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Salvianolic acid B inhibits hydrogen peroxide-induced endothelial cell apoptosis through regulating PI3K/Akt signaling.

Liu CL, Xie LX, Li M, Durairajan SS, Goto S, Huang JD - PLoS ONE (2007)

Bottom Line: By examining the effect of Sal B on H(2)O(2)-induced apoptosis in rat cerebral microvascular endothelial cells (rCMECs), we found that Sal B pretreatment significantly attenuated H(2)O(2)-induced apoptosis in rCMECs.We next examined the signaling cascade(s) involved in Sal B-mediated anti-apoptotic effects.On the other hand, blockage of the PI3K/Akt pathway abrogated the protective effect conferred by Sal B and potentated H(2)O(2)-induced apoptosis, suggesting that Sal B prevents H(2)O(2)-induced apoptosis predominantly through the PI3K/Akt (upstream of ERK) pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China.

ABSTRACT

Background: Salvianolic acid B (Sal B) is one of the most bioactive components of Salvia miltiorrhiza, a traditional Chinese herbal medicine that has been commonly used for prevention and treatment of cerebrovascular disorders. However, the mechanism responsible for such protective effects remains largely unknown. It has been considered that cerebral endothelium apoptosis caused by reactive oxygen species including hydrogen peroxide (H(2)O(2)) is implicated in the pathogenesis of cerebrovascular disorders.

Methodology and principal findings: By examining the effect of Sal B on H(2)O(2)-induced apoptosis in rat cerebral microvascular endothelial cells (rCMECs), we found that Sal B pretreatment significantly attenuated H(2)O(2)-induced apoptosis in rCMECs. We next examined the signaling cascade(s) involved in Sal B-mediated anti-apoptotic effects. We showed that H(2)O(2) induces rCMECs apoptosis mainly through the PI3K/ERK pathway, since a PI3K inhibitor (LY294002) blocked ERK activation caused by H(2)O(2 )and a specific inhibitor of MEK (U0126) protected cells from apoptosis. On the other hand, blockage of the PI3K/Akt pathway abrogated the protective effect conferred by Sal B and potentated H(2)O(2)-induced apoptosis, suggesting that Sal B prevents H(2)O(2)-induced apoptosis predominantly through the PI3K/Akt (upstream of ERK) pathway.

Significance: Our findings provide the first evidence that H(2)O(2) induces rCMECs apoptosis via the PI3K/MEK/ERK pathway and that Sal B protects rCMECs against H(2)O(2)-induced apoptosis through the PI3K/Akt/Raf/MEK/ERK pathway.

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Role of PI3K signaling.(A) Effects of PI3K or MEK inhibition on phosphorylated Akt (Ser-473) and phosphorylated ERK1/2 expression in the presence or absence of H2O2 (200 µM). rCMECs were incubated with LY294002 (50 µM) or U0126 (10 µM) for 1 h and then exposed to H2O2 for 30 min. Blot shown is representative of at least three independent experiments. (B) Phosphorylated Akt (Ser-473) and phosphorylated c-Raf (Ser-259) expressions in rCMECs incubated with Sal B (20 µM) for the indicated times. (C) Effect of PI3K inhibition on phosphorylated Akt (Ser-473) and phosphorylated c-Raf (Ser-259) expressions induced by Sal B. rCMECs were incubated with LY294002 (50 µM) and/or Sal B (20 µM) for 1 h. (D) Effect of c-Raf inhibition on phosphorylated Akt (Ser-473) and phosphorylated ERK1/2 activation induced by H2O2. rCMECs were incubated with GW5074 (5 µM) and/or H2O2 (200 µM) for 1 h. (E) Effect of PI3K inhibition on H2O2-induced apoptosis in the presence or absence of Sal B (20 µM). rCMECs were incubated with LY294002 (50 µM) and/or Sal B (20 µM) for 1 h and then exposed to H2O2 for 12 h. LY, indicates LY294002. S, indicates Sal B. *P<0.05; **P<0.01 versus H2O2 alone; n.s., not significant. Data are representative of three independent experiments.
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pone-0001321-g003: Role of PI3K signaling.(A) Effects of PI3K or MEK inhibition on phosphorylated Akt (Ser-473) and phosphorylated ERK1/2 expression in the presence or absence of H2O2 (200 µM). rCMECs were incubated with LY294002 (50 µM) or U0126 (10 µM) for 1 h and then exposed to H2O2 for 30 min. Blot shown is representative of at least three independent experiments. (B) Phosphorylated Akt (Ser-473) and phosphorylated c-Raf (Ser-259) expressions in rCMECs incubated with Sal B (20 µM) for the indicated times. (C) Effect of PI3K inhibition on phosphorylated Akt (Ser-473) and phosphorylated c-Raf (Ser-259) expressions induced by Sal B. rCMECs were incubated with LY294002 (50 µM) and/or Sal B (20 µM) for 1 h. (D) Effect of c-Raf inhibition on phosphorylated Akt (Ser-473) and phosphorylated ERK1/2 activation induced by H2O2. rCMECs were incubated with GW5074 (5 µM) and/or H2O2 (200 µM) for 1 h. (E) Effect of PI3K inhibition on H2O2-induced apoptosis in the presence or absence of Sal B (20 µM). rCMECs were incubated with LY294002 (50 µM) and/or Sal B (20 µM) for 1 h and then exposed to H2O2 for 12 h. LY, indicates LY294002. S, indicates Sal B. *P<0.05; **P<0.01 versus H2O2 alone; n.s., not significant. Data are representative of three independent experiments.

Mentions: We next examined the effect of Akt inhibition on ERK phosphorylation in rCMECs exposed to H2O2. Treatment with LY294002, a specific inhibitor of Akt upstream kinase PI3K [24], resulted in the blockage of H2O2-induced ERK phosphorylation, as well as basal and H2O2-induced Akt phosphorylation. The basal level of ERK phosphorylation was also diminished (Fig. 3A). In the presence of U0126, basal and H2O2-induced ERK phosphorylation were blocked. However, U0126 had no effect on either basal or H2O2-induced Akt phosphorylation (Fig. 3A). These data clearly illustrate that PI3K acts upstream of ERK in the H2O2-induced signaling cascade. Previous studies have shown that Akt inhibited activation of the MEK/ERK signaling pathway by phosphorylating c-Raf at residue Ser-259 [25]. To investigate whether in rCMECs Sal B inhibited H2O2-induced MEK/ERK activation through Akt, we therefore evaluated the effect of Sal B on Akt activation. Results showed that the phosphorylation of Akt peaked at 15 min in the cells incubated with Sal B alone, and then returned to basal level over 60 min (Fig. 3B). An elevated level of phosphorylated c-Raf at Ser-259 was also triggered by Sal B alone (Fig. 3B). Furthermore, LY294002 treatment completely blocked expressions of phosphorylated Akt (Ser-473) and c-Raf (Ser-259) induced by Sal B (Fig. 3C). This indicates PI3K is required for Sal B-induced Akt activation and c-Raf deactivation. Since c-Raf is known to lie downstream of Akt, and upstream of ERK [25], [26], we then sought to confirm that this was also the case in rCMECs. GW5074, a selective inhibitor of c-Raf, inhibits the Raf/MEK/ERK cascade in in vitro assays by 90% at 5 µM [27]. Treatment with GW5074 had no effect on either basal or H2O2-induced Akt phosphorylation (Fig. 3D), but blocked H2O2-induced ERK phosphorylation (Fig. 3D). To further determine if the anti-apoptotic effects of Sal B were due to its effect on Akt, rCMECs were incubated with LY294002, with and without Sal B prior to H2O2 treatment. Inhibition of PI3K completely ablated the anti-apoptotic effect of Sal B, as well as H2O2-induced apoptosis was potentiated (Fig. 3E). Thus, these results indicate that Sal B prevents H2O2-induced rCMECs apoptosis, at least in part, by altering PI3K/Akt/Raf/MEK/ERK activation.


Salvianolic acid B inhibits hydrogen peroxide-induced endothelial cell apoptosis through regulating PI3K/Akt signaling.

Liu CL, Xie LX, Li M, Durairajan SS, Goto S, Huang JD - PLoS ONE (2007)

Role of PI3K signaling.(A) Effects of PI3K or MEK inhibition on phosphorylated Akt (Ser-473) and phosphorylated ERK1/2 expression in the presence or absence of H2O2 (200 µM). rCMECs were incubated with LY294002 (50 µM) or U0126 (10 µM) for 1 h and then exposed to H2O2 for 30 min. Blot shown is representative of at least three independent experiments. (B) Phosphorylated Akt (Ser-473) and phosphorylated c-Raf (Ser-259) expressions in rCMECs incubated with Sal B (20 µM) for the indicated times. (C) Effect of PI3K inhibition on phosphorylated Akt (Ser-473) and phosphorylated c-Raf (Ser-259) expressions induced by Sal B. rCMECs were incubated with LY294002 (50 µM) and/or Sal B (20 µM) for 1 h. (D) Effect of c-Raf inhibition on phosphorylated Akt (Ser-473) and phosphorylated ERK1/2 activation induced by H2O2. rCMECs were incubated with GW5074 (5 µM) and/or H2O2 (200 µM) for 1 h. (E) Effect of PI3K inhibition on H2O2-induced apoptosis in the presence or absence of Sal B (20 µM). rCMECs were incubated with LY294002 (50 µM) and/or Sal B (20 µM) for 1 h and then exposed to H2O2 for 12 h. LY, indicates LY294002. S, indicates Sal B. *P<0.05; **P<0.01 versus H2O2 alone; n.s., not significant. Data are representative of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2117346&req=5

pone-0001321-g003: Role of PI3K signaling.(A) Effects of PI3K or MEK inhibition on phosphorylated Akt (Ser-473) and phosphorylated ERK1/2 expression in the presence or absence of H2O2 (200 µM). rCMECs were incubated with LY294002 (50 µM) or U0126 (10 µM) for 1 h and then exposed to H2O2 for 30 min. Blot shown is representative of at least three independent experiments. (B) Phosphorylated Akt (Ser-473) and phosphorylated c-Raf (Ser-259) expressions in rCMECs incubated with Sal B (20 µM) for the indicated times. (C) Effect of PI3K inhibition on phosphorylated Akt (Ser-473) and phosphorylated c-Raf (Ser-259) expressions induced by Sal B. rCMECs were incubated with LY294002 (50 µM) and/or Sal B (20 µM) for 1 h. (D) Effect of c-Raf inhibition on phosphorylated Akt (Ser-473) and phosphorylated ERK1/2 activation induced by H2O2. rCMECs were incubated with GW5074 (5 µM) and/or H2O2 (200 µM) for 1 h. (E) Effect of PI3K inhibition on H2O2-induced apoptosis in the presence or absence of Sal B (20 µM). rCMECs were incubated with LY294002 (50 µM) and/or Sal B (20 µM) for 1 h and then exposed to H2O2 for 12 h. LY, indicates LY294002. S, indicates Sal B. *P<0.05; **P<0.01 versus H2O2 alone; n.s., not significant. Data are representative of three independent experiments.
Mentions: We next examined the effect of Akt inhibition on ERK phosphorylation in rCMECs exposed to H2O2. Treatment with LY294002, a specific inhibitor of Akt upstream kinase PI3K [24], resulted in the blockage of H2O2-induced ERK phosphorylation, as well as basal and H2O2-induced Akt phosphorylation. The basal level of ERK phosphorylation was also diminished (Fig. 3A). In the presence of U0126, basal and H2O2-induced ERK phosphorylation were blocked. However, U0126 had no effect on either basal or H2O2-induced Akt phosphorylation (Fig. 3A). These data clearly illustrate that PI3K acts upstream of ERK in the H2O2-induced signaling cascade. Previous studies have shown that Akt inhibited activation of the MEK/ERK signaling pathway by phosphorylating c-Raf at residue Ser-259 [25]. To investigate whether in rCMECs Sal B inhibited H2O2-induced MEK/ERK activation through Akt, we therefore evaluated the effect of Sal B on Akt activation. Results showed that the phosphorylation of Akt peaked at 15 min in the cells incubated with Sal B alone, and then returned to basal level over 60 min (Fig. 3B). An elevated level of phosphorylated c-Raf at Ser-259 was also triggered by Sal B alone (Fig. 3B). Furthermore, LY294002 treatment completely blocked expressions of phosphorylated Akt (Ser-473) and c-Raf (Ser-259) induced by Sal B (Fig. 3C). This indicates PI3K is required for Sal B-induced Akt activation and c-Raf deactivation. Since c-Raf is known to lie downstream of Akt, and upstream of ERK [25], [26], we then sought to confirm that this was also the case in rCMECs. GW5074, a selective inhibitor of c-Raf, inhibits the Raf/MEK/ERK cascade in in vitro assays by 90% at 5 µM [27]. Treatment with GW5074 had no effect on either basal or H2O2-induced Akt phosphorylation (Fig. 3D), but blocked H2O2-induced ERK phosphorylation (Fig. 3D). To further determine if the anti-apoptotic effects of Sal B were due to its effect on Akt, rCMECs were incubated with LY294002, with and without Sal B prior to H2O2 treatment. Inhibition of PI3K completely ablated the anti-apoptotic effect of Sal B, as well as H2O2-induced apoptosis was potentiated (Fig. 3E). Thus, these results indicate that Sal B prevents H2O2-induced rCMECs apoptosis, at least in part, by altering PI3K/Akt/Raf/MEK/ERK activation.

Bottom Line: By examining the effect of Sal B on H(2)O(2)-induced apoptosis in rat cerebral microvascular endothelial cells (rCMECs), we found that Sal B pretreatment significantly attenuated H(2)O(2)-induced apoptosis in rCMECs.We next examined the signaling cascade(s) involved in Sal B-mediated anti-apoptotic effects.On the other hand, blockage of the PI3K/Akt pathway abrogated the protective effect conferred by Sal B and potentated H(2)O(2)-induced apoptosis, suggesting that Sal B prevents H(2)O(2)-induced apoptosis predominantly through the PI3K/Akt (upstream of ERK) pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China.

ABSTRACT

Background: Salvianolic acid B (Sal B) is one of the most bioactive components of Salvia miltiorrhiza, a traditional Chinese herbal medicine that has been commonly used for prevention and treatment of cerebrovascular disorders. However, the mechanism responsible for such protective effects remains largely unknown. It has been considered that cerebral endothelium apoptosis caused by reactive oxygen species including hydrogen peroxide (H(2)O(2)) is implicated in the pathogenesis of cerebrovascular disorders.

Methodology and principal findings: By examining the effect of Sal B on H(2)O(2)-induced apoptosis in rat cerebral microvascular endothelial cells (rCMECs), we found that Sal B pretreatment significantly attenuated H(2)O(2)-induced apoptosis in rCMECs. We next examined the signaling cascade(s) involved in Sal B-mediated anti-apoptotic effects. We showed that H(2)O(2) induces rCMECs apoptosis mainly through the PI3K/ERK pathway, since a PI3K inhibitor (LY294002) blocked ERK activation caused by H(2)O(2 )and a specific inhibitor of MEK (U0126) protected cells from apoptosis. On the other hand, blockage of the PI3K/Akt pathway abrogated the protective effect conferred by Sal B and potentated H(2)O(2)-induced apoptosis, suggesting that Sal B prevents H(2)O(2)-induced apoptosis predominantly through the PI3K/Akt (upstream of ERK) pathway.

Significance: Our findings provide the first evidence that H(2)O(2) induces rCMECs apoptosis via the PI3K/MEK/ERK pathway and that Sal B protects rCMECs against H(2)O(2)-induced apoptosis through the PI3K/Akt/Raf/MEK/ERK pathway.

Show MeSH
Related in: MedlinePlus