| BackgroundHeat stroke (HS) is an acute heat-related illness characterized clinically by severe hyperthermia and central nervous system dysfunction after exposure to high environmental temperatures (classical heat stroke, CHS) or performing strenuous work (exertional heat stroke, EHS). Heat-related morbidity and mortality have profoundly increased over the years with exacerbation of global warming and urban heat island effect worldwide. However, the pathogenesis of HS remains unclear, and no specific treatment is currently available. Therefore, animal models are required to further delineate the pathophysiological mechanisms and test new drugs. The conventional method to establish an animal model of CHS is placing rats into an artificial climate chamber (ACC), which simulates a hot and humid environment, thus promoting HS. The large bulk and expense of ACC limits its application, and many researchers substitute ACC with warm blankets. Animal temperature controller (ATC) is a device with a temperature control system and a warm blanket that could be used to induce HS in animals. However, whether the model established by ATC resembles that of ACC has not been investigated.As to the mechanism of HS, researchers have propounded many theories, such as direct cytotoxic effects of heat, gut-derived endotoxemia, dysfunction of temperature regulation, and abnormal responses to heat stress. Endotoxemia under the condition of heat stress is one of the hotspot in recent years. This theory claims that when the body temperature elevated to a certain extent, the permeability of intestinal mucosa increases, which leads to the leakage of endotoxin from gut cavity to blood circulation. When the immunocytes are activated by endotoxin, they will release a series of inflammatory factors, such as tumor necrosis factor-alpha (TNF-a), interleukin-1 beta (IL-1β) and interferon-gamma (IFN-y). Together with endotoxin, these inflammatory factors will trigger inflammatory cascade reaction, and ultimately lead to the occurrence of HS and its complications. Some studies have found that measures that inhibit or eliminate endotoxin or some inflammatory factors may decrease the morbidity and mortality of HS. These theories constitute the basic skeleton of doctrine of endotoxemia in HS. Gut-derived endotoxemia and inflammatory reaction play a crucial role in the occurrence and development of HS. While both of them have an intimate relation with Kupffer cells (KCs).KCs are the resident macrophages of the liver, adhering to the endothelial lining, and are a critical component of the mononuclear phagocytic system. Under normal state, the endotoxin in portal vein absorbed from gut lumen could almost all be eliminated by KCs. Studies have found that the phagocytosis of KCs is inhibited, while its secretion of cytokines is markedly increased in some diseases, such as ischemia/reperfusion injury of liver, obstructive jaundice, hepatic fibrosis, hemorrhagic shock and pancreatitis, which results in the extension of inflammatory reaction and aggravation of disease. On the contrary, if the secretory function of KCs was inhibited, the inflammatory reaction will be alleviated and the condition will be improved.It is unclear what the relationship between KCs and the occurrence and development of HS is. What change will happen on the phagocytotic and secretory function of KCs under the condition of continuous heat stress and endotoxin stimulation? What is the relation between these alteration and the formation of endotoxemia and the breakout of inflammatory reaction? What is the underlying mechanisms of these alteration? However, all these problems have not been reported. We speculate that there may be some relation between KCs and the occurrence and development of HS. Reasons are as follows:First, the formation of endotoxemia not only has relation with the leakage of endotoxin through intestinal mucosa, but also may have relation with the decreased phagocytotic function of KCs. Therefore, we assume that the phagocytotic function of KCs is weakened, facilitating the leakage of endotoxin from reticuloendothelial system and activating inflammatory cells in blood circulation or tissue, further triggering inflammatory cascade reaction and finally resulting multiple organ inflammatory damage. Second, some studies have found that the serum levels of TNF-α, IL-1β and INF-y are predominately elevated in HS patients. However, the decline of body temperature, even dropped to normal, could not prevent the release of these inflammatory factors. Since KCs have great secretory capability of inflammatory factors, we speculate that phagocytosis of endotoxin by KCs in HS is significantly weakened, but the secretion of TNF-a, IL-1β and INF-y by KCs is markedly stimulated, which promotes the process of inflammation.Salvia miltiorrhiza is a kind of herbs, and has many pharmacological effects. Tanshinone II A is one of its active ingredient, and has the effects of improve blood circulation,anti-microbial, and anti-inflammation. Since tanshinone ⅡA is fat soluble and difficult to dissolve in water, one of its hydrosoluble form, sodium tanshinone ⅡA sulfonate (STS), is extensively used clinically. Studies have found that STS could inhibit the secretory function and alleviate liver injury caused by inflammation. On these grounds, we assume that STS may regulate the function of KCs, namely improving the phagocytosis and inhibiting the secretion.AimsTherefore, the aims of the present study were:(1) to investigate whether ATC resembles ACC in the establishment of a rat model for CHS; (2) to determine whether the function of KCs change in HS; (3) and to determine whether STS could regulate the function of KCs in HS.MethodsAnimal temperature controller resembles artificial climate chamber in the establishment of a rat model for classical heat stroke1. Twenty-four SPF SD rats were randomly assigned to a room temperature control (C-C) group, a conscious heat stroke (HS-C) group, and an anesthetic heat stroke (HS-A) group. The rats in the HS-C and HS-A groups were placed into pre-warmed ACC and ATC (35℃) respectively.2. The systolic blood pressure (SBP, represented by the caudal artery systolic pressure) and core body temperature (Tc, represented by the rectal temperature) were monitored every 5 min until HS onset. The moment at which SBP decreased from its peak value was considered to be the time of onset of HS.3. Blood samples were collected at baseline before the experiments began and at the end of heat exposure. White blood cell (WBC) counts and serum levels of C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-a) and interleukin-1 beta (IL-1β) were measured by ELIS A kits.4. Tissue samples from the brains, lungs, livers, small intestines and kidneys were taken from the rats immediately following the conclusion of HS induction after euthanasia. Lesions in these organs were examined using hematoxylin-eosin (HE) staining.The function alteration of KCs in HS and the effect of STS on function of KCs 1. Fifty-seven SPF SD rats were assigned to 4 groups, namely normal control group (n = 3), HS model group, STS treatment group, and normal saline (NS) treatment group (n= 18, respectively). According to the time after been established model or given drugs, the latter three groups were randomly divided into 6 subgroups respectively, i.e. Oh,2h,6h,12h,24h,48h (n= 3, respectively).2. Rats in NC group were placed in room temperature. Rats in HS, STS and NS groups were established HS model using ATC. After the onset of HS, rats in STS group were given STS (2 mg/100g body weight) through femoral vein injection. While rats in NS group were given equal volume NS.3. At the corresponding time points, rats were injected Indian ink (0.1 ml/100g body weight) through femoral vein after been anesthesia. KCs’phagocytic index was calculated after hepatic tissue section stained by HE staining. Levels of endotoxin in portal and hepatic vein were measured by limulus reagent kits. Peripheral serum levels of TNF-a, IL-1β and IFN-y were measured by ELISA kits. RT-PCR was used to determine the mRNA levels of TNF-a, IL-1β and IFN-y in liver tissue.Statistical analysisData are expressed as the mean± SD. Data from multiple groups were compared using one-way analysis of variance (ANOVA) followed by a least significant difference (LSD) t-test. Mortality between the HS-A and the HS-C group was tested with a Pearson Chi-square test. Statistical significance was defined as P< 0.05. All analyses were performed with SPSS 19.0.ResultsAnimals experimentAnimal temperature controller resembles artificial climate chamber in the establishment of a rat model for classical heat stroke1. After exposed to heat stress, rats’heart and respiratory rate were obviously raised in HS-A and HS-C groups. Skin and mucous membrane of these rats became cyanosis, and secretion of their salivary glands increased. Rats in HS-C group developed depression. Rats in both groups all presented limbs twitch. At the end of the experiment, there were 2 and 1 rat dead, respectively, in HS-C and HS-A groups.2. The onset time of the HS-A group was predominantly shorter than that of the HS-C group (P< 0.01). In addition, the SBP and Tc of the HS-A group at the end of heat stress were significantly lower than those of the HS-C group (P< 0.01).3. HS dramatically elevated the peripheral WBC counts, serum levels of CRP, TNF-α and IL-1β in comparison to the sham-heated C-C group (P< 0.01). The serum levels of CRP, TNF-α and IL-1β in the HS-A group were lower than the levels in the HS-C group (P< 0.01). However, no difference was observed in WBC counts between the two groups.4. The pathological changes of major organs in the HS-A group were similar to those in the HS-C group.The function alteration of KCs in HS and the effect of STS on function of KCs1. Phagocytosis of Indian ink and gut-derived endogenous endotoxin by KCs in HS group was predominantly weakened than NC group (P< 0.01). STS could significantly increase phagocytotic index of KCs, and improve the phagocytosis of endotoxin (P< 0.05). While NS intervention has no difference with HS group (P> 0.05).2. Peripheral serum levels of TNF-α, IL-1β, and IFN-γ, and mRNA levels of TNF-α, IL-1β, and IFN-γ in KCs in HS group were predominantly increased than NC group (P < 0.01). STS treatment could significantly decrease the serum levels of these inflammatory factors and down-regulate the expression of mRNA of these factors (P< 0.05). While NS intervention has no difference with HS group (P> 0.05).ConclusionWe have demonstrated for the first time that ATC resembles ACC in the establishment of a rat model for CHS. The ATC presents a suitable substitute of ACC for future study of the mechanisms in HS, as well as for testing novel therapeutic interventions. Furthermore, we found that KCs’ phagocytotic function was inhibited and its secretory function was activated in HS. While STS could improve the phagocytotic function and inhibit the secretion of inflammatory factors. In conclusion, our study suggests that STS could regulate KCs’ function in HS and can be therapeutic for the treatment of HS. |
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