Antioxidant activity of phenylethanoid glycosides on glutamate-induced neurotoxicity

Figures & data

Figure 1. Protective effect of acteoside and isoacteoside on glutamate-induced cytotoxicity in PC12 cells. (a) Chemical structure of acteoside and isoacteoside. (b) Effects of acteoside and isoacteoside on the cell viability measured by MTT assay. Values represent mean ± SD of 3 independent experiments. #p < 0.05 vs control, *p < 0.05, **p < 0.01 vs glutamate group.

Figure 2. Effects of acteoside and isoacteoside on glutamate-induced apoptosis in PC12 cells. Cells were incubated with indicated concentrations of acteoside and isoacteoside for 12 h before exposure to 10 mM glutamate for 24 h. (a-d), Nuclear morphological changes in the cells were observed after Hoechst 33342 staining in control (a), glutamate (b), glutamate+acteoside (c) and glutamate+isoacteoside (d) groups. (e), DNA degradation with agarose gel electrophoresis analysis. Lane 1: DNA marker; Lane 2: normal; Lane 3: glutamate; Lane 4, 5: pre-incubated 12 h with 100 μM acteoside and isoacteoside and then treated with glutamate for 24 h. (f), Representative patterns of flow cytometric distribution of PC12 cells after Annexin V-PI double staining and the quantitative analysis of apoptotic cells. ##p < 0.01 vs the control group. **p < 0.01 vs glutamate group.

Table 1. Effects of acteoside and Isoacteoside on lipid peroxidation and antioxidant enzyme activities in PC12 cells.

Treatment	MDA(mmol/mg protein)	SOD (U/mg protein)	GSH-PX (U/mg protein)
Control	0.94 ± 1.10	44.08 ± 0.95	56.53 ± 2.61
Glu	2.23 ± 0.17*	11.67 ± 0.35**	21.12 ± 0.28**
L-1(50 uM) + Glu	1.57 ± 0.41^##	19.75 ± 3.39	28.16 ± 1.73
L-1(100 uM)+Glu	1.07 ± 0.60^##	30.48 ± 0.40^##	32.88 ± 1.29^#
L-2(50 uM) + Glu	1.20 ± 0.30^##	17.10 ± 2.2	30.25 ± 2.20
L-2(100 uM)+Glu	1.25 ± 0.04^##	32.12 ± 2.15^#	37.95 ± 1.29^##

L-1, acteoside; L-2, Isoacteoside; Glu, glutamate. *P < 0.05,**p < 0.01 vs the control group. #P < 0.05, ##p < 0.01 vs the glu-treated group.

Figure 3. Effect of acteoside and isoacteoside on glutamate-induced changes of ROS level and concentration of Ca²⁺ in PC12 cells. (a), Effects of acteoside and isoacteoside on production of intracellular ROS. (b), Effect of acteoside and isoacteoside on the concentration of Ca²⁺ in glutamate-treated PC12 cells. Values represent mean ± SD; ^##P < 0.01 vs control group; *P < 0.05, **P < 0.01 vs glutamate group.

Figure 4. Effects of acteoside and isoacteoside on the changes of protein expression induced by glutamate in PC12 cells. (a), Assessment of Bcl-2, Bax protein levels and the ratio of Bax/Bcl-2 by western blotting. (b), Relative level of activated caspase-3 subunit. ^##P < 0.01 vs control; *P < 0.05, **P < 0.01 vs glutamate group.

Table 2. Protective effect of acteoside against Y-maze test in glutamate-induced amnesia.

Groups		Correct numbers
	Dose (g/kg)	learning capacity memory(24 h) memory (48 h)
Control	-	15.3 ± 2.8	16.3 ± 2.2	18.6 ± 1.4
glutamate	6.0	12.1 ± 2.7^#	12.7 ± 3.4^#	15.2 ± 2.9^##
L-1+ glu	0.1	14.4 ± 2.4	16.8 ± 2.6*	19.0 ± 1.6**
L-1+ glu	0.2	15.7 ± 2.3*	17.6 ± 2.3*	17.9 ± 2.2*

L-1, acteoside; Glu, glutamate. ^##P < 0.01 vs control group; *P < 0.05 or **P < 0.01 vs glutamate group.

Figure 5. Effect of acteoside on morphological changes of CA1 neuron in hippocampus. (a-d), representative images obtained by HE staining (400×) of control group (a), glutamate group (6 g/kg) (b), glutamate+ acteoside (0.1 g/kg) group (c), and glutamate+ acteoside (0.2 g/kg) group (d). (e), ultrastructural images of nuclei (upper), organells (middle) and synapse (below) in CA1 region of mice from control group, glutamate group (6 g/kg), glutamate+ acteoside (0.1 g/kg) group, and glutamate+ acteoside (0.2 g/kg) group (from left to right).