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Inhibitory effects of lithospermic acid on proliferation and migration of rat vascular smooth muscle cells.

Chen L, Wang WY, Wang YP - Acta Pharmacol. Sin. (2009)

Bottom Line: To understand the effects of lithospermic acid (LA), a potent antioxidant from the water-soluble extract of Salvia miltiorrhiza, on the migration and proliferation of rat thoracic aorta vascular smooth muscle cells (VSMCs).Furthermore, LA attenuated LPS-induced VSMC migration by inhibiting MMP-9 expression and its enzymatic activity.LA is able to inhibit FBS-induced VSMC proliferation and LPS-induced VSMC migration, which suggests that LA may have therapeutic effects in the prevention of atherosclerosis, restenosis and neointimal hyperplasia.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of New Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

Aim: To understand the effects of lithospermic acid (LA), a potent antioxidant from the water-soluble extract of Salvia miltiorrhiza, on the migration and proliferation of rat thoracic aorta vascular smooth muscle cells (VSMCs).

Methods: VSMC migration, proliferation, DNA synthesis and cell cycle progression were investigated by transwell migration analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, bromodeoxyuridine (BrdU) incorporation assay, and flow cytometric detection, respectively. Intracellular reactive oxygen species (ROS) generation was detected using 2',7'-dichlorofluorescin diacetate (DCFH-DA). The expression of cyclin D1 protein and matrix metalloproteinase-9 (MMP-9) protein, as well as the phosphorylation state of ERK1/2, were determined using Western blots. The activity of MMP-9 and the expression of MMP-9 mRNA were assessed by gelatin zymography analysis and RT-PCR, respectively.

Results: LA (25-100 micromol/L) inhibited both lipopolysaccharide (LPS)- and fetal bovine serum (FBS)-induced ROS generation and ERK1/2 phosphorylation. By down-regulating the expression of cyclin D(1) and arresting cell cycle progression at the G(1) phase, LA inhibited both VSMC proliferation and DNA synthesis as induced by 5% FBS. Furthermore, LA attenuated LPS-induced VSMC migration by inhibiting MMP-9 expression and its enzymatic activity.

Conclusion: LA is able to inhibit FBS-induced VSMC proliferation and LPS-induced VSMC migration, which suggests that LA may have therapeutic effects in the prevention of atherosclerosis, restenosis and neointimal hyperplasia.

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Effects of LA on FBS-induced proliferation and DNA synthesis in VSMCs. (A) Proliferation activities were measured by MTT assay in the absence (left) or presence (right) of 5% FBS. Relative proliferation activities were expressed using untreated control cells as a standard. n=6. (B) DNA synthesis was measured by BrdU incorporation assay. The left part of the diagram shows BrdU incorporation of quiescent and FBS-stimulated VSMCs. On the right side, a concentration-dependent decrease of BrdU incorporation in LA-treated VSMCs is shown. n=6. Values are presented as mean±SEM. cP<0.01 vs control. eP<0.05, fP<0.01 vs FBS.
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fig4: Effects of LA on FBS-induced proliferation and DNA synthesis in VSMCs. (A) Proliferation activities were measured by MTT assay in the absence (left) or presence (right) of 5% FBS. Relative proliferation activities were expressed using untreated control cells as a standard. n=6. (B) DNA synthesis was measured by BrdU incorporation assay. The left part of the diagram shows BrdU incorporation of quiescent and FBS-stimulated VSMCs. On the right side, a concentration-dependent decrease of BrdU incorporation in LA-treated VSMCs is shown. n=6. Values are presented as mean±SEM. cP<0.01 vs control. eP<0.05, fP<0.01 vs FBS.

Mentions: We next studied the effect of LA on the proliferation of VSMCs using the MTT assay. When growth-arrested cells were treated with LA (25, 50, 100 μmol/L) in the presence of 0.1% FBS, no significant difference was observed in cell viability (Figure 4A, left panel), suggesting that LA did not show significant cytotoxicity up to 100 μmol/L. The absence of cytotoxicity was further confirmed with a trypan blue exclusion assay (data not shown). Moreover, we found that FBS induced a 2.21-fold increase in the proliferation of VSMCs. Treatment of cells with LA 2 h before stimulation with FBS reduced cell proliferation in a concentration-dependent manner (Figure 4A, right panel). We further examined the effect of LA on DNA synthesis, as induced by 5% FBS in VSMCs using the BrdU incorporation assay. BrdU incorporation was markedly increased in VSMCs exposed to 5% FBS for 48 h (P<0.01), indicating an increase in DNA synthesis. However, this effect was abolished in VSMCs pretreated with LA (Figure 4B).


Inhibitory effects of lithospermic acid on proliferation and migration of rat vascular smooth muscle cells.

Chen L, Wang WY, Wang YP - Acta Pharmacol. Sin. (2009)

Effects of LA on FBS-induced proliferation and DNA synthesis in VSMCs. (A) Proliferation activities were measured by MTT assay in the absence (left) or presence (right) of 5% FBS. Relative proliferation activities were expressed using untreated control cells as a standard. n=6. (B) DNA synthesis was measured by BrdU incorporation assay. The left part of the diagram shows BrdU incorporation of quiescent and FBS-stimulated VSMCs. On the right side, a concentration-dependent decrease of BrdU incorporation in LA-treated VSMCs is shown. n=6. Values are presented as mean±SEM. cP<0.01 vs control. eP<0.05, fP<0.01 vs FBS.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4007173&req=5

fig4: Effects of LA on FBS-induced proliferation and DNA synthesis in VSMCs. (A) Proliferation activities were measured by MTT assay in the absence (left) or presence (right) of 5% FBS. Relative proliferation activities were expressed using untreated control cells as a standard. n=6. (B) DNA synthesis was measured by BrdU incorporation assay. The left part of the diagram shows BrdU incorporation of quiescent and FBS-stimulated VSMCs. On the right side, a concentration-dependent decrease of BrdU incorporation in LA-treated VSMCs is shown. n=6. Values are presented as mean±SEM. cP<0.01 vs control. eP<0.05, fP<0.01 vs FBS.
Mentions: We next studied the effect of LA on the proliferation of VSMCs using the MTT assay. When growth-arrested cells were treated with LA (25, 50, 100 μmol/L) in the presence of 0.1% FBS, no significant difference was observed in cell viability (Figure 4A, left panel), suggesting that LA did not show significant cytotoxicity up to 100 μmol/L. The absence of cytotoxicity was further confirmed with a trypan blue exclusion assay (data not shown). Moreover, we found that FBS induced a 2.21-fold increase in the proliferation of VSMCs. Treatment of cells with LA 2 h before stimulation with FBS reduced cell proliferation in a concentration-dependent manner (Figure 4A, right panel). We further examined the effect of LA on DNA synthesis, as induced by 5% FBS in VSMCs using the BrdU incorporation assay. BrdU incorporation was markedly increased in VSMCs exposed to 5% FBS for 48 h (P<0.01), indicating an increase in DNA synthesis. However, this effect was abolished in VSMCs pretreated with LA (Figure 4B).

Bottom Line: To understand the effects of lithospermic acid (LA), a potent antioxidant from the water-soluble extract of Salvia miltiorrhiza, on the migration and proliferation of rat thoracic aorta vascular smooth muscle cells (VSMCs).Furthermore, LA attenuated LPS-induced VSMC migration by inhibiting MMP-9 expression and its enzymatic activity.LA is able to inhibit FBS-induced VSMC proliferation and LPS-induced VSMC migration, which suggests that LA may have therapeutic effects in the prevention of atherosclerosis, restenosis and neointimal hyperplasia.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of New Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

Aim: To understand the effects of lithospermic acid (LA), a potent antioxidant from the water-soluble extract of Salvia miltiorrhiza, on the migration and proliferation of rat thoracic aorta vascular smooth muscle cells (VSMCs).

Methods: VSMC migration, proliferation, DNA synthesis and cell cycle progression were investigated by transwell migration analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, bromodeoxyuridine (BrdU) incorporation assay, and flow cytometric detection, respectively. Intracellular reactive oxygen species (ROS) generation was detected using 2',7'-dichlorofluorescin diacetate (DCFH-DA). The expression of cyclin D1 protein and matrix metalloproteinase-9 (MMP-9) protein, as well as the phosphorylation state of ERK1/2, were determined using Western blots. The activity of MMP-9 and the expression of MMP-9 mRNA were assessed by gelatin zymography analysis and RT-PCR, respectively.

Results: LA (25-100 micromol/L) inhibited both lipopolysaccharide (LPS)- and fetal bovine serum (FBS)-induced ROS generation and ERK1/2 phosphorylation. By down-regulating the expression of cyclin D(1) and arresting cell cycle progression at the G(1) phase, LA inhibited both VSMC proliferation and DNA synthesis as induced by 5% FBS. Furthermore, LA attenuated LPS-induced VSMC migration by inhibiting MMP-9 expression and its enzymatic activity.

Conclusion: LA is able to inhibit FBS-induced VSMC proliferation and LPS-induced VSMC migration, which suggests that LA may have therapeutic effects in the prevention of atherosclerosis, restenosis and neointimal hyperplasia.

Show MeSH
Related in: MedlinePlus