Spirodelae Herba ethanol extract attenuates neurotoxicity in hippocampal cells and improves scopolamine-induced cognitive impairment in mice

Abstract

Background: Spirodelae Herba (SH) is an herb that has been used in traditional medicine in East Asia. Whereas its anti-inflammatory, anti-allergic, and antioxidant activities have recently been demonstrated, the effects of SH ethanol extract (SHE) on neurotoxicity in hippocampal neurons, neuroinflammation in microglia, and cognitive impairment in mice have not been studied.

Methods: In this study, we explored the protective effect of SHE on neurotoxicity related to oxidative stress and the related molecular mechanisms in a hippocampal cell model. We also examined the inhibitory effect of SHE on neuroinflammation and its related mechanisms in endotoxin-stimulated microglia. We also explored the ameliorative effect of SHE on cognitive impairment in mice through behavioral tests and examined histopathological changes in the hippocampus and cortex using Nissl staining. In addition, we conducted a comprehensive analysis of the related mechanisms, including the microbiota-gut-brain axis.

Results: SHE inhibited glutamate-induced neurotoxicity in HT22 cells and induced changes in related mechanisms. SHE effectively inhibited lipopolysaccharide-induced neuroinflammation in BV2 cells and regulated the activation of related mechanisms. In addition, SHE administration significantly alleviated scopolamine (SCO)-induced decreases in memory and learning ability in mice. SHE suppressed damage to hippocampal neurons in the mice's brain and significantly increased the expression of the brain-derived neurotrophic factor and its related pathway proteins in hippocampal tissue. Furthermore, microbiome analysis revealed that SHE administration normalized SCO-induced gut microbiota imbalance (dysbiosis). These findings indicate that the cognitive improvement effects of SHE may be mediated through the modulation of the gut microbiota composition and the microbiota-gut-brain axis.

Conclusion: The results of this study demonstrate the neuroprotective and anti-neuroinflammatory effects of SHE and its strong potential as a preventive and therapeutic agent for cognitive impairment.

Keywords: Spirodelae Herba; antioxidant; cognitive impairment; microbiota–gut–brain axis; neuroinflammation; neuronal protection.

FIGURE 1

Effects of Spirodelae Herba ethanol extract (SHE) on glutamate-induced (A–D) cytotoxicity and (E) intracellular reactive oxygen species (ROS) production in HT22 cells. Control cells were incubated with the vehicle alone. All experiments were repeated at least three times, and similar results were obtained. (A–C, E) Data are expressed as the mean ± standard error of the mean of six independent experiments. (D) Scale bar = 20 μm. (E) Scale bar = 200 μm. Con, control; LDH, lactate dehydrogenase; Glu, glutamate. ^# p < 0.05 (vs. control) and ^† p < 0.001 (vs. glutamate).

FIGURE 2

Effects of Spirodelae Herba ethanol extract (SHE) on (A) apoptosis, (B) Nrf-2 pathway activation, (C) ERK/CREB/BDNF activation, and (D) ERK/CREB/BDNF activation with U0126 in glutamate-exposed HT22 cells. (A) Data are expressed as the mean ± standard error of the mean of six independent experiments. (B–D) Histograms show the expression levels of proteins relative to those of the housekeeping protein. Data are expressed as the mean ± standard error of the mean values of three independently obtained protein samples from Western blot analysis. Con, control; Glu, glutamate; Nrf-2, nuclear factor-E2-related factor 2; HO, heme oxygenase; NQO1, NAD(P)H quinone oxidoreductase 1; ERK, extracellular signal-regulated kinase; CREB, cAMP response element-binding protein; BDNF, brain-derived neurotrophic factor; LDH, lactate dehydrogenase. ^# p < 0.05 (vs. control), ^* p < 0.05, ^** p < 0.01, ^† p < 0.001 (vs. glutamate), and ^‡ p < 0.05 (vs. glutamate + SHE 30).

FIGURE 3

Effects of Spirodelae Herba ethanol extract (SHE) on (A) viability, (B) secretion of NO, (C) production of inflammatory cytokines, and (D, E) activation of NF-κB/MAPK pathways in LPS-stimulated BV2 cells. (A–C) Data are expressed as the mean ± standard error of the mean of six independent experiments. (D, E) Histograms show the expression levels of proteins relative to those of the housekeeping protein. Data are expressed as the mean ± standard error of the mean values of three independently obtained protein samples from Western blot analysis. Con, control; LPS, lipopolysaccharide; TNF, tumor necrosis factor; IL, interleukin; MCP, monocyte chemoattractant protein; NF, nuclear factor; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH₂-terminal kinase. ^# p < 0.05 (vs. control), ^* p < 0.05, ^** p < 0.01, and ^† p < 0.001 (vs. LPS).

FIGURE 4

Effects of Spirodelae Herba ethanol extract (SHE) on scopolamine (SCO)-induced cognitive impairment in mice. Spatial memory function in mice was determined by the (A) escape latency, (B) escape distance, and (C) swimming trajectory in the Morris water maze test. (C) Images represent the swimming trajectory of mice in each group on day 6. Working memory ability was examined by (D) step-through latency in the passive avoidance test. (A, B, D) Data are expressed as the mean ± standard error of the mean of eight mice. ^# p < 0.05 (vs. normal), ^* p < 0.05, ^** p < 0.01, and ^† p < 0.001 (vs. SCO).

FIGURE 5

Effects of Spirodelae Herba ethanol extract (SHE) on scopolamine (SCO)-induced (A) neuronal damage and depression of the (B) ERK/CREB/BDNF and (C) PI3K/Akt/GSK-3β signaling pathways in mice brains. (A) Scale bars = 50 μm. Data are expressed as the mean ± standard error of the mean of six randomly selected fields. (B, C) Histograms show the expression levels of proteins relative to those of the housekeeping protein. Data are expressed as the mean ± standard error of the mean values of three independently obtained protein samples from Western blot analysis. ERK, extracellular signal-regulated kinase; CREB, cAMP response element-binding protein; BDNF, brain-derived neurotrophic factor; PI3K, phosphoinositide 3-kinases; Akt, protein kinase B; GSK, glycogen synthase kinase. ^# p < 0.05 (vs. normal), ^* p < 0.05, ^** p < 0.01, and ^† p < 0.001 (vs. SCO).

FIGURE 6

Effects of Spirodelae Herba ethanol extract (SHE) on gut microbiota diversity and composition in the scopolamine (SCO)-induced cognitive impairment mouse model. (A) Shannon and (B) evenness indices indicating the alpha diversity of gut microbiota. (C) Principal coordinates analysis based on Bray–Curtis dissimilarity showing the beta diversity of gut microbiota. (D) Relative abundance of major bacterial phyla. (E) Relative abundance of Firmicutes. (F) Relative abundance of Bacteroidetes. (G) Ratio of Bacteroidetes to Firmicutes in the normal, SCO, and SCO + SHE 100 groups. ^# p < 0.05 (vs. normal).

FIGURE 7

Structures of compounds 1–6 and high-performance liquid chromatograms of the six standard compounds derived from SHE at UV wavelengths of 320 nm. Isoorientin (1), orientin (2), vitexin (3), cynaroside (4), cosmosiin (5), and luteolin (6) were identified.

FIGURE 8

Effects of the main components of Spirodelae Herba ethanol extract (SHE) on glutamate-induced (A–C) cytotoxicity and (D, E) intracellular reactive oxygen species (ROS) production in HT22 cells and (F) structure–activity relationship of the six main components. (A–E) Data are expressed as the mean ± standard error of the mean of six independent experiments. (D, E) Scale bar = 200 μm. (F) Red zone: activity elements; Blue zone: inactivity elements. Con, control; Glu, glutamate; LDH, lactate dehydrogenase. ^# p < 0.05 (vs. control), ^* p < 0.05, ^** p < 0.01, and ^† p < 0.001 (vs. glutamate).