Anti-Oxidant Activity of Gallotannin-Enriched Extract of Galla Rhois Can Associate with the Protection of the Cognitive Impairment through the Regulation of BDNF Signaling Pathway and Neuronal Cell Function in the Scopolamine-Treated ICR Mice

Abstract

The antibacterial, anti-inflammatory, anti-metastatic/anti-invasion activities and laxative activity of Galla Rhois (GR) are well-known, although the neuropreservation effects of their extracts are still to be elucidated. To investigate the novel therapeutic effects and molecular mechanism of GR on alleviation of cognitive impairment, two different dosages of gallotannin-enriched GR (GEGR) were administered to Korl:ICR mice for three weeks, and to induce memory impairment, scopolamine (SP) was administered during the last seven days of the GEGR treatment period. GEGR showed the high level of the free radical scavenging activity to DPPH and suppressive activity to reactive oxygen species (ROS) in B35 cells as well as enhanced SOD and CAT activity in brains of the SP-induced model. Latency time for memory impairment assessed by the passive avoidance test significantly protected in the SP+GEGR treated group as compared to the SP+Vehicle treated group. Moreover, similar protective effects were observed on the secretion of BDNF in SP+GEGR treated mice. The expression of TrkB receptor, and phosphorylation of PI3K on the TrkB receptor signaling pathway were dramatically protected in the SP-induced model after GEGR treatment, whereas the expression of p75^NTR receptor, the phosphorylation of JNK, and expression of Bax/Bcl-2 on the p75^NTR receptor signaling pathway was significantly protected in the same group. Furthermore, the GEGR treated SP-induced model showed decreased number of dead neural cells and suppressed acetylcholine esterase (AChE) activity and inhibited inflammatory responses. Taken together, these results indicate that the anti-oxidant activity of GEGR contributes to improving the neuronal cell function and survival during cognitive impairment in the SP-induced model through regulation of BDNF secretion and their receptor signaling pathway.

Keywords: BDNF; acetylcholinesterase; cognitive impairment; galla rhois; scopolamine.

Figure 1

Anti-oxidant activity and BDNF secretion activity of GEGR in vitro. (A) Free radical scavenging activity of GEGR. DPPH radical scavenging activity was assayed in a mixture containing 0.1 mM DPPH within a range of GEGR concentrations from 1 to 2000 μg/mL. (B) Determination of intracellular ROS production. After 2’,7’-dichlorofluorescein diacetate (DCFH-DA) treatment, green fluorescence in cells of each subset group was observed using a fluorescent microscope. B35 cells in each square of a 200× magnification image (left column) were also examined under 400× magnification (right column). (C) Detection of superoxide dismutase (SOD), Nrf2, and p-Nrf2 protein. Total tissue homogenates were prepared from brains of scopolamine (SP)-injected mice treated with Vehicle or GEGR as described in Materials and Methods. Three proteins were detected with specific antibody and quantified using an imaging densitometer. (D) Detection of brain-derived neurotrophic factor (BDNF) protein. Total tissue homogenates were prepared from brains of SP-injected mice treated with Vehicle or GEGR as described in Materials and Methods. BDNF protein was detected with specific antibody and quantified using an imaging densitometer. Three samples were assayed in duplicate by western blotting. * p < 0.05 compared with the No treated group. ^# p < 0.05 compared with the SP+Vehicle treated group. Abbreviation: DPPH, 2,2-diphenyl-1-picrylhydrazyl radical; IC₅₀, half maximum inhibitory concentration; GEGR, gallotannin-enriched extract of Galla Rhois; ROS, reactive oxygen species.

Figure 1

Anti-oxidant activity and BDNF secretion activity of GEGR in vitro. (A) Free radical scavenging activity of GEGR. DPPH radical scavenging activity was assayed in a mixture containing 0.1 mM DPPH within a range of GEGR concentrations from 1 to 2000 μg/mL. (B) Determination of intracellular ROS production. After 2’,7’-dichlorofluorescein diacetate (DCFH-DA) treatment, green fluorescence in cells of each subset group was observed using a fluorescent microscope. B35 cells in each square of a 200× magnification image (left column) were also examined under 400× magnification (right column). (C) Detection of superoxide dismutase (SOD), Nrf2, and p-Nrf2 protein. Total tissue homogenates were prepared from brains of scopolamine (SP)-injected mice treated with Vehicle or GEGR as described in Materials and Methods. Three proteins were detected with specific antibody and quantified using an imaging densitometer. (D) Detection of brain-derived neurotrophic factor (BDNF) protein. Total tissue homogenates were prepared from brains of SP-injected mice treated with Vehicle or GEGR as described in Materials and Methods. BDNF protein was detected with specific antibody and quantified using an imaging densitometer. Three samples were assayed in duplicate by western blotting. * p < 0.05 compared with the No treated group. ^# p < 0.05 compared with the SP+Vehicle treated group. Abbreviation: DPPH, 2,2-diphenyl-1-picrylhydrazyl radical; IC₅₀, half maximum inhibitory concentration; GEGR, gallotannin-enriched extract of Galla Rhois; ROS, reactive oxygen species.

Figure 2

SOD activity, catalase (CAT) activity, and ROS concentration in brain of SP+GEGR treated mice. (A) Total tissue homogenates were prepared from brains of SP-injected mice treated with Vehicle or GEGR as described in Materials and Methods. SOD protein were detected from total protein (50 μg per sample) using specific protein. (B) The SOD activity level was measured in homogenates of brain tissue collected from each subset group as described in Materials and Methods. One SOD unit is defined as the amount of the enzyme in 20 μL of the sample solution that inhibits by 50% the reduction reaction of water-soluble tetrazolium salt-1 (WST-1) with superoxide anion. (C) The levels of SOD transcripts in the total messenger RNA (mRNA) of brain were measured by quantitative real-time (RT)-PCR analyses using specific primers. The mRNA level of SOD gene was calculated, based on the intensity of actin as an endogenous control. (D) Total tissue homogenates were prepared from brains of SP-injected mice treated with Vehicle or GEGR as described in Materials and Methods. Nrf2 and p-Nrf2 protein were detected from total protein (50 μg per sample) using specific protein. (E) The CAT activity was measured in homogenates of brain tissue collected from each subset group as described in Materials and Methods. Catalase 1 unit is defined as the amount of enzyme required to decompose 1 μmole of H₂O₂ per min at pH 7.0 and 25 °C. (F) ROS level was measured in homogenates of brain tissue collected from each subset group as described in Materials and Methods. This assay kit has a detection sensitivity limit of 10 pM for 2’,7’-dichlorofluorescein (DCF) and 40 nM for H₂O₂ respectively. Three samples were assayed in duplicate by western blotting. Data presented are means ± SD of duplicates. * p < 0.05 compared with the No group. ^# p < 0.05 compared with the SP+Vehicle treated group. Abbreviation: DP, donepezil; GEGR, gallotannin-enriched extract of Galla Rhois; SOD, superoxide dismutase; SP, scopolamine.

Figure 3

Alteration of cognition in SP+GEGR treated mice. A passive avoidance test was used to determine step-through latency for 300 s. Behavioral changes in mice were measured after SP injection. Ten to twelve mice per group underwent cognitive defect testing. Data represent the means ± SD of duplicates and analyzed by Kruskal-Wallis and Mann-Whitney U test. * p < 0.05 compared with the No treated group. ^# p < 0.05 compared with the SP+Vehicle treated group. Abbreviation: DP, donepezil; GEGR, gallotannin-enriched extract of Galla Rhois; SP, scopolamine.

Figure 4

Secretion level of BDNF protein. Total tissue homogenates were prepared from brains of SP-injected mice treated with Vehicle or GEGR as described in Materials and Methods. Total protein (50 μg per sample) was immunoblotted with antibodies for the BDNF protein. After determining the individual band intensity level using an imaging densitometer, the relative levels of the proteins were calculated based on the intensity of actin. Three samples were assayed in duplicate by western blotting. Data are reported as mean ± SD values. * p < 0.05 compared with the No treated group. ^# p < 0.05 compared with the SP+Vehicle treated group. Abbreviation: DP, donepezil; GEGR, gallotannin-enriched extract of Galla Rhois; SP, scopolamine.

Figure 5

Alteration of the tropomyosin receptor kinase B (TrkB) receptor signaling pathway. Total tissue homogenates were prepared from the brain of Vehicle or GEGR treated SP-injected as described in Materials and Methods. Total protein (50 μg per sample) was immunoblotted with TrkB, p-TrkB, PI3K, p-PI3K, ERK, p-ERK, or β-actin antibodies. The intensity of each band was determined by using an imaging densitometer, and the relative levels of the proteins were based on the intensity of actin. Three samples were assayed in duplicate by western blotting. Data are reported as mean ± SD values. * p < 0.05 compared with the No treated group. ^# p < 0.05 compared with the SP+Vehicle treated group. Abbreviation: DP, donepezil; GEGR, gallotannin-enriched extract of Galla Rhois; SP, scopolamine.

Figure 6

Alteration of the p75^{neurotrophin receptor} (p75^NTR) receptor signaling pathway. Total tissue homogenates were prepared from brains of Vehicle or GEGR treated SP-injected mice as described in Materials and Methods. Total protein (50 μg per sample) was immunoblotted with p75^NTR, JNK, p-JNK, Bax, Bcl-2, or β-actin antibodies. After determining individual band intensities using an imaging densitometer, the relative levels of the proteins were based on the intensity of actin. Three samples were assayed in duplicate by western blotting. Data are reported as mean ± SD values. * p < 0.05 compared with the No treated group. ^# p < 0.05 compared with the SP+Vehicle treated group. Abbreviation: DP, donepezil; GEGR, gallotannin-enriched extract of Galla Rhois; SP, scopolamine.

Figure 7

Observation of Nissl-stained neurons in the hippocampus of mice. After SP injection into mice pretreated with GEGR for three weeks, brain tissues were collected from each group and histological changes were assessed as described in Materials and Methods. Slides bearing sections of brain tissue were stained with Nissl and then observed at 400× magnification. Abbreviation: DP, donepezil; GEGR, gallotannin-enriched extract of Galla Rhois; SP, scopolamine; CA, cornu ammonis.

Figure 8

Measurement of acetylcholinesterase (AChE) activity in SP+GEGR treated mice. After SP treatment, AChE activity was measured in homogenates of brain tissues collected from mice in each group by using an acetylcholinesterase assay kit. This assay can detect as little as 0.1 mU AChE in a 100 µL assay volume (1 mU/mL). Data are reported as mean ± SD values. * p < 0.05 compared with the No treated group. ^# p < 0.05 compared with the SP+Vehicle treated group. Abbreviation: DP, donepezil; GEGR, gallotannin-enriched extract of Galla Rhois; SP, scopolamine.

Figure 9

Measurement of inflammatory cytokines and their mediators in SP+GEGR treated mice. The levels of COX-2 (A), IL-1β (B), and IL-10 (C) transcripts in the total mRNA of brain were measured by quantitative real time-PCR analyses using specific primers. The mRNA level of each gene was calculated based on the intensity of actin as an endogenous control. Three samples were assayed in duplicate by qRT-PCR analyses. The values of data represent the mean ± SD. * p < 0.05 compared with the No treated group. ^# p < 0.05 compared with the SP+Vehicle treated group. Abbreviation: DP, donepezil; GEGR, gallotannin-enriched extract of Galla Rhois; SP, scopolamine.