Lupeol Treatment Attenuates Activation of Glial Cells and Oxidative-Stress-Mediated Neuropathology in Mouse Model of Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) signifies a major cause of death and disability. TBI causes central nervous system (CNS) damage under a variety of mechanisms, including protein aggregation, mitochondrial dysfunction, oxidative stress, and neuroinflammation. Astrocytes and microglia, cells of the CNS, are considered the key players in initiating an inflammatory response after injury. Several evidence suggests that activation of astrocytes/microglia and ROS/LPO have the potential to cause more harmful effects in the pathological processes following traumatic brain injury (TBI). Previous studies have established that lupeol provides neuroprotection through modulation of inflammation, oxidative stress, and apoptosis in Aβ and LPS model and neurodegenerative disease. However, the effects of lupeol on apoptosis caused by inflammation and oxidative stress in TBI have not yet been investigated. Therefore, we explored the role of Lupeol on antiapoptosis, anti-inflammatory, and antioxidative stress and its potential mechanism following TBI. In these experiments, adult male mice were randomly divided into four groups: control, TBI, TBI+ Lupeol, and Sham group. Western blotting, immunofluorescence staining, and ROS/LPO assays were performed to investigate the role of lupeol against neuroinflammation, oxidative stress, and apoptosis. Lupeol treatment reversed TBI-induced behavioral and memory disturbances. Lupeol attenuated TBI-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 heme-oxygenase 1 (HO-1) in the mouse brain. Similarly, our results indicated that lupeol treatment inhibited glial cell activation, p-NF-κB, and downstream signaling molecules, such as TNF-α, COX-2, and IL-1β, in the mouse cortex and hippocampus. Moreover, lupeol treatment also inhibited mitochondrial apoptotic signaling molecules, such as caspase-3, Bax, cytochrome-C, and reversed deregulated Bcl2 in TBI-treated mice. Overall, our study demonstrated that lupeol inhibits the activation of astrocytes/microglia and ROS/LPO that lead to oxidative stress, neuroinflammation, and apoptosis followed by TBI.

Keywords: cognitive impairment; glia activation; lupeol; neurodegenerative diseases; neuroinflammation; oxidative stress; traumatic brain injury.

Figure 1

Lupeol ameliorates TBI-induced microglia/astrocytes activation in mouse brain: (A) Western blot analysis showing the expression of GFAP and Iba-1 in the experimental mice (cortex and hippocampus regions); (B,C) confocal photomicrographs showing the immunoreactivity of GFAP and Iba-1 (FITC—green), stained with DAPI (blue) in the cortex and DG region of hippocampus in different mice groups. The cropped bands were quantified using Image software, and the differences are represented in the histogram. Asterisk (*) sign indicates significant difference from the normal saline-treated group; hash (#) sign indicates significant difference from the TBI-treated group. Bar = 50µm. The density values are expressed in arbitrary units as the mean ± SEM of the indicated proteins (n = 6 animals per group). Significance = p ≤ 0.05.

Figure 2

Lupeol treatment inhibited TBI-induced oxidative stress and ameliorated ROS/LPO production in the cortical and hippocampal region of mouse brains: (A) Western blot analysis representing the expression of Nrf2 and HO-1 in the cortex and hippocampus of mouse brain; (B,C) the analysis of the generation of ROS and LPO production in vivo in the mouse brain (cortex and hippocampus); (D) immunofluorescence analysis of Nrf2 immunoreactivity (FITC—green), stained with DAPI (blue) in the cortex and DG region of hippocampus in different experimental mice groups. The cropped bands were quantified using Image software, and the differences are represented in the histogram. Asterisk (*) sign indicates significant difference from the normal saline-treated group; hash (#) sign indicates significant difference from the TBI-treated group, while the phi (Φ) sign indicates no significant from normal saline-treated control group. Bar = 50 µm. The density values are expressed in arbitrary units as the mean ± SEM of the indicated proteins (n = 6 animals per group). Significance = p ≤ 0.05.

Figure 3

Lupeol alleviates the expression of p-NF-kB and inflammatory cytokines in TBI-treated mouse brain: (A) Western blot analysis of p-NF-kB, TNF-α, COX2, and IL-1β expression in the cortex and hippocampus of mouse; (B) immunofluorescence analysis of TNF-α immunoreactivity (FITC—green), stained with DAPI (blue) in the cortex and DG region of hippocampus in different experimental mice groups. The cropped bands were quantified using Image software, and the differences are represented in the histogram. Asterisk (*) sign indicates significant difference from the normal saline-treated group; hash (#) sign indicates significant difference from the TBI-treated group. Bar = 50µm. The density values are expressed in arbitrary units as the mean ± SEM of the indicated proteins (n = 6 animals per group). Significance = p ≤ 0.05.

Figure 4

Lupeol reversed the TBI-induced apoptotic cell death in the mouse brain. (A) Western blot analysis of proapoptotic protein caspase-3, Bax, cytochrome-C, and antiapoptotic protein expression (Bcl2) in the cortex and hippocampus of mice; (B) immunofluorescence images of the colocalized reactivity of GFAP (FITC—green), Casp3 (red), stained with DAPI (blue) in the cortex region; (C) immunofluorescence images of the colocalized reactivity of GFAP (FITC—green), Casp3 (red), stained with DAPI (blue) in the hippocampus region. The zoom represents high magnification. The cropped bands were quantified using Image software, and the differences are represented in the histogram. Asterisk (*) sign indicates significant difference from the normal saline-treated group; hash (#) sign indicates significant difference from the TBI-treated group. Bar = 50µm. The density values are expressed in arbitrary units as the mean ± SEM of the indicated proteins (n = 6 animals per group). Significance = p ≤ 0.05.

Figure 4

Lupeol reversed the TBI-induced apoptotic cell death in the mouse brain. (A) Western blot analysis of proapoptotic protein caspase-3, Bax, cytochrome-C, and antiapoptotic protein expression (Bcl2) in the cortex and hippocampus of mice; (B) immunofluorescence images of the colocalized reactivity of GFAP (FITC—green), Casp3 (red), stained with DAPI (blue) in the cortex region; (C) immunofluorescence images of the colocalized reactivity of GFAP (FITC—green), Casp3 (red), stained with DAPI (blue) in the hippocampus region. The zoom represents high magnification. The cropped bands were quantified using Image software, and the differences are represented in the histogram. Asterisk (*) sign indicates significant difference from the normal saline-treated group; hash (#) sign indicates significant difference from the TBI-treated group. Bar = 50µm. The density values are expressed in arbitrary units as the mean ± SEM of the indicated proteins (n = 6 animals per group). Significance = p ≤ 0.05.

Figure 5

Lupeol improved memory, learning, and cognitive behavior in TBI-treated 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. Asterisk (*) sign indicates significant difference from the normal saline-treated group; hash (#) sign indicates significant difference from the TBI-treated group. The density values are expressed in arbitrary units as the mean ± SEM of the indicated proteins (n = 6 animals per group). Significance = * p ≤ 0.05, # p ≤ 0.05.

Figure 6

Suggested graphical representation of possible Lupeol neuroprotective effect against TBI-induced neurotoxicity. Lupeol (a triterpenoid) reduced TBI-induced neuroinflammation, oxidative stress, neuroapoptosis, memory impairment, and neurodegeneration induced by TBI in mouse brain. (Upward direction of arrow indicates activation/upregulation, while downward direction indicates downregulation).

Figure 7

Schematic diagram of the experimental design showing the duration of the TBI and/or lupeol administration in adult mice and the behavioral analysis.