Fig 1.
Autophagy prevents ox-LDL-induced foam cell formation in THP-1 cells.
THP-1 cells were treated with the vehicle solution (control), control + CQ (10 μM), ox-LDL (100 μg/ml), ox-LDL (100 μg/ml) + CQ (10 μM), ox-LDL (100 μg/ml) + Rap (20 μM) for 36 h, respectively. (A) LC3-I (18 kDa), LC3-II (16 kDa), and p62 (62 kDa) protein levels were detected by western blot analysis. Each lane contained 20 μg proteins for all experiments. (B) and (C) The LC3-II/LC3-I ratio and p62 level were quantified with Sigma Scan Pro5 software. Each lane was normalized to that of GAPDH (kDa). (D) Oil red O staining was used to evaluate THP-1 foam cell formation (magnification × 200). (D) Intracellular total cholesterol content was determined by enzymatic assay. All the data were shown as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.
Fig 2.
NC rescues the impaired autophagy flux in ox-LDL-treated THP-1 cells.
(A) THP-1 cells were treated with different concentration of NC (0, 1, 5, 10, 20 μM) in the presence of ox-LDL (100 μg/ml) for 36 h. Cell lysates were analyzed by western blotting assay for LC3-I (18 kDa), LC3-II (16 kDa), and p62 (62 kDa) protein levels. Each lane was loaded with 20 μg proteins in all experiments. (B) and (C) The LC3-II and p62 levels were quantified with Sigma Scan Pro5 software. (D) Confocal images of representative images of GFP and RFP fluorescent puncta in THP-1 cells transfected with GFP-RFP-LC3II for 24 h, and then treated with indicated reagents for 36 h. (E) Quantification of GFP/RFP double-positive and RFP single-positive puncta in each cell treated with indicated reagents for 36 h (n = 23 cells/group). All the data were shown as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.
Fig 3.
NC reduces ox-LDL accumulation in THP-1 cells via activation of autophagy.
(A) Representative photomicrographs of THP-1 cells loaded with Dil-ox-LDL. Cells were treated with ox-LDL (100 μg/ml), ox-LDL (100 μg/ml) + NC (10 μM), ox-LDL (100 μg/ml) + NC (10 μM) + 3-MA (10 mM), ox-LDL (100 μg/ml) + NC (10 μM) + CQ (20 μM), and ox-LDL (100 μg/ml) + vehicle for 36 h. After washing 3 times, cell lysates were collected for the measurement of fluorescence. Nuclei were counterstained with DAPI. (B) Quantification of fluorescence intensity from experiments as described in (A). (C) TEM was used to evaluate foam cell formation and autophagy alteration. THP-1 cells were treated with vehicle, ox-LDL (100 μg/ml), ox-LDL + NC (10 μM)), ox-LDL (100 μg/ml) + NC (10 μM) + 3-MA, ox-LDL (100 μg/ml) + NC (10 μM) + CQ (20 μM), and ox-LDL (100 μg/ml) + vehicle for 36 h, respectively. Mitochondria (M), the nucleus (N), lysosomes (L), autophagosomes (APs), autophagolysosomes (ALs), and lipid droplets (LDs) were indicated. (D, E, and F) Average number of APs, ALs, and LDs was quantified as described in Methods section (n = 12 cells/group). All the data were shown as mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.01.
Fig 4.
NC enhances intracellular ox-LDL degradation through facilitation of lipophagy.
(A) Representative photomicrographs of colocalization of lipid droplets (LDs) with LC3-II in THP-1 cells. After THP-1 cells were transfected with GFP-LC3II for 24 h, cells were treated with ox-LDL (100 μg/ml), ox-LDL (100 μg/ml) + NC (10 μM), ox-LDL (100 μg/ml) + NC (10 μM) + 3-MA (10 mM), ox-LDL (100 μg/ml) + NC (10 μM) + CQ (20 μM), and ox-LDL (100 μg/ml) + vehicle for 36 h. After washing with PBS, cells were fixed with 4% paraformaldehyde, and then stained with Nile Red (10 ng/ml) for 30 min to evaluate the accumulation of LDs. The colocalization of LDs with LC3II was examined by immunocytochemistry as described in Methods section. (B) The percentage of colocalization of LDs with LC3-II was quantified with Image J software (n = 16 cells/group). (C) Representative photomicrographs of LD accumulation in THP-1cells. Cells were incubated with ox-LDL (100 μg/ml) conjugate without or with NC of 36 h, or pre-incubated with ox-LDL for 4 h, and then treated without or with NC for additional 36 h. The intracellular LD accumulation was evaluated by Nile Red (10 ng/ml) staining. (D) Average number of LDs in THP-1 cells was quantified (n = 12 cells/group). All the data were shown as mean ± SEM of 3 independent experiments. NS: no significant difference. *P < 0.05, **P < 0.01.
Fig 5.
NC promotes cholesterol efflux via restoring autophagy flux.
(A)and (B) THP-1 cells were incubated in medium containing 100 μg/ml ox-LDL that was labeled with 0.5 μ Ci/mL 3H-cholesterol (PerkinElmer) for an additional 30 h and then treated with vehicle, NC (10 μM), NC (10 μM) +3-MA (10 mM), and NC (10 μM) + CQ (20 μM) for additional 6 h. Subsequently, ApoA1- or HDL-mediated cholesterol efflux was analyzed by liquid scintillation counting assay. The efflux is expressed as the percentage of effluxed 3H-cholesterol/total cell cholesterol 3H-cholesterol content (effluxed 3H-cholesterol + intracellular 3H-cholesterol) × 100%. All the data were shown as mean ± SEM of 3 independent experiments. NS: no significant difference. *P < 0.05, **P < 0.01.
Fig 6.
NC rescues autophagy flux via inhibiting the PI3K /m-TOR pathway.
(A) THP-1 cells were treated with vehicle, ox-LDL (100 μg/ml), ox-LDL (100 μg/ml) +NC (10 μM), ox-LDL (100 μg/ml) +NC (10 μM) + 740Y-P (20 μM), and ox-LDL (100 μg/ml) + vehicle for 36 h. Cell lysates were collected and analyzed by western blotting assay for PI3K (85 kDa), p-mTOR (289 kDa), p-p70S6K (70 kDa), LC3-I (18 kDa), LC3-II (16 kDa), and p62 (62 kDa) protein levels. Each lane was loaded with 20 μg proteins for all experiments. (B, C, D, and E) The relative optical density values of PI3K, p-mTOR, p-p70S6K, LC3-II, LC3-I, and p62 to GAPDH, respectively, were quantified with Sigma Scan Pro5 software. All the data were shown as mean ± SEM of 3 independent experiments. NS: no significant difference. *P < 0.05, **P < 0.01.