insulin, angiotonin I and renin activities were significantly higher in the parachuting group.Increased levels of serum growth hormone,insulin,angiotonin I and renin activities are the commonest feature of human stress response[20，21].The higher levels of these hormones following parachuting thus indicate that the parachutists are stressed.However,angiotonin I and renin activities increased after parachuting,while angiotonin II did not.It remained to be further investigated whether these changes are due to the short parachuting.
The plasma of subjects in the parachuting group also contained higher concentrations of MDA (Table 3).MDA is generally formed following oxidative damage to membrane lipids,usually from reactive oxygen species which have been implicated as playing a central role in the pathophysiology of clinical infections and other diseases[22].Organisms have developed a complex system not only to avoid contact with these oxidants,but also to defend themselves from injury once oxidants are formed.These strategies include highly specific and effective enzymatic activities such as SOD,GSH-Px,and GST[23].In this study no difference in these enzymatic activities was observed between the parachuting and control groups,showing that their modulation does not appear to play a role in the protection of paratroops from increased oxidative damage during parachuting.The observed stress following parachuting and the increased oxidative stress may serve to promote the measured production and release of TNF-α and HSP70 (Table 4).TNF-α is a proinflammatory cytokine and plays an important regulatory role in a variety of infectious,inflammatory and immune conditions.Its production may further potentiate cellular pro-oxidant release and its interaction with other cytokines may mediate many physiogical and pathophysiogical processes[23,24].However,its precise functions and roles in the parachuting stress response are still unclear.
The higher levels of plasma HSP70 after parachuting mimic those caused by other stresses including environmental,physiological and psychological stresses.The induced synthesis of HSPs may represent an adaptive mechanism since HSP prodution beyond its normal (relatively low) constitutive levels can be induced by increased temperature and/ or exposure to a large number of xenobiotics as a means of protection against both heat and oxidant damages[18].Several studies have demonstrated that the thermosensitivity of cells is altered when the expression of HSP70 is enhanced or reduced.Further,many papers have reported that HSP70 plays an important role in protecting organisms against the damage of other stressors[7].Whether plasma HSP70 can potentially be used as a biomonitor to assess if parachutists are experiencing abnormal stress and to evaluate the health condition and to select paratroops remains to be shown.
In summary,our results show significantly higher levels of a number of markers of the stress response following parachuting.In this stress response,the sensing mechanisms and the signal transduction pathways involved remain to be studied.Future research on the relationship between the differences in these biomarkers and parachutists' performance will be needed in order to establish the significance of these indexes in evaluating the health and in selecting parachutists.
[Acknowledgements：We are particularly grateful to the parachutists who voluntarily accepted to participate in the study and to the many members of the medical personnel of 457 Hospital for their availability and their generous help in the sampling of parachutists.We also thank Dr.D Joanisse(Laval University) for his critical review of our manuscript.]
*Foundation item:supported by National Natural Scientific Foundation of China (39700118)
References
[1]Mellen PF,Sohn SS.Military parachute mishap fatalities: a retrospective study[J].Aviat Space Environ Med,1990,61(12):1149～1152
[2]Pirson J,Verbiest E.A study of some factors influencing military parachute landing injuries[J].Aviat Space Environ Med，1985,56(6):564～567
[3]Owasoyo JO,Neri DF,Lamberth JG.Tyrosine and its potential use as a countermeasure to performance decrement in military sustained operations[J].Aviat Space Environ Med，1992,63(5):364～369
[4]Lindquist S.The heat shock response[J].Annu Rev Biochem，1986,55(4):1151～1191
[5]Lindquist S,Craig EA.The heat～shock proteins[J].Annu Rev Genet，1988, 22(2):631～677
[6]Craig EA.The heat-shock response[J].CRC Crit Biochem，1985,18(3):239～280
[7]Wu TC,Tanguay RM,Wu Y et al.Presence of antibodies to heat stress proteins and its possible significance in workers exposed to high temperature and carbon monoxide[J].Biomed Environ Sci，1996,9(4):370～379
[8]Mizzen LA,Chang C,Garrels JI et al.Identification,characterization,and purification of two mammalian stress proteins present in mitochondria,grP75,a member of the HSP70 family and Hsp58,a homolog of bacterial groE protein[J].J Biol Chem，1989, 264(34):20664～20675
[9]Laszlo A.Evidence for two states of thermotolerance in mammalian cells[J].Int J Hyperthermia，1988, 4(5):513～526
[10]Mosser DD,Bols NC.Relationship between heat shock protein synthesis and thermotolerance in rainbow trout fibroblasts[J].J Com Physiol，1988, 158(4):457～467
[11]Gething MJ,Sambrook J.Protein folding in the cell[J].Nature，1992, 355(6355):33～45
[12]Hightower LE.Heat shock,stress proteins,chaperones,and Proteotoxicity[J].Cell，1991,66(2):191～197
[13]Xu JY,Gorny MK,Zolla-Pazner S.An immuno-dot blot for the detection of antibody to HIV[J].J Immunol Meth,1989,120(2):179～183
[14]Sternberg J and Jeppesen.Dot-blotting-a novel screening assay for antibodies in hybridoma cultures[J].J Immunol Meth，1983,64(1):39～43
[15]Tanguay RM,Wu Y,Khandjian EW.Tissue-specific expression of heat shock stress proteins of the mouse in the absence of stress[J].Dev Genet，1993,14(2):112～118
[16]Carbajal ME,Duband JL,Lettre F et al.Cellular localization of Drosophila 83-kilodalton heat shock protein in normal,heat shocked,and recovering cultured cells with a specific antibody[J].Biolchem Cell Biol,1986,64(8):816～825
[17]SAS Institute.SAS/STAT user guide,release 6.03.Carry[H],NC.SAS Institute Inc
[18]Mager WH,De Kruijff AJJ.Stress-induced transcription activation[J].Microbiol Rev,1995,59(3):506～531
[19]Macario AJL.Heat-shock proteins and molecular chaperones: implications for pathogenesis,diagnostics,and therapeutics[J].Int J Clin Lab Res,1995,25(2):59～70
[20]Zimet GD,Owens R,Dahms W et al.Psychosocial outcome of children evaluated for short stature[J].Arch Pediatr Adolesc Med,1997,151(10):1017～1023
[21]Biolo G,Toigo G,Ciocchi B et al.Metabolic response to injury and sepsis: changes in protein metabolism[J].Nutrition,1997,13(9 Supp 1):52s～57s
[22]Miller RA and Britigan BE.The formation and biological significance of phagocyte-derived oxidants[J].J Invest Med,1995,43(1):39～49
[23]Miller RA,Britigan BE.Role of oxidants in microbial pathophysiology[J].Clin Microbiol Rev,1997,10(1):1～18
[24]Liu JL,Marino MW,Wong G et al.TNF is a potent anti-inflammatory cytokine in autoimmune-mediated demyelination[J].Nature Med，1998,4(1):78～83
Received date:1999-01-11