Due to the effects of estrogen on HS (25), 72 male adult (25-week-old) pathogen-free Wistar rats (weighing 250-220 g) purchased from the Experimental Animal Center of the Guangzhou General Hospital of Guangzhou Military Command (Animal Quality Certification No. SCXK2006-0015, Guangzhou, China) were used in this study. The rats were housed in an environment of 25±0.5°C with 35±5% humidity in individual wire hanging cages with a normal light-dark cycle (14:10 h; 06:00-20:00 light cycle and 20:00-06:00 dark cycle) and were allowed free access to food and water. Prior to each experiment, the rats were allowed to adapt to the new living environment for at least 2 weeks. This study was carried out in strict accordance with the guidelines of the National Health and Medical Research Council for the Care and Use of Animals for Experimental Purposes in China. The protocol was approved by the Institutional Animal Care and Use Committee of Guangzhou General Hospital of Guangzhou Military Command.

A rat macrophage cell line (RMa-bm) obtained from ScienCell (Carlsbad, CA, USA) was cultured in macrophage medium (MaM) (Cat. No. 1921) according to the manufacturer’s instructions (ScienCell) and maintained at 37°C in 5% carbon dioxide (CO₂)-enriched air in a cell incubator (HF90; Healforce, Shanghai, China).

After the rats were allowed to acclimatize for 2 weeks at an ambient temperature (25±0.5°C) and humidity (35±5%), the 72 rats were randomly divided into 3 groups. Prior to the induction of HS, 2 groups of rats (n=24/group) were intravenously injected with phosphate-buffered saline (PBS) (4 ml/kg body weight) twice daily for 3 days through the tail vein, and another group (n=24) was intravenously injected with XBJ (4 ml/kg body weight; Chinese medicine accurate character Z20040033; Chase Sun Pharmaceutical Co., Ltd., Tianjin, China) twice daily for 3 days, as previously described (23). For the HS modelestab, the rats were generally anesthetized with pentobarbital sodium (50 mg/kg; Merck KGaA, Darmstadt, Germany) by intraperitoneal injection to abolish corneal and pain reflexes and to minimize their suffering. A trocar (24 G) was cannulated in the right femoral artery of each rat to monitor mean arterial pressure (MAP). The rats in the group pre-treated with PBS (n=24) and the group pre-treated with XBJ (n=24) were placed in an incubator with a stable temperature (40.0±0.5°C) and relative humidity (60±5%); these groups were termed the HS group and XBJ group, respectively. The rats in the group pre-treated with PBS (n=24) were exposed to a temperature of 25±0.5°C with a humidity of 35±5%; this was used as the sham-operated group. A multi-parameter Physiological Monitor (Infinity^® Delta XL; Dräger, Lübeck, Germany) was used to monitor rectal temperature (Tc) at intervals of 10 min. When the MAP decreased by 25 mmHg and the core temperature was >42°C, the rats were withdrawn from the chamber, as previously described (26).

During the preparation of the model of HS, 8 rats from each group were randomly selected at 30, 60 and 90 min for the measurement of Ang II expression. Blood samples were collected through the orbital vein and serum was separated by centrifugation and stored at −80°C. Following blood collection, the chests of the rats were opened immediately and the hearts were excised. Left ventricular myocardial tissue was isolated and weighed. A total of 100 mg myocardial tissues was collected and boiled in 1 ml of acetic acid (0.5 mM) for 10 min. The tissues were cut into sections and homogenized under ice-cold conditions followed by centrifugation (12,000 rpm) at 4°C for 20 min. The supernatants were then collected for analysis. Ang II levels in the myocardial tissue or serum were determined using the rat Ang II ELISA kit (Biorbyt Ltd., Cambridge, UK) according to standard protocols.

The rat macrophages were pre-treated with XBJ at concentrations of 10, 25, or 50 mg/ml for 24 h. Subsequently, LPS (10 mg/ml; Sigma, St. Louis, MO, USA) was added followed by incubation for 24 h. Total cell RNA or protein was harvested according to standard protocols for further analysis.

Total RNA was extracted from the right common carotid arteries of the rats or from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and 5 μg of total RNA were reverse-transcribed into cDNA using M-MLV reverse transcriptase (Clontech, Palo Alto, CA, USA). cDNA was used as the template for RT-qPCR. For rat Arap1, the sense primer was 5′-ccagaaagcgagtactataagctgc-3′ and the antisense primer was 5′-cttaatggaaagtgttggggttgg-3′. Rat GAPDH primers (sense, 5′-acagcaacagggtggtggac-3′ and antisense, 5′-tttgagggtgcagcgaactt-3′) were used as the controls. The RT-qPCR mixture system contained 5 μl SsoFast™ EvaGreen Supermix (Bio-Rad, Hercules, CA, USA), 1 μl of cDNA (diluted in 1:50) and 2 μl of each of the forward and reverse primers (1 μM) to a final volume of 10 μl. The PCR procedure was as follows: 94°C for 4 min; 94°C for 20 sec, 55°C for 30 sec and 72°C for 20 sec; 2 sec for plate reading for 35 cycles; and melting curve from 65 to 95°C. GAPDH was used as the control for normalizing gene expression. Three independent experiments were performed. The data obtained were calculated using the 2^−ΔΔCt method and statistical analysis was carried out as previously described (27), followed by an unpaired sample t-test.

Membrane protein was isolated using the Membrane Protein Extraction kit (Sangon, Shanghai, China) as per the manufacturer’s instructions. Briefly, the cells or tissues (200 mg, cut into small sections) were washed with wash buffer at least 3 times. Subsequently, 1 ml of extract buffer containing DTT (1 μg/ml) was added and homogenized under ice-cold conditions followed by centrifugation (14,000 rpm) at 4°C for 10 min. The supernatants were collected, bathed at 37°C for 10 min, and then centrifuged (13,000 rpm) at room temperature for 5 min. The samples were divided into 2 layers: the upper layer containing cytoplasmic proteins was collected and the bottom layer containing membrane proteins was dissolved in 500 μl of ice-cold sterile water for 5 min at 4°C followed by incubation in a water bath at 37°C for 5 min. Following centrifugation (13,000 rpm) at room temperature for 5 min, the bottom layer was collected and dissolved in ice-cold sterile water again following the above steps. Finally, the membrane extracts were collected and the protein concentration was measured using the BCA kit (Pierce Biotechnology, Inc., Rockford, IL, USA). For SDS-PAGE, a total of 100 μl membrane protein was mixed with 0.9 ml acetone, and incubated under ice-cold conditions for 20 min followed by centrifugation (10,000 rpm) for 20 min. The supernatants were removed, and 100 μl of loading buffer and 2 μl of β-mercaptoethanol were added to dissolve the sediments for SDS-PAGE. Nuclear proteins were extracted using an extraction kit (Sangon) according to the manufacturer’s instructions. Briefly, the cells were lysed in cytoplasmic buffer containing protease inhibitors, mixed and incubated for 15 min at 4°C, followed by centrifugation at 12,000 rpm for 20 min at 4°C. Cell sediments were collected and were resuspended in nuclear buffer for 10 min at 4°C. The sample was then centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant containing nuclear proteins was collected for analysis.

Proteins separated by 12% SDS-PAGE were electrotransferred onto a nitrocellulose membrane (Amersham, Little Chalfont, UK). The membrane was then incubated with 2% non-fat dry milk in Tris-buffered saline (TBS) to block non-specific binding at room temperature for 1 h. Subsequently, the membrane was incubated with the following primary antibodies: rabbit anti-rat AT1 receptor polyclonal IgG (1:1,000) antibody, goat anti-rat Arap1 polyclonal IgG (1:1,000; both from Abcam, Cambridge, UK), goat anti-rat Histone polyclonal IgG (1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) rabbit anti-rat NF-κB p65 polyclonal IgG (1:1,000; Santa Cruz Biotechnology, Inc.), goat anti-rat IκBα and p-IκBα monoclonal IgG (1:2,000; Cell Signaling Technology, Inc., Boston, MA, USA), rabbit anti-rat GAPDH polyclonal IgG and caveolin-1 (1:1,000; Abcam) antibody diluted in blocking buffer overnight at 4°C. Subsequently, the membrane was incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or rabbit anti-goat IgG (Boster Biological Technology Co., Ltd, Wuhan, China) diluted in blocking buffer according to the corresponding primary antibodies for 1 h. Finally, 4-chloro-1-naphthol (4-CN) as an HRP substrate was used for protein visualization. The protein gray intensity was detected by using BandScan 0.5 software (ProZyme, San Leandro, CA, USA).

Data are expressed as the means ± standard deviation (SD). The statistical significance of differences between 2 groups was determined by the Student’s t-test, and differences among multiple groups were determined by one-way ANOVA. A value of P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS version 11.5 software (SPSS Inc., Chicago, IL, USA).

A previous study (12) revealed that arterial hypotension occurs during HS. In our previous data (23), we demonstrated that XBJ alleviated the decrease in MAP during HS in a rat model. Ang II, a type of vasoconstrictor, increases blood pressure (16). In this study, in order to investigate the levels of Ang II in rats during HS and the effects of XBJ on Ang II, we evaluated Ang II levels in serum or myocardial tissue. The results revealed that the serum levels of Ang II in the HS or XBJ groups were markedly increased compared with those in the sham-operated group at 30, 60 and 90 min (p<0.05) (Fig. 1A). However, there was no significant difference in the serum levels of Ang II between the HS and XBJ groups. Furthermore, the Ang II serum levels increased from 30 to 90 min in the HS and XBJ groups in a time-dependent manner (p<0.05), while the Ang II levels remained stable in the sham-operated group (Fig. 1A). Similar results were obtained in the myocardial tissue (Fig. 1B). These data indicated that the Ang II levels were upregulated during HS, and that pre-treatment with XBJ had no obvious effect on the Ang II levels in the rats suffering from HS. Therefore, it is intriguing that the arterial pressure still decreased as shown in our previous study (23) during HS, while the Ang II levels increased. However, our previous data (23) demonstrated that pre-treatment with XBJ still attenuated hypotension. Collectively, our data indicated that XBJ alleviated the decrease in blood pressure and that this decrease was independent of the Ang II levels in the rats suffering from HS.

The Ang II levels were increased during HS; however, blood pressure was still downregulated. We hypothesized that Ang II signal transduction is inhibited during HS. In order to examine our hypothesis, we analyzed the Ang II corresponding receptor, AT1 receptor, which has been suggested to be desensitized by elevated levels of Ang II (16,17,28). Hence, we detected the membrane and cytoplasmic levels of the AT1 receptor in arterial tissue. The results revealed that AT1 receptor membrane expression was decreased by 66.4% in the HS group in comparison with the sham-operated group, while pre-treatment with XBJ increased AT1 receptor membrane expression by 40.3% when compared with the HS group (p=0.0437) (Fig. 2A and B). There was a significant accumulation of cytoplasmic AT1 receptor (3.9-fold increase) in the HS group as compared with the sham-operated group, while pre-treatment with XBJ markedly attentuated this effect (57.4% decrease; p=0.0202) (Fig. 2C and D). These data suggest that pre-treatment with XBJ promotes AT1 receptor surface expression.

Arap1 has been suggested as the most relevant mediator of AT1 receptor trafficking to the cell membrane (20,21); therefore, we wished to investigate whether Arap1 expression is altered during HS and the effects of XBJ on Arap1 expression. The results from RT-qPCR indicated that the Arap1 transcription levels in the HS group were significantly decreased by 77.6% compared with the sham-operated group (Fig. 3A). These results were confirmed by western blot analysis (decrease of 61.4%; p=0.0132) (Fig. 3B and C). This may explain why hypotension still occurs while Ang II levels increase during HS. We also hypothesized that XBJ positively regulates Arap1 expression. As expected, pre-treatment with XBJ attenuated the effects of HS on Arap1 expression, indicating that XBJ accelerated Ang II signal transduction through the upregulation of Arap1.

The main hazard of heat stress is dysregulated intestinal permeability leading to the translocation of bacteria and endotoxins into the circulation, which then results in a dramatic inflammatory response and cytokine secretion (5,6). Pro-inflammatory cytokines have been shown to have an inhibitory effect on Arap1 expression (22). Our previous study demonstrated that pre-treatment with XBJ has an inhibitory effect on pro-inflammatory cytokine secretion in a rat model of HS (23). In this study, we wished to investigate whether XBJ inhibits cytokine expression in an in vitro cell model. Rat macrophages were pre-treated with XBJ at concentrations of 10, 25 and 50 mg/ml, and then stimulated with LPS; the levels of IL-1β or TNFα were then determined. The results revealed that the XBJ concentration of 50 mg/ml resulted in a significant decrease in the IL-1β levels by 33.6% (p=0.0308; Fig. 4A) and in the TNFα levels by 58.2% (p=0.0237; Fig. 4B). However, XBJ at concentrations of 10 and 25 mg/ml had no effect on the release of pro-inflammatory cytokines (p>0.05; Fig. 4).

It has been shown that bacterial LPS regulates the expression of pro-inflammatory cytokines through the NF-κB signaling pathway (29). Thus, we hypothesized that XBJ may modify pro-inflammatory cytokine secretion through the NF-κB signaling pathway. As the concentration of 50 mg/ml of XBJ effectively suppressed the secretion of IL-1β or TNFα, we used this sample to analyze the activation status of the NF-κB signaling pathway. As is already known, NF-κB dimers are inhibited by IκB (α, β or ɛ) in the cytoplasm, while the phosphorylation of IκB results in NF-κB nuclear translocation and activation (30). We detected the phosphorylation status of IκB, and found that pre-treatment with XBJ decreased the phosphorylation levels of IκBα by 69.8% (p=0.0297) [Fig. 5A (top panel) and B]. By contrast, the levels of unphosphorylated IκBα were increased by 2.56-fold (p=0.0142) [Fig. 5A (middle panel) and C]. Additionally, the protein levels of p65, a subunit of NF-κB, were decreased by 76.6% in the nuclear extracts of the XBJ-pre-treated cells upon LPS stimulation (Fig. 5D and E). These results suggest that XBJ inhibits NF-κB signaling pathway transduction induced by LPS.