Purified Cornel Iridoid Glycosides Attenuated Oxidative Stress Induced by Cerebral Ischemia-Reperfusion Injury via Morroniside and Loganin Targeting Nrf2/NQO-1/HO-1 Signaling Pathway

Abstract

Oxidative stress significantly contributes to the exacerbation of brain damage during cerebral ischemia-reperfusion injury (CIR/I). In our previous study, purified cornel iridoid glycoside (PCIG), consisting of morroniside (MOR) and loganin (LOG), showed neuroprotective effects against CIR/I. To further explore the antioxidative effects and underlying molecular mechanisms, we applied PCIG, MOR, and LOG to rats injured by middle cerebral artery occlusion/reperfusion (MCAO/R) as well as H₂O₂-stimulated PC12 cells. Additionally, the molecular docking analysis was performed to assess the interaction between the PCIG constituents and Kelch-like ECH-associated protein 1 (Keap1). The results showed that the treated rats experienced fewer neurological deficits, reduced lesion volumes, and lower cell death accompanied by decreased levels of malondialdehyde (MDA) and protein carbonyl, as well as increased activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). In H₂O₂-stimulated PC12 cells, the treatments decreased reactive oxygen species (ROS) production, mitigated mitochondrial dysfunction, and inhibited mitochondrial-dependent apoptosis. Moreover, the treatments facilitated Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation into the nucleus and selectively increased the expression of NAD(P)H quinone oxidoreductase 1 (NQO-1) and heme oxygenase 1 (HO-1) through MOR and LOG, respectively. Both MOR and LOG demonstrated strong binding affinity to Keap1. These findings suggested that PCIG, rather than any individual components, might serve as a valuable treatment for ischemic stroke by activating the Nrf2/NQO-1 and Nrf2/HO-1 signaling pathway.

Keywords: Nrf2 signaling pathway; cerebral ischemia-reperfusion injury; loganin; morroniside; oxidative stress; purified cornel iridoid glycosides.

Figure 1

The composition of purified cornel iridoid glycosides.

Figure 2

Protective effect of MOR, LOG and PCIG on cerebral ischemia-reperfusion injury in rats. (A) Timeline flowchart for the animal experiments. (B) Representative brain sections with TTC staining. (n = 3). (C) Infarct volume ratio based on TTC staining. (n = 3). (D) Neurological scores. (n = 8). (E) Brain water content of the rats. (n = 5). The columns and error bars were represented as means ± SD. ### p < 0.001 vs. the SHAM group, *** p < 0.001 vs. the MCAO/R group.

Figure 3

Histopathological analysis on brain tissues of the rats involved in the SHAM (A), MCAO/R (B), MOR (C), LOG (D), L-PCIG (E), M-PCIG (F) and H-PCIG (G) groups. Representative (a) H&E staining. (b) TUNEL staining. (H) Number of green fluorescence cells based on TUNEL staining. (n = 3).

Figure 4

Biochemical analysis on rat brains collected from SHAM, MCAO/R, MOR, LOG, L-PCIG, M-PCIG and H-PCIG groups. Levels of (A) protein carbonyl; (B) MDA; (C) SOD activity; (D) GSH; (E) GSH-Px. The columns and error bars were represented as means ± SD. ### p < 0.001 vs. the SHAM group, * p < 0.05 vs. the MCAO/R group, ** p < 0.01 vs. the MCAO/R group, *** p < 0.001 vs. the MCAO/R group (n = 5).

Figure 5

Protective effects of MOR, LOG and PCIG on PC12 cells against H₂O₂-induced cell apoptosis. Viability of (A) PC12 cells treated with the samples. (B) H₂O₂-stimulated PC12 cells were treated with the samples. (C) mitochondrial membrane potential of the cells. The columns and error bars were represented as means ± SD. ### p < 0.001 vs. the CON, *** p < 0.001 vs. the H₂O₂ (n = 3).

Figure 6

Protective effects of MOR, LOG and PCIG on PC12 cells against H₂O₂-induced cell apoptosis. The protein levels of (A,D) Caspase-3, (B,C,E) BAX, BCL-2 and BAX/BCL-2 in PC12 cells treated with MOR and PCIG. The protein levels of (F,I) Caspase-3, (G,H,J) BAX, BCL-2 and BAX/BCL-2 in PC12 cells treated with LOG and PCIG. The columns and error bars were represented as means ± SD. ## p < 0.01 vs. the CON, ### p < 0.001 vs. the CON, ** p < 0.01 vs. the H₂O₂, *** p < 0.001 vs. the H₂O₂ (n = 3).

Figure 7

Effects of MOR, LOG and PCIG on reducing oxidative stress induced by H₂O₂. (A) Representative image of cells with ROS fluorescence. (B) Intensity of ROS fluorescence. Levels of (C) MDA. (D) Protein carbonyl. (E) SOD activity. (F) GSH. The columns and errors bars were represented as means ± SD (n = 3). (* p < 0.05 vs. H₂O₂, ** p < 0.01 vs. H₂O₂, *** p < 0.001 vs. H₂O₂; ### p < 0.001 vs. CON).

Figure 8

MOR, LOG and PCIG enhanced antioxidative activities in H₂O₂-stimulated PC12 cells (A) Nrf2 nuclear translocation. The protein levels of (B,D) NQO-1, (C,E) HO-1 in H₂O₂-stimulated PC12 cells treated with MOR and PCIG. The protein levels of (F,H) NQO-1, (G,I) HO-1 in H₂O₂-stimulated PC12 cells treated with LOG and PCIG. The columns and errors bars were represented as means ± SD. ### p < 0.001 vs. the CON, * p < 0.05 vs. the H₂O₂, ** p < 0.01 vs. the H₂O₂, *** p < 0.001 vs. the H₂O₂ (n = 3).

Figure 8

MOR, LOG and PCIG enhanced antioxidative activities in H₂O₂-stimulated PC12 cells (A) Nrf2 nuclear translocation. The protein levels of (B,D) NQO-1, (C,E) HO-1 in H₂O₂-stimulated PC12 cells treated with MOR and PCIG. The protein levels of (F,H) NQO-1, (G,I) HO-1 in H₂O₂-stimulated PC12 cells treated with LOG and PCIG. The columns and errors bars were represented as means ± SD. ### p < 0.001 vs. the CON, * p < 0.05 vs. the H₂O₂, ** p < 0.01 vs. the H₂O₂, *** p < 0.001 vs. the H₂O₂ (n = 3).

Figure 9

Three-dimensional and partially enlarged graphics of molecular docking analysis for the compounds binding to Keap1 protein (PDB code: 4L7B). The interaction between the Keap1 and (A) morroniside; (B) loganin.

Figure 10

Schematic presentation of the treatment pathway of cerebral ischemia-reperfusion injury with purified cornel iridoid glycosides.