PURPOSE: The incidence of heatstroke (HS) is not particularly high; however, once it occurs, the consequences are serious. It is reported that calcitonin gene-related peptide (CGRP) is protective against brain injury in HS rats, but detailed molecular mechanisms need to be further investigated. In this study, we further explored whether CGRP inhibited neuronal apoptosis in HS rats via protein kinase A (PKA)/p-cAMP response element-binding protein (p-CREB) pathway. METHODS: We established a HS rat model in a pre-warmed artificial climate chamber with a temperature of (35.5 ± 0.5) °C and a relative humidity of 60% ± 5%. Heatstress was stopped once core body temperature reaches above 41 °C. A total of 25 rats were randomly divided into 5 groups with 5 animals each: control group, HS group, HS+CGRP group, HS+CGRP antagonist (CGRP8-37) group, and HS+CGRP+PKA/p-CREB pathway blocker (H89) group. A bolus injection of CGRP was administered to each rat in HS+CGRP group, CGRP8-37 (antagonist of CGRP) in HS+CGRP8-37 group, and CGRP with H89 in HS+CGRP+H89 group. Electroencephalograms were recorded and the serum concentration of S100B, neuron-specific enolase (NSE), neuron apoptosis, activated caspase-3 and CGRP expression, as well as pathological morphology of brain tissue were detected at 2 h, 6 h, and 24 h after HS in vivo. The expression of PKA, p-CREB, and Bcl-2 in rat neurons were also detected at 2 h after HS in vitro. Exogenous CGRP, CGRP8-37, or H89 were used to determine whether CGRP plays a protective role in brain injury via PKA/p-CREB pathway. The unpaired t-test was used between the 2 samples, and the mean ± SD was used for multiple samples. Double-tailed p < 0.05 was considered statistically significant. RESULTS: Electroencephalogram showed significant alteration of θ (54.50 ± 11.51 vs. 31.30 ± 8.71, F = 6.790, p = 0.005) and α wave (16.60 ± 3.21 vs. 35.40 ± 11.28, F = 4.549, p = 0.020) in HS group compared to the control group 2 h after HS. The results of triphosphate gap terminal labeling (TUNEL) showed that the neuronal apoptosis of HS rats was increased in the cortex (9.67 ± 3.16 vs. 1.80 ± 1.10, F = 11.002, p = 0.001) and hippocampus (15.73 ± 8.92 vs. 2.00 ± 1.00, F = 4.089, p = 0.028), the expression of activated caspase-3 was increased in the cortex (61.76 ± 25.13 vs. 19.57 ± 17.88, F = 5.695, p = 0.009) and hippocampus (58.60 ± 23.30 vs. 17.80 ± 17.62, F = 4.628, p = 0.019); meanwhile the expression of serum NSE (5.77 ± 1.78 vs. 2.35 ± 0.56, F = 5.174, p = 0.013) and S100B (2.86 ± 0.69 vs. 1.35 ± 0.34, F = 10.982, p = 0.001) were increased significantly under HS. Exogenous CGRP decreased the concentrations of NSE and S100B, and activated the expression of caspase-3 (0.41 ± 0.09 vs. 0.23 ± 0.04, F = 32.387, p < 0.001) under HS; while CGRP8-37 increased NSE (3.99 ± 0.47 vs. 2.40 ± 0.50, F = 11.991, p = 0.000) and S100B (2.19 ± 0.43 vs. 1.42 ± 0.30, F = 4.078, p = 0.025), and activated the expression caspase-3 (0.79 ± 0.10 vs. 0.23 ± 0.04, F = 32.387, p < 0.001). For the cell experiment, CGRP increased Bcl-2 (2.01 ± 0.73 vs. 2.15 ± 0.74, F = 8.993, p < 0.001), PKA (0.88 ± 0.08 vs. 0.37 ± 0.14, F = 20.370, p < 0.001), and p-CREB (0.87 ± 0.13 vs. 0.29 ± 0.10, F = 16.759, p < 0.001) levels; while H89, a blocker of the PKA/p-CREB pathway reversed the expression. CONCLUSIONS CGRP can protect against HS-induced neuron apoptosis via PKA/p-CREB pathway and reduce activation of caspase-3 by regulating Bcl-2. Thus CGRP may be a new target for the treatment of brain injury in HS.
Animals ; Rats ; Apoptosis ; Brain Injuries/pathology* ; Calcitonin Gene-Related Peptide/metabolism* ; Caspase 3 ; Isoquinolines ; Proto-Oncogene Proteins c-bcl-2 ; Rats, Sprague-Dawley ; Sulfonamides ; Heat Stroke/pathology*

OBJECTIVETo investigate the protective effect of ulinastatin (UTI) against acute lung injury induced by heatstroke in mice.

METHODSSixty C57/BL6 mice were randomly divided into 6 groups, with 10 mice in each: control group, heatstroke group, UTI pretreatment group, saline pretreatment group, UTI post-treatment group, saline post-treatment group. The control mice were housed at a controlled room temperature of (22∓1) degrees; celsius, and the other groups were placed inside a temperature and humidity controlled chamber pre-set at 37 degrees; celsius and 60%. The two UTI groups were intraperitoneally injected with UTI at 5×10(4) U/kg 10 min before or after heat stress, and the two saline groups were given then equal amounts of saline in the same manner. The core body temperature of mice was monitored by a mercury thermometer every 30 min in the first 1.5 h during heating. The core temperature was measured, then every 15 min until it reached 42.7 degrees; celsius, which was taken as the onset of heatstroke. The animals were allowed to recover passively at ambient temperature for 6 h. The lung histopathological changes, protein concentration in BALF, lung wet/dry weight ratios, lung water content, and pulmonary microvascular permeability were assayed after 6 h of recovery at 37 degrees;celsius.

RESULTSCompared with the control group, the heatstroke model group and two saline groups displayed more severe lung damage and pathological morphology changes, and the lung wet/dry weight ratio, protein concentration in BALF, lung water content and pulmonary microvascular permeability were also significantly increased. These effects were significantly alleviated in UTI treated group. Pretreat ment with UTI significantly prolonged the time to Tc≥42.7 degrees; celsius but had no effect on lung injury induced by heatstroke.

CONCLUSIONUTI can reduce the pulmonary edema and inflammatory exudation in acute lung injury caused by heatstroke.

Acute Lung Injury ; drug therapy ; physiopathology ; Animals ; Body Temperature ; Bronchoalveolar Lavage Fluid ; chemistry ; Edema ; prevention & control ; Glycoproteins ; therapeutic use ; Heat Stroke ; physiopathology ; Lung ; pathology ; Mice ; Mice, Inbred C57BL

The present study is to assess the prophylactic effect of quinacrine (QA) , an anti-malarial drug, against heatstroke in rats. Conscious rats were orally given equal volume normal saline or QA (dissolved in normal saline and final dosage for rats was 4.5, 9.0 and 18 mg x kg(-1)). An hour later rats were put into a warm water circulated hot chamber (41.0 +/- 0.5) degrees C. Rectal temperature (core temperature, T(co)) of rats in hot chamber was continuously monitored by a thermocouple. T(co) and survival time of rats showed that QA pre-treatment postponed the hyperthermia, and increased the survival time of rats in hot chamber. Primary striatum neurons' culture from new born rats was maintained with D-MEM and 10% FBS. After immuno-cytochemistry identification with antibodies against neural specific proteins, culture received 20 micromol x L(-1) QA only for 1 h and followed by 43.0 degrees C heat treatment for another hour, or 20 micromol x L(-1) QA for 1 h followed by 43.0 degrees C heat treatment for another hour. Control culture received heat treatment only. Cultures were labeled with the fluorescent indicator DPH and the relative membrane fluidity of neurons was measured with the help of fluorescent polarized spectrophotometer. [3H] Arachidonic acid (AA) labeled membrane of E. Coli cells was used as substrate to determine cPLA2 activity of neurons. Gas chromatography and mass spectrum were also employed to detect on the level of fatty acids level in rat striatum neurons. Results from cells indicated that inhibition of cPLA2, reduction the release of active fatty acids such as AA, and possibly, stabilization of the cell membrane which was disturbed by hot treatment, may contribute to the mechanism underlying heat protection and heatstroke preventive effects of quinacrine.
Animals ; Cells, Cultured ; Corpus Striatum ; drug effects ; pathology ; Fatty Acids ; metabolism ; Heat Stroke ; metabolism ; physiopathology ; prevention & control ; Hot Temperature ; adverse effects ; Male ; Membrane Fluidity ; drug effects ; Neurons ; enzymology ; metabolism ; physiology ; Phospholipases A2 ; metabolism ; Quinacrine ; pharmacology ; Rats ; Rats, Wistar