Neuroprotective Effects of a Multi-Herbal Extract on Axonal and Synaptic Disruption in Vitro and Cognitive Impairment in Vivo

Abstract

Background: Alzheimer's disease (AD) is a multifactorial disorder characterized by cognitive decline. Current available therapeutics for AD have limited clinical benefit. Therefore, preventive therapies for interrupting the development of AD are critically needed. Molecules targeting multifunction to interact with various pathlogical components have been considered to improve the therapeutic efficiency of AD. In particular, herbal medicines with multiplicity of actions produce cognitive benefits on AD. Bugu-M is a multi-herbal extract composed of Ganoderma lucidum (Antler form), Nelumbo nucifera Gaertn., Ziziphus jujuba Mill., and Dimocarpus longan, with the ability of its various components to confer resilience to cognitive deficits.

Objective: To evaluate the potential of Bugu-M on amyloid-β (Aβ) toxicity and its in vitro mechanisms and on in vivo cognitive function.

Methods: We illustrated the effect of Bugu-M on Aβ_25-35-evoked toxicity as well as its possible mechanisms to diminish the pathogenesis of AD in rat cortical neurons. For cognitive function studies, 2-month-old female 3×Tg-AD mice were administered 400 mg/kg Bugu-M for 30 days. Behavioral tests were performed to assess the efficacy of Bugu-M on cognitive impairment.

Results: In primary cortical neuronal cultures, Bugu-M mitigated Aβ-evoked toxicity by reducing cytoskeletal aberrations and axonal disruption, restoring presynaptic and postsynaptic protein expression, suppressing mitochondrial damage and apoptotic signaling, and reserving neurogenic and neurotrophic factors. Importantly, 30-day administration of Bugu-M effectively prevented development of cognitive impairment in 3-month-old female 3×Tg-AD mice.

Conclusion: Bugu-M might be beneficial in delaying the progression of AD, and thus warrants consideration for its preventive potential for AD.

Keywords: Alzheimer’s disease; amyloid-β; axon; cognition; dementia; herbal; mild cognitive impairment; natural product; prevention; synapse.

Fig. 1

Bugu-M rescues Aβ-induced neurotoxicity in neurons. A) A representative image of Aβ_25–35 after incubation at 37°C for 7 days. Scale bar, 200 nm. Rat cortical neurons were cultured in Bugu-M (B) or Aβ_25–35 (C) at indicated concentrations for 72 h. (D) Rat cortical neurons were cultured in Aβ_25–35 at 20 μM with or without Bugu-M (12.5, 25, 50, and 100 μg/mL) for 72 h. Cell viability was measured using the MTT assay (B-D). E) Rat cortical neurons were cultured in Aβ_25–35 at 20 μM in the absence or presence of Bugu-M (100 μg/mL) for 72 h. Cytotoxicity was measured by LDH release assay. F) Rat cortical neurons were pretreated with 100 μg/mL Bugu-M for 1 h prior to Aβ_25–35 incubation for 72 h. Neurite outgrowth was analyzed by immunofluorescent labeling of β-III tubulin. Confocal images are single optical slices, and camera and microscope setting were equivalent for comparisons between groups. Shown are representative images. Scale bar, 20 μm. G) Quantification of total length of axons and minor neurites by NeuronJ software. Data are expressed as mean±SEM (n = 3). Statistic significance between groups were analyzed by one-way ANOVA and Dunnett’s multiple comparisons test. For all tests: ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 versus the untreated control group; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 versus the Aβ group.

Fig. 2

Bugu-M alleviates Aβ-triggered apoptotic signaling and axonal disruption in neurons. Rat cortical neurons were pretreated with Bugu-M (50 and 100 μg/mL) (A, B) or 100 μg/mL Bugu-M (C) for 1 h prior to Aβ_25–35 incubation for 72 h. A, D) Whole lysates, (B) mitochondrial fractions and (C) membrane-enriched fractions analyzed by immunoblotting for apoptosis (cleaved caspase-3, caspase-6-cleaved α-tubulin (TubΔCsp6)), mitochondrial dysfunction (HSP60, HSP40, HSP90), axon (Tau, NfL), dendrite (MAP2), and TDP-43 pathology related proteins. GAPDH and β-actin were used as a loading control. Total TDP-43 shows C-terminal TDP-43 fragments (CTFs), while phospho-TDP-43 reveals high molecular weight smears (asterisk) and low levels of CTFs. Densitometric quantification of immunoreactive bands was determined using ImageJ, and the fold changes to control bands are analyzed in Supplementary Figure 3D.) Quantification of Tau by ELISA in cell culture supernatants. Data are represented as mean±SEM (n = 3). Statistical significance between groups were calculated by one-way ANOVA and Dunnett’s multiple comparisons test. ^##p < 0.01 versus the untreated control group; ^**p < 0.01 versus the Aβ group.

Fig. 3

Bugu-M reverses Aβ-evoked synaptic disruption in neurons. A) Rat cortical neurons were pre-incubated with Bugu-M (50 and 100 μg/mL) for 1 h before Aβ_25–35 stimulation for 72 h. Insoluble fractions were subject to immunoblotting for PSD-95 and synaptophysin. GAPDH was used as a loading control. B) Rat cortical neurons were pretreated with 100 μg/mL Bugu-M for 1 h and then incubated with Aβ_25–35 for 72 h. Synaptic integrity was assessed by immunofluorescence co-labeling of PSD-95 (green, a) and β-III tubulin (red, b), and higher magnification images are shown in a-c (merge). Confocal images are single optical slices, and camera and microscope setting were equivalent for comparisons between groups. Shown are representative images. Scale bar, 10 μm. (C) PSD-95 immunoreactivity was quantified by ImageJ. Data are shown as mean±SEM (n = 3). Statistical significance between groups were analyzed by one-way ANOVA and Fisher‘s LSD test. ^#p < 0.05 versus the untreated control group; ^**p < 0.01 versus the Aβ group. D) Rat cortical neurons were pre-incubated with 100 μg/mL Bugu-M for 1 h followed by Aβ_25–35 incubation for 72 h. The membrane-enriched fractions were subject to Westen blot analysis for presynaptic proteins (synaptophysin, synaptogyrin-3, GAP43, SNAP25, VAMP2, and SV2A), postsynaptic proteins (PSD-95 and neurogranin), and glutamate receptors (AMPAR1 and NMDAR1). GAPDH was used as a loading control. Densitometric quantification of immnoreactive bands was conducted using ImageJ, and the fold changes to control bands are analyzed in Supplementary Figure 4.

Fig. 4

Bugu-M activates glutamate receptor signaling in Aβ-treated neurons. Rat cortical neurons were pre-incubated with Bugu-M (100 μg/mL) for 1 h before Aβ_25–35 incubation for 72 h. Whole lysates were analyzed by immunoblotting for glutamate receptors (mGluR1, mGluR2), CamKII, Phospho-CamKII, phospho-PKC, neurogenesis related factors (BDNF, DCX) (A), and phospho-CREB (B). GAPDH was used as a loading control. Quantification of immnoreactive bands was conducted using ImageJ, and the fold changes to control bands are analyzed in Supplementary Figure 5C.) Immunolabeling of DCX in rat cortical neurons. Scale bar, 10 μm. Confocal images are maximum projections, and camera and microscope setting were equivalent for comparisons between groups. Shown are representative images.

Fig. 5

Bugu-M abrogates Aβ-induced suppression of PI3K signaling in neurons. Rat cortical neurons were pre-incubated with Bugu-M (100 μg/mL) for 1 h followed by Aβ_25–35 stimulation for 72 h. Membrane-enriched fractions were subject to Western blot analysis for phospho-PI3K, PI3K, Phospho-Akt, Akt, phospho-GSK-3β, and GSK-3. β-actin was used as a loading control. Quantification of immnoreactive bands was conducted by ImageJ, and the fold changes to control bands are analyzed in Supplementary Figure 6.

Fig. 6

Bugu-M improves T-maze performance in 3-month 3×Tg-AD mice. A) Illustration of experimental timeline. Acclimation was initiated 6 weeks of age. Bugu-M treatment was administered until the mice were 8 weeks of age. Bugu-M or water administration, and behavioral testing are shown along the experimental timeline of 30 days. B) Changes in body weight after Bugu-M treatment. Mice were treated with 400 mg/kg Bugu-M or distilled water by gavage for 30 days. Data are expressed as mean±SEM (n = 10 per group). No significance between groups were observed by two-way ANOVA. Spatial working memory was tested in T-maze by movement track (C), time spent in novel arm (D), distanced moved in novel arm (E), spontaneous alternation (F), and the frequency of entries into novel and familiar arms (G). Data are represented as mean±SEM (n = 3–10 per group). Statistic significance between groups were analyzed by one-way ANOVA and Fisher’s LSD test (D) or Dunnett’s multiple comparisons test (E, F), except the frequency of entries into novel and familiar arms was analyzed using Chi-square tests. ^#p < 0.05 and ^####p < 0.0001 versus NonTg control; ^*p < 0.05 and ^**p < 0.01 versus 3×Tg-AD group.

Fig. 7

Bugu-M improves MWM performance in 3-month 3×Tg-AD mice. MWM test for movement track (A), the assessment of escape latencies to arrive visible platform over days 1–3 (B) and with hidden platform on day 4 after removing the platform (C), and goal- versus non-goal-directed strategies (D) for 3×Tg-AD and NonTg mice. Data are presented as mean±SEM (n = 8–10 per group). For escape latencies, differences among groups were determined by one-way ANOVA and Fisher’s LSD test. ^#p < 0.05 and ^##p < 0.01 versus NonTg control; ^*p < 0.05 versus 3×Tg-AD group. The frequency of use of various search strategies (operationally defined as per [42]) was calculated across trials and analyzed using logistic regression. Statistical results are provided below each panel.

Fig. 8

Proposed mechanisms of Bugu-M acting on Aβ_25–35 neurotoxicity in rat cortical neurons. Aβ results in a multitude of biochemical alterations that closely mirror the pathophysiological and functional changes that characterize the progression of AD. Decrease of NfL and tau at the presynaptic nerve terminal leads to cytoskeletal disruption, concomitant with axonal damage and thus limiting the rate of axonal transport and damages presynaptic vesicle cycling. Of note, Aβ gives rise to synaptic protein depletion along with decrease in glutamate receptor (NMDAR, AMPAR, and mGluR) expression, both of which result in synaptic dysfunction and neurotoxicity. These are manifested by downstream dysregulation of CAMKII, PKC, PI3K/Akt, and CREB-associated pathways related to neurogenesis, synaptic function, and apoptosis. Bugu-M, a multi-component agent, sustained cytoskeletal and axonal integrity and its associated protein levels when given at 100 μg/mL for 72 h. In parallel, Bugu-M rescues Aβ damage by reserving synaptic proteins, remaining glutamate receptor levels, activating CREB, promoting neurogenesis, decreasing apoptosis, and modulating mitochondrial function.