Icariside II attenuates cerebral ischemia/reperfusion-induced blood-brain barrier dysfunction in rats via regulating the balance of MMP9/TIMP1

Abstract

Cerebral ischemia/reperfusion (I/R) results in harmful consequences during ischemic stroke, especially the disruption of the blood-brain barrier (BBB), which leads to severe hemorrhagic transformation through aggravation of edema and brain hemorrhage. Our previous study demonstrated that icariside II (ICS II), which is derived from Herba Epimedii, attenuates cerebral I/R injury by inhibiting the GSK-3β-mediated activation of autophagy both in vitro and in vivo. However, the effect of ICS II on the BBB remains unclear. Thus, in this study, we investigated the regulation of BBB integrity by ICS II after cerebral I/R injury and further explored the underlying mechanism in rats. Cerebral I/R injury was induced by middle cerebral artery occlusion (MCAO), and the treatment groups were administered ICS II at a dose of 16 mg/kg by gavage twice a day for 3 days. The results showed that ICS II effectively prevented BBB disruption, as evidenced by Evans Blue staining. Moreover, ICS II not only significantly reduced the expression of MMP2/9 but also increased TIMP1 and tight junction protein (occludin, claudin 5, and ZO 1) expression. Intriguingly, ICS II may directly bind to both MMP2 and MMP9, as evidenced by molecular docking. In addition, ICS II also inhibited cerebral I/R-induced apoptosis and ameliorated the Bax/Bcl-2 ratio and cleaved-caspase 3 level. Collectively, our findings reveal that ICS II significantly ameliorates I/R-induced BBB disruption and neuronal apoptosis in MCAO rats by regulating the MMP9/TIMP1 balance and inhibiting the caspase 3-dependent apoptosis pathway.

Keywords: MCAO rats; Stroke; apoptosis; blood–brain barrier; cerebral ischemia/reperfusion; icariside II; molecular docking; tight junction.

Fig. 1. Cortical blood flow before and after MCAO and the protective effect of ICS II against BBB damage after ischemic brain injury induced by cerebral I/R in rats.

a Representative images of cortical blood flow. b Quantitative analysis of relative cortical blood flow (%). **P < 0.01 vs before occlusion, ^##P < 0.01 vs occlusion, n = 5. c Representative images of Evans blue extravasation in the rat brain. d Quantitative analysis of Evans blue dye extravasation into the brain hemisphere. **P < 0.01 vs sham; ^##P < 0.01 vs MCAO; n = 6 per group.

Fig. 2. ICS II suppressed damage to the hippocampus, cortex and striatum after cerebral I/R injury.

a The hippocampal CA1, CA3 and DG regions. b Striatum and cortex (×400, scale bar = 50 μm). n = 6 per group.

Fig. 3. ICS II protected against cerebral I/R injury-induced neuronal loss in the hippocampus and cortex.

a The hippocampal CA1, CA3, and DG regions and cortex. b Quantitative analysis of neuronal density in the CA1 region. c Quantitative analysis of neuronal density in the CA3 region. d Quantitative analysis of neuronal density in the DG region. e Quantitative analysis of neuronal density in the cortex. *P < 0.05, **P < 0.01 vs sham; ^#P < 0.05, ^##P < 0.01 vs MCAO; n = 6 per group.

Fig. 4. ICS II attenuated MMP2/9 expression in cerebral I/R-induced injury.

a Representative images of immunohistochemical staining of MMP9 in the cortex and striatum. b Quantitative analysis of MMP9 in the cortex and striatum. c Representative images of immunoblotting of MMP9 in the penumbra. d Quantitative analysis of MMP9 in the penumbra. e Representative images of immunohistochemical staining of MMP2 in the cortex and striatum. f Quantitative analysis of MMP2 in the cortex and striatum. g Representative images of immunoblotting of MMP2 in the penumbra. h Quantitative analysis of MMP2 in the penumbra. Five random microscope fields per section were chosen. The average was calculated. **P < 0.01 vs sham; ^#P < 0.05, ^##P < 0.01 vs MCAO; n = 6 per group.

Fig. 5. ICS II increased TIMP1 expression in cerebral I/R-induced injury.

a Representative images of immunohistochemical staining of TIMP1 in the cortex and striatum. b Quantitative analysis of TIMP1 in the cortex and striatum. c Representative images of immunoblotting of TIMP1 in the penumbra. d Quantitative analysis of TIMP1 in the penumbra. e Quantitative analysis of active MMP9/TIMP1 in the penumbra. f Quantitative analysis of active MMP2/TIMP1 in the penumbra. Five random microscope fields per section were chosen. The average was calculated. **P < 0.01 vs sham; ^#P < 0.05, ^##P < 0.01 vs MCAO; n = 6 per group.

Fig. 6. ICS II improved BBB integrity after cerebral I/R by upregulating the expression of tight junction-related proteins.

a Representative images of immunoblotting staining of claudin 5, occludin and ZO 1 in the penumbra. b Quantitative analysis of occludin. c Quantitative analysis of claudin 5. d Quantitative analysis of ZO 1. *P < 0.05, **P < 0.01 vs sham; ^#P < 0.05, ^##P < 0.01 vs MCAO; n = 6 per group.

Fig. 7. ICS II inhibited cerebral I/R-induced neural apoptosis in rats.

a Representative images of TUNEL staining in the hippocampus and cortex. b Quantitative analysis of TUNEL-positive cells in the hippocampus and cortex. c Representative images of immunoblotting of Bax and Bcl-2. d Representative images of immunoblotting of caspase 3 and cleaved-caspase 3 in the penumbra. e Quantitative analysis of Bax/Bcl-2. f Quantitative analysis of cleaved-caspase 3/caspase 3. Three to five random microscope fields per section were chosen. The average was calculated. **P < 0.01 vs sham; ^##P < 0.01 vs MCAO; n = 6 per group.

Fig. 8. ICS II bound and inhibited MMP2/9.

a A visual of the binding sites between ICS II and MMP2. b A close-up of the molecule binding pocket from the side. c The crystal structure of ICS II (gray) displaying MMP2 (yellow and purple) bound to the docking pocket. d Amino acid residues. e A visual of the binding sites between ICS II and MMP9. f A close-up of the molecule binding pocket from the side. g The crystal structure of ICS II (gray) displaying MMP9 (yellow and purple) bound to the docking pocket. h Amino acid residues.

Fig. 9. A schematic showing the underlying mechanisms of the inhibitory effects of ICS II on cerebral I/R-induced apoptosis in rats.

ICS II attenuates cerebral I/R-induced BBB dysfunction by inhibiting neuronal apoptosis in rats in a manner involving regulation of the MMP9/TIMP1 balance.