Briefly, AR42J cells were treated or not with DCQD prior for 30 min and then stimulated with cerulein (10?8 M) for 24 h. Then cells were collected and resuspended in the culture medium at a density of 1��106 cells/mL, stained with 5 ��L of Annexin V-FITC and blog post 5 ��L propidium iodide (PI) in 300 ��L binding buffer (10 mM HEPES, pH 7.4, 140 mM NaOH, and 2.5 mM CaCl2) according to the manufacturer’s instructions for 15 min at room temperature in the dark. Quantification of apoptotic cells was analysis by flow cytometry (FACScan, Becton Dickinson, USA). 2.5. Measurement of ROS generation The generation of ROS in cells was determined using a FACScan flow cytometry following the manufacturer’s instructions. Briefly, AR42J cells were pretreated with DCQD 30 min before stimulated with cerulein for 24 h.

Cells were collected and incubated with 10 ��M/L DCFH-DA 30 min in the dark and then washed twice with PBS. Intracellular low-molecular-weight peroxides oxidize DCFH-DA to the highly fluorescent compound dichlorofluorescein (DCF). Then the cells were harvested and the pellets were suspended in 300 ��l PBS at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. 2.6. Animal models and treatment with DCQD Sprague-Dawley rats were divided randomly into sham-operated group, AP group and DCQD- treated group (n=6). While the rats were under ether anesthesia and laparotomy, pancreatitis was induced by retrograde perfusion into the biliopancreatic duct of 3.5% sodium taurocholate (Sigma, St. Louis, MO, USA) (1 mL/kg body weight) at a rate of 0.2 mL/min with a microinfusion pump [17].

The entire procedure from induction of anesthesia to closure of the incisions requires ~30 min for each animal. The same procedure was applied to sham-operated group but receiving Batimastat an intraductal perfusion of saline (NaCl 0.9%) instead of sodium taurocholate. In DCQD- treated group, the rats recovered from anesthesia and were administered intragastrically DCQD 20 g/kg body weight (equivalent to 2 g/mL crude herbs) 2 h after operation. In the sham-operated group and AP group, rats were given equal volume of saline. After 48 h, blood were obtained from the vena caudalis and centrifuged to obtain serum for amylase examination. The animals were sacrificed by exsanguination while under ether anesthesia and the pancreatic tissues were rapidly collected for pathological and apoptotic examinations. Tissue homogenate was collected for NO and iNOS concentration measurement. 2.7. Amylase and NO and iNOS Assay Serum was collected from the rats for amylase activity (U/L) measurement by an enzymatic assay kit from Sigma (St. Louis, MO, USA) according to manufacturer’s instructions.