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Objective:To investigate the effects of three monomer components stilbene glycoside, emodin, catechin of Polygonum multiflorum on damages of liver tissue and cell.Methods:A total of 48 rats were randomly divided into four groups of stilbene glycoside, emodin, catechin-and normal saline(control)-treated groups(n=12, each). Rats were gavaged with the same dose of 1 g/kg for stilbene glycoside, emodin, catechin groups and normal saline for control group for 28 days.During the administration, the general state of rat was observed.After the last administration, serum biochemical indexes related to liver function were detected.Pathological morphological changes of liver tissue were observed by hatmatoxylin-Eosin(HE)staining.The expression levels of apoptosis-related proteins in liver tissue were determined by Western blot.Cells of human normal liver cell line LO2 divided into the stilbene glycoside-, emodin-, catechin-treated cells groups were cultured with 7 different concentrations of interfering agents for 24 h and 48 h respectively, and cultured with normal saline in the control group for the same time.Cell proliferation was observed using the cholecystokinin octapeptide(CCK8), and the mRNA expressions of apoptosis-related factors were detected using PCR.Results:After 28 days of feeding, there was no significant difference in body weight and food intake among the four groups( P>0.05). Pathological liver damage and abnormal liver biochemical indexes were observed in rat liver tissues in the emodin and catechin groups( P<0.05). Compared with the control group, the expression levels of anti-apoptotic proteins Bcl-2 were decreased(0.34±0.03, 0.41±0.07 vs.0.45±0.04, P<0.05), the expression levels of apoptotic proteins Caspase-3 and Bax were increased(0.76±0.03, 0.27±0.06 vs.0.03±0.00; 0.44±0.03, 0.15±0.04 vs.0.02±0.00, P<0.01), and those in the stilbene glycoside group were normal.Compared with the control group, emodin and catechin-treated cell groups showed the concentration-and time-dependent proliferation inhibition in LO2 normal hepatocytes( P<0.05)after treatment.And the mRNA expression of hepatocyte apoptosis factors Bax and Caspase-3 were increased(1.74±0.05, 1.29±0.01 vs.0.89±0.12, 1.21±0.07, 1.25±0.01 vs.0.97±0.07, P<0.01), and that of anti-apoptotic proteins Bcl-2 was decreased(0.06±0.06, 0.56±0.11 vs.1.39±0.18, P<0.01). Compared with the control group, the survival rate had no significant difference in the stilbene glycoside treated-cell group( P>0.05), while its mRNA expression of hepatocyte apoptosis factors was reduced( P<0.05). Conclusions:No obvious liver damage is found in rats treated with the stilbene glycoside of Polygonum multiflorum, but the emodin and catechin cause damages of liver tissue and cell in vivo and in vitro.
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Objective To investigate the relationship between different tidal volume (VT) mechanical ventilation (MV) and autophagy and mitochondrial damage in rats. Methods A total of 120 clean-grade male Sprague-Dawley (SD) rats were divided into five groups (n = 24) by random number table method, and then given 0 (spontaneous breathing), 10, 20, 30, 40 mL/kg VT for MV. The rats in each group were subdivided into four subgroups of 1, 2, 3, and 4 hours according to ventilation time, with 6 rats in each subgroup. The lung tissue and bronchoalveolar lavage fluid (BALF) were harvested, and alveolar macrophages (AMs) and type Ⅱ alveolar epithelial cells (AECⅡ) were cultured in vitro. The mRNA and protein expressions of autophagy-associated protein microtubule-associated protein 1 light chain 3B -Ⅱ (LC3B -Ⅱ) and autophagy-related genes Beclin1 and p62 were determined by reverse transcription-polymerase chain reaction (RT-PCR) or Western Blot. Lung autophagosome formation was observed under transmission electron microscope. The levels of adenosine triphosphate (ATP), reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in lung tissue were determined for assessing mitochondrial damage. Results There were no significant differences in the mRNA and protein expressions of LC3B -Ⅱ, p62 and Beclin1 at 1 hour after ventilation among the groups. With the prolonged ventilation time, the mRNA and protein expressions of LC3B -Ⅱ, p62 and Beclin1 in MV groups were increased gradually, peaked at 2-3 hours, and they were increased significantly in 30 mL/kg VT group as compared with those in spontaneous respiration group with statistical significances [ventilation for 2 hours: LC3B -Ⅱ mRNA (2-ΔΔCt) was 2.44±0.24 vs. 1.12±0.04, LC3B -Ⅱ/LC3B -Ⅰ was 1.42±0.16 vs. 0.57±0.03, p62 mRNA (2-ΔΔCt) was 2.96±0.14 vs. 1.14±0.02, Beclin1 mRNA (2-ΔΔCt) was 2.80±0.13 vs. 1.14±0.02; ventilation for 3 hours: p62/β-actin was 1.14±0.15 vs. 0.55±0.04, Beclin1/β-actin was 1.27±0.06 vs. 0.87±0.04, all P < 0.05]. Autophagosomes and autolysosomes were found in AECⅡ after ventilation for 2 hours at 30 mL/kg VT by transmission electron microscopy, but not in AECⅠ. Compared with spontaneous breathing group, ATP synthesis in AMs was significantly decreased at 2 hours of ventilation in 30 mL/kg VT group (A value: 0.82±0.05 vs. 1.00±0.00, P < 0.05), ROS accumulate in AMs and AECⅡ were significantly increased [ROS in AMs: (33.83±4.00)% vs. (6.90±0.62)%, ROS in AECⅡ: (80.68±0.90)% vs. (2.16±0.19)%, both P < 0.05]. With the increase in VT and the prolongation of ventilation time, ATP and ROS levels in AMs and AECⅡ were gradually decreased, the ATP (A value) in AMs at 4 hours of ventilation in 40 mL/kg VT group was 0.41±0.05, the ROS in AMs was (12.95±0.88)%, and the ROS in AECⅡ was (40.43±2.29)%. With the increase in VT and the prolongation of ventilation time, MMP levels were gradually increased, the MMP (green/red fluorescence intensity ratio) in AMs at 2 hours of ventilation in 30 mL/kg VT group was 1.11±0.17, the MMP in AECⅡwas 0.96±0.04, and the MMP (green/red fluorescence intensity ratio) at 4 hours of ventilation in 40 mL/kg VT group was 0.51±0.07 and 0.49±0.06, respectively. Conclusion The MV with high VT could induce autophagy activation and mitochondrial damage in lung tissue of rats, and the longer the ventilation time, the more obvious autophagy in the lung.
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OBJECTIVE@#To investigate the relationship between different tidal volume (VT) mechanical ventilation (MV) and autophagy and mitochondrial damage in rats.@*METHODS@#A total of 120 clean-grade male Sprague-Dawley (SD) rats were divided into five groups (n = 24) by random number table method, and then given 0 (spontaneous breathing), 10, 20, 30, 40 mL/kg VT for MV. The rats in each group were subdivided into four subgroups of 1, 2, 3, and 4 hours according to ventilation time, with 6 rats in each subgroup. The lung tissue and bronchoalveolar lavage fluid (BALF) were harvested, and alveolar macrophages (AMs) and type II alveolar epithelial cells (AEC II) were cultured in vitro. The mRNA and protein expressions of autophagy-associated protein microtubule-associated protein 1 light chain 3B-II (LC3B-II) and autophagy-related genes Beclin1 and p62 were determined by reverse transcription-polymerase chain reaction (RT-PCR) or Western Blot. Lung autophagosome formation was observed under transmission electron microscope. The levels of adenosine triphosphate (ATP), reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in lung tissue were determined for assessing mitochondrial damage.@*RESULTS@#There were no significant differences in the mRNA and protein expressions of LC3B-II, p62 and Beclin1 at 1 hour after ventilation among the groups. With the prolonged ventilation time, the mRNA and protein expressions of LC3B-II, p62 and Beclin1 in MV groups were increased gradually, peaked at 2-3 hours, and they were increased significantly in 30 mL/kg VT group as compared with those in spontaneous respiration group with statistical significances [ventilation for 2 hours: LC3B-II mRNA (2-ΔΔCt) was 2.44±0.24 vs. 1.12±0.04, LC3B-II/LC3B-I was 1.42±0.16 vs. 0.57±0.03, p62 mRNA (2-ΔΔCt) was 2.96±0.14 vs. 1.14±0.02, Beclin1 mRNA (2-ΔΔCt) was 2.80±0.13 vs. 1.14±0.02; ventilation for 3 hours: p62/β-actin was 1.14±0.15 vs. 0.55±0.04, Beclin1/β-actin was 1.27±0.06 vs. 0.87±0.04, all P < 0.05]. Autophagosomes and autolysosomes were found in AEC II after ventilation for 2 hours at 30 mL/kg VT by transmission electron microscopy, but not in AEC I. Compared with spontaneous breathing group, ATP synthesis in AMs was significantly decreased at 2 hours of ventilation in 30 mL/kg VT group (A value: 0.82±0.05 vs. 1.00±0.00, P < 0.05), ROS accumulate in AMs and AEC II were significantly increased [ROS in AMs: (33.83±4.00)% vs. (6.90±0.62)%, ROS in AEC II: (80.68±0.90)% vs. (2.16±0.19)%, both P < 0.05]. With the increase in VT and the prolongation of ventilation time, ATP and ROS levels in AMs and AEC II were gradually decreased, the ATP (A value) in AMs at 4 hours of ventilation in 40 mL/kg VT group was 0.41±0.05, the ROS in AMs was (12.95±0.88)%, and the ROS in AEC II was (40.43±2.29)%. With the increase in VT and the prolongation of ventilation time, MMP levels were gradually increased, the MMP (green/red fluorescence intensity ratio) in AMs at 2 hours of ventilation in 30 mL/kg VT group was 1.11±0.17, the MMP in AEC II was 0.96±0.04, and the MMP (green/red fluorescence intensity ratio) at 4 hours of ventilation in 40 mL/kg VT group was 0.51±0.07 and 0.49±0.06, respectively.@*CONCLUSIONS@#The MV with high VT could induce autophagy activation and mitochondrial damage in lung tissue of rats, and the longer the ventilation time, the more obvious autophagy in the lung.