| I. Background and PurposeHeat stroke is an acute cause the disease, and the characteristics is the high morbidity and high mortality rate. Its’typically symptom is high fever (>40℃), no sweat and disturbance of consciousness. Due to the pathogenesis is unclear, we lack of effective treatments in the clinical. Despite we take active cooling and appropriate measures such as viscera support, its mortality is still high. Epidemiological data showed that during the period of extremely hot, American urban heat stroke incidence is20/10, ten thousand people, its potential high case fatality rate is10%-70%, and in Saudi Arabia, the fatality rate is about50%. Epidemiological investigation is still a lack of system in our country. In military training, it can lead to military personnel downsizing or death. According to incomplete statistics, heat stroke in our country, the case fatality rate is as high as10%15%, and once for the development of heat stroke combined multiple organ dysfunction syndrome (MODS), the case fatality rate can reach more than40%. In recent years, increasingly attention by people, in related research.. The heat stroke pathogenesis is still not entirely clear. The popular theories at home and abroad are:direct damage, hot regulate dysfunction, abnormal heat stress and enterogenous endotoxin. Among them, the heat stress condition of enterogenous endotoxin blood disease is the hot spot in the study of pathogenesis in recent years. Kupffer cells (KC) is fixed macrophages in the liver,2.1%of liver volume, bear the body function of mononuclear macrophages of80%-90%. On the one hand, KC has the function of removing endotoxin (phagocytosis), on the other hand, it can be activated by endotoxin (secretion), produce the active substances such as a large number of cytokines and inflammatory mediators. In animal models of heat stress, the endotoxin of the door in the venous blood obviously increases, but has not found the existence of endotoxin in the artery system. While the liver and serum inflammatory mediators have showed a trend of increase, the important source of local and systemic inflammatory may be contrubited to the KC. Therefore in heat stress animal model, research the changes of KC function and its effect on the prognosis of rats can be further important for KC dysfunction pathological physiological process in the heatstroke. for the prevention and control in the development of severe heatstroke in the new drug targets and a possible new approach for treatment to increase the ferocity of our army in hot environment is important.Ⅱ.Methods1. The establishment of the heat stress model in ratsPrepare and grouping of experimental animals Experimentalanimals prepare SPF male Wistar rats and grouping80(general hospital of guangzhou military area commands wuhan), weight of250～300g, were randomly divided into normal control group (NC),41℃group,42℃group,43℃group,44℃group, each group14rats, and number of each rat in turn. Within20min before hot blow, every10min1core body temperature measurement.Method of heat stress In addition to the normal control group (NC) at room temperature environment, the rest of the experimental group rats were put into preheated to30℃hot climate simulation animal tank, and within30min to high temperature tank temperature is39℃, humidity65%of experimental conditions.At this time, heat stress begin. during the peirod, connect the Powlalr8sp physiological recorder, place the thermocouple into rectal each10min. The rectal core body temperature (Tc), represents the core body temperature, each measuring time of1min.When groups of rats to each preset core body namely,41℃,42℃and43℃,44℃when out in the rat.Fasting ban water during the experiment.Recovery processing In recovery, don’t do special handling, the rats in the ambient temperature of25℃at room temperature environment, free food, water, and put back the original breeding environment continue to monitor the Tc change within8h and72h within the survival situation.2. Phagocytosis test in vivoInject of India ink (1:3diluted saline,0.2ml/only)into the tail vein, and was followed by2min (T1) and12min (T2) orbital blood, the type721spectrophotometer:600nm wavelength, distilled water zero, OD1, OD2metering density values Calculation index K value:K=(Od1-109log OD2)/(T2-T1).400times the liver by eosin staining, microscope vision observation consuming carbon kupffer cells, comparing each density differences. 3.Endotoxin, secretion, liver function testIn the portal vein and peripheral vascular blood, ELLSA detecting endotoxin levels in the blood, the TNF alpha, beta, IL-6, IL-1level changes.4. KC purification in vitroBy in situ perfusion and collagen enzyme preparation into hepatic cells in vitro perfusion method after the suspension, the liver cell suspension centrifugal5min85g (4℃), from the qing to another50ml centrifuge tube, the liquid centrifuge tube to45ml suspension cells,550g centrifugal6min (4℃), remove the supernatant on cells, will precipitate heavy suspension to12ml and fully blending.Another four15ml centrifuge tube,60°, along the wall to join50%respectively percoll3.8ml,25%percoll5ml, cell suspension3ml.800g centrifugal20min (4℃), slowly to the upper liquid absorption, return50%percoll layer liquid culture medium dilution with such as volume,950g centrifuge for10min (4℃). After the last centrifugal kupffer cells (KC) serum-free culture medium heavy precipitation in1000r centrifugal5min after the suspension, to go to qing precipitation, repeat the above for three times.After precipitation with containing20%fetal bovine serum culture medium heavy suspension and into a culture flask. After6h with warm PBS gently wash three times, remove adherent cells.Adherent cells is the high purity of kupffer cells (KC).5. Heat stress of KCNew extraction of KC in accordance with the programmed temperature gradient in the cell culture box are hot crack, respectively, namely, N,41℃,42℃and43℃,44℃, time is set to1h.And according to the detection of endotoxin in the body content, such as thermal stress at the same time to join the proportion of LPS.After using flow cytometry and ELLSA detecting phagocytosis and secretion function respectively.Ⅲ. results1. Result of heat stress model With varying degrees of heat stress, Tc present different characteristic changes of a rat.During the heat stress,41℃and42℃group of bipolar type Tc rise in rats.44℃and43℃and group performance for three phase type Tc rise in rats.From heat stress,41℃and42℃group Tc levels back to normal within1h of rats,43℃and44℃group and appear low Tc, with44℃group is obvious.41℃and42℃group rats death does not appear within72h,43℃and44℃group and different proportions of rats death, with high mortality rate of44℃group (P <0.01). Survival analysis showed that44℃group rats significantly worse prognosis in43℃group rats (P<0.01).2. The results of phagocytosis in vivo The control index K value is (1.516±0.583),41℃index K value is (2.553±0.485),42℃index K value is (2.167±0.469),43℃index K value is (1.624±0.451), and44℃index K value is (0.923±0.351).41℃and42℃group enhanced the phagocytosis than the control group,43℃,44℃group phagocytosis decreased significantly, the K value between groups had significant difference (P<0.05).3. The endotoxin, secretion and the liver function result Control group portal vein endotoxin11.49±0.43U/L, the extra blood is6.35±0.33u/l;IL-beta, IL-6, the content of TNF alpha, peripheral blood endotoxin19.2±4.1pg/ml, the content of IL-1beta, IL-6, TNF alpha is11.1±0.52ng/l,43.34±0.62ng/l,93.15±0.73ng/l;41℃portal vein endotoxin13.32±0.46u/l, the extra blood is9.56±0.55u/l;IL-beta, IL-6, the content of TNF alpha is21.21±0.34ng/l,81.24±0.58ng/l,156.15±0.64ng/l;42℃portal vein endotoxin29.79±0.61u/l, the extra blood is26.98±0.64u/l; IL-beta, IL-6, the content of TNF alpha is52.53±0.55ng/l,200.12±0.61ng/l,200.12±0.61ng/l;43℃portal vein endotoxin63.35±0.72u/l, the extra blood is61.56±0.70u/l; IL-beta, IL-6, the content of TNF alpha is106.43±0.47ng/l,414.24±2.68ng/l,414.84±3.71ng/l;44℃portal vein endotoxin85.16±0.79u/l, the extra blood is86.65±0.75u/l; IL-beta,IL-6,the content of TNF alpha is144.86±0.67ng/l,584.23±3.47ng/l,929.15±6.39ng/l.Groups of endotoxin in the portal vein and peripheral blood endotoxin approaching, each group of endotoxin increases gradually, there were significant differences (P<0.05).4. The result of KC phagocytosis in vitro Using flow cytometry technology, index is expressed as the following figure in Q2/Q2+Q4.Experiment of the control index was48.13±0.44%,41℃group index was88.23±0.36%,42℃group index was80.65±0.42%,43℃group index was50.12±0.31%,44℃group index was27.23±0.42%.The control group, the phagocytosis of41℃and42℃group activation, index increased gradually;43℃group,44℃phagocytosis decreased, index gradually reduced.There were significant differences between groups (P<0.05).5. The result of KC secrtion in vitro Control group IL-beta, IL-6, content of TNF alpha, is5.1±0.49ng/l,17.34±0.55ng/l,43.45±0.53ng/l;41℃group IL-beta, IL-6, content of TNF alpha, is11.98±0.44ng/l,38.24±0.55ng/l,78.98±0.74ng/l;42℃group IL-beta, IL-6, content of TNF alpha, is27.53±0.35ng/l,95.22±0.51ng/l,98,65±0.54ng/l;43℃group IL-beta, IL-6, content of TNF alpha is60.43±0.47ng/l,201.24±0.68ng/l,199.84±0.71ng/l;44℃IL-beta, IL-6, content of TNF alpha is78.86±0.67ng/l,221.23±1.47ng/l,.476.15±3.39ng/l.Groups of inflammatory factor levels increase gradually, the content has a significant difference (P<0.05).IV. conclusionWith the augmentation of the heat stress degree, the bacterial endotoxin in rats was rising, small dose of endotoxin can strengthen the function of mononuclear macrophage system, activate the KC, while high endotoxin content in immunosuppression, KC low phagocytosis, and enhanced secretion.Other research shows that the recovery of low temperature and heat stress period at maximum core TiWenShui pp.47-53positive correlation.The thermal stress at the core body temperature is higher, the deeper the recovery degree of hypothermia. Normal animals, heat receptor stimulation from outside the core body temperature drop and transmitted to the hypothalamus information, make its comprehensive and stimulate the corresponding activities, to maintain the temperature regulating control and prevent the occurrence of fatal hypothermia.Due to the high level of thermal stress in rats to this lack of ability, and therefore cannot recover from autonomy at low temperature, become not controlled hypothermia. Because there are many complications after heat stress, can be detected in the process of heat stress to restore the rise of a variety of cytokines, we believe that the occurrence of hypothermia may be associated with the rise of cytokines, and production of cytokines and the function of the KC change, therefore, the function of the KC change for thermal recovery of low temperature stress in rats and prognosis is influential. |
PDF Full Text Request