Lycium barbarum polysaccharide prevents focal cerebral ischemic injury by inhibiting neuronal apoptosis in mice

Abstract

Aims of the study: To investigate the neuroprotective effect of Lycium barbarum polysaccharide (LBP) on focal cerebral ischemic injury in mice and to explore its possible mechanism.

Materials and methods: Male ICR mice were used to make the model of middle cerebral artery occlusion (MCAO) after intragastric administration with LBP (10, 20 and 40 mg/kg) and Nimodipine (0.4 mg/kg) for seven successive days. After 24 h of reperfusion, neurological scores were estimated and infarct volumes were measured by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Morphological changes in ischemic brains were performed for hematoxylin-eosin (HE) staining. The number of apoptotic neurons was detected by TUNEL staining. The Bax, Bcl-2 protein expression and CytC, Caspase-3, -9 and cleaved PARP-1 activation were investigated by immunofluorescence and western-blot analysis.

Results: LBP (10, 20 and 40 mg/kg) treatment groups significantly reduced infract volume and neurological deficit scores. LBP also relieved neuronal morphological damage and attenuated the neuronal apoptosis. LBP at the dose of 40 mg/kg significantly suppressed overexpression of Bax, CytC, Caspase-3, -9 and cleaved PARP-1, and inhibited the reduction of Bcl-2 expression.

Conclusions: Based on these findings we propose that LBP protects against focal cerebral ischemic injury by attenuating the mitochondrial apoptosis pathway.

Figure 1. Protective effect of LBP against cerebral ischemic injury in MCAO mice brains.

A TTC staining of representative coronal sections at 24B Quantitative analysis of infarct volumes at 24 h after reperfusion. C Quantification of neurologic scores at 24 h after reperfusion. Data are expressed as mean±SEM (n = 6). ^##P<0.01, vs. sham-operated group, * P<0.05, **P<0.01 vs. vehicle group.

Figure 2. Effect of LBP treatment on morphological changes in ischemic penumbra of the cortex at 24-eosin staining (400×).

A Sham-operated group. B Vehicle group. C LBP 10 mg/kg group. D LBP 20mg/kg group. E LBP 40mg/kg group. F Nimodipine 0.4mg/kg group.

Figure 3. LBP reduces the number of Tunel positive neurons after focal cerebral ischemic injury.

A TUNEL staining of representative sections in mice ischemic penumbra of the cortex at 24B Quantitative analysis of apoptosis cells in cortex in different groups at 24 h after reperfusion. Data are expressed as mean±SEM (n = 6). ^##P<0.01 vs. sham-operated group; *P<0.05, **P<0.01 vs. vehicle group.

Figure 4. Effects of LBP (40 mg/kg) on the caspase-3 activities in left hemisphere after 2 h of MCAO and 24 h of reperfusion.

Data are expressed as mean±SEM (n = 6). ^##P<0.01, vs. sham-operated group, *P<0.05 vs. vehicle group.

Figure 5. Effects of LBP on the expression of Bax protein.

A Representative photomicrographs of Bax immunofluorescence staining (400×). B Quantification of Bax protein fluorescence intensity in different groups. C Representative Western blot band of Bax protein expression in the ischemic cortex at 24 h after reperfusion. D Effect of LBP (40 mg/kg) on the Bax expression in MCAO mice cortex at 24 h after reperfusion. Data are expressed as mean±SEM (n = 6). ^#P<0.05, ^##P<0.01 vs. sham-operated group; *P<0.05, **P<0.01 vs. vehicle group.

Figure 6. Effects of LBP on the expression of CytC.

A Representative photomicrographs of CytC immunofluorescence staining (400×). B Quantification of CytC fluorescence intensity in different groups. C Representative Western blot band of CytC activation in the ischemic cortex at 24 h after reperfusion. D Effect of LBP (40 mg/kg) on the CytC activation in MCAO mice cortex at 24 h after reperfusion. Data are expressed as mean±SEM (n = 6). ^##P<0.01 vs. sham-operated group; *P<0.05, **P<0.01 vs. vehicle group.

Figure 7. Effects of LBP on the expression of Caspase-3.

A Representative photomicrographs of Caspase-3 immunofluorescence staining (400×). B Quantification of Caspase-3 fluorescence intensity in different groups. C Representative Western blot band of Caspase-3 activation in the ischemic cortex at 24 h after reperfusion. D Effect of LBP (40 mg/kg) on the Caspase-3 activation in MCAO mice cortex at 24 h after reperfusion. Data are expressed as mean±SEM (n = 6). ^##P<0.01 vs. sham-operated group; **P<0.01 vs. vehicle group.

Figure 8. Effects of LBP on the expression of Bcl-2 protein.

A Representative photomicrographs of Bcl-2 immunofluorescence staining (400×). B Quantification of Bcl-2 protein fluorescence intensity in different groups. C Representative Western blot band of Bcl-2 protein expression in the ischemic cortex at 24 h after reperfusion. D Effect of LBP (40 mg/kg) on the Bcl-2 expression in MCAO mice cortex at 24 h after reperfusion. Data are expressed as mean±SEM (n = 6). ^#P<0.05, ^##P<0.01 vs. sham-operated group; **P<0.01 vs. vehicle group.

Figure 9. Effects of LBP on the expression of Caspase-9.

A Representative Western blot band of Caspase-9 activation in the ischemic cortex at 24 h after reperfusion. B Effect of LBP (40 mg/kg) on the Caspase-9 activation in MCAO mice cortex at 24 h after reperfusion. Data are expressed as mean±SEM (n = 6). ^##P<0.01 vs. sham-operated group; *P<0.05, **P<0.01 vs. vehicle group.

Figure 10. Effects of LBP on the expression of Cleaved PARP-1.

A Representative Western blot band of Cleaved PARP-1 activation in the ischemic cortex at 24 h after reperfusion. B Effect of LBP (40 mg/kg) on the Cleaved PARP-1 activation in MCAO mice cortex at 24 h after reperfusion. Data are expressed as mean±SEM (n = 6). ^##P<0.01 vs. sham-operated group; *P<0.05, **P<0.01 vs. vehicle group.