Neuroprotection by Brazilian Green Propolis against In vitro and In vivo Ischemic Neuronal Damage

Abstract

We examined whether Brazilian green propolis, a widely used folk medicine, has a neuroprotective function in vitro and/or in vivo. In vitro, propolis significantly inhibited neurotoxicity induced in neuronally differentiated PC12 cell cultures by either 24 h hydrogen peroxide (H(2)O(2)) exposure or 48 h serum deprivation. Regarding the possible underlying mechanism, propolis protected against oxidative stress (lipid peroxidation) in mouse forebrain homogenates and scavenged free radicals [induced by diphenyl-p-picrylhydrazyl (DPPH). In mice in vivo, propolis [30 or 100 mg/kg; intraperitoneally administered four times (at 2 days, 1 day and 60 min before, and at 4 h after induction of focal cerebral ischemia by permanent middle cerebral artery occlusion)] reduced brain infarction at 24 h after the occlusion. Thus, a propolis-induced inhibition of oxidative stress may be partly responsible for its neuroprotective function against in vitro cell death and in vivo focal cerebral ischemia.

Figure 1

Typical photographs illustrating the effect of propolis on serum deprivation-induced cell damage in PC12 cell cultures [Hoechst 33342 and propidium iodide (PI) single or dual staining]. Differentiated PC12 cells were immersed in serum-free DMEM supplemented with 0.1% BSA, and then propolis was added to the cell cultures. Cells were maintained in this condition for 2 days. Viable cells are Hoechst 33342-positive and PI-negative, whereas dead cells are Hoechst 33342-positive and PI-positive. At 4 μg/ml, propolis (extract with ethanol) decreased the number of cells stained by PI (versus vehicle treatment). (A, D and H) Control (vehicle treatment). (B, E and I) Vehicle treatment + serum deprivation. (C, F and J) Propolis treatment + serum deprivation. (A–C) Hoechst 33342 staining. (D–F) PI staining. (G–I) Merged images (Hoechst 33342 + PI dual staining).

Figure 2

Propolis and Trolox reduced (A) DPPH-induced free radicals and (B) lipid peroxidation in mouse forebrain homogenate. DPPH, diphenyl-p-picrylhydrazyl; TBARS, thiobarbituric acid-reactive substance. Values represent the mean ± SEM of 4–8 independent experiments. ¹extract with ethanol; ²extract with water. ^*P < 0.05; ^#P < 0.01 versus control (vehicle-treated group).

Figure 3

2,3,5-Triphenyltetrazolium chloride (TTC) staining of coronal brain sections (thickness, 2 mm) from representative mice at 24 h after permanent middle cerebral artery (MCA) occlusion. Damaged tissue shows as white areas. Sections are arranged from rostral (top) to caudal (bottom). Left and right sections are from vehicle-injected mouse (control) and propolis-treated mouse, respectively. Propolis (extract with water, 100 mg/kg) was administered i.p. four times (at 2 days, 1 day and 60 min before, and at 4 h after the occlusion).

Figure 4

(A) Propolis reduced the brain infarct area at 24 h after permanent middle cerebral artery (MCA) occlusion in mice. The brains were removed and the forebrains sliced into five coronal 2 mm sections. Propolis, which was extracted with water, was administered i.p. at 30 or 100 mg/kg four times (at 2 days, 1 day and 60 min before, and at 4 h after the occlusion). Infarct areas were revealed by 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining. Values represent the mean ± SEM of 11 or 12 independent experiments. ^*P < 0.05, ^#P < 0.01 versus control (vehicle treatment). (B) Propolis reduced the infarct volume at 24 h after permanent MCA occlusion in mice. Values represent the mean ± SEM of 11 or 12 independent experiments. ^#P < 0.01 versus control (vehicle treatment).