| Background and objectivesStress is a systemic non-specific adaptative reaction induced by internal and external environmental factors, society and psychological factors threats. Military stress means the reactions to extremely bad living environments, over- load mind tension, and heavy work load when soldiers perform military command, which includes the psychological or physiological changes. Moderate stress usually can activate thinking, and improve soldiers'alertness and working effectiveness. It is called military stress disturbance when stress pressure change soldier's cognition, mood, and behavior, and cut down soldier's working effectiveness.The heart is one of the primary target organs of chronic stress and mental strains. It has been reported that many types of highly pathogenic heart disease are closely related to stress. Besides various stress hormones, the production, and the release of inflammatory cytokines link the stress with heart disease.It is well known that Toll like receptors (TLRs) are membrane receptors and play an important role in infection defence. It could be activated by danger signals such as stress, injury and necrosis, etc. They are widely expressed in many organs including lung, heart, brain, and etc. TLR4 is mainly expressed in heart. TLR4 activation can transduce signal into intra-cellular and activate NF-?B with the help of a series of enzymes, then, NF-?B enters nucleus and induces definitive gene experssions which were responsible for the production of inflammatory cytokines.To our knowledge, the role of TLR4/NF-?B pathway in heart from chronic stress is not clearly clarified yet. Therefore, this study was designed to establish a chronic stress rat model, to observe the effect of myocardial injury induced by chronic stress, to measure the relationship between TLR4/NF-?B signal pathway and myocardial injury. Further, a specific NF-?B antagonist NAC was used to investigate the related effects when NF-?B was arrest. Based on these findings, we expected to illuminate the role of TLR4/NF-?B in chronic stress and provide some experimental evidences for prevention of heart injury.Materials and methodsPart 1: The effect of acute military stress on soldiers'cardiovascular systemSelected 64 healthy male soldiers, aged from 18 - 20 years old as experiment group, meanwhile, selected 10 healthy soldiers in corresponding periods as control group. Firstly, soldiers were required to write SCL-90 scale. Then other experimental indices including BP, HR, EKG, were tested. All soldiers were required to finish three kilometer cross-country armed runing, and then were required to finish 100 simple mathematic questions within five minutes. After training, all these experimental indices were re-tested. Meanwhile, all soldiers were asked to re-write SCL-90 scale, and all soldiers'plasma blood routine, C-RP, ACTH, myocardial enzyme activation, corticosterone, TNF-?and IL-6 were measured. Control group did not experience the stress.Part 2: The study on rat myocardial injury induced by chronic stress and the prevention effect of NACSpecific pathogen-free male Sprague Dawley rats, aged 6–7 weeks, weighed 180–220 g were used in our experiment. The rats were randomly divided into seven groups including a control; 1-day, 1-, 2-, 3-, 4-week stress; 2-week stress plus NAC groups (n=8 in per group).The rats in stress groups were stressed by restraint for 6h plus forced swimming for 15 min each day. NAC in a dose of 150mg/kg/d was given by irrigation for 2 weeks. Rats were weighed every 3 days and were observed for behavior changes every day. The rats in the different groups were anesthetized by an intraperitoneal injection of 10% ketamine (0.1 ml/100 g) and then killed by cervical dislocation on the second day after the last stress. Continuous recording HR, EKG and blood pressur for 5 minutes were performed before rats were sacrificed. The rats in the control group were killed together on the second day after the chronic stress for 4 weeks. The blood sample and the apical region myocardium were collected for later use.Plasma creatine phosphokinase (CK) was measured using a double-antibody sandwich enzyme-linked immunosorbentassay (ELISA) in accordance with the manufacturer's instructions. The activities of plasma lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in the serum samples were measured using DXC800 automatic biochemical analyzer. The myocardial tissue slides were stained with hematoxylin and eosin, and then observed using an optical microscope. Semiquantitative analysis was performed according to the degree of myocardial edema, inflammation, and hemorrhage in corresponding period. The myocardial ultrastructure was examined using a HT7700 transmission electron microscope. Myocardial cell apoptosis were examined by Tunel test, the iNOS expression were detected by immunohistochemistry, respectively.Part 3: The expressions and roles of TLR4 signal pathway in myocardium in a chronic stress rat modelExperimental protocol was the same as part 2. Myocardial TLR4 mRNA were detected by RT-PCR method, myocardial NF-kB DNA binding activity were detected by EMSA method, the contents of TNF-?and IL-6 in myocardial homogenate were measured by ELISA method, and the expressions of TNF-?and IL-6 were detected by immunohistochemistry, respectively. Statistical methods: All the data were expressed as means±SE of the mean (s.e.m). The differences between groups and among groups were examined by t-test and one way analysis of variance (ANOVA), respectively. A p value of less than 0.05 was considered statistically significant.ResultsPart 1:Soldiers'heart rate (82.17±15.38 vs 69.70±10.19times/min, P<0.01) increased and DBP (78.90±8.407 vs 4.68±8.68mmHg, P<0.01) elevated after acute military training. C-RP level (2.05±9.36 vs 1.56±0.95 mg/L, P<0.05) increased after stress and were significantiy different from these of control group. Among these, 6 soldiers'EKG showed tachycardia; One soldier's ECG showed atrial premature beats, short bigeminal rhythm and ST segment elevated (V1 lead more than 0.2mV), respectively. The score of SCL-90 scale before and after stress had no significant differences, The level of blood routine, plasma ACTH, myocardial enzymes, corticosterone, TNF-?and IL-6 of experiment group were not significantly different from that of control group.Part 2:1. A rat restraint plus forced swimming chronic stress model was established successfully .The obvious changes of behavior and the decreases of body weight were observed in stress group. The plasma corticosterone level increased gradually from 2 week and was significantly different (80.35±15.63 in 2w,88.64±13.21 in 3w,85.68±26.38 in 4w nmol/L, P<0.05,P<0.05,P<0.05) compared with those in control group (63.04±9.91 nmol/L).2. EKG changes in rat chronic stress model : Among those groups, 3 rats were recorded with supraventricular tachycardia in 1d stress group; 1 rat with supraventricular tachycardia in 4w stress group; 1 rat with premature ventricualr contraction was observed in 3w stress group and 4w stress group, respectively. Furthermore, 1 rat of atrial fibrilation was recorded in 2w stress plus NAC group.3. The changes of myocardial enzymes, pathological and ultrastrcture: The results showed that serum CK was significantly elevated after the stress for one day and one week(595.51±53.78,574.61±50.33 vs 466.95±83.19u/L.P<0.05, P<0.05), The serum LDH activity significantly increased after the stress for 1 week(4132.67±716.99vs1616.80±672.99 u/L,P?0.01). And the AST activity markedly enhanced after the stress exposure for 1 day and 1 week (370.5±125.02, 260.40±98.77 vs 131.6±36.24 u/L. P<0.01, P<0.01), respectively. However, no significant differences could be seen in these myocardial enzymes among the other stress groups and the control group.Significant myocardial swelling, interstitial vascular dilatation and congestion, edema in part of subendocardial regions, and mucoid degeneration in part of endocardium and valves were observed after the chronic stress for 1-3 weeks. After the chronic stress for 2 week, the total myocardial injury score of stress group was higher than that of control group (7.3±1.6 vs 2.7±0.5,p<0.01). Thickened endocardium, degenerated endothelial cells, concentrated nucleus of cardiomyocytes and endothelial cells, hydropic cardiocytes, dissolved and even disappeared matrix components, swollen cytoplasm and mitochondria, thickened subcutaneous connective tissue, and visible lipidoses could be seen at the chronic stress groups for 2-4 weeks.4. The rates of myocardial cell apoptosis in 1d, 1-,2-,3-,4-week stress groups increased, compared with control group ( 27.13%±5.12%, 34.82%±7.98%, 31.51%±15.31%, 21.15%±2.60%, 25.83%±3.77% vs 10.31%±2.01%. P<0.01, P<0.01, P<0.01, P<0.05, P<0.01). The iNOS expression was found in each stress group, and the highest level was found during 1 to 3 weeks.5. After NAC treatment, the pathologic changes of rat myocardial cell improved obviously, only mild degeneration but no other special pathological changes were found in the myocardial cells of NAC treatment group. NAC markedly decreased the total myocardial injury score compared with the 2-week stress group(4.2±0.5 vs 7.3±1.6,p<0.01). Meanwhile, there was a significant difference between NAC plus 2-week stress group and control group (4.2±0.5 vs 2.7±0.5, p<0.01). NAC treatment decreased the rate of myocardial cell apoptosis compared with 2 week stress group, the difference was significant. (16.01%±2.66% vs 31.51%±15.31% , P<0.05).Furthmore, NAC treatment also reduced the iNOS expression compared with that of 2w stress group.Part 3:1. The expression of TIR4 mRNA, the activity of NF-?B and the level of downstream cytokins increased in myocardium of rat chronic stress model. The expression of TLR4 mRNA peaked after the chronic stress for 1 week and persisted for 3 weeks. After that, the expression decreased gradually and restored to a normal level until 4-week post stress exposure. There were significant differences between the normal control group and 1-, 2-, 3-week stress groups(1.15±0.30, 1.10±0.19, 1.18±0.29 vs 0.88±0.10. P<0.05,P<0.05,P<0.05).Compared with the control, the NF-?B DNA-binding activity increased at 1 day stress group. The NF-?B activity was attenuated in 1w stress group, The NF-?B DNA-binding activity was again increased in 2w stress group. Then the activity gradually declined and restored to a normal level after 4-week stress exposure,There were significant differences between the control and 1day and 2-week stress groups(7.75×105±3.31×105, 6.96×105±3.08×105 vs 2.65×105±1.47×105. P<0.01,P<0.01). The levels of myocardial TNF-?and IL-6 gradually elevated after the chronic stress, and they peaked after the chronic stress for 4 week. Also, there were significant differences between the control group and 4-week stress group (IL-6, 2174.28±742.14 vs 1285.14±462.4 pg/g protein,P<0.05. TNF-a, 6.69±1.90 vs 3.80±2.35 ng/g protein, P<0.05). The expressions of TNF-?and IL-6 in myocardial cytoplasm were observed by immunohistochemistry in every stress group2. The NF-?B DNA-binding activity and the level of myocardial IL-6 and TNF-a were decreased by the treatment of NAC significantly (IL-6, 801.42±115.58 vs 1835.95±834.95 pg/g protein, P<0.01. TNF-a, 1.30±0.60 vs 5.48±1.88 ng/g protein, P<0.01). Conclusions1. After training, the soldiers'HR increased, DBP and plasma C-RP elevated. Cardiac arrhythmias such as sinus tachycardia, frequently atrial premature beats and ST segment elevated were also recorded after acute military training stress. Our results implicated that acute military stress could induce soldiers'cardiovascular changes.2. The rat chronic stress model (restraint plus forced swimming) was established successfully. The obvious changes of behavior such as anxiety, lassitude, weakly responsive and the decreased of body weight were observed in the stress groups. The plasma corticosterone level of the stress group increased gradually from 2 week after stress and is significantly different from that of control group.3. The rat plasma myocardial enzyme level elevated, myocardial pathological changes appeared, myocardial cell apoptosis increased, and arrhythmias such as supraventricular tachycardia, premature ventricular beat and atrial fibrilation were observed in chronic stress rats. The results showed that chronic stress could induce myocardial injury.4. The TLR4mRNA expression, NF-?B DNA binding activity, and the level of IL-6 and TNF-?increased in rat myocardium after chronic stress. NAC inhibited NF-?B and associated cytokine signal pathway, decrease the myocardial cell apoptosis. Our results illustrated that the activation of TLR4/NF-?B signal pathway may be an important mechanismn of myocardial cell injury from chronic stress. It is also recommanded that TLR4/NF-?B signal pathway might be a new possible target for the prevention and treatment of myocardial injury induced by chronic stress. |
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