A total of 100 male C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed under pathogen-free conditions. Mice were maintained in an environment with a 12 h light/dark cycle and free access to food and water. The mouse model of ischemia-reperfusion injury was generated according to a protocol described in a previous study (29). The mice were randomly divided into two groups (n=40 per group). In the first group the animals received 10 mg/kg berberine orally once per day, whilst the second group received the same volume of PBS. The treatment continued for 30 days. On day 30, the myocardial function of the mice was then analyzed to evaluate the efficacy of berberine.

The current study was approved by the Tab of Animal Experimental Ethical Inspection of the General Hospital of the Chinese People's Armed Police Forces (Beijing, China) and performed in accordance with their recommendations. All surgery and experiments were performed under anesthetic to minimize pain.

Myocardial cells were isolated from experimental mice on day 30, in accordance with a previous study (30), and cultured in minimum essential medium with 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Myocardial cells were treated by a promoter of NF-κB (NF-κBPr; 1:800; NEMO; cat no., AF2684; R&D Systems, Inc., Minneapolis, MN, USA) or PI3K inhibitor (PI3KI; 1:500; cat. no., HY-17645; MedChemExpress, Monmouth Junction, NJ, USA) and cultured at 37°C with 5% CO₂ in a humidified atmosphere. Then, the myocardial cells were treated with 10 mg/ml berberine (cat. no., PHR1502; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 24 h to analyze its effect in vitro.

In the protein detection assay, mouse C-reactive protein (CRP; cat. no., MCRP00), procalcitonin (cat. no., DY8350-05) and high mobility group box 1 protein (HMGB1; cat. no., AF1690) (all R&D Systems, Inc.). ELISA kits were used to determine inflammatory responses. The procedures were conducted according to the manufacturer's protocol. The final results were recorded at 450 nm using a microplate reader.

Myocardial cells isolated from berberine treated mice were homogenized in a radioimmunoprecipitation assay buffer containing protease inhibitors (Gibco; Thermo Fisher Scientific, Inc.). Then, the myocardial cells were centrifuged at 2,000 × g at 4°C for 10 min and 2 µg/lane of protein was separeated by a 12% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc). After blocking with 5% skimmed milk for 12 h at 4°C, the polyvinylidene fluoride membranes were incubated with goat anti-mouse primary antibodies directed against the following proteins: Interleukin (IL)-6 (1:2,000; cat. no., ab6672), IL-10 (1:2,000; cat. no., ab34843), NF-κB p65 (1:1,000; cat. no., ab16502), inhibitor of NF-κB kinase subunit β (1:1,000; IKK-β; cat. no., ab3204), NF-κB inhibitor α (1:1,000; IκBα; cat. no., ab309300), apoptotic protease-activating factor 1 (1:2,000; Apaf-1; cat. no., ab32372), caspase-3 (1:2,000; cat. no., ab2172), caspase-9 (1:2,000; cat. no., ab32539), PI3K (1:2,000; cat. no., ab189403), AKT (1:2,000; cat. no., ab8805), Bcl2-associated agonist of cell death (Bad; 1:2,000; cat. no., ab32445), Bcl-2-like protein 1 (Bcl-xl; 1:2,000; cat. no., ab32370), β-actin (1:2,000; cat. no., ab8226) and cellular tumor antigen p53 (p53; 1:2,000; cat. no., ab61241; all Abcam, Cambridge, UK) for 2 h at 37°C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no., HAF010; Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 1 h at 37°C at a 1:5,000 dilution. The results were visualized using an enhanced chemiluminescence detection system (cat. no., D5905-50TAB; Sigma-Aldrich; Merck KGaA). Protein expression was analyzed using BandScan software (version, 5.0; Glyko, Inc.; BioMarin Pharmaceutical, Inc., San Rafael, CA, USA).

Apoptosis rates of the myocardial cells were evaluated using an Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (BD Biosciences, San Jose, CA, USA). Myocardial cells were collected and suspended with Annexin V-FITC and PI according to the manufacturer protocol. Fluorescence was measured with a fluorescence-activated cell sorting flow cytometer using FCS Express™ IVD software (version 4; De Novo Software, Los Angeles, CA, USA).

NF-κB activity in the myocardial cells was analyzed according to a previously described method (31). The heart rates of the experimental mice treated with berberine or PBS were measured using a cardiotachometer on day 0 and 30 after treatment. The results were recorded to analyze the effects of berberine on myocardial function.

Blood samples were collected from experimental animals on day 30. An autohumalyzer (Dimension^® RxL Max^®; Siemens Healthineers, Erlangen, Germany) was used to assess total cholesterol according to the manufacturer's protocol.

The total lipid content from total cholesterol was determined by high-performance liquid chromatography as reported previously (32). In brief, the supernatant (200 µl) of the blood samples were transferred into a fresh 1.5 ml amber microcentrifuge tube and prepared with 12 mM potassium ferricyanide solution in 3.3 M sodium hydroxide to start the thiochrome reaction. The thiochrome reaction quenched by 25 µl of 1M phosphoric acid; the neutralized samples were filtered and analyzed. The samples were kept at 8°C in the autosampler and 50 µl was injected into an Agilent Eclipse Plus C18 (4.6×150 mm, 5 mm; Agilent Technologies, Inc., Santa Clara, CA, USA) column protected by a SecurityGuard C18 (4×3 mm) guard column (Phenomenex, Inc., Torrance, CA, USA) at 40°C. A total of 150 mM potassium phosphate dibasic (solvent A; pH 7.0) and methanol (solvent B) served as the mobile phase at a flow rate of 1.5 ml/min and an 8 min gradient as follows: 0 min (85% solvent A), 1 min (80% solvent A), 3 min (80% solvent A), 6 min (50% solvent A), 7 min (85% solvent A), 8 min (85% solvent A). The samples were analyzed using Agilent 1200 HPLC System equipped with a fluorescence detector and operated by ChemStation Rev. B.02.01 SR1 (both Agilent Technologies, Inc.).

In addition, the percentage of lymphocytes was analyzed by flow cytometric analysis using conjugated antibodies directed against CD8-FITC (cat. no. ANT-282; Prospec-Tany TechnoGene, Ltd., East Brunswick, NJ, USA), CD4-PE (cat. no. A07751; Beckman Coulter, Inc., Brea, CA, USA) and CD3-PC5 (cat. no. A07749; Beckman Coulter, Inc.) (all 1:1,000) at 4°C for 12 h according to a previous study (33). Briefly, peripheral blood mononuclear cells were aseptically separated by density gradient centrifugation (speed, 8,000 × g) at 4°C for 10 min. Cells were blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) at 37°C for 2 h and then incubated with the aforementioned antibodies and washed three times with PBS. The percentage of lymphocytes analyses was performed using a FACSCalibur™ flow cytometer (BD Biosciences) equipped with 488 nm argon laser. A minimum of 10,000 events was acquired and analyzed using BD CellQuest Software (version 3.3; BD Biosciences).

Paraffin-embedded 4-µm-thick sections of myocardial tissue were prepared and epitope retrieval was performed as described previously (34). The paraffin-embedded sections were treated with hydrogen peroxide (3%) for 10–15 min, and were subsequently blocked using 5% skimmed milk powder for 10–15 min at 37°C. Finally, the sections were stained with hemotoxylin and eosin at 4°C for 12 h. All sections were washed three times with PBS. The area of myocardial injury, circumference fragmentation and segmentation of myocardial cells were determined in six random fields through a fluorescence microscope (BZ-9000; Keyence Corporation, Osaka, Japan).

All data are presented as the mean ± standard error of the mean from three independent experiments. Unpaired data was analyzed using a Student's t-test. Data between multiple groups were compared using one-way analysis of variance followed by a post hoc Tukey's range test. P<0.05 was considered to indicate a statistically significant difference using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA).

The effects of berberine on inflammatory responses in a mouse model of ischemia-reperfusion injury were investigated. As shown in Fig. 1A, CRP was upregulated in ischemia-reperfusion injury mice and significantly downregulated by berberine. In addition, berberine significantly decreased the percentage of lymphocytes in the serum of ischemia-reperfusion injury mice compared with the control group (Fig. 1B). Plasma levels of procalcitonin were significantly decreased in mice treated with berberine compared to the control group (Fig. 1C). Furthermore, the expression of HMGB1 was significantly downregulated by berberine in the serum of mice with ischemia-reperfusion injury compared with the control group (Fig. 1D). Collectively, these results suggest that berberine markedly decreases inflammatory responses in the mouse model of ischemia-reperfusion injury.

The molecular mechanisms underlying ischemia-reperfusion injury-induced inflammatory responses were investigated in the myocardial cells of the mouse model of ischemia-reperfusion injury. The results revealed that berberine significantly downregulated the expression of IL-6 and IL-10 in the serum of mice with ischemia-reperfusion injury (Fig. 2A and B). The levels of NF-κB p65, IKK-β and IκBα were decreased by berberine treatment in the myocardial cells of the experimental mice (Fig. 2C). NF-κB activity was also significantly inhibited by berberine compared to the control (Fig. 2D). The stimulation of NF-κB (NF-κBPr) activity significantly blocked berberine-suppressed IL-6 and IL-10 expression in the myocardial cells (Fig. 2E and F). This data indicates that berberine regulates the expression of inflammatory factors through the NF-κB signaling pathway.

The apoptosis rate of myocardial cells in ischemia-reperfusion injury mice and the mechanism of the antiapoptotic effects of berberine were investigated in the present study. Berberine significantly inhibited the apoptosis of myocardial cells induced by ischemia-reperfusion injury compared with the control group (Fig. 3A). Berberine also significantly inhibited the expression of Apaf-1 and cleaved caspase-3 and caspase-9 in myocardial cells compared with the control group (Fig. 3B). In addition, the expression of Bad, Bcl-xl and p53 was upregulated by berberine in the myocardial cells of the mouse model of ischemia-reperfusion injury (Fig. 3C). In addition, the expression of PI3K and AKT was significantly increased by berberine in myocardial cells (Fig. 3D). Furthermore, the inhibition of PI3K significantly inhibited the antiapoptotic effect of berberine on myocardial cells (Fig. 3E). Treatment with the PI3K inhibitor (PI3KI) decreased Bcl-xl and p53 levels in the myocardial cells in vitro (Fig. 3F). These results suggest that berberine inhibits the apoptosis of myocardial cells through the PI3K/AKT-mediated mitochondrial apoptosis signaling pathway.

The benefits of berberine on the myocardial function of experimental mice with ischemia-reperfusion injury were analyzed. The results revealed that the blood lipid content was significantly decreased by berberine in the mouse model of ischemia-reperfusion injury (Fig. 4A). In addition, heart rate was significantly decreased by berberine in mice with ischemia-reperfusion injury (Fig. 4B). Furthermore, the area of myocardial infarction was significantly decreased in the myocardial tissue of mice treated with berberine (Fig. 4C). Additionally, as determined by histological analysis, the circumference fragmentation and segmentation of myocardial cells were markedly improved by treatment with berberine (Fig. 4D). Taken together, these results suggest that berberine markedly improves myocardial function in mice with ischemia-reperfusion injury.