Propofol induces nuclear localization of Nrf2 under conditions of oxidative stress in cardiac H9c2 cells

<div><p>Oxidative stress contributes to myocardial ischemia-reperfusion injury, which causes cardiomyocyte death and precipitate life-threatening heart failure. Propofol has been proposed to protect cells or tissues against oxidative stress. However, the mechanisms underlying its beneficial effects are not fully elucidated. In the present study, we employed an <i>in vitro</i> oxidative injury model, in which rat cardiac H9c2 cells were treated with H₂O₂, and investigated roles of propofol against oxidative stress. Propofol treatment reduced H₂O₂-induced apoptotic cell death. While H₂O₂ induced expression of the antioxidant enzyme HO-1, propofol further increased HO-1 mRNA and protein levels. Propofol also promoted nuclear localization of Nrf2 in the presence of H₂O₂. Knockdown of Nrf2 using siRNA suppressed propofol-inducible Nrf2 and expression of Nrf2-downstream antioxidant enzyme. Knockdown of Nrf2 suppressed the propofol-induced cytoprotection. In addition, Nrf2 overexpression induced nuclear localization of Nrf2 and HO-1 expression. These results suggest that propofol exerts antioxidative effects by inducing nuclear localization of Nrf2 and expression of its downstream enzyme in cardiac cells. Finally, we examined the effect of propofol on cardiomyocytes using myocardial ischemia-reperfusion injury models. The expression level of Nrf2 protein was increased at 15 min after reperfusion in the ischemia-reperfusion and propofol group compared with ischemia-reperfusion group in penumbra region. These results suggest that propofol protects cells or tissues from oxidative stress via Nrf2/HO-1 cascade.</p></div

Propofol upregulate HO-1 expression and nuclear localization of Nrf2 under oxidative stress conditions.

<p><b>A</b>: mRNA expression profile for HO-1. n = 3. The data are expressed as mean ± SEM. <b>B</b>: Western blot analysis of HO-1 in H9c2 cells. <b>C</b>: Quantification of the density of expression level of HO-1. n = 3. The data are expressed as mean ± SEM. <b>D</b>: Western blot analysis of Nrf2 in H9c2 cells. <b>E</b>: Quantification of the density of expression level of total Nrf2. n = 3. The data are expressed as mean ± SEM. <b>F</b>: Western blot analysis of Nrf2 in total lysates, nuclear lysates and cytoplasmic lysates of H9c2 cells. <b>G</b>: Western blot analysis of Nrf2 in nuclear lysates and cytoplasmic lysates of H9c2 cells. <b>H</b>, <b>I</b>: Quantification of the density of expression level of nuclear Nrf2 and cytoplasmic Nrf2. <b>J</b>: Representative immunocytochemical labeling of Nrf2 in H9c2 cells treated with H₂O₂ and propofol (PR). Scale bar, 50 μm. Control (con) means no treatment. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Effects of propofol on myocardial ischemia-reperfusion injury.

<p><b>A-B</b>: Scheme of ischemia-reperfusion injury and propofol administration. <b>C</b>: Expression profiles of HO-1 mRNA in penumbra and infarction region of ischemia-reperfusion (I/R) group rats and I/R+propofol group rats at 15 min or 90 min after reperfusion. Rep, reperfusion. n = 4. The data are expressed as mean ± SEM. <b>D</b>: Western blot analysis of Nrf2 in penumbra and infarction region of I/R group rats and I/R+propofol group rats at 15 min after reperfusion. n = 3. The data are expressed as mean ± SEM.</p

Nrf2 overexpression induce nuclear localization of Nrf2 and HO-1 expression.

<p><b>A</b>: Western blot analysis of Nrf2 in HEK293T cells using anti-c-Myc antibody. <b>B</b>: Western blot analysis of Nrf2 in H9c2 cells. Upper band is nuclear Nrf2 (N-Nrf2) and lower band is cytoplasmic Nrf2 (C-Nrf2). <b>C</b>: Quantification of the density of expression level of nuclear Nrf2. n = 3. The data are expressed as mean ± SEM. <b>D</b>: Western blot analysis of Nrf2 and HO-1 in Nrf2 overexpressed H9c2 cells. The number means intensity of nuclear Nrf2 (arrows). <b>E</b>: Quantification of the density of expression level of nuclear Nrf2. n = 3. The data are expressed as mean ± SEM. *<i>P</i> < 0.05.</p

Nrf2 inhibition suppresses propofol-induced cytoprotection.

<p><b>A-D</b>: Effect of Nrf2, HO-1, Nqo-1 or scramble siRNA on propofol-induced cytoprotection, after at 24 h after treatment with H₂O₂. Cytotoxicity was measured using an LDH assay. n = 3. The data are expressed as mean ± SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01. N.S., not significant.</p

Knockdown of Nrf2 by siRNA suppresses propofol-induced antioxidant expression.

<p><b>A</b>: Expression of Nrf2 mRNA in H9c2 cells transfected with Nrf2 and scramble (control) siRNA. n = 3. The data are expressed as mean ± SEM. <b>B-D</b>: Expression profiles of Nrf2 (B), HO-1 (C) and Nqo-1 (D) mRNAs in H9c2 cells transfected with scramble, Nrf2 and HO-1 siRNA. n = 3. The data are expressed as mean ± SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Propofol inhibits H₂O₂-induced H9c2 cell death.

<p><b>A</b>: Effect of H₂O₂ on cell viability. Cytotoxicity was measured using an LDH assay. n = 3. <b>B</b>: Effect of propofol (PR) on cell viability. Scale bar, 200 μm. <b>C</b>: Quantitative analysis of PR-induced cytotoxicity in H9c2 cells. n = 3. <b>D</b>: Diagram of the scheme of propofol (PR) and H₂O₂ treatment. <b>E</b>: Effect of PR on cell viability under oxidative stress conditions. Arrows, dead cells; arrowheads, surviving cells. Scale bar, 250 μm. <b>F</b>: Quantification of dead H9c2 cells. n = 3. The data are expressed as mean ± SEM. <b>G</b>: TUNEL staining. Arrowheads, TUNEL-positive cells. Scale bar, 200 μm. <b>H</b>: Quantification of TUNEL-positive cells. n = 3. The data are expressed as mean ± SEM. Control (con) means no treatment. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p