The six components of DJC were provided and identified by The First Affiliated Hospital of Anhui University of Chinese Medicine. All these herbs except leech were extracted with boiled water for three times, followed by concentration of the mixed extracts. After grinding into fine powder, leech was mixed with the above mentioned extracts. Then the mixture was dried, grinded and capsulized for clinical use, with each capsule containing 0.4 g extract prepared from 8 g herbal medicine. For use in this experiment, DJC powder was dissolved in distilled water and given to rats intragastrically.
Animals and induction of diabetes
Sixty male Wistar rats aged 7 weeks (weight: 200–220 g) were obtained from the Experimental Animal Center of Nanjing Medical University and maintained in a temperature and humidity controlled room with a 12 h light–dark cycle. Rats were fed a standard diet with free access to drinking water. After adaptive feeding for 1 week, all rats except 10 randomly allocated to Control group were injected intraperitoneally with streptozotocin (STZ, 50 mg/kg, Sigma, MO, USA) to induce type 1 diabetes. Ten days later, random blood glucose (RBG) was determined, and rats with RBG levels ≥16.7 mmol/L were defined as successful diabetic rats . This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Committee on the Care and Use of Laboratory Animals of Anhui University of Chinese Medicine.
Animal grouping and drug dosing
Thirty-six diabetic rats were randomly allocated to 3 groups, namely diabetic group (Model), low dose of DJC group (DJCL, 0.63 g/kg · d−1) and high dose of DJC group (DJCH, 1.26 g/kg · d−1). The low dose of Danzhi Jiangtang capsule was extrapolated from human dose through normalization to body surface area (equivalent dose). Rats in DJCH and DJCL groups were administered intragastrically with DJC for 6 weeks, while those in Control and Model groups were given distilled water of the same volume. Doses of DJC were adjusted according to the changes in body weight monitored biweekly. RBG was measured biweekly by glucose oxidase method using a commercial glucometer (Sinocare Incorporation, Changsha China).
At the end of the experiment, rats were fasted overnight and anaesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg body weight). Blood samples were collected into EDTA-coated tubes and plasma was separated by centrifugation at 3000 g, 4 °C for 10 min (Eppendorf Corporation, Hamburg, Germany). Fasting plasma glucose (FPG) was determined by glucose oxidase method (Nanjing Jiancheng Bioengineering Company, Nanjing China) and fasting plasma insulin (FINS) was measured by enzyme-linked immunosorbent assay (Hufeng Biotech, Shanghai, China). Plasma levels of Total antioxidant capacity (TAC) and superoxide dismutase (SOD) activity were evaluated by ferric reducing antioxidant power test and xanthine oxidase method, respectively . Malondialdehyde (MDA) content in plasma was determined by thiobarbituric acid method (Nanjing Jiancheng Bioengineering Company, Nanjing, China). Pancreatic tissues were homogenized in phosphate buffered saline (PBS) and the levels of SOD activity and MDA content in the homogenates were determined as described above. Activities of Caspase-3 and Caspase-9 were determined with Colorimetric assay kit (Beyotime institute of Biotechnology, Haimen, China). Protein concentrations in pancreatic homogenates were measured by Bradford’s method (Beyotime institute of Biotechnology, Haimen, China). All biochemical analyses were performed with a microplate reader (Thermo Fisher Scientific Incorporation, MA, USA).
Tissues from the same region of pancreas were fixed in formalin at room temperature overnight, followed by dehydration and embedding in paraffin. After deparaffinage and rehydration, sections of 5 μm (Leica RM2245, Leica Biosystems, Nussloch, Germany) were subjected to hematoxylin and eosin (H&E) staining. Samples were examined under an Olympus IX51 microscope (Olympus Corporation, Tokyo, Japan) by a pathologist.
Evaluation of pancreatic beta cell apoptosis
Pancreatic beta cell apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. Paraffin sections (5 μm) were incubated with proteinase K (20 μg/ml, Invitrogen, CA, USA) at 37 °C for 20 min. Endogenous peroxidase activity was blocked by incubation with 3 % hydrogen peroxide in methanol. After rinsing with PBS, sections were subjected to TUNEL staining according to the manufacturer’s instructions (Roche Biochemicals, Mannheim, Germany). The conjugated horseradish peroxidase was visualized with diaminobenzidine (DAB, Sigma, MO, USA), followed by counterstaining with hematoxylin. Six randomly selected islets of each section were analyzed under high magnification (×400) with Image-Pro Plus 6.0 (Media Cybernetics MD, USA) for TUNEL-positive (apoptotic) cells, with the results expressed as percentage of apoptotic cells over total cells. The apoptotic rate of each animal represents the mean of 6 randomly selected islets.
Paraffin sections were incubated with 3 % hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. After antigen retrieval with microwave heating and blocking with goat serum, sections were incubated with monoclonal anti-body against Bax and Bcl-2 (Santa Cruz, CA, USA) at 4 °C overnight. Then the sections were incubated with biotinylated secondary antibody (Zhongshan Golden Bridge., Beijing, China) for 30 min, followed by incubation with horseradish peroxidase-conjugated streptavidin for 30 min at room temperature. The peroxidase was visualized by incubation with DAB solution in dark for 10 min and then the sections were counterstained with hematoxylin. Six randomly selected islets of each section were captured under high magnification (×400) and analyzed with Image-Pro Plus 6.0 for mean optical density . The expression level of each animal represents the mean of 6 randomly selected islets.
Western blot analysis
Pancreatic levels of Bcl-2, Bax and pancreatic duodenal homeobox-1 (PDX-1) protein were evaluated by western blot analysis. Total proteins were extracted with RIPA lysis buffer (Beyotime institute of Biotechnology, Haimen, China) supplemented with 1 mmol/L phenylmethanesulfonyl fluoride (PMSF, Sigma, MO, USA), followed by determination of protein concentrations by bicinchoninic acid (BCA) method (Beyotime institute of Biotechnology, Haimen, China). Then protein samples were subjected to SDS-polyacrylamide gel electrophoresis (Bio-Rad, CA, USA) and transferred to nitrocellulose membrane (Millipore, MA, USA). After blocking with 5 % skimmed milk in TBS containing 0.2 % Tween-20 (TBST), the membrane was incubated overnight at 4 °C with primary antibodies against Bcl-2, Bax, PDX-1 (Santa Cruz, CA, USA) and β-actin (Beyotime institute of Biotechnology, Haimen, China), followed by incubation with horseradish peroxidase (HRP) conjugated secondary antibody (Beyotime institute of Biotechnology, Haimen, China) for 1 h at room temperature. Bound HRP was visualized with an enhanced chemiluminescence substrate (ECL, Beyotime institute of Biotechnology, Haimen, China) and protein bands were analyzed for integrated optical density (IOD) with Quantity One software (Bio-Rad, CA, USA). The levels of PDX-1, Bcl-2 and Bax were normalized to that of β-actin.
Data were presented as mean ± SD. Comparisons among groups were performed by one-way ANOVA followed by the least significant difference (LSD) test. A value of P < 0.05 was considered statistically significant. All statistical analysis was performed with SPSS statistical software package, version 21.0 (SPSS Incorporation, IL, USA).