Therapeutic Effect of Icaritin on Cerebral Ischemia-Reperfusion-Induced Senescence and Apoptosis in an Acute Ischemic Stroke Mouse Model

Abstract

An ischemic stroke is brain damage caused by interruption of blood supply to the brain that can cause death and long-term disability. New medical strategies or therapies are urgently needed for ischemic stroke. Icaritin (ICT) is a metabolite of icariin (ICA), which are two active flavonoid components extracted from Herba epimedii and considered neuroprotective agents in animal models of Alzheimer's disease and ischemic stroke. The therapeutic effect of ICT on ischemic still remains to be clarified. The aim of this study was to investigate the therapeutic effect of ICT on cerebral ischemia-reperfusion-associated senescence and apoptosis in a middle cerebral artery occlusion (MCAO) mouse model (ischemia for 50 min and reperfusion for 24 h). Administration of ICT after ischemia significantly reduced MCAO-induced neurological damage, infarct volume, and histopathological changes in the brain of acute ischemic stroke mice. ICT treatment could also reduce neuronal apoptosis and senescence and reversed the expression of apoptosis- and senescence-related signaling proteins. These findings suggest that ICT may have therapeutic potential to ameliorate acute ischemic stroke.

Keywords: apoptosis; icaritin; ischemic stroke; senescence.

Figure 1

The therapeutic effects of icaritin (ICT) on body weight loss, neurological severity, and infarct volume in mice with acute cerebral ischemia-reperfusion. (A) Experimental flow chart of the therapeutic effect exerted by ICT in an acute cerebral ischemia/reperfusion injury mouse model. (B–E) Mice were treated with ICT (60 mg/kg) and edaravone ((E); 3 mg/kg, as a positive control) after ischemia. The average body weight loss percentage of mice in each group was calculated (B). The modified neurological severity score (mNSS) in each group was evaluated (C). Photographs of the mouse cerebral infarct areas in each group (D). Quantification of infarct volume (E). Data are presented as mean ± SD (n = 6). * p < 0.05 compared to the MCAO group.

Figure 2

Effects of ICT treatment on histopathological changes in the left cerebral hemisphere of mice with acute cerebral ischemia-reperfusion. H&E staining was used to detect histopathological changes in both the cortex and hippocampus under 40× and 200× magnification. Black scale bar = 100 μm; white scale bar = 500 μm.

Figure 3

Effects of ICT treatment on neuronal cell apoptosis in the left cerebral hemisphere of mice with acute cerebral ischemia-reperfusion. TUNEL staining was used to detect neuronal apoptotic cells in the hippocampus and cortex. TUNEL-positive cells were stained a fluorescent green color, whereas cell nuclei were stained a fluorescent blue color. Scale bar = 75 μm.

Figure 4

Effects of ICT treatment on the levels of protein expression of apoptotic markers in the left cerebral hemisphere of mice with acute cerebral ischemia-reperfusion. The levels of protein expression of apoptotic markers (cleaved caspase-3, cleaved PARP, Bcl-2, and Bax) in the hippocampus and cortex areas were determined by Western blotting. Protein expression was quantified by densitometry. Data are presented as mean ± SD (n ≥ 4). * p < 0.05 compared to sham group; # p < 0.05 compared to I/R group.

Figure 5

Effects of ICT treatment on the expression of senescence markers induced by MCAO in the cerebral hippocampus and cortex of mice. Immunohistochemical images of the expression of senescence-associated β-galactosidase (SA-β-gal) in the hippocampus and cortex of mouse brain sections. Images scale bar = 100 µm.

Figure 6

Effects of ICT treatment on cell senescence in mouse brain tissue induced by ischemia-reperfusion. Senescence-associated proteins (p53, p27, and p21) were determined by Western blotting. Data are presented as mean ± SD (n ≥ 4). * p < 0.05 compared with sham group; # p < 0.05 as with I/R group.