The QHBDY was prepared by the Chinese medicine laboratory of The Third Xiangya Hospital of Central South University (Changsha, China). The caspase-3 colorimetric assay kit and Annexin V-FITC and propidium iodide (PI) apoptosis assay kit were purchased from Kaiji Biotech (Nanjing, China). The antibody against caspase-3 was obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-Hsp70 antibody was purchased from Seajet Scientific Inc. (Beijing, China). The anti-GAPDH antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The cell culture media and reagents were obtained from Invitrogen (Carlsbad, CA, USA). The IEC-18 cells were from American Type Culture Collection (ATCC; Baltimore, MD, USA). The Sprague Dawley (SD) rats were provided by the Experimental Animal Center of The Third Xiangya Hospital of Central South University. Both normal and burn serum were collected from the rat heart.

The animal experiments were approved by the Institutional Animal Care and Use Committee of Central South University. The methods for the model construction of severely burned rats were previously described by Walker and Mason (13). Briefly, 136 healthy SD rats weighing 180–220 g were divided into three groups randomly: normal group (n=8), burned group (n=32) and treatment group (n=96). According to the treatment dosage of QHBDY, the treatment group was subdivided into groups of 0.5 ml/100 g, 1 ml/100 g or 1.5 ml/100 g (n=32 for each group). Under anesthesia, the nude skin was scalded with a 97°C water bath for 18 seconds to cause a 3rd degree burn encompassing 30% total body surface area (TBSA). The rats were resuscitated with an intraperitoneal injection of Ringer’s lactate in the volume of 4 ml/kg/1% TBSA, placed in individual cages and allowed free access to food and water. The normal group had the same procedure with the exception that the water temperature was 37°C. The rats in treatment group were given 1.0 g/l QHBDY by oral gavage twice within 24 h before burning. After burning, 1.0 g/l QHBDY was administered again within 5 minutes. The burn area calculation and control was performed according to the rat’s total body surface area with the formula: Area (cm²) = K × W^2/3, W is the weight (g), K=10. The desired burn area was 30% of the TBSA.

IEC-18 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO₂. Serum and QHBDY were diluted with 1% saline solution (vol/vol) into appropriate concentrations, which were then added to treat cells for 24 h

Monolayers of IEC-18 cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed by ice-cold lysis buffer [150 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 50 mM sodium fluoride, 0.2% SDS, 100 mM sodium vanadate and 1 mM phenylmethylsulfonyl fluoride]. After 30–60 min on ice, lysates were cleared of cellular debris by centrifugation (13,000 × g) for 1 min at 4°C. Protein (50 μg; determined by the Bradford protein assay) was diluted in 5X SDS-PAGE sample buffer [150 mM Tris base (pH 6.8), 30% glycerol, 4% SDS, 7.5 mM dithiothreitol (DTT) and 0.01% bromophenol blue] and separated on 10% SDS polyacrylamide gel. Following electrophoresis, the separated proteins were transferred to nitrocellulose (NC) membranes. The membranes were incubated overnight in 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 to saturate the nonspecific binding sites. The membranes were then incubated with anti-Hsp70 primary antibody (dilution, 1:400) for 1 h at 37°C. The protein bands were visualized using horseradish peroxidase-conjugated secondary antibody (Goat Anti-Rabbit IgG, Santa Cruz) with a chemiluminescence-based detection system (ECL western blotting kit; Pierce Biotechnology, Inc. Rockford, IL, USA). For molecular weight determinations, multicolored protein markers were used. To verify equal protein loading, membranes were stripped and reprobed with anti-GAPDH monoclonal antibody (dilution, 1:1,000). The intensity of protein bands was quantified using a ScanJet 4C Flatbed Scanner (Hewlett-Packard, Palo Alto, CA, USA) with NIH Image v1.52 software (http://rsb.info.nih.gov/nih-image/). Lightly exposed films were used for quantification.

The tissues from the small intestine were fixed in 10% paraformaldehyde solution, then embedded in paraffin or frozen. The sections (4-mm thick) were cut from tissue blocks and mounted on slides, then sections were baked at 60–65°C for 4 h. The slides were incubated in xylene and graded ethanol to remove the paraffin. Antigen retrieval was performed with a high temperature and high pressure citrate buffer. Goat serum was used to block nonspecific staining and H₂O₂ to quench endogenous peroxidase activity. The slides were then incubated with primary antibody (caspase-3, 1:500; Hsp70, 1:500) for 1 h at 37°C. After washing with PBS, the secondary antibody (Goat Anti Rabbit IgG-HRP, Maixin, Inc., Fuzhou, China) was added and the slides were incubated at 37°C for 1 h. The staining was visualized using a DAB staining kit (Maixin, Inc.) according to the manufacturer’s instructions and samples were counterstained with hematoxylin and eosin (H&E) before viewing under a microscope (Nikon, Tokyo, Japan). Under high-magnification microscopy, five visual fields were randomly selected to assess the optical density of positively immunostained cells. The average gray value was then calculated for quantitative analysis.

The tissue samples were fixed in 10% paraformaldehyde solution for 24 h, then embedded in paraffin. The paraffin-embedded tissues were cut into 4-μm sections. TUNEL assays were performed using the In Situ Cell Death Detection kit (Kaiji Biotech) according to the manufacturer’s instructions. The number of apoptotic cells was counted under an optical microscope.

The caspase-3 activity assay was performed using the caspase-3 activity assay kit according to the manufacturer’s instructions.

For measuring the apoptosis rate, cells were dual-stained with PI and Alexa Fluor 488-Annexin V using an Annexin V-FITC and propidium iodide (PI) apoptosis assay kit (Kaiji Biological Inc.) according to the manufacturer’s instructions. The stained cells were analyzed by FCM (FC 500 MPL system; Beckman Coulter, Inc., Miami, FL, USA).

The data were analyzed for statistical significance using a Student’s t-test. P<0.05 was considered to indicate a statistically significant result.

The burned SD rats in the three treatment groups were treated with QHBDY at dosages of 0.5 ml/100 g (QA group), 1 ml/100 g (QB group) and 1.5 ml/100 g (QC group), and the apoptosis rate of the mucosa of the small intestine was observed at time points of 6, 12, 24 and 48 h (Fig. 1). As shown in Fig. 2, the apoptosis rates in the treatment groups were lower than in the burned group (B group), and the apoptosis rates in the 1 ml/100 g and 1.5 ml/100 g groups were statistically different from that of the burned group (P<0.05).

The burned SD rats in three treatment groups were treated with QHBDY at dosages of 0.5, 1 and 1.5 ml/100 g. Tissues were collected from the small intestine following treatment times of 6, 12, 24 and 48 h for immunohistochemical staining (Fig. 3). The expression level of Hsp70 protein was measured using the analysis software of the microscope. As shown in Fig. 4, there was a statistical difference in the level of Hsp70 protein expression between the 1.5 ml/100 g treatment group and the burned group (P<0.05) at 6, 12, 24 and 48 h. However, no differences were identified between the other two treatment groups and the burn group.

At 6, 12, 24 and 48 h after the treatment of the burned SD rats with QHBDY at three different dosages, the tissues were collected from the small intestine to investigate the effect of QHBDY on caspase-3 expression (Fig. 5). The results show that QHBDY at the dosage of 1.5 ml/100 g was able to decrease the expression of caspase-3 following treatment for 6, 12, 24 and 48 h compared with that in the burned group (Fig. 6).

In order to study the effect of burn serum on caspase-3 protein activity, burn serum was added to IEC-18 cells for 24 h at three different concentrations (1:150, 1:100 and 1:80), or normal serum (1:100) was added to IEC-18 cells as a control. As shown in Fig. 7, following treatment with burn serum, the relative activity of caspase-3 protein increased; the relative activity of caspase-3 protein in the normal serum group was 2.48±0.63, but in the three burn serum treatment groups, the relative caspase-3 activities were 3.34±0.79, 6.40±1.75 and 9.45±2.56 at 1:150, 1:100 and 1:80 concentrations, respectively. There were statistically significant differences between the three burn serum treatment groups and the normal serum treatment group, and the relative caspase-3 activity in the 1:80 burn serum treatment group was the most elevated compared with that in the normal serum group (P<0.01).

The cell apoptosis rates in the four groups of cells were compared by FCM. Fig. 8 shows that following treatment with burn serum, the cell apoptosis rate increased; the cell apoptosis rate in the normal serum group was 7.75±1.85%, but in three burn serum treatment groups, the cell apoptosis rates were 8.64±2.36, 10.7±2.86 and 15.89±3.98% at 1:150, 1:100 and 1:80 concentrations, respectively. The cell apoptosis rates in the 1:100 burn serum treatment groups were significantly different from that in the normal serum treatment group (P<0.05), and the difference was most evident between the 1:80 burn serum treatment and normal serum groups (P<0.01).

Three different concentrations of QHBDY (1:100, 1:80 and 1:60) were used to treat IEC-18 cells for 24 h, and 1% saline solution was selected as control. In this experiment, the results indicate that QHBDY was able to decrease the relative activity of caspase-3. There was a statistically significant difference in caspase-3 activity between the 1:60 treatment group and the control (P<0.05; Fig. 9).

As shown in Fig. 10, treatment with QHBDY decreased the IEC-18 cell apoptosis rates; the cell apoptosis rate in the 1:80 and 1:60 treatment groups were statistically significantly different from the control (P<0.05).

According to the experimental results of caspase-3 relative activity and IEC-18 apoptosis rate, burn serum at a concentration of 1:80 and QHBDY at a concentration of 1:60 were selected to treat IEC-18 cells together for 24 h. The results show that compared with the burn serum treatment group, the caspase-3 relative activity in the co-treatment group decreased and there was a statistically significant difference between the two groups (P<0.05; Fig. 11). The cell apoptosis rate was also statistically different between the co-treatment group and the burn serum treatment group (P<0.05); the cell apoptosis rate in the co-treatment group was lower than that in the burn serum treatment group (Fig. 12).

After treatment with burn serum and QHBDY for 24 h, the IEC-18 cells were collected and protein was extracted in order to perform western blot analysis. GAPDH was used for quantification. The western blot images are shown in Fig. 13. The relative optical density of Hsp70 was 0.29±0.02 in the burn serum group and 0.39±0.03 in the co-treatment group. There was a statistically significant difference between the two groups (P<0.05; Fig 14).