| AIM: To investigate the damage of pancreatic acinar cell induced by lipopolysaccharide (LPS) and sodium deoxycholate(SDOC), and the alteration of the content of maloidialdehyde (MDA), the activity of superoxide dismutase (SOD) and phospholipase (PLA2), the concentration of [Ca2+]i and the activity of NF- K B in pancreatic acinar cell, further to explore the possible mechanism of pancreatic acinar cell damage induced by LPS and SDOC. Moreover, to investigate the protective effect of tetrandrine (Tet) on LPS- or SDOC-induced pancreatic acinar cell damage and its mechanism. METHODS: Rat pancreatic acinar cells were isolated by collagenase digestion and were treated with varying concentration of LPS (from 1 mg/L to 20 mg/L), SDOC (from 10 umol/L to 100 umol/L), Tet (50 umol/L and 100 umol/L), or culture media , respectively. At different time points (30 min,1 h, 2 h, 4 h, 10 h) , cell viability was determined by MTT and the supernatant of cells was collected to measure the content of MDA, the activity of SOD and PLA2. Some cells were loaded with Fluo-3/AM, then exposed to varying doses of LPS and SDOC in the absence or presence of Ca2+ in extracellular fluid. The dynamic change of [Ca2+]i in single pancreatic acinar cell was determined by laser scanning confocal microscopy. The nuclear translocation of the subunit p6S of NF- K B was visualized by immunofluorescence staining and nuclei protein was extracted to perform electrophoretic mobility shift assay (EMSA) which was used to assay the activity of NF- K. B binding to the DNA sequence containing recognition site of NF- K B. RESULTS: (D LPS and SDOC initiated cell damage in a time- and concentration-dependent manner( P<0.05), and 1 mmol/L egtazic acid (EGTA) could decrease the cell mortality( P<0.05); LPS induced a slight rise of [Ca2+]i in the calcium-free extracellular fluid containing 1 mmol/L EGTA. Addition of extracellular calcium in the presence of LPS resulted in a more immediate andremarkable rise of [Ca2+]j, which reached the peak value within 150 s and maintained the value for 100 s. SDOC did not induce a rise of [Ca2+]i in the calcium-free extracellular fluid, but caused a rapid and remarkable rise of [Ca2+]j when adding calcium into the media to recover a normal concentration of [Ca2+]e. The increase in intracellular [Ca2+]; preceded the pathological and biological alteration of pancreatic acinar cells. In the supernatant of LPS and SDOC groups, the content of MDA and the activity of PLA2 increased ( /*<0.05) and the activity of SOD decreased (P<0.05). (2) NF- K B p65 immunofluorescence staining showed that the intensity in cytoplasma increased and nuclear translocation occurred at 30 min and its zenith was at 1 h after LPS (10 mg/L) or SDOC (50 umol/L) treatment and the intensity of immunofluorescence staining still kept a higher level at 4h . NF- K B DNA binding activity showed the same alteration phase as p65 immunofluorescence staining. NF- K B activation preceded the pathological alteration of pancreatic acinar cells. A Ca2* chelator EGTA inhibited the LPS- or SDOC- induced NF- K B activation. (3) Tet attenuated LPS- or SDOC-induced cell damage (50umol/L, P<0.05; 100umol/L, P<0.01) and inhibited the elevation of cytoplasmic free calcium of rat pancreatic acinar cells. In the supernatant, let decreased the content of MDA and the activity of PLAa (P<0.05) and increased the activity of SOD (P<0.05). NF- K B p65 immunofluorescence staining in the cytoplasma or the nuclear, and NF- K B DNA binding activity were lessened by Tet. CONCLUSION: (1) LPS and SDOC initiated cell damage in a time-, concentration-and calcium-dependent manner. LPS and SDOC induced the calcium overload in the cytoplasm of pancreatic acinar cell in a different manner. LPS not only induced the discharge of Ca2+ from the inteacellular calcium store but also the influx of Ca2+ from the extracellular fluid. Whereas, SDOC only induced the influx of Ca2+ from the extracellular fluid. Calcium overload as an early pathogenetic event took part in the damage of pancreatic acinar cells by...
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