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

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.

Figure 1

Chemical structure of cyclo(His-Pro).

Figure 2

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.

Figure 3

Cyclo(His-Pro) triggers nuclear translocation of Nrf2 leading to changes in the antioxidant gene expression pattern in PC12 cells. Cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs prior to treatment with 100 μM H₂O₂. (A) Nrf2 protein in cytosol (white bars) and nuclear (grey bars) fractions. The nuclear and cytosolic extracts containing 40 |ag of proteins were subjected to Western blotting analysis with the indicated antibodies. Anti-α-tubulin and anti-lamin B antibodies were used as markers for the cytosolic and nuclear extracts, respectively. Quantification of the scanned band intensities revealed a ∼2-fold increase in Nrf2 intensity in the nuclear fraction (lower panel). (B) Real-time PCR analyses of the samples revealing changes in the expression of several genes, whose values were normalized to β-actin expression, and presented as 2^−ΔΔ. Relative mRNA abundance of each gene in untreated cells was assumed to be 1.0 (control). Results are given as mean ± S.D. values for n= 3 independent experiments. *P< 0.05 versus control; #P< 0.05 versus H₂O₂-treated cells.

Figure 4

Knockdown of Nrf2 by RNA interference prevents cyclo(His-Pro)-mediated regulation of antioxidant gene expression in PC12 cells. Cells were transfected with 100 nM siRNA or 100 nM scRNA (scrambled RNA), as described in Experimental, followed by incubation with 50 μM cyclo(His-Pro) for 24 hrs. (A) Nrf2 mRNA abundance, as revealed by real-time PCR. (B) Nrf2 in nuclear extracts analysed by Western blotting, revealing a ∼50% decrease in Nrf2 protein content. Results are given as mean ± S.D. values for n = 3 independent experiments. *P< 0.05 versus control. #P < 0.05 versus siRNA values. (C) Real-time PCR analyses showing expression of Nrf2 downstream genes. Changes in gene expression were normalized to p-actin expression and presented as 2^−ΔΔCT. Relative mRNA abundance of each gene in untreated cells was assumed to be 1.0 (control). (D) Cell viability (MTT reduction), ROS, NO and glutathione (GSH) were assessed in Nrf2-silenced cells treated with 50 μM cyclo(His-Pro) for 24 hrs prior to H₂O₂ treatment. Control values (100%) are indicated in the Figure 2 legend. Results are given as mean ± S.D. values for n = 3 independent experiments. *P<0.05 versus CHP+H₂O₂- Nrf2scRNA.

Figure 5

p-38 MAPK mediates cyclo(His-Pro)-induced Nrf2 translocation and antioxidant gene expression in PC12 cells. Cells were pre-incubated in the presence of 10 μM of SB203580, a well-known compound proved to be selective for p-38 MAPK at such concentration. After 2 hrs, 50 μM of cyclo(His-Pro) was added, and cells were incubated for further 24 hrs followed by 100 μM H₂O₂ treatment. (A) Involvement of p-38 MAPK in cyto-protection by cyclo(His-Pro), as assumed by Western blotting. Results are given as mean ± S.D. values for n = 3 independent experiments. *P< 0.05 versus control; #P< 0.05 versus H₂O₂-treated cells; §P< 0.05 versus CHP-treated cells. (B) Nrf2 protein is decreased in the nuclear extracts of cells treated with the p-38 MAPK inhibitor, as assessed by Western blotting. (C) Real-time PCR analyses of the samples revealing changes in the expression of Nrf2 downstream genes, whose values were normalized to (J-actin expression, and presented as 2^−AACt. Relative mRNA abundance of each gene in untreated cells was assumed to be 1.0 (control). (D) Cell viability (MTT reduction), ROS, NO and glutathione (GSH) were assessed in the SB203580-treated cells incubated with 50 μM cyclo(His-Pro) for 24 hrs prior to H₂O₂ treatment. Control values (100%) are indicated in the Figure 2 legend. Results are given as mean ± S.D. values for n = 3 independent experiments. *P < 0.05 versus CHP+H₂O₂-treated cells.

Figure 6

Cyclo(His-Pro) attenuates ROS production and prevents glutathione depletion caused by glutamate, rotenone, paraquat and p-amyloid in PC12 cells. Cells were incubated for 24 hrs with 10 mM glutamate (A), 100 μM rotenone (B), 100 μM paraquat (C) or 25 μM β-amyloid 25–35 (D), either in the absence or in the presence of 100 μM cyclo(His-Pro). After 24 hrs, cells were harvested and used to assess cell viability, GSH concentration, NO and ROS production. Control values (100%; mean ± S.D., n = 3) are indicated in the Figure 2 legend. *P < 0.05 versus control; #P < 0.05 versus injured cells.