Lai Fu Cheng Qi decoction was provided by the General Hospital Affiliated to Tianjin Medical University (Tianjin, China). The prescription consisted of 20 g Rheum palmatum L., 15 g Mirabilite, 15 g Magnolia officinalis Rehd et Wils., 10 g Fructus Aurantii, 15 g Semen Raphani, 10 g Radix Aucklandiae and 10 g Achyranthes bidentata. Sodium taurocholate was purchased from Beijing Bioxin Biological Technology Co., Ltd. (Beijing, China). An ATPase detection kit was purchased from Nanjing Jiancheng Biological Engineering Institute (#A070-1; Nanjing, China) and a bicinchoninic acid (BCA) protein assay kit was provided by Beijing Gainingjinnuo Biotechnology Co., Ltd. (Beijing, China). A Tissue Mitochondria Isolation Kit was purchased from the Beyotime Institute of Biotechnology (#C3606; Haimen, China) and a TUNEL in situ cell apoptosis detection kit was purchased from Beijing Dingguochangsheng Biotechnology Co., Ltd. (#TUNEL-A1; Beijing, China). Serum biochemical indexes were detected using a Vitros 250 automatic biochemical analyzer (Johnson & Johnson, Rochester, NY, USA). A Cell Lab Quanta SC (CLQSC) Flow Cytometer was obtained from Beckman Coulter (Miami, FL, USA) and an Olympus CK40 inverted microscope was purchased from Olympus Corporation (Tokyo, Japan).

In total, 30 male Sprague-Dawley rats with a body mass of 200–250 g were provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China). The animals were housed in standard conditions with free access to food and water. All animal experiments were conducted according to the ethical guidelines of Tianjin Medical University, and all efforts were made to minimize animal suffering. The study was approved by the ethics committee of Tianjin Medical University.

The animals were randomly divided into three groups, which included the sham (n=10), SAP (n=10) and the Lai Fu Cheng Qi decoction treatment group (n=10).

SAP was induced in the SAP and decoction treatment groups by a retrograde pancreatic duct injection of 5% sodium taurocholate (1 ml/kg body weight). Briefly, an incision was made in the abdomen at ~1 cm under the xiphoid midline following anesthetization using 10% chloral hydrate (3 ml/kg; China Pharmaceutical Group Shanghai Reagent Company, Shanghai, China). Once the biliopancreatic duct and the duodenal papilla opening was located, a syringe needle was placed (from the intestinal wall contralaterally to the duodenal nipple) into the biliopancreatic duct through the duodenal papillary opening on the cholo-pancreatic duct. After clipping the cholangitic porta hepatis with a small artery clamp, 5% sodium taurocholate was slowly infused (0.1 ml/min) retrogradely into the pancreatic duct. At 5 min after the injection, the syringe and arterial clamp were removed, the incision was sutured, and the abdomen was closed. In the sham group, an incision was made in the abdomen and the pancreatic tissue was marginally rotated several times prior to closing the abdomen. The abdomen was closed without a sodium taurocholate injection.

At 8 and 16 h after model preparation, the rats in the decoction treatment group received an intragastric administration of Lai Fu Cheng Qi decoction (2.5 ml/kg body weight). As controls, the rats in the sham and SAP groups received an equal volume of saline by intragastric administration.

General conditions of the rats following modeling were observed. At 24 h after modeling, three rats in the SAP group and one rat in the decoction treatment group had died. The remaining rats in all three groups were euthanized by intraperitoneal injection of 10% chloral hydrate at 24 h after modeling. The abdomen was opened by surgery, and the pancreas and heart were removed. In addition, blood from the abdominal aorta was collected. The aforementioned procedures were all performed under sterile conditions. The heart tissue was dissected into four sections on ice, and these four sections were used for the detection of Na⁺-K⁺-ATPase activity, mitochondrial membrane potential, myocardial cell apoptosis and pathological examination.

Arterial blood was naturally coagulated at room temperature for 1 h. The blood was subsequently centrifuged at 1006.2 × g for 10 min at 4°C to collect the serum. The CK-MB and LDH concentrations in the serum were detected using the Vitros 250 automatic biochemical analyzer.

One section of the heart tissue was ground into a homogenate, and the Na⁺-K⁺-ATPase activity was detected using a spectrophotometric method, as previously described (15,16). The experiment was conducted according to the instructions provided in the ATPase detection kit. Briefly, protein levels in the heart homogenate were determined using the BCA protein assay kit. The optical density (OD) of the color developed was measured at a wavelength of 562 nm in a spectrophotometer (Beijing Puxi General Instrument Co., Ltd., Beijing, China). The Na⁺-K⁺-ATPase activity was expressed as U/mgprot and was calculated based on the following formula: Na⁺-K⁺-ATPase activity = [(OD of test tube − OD of control tube)/(OD of standard tube − OD of control tube)] × concentration of standard tube (0.02 μmol/ml) × 6 × 7.8/protein level of the homogenate (mgprot/ml).

Using one section of the heart tissue, mitochondria were isolated using the myocardial mitochondria isolation kit and were used to detect the mitochondrial membrane potential. Briefly, the isolated mitochondria were resuspended in 0.5 ml phosphate-buffered saline (Wuhan Boster Biotechnology, Ltd., Wuhan, China), and 10 μl rhodamine 123 working solution (Sigma-Aldrich, St. Louis, MO, USA) was added and incubated with the mitochondria at 37°C in 5% CO₂ for 15 min. Following incubation, the mitochondrial membrane potential was analyzed on a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 633 nm.

A TUNEL assay was performed according to the instructions of the apoptosis detection kit. The nuclei of the apoptotic cells exhibited brown/yellow staining, while those of non-apoptotic cells and the negative control were stained blue. The number of apoptotic cells was counted in each group and the apoptotic index (apoptotic cell number/total cell number × 100%) was calculated.

Pancreatic tissues and one section of the heart tissue were fixed in 4% paraformaldehyde and embedded in paraffin (Beijing Dingguochangsheng Biotechnology Co., Ltd.). The tissues were sliced into sections and stained with HE (Beijing CellChip Biotechnology Co. Ltd., Beijing, China). Briefly, the tissue sections were dewaxed in xylene (Tianjin East Tengen Fine Chemical Reagent Co., Tianjin, China) and rehydrated in graded alcohols. After washing with running water and distilled water, the sections were stained with hematoxylin for 3–5 min. Following a second wash with running water, the sections were differentiated with 1% HCl in 70% alcohol. Subsequently, the sections were stained with eosin for 1–4 min after washing with running water. Following dehydration and differentiation in alcohol, the sections were mounted on cover slips and observed under light microscopy.

The morphology of the pancreas and heart was evaluated by professional pathologists and the severity of the pathological changes was scored according to previous studies (17,18). According to the pathological changes in edema, infiltration of inflammatory cells, hemorrhage and necrosis in the pancreatic tissues, the following pathological scores were proposed: 0, no pathological changes; 1, mild pathological changes; 2, moderate pathological changes; 3, severe pathological changes; and 4, very severe pathological changes. According to the pathological changes with regard to infiltration of inflammatory cells and myocardial necrosis in the heart tissues, the following pathological scores were proposed: 0, no pathological changes; 1, mild pathological changes; 2, moderate pathological changes; 3, severe pathological changes; 4, very severe pathological changes; and 5, extremely severe pathological changes. The pathological scores of the pancreas (maximum, 16) and heart (maximum, 20) were the total pathological scores, inlcuding the scores for edema, infiltration of inflammatory cells, hemorrhage and necrosis.

Data are expressed as the mean ± standard deviation and SPSS statistical software, version 13.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. The mortality rates were analyzed using Fisher’s exact probability for a 2×2 contingency table. Comparisons between groups were analyzed using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Following the induction of SAP in the rats, the general conditions of the rats were observed. In the sham group, the general conditions of the rats prior to and following surgery were good and did not change significantly. However, the general conditions of the rats in the SAP group were relatively poor. Rats in the SAP group exhibited symptoms of piloerection, laziness, depression, lags in responses and shortness of breath. Three rats had died at 24 h after modeling; thus, the mortality rate was 30% in the SAP group. Following treatment with Lai Fu Cheng Qi decoction, the general conditions of the rats in the decoction treatment group were improved. For example, the symptoms of depression and the lags in responses gradually disappeared. Only one rat had died at 24 h after modeling; thus, the mortality rate was 10% in the decoction treatment group. However, there was no statistically significant difference in the morbidity rate between the SAP and decoction treatment groups (data not shown), indicating that the general conditions of the SAP rats were improved by treatment with Lai Fu Cheng Qi decoction.

To determine the effect of Lai Fu Cheng Qi decoction on the serum levels of CK-MB and LDH, the concentrations of CK-MB and LDH in the serum were measured using an automatic biochemical analyzer (Table I). Compared with the sham group, the serum levels of CK-MB and LDH in the SAP and decoction treatment groups increased significantly (P<0.01). However, the levels of CK-MB and LDH in the decoction treatment group were significantly lower compared with those in the SAP group (P<0.01). These results indicated that SAP increased the levels of CK-MB and LDH in the serum; however, this increase was inhibited by administration of Lai Fu Cheng Qi decoction.

To evaluate the effect of Lai Fu Cheng Qi decoction on Na⁺-K⁺-ATPase activity, the Na⁺-K⁺-ATPase activity of the myocardial cells was detected using a spectrophotometric method. As shown in Table II, the Na⁺-K⁺-ATPase activity of the myocardial cells in the SAP group was significantly lower compared with that of the sham group (P<0.01). In addition, the Na⁺-K⁺-ATPase activity in the decoction treatment group was significantly lower compared with that in the sham group (P<0.01). However, the decoction treatment group had a significantly higher level of Na⁺-K⁺-ATPase activity when compared with the SAP group (P<0.01). These results indicated that SAP reduced the Na⁺-K⁺-ATPase activity of myocardial cells, while Lai Fu Cheng Qi decoction was able to reverse the effect of SAP by increasing the Na⁺-K⁺-ATPase activity.

To analyze the effect of Lai Fu Cheng Qi decoction on the mitochondrial membrane potential, mitochondria of the myocardial cells were isolated and the mitochondrial membrane potential was measured by flow cytometric analysis. Representative flow cytometry results are shown in Fig. 1. Cells that did not exhibit a decrease in mitochondrial membrane potential were counted and the percentage was calculated (Table III). In the SAP group, the ratio of cells without a decrease in mitochondrial membrane potential was significantly lower compared with the sham group (P<0.01). Following treatment with Lai Fu Cheng Qi decoction, the ratio of cells without a decrease in mitochondrial membrane potential was significantly higher when compared with the SAP group (P<0.05). Compared with the rats in the sham group, the rats in the decoction treatment group exhibited a significantly lower ratio of cells without a decrease in mitochondrial membrane potential. These results indicated that the mitochondrial membrane potential of myocardial cells decreased following SAP induction, while Lai Fu Cheng Qi decoction was able to reverse the effect of SAP by increasing the mitochondrial membrane potential.

To investigate the effect of Lai Fu Cheng Qi decoction on the rate of myocardial apoptosis in rats with SAP, a TUNEL assay was performed. Representative TUNEL assay results are shown in Fig. 2. The nuclei of the apoptotic cells were stained brown/yellow, while those of non-apoptotic cells and the negative control were stained blue. In the sham group (Fig. 2A), the majority of the nuclei in the myocardial cells were stained blue and only a few myocardial cells exhibited brown/yellow staining in the nuclei. However, in the SAP group, a large number of myocardial cells exhibited brown/yellow staining in the nuclei (Fig. 2B). In addition, the number of cells with brown/yellow staining in the decoction treatment group was higher compared with that in the sham group, whereas the number was lower when compared with the SAP group (Fig. 2C). The number of apoptotic cells was counted in each group and the apoptotic index (apoptotic cell number/total cell number × 100%) was calculated. As shown in Table IV, the apoptosis index in the SAP group was significantly higher when compared with the sham group (P<0.01). Although the apoptosis index of the decoction treatment group was significantly higher compared with that of the sham group (P<0.01), when compared with the SAP group, the apoptosis index of the decoction treatment group decreased significantly (P<0.01). These results indicated that Lai Fu Cheng Qi decoction treatment alleviated the rate of apoptosis induced by SAP in myocardial cells.

To detect the effects of Lai Fu Cheng Qi decoction on the pathological changes to the pancreas and heart, HE staining was performed. Representative HE staining results are shown in Fig. 3. The morphology of the pancreas and heart was evaluated by professional pathologists. The sham group exhibited a normal morphology of the pancreas (Fig. 3A), and no marked pathological changes were observed. By contrast, the SAP group revealed morphological changes of interstitial edema, interstitial hemorrhage, hemorrhage, necrosis and fat necrosis. The necrotic area was infiltrated with inflammatory cells, which consisted primarily of neutrophils, and the lobular structure was destructed. Following treatment with Lai Fu Cheng Qi decoction, the pathological features of the pancreas in the decoction treatment group were found to improve. Similarly, as shown in Fig. 3B, the sham group exhibited a normal morphology of the myocardial tissue and no significant pathological changes were observed. However, in the SAP group, the myocardial fibers were significantly degenerated and swelled. The majority of the myocardial fiber structures had disappeared and a number of the myocardial membranes were broken. The myocardial fibers were arranged disorderly and the blood vessels exhibited congestion. The aforementioned pathological changes were consistent with the pathological features of myocardial injury induced by SAP. Following treatment with Lai Fu Cheng Qi decoction, the pathological features of the heart in the decoction treatment group were improved. However, edema and mild degeneration of the myocardial fibers was observed in the decoction treatment group, and a number of myocardial fibers had lost cross striations.

In addition, the severity of the pathological changes was scored. The pathological scores of the pancreas and heart are shown in Table V. When compared with the sham group, the pathological scores of the pancreas and heart in the SAP and decoction treatment groups were significantly higher (P<0.01). However, following treatment with Lai Fu Cheng Qi decoction, the pathological scores of the pancreas and the heart in the decoction treatment group were significantly lower compared with those in the SAP group (P<0.01).

Collectively, these results indicated that SAP induced injury in the pancreas and the heart; however, these injuries were alleviated following treatment with Lai Fu Cheng Qi decoction.