ABHD2 knockdown resulted in decline in cell viability and aggravated PA-induced decline in cell viability in A549 cells
We treated A549 cells with ABHD2 siRNA followed by functional assays. Immunoblot analysis confirmed specific knockdown of ABHD2 by siRNA (Fig. 1A). A549 cells with/without ABHD2 knockdown were exposed to 0, 50, 100, 150, 200, or 300 umol/L PA for 24 h. CCK-8 analysis showed that PA treatment inhibited A549 cells growth. Cell viability was gradually reduced with increasing concentrations of PA in a dose-dependent manner (p < 0.05). Meanwhile, the cell viability of A549 cells with ABHD2 knockdown was lower than cells without ABHD2 knockdown under different concentrations of PA (p < 0.05) (Fig. 1B). By calculation, IC50 was 179umol/L in cells without ABHD2 knockdown and 142umol/L in cells with ABHD2 knockdown. Therefore, according to the IC50 calculated by us, relevant literature and observation of cell state, we selected the condition for treatment of cells with/without ABHD2 knockdown at 0,50,100umol/L PA for 24 h in the following experiments.
ABHD2 knockdown resulted in lipid accumulation and augmented PA-induced lipid accumulation in A549 cells.
After treatment of A549 cells with different concentrations of PA for 24 h, intracellular lipid accumulation was assessed by Oil Red O staining and cells were observed under inverted microscope. From morphological observation and quantitative analysis, the result indicated that ABHD2 knockdown caused abnormal accumulation of LDs in A549 cells without PA intervention. Meanwhile, with the increase of PA concentration, the cells with/without ABHD2 knockdown showed gradually increased accumulation of LDs. Accumulation of LDs of cells with ABHD2 knockdown was more than cells without ABHD2 knockdown under different concentrations of PA. (p < 0.05) (Fig. 1C,1D)
We next found similar results when measured intracellular triglyceride content. The result indicated that ABHD2 knockdown increased intracellular triglycerides levels in A549 cells without PA intervention. Meanwhile, PA caused a dose-dependent increase of intracellular triglycerides levels in A549 cells with/without ABHD2 knockdown. Intracellular triglycerides levels of cells with ABHD2 knockdown was higher than cells without ABHD2 knockdown under different concentrations of PA. (p < 0.05) (Fig. 1E)
ABHD2 knockdown resulted in lipid peroxidation and augmented PA-induced lipid peroxidation in A549 cells.
Oxidative stress is one of the main causes of lipotoxicity. To investigate the effect of ABHD2 knockdown on ROS accumulation in A549 cells, the fluorescence intensity of DCF were evaluated by fluorescence microscope. The result showed that ABHD2 knockdown caused abnormal accumulation of fluorescence intensity of DCF in A549 cells without PA intervention. Meanwhile, with the increase of PA concentration, the cells with/without ABHD2 knockdown showed gradually increased fluorescence intensity of DCF. Fluorescence intensity of DCF of cells with ABHD2 knockdown was more than cells without ABHD2 knockdown under different concentrations of PA. The result indicated that ABHD2 knockdown could induce the accumulation of intracellular ROS without PA intervention. Intracellular ROS level of cells with ABHD2 knockdown was more than cells without ABHD2 knockdown under different concentrations of PA. (Fig. 2A,2B)
MDA is a terminal product of lipid peroxidation. To directly assess oxidative damage and lipid peroxidation, MDA level was determined. The result indicated that ABHD2 knockdown substantially increased intracellular MDA levels in A549 cells without PA intervention. Meanwhile, PA caused a dose-dependent increase of intracellular MDA levels in A549 cells with/without ABHD2 knockdown. Intracellular MDA levels of cells with ABHD2 knockdown was higher than cells without ABHD2 knockdown under different concentrations of PA (p < 0.05) (Fig. 2C)
ABHD2 knockdown resulted in apoptosis and augmented PA-induced apoptosis in A549 cells.
More recently extensive evidences have been reported that pulmonic lipotoxicity is associated with apoptosis. The apoptosis assay using annexin V-FITC and PI staining was conducted to observe cells under fluorescence microscope. The result showed that ABHD2 knockdown caused abnormal accumulation of the green and red fluorescence intensity in A549 cells without PA intervention. Meanwhile, with the increase of PA concentration, the cells with/without ABHD2 knockdown showed gradually increased the green and red fluorescence intensity. The green and red fluorescence intensity of cells with ABHD2 knockdown was more than cells without ABHD2 knockdown under different concentrations of PA. The result indicated that ABHD2 knockdown could induce apoptosis without PA intervention in A549 cells. Moreover, apoptosis level of cells with ABHD2 knockdown was more than cells without ABHD2 knockdown under different concentrations of PA. (Fig. 2E,2F)
Caspase3 is involved in cells apoptosis. The result of Caspase3 activity levels indicated that ABHD2 knockdown substantially increased intracellular Caspase3 activity levels in A549 cells without PA intervention. Meanwhile, PA caused a dose-dependent increase of intracellular Caspase3 activity levels in A549 cells with/without ABHD2 knockdown. Intracellular Caspase3 activity levels of cells with ABHD2 knockdown was higher than cells without ABHD2 knockdown under different concentrations of PA. (p < 0.05) (Fig. 2D)
The result showed that ABHD2 knockdown could increase apoptosis level without PA intervention. Moreover, apoptosis level of cells with ABHD2 knockdown was more than cells without ABHD2 knockdown under different concentrations of PA.
Abnormal lipid metabolism and lipid peroxidation in serum of ABHD2KO mice
Serum TG, TC and MDA levels were higher in ABHD2KO mice at the age of 3 months, in comparison with B6 mice at the same age, while there was no statistical difference. Moreover, serum TG, TC and MDA levels were significantly higher in ABHD2KO mice at the age of 6 and 12 months, in comparison with B6 mice at the same age. (p < 0.05) (Fig. 3C,3D,3E)
The result showed that abnormal lipid metabolism and lipid peroxidation were found in serum of ABHD2KO mice.
Abnormal lipid metabolism and lipid peroxidation in lung tissues of ABHD2KO mice
TG and MDA levels in lung tissues were higher in ABHD2KO mice at the age of 3 months, in comparison with B6 mice at the same age, while there was no statistical difference. Moreover, TG and MDA levels in lung tissues were significantly higher in ABHD2KO mice at the age of 6 and 12 months, in comparison with B6 mice at the same age. (p < 0.05) (Fig. 4B,4C)
Lipid accumulation of lung tissues of mice respectively was assessed by Oil Red O staining. As illustrated in Fig. 4A, it was found that few accumulation of lipid droplets in lung tissues of 3-month-old B6 and ABHD2KO mice were presented. Meanwhile, few accumulation of lipid droplets in the lung tissues of 6-month-old B6 mice were presented, while the lung tissues of 6-month-old ABHD2KO mice had abnormal accumulation of lipid droplets. On the other hand, the lung tissues of 12-month-old B6 mice had abnormal accumulation of lipid droplets, while the lung tissues of 12-month-old ABHD2KO mice showed more abnormal accumulation of lipid droplets than wild-type mice of the same age.
The result showed that abnormal lipid metabolism and lipid peroxidation were found in lung tissues of ABHD2KO mice.
Emphysematous changes in lung tissues of ABHD2KO mice
HE dyeing was carried out lung tissues of mice respectively. It observed no obvious emphysematous changes in lung tissues of 3-month-old B6 and ABHD2KO mice. Moreover, the lung tissues of 6-month-old B6 mice showed no obvious emphysematous changes, while the lung tissues of 6-month-old ABHD2KO mice showed emphysematous changes. On the other hand, the lung tissues of 12-month-old B6 mice showed senile emphysematous changes, while the lung tissues of 12-month-old ABHD2KO mice showed serious emphysematous changes than wild-type mice of the same age. (Fig. 3A)
Increased apoptosis level in lung tissues of ABHD2KO mice
TUNEL staining was performed to detect the apoptosis of the lung tissues of mice, and the nucleus of TUNEL-positive cells was stained darkly brown. As illustrated in Fig. 5A, it was found that few TUNEL-positive cells in lung tissues of 3-month-old B6 and ABHD2KO mice were presented. Meanwhile, few TUNEL-positive cells in the lung tissues of 6-month-old B6 mice were presented, while the lung tissues of 6-month-old ABHD2KO mice had many deeply brown stained cells. On the other hand, the lung tissues of 12-month-old B6 mice had some TUNEL-positive cells, while the lung tissues of 12-month-old ABHD2KO mice showed more TUNEL-positive cells than wild-type mice of the same age.
Caspase3 is involved in cells apoptosis. The result of Caspase3 activity levels indicated that Caspase3 activity levels in lung tissues were higher in ABHD2KO mice at the age of 3 months, in comparison with B6 mice at the same age, while there was no statistical difference. Moreover, Caspase3 activity levels in lung tissues were significantly higher in ABHD2KO mice at the age of 6 and 12 months, in comparison with B6 mice at the same age. (p < 0.05) (Fig. 5B)