[Effect of autophagy and mitochondrial coenzyme Q on exocrine function of pancreas in rats with acute sepsis].

Objective: To investigate the effects of autophagy on exocrine function of pancreas in rats with acute sepsis, and to determine whether the mitochondrial coenzyme Q (Mito Q) can prevent exocrine dysfunction of pancreas mediated by autophagy.

Methods: Experiment I: 30 Sprague-Dawley (SD) rats were randomly divided into three groups, with 10 rats in each group. All the rats were given lipopolysaccharide (LPS, 10 mg/kg) intraperitoneally, and Wortmannin (2 mg/kg), the specific inhibitor of autophagy (LPS + Wortmannin group), Mito Q (6.5 μmol/kg, LPS + Mito Q group), or the same volume of normal saline (LPS group) was respectively injected via the tail vein 1 hour later. Survival rate was assessed within 12 hours after LPS injection. Experiment II: another 100 male SD rats were randomly divided into ten groups with 10 rats in each group: namely control 4, 6 and 12 hours groups, LPS 4, 6 and 12 hours groups, and LPS + Wortmannin 4 hours group, Wortmannin 4 hours group, LPS + Mito Q 6 hours group, and Mito Q 6 hours group. The protocols of model reproduction and drug administration were the same as in the experiment I. Blood samples were collected at each time point, and the amylase content was determined with the velocity method. The levels of reactive oxygen species (ROS) in the pancreases were measured with enzyme-linked immunosorbent assay (ELISA). The expression of the autophagy-related protein LC3 was determined with Western Blot. The pathological changes in the pancreas were observed with microscopy.

Results: (1) The survival time in the LPS + Wortmannin group was significantly shorter than that in the LPS group (hours: 7.50±0.64 vs. 11.90±0.13, χ (2) = 19.847, P = 0.001). There was no significant difference in the survival time between LPS + Mito Q and LPS groups (hours: 11.60±0.24 vs. 11.90±0.13, χ (2) = 1.055, P = 0.137). (2) The serum amylase in the LPS 6 hours, LPS + Wortmannin 4 hours, and LPS + Mito Q 6 hours groups were significantly higher than those in the control group at the same time points (U/L: 2 881.00±550.12 vs. 2 099.20±249.57, 3 672.00±779.24 vs. 2 081.36±245.18, 2 975.20±687.03 vs. 2 099.20±249.57, all P < 0.05), and were significantly lowered in LPS 12 hours group (U/L: 794.00±218.71 vs. 2 086.80±261.75, P < 0.01). The pancreatic ROS in the LPS 6 hours and 12 hours groups, LPS + Wortmannin 4 hours group, and LPS + Mito Q 6 hours group were significantly higher than those of the control group at the same time points (kU/L: 3.18±1.06 vs. 1.78±0.37, 3.63±1.08 vs. 1.85±0.41, 3.14±0.98 vs. 1.65±0.34, 3.17±1.03 vs. 1.78±0.37, all P < 0.05). The serum amylase and pancreatic ROS in LPS + Wortmannin 4 hours group were significantly higher than those of the LPS group at the same time points (U/L: 3 672.00±779.24 vs. 2 432.20±442.85, kU/L: 3.14±0.98 vs. 1.87±0.42, both P < 0.05), but there were no differences in above two parameters between LPS + Mito Q 6 hours group and LPS group (U/L: 2 975.20±687.03 vs. 2 881.00±550.12, kU/L: 3.17±1.03 vs. 3.18±1.06, both P > 0.05). Light microscopy showed that obvious pathological changes were found in the pancreas in the LPS 6 hours and 12 hours groups, LPS + Wortmannin 4 hours group, and LPS + Mito Q 6 hours group. Electron microscopy showed that the number of autophagic vacuoles increased 6 hours after LPS administration. There was no difference at any time point in the number of autophagic vacuoles between LPS + Mito Q 6 hours group and LPS 6 hours group, and the autophagic vacuoles were not found after Wortmannin intervention. It was demonstrated by Western Blot that the levels of LC3 protein in the LPS 6 hours and 12 hours groups, and LPS + Mito Q 6 hours group were significantly higher than those of the control group at the same time points (A value: 0.34±0.02 vs. 0.17±0.02, 0.37±0.03 vs. 0.18±0.04, 0.36±0.02 vs. 0.17±0.02, all P < 0.05), but there were no differences between LPS 12 hours group or LPS + Mito Q 6 hours group and LPS 6 hours group (both P > 0.05).

Conclusions: Autophagy prevents exocrine dysfunction of pancreas in septic rats, and the autophagic capacity or autophagosome-formation rate may determine the development of exocrine pancreatic dysfunction. The mitochondria-targeted antioxidant Mito Q does not prevent exocrine dysfunction of pancreas.