ABSTRACT
Objective:To investigate the protective effect and potential mechanism of mitochondrial coenzyme Q (MitoQ) on mitochondria-dependent apoptosis in type Ⅱ alveolar epithelial cells induced by lipopolysaccharide (LPS).Methods:The type Ⅱ lung epithelial cell line (A549) were cultured with different concentrations of LPS in vitro, a cell model of acute lung injury (ALI) was reproduced, the optimal concentration of LPS was obtained according to the half maximal inhibitory concentration (IC 50). The cells were pretreated with different concentrations of MitoQ to determine the best intervention concentration of MitoQ. The cells were divided into four groups: the cells in blank control group were cultured in DMEM; the cells in LPS group were stimulated with 10 mg/L of LPS for 24 hours; the cells in MitoQ+LPS group were pretreated with 1 μmol/L MitoQ for 60 minutes, and then were co-cultured with 10 mg/L of LPS for 24 hours; and the cells in MitoQ+phosphatidylinositol 3-kinase (PI3K) selective inhibitor LY294002+LPS group were pretreated with 1 μmol/L MitoQ and 20 μmol/L LY294002 for 60 minutes, and then were co-cultured with 10 mg/L of LPS for 24 hours. Cell viability was measured using cell counting kit-8 (CCK-8). The cell apoptosis rate was determined by flow cytometry and TdT-mediated dUTP-nick end labeling (TUNEL) method. The protein expression levels of apoptosis protein Bax, anti-apoptotic protein Bcl-2 and PI3K-serine/threonine kinase (Akt) protein PI3K expression and Akt phosphorylation level were detected by Western blotting. Results:According to the inhibition rate curve, the IC 50 of LPS on A549 cells was 11.06 mg/L. Therefore, 10 mg/L was selected as the stimulating concentration of LPS. After stimulation with 10 mg/L LPS, the cell viability first increased and then decreased with the increase in MitoQ pretreatment concentration. According to the cell viability curve, 1 μmol/L was selected as the optimum concentration of MitoQ. Compared with LPS group, after pretreated with 1 μmol/L MitoQ, cell mitochondrial dependent apoptosis was significantly attenuated, which was characterized by the apoptosis rate was significantly decreased [flow cytometry: (8.73±0.25)% vs. (18.10±0.70)%, TUNEL: (12.30±0.82)% vs. (21.43±0.86)%, both P < 0.05], the expression of Bax was significantly down-regulated (Bax/β-actin: 0.58±0.03 vs. 1.06±0.10, P < 0.05) and Bcl-2 level was significantly up-regulated (Bcl-2/β-actin: 1.03±0.06 vs. 0.53±0.07, P < 0.05), meanwhile the expression of PI3K and Akt phosphorylation level were significantly increased [PI3K protein (PI3K/β-actin): 1.20±0.02 vs. 0.96±0.04, phosphorylated Akt (p-Akt) protein (p-Akt/t-Akt): 1.22±0.08 vs. 0.92±0.04, both P < 0.05]. Pretreatment with LY294002 could inhibit the anti-apoptotic effect of MitoQ on cells, it was characterized by the apoptotic rate was significantly increased as compared with MitoQ+LPS group [flow cytometry: (14.50±0.57)% vs. (8.73±0.25)%, TUNEL: (16.50±0.53)% vs. (12.30±0.82)%, both P < 0.05], the expression of Bax was significantly up-regulated (Bax/β-actin: 0.95±0.03 vs. 0.58±0.03, P < 0.05) and Bcl-2 level was significantly down-regulated (Bcl-2/β-actin: 0.62±0.03 vs. 1.03±0.06, P < 0.05), meanwhile the expression of PI3K and Akt phosphorylation level were significantly decreased [PI3K protein (PI3K/β-actin): 0.90±0.05 vs. 1.20±0.02, p-Akt protein (p-Akt/t-Akt): 0.89±0.02 vs. 1.22±0.08, both P < 0.05]. Conclusion:MitoQ improved LPS induced mitochondria-dependent apoptosis of A549 cells by significantly activating PI3K/Akt signal pathway, which provided a new treatment for LPS induced ALI.
ABSTRACT
ObjectiveTo 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 ExperimentⅠ: 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 (6.5μmol/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. ExperimentⅡ: 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 experimentⅠ. 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.Results① 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,χ2= 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,χ2= 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, allP 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, allP 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.
ABSTRACT
Objective To investigate the effects of autophagy on cardiac function and to determine whether the mitochondrial coenzyme Q (MitoQ) prevents cardiac dysfunction,mediated by autophagy,in rats with acute sepsis.Methods Forty-five Sprague Dawley (SD) rats were randomly divided into 9 groups (n =5,each group):control group,4 h lipopolysaccharide(LPS) group,6 h LPS group,12 h LPS group,4 h LPS + Wortmannin group,4 h LPS + MitoQ group,6 h LPS + MitoQ group,MitoQ group and Wortmannin group.Rats in LPS + Wortmannin group and LPS + MitoQ group were intraperitoneally given LPS(10 mg/kg) and followed by an injection of Wortmannin(2 mg/kg) and MitoQ (6.5 μmol/kg) via tail vein 1 hour later,respectively.Rats in each group were given the same amount of normal sodium in addition to different intervention drugs.The cardiac function parameters were measured by a BL-420E + biosignal collection system.Blood samples from abdominal aorta were taken at each time point,and creatine kinase MB isoenzyme (CK-MB) content was detected by using the velocity method.The content of reactive oxygen species (ROS) in isolated myocardial tissues in rats was measured by enzyme-linked immunoadsorbent assay(ELISA).The protein expression of microtubule-associated protein 1 light chain 3 (LC3) was detected by Western blot method.The pathological changes of myocardial tissue were observed by light and electronic microscopy.Results Compared with the control group,the left ventricular systolic pressure(LVSP),the rate of the rise in left ventricular pressure (± dp/dt max) were significantly decreased in 6 h LPS group,6 h LPS + MitoQ group and 4 h LPS + Wortmannin group(P ＜0.05),left ventricular end-diastolic pressure(LVEDP) was significantly increased in these 3 groups(P ＜0.05).The contents of CKMB and ROS in 6 h LPS group,6 h LPS =MitoQ group and 4 h LPS + Wortmannin group were higher than those in the control group(P ＜ 0.05).Electron microscopy showed that the number of autophagic vacuoles increased 6 h after LPS was administered,but did not increase significantly thereafter to 12 h.There was no difference at any time point in the number of autophagic vacuoles in the group given MitoQ and LPS.Immunoblotting demonstrated that the levels of LC3Ⅱ protein in the LPS 6 h group and LPS + MitoQ 6 h group were higher than those in the control group(P ＜0.05),but there was no difference between the LPS 12 h and LPS 6 h groups (P ＞ 0.05).Conclusions The mitochondria-targeted antioxidant MitoQ does not prevent cardiac dysfunction.However,autophagy prevents cardiac dysfunction,and the autophagic capacity or autophagosome-formation rate may determine whether cardiac dysfunction develops.