Table 1.
Primers used for Real-time PCR.
Figure 1.
Vasorelaxant effects of TMP on rat aortic rings.
The freshly isolated rat aortic rings were treated with (A) TMP, (B) crocin, (C) ferulic acid and (D) chlorogenic acid at increasing concentrations. After the treatments, both endothelium-intact (E+) and endothelium-denuded (E−) ring relaxation were measured as described in the Methods. Data are mean ± SEM (n = 4–7). ##P<0.01 vs. E+; #P<0.05 vs. E+.
Figure 2.
TMP increased NO production in endothelial cells exposed to high glucose.
DAF-FM indicator was used to detect NO levels in HUVEC treated with (A) 5.6 mmol/L glucose (Control), (B) High glucose (30 mmol/L) and (C) High glucose+TMP (30 µmol/L). The treated cells were photographed by fluorescence microscopy (A–C). (D) bEnd.3 and (E) HUVEC were treated as indicated for 48 hrs, and NO production was measured using flexstation3 and quantified with the changes in fluorescence intensity. “F0” represents fluorescence of Control group and “F” depicts fluorescence of other treated groups. Data are mean ± SEM (n = 4). #P<0.05 vs. Control; ##P<0.01 vs. Control; *P<0.01 vs. High glucose.
Figure 3.
TMP decreased ROS generation in high glucose-treated endothelial cells.
CM-H2DCFDA indicator was used to detect ROS levels in HUVEC treated with (A) 5.6 mmol/L glucose (Control), (B) High glucose (30 mmol/L) and (C) High glucose+TMP (30 µmol/L). (D) bEnd.3 and (E) HUVEC were treated as indicated for 48 hrs, and ROS production was measured using flexstation3 and quantified with the changes in fluorescence intensity. “F0” represents fluorescence of Control group and “F” depicts fluorescence of other treated groups. Data are mean ± SEM (n = 4). #P<0.05 vs. Control; ##P<0.01 vs. Control; **P<0.01 and $$P<0.01 vs. High glucose.
Figure 4.
TMP decreased superoxide anions in high glucose-treated endothelial cells.
Superoxide anions were detected in HUVEC treated with (A) 5.6 mmol/L glucose (Control), (B) High glucose (30 mmol/L), (C) Control+ TMP (30 µmol/L) and (D) High glucose+TMP (30 µmol/L) by using MitoSOX™ Red. Fluorescence intensity was recorded by fluorescence microscopy, and the quantification data are shown (E). Data are mean ± SEM (n = 4). ##P<0.01 vs. Control; *P<0.05 vs. High glucose.
Figure 5.
The protective effects of TMP against mitochondrial dysfunction in endothelial cells.
The bEnd.3 and HUVEC cells were treated with Control (5.6 mmol/L glucose, CTR), High glucose (30 mmol/L, HG), Control+ TMP (30 µmol/L) and High glucose+TMP (30 µmol/L) for 48 hrs. Levels of complex III expression were analyzed by Western blotting in bEnd.3 (A and B) and HUVEC cells (C and D). Representative blots (A and C) and quantification data (B and D) are shown. Mitochondrial potential were determined by JC-1 indicator in the treated bEnd.3 (E) and HUVEC cells (F). Data are mean ± SEM (n = 4). #P<0.05 vs. Control group; ##P<0.01 vs. Control; *P<0.05 vs. High glucose; **P<0.01 vs. High glucose.
Figure 6.
TMP up-regulated mitochondrial biogenesis-related factors.
The bEnd.3 and HUVEC cells were treated with Control (5.6 mmol/L glucose, CTR), High glucose (30 mmol/L, HG), Control+ TMP (30 µmol/L) and High glucose+TMP (30 µmol/L) for 48 hrs. Levels of PGC-1α expression were analyzed by Western blotting in bEnd.3 (A and B) and HUVEC cells (C and D). Representative blots (A and C) and quantification data (B and D) are shown. Levels of PGC-1α, NRF-1 and TFAM mRNA were determined by quantitative RT-PCR in the treated bEnd.3 (E) and HUVEC (F). Data are mean ± SEM (n = 4). #P<0.05 vs. Control; ##P<0.01 vs. Control; *P<0.05 vs. High glucose; **P<0.01 vs. High glucose.
Figure 7.
TMP up-regulated SIRT1 expression.
The bEnd.3 and HUVEC cells were treated with Control (5.6 mmol/L glucose, CTR), High glucose (30 mmol/L, HG), Control+ TMP (30 µmol/L) and High glucose+TMP (30 µmol/L) for 48 hrs. The mRNA levels of SIRT1 were determined by RT-PCR (A and B). Levels of SIRT1 expression were analyzed by Western blotting in bEnd.3 (C and D) and HUVEC cells (E and F). Representative blots (C and E) and quantification data (D and F) are shown. Data are mean ± SEM (n = 4). #P<0.05 vs. Control; ##P<0.01 vs. Control; *P<0.05 vs. High glucose; **P<0.01 vs. High glucose.
Figure 8.
EX-527 abolished TMP-induced PGC-1α up-regulation.
Levels of PGC-1α mRNA were determined by quantitative RT-PCR in the treated bEnd.3 (A) and HUVEC cells (B). Data are mean ± SEM (n = 4). #P<0.05 vs. Control; *P<0.05 vs. High glucose; $P<0.05 vs. High glucose +TMP; $$P<0.01 vs. High glucose +TMP.