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Cyclo(His-Pro) promotes cytoprotection by activating Nrf2-mediated up-regulation of antioxidant defence.

Minelli A, Conte C, Grottelli S, Bellezza I, Cacciatore I, Bolaños JP - J. Cell. Mol. Med. (2008)

Bottom Line: Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells.Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown.These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento Medicina Sperimentale Scienze Biochimiche, Università di Perugia, Italy. aminelli@unipg.it <aminelli@unipg.it>

ABSTRACT
Hystidyl-proline [cyclo(His-Pro)] is an endogenous cyclic dipeptide produced by the cleavage of thyrotropin releasing hormone. Previous studies have shown that cyclo(His-Pro) protects against oxidative stress, although the underlying mechanism has remained elusive. Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells. Cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion caused by glutamate, rotenone, paraquat and beta-amyloid treatment. Moreover, real-time PCR analyses revealed that cyclo(His-Pro) induced the expression of a number of ARE-related genes and protected cells against hydrogen peroxide-mediated apoptotic death. Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown. Finally, pharmacological inhibition of p-38 MAPK partially prevented both cyclo(His-Pro)-mediated Nrf2 activation and cellular protection. These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence.

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Cyclo(His-Pro) protects PC12 cells against H2O2-induced apoptotic death, oxidative/nitrosative stress and calcium accumulation. (A) PC12 cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs, then H2O2 was added at the indicated concentrations and the analyses performed as described. Reduced MTT data in control cells (absorbance at 550 nm = 1.66 ± 0.2) were considered as 100%. Results are expressed as the mean ± S.D. (n= 8). *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells. (B) PC12 cells were incubated with increasing cyclo(His-Pro) concentrations for 24 hrs, then 100 μM or 500 μM H2O2 were added and the analyses performed as described. Results are expressed as the mean ± S.D. (n= 8). *P< 0.05 versus control cells (100 μM H2O2); #P< 0.05 versus control cells (500 μM H2O2). (C) PC12 cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs, then 100 (jlM H2O2 was added and condensed and/fragmented nuclei were determined by Hoechst staining and expressed as percentages of control (no treatment). Results are given as the mean ± S.D. (n= 8). *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells. (D). PC12 cells were treated with 100 (jlM H2O2, then 50 μM cyclo(His-Pro) was added after the indicated time intervals. Reduced MTT data in H2O2-treated cells were considered as 100%. Results are expressed as the mean ± S.D. (n= 5). *P< 0.05 versus H2O2-treated cells. (E) Cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs, then 100 μM of H2O2 was added and the analyses performed as described. Values (mean ± S.D., n = 7), given as percentages of control (no treatment; considered as 100%), were: NO (absorbance values at 550 nm): 0.8 ± 0.02; glutathione (GSH, in μM): 4.5 ± 0.1; ROS (DCF fluorescence): 0.9 ± 0.2; *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells. (F) Cells were incubated for 24 hrs either in the absence or in the presence of 50 /jJM cyclo(His-Pro), loaded with 3 μM Fura-2/AM in Krebs-Ringer HEPES (KRH), and incubated with 100 μM H2O2. Intracellular Ca2+ was measured by Fura-2/AM F340/F380 fluorescence ratio. Data are presented as mean ± S.D., (n= 5). *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells.
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fig02: Cyclo(His-Pro) protects PC12 cells against H2O2-induced apoptotic death, oxidative/nitrosative stress and calcium accumulation. (A) PC12 cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs, then H2O2 was added at the indicated concentrations and the analyses performed as described. Reduced MTT data in control cells (absorbance at 550 nm = 1.66 ± 0.2) were considered as 100%. Results are expressed as the mean ± S.D. (n= 8). *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells. (B) PC12 cells were incubated with increasing cyclo(His-Pro) concentrations for 24 hrs, then 100 μM or 500 μM H2O2 were added and the analyses performed as described. Results are expressed as the mean ± S.D. (n= 8). *P< 0.05 versus control cells (100 μM H2O2); #P< 0.05 versus control cells (500 μM H2O2). (C) PC12 cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs, then 100 (jlM H2O2 was added and condensed and/fragmented nuclei were determined by Hoechst staining and expressed as percentages of control (no treatment). Results are given as the mean ± S.D. (n= 8). *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells. (D). PC12 cells were treated with 100 (jlM H2O2, then 50 μM cyclo(His-Pro) was added after the indicated time intervals. Reduced MTT data in H2O2-treated cells were considered as 100%. Results are expressed as the mean ± S.D. (n= 5). *P< 0.05 versus H2O2-treated cells. (E) Cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs, then 100 μM of H2O2 was added and the analyses performed as described. Values (mean ± S.D., n = 7), given as percentages of control (no treatment; considered as 100%), were: NO (absorbance values at 550 nm): 0.8 ± 0.02; glutathione (GSH, in μM): 4.5 ± 0.1; ROS (DCF fluorescence): 0.9 ± 0.2; *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells. (F) Cells were incubated for 24 hrs either in the absence or in the presence of 50 /jJM cyclo(His-Pro), loaded with 3 μM Fura-2/AM in Krebs-Ringer HEPES (KRH), and incubated with 100 μM H2O2. Intracellular Ca2+ was measured by Fura-2/AM F340/F380 fluorescence ratio. Data are presented as mean ± S.D., (n= 5). *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells.

Mentions: In a previous study, we reported that cyclo(His-Pro) can prevent PC12 cells from serum deprivation-mediated apoptotic death [5]. To investigate whether such cytoprotection could be ascribed an antioxidant effect, we first assessed the effect of cyclic dipeptide on H2O2-mediated PC12 cell injury. Cytotoxicity induced by H2O2 was initially measured by determining MTT reduction in cells incubated with increasing concentrations (50–500 μM) of H2O2. As shown in Figure 2A, treatment of PC12 cells with cyclo(His-Pro) abolished H2O2-mediated cytotoxicity, an effect that was dose-dependent (Fig. 2B). Analogues of cyclo(His-Pro), i.e. cyclo-(His-His), cyclo-(Leu-Pro) and cyclo-(Gly-His) showed no cytoprotection (data not shown). Morphological analysis of PC12 cells treated with 100 μM H2O2 exhibited the typical phenotypic changes due to apoptotic cell death, i.e. cell shrinkage and membrane bleb-bing (not shown). Furthermore, quantification of cells exhibiting fragmented DNA revealed that cyclo(His-Pro) fully prevented apoptotic cell death (Fig. 2C). Treatment of PC12 cells with cyclo(His-Pro) after H2O2- injury resulted in cytoprotection, at least up to 12 hrs (Fig. 2D). Next, we assessed whether cyclo(His-Pro) modifed cellular redox status. Thus, H2O2 induced a significant increase in ROS and NO production, as well as a marked decrease in intracellular GSH concentrations (Fig. 2E), an effect that was fully prevented by cyclo(His-Pro) (50 μM). Since oxidative stress is accompanied by the occurrence of large increases in intracellular calcium [17–19], we next determined intracellular calcium levels by fluorescence using Fura-2 AM. As shown in Figure 2E, H2O2 (100 μM) increased the fluorescence ratio (F340/F380) from 100% in controls to 248 ± 20% in H2O2-exposed cells, whereas pre-treatment with cyclo(His-Pro) (50 μM), led F340/F380 ratio to decrease to 158 ± 18%. As negative control, we assessed the F340/F380 ratio in calcium-free Krebs’ solution, resulting in no alterations under either condition (Fig. 2E).


Cyclo(His-Pro) promotes cytoprotection by activating Nrf2-mediated up-regulation of antioxidant defence.

Minelli A, Conte C, Grottelli S, Bellezza I, Cacciatore I, Bolaños JP - J. Cell. Mol. Med. (2008)

Cyclo(His-Pro) protects PC12 cells against H2O2-induced apoptotic death, oxidative/nitrosative stress and calcium accumulation. (A) PC12 cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs, then H2O2 was added at the indicated concentrations and the analyses performed as described. Reduced MTT data in control cells (absorbance at 550 nm = 1.66 ± 0.2) were considered as 100%. Results are expressed as the mean ± S.D. (n= 8). *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells. (B) PC12 cells were incubated with increasing cyclo(His-Pro) concentrations for 24 hrs, then 100 μM or 500 μM H2O2 were added and the analyses performed as described. Results are expressed as the mean ± S.D. (n= 8). *P< 0.05 versus control cells (100 μM H2O2); #P< 0.05 versus control cells (500 μM H2O2). (C) PC12 cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs, then 100 (jlM H2O2 was added and condensed and/fragmented nuclei were determined by Hoechst staining and expressed as percentages of control (no treatment). Results are given as the mean ± S.D. (n= 8). *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells. (D). PC12 cells were treated with 100 (jlM H2O2, then 50 μM cyclo(His-Pro) was added after the indicated time intervals. Reduced MTT data in H2O2-treated cells were considered as 100%. Results are expressed as the mean ± S.D. (n= 5). *P< 0.05 versus H2O2-treated cells. (E) Cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs, then 100 μM of H2O2 was added and the analyses performed as described. Values (mean ± S.D., n = 7), given as percentages of control (no treatment; considered as 100%), were: NO (absorbance values at 550 nm): 0.8 ± 0.02; glutathione (GSH, in μM): 4.5 ± 0.1; ROS (DCF fluorescence): 0.9 ± 0.2; *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells. (F) Cells were incubated for 24 hrs either in the absence or in the presence of 50 /jJM cyclo(His-Pro), loaded with 3 μM Fura-2/AM in Krebs-Ringer HEPES (KRH), and incubated with 100 μM H2O2. Intracellular Ca2+ was measured by Fura-2/AM F340/F380 fluorescence ratio. Data are presented as mean ± S.D., (n= 5). *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells.
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fig02: Cyclo(His-Pro) protects PC12 cells against H2O2-induced apoptotic death, oxidative/nitrosative stress and calcium accumulation. (A) PC12 cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs, then H2O2 was added at the indicated concentrations and the analyses performed as described. Reduced MTT data in control cells (absorbance at 550 nm = 1.66 ± 0.2) were considered as 100%. Results are expressed as the mean ± S.D. (n= 8). *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells. (B) PC12 cells were incubated with increasing cyclo(His-Pro) concentrations for 24 hrs, then 100 μM or 500 μM H2O2 were added and the analyses performed as described. Results are expressed as the mean ± S.D. (n= 8). *P< 0.05 versus control cells (100 μM H2O2); #P< 0.05 versus control cells (500 μM H2O2). (C) PC12 cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs, then 100 (jlM H2O2 was added and condensed and/fragmented nuclei were determined by Hoechst staining and expressed as percentages of control (no treatment). Results are given as the mean ± S.D. (n= 8). *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells. (D). PC12 cells were treated with 100 (jlM H2O2, then 50 μM cyclo(His-Pro) was added after the indicated time intervals. Reduced MTT data in H2O2-treated cells were considered as 100%. Results are expressed as the mean ± S.D. (n= 5). *P< 0.05 versus H2O2-treated cells. (E) Cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs, then 100 μM of H2O2 was added and the analyses performed as described. Values (mean ± S.D., n = 7), given as percentages of control (no treatment; considered as 100%), were: NO (absorbance values at 550 nm): 0.8 ± 0.02; glutathione (GSH, in μM): 4.5 ± 0.1; ROS (DCF fluorescence): 0.9 ± 0.2; *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells. (F) Cells were incubated for 24 hrs either in the absence or in the presence of 50 /jJM cyclo(His-Pro), loaded with 3 μM Fura-2/AM in Krebs-Ringer HEPES (KRH), and incubated with 100 μM H2O2. Intracellular Ca2+ was measured by Fura-2/AM F340/F380 fluorescence ratio. Data are presented as mean ± S.D., (n= 5). *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells.
Mentions: In a previous study, we reported that cyclo(His-Pro) can prevent PC12 cells from serum deprivation-mediated apoptotic death [5]. To investigate whether such cytoprotection could be ascribed an antioxidant effect, we first assessed the effect of cyclic dipeptide on H2O2-mediated PC12 cell injury. Cytotoxicity induced by H2O2 was initially measured by determining MTT reduction in cells incubated with increasing concentrations (50–500 μM) of H2O2. As shown in Figure 2A, treatment of PC12 cells with cyclo(His-Pro) abolished H2O2-mediated cytotoxicity, an effect that was dose-dependent (Fig. 2B). Analogues of cyclo(His-Pro), i.e. cyclo-(His-His), cyclo-(Leu-Pro) and cyclo-(Gly-His) showed no cytoprotection (data not shown). Morphological analysis of PC12 cells treated with 100 μM H2O2 exhibited the typical phenotypic changes due to apoptotic cell death, i.e. cell shrinkage and membrane bleb-bing (not shown). Furthermore, quantification of cells exhibiting fragmented DNA revealed that cyclo(His-Pro) fully prevented apoptotic cell death (Fig. 2C). Treatment of PC12 cells with cyclo(His-Pro) after H2O2- injury resulted in cytoprotection, at least up to 12 hrs (Fig. 2D). Next, we assessed whether cyclo(His-Pro) modifed cellular redox status. Thus, H2O2 induced a significant increase in ROS and NO production, as well as a marked decrease in intracellular GSH concentrations (Fig. 2E), an effect that was fully prevented by cyclo(His-Pro) (50 μM). Since oxidative stress is accompanied by the occurrence of large increases in intracellular calcium [17–19], we next determined intracellular calcium levels by fluorescence using Fura-2 AM. As shown in Figure 2E, H2O2 (100 μM) increased the fluorescence ratio (F340/F380) from 100% in controls to 248 ± 20% in H2O2-exposed cells, whereas pre-treatment with cyclo(His-Pro) (50 μM), led F340/F380 ratio to decrease to 158 ± 18%. As negative control, we assessed the F340/F380 ratio in calcium-free Krebs’ solution, resulting in no alterations under either condition (Fig. 2E).

Bottom Line: Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells.Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown.These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento Medicina Sperimentale Scienze Biochimiche, Università di Perugia, Italy. aminelli@unipg.it <aminelli@unipg.it>

ABSTRACT
Hystidyl-proline [cyclo(His-Pro)] is an endogenous cyclic dipeptide produced by the cleavage of thyrotropin releasing hormone. Previous studies have shown that cyclo(His-Pro) protects against oxidative stress, although the underlying mechanism has remained elusive. Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells. Cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion caused by glutamate, rotenone, paraquat and beta-amyloid treatment. Moreover, real-time PCR analyses revealed that cyclo(His-Pro) induced the expression of a number of ARE-related genes and protected cells against hydrogen peroxide-mediated apoptotic death. Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown. Finally, pharmacological inhibition of p-38 MAPK partially prevented both cyclo(His-Pro)-mediated Nrf2 activation and cellular protection. These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence.

Show MeSH
Related in: MedlinePlus