Morroniside protects OLN-93 cells against H₂O₂-induced injury through the PI3K/Akt pathway-mediated antioxidative stress and antiapoptotic activities

Figures & data

Figure 1. MR protects OLN-93 cells against H₂O₂-induced death. (a) Effects of MR on the viability of OLN-93 cell. MR did not influence the viability of cell at concentrations of 100, 200, 300, and 400 μM; (b–d) Protective effects of MR pretreatment on H₂O₂-induced cytotoxicity in OLN-93 cells. Cells were pretreated with different concentrations of MR (100–400 µM) for 24 h and then incubated in the absence or presence of 100 µM H₂O₂ for 12 h. (b) Viability was assessed by the MTT reduction assay. (c) MR (200 µM) inhibited the release of lactate dehydrogenase (LDH) in OLN-93 cells induced by H₂O₂. (d) MR suppressed the changes of morphological induced by H₂O₂. Scale bar, 50 µm. ^#P< 0.01 vs. the control group; *P< 0.05, **P< 0.01 vs. the only H₂O₂ treatment group

Figure 2. MR inhibits H₂O₂-induced apoptosis of OLN-93 cells. (a–d) Apoptotic nuclei of OLN-93 cells were stained by Hoechst 33342. The arrow indicates condensed nuclear cells. (a) Control group; (b) cells induced by H₂O₂ (100 µM) with condensed nuclei; (c) cells administrated with 200 µM MR for 24 h displayed attenuated sensitivity to H₂O₂ (100 µM), as evidenced by fewer cells with condensed nuclei. (e–h) Apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). (e) Control group; (f) cells induced by H₂O₂ (100 µM) with condensed nuclei; (g) cells were administrated with 200 µM MR followed by 100 µM H₂O₂ for 12 h. Quantitative analyses are shown in panels (d) and (h). Scale bar, 20 µm. *P< 0.01 vs. the control group; ^#P< 0.01 vs. the only H₂O₂ treatment group

Figure 3. MR inhibits the elevation of intracellular ROS level induced by H₂O₂. (a) Control group; (b) OLN-93 cells incubated with 200 µM MR; (c) cells exposed to 100 µM H₂O₂; (d) cells pretreated with 200 µM MR for 24 h prior to induced by H₂O₂ (100 µM) for 12 h. Scale bar, 50 µm. Quantitative analysis is shown in E. *P< 0.01 vs. the control group and the 200 µM MR group, respectively; ^#P< 0.05 vs. the only H₂O₂ treatment group

Figure 4. Effect of MR on H₂O₂-induced lipid oxidation. Cells were pretreated with 200 µM MR for 24 h followed by 100 µM H₂O₂ for 12 h. *P< 0.05 vs. the control group; ^#P< 0.05 vs. the only H₂O₂ treatment group

Figure 5. Inhibition of H₂O₂-induced reduction in mitochondrial membrane potential (MMP) by MR. (a) Cells were administrated with 200 µM MR for 24 h prior to exposure to H₂O₂ (100 µM) for 12 h. Aggregated (red) or monomeric (green) cell morphological images were taken under a fluorescence microscope. (b) The ratio of aggregated and monomeric JC-1 was used to quantify the change of MMP. Bar diagram showing the loss of MMP by MR. Scale bar, 50 µm. *P< 0.01 vs. the control group; ^#P< 0.01 vs. the only H₂O₂ treatment group

Figure 6. Effect of MR on the expression of oxidation-related proteins. (a) Cells were pretreated with 200 µM MR for 24 h, followed by incubation in the presence of 100 µM H₂O₂ for 12 h. The cells were analyzed by SDS-PAGE followed by western blot analysis. (b–c) The levels of SOD2 and iNOS were quantified by densitometric analysis.*P< 0.01 vs. the control group; #P< 0.01 vs. the only H₂O₂ treatment group

Figure 7. Effect of MR on the expression of apoptosis-related proteins in OLN-93 cells. (a) Cells were pretreated with 200 µM MR for 24 h and then incubated in the presence of 100 µM H₂O₂ for 12 h. The cells were analyzed by SDS-PAGE followed by western blot analysis. (b–d) The levels of cleaved-caspase3, bcl-2, and Bax were quantified by the densitometric analysis. *P< 0.05 vs. the control group; #P< 0.05 vs. the only H₂O₂ treatment group

Figure 8. MR rescues H₂O₂-induced downregulation of p-AKT in OLN-93 cells. (a) Cells were pretreated with 200 µM MR for 24 h and then incubated in the presence of 100 µM H₂O₂ for 12 h. The cells were analyzed by SDS-PAGE followed by western blot analysis. (b) Relative levels of p-AKT versus total AKT in each sample as determined by blot densitometry. Densitometric analysis of the immunoblot was expressed as a fold of control. *P< 0.05 vs. the control group; ^#P< 0.05 vs. the only H₂O₂ treatment group

Figure 9. LY294002 blocks the protective role of MR on H₂O₂-induced apoptosis in OLN-93 cells. (a) OLN-93 cells were preincubated with LY294002 for 1 h and then pretreated with MR for 24 h, followed by incubation with H₂O₂ for 12 h. Apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). Quantitative analyses are shown in panel B. Scale bar, 50 µm. ^**P<0.01 vs. the control group; ^##P<0.01 vs. the only H₂O₂ treatment group; ^&P<0.05 and ^$P<0.05 vs. MR+H₂O_2.

Figure 10. LY294002 blocks inhibitory effects of MR on H₂O₂-induced ROS overproduction. OLN-93 cells were preincubated with LY294002 for 1 h and then pretreated with MR for 24 h, followed by incubation with H₂O₂ for 12 h. ^**P<0.01 vs. the control group; ^##P<0.01 vs. the only H₂O₂ treatment group; ^&&P<0.01 and ^$$P<0.01 vs. MR+H₂O_2.

Figure 11. MR modulates the expression of apoptosis-related proteins by increasing Akt phosphorylation. Cells were preincubated with LY294002 for 1 h and then pretreated with MR for 24 h, followed by incubation with H₂O₂ for 12 h. The cells were analyzed by SDS-PAGE followed by western blot analysis (a and f). Relative levels of p-AKT versus total AKT in each sample as determined by blot densitometry (b). Densitometric analysis of the immunoblot was expressed as a fold of control (b–f). ^**P<0.01 vs. the control group; ^##P<0.01 vs. the only H₂O₂ treatment group; ^&&P<0.05 and ^$$P<0.05 vs. MR+H₂O_2.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.