Hesperetin, a Citrus Flavonoid, Attenuates LPS-Induced Neuroinflammation, Apoptosis and Memory Impairments by Modulating TLR4/NF-κB Signaling

Abstract

Glial activation and neuroinflammation play significant roles in apoptosis as well as in the development of cognitive and memory deficits. Neuroinflammation is also a critical feature in the pathogenesis of neurodegenerative disorders such as Alzheimer and Parkinson's diseases. Previously, hesperetin has been shown to be an effective antioxidant and anti-inflammatory agent. In the present study, in vivo and in vitro analyses were performed to evaluate the neuroprotective effects of hesperetin in lipopolysaccharide (LPS)-induced neuroinflammation, oxidative stress, neuronal apoptosis and memory impairments. Based on our findings, LPS treatment resulted in microglial activation and astrocytosis and elevated the expression of inflammatory mediators such as phosphorylated-Nuclear factor-κB (p-NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in the cortical and hippocampal regions and in BV2 cells. However, hesperetin cotreatment markedly reduced the expression of inflammatory cytokines by ameliorating Toll-like receptor-4 (TLR4)-mediated ionized calcium-binding adapter molecule 1/glial fibrillary acidic protein (Iba-1/GFAP) expression. Similarly, hesperetin attenuated LPS-induced generation of reactive oxygen species/lipid per oxidation (ROS/LPO) and improved the antioxidant protein level such as nuclear factor erythroid 2-related factor 2 (Nrf2) and Haem-oxygenase (HO-1) in the mouse brain. Additionally, hesperetin ameliorated cytotoxicity and ROS/LPO induced by LPS in HT-22 cells. Moreover, hesperetin rescued LPS-induced neuronal apoptosis by reducing the expression of phosphorylated-c-Jun N-terminal kinases (p-JNK), B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), and Caspase-3 protein and promoting the Bcl-2 protein level. Furthermore, hesperetin enhanced synaptic integrity, cognition, and memory processes by enhancing the phosphorylated-cAMP response element binding protein (p-CREB), postsynaptic density protein-95 (PSD-95), and Syntaxin. Overall, our preclinical study suggests that hesperetin conferred neuroprotection by regulating the TLR4/NF-κB signaling pathway against the detrimental effects of LPS.

Keywords: LPS; hesperetin; memory Impairments; microglia/astrocytes; neurodegeneration; neuroinflammation; reactive oxygen species (ROS); tumor necrosis factor (TNF).

Figure 1

Schematic diagram of the experimental design showing the duration of the lipopolysaccharide (LPS) and/or hesperetin administration in adult mice and behavioral analysis.

Figure 2

Hesperetin ameliorates TLR4/gliosis-mediated neuroinflammation in the LPS-treated mouse brain: (A) Western blot analysis showing the expression of TLR4, GFAP, and Iba-1 in the experimental mice (cortex and hippocampus regions); (B) confocal photomicrographs showing the immunoreactivity of GFAP in the cortex and DG region of hippocampus in different experimental mice groups. The data are presented as the mean ± SEM of 7–10 mice per group and are representative of three independent experiments, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

Hesperetin mitigates the expression of p-NF-κB and inflammatory cytokines in LPS-treated mouse brains and BV2 cells: (A) Western blot analysis of p-NF-κB, TNF-α, and IL-1β expression in the cortex and hippocampus of mice; (B) immunofluorescence analysis of TNF-α immunoreactivity in the cortex and DG region of the hippocampus. The data are presented as the mean ± SEM of 7–10 mice per group and are representative of three independent experiments, * p ≤ 0.05, ** p ≤ 0.01.

Figure 4

(A) Western blot analysis of the TLR4 and Iba-1 expression in BV2 cells treated with hesperetin and TAK-242 (TLR4 inhibitor) and their respective histograms; (B) Western blot analysis of p-NF-κB and TNF-α protein expression level in BV2 cell line treated with LPS and/or hesperetin/BAY (NF-κB inhibitor). The data are shown as the mean ± SEM of triplicate wells for in vitro experiments are representative of three independent experiments, * p ≤ 0.05, ** p ≤ 0.01.

Figure 5

Effects of hesperetin against LPS-induced oxidative stress in the cortical and hippocampal region of mouse brains and in HT-22 cells: (A,B) the analysis of the generation of ROS and LPO production in vivo in the mouse brain (cortex and hippocampus regions); (C) Western blot analysis of Nrf2 and HO-1 in the cortex and hippocampus of adult mouse brains. The data are presented as the mean ± SEM of 8–10 mice per group and are representative of three independent experiments, * p ≤ 0.05, ** p ≤ 0.01.

Figure 6

The protective effects of hesperetin in LPS-treated in HT-22 cells: (A) the MTT assay conducted in HT-22 cells representing the cell viability in different treated groups (number of experiments = 3); (B,C) Analysis of the ROS generation and LPO production in HT-22 Cells. The data are shown as the mean ± SEM of triplicate wells for in vitro experiments are representative of three independent experiments, * p ≤ 0.05.

Figure 7

Hesperetin reversed the LPS-induced apoptotic cell death in the mouse brain and enhanced cell viability in vitro: (A) Western blot analysis of stress kinase p-JNK and pro-apoptotic protein Bax and cleaved-Caspase-3 and anti-apoptotic protein expression differential experiential groups and their histograms respectively; (B) photomicrograph of Nissl’s staining in the cortex and DG regions of the hippocampus in the mouse brain. The data are presented as the mean ± SEM of 7–10 mice per group and are representative of three independent experiments, * p ≤ 0.05, ** p ≤ 0.01.

Figure 8

Hesperetin attenuated the detrimental effects of LPS on the synaptic and memory-related proteins in the mouse brain: (A) Western blot analysis representing the expression level of p-CREB, Syntaxin, and PSD-95 in the cortex and hippocampus of the experimental groups; (B) confocal immunofluorescent photomicrographs of p-CREB reactivity in the cortex and DG region of hippocampus of the experimental groups. The data are presented as the mean ± SEM of 7–10 mice per group and are representative of three independent experiments, * p ≤ 0.05, ** p ≤ 0.01.

Figure 9

Hesperetin improved memory, learning, and cognitive behavior in LPS-treated adult mice: (A) mean escape latency to reach the hidden platform during training (5 days) with its representative trajectories at day-6 and (B) at the 6th day after training; (C,D) the time spent in the target quadrant where the hidden platform was previously present and the number of crossings over that location in the absence of a platform; (E) the Y-Maze analysis representing the spontaneous alteration behaviors of mice and its representative trajectories. The data are presented as the mean ± SEM of 8–10 mice per group and are representative of three independent experiments, * p ≤ 0.05, ** p ≤ 0.01.