| BackgroundHeat stroke (HS), which results from exposure to a high environmental temperature (classic heat stroke, CHS) or strenuous exercise (exertional heat stroke, EHS), is characterized by a Tc greater than 40℃ and neurologic abnormalities. CHS and EHS can lead to multiple organ dysfunction syndrome, which is associated with a systemic inflammatory response and disseminated intravascular coagulation (DIC). The mortality rate in HS patients is increasing, and approximately 30% of survivors experience permanent deficits in neurological and peripheral tissue function. A recent study found that the majority of CHS reactions could be mimicked by exposing anesthetized rats to a high Ta. In these rats, arterial hypotension, hyperpyrexia, a hypercoagulable state, activated inflammation and tissue injury occurred during HS. Although more research has been undertaken in recent years, specific and effective therapies for HS remain lacking.Heat stroke and its progression to multiorgan-dysfunction syndrome are due to a complex interplay. Recently, studies have demonstrated that hyperpyrexia lead to systemic response among the inflammatory and coagulation responses. So, systemic inflammatory response plays an important role, and endothelial cell injury and coagulation disorder are key factor in the pathophysiology of HS. Some theory deem that intestinal mucosal permeability to endotoxin is increased, and this alteration allows leakage of endotoxins and increases production of inflammatory cytokines in heat stroke. The endothelium, which covering the entire luminal surface of blood vessels, normally performs a variety of roles concerned with vascular homeostasis. This is achieved through the regulation of vascular tone, blood flow, coagulation, the traffic of leukocytes. Recent studies suggest that endothelial-cell can induce significant apoptosis during acute-phase response to heat stress, and apoptotic endothelial cells exhibit disordered coagulation because of the loss of anticoagulant membrane components, which subsequently leads to procoagulant activation, results in multiorgan dysfunction syndrome in HS finally.Sodium tanshinone ⅡA sulfonate is a water-soluble derivative of tanshinone IIA. The latter is one of the major lipophilic components extracted from the dry root or rhizome of Salvia miltiorrhiza Bge, known as ’Danshen’ in traditional Chinese medicine. Tanshinone IIA has poor water solubility; therefore, STS was developed to increase the bioavailability and has been used successfully to treat patients with cardiovascular disorders. STS has been reported to has a wide range of pharmacological activities, such as anti-inflammatory properties, antioxidant capacity, and the ability to inhibit apoptosis. Moreover, STS has been shown to depress cardiomyocyte hypertrophy and protect human vascular endothelial cells in vivo. Therefore, it is likely that HS-induced inflammatory and endothelial cell injuries could be reduced by STS treatment. In fact, proinflammatory cytokines and endothelial cell injury have been shown to initiate coagulation disorders and ultimately lead to DIC in HS. It is likely that HS-induced DIC and multiple organ damage could also be reduced by STS treatment. To evaluate this hypothesis, following STS administration into the femoral veins of rats at the time of onset of HS, we assessed the therapeutic effects of STS on inflammation, aortic endothelial cell apoptosis, DIC and multiple organ damage.AimTherefore, the aims of the present study were:(1) to investigate whether STS treatment after HS could improve the inflammation; (2) to determine whether STS treatment after HS could attenuate aortic endothelial cell damage; (3) and to determine whether STS treatment after HS could ameliorate DIC and multiple organ damage.MethodsAnimals and Induction of heat strokeAdult male Sprague-Dawley rats weighing 200 to 260 g were obtained from the Animal Resource Center of Hubei province (Wuhan, China). The animals were housed individually at an ambient temperature (Ta) of 25 ± 1℃, a relative humidity of 50 ± 10%, and a 12-hour light/dark cycle for one week prior to the start of the experiments. Pelleted rat chow and tap water were provided ad libitum. The rats were anesthetized by intraperitoneal injections of 3% chloral hydrate (3 ml/kg). The anesthetic administration was completed when the corneal reflex and pain reflexes induced by tail pinch were abolished in the rats. Adequate anesthesia was maintained throughout the course of all the experiments. HS was induced by placing the rats in a warm blanket in an animal temperature controller preset to 35℃. The core body temperature (Tc, represented by the rectal temperature) of rats was monitored every 5 min after the administration of anesthesia until HS onset. At the moment at which the Tc reached 43.5℃, which was considered the time of onset of HS.Experimental designThe rats were randomly divided into the following groups. In the normothermic control (NC) group,8 rats were placed into a temperature-controlled room (26℃) after the administration of anesthesia, and the rats were maintained in that room throughout the entire experiment. In the heat stroke (HS) groups, the core body temperature (Tc, represented by the rectal temperature) of 32 rats was monitored every 5 min after the administration of anesthesia until HS onset. At the moment at which the Tc reached 43.5℃, which was considered the time of onset of HS, the rats were removed from the ATC and allowed to recover at 26℃ for 0 h,2 h,6 h or 12 h (HS-0, HS-2, HS-6, and HS-12 groups, respectively). In the STS-treated heat stroke (STS-HS) groups,32 rats were exposed to the same heat treatment described above. Immediately after the onset of HS, the rats received intravenous injections of STS (5,10,20 or 40 mg per kilogram of body weight in groups STS-5, STS-10, STS-20, and STS-40, respectively) via the femoral vein and were the placed at a temperature of 26℃ to recover for 6 h.Sample collection and measurementWhole blood was obtained from abdominal aorta. The serum levels of TNF-a, IL-6, and IL-1β were measured using commercially available ELISA kits and according to manufacture’s instructions. The serum levels of Cr, BUN, ALT, AST, ALP, and LDH were determined with an automatic biochemical analyzer. The plasma levels of PT, aPTT, and D-dimer were measured using automated coagulation instruments. The platelet count was determined by automated blood cell counting instruments.Tissues specimens from livers, kidneys, adrenal glands, small intestines, spleens, and lungs were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 4μm thickness, and stained with hematoxylin and eosin (H&S), and imaged by a microscope with digital camera system. For the detection and quantification of apoptotic endothelial cells in aortic sections, we used terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technology in combination with immunofluorescence for CD31.Statistical analysisData are expressed as mean±SD. The statistical analysis of the data was conducted using the one-way analysis of variance (ANOVA) followed by LSD post hoc test (equal variances) or Dunnett’s T3 post hoc test (unequal variances). Statistical significance was defined as P<0.05 or P<0.01. All analyses were performed with SPSS 19.0.ResultsSTS attenuated the levels of TNF-a, IL-1β and IL-6 during HSThe serum levels of TNF-a, IL-1β and IL-6 in the NC group, HS groups, and STS-HS groups showed obvious differences. The serum levels of TNF-a, IL-1β and IL-6 in all the HS groups (HS-0, HS-2, HS-6, and HS-12 groups) were significantly higher than those of the NC group. The serum levels of TNF-a, IL-1β and IL-6 in the HS-6 group were higher than those of the other HS groups. Treatment with STS (5-40 mg/kg, i.v.) significantly attenuated the increased serum levels of IL-1β, TNF-a and IL-6 after HS following recovery for 6 h. Moreover, the serum levels of IL-1β, TNF-a and IL-6 were maintained at an extremely low level in the rats treated with STS at 40 mg/kg (P<0.05).STS attenuated the numbers of apoptotic aortic endothelial cells during HSThe numbers of apoptotic aortic endothelial cells in the NC group, HS groups, and STS-HS groups showed obvious differences. The numbers of apoptotic aortic endothelial cells in all the HS groups (HS-0, HS-2, HS-6, and HS-12 groups) were significantly higher than those of the NC group. The numbers of apoptotic aortic endothelial cells in the HS-6 group were higher than those of the other HS groups. Treatment with STS (5-40 mg/kg, i.v.) significantly attenuated the increased number of apoptotic aortic endothelial cells after HS following recovery for 6 h. Moreover, the number of apoptotic aortic endothelial cells was maintained at an extremely low level in the rats treated with STS at 40 mg/kg. (P<0.05)STS improved aortic endothelium-dependent vasodilation during HSConcentration-dependent vasodilation in response toAch was significantly decreased in HS-6 group compared with NC group. STS (40 mg/kg) treatment of the HS-6 group appreciably improved the vascular endothelium-dependent relaxation in response to Ach compared with the HS-6 group. (P<0.01)STS attenuated DIC during HSThe plasma PT, aPTT, platelet count and D-dimer levels in the NC group, HS-6 group, and STS-6-40 group (40 mg/kg STS-treated heat stroke group with recovery for 6 h) showed obvious differences. The plasma PT, aPTT, and D-dimer values in the HS-6 group were significantly higher than those of the NC group. In contrast, the platelet count was significantly lower in the HS-6 group than in the NC group. In addition, STS (40 mg/kg) treatment of the HS-6 group appreciably attenuated the HS-induced increased plasma levels of PT, aPTT, and D-dimer and the decreased platelet count. (P<0.01)The histopathologic findings showed DIC in vital organs of the HS-6 group. Intravascular thrombus formation was observed in small- and medium-sized blood vessels, and interstitial space hemorrhage was observed in the lungs, kidneys, adrenal glands and livers. However, the hemorrhage and thrombosis were significantly alleviated in the STS-6-40 group.STS attenuated multiple organ damage during HSThe serum levels of ALT, AST, ALP, BUN, Cr, and LDH in the NC, HS-6, and STS-6-40 groups showed obvious differences. The serum levels of ALT, AST, ALP, BUN, Cr, and LDH in the HS-6 group were significantly higher than in the NC group. However, STS (40 mg/kg) treatment significantly attenuated the HS-induced (recovery for 6 h) increased serum levels of ALT, AST, ALP, BUN, Cr, and LDH (P<0.01).The histopathologic findings revealed unremarkable damage to the liver, spleen, lung, kidney, and small intestines in the NC group. The damage observed in the major organs of the HS-6 group was extensive; however, a significant decrease in this damage was observed in the STS-6-40 group. The injury to the liver was multifocal in the HS-6 group, including hepatocellular architecture disruption, hepatic cell degenerative changes, hepatic sinusoid congestion and/or hemorrhage, thrombi, and increased inflammatory cells. The architecture of spleen tissues had become disordered, and the boundary between the red and white pulp was unclear in the HS-6 group. Moreover, the cellularities of the periarterial lymphatic sheath and the marginal zone were also indistinct, although there was extravasated blood in the marginal zone. The lung tissues in the HS-6 group exhibited substantial morphological changes, including pulmonary edema, alveolar collapse, inflammatory cell infiltration, and extensively thickened pulmonary alveolar septa. Furthermore, vascular congestion, hemorrhage, and thrombosis were observed. The kidney injury manifested as tubular epithelial cell edema, hemorrhage, thrombosis, and inflammatory cell infiltration in the HS-6 group. In the small intestines, the bowel walls were swollen and thickened, and the villous architecture exhibited disorder, degeneration, capillary exposure, congestion, and inflammatory cell infiltration. The pathologic impairments of the liver, spleen, lung, kidney and small intestines were significantly alleviated in the STS-6-40 group.ConclusionWe showed that HS induced increased inflammatory mediators, aortic endothelial cell damage, DIC and multiple organ damage in an experimental rat model. STS treatment after HS improved the inflammation, aortic endothelial cell damage, DIC and multiple organ damage. |
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