Neuroinflammation is a fundamental feature in the pathogenesis of amyotrophic lateral sclerosis (ALS) and arises from the activation of astrocytes and microglial cells. Previously, we reported that Miyako Bidens pilosa extract (MBP) inhibited microglial activation and prolonged the life span in a human ALS-linked mutant superoxide dismutase-1 (SOD1^G93A) transgenic mouse model of ALS (G93A mice). Herein, we evaluated the effect of MBP on microglial activation in the spinal cord of G93A mice and lipopolysaccharide-stimulated BV-2 microglial cells. The administration of MBP inhibited the upregulation of the M1-microglia/macrophage marker (interferon-γ receptor (IFN-γR)) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) in G93A mice. However, MBP did not affect the increase in the M2-microglia/macrophage marker (IL-13R) and anti-inflammatory cytokines (transforming growth factor (TGF)-β and IL-10) in G93A mice. BV-2 cell exposure to MBP resulted in a decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction activity and bromodeoxyuridine incorporation, without an increase in the number of ethidium homodimer-1-stained dead cells. Moreover, MBP suppressed the production of lipopolysaccharide-induced pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in BV-2 cells. These results suggest that the selective suppression of M1-related pro-inflammatory cytokines is involved in the therapeutic potential of MBP in ALS model mice.

Neuropathic pain, characterized by chronic allodynia, remains difficult to manage with current pharmacotherapies. Microglial activation plays a pivotal role in the development and maintenance of neuropathic pain and represents a promising therapeutic target. We previously demonstrated that Miyako Bidens pilosa extract powder (MBP), derived from Miyako Island, Okinawa, suppresses glial activation in a mouse model of amyotrophic lateral sclerosis. In this study, we investigated the analgesic potential of MBP in a mouse model of neuropathic pain. Neuropathic pain was induced in male ICR mice by partial sciatic nerve ligation (PSNL). Mice were orally administered MBP (2 g/kg) or vehicle daily. Mechanical allodynia was assessed using von Frey filaments. On postoperative day 7, MBP-treated mice exhibited significantly reduced allodynia compared to vehicle-treated mice. MBP also attenuated thermal hyperalgesia on postoperative day 7. Lumbar spinal cords (L5) were subjected to immunohistochemical analysis for ionized calcium-binding adaptor molecule 1 (Iba1), a microglial marker. MBP significantly decreased the number of Iba1-positive microglia in the ipsilateral dorsal horn. These results suggest that MBP alleviates neuropathic pain, at least in part, by suppressing microglial activation in the spinal cord. MBP may represent a novel plant-derived therapeutic candidate for treating neuropathic pain.

Oxidative stress and proteotoxic endoplasmic reticulum (ER) stress are interconnected cellular stress processes implicated in neuronal injury. J147 reportedly exerts neuroprotective effects through ATP synthase α-subunit (ATP5A)-associated mitochondrial mechanisms, but whether it protects cells from death under proteotoxic ER stress through the same mechanism remains unclear. Here, we directly compared curcumin, CNB-001, and J147 under glutamate-induced oxytosis and tunicamycin (TM)-induced ER stress in murine hippocampal HT22 cells. MTT reduction assays showed that all three compounds attenuated glutamate-induced cell death, whereas only J147 protected cells from TM-induced cytotoxicity at a submicromolar range. LIVE/DEAD staining confirmed the protective effects of J147 against both stressors. At the molecular level, J147 suppressed the TM-induced upregulation of the pro-apoptotic transcription factor C/EBP homologous protein (CHOP) at the mRNA and protein levels without suppressing the adaptive ER chaperone glucose-regulated protein 78. ATP5A knockdown attenuated glutamate-induced oxytosis but did not protect against TM-induced cell death, and J147 retained its protective effect after ATP5A knockdown. These findings support stressor-dependent protective actions of J147 involving an ATP5A-associated mechanism against oxidative stress and an ATP5A-independent mechanism associated with suppression of CHOP induction. J147 may therefore provide a useful pharmacological tool for investigating neuronal responses to interconnected oxidative and proteotoxic stress.

Neuritogenesis is essential for neuronal development and circuit formation. Although cAMP signaling downstream of Gs-coupled receptors is considered pro-neuritogenic, activation of these Gs-coupled receptors can produce divergent cellular outcomes. We previously showed that prostaglandin E₂ (PGE₂) induces neurite outgrowth in NSC-34 motor neuron-like cells predominantly through Gs-coupled E-prostanoid receptor 2 (EP2) signaling. The I-prostanoid receptor (IP) is also Gs-coupled, but whether its ligand PGI₂ elicits neuritogenesis remains unclear. Here, we compare the neuritogenic and signaling responses to PGE₂ and PGI₂ in NSC-34 cells. PGE₂ and the EP2 agonist butaprost increased the proportion of neurite-bearing cells, whereas PGI₂ and the IP agonist beraprost had no effect. PGI₂ and PGE₂ induced comparable cAMP accumulation and protein kinase A substrate phosphorylation, and elicited peak cAMP response element binding protein (CREB) phosphorylation at 1 h. However, only PGE₂ maintained significant CREB phosphorylation at 3–6 h. RNA sequencing at 4 h revealed broadly concordant transcriptional responses, while direct comparison identified Fst as the only gene expressed at higher levels under PGE₂ than under PGI₂. These findings suggest that the temporal profile of CREB phosphorylation, rather than the magnitude of early cAMP-PKA signaling, may be associated with differences in neuritogenic outcomes of Gs-coupled prostanoid signaling.