Fig 2: Role of H2S in ox-LDL-induced Lp-PLA2 expression levels in THP-1 cells. Relative Lp-PLA2 protein expression levels were determined via western blotting in (A) cells pretreated with NaHS (exogenous H2S donor) for 24 h prior to incubation with 50 µg/ml ox-LDL for 24 h and (B) cells pretreated with 100 µM NaHS for 24 h prior to incubation with 50 µg/ml ox-LDL for the indicated times. Relative mRNA expression levels of Lp-PLA2 were determined via reverse transcription-PCR in (C) cells pretreated with NaHS for 24 h prior to incubation with 50 µg/ml ox-LDL for 24 h and (D) cells pretreated with 100 µM NaHS for 24 h prior to incubation with 50 µg/ml ox-LDL for the indicated times. Levels of Lp-PLA2 activity were determined with a Lp-PLA2 activity kit in (E) cells pretreated with NaHS for 24 h prior to incubation with 50 µg/ml ox-LDL for 24 h and (F) cells pretreated with 100 µM NaHS for 24 h prior to incubation with 50 µg/ml ox-LDL for the indicated times. Relative (G) protein and (H) expression levels, as well as (I) activity levels of Lp-PLA2 were determined in cells pretreated with 100 µM NaHS for 24 h or 3 mM PPG for 2 h prior to incubation with 50 µg/ml ox-LDL for 24 h. The data are presented as the mean ± SEM (n=5). !P<0.01 vs. control; &P<0.01 vs. 50 µg/ml ox-LDL + 0 µM NaHS; *P<0.05, **P<0.01 vs. 100 µM NaHS pretreated for 0 h + 50 µg/ml ox-LDL; ?P<0.01 vs. ox-LDL; aP<0.01 vs. ox-LDL + PPG. ox-LDL, oxidized low-density lipoprotein; Lp-PLA2, lipoprotein-associated phospholipase A2; PPG, DL-propargylglycine.

Fig 3: Abundance of PON1, LCAT and PAF-AH is decreased in EL-HDL.HDL proteins (10 µg/lane) were separated by 12% SDS-PAGE followed by Western blotting analyses of A) PON1, B) LCAT, and C) PAF-AH. Results show representative Western blots and corresponding densitometric analyses. Densitometric values of PON1, LCAT and PAF-AH were normalized to the corresponding apoA-I signals. Results obtained by densitometry are means + SEM of 4 independent modifications of human HDL, each loaded in duplicates and analyzed by two-tailed unpaired t-test. ***P < 0.001.

Fig 4: ox-LDL induces Lp-PLA2 expression and inhibits CSE expression in THP-1 cells. Cells were treated with 50 µg/ml ox-LDL for 24 h. Relative protein expression levels of (A) Lp-PLA2 and (B) CSE were determined via western blot analysis. Relative mRNA expression levels of (C) Lp-PLA2 and (D) CSE were determined via reverse transcription-PCR. (E) Activity levels of Lp-PLA2 were determined using a Lp-PLA2 activity kit. (F) CSE activity was determined with a methylene blue spectrophotometric assay. The data are presented as the mean ± SEM (n=5). **P<0.01 vs. control. ox-LDL, oxidized low-density lipoprotein; Lp-PLA2, lipoprotein-associated phospholipase A2; CSE, cystathionine ?-lyase.

Fig 5: H2S decreases lipid accumulation in macrophages by inhibiting the activity of Lp-PLA2. THP-1 cells were preincubated with PMA for 72 h to establish a macrophage model. THP-1 macrophages were pretreated with SB203580 (20 mM) and SB202190 (20 mM) for 30 min, PPG (3 mM) for 2 h, NaHS (100 µM) for 24 h or Lp-PLA2 siRNA (30 nM) for 48 h, prior to incubation with ox-LDL (50 µg/ml) for 24 h. Cells exhibiting lipid accumulation were observed and counted via light microscopy following ORO staining (magnification, ×40) in the (A) Control, (B) ox-LDL, (C) ox-LDL + PPG, (D) ox-LDL + SB203580, (E) ox-LDL + SB202190, (F) ox-LDL + NaHS and (G) ox-LDL + Lp-PLA2 siRNA groups. Images are representative of six independent repeats. (H) Quantitative analysis of the positive area of ORO (%) in each group. Detection of Lp-PLA2 siRNA transfection by (I) western blotting and (J) RT-PCR. THP-1 macrophages were pretreated with Lp-PLA2 siRNA (30 nM) or NC siRNA(30 nM) for 48 h. (K) Western blotting and (L) RT-PCR were conducted to detect the effect of Lp-PLA2 siRNA on inhibiting the expression of Lp-PLA2 in THP-1 macrophages induced by ox-LDL. !P<0.01 vs. control; *P<0.05 vs. ox-LDL; **P<0.01 vs. ox-LDL + PPG; &P<0.01 vs. ox-LDL + Lp-PLA2 siRNA. Lp-PLA2, lipoprotein-associated phospholipase A2; PMA, phorbol-12-myristate-13-acetate; PPG, DL-propargylglycine; ox-LDL, oxidized low-density lipoprotein; ORO, oil red O; RT, reverse transcription; siRNA, small interfering RNA; NC, negative control.