Oral administration of osthole mitigates maladaptive behaviors through PPARα activation in mice subjected to repeated social defeat stress.

Psychological stress induces neuroinflammatory responses, which are associated with the pathogenesis of various psychiatric disorders, such as posttraumatic stress disorder and anxiety. Osthole-a natural coumarin isolated from the seeds of the Chinese herb Cnidium monnieri-exerts anti-inflammatory and antioxidative effects on the central nervous system. However, the therapeutic benefits of osthole against psychiatric disorders remain largely unknown. We previously demonstrated that mice subjected to repeated social defeat stress (RSDS) in the presence of aggressor mice exhibited symptoms of posttraumatic stress disorder, such as social avoidance and anxiety-like behaviors. In this study, we investigated the therapeutic effects of osthole and the underlying molecular mechanisms. Osthole exerted therapeutic effects on cognitive behaviors, mitigating anxiety-like behaviors and social avoidance in a mouse model of RSDS. The anti-inflammatory response induced by the oral administration of osthole was strengthened through the upregulation of heme oxygenase-1 expression. The expression of PPARα was inhibited in mice subjected to RSDS. Nonetheless, osthole treatment reversed the inhibition of PPARα expression. We identified a positive correlation between heme oxygenase-1 expression and PPARα expression in osthole-treated mice. In conclusion, osthole has potential as a Chinese herbal medicine for anxiety disorders. When designing novel drugs for psychiatric disorders, researchers should consider targeting the activation of PPARα.

Ramelteon protects against social defeat stress-associated abnormal behaviors.

Psychological stress affects the neuroendocrine regulation, which modulates mental status and behaviors. Melatonin, a hormone synthesized primarily by the pineal gland, regulates many brain functions, including circadian rhythms, pain, sleep, and mood. Selective pharmacological melatonin agonist ramelteon has been clinically used to treat mood and sleep disorders. Posttraumatic stress disorder (PTSD) is a psychiatric condition associated with severe trauma; it is generally triggered by traumatic events, which lead to severe anxiety and uncontrollable trauma recall. We recently reported that repeated social defeat stress (RSDS) may induce robust anxiety-like behaviors and social avoidance in mice. In the present study, we investigated whether melatonin receptor activation by melatonin and ramelteon regulates RSDS-induced behavioral changes. Melatonin treatment improved social avoidance and anxiety-like behaviors in RSDS mice. Moreover, treatment of the non-selective MT1/MT2 receptor agonist, ramelteon, markedly ameliorated RSDS-induced social avoidance and anxiety-like behaviors. Moreover, activating melatonin receptors also balanced the expression of monoamine oxidases, glucocorticoid receptors, and endogenous antioxidants in the hippocampus. Taken together, our findings indicate that the activation of both melatonin and ramelteon regulates RSDS-induced anxiety-like behaviors and PTSD symptoms. The current study also showed that the regulatory effects of neuroendocrine mechanisms and cognitive behaviors on melatonin receptor activation in repeated social defeat stress.

LPS priming-induced immune tolerance mitigates LPS-stimulated microglial activation and social avoidance behaviors in mice.

In this study, we investigated the regulatory mechanisms underlying the effects of LPS tolerance on the inflammatory homeostasis of immune cells. LPS priming-induced immune tolerance downregulated cyclooxygenase-2, and lowered the production of prostaglandin-E2 in microglial cells. In addition, LPS tolerance downregulated the expression of suppressor of cytokine signaling 3, and inducible nitric oxide synthase/nitric oxide; suppressed the LPS-mediated induction of tumor necrosis factor-α, interleukin (IL)-6, and IL-1; and reduced reactive oxygen species production in microglial cells. LPS stimulation increased the levels of the adaptive response-related proteins heme oxygenase-1 and superoxide dismutase 2, and the levels of heme oxygenase-1 (HO-1) enhanced after LPS priming. Systemic administration of low-dose LPS (0.5 mg/kg) to mice for 4 consecutive days attenuated high-dose LPS (5 mg/kg)-induced inflammatory response, microglial activation, and proinflammatory cytokine expression. Moreover, repeated exposure to low-dose LPS suppressed the recruitment of peripheral monocytes or macrophages to brain regions and downregulated the expression of proinflammatory cytokines. Notably, LPS-induced social avoidance behaviors in mice were mitigated by immune tolerance. In conclusion, immune tolerance may reduce proinflammatory cytokine expression and reactive oxygen species production. Our findings provide insights into the effects of endotoxin tolerance on innate immune cells and social behaviors.

Inhibitory Effects of Urolithins, Bioactive Gut Metabolites from Natural Polyphenols, against Glioblastoma Progression.

We previously reported that proinflammatory cytokines, particularly tumor necrosis factor (TNF)-α, promoted tumor migration, invasion, and proliferation, thus worsening the prognosis of glioblastoma (GBM). Urolithins, the potent metabolites produced by the gut from pomegranate polyphenols, have anticancer properties. To develop an effective therapy for GBM, this study aimed to study the effects of urolithins against GBM. Urolithin A and B significantly reduced GBM migration, reduced epithelial-mesenchymal transition, and inhibited tumor growth. Moreover, urolithin A and B inhibited TNF-α-induced vascular cell adhesion molecule (VCAM)-1 and programmed death ligand 1 (PD-L1) expression, thereby reducing human monocyte (HM) binding to GBM cells. Aryl hydrocarbon receptor (AhR) level had higher expression in patients with glioma than in healthy individuals. Urolithins are considered pharmacological antagonists of AhR. We demonstrated that the inhibition of AhR reduced TNF-α-stimulated VCAM-1 and PD-L1 expression. Furthermore, human macrophage condition medium enhanced expression of PD-L1 in human GBM cells. Administration of the AhR antagonist attenuated the enhancement of PD-L1, indicating the AhR modulation in GBM progression. The modulatory effects of urolithins in GBM involve inhibiting the Akt and epidermal growth factor receptor pathways. The present study suggests that urolithins can inhibit GBM progression and provide valuable information for anti-GBM strategy.

Bradykinin B1 Receptor Affects Tumor-Associated Macrophage Activity and Glioblastoma Progression.

Bradykinin is a small active peptide and is considered an inflammatory mediator in several pathological conditions. Bradykinin exerts its effects by coupling to its receptors, including bradykinin B1 (B1R) and bradykinin B2. B1R has been implicated in the development of various cancers. Our previous study reported that B1R promoted glioblastoma (GBM) development by supporting the migration and invasion of GBM cells. However, the mechanisms underlying the effects of B1R on tumor-associated macrophages (TAMs) and GBM progression remain unknown. Accordingly, to explore the regulatory effects of B1R overexpression (OE) in GBM on tumor-associated immune cells and tumor progression, we constructed a B1R wild-type plasmid and developed a B1R OE model. The results reveal that B1R OE in GBM promoted the expression of ICAM-1 and VCAM-1-cell adhesion molecules-in GBM. Moreover, B1R OE enhanced GBM cell migration ability and monocyte attachment. B1R also regulated the production of the protumorigenic cytokines and chemokines IL-6, IL-8, CXCL11, and CCL5 in GBM, which contributed to tumor progression. We additionally noted that B1R OE in GBM increased the expression of CD68 in TAMs. Furthermore, B1R OE reduced the level of reactive oxygen species in GBM cells by upregulating heme oxygenase-1, an endogenous antioxidant protein, thereby protecting GBM cells from oxidative stress. Notably, B1R OE upregulated the expression of programmed death-ligand 1 in both GBM cells and macrophages, thus providing resistance against T-cell response. B1R OE in GBM also promoted tumor growth and reduced survival rates in an intracranial xenograft mouse model. These results indicate that B1R expression in GBM promotes TAM activity and modulates GBM progression. Therefore, B1R could be an effective target for therapeutic methods in GBM.

Role of Zerumbone, a Phytochemical Sesquiterpenoid from Zingiber zerumbet Smith, in Maintaining Macrophage Polarization and Redox Homeostasis.

Macrophages and microglia are highly versatile cells that can be polarized into M1 and M2 phenotypes in response to diverse environmental stimuli, thus exhibiting different biological functions. In the central nervous system, activated resident macrophages and microglial cells trigger the production of proinflammatory mediators that contribute to neurodegenerative diseases and psychiatric disorders. Therefore, modulating the activation of macrophages and microglia by optimizing the inflammatory environment is beneficial for disease management. Several naturally occurring compounds have been reported to have anti-inflammatory and neuroprotective properties. Zerumbone is a phytochemical sesquiterpenoid and also a cyclic ketone isolated from Zingiber zerumbet Smith. In this study, we found that zerumbone effectively reduced the expression of lipocalin-2 in macrophages and microglial cell lines. Lipocalin-2, also known as neutrophil gelatinase-associated lipocalin (NGAL), has been characterized as an adipokine/cytokine implicated in inflammation. Moreover, supplement with zerumbone inhibited reactive oxygen species production. Phagocytic activity was decreased following the zerumbone supplement. In addition, the zerumbone supplement remarkably reduced the production of M1-polarization-associated chemokines CXC10 and CCL-2, as well as M1-polarization-associated cytokines interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α. Furthermore, the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 and the production of NO were attenuated in macrophages and microglial cells supplemented with zerumbone. Notably, we discovered that zerumbone effectively promoted the production of the endogenous antioxidants heme oxygenase-1, glutamate-cysteine ligase modifier subunit, glutamate-cysteine ligase catalytic subunit, and NAD(P)H quinone oxidoreductase-1 and remarkably enhanced IL-10, a marker of M2 macrophage polarization. Endogenous antioxidant production and M2 macrophage polarization were increased through activation of the AMPK/Akt and Akt/GSK3 signaling pathways. In summary, this study demonstrated the protective role of zerumbone in maintaining M1 and M2 polarization homeostasis by decreasing inflammatory responses and enhancing the production of endogenous antioxidants in both macrophages and microglia cells. This study suggests that zerumbone can be used as a potential therapeutic drug for the supplement of neuroinflammatory diseases.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-glucoside Attenuates Reactive Oxygen Species-Dependent Inflammation and Apoptosis in Porphyromonas gingivalis-Infected Brain Endothelial Cells.

We recently reported that the periodontopathic bacteria Porphyromonas gingivalis (P. gingivalis) initiates an inflammatory cascade that disrupts the balance of reactive oxygen species (ROS), resulting in apoptotic cell death in brain endothelial cells. An extract from Polygonum multiflorum Thunb., 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-glucoside (THSG) has been well-reported to diminish the inflammation in many disease models. However, the effects of THSG in the area of the brain-oral axis is unknown. In this study, we examined the effects of THSG in P. gingivalis-stimulated inflammatory response and apoptotic cell death in brain endothelial cells. THSG treatment remarkably lessened the upregulation of IL-1ß and TNF-α proteins in bEnd.3 cells infected with P. gingivalis. Treatment of THSG further ameliorated brain endothelial cell death, including apoptosis caused by P. gingivalis. Moreover, the present study showed that the inhibitory effects on NF-κB p65 and antiapoptotic properties of THSG is through inhibiting the ROS pathway. Importantly, the ROS inhibitory potency of THSG is similar to a ROS scavenger N-Acetyl-L-Cysteine (NAC) and NADPH oxidase inhibitor apocynin. Furthermore, the protective effect of THSG from P. gingivalis infection was further confirmed in primary mouse brain endothelial cells. Taken together, this study indicates that THSG attenuates an ROS-dependent inflammatory response and cell apoptosis in P. gingivalis-infected brain endothelial cells. Our results also suggest that THSG could be a potential herbal medicine to prevent the risk of developing cerebrovascular diseases from infection of periodontal bacteria.

Porphyromonas gingivalis Induces Proinflammatory Cytokine Expression Leading to Apoptotic Death through the Oxidative Stress/NF-κB Pathway in Brain Endothelial Cells.

Porphyromonas gingivalis, a periodontal pathogen, has been proposed to cause blood vessel injury leading to cerebrovascular diseases such as stroke. Brain endothelial cells compose the blood-brain barrier that protects homeostasis of the central nervous system. However, whether P. gingivalis causes the death of endothelial cells and the underlying mechanisms remain unclear. This study aimed to investigate the impact and regulatory mechanisms of P. gingivalis infection in brain endothelial cells. We used bEnd.3 cells and primary mouse endothelial cells to assess the effects of P. gingivalis on endothelial cells. Our results showed that infection with live P. gingivalis, unlike heat-killed P. gingivalis, triggers brain endothelial cell death by inducing cell apoptosis. Moreover, P. gingivalis infection increased intracellular reactive oxygen species (ROS) production, activated NF-κB, and up-regulated the expression of IL-1ß and TNF-α. Furthermore, N-acetyl-L-cysteine (NAC), a most frequently used antioxidant, treatment significantly reduced P. gingivalis-induced cell apoptosis and brain endothelial cell death. The enhancement of ROS production, NF-κB p65 activation, and proinflammatory cytokine expression was also attenuated by NAC treatment. The impact of P. gingivalis on brain endothelial cells was also confirmed using adult primary mouse brain endothelial cells (MBECs). In summary, our results showed that P. gingivalis up-regulates IL-1ß and TNF-α protein expression, which consequently causes cell death of brain endothelial cells through the ROS/NF-κB pathway. Our results, together with the results of previous case-control studies and epidemiologic reports, strongly support the hypothesis that periodontal infection increases the risk of developing cerebrovascular disease.