Neuroinflammation and oxidative stress play a crucial role in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease. The triggering receptor expressed on myeloid cells 2 (TREM2), highly expressed by microglia in the central nervous system (CNS), can modulate neuroinflammatory responses. Currently, there are no approved drugs specifically targeting TREM2 for CNS diseases. Aspidosperma alkaloids have shown potential as anti-inflammatory and neuroprotective agents. This study aimed to elucidate the potential therapeutic effect of Hecubine, a natural aspidosperma-type alkaloid, as a TREM2 activator in lipopolysaccharide (LPS)-stimulated neuroinflammation in in vitro and in vivo models. In this study, molecular docking and cellular thermal shift assay (CTSA) were employed to investigate the interaction between Hecubine and TREM2. Enzyme-linked immunosorbent assay (ELISA), quantitative PCR, immunofluorescence, Western blotting, and shRNA gene knockdown were used to assess the anti-neuroinflammatory and antioxidant effects of Hecubine in microglial cells and zebrafish. Our results revealed that Hecubine directly interacted with TREM2, leading to its activation. Knockdown of TREM2 mRNA expression significantly abolished the anti-inflammatory and antioxidant effects of Hecubine on LPS-stimulated proinflammatory mediators (NO, TNF-α, IL-6, and IL-1β) and oxidative stress in microglia cells. Furthermore, Hecubine upregulated Nrf2 expression levels while downregulating TLR4 signaling expression levels both in vivo and in vitro. Silencing TREM2 upregulated TLR4 and downregulated Nrf2 signaling pathways, mimicking the effect of Hecubine, further supporting TREM2 as the drug target by which Hecubine inhibits neuroinflammation. In conclusion, this is the first study to identify a small molecule, namely Hecubine directly targeting TREM2 to mediate anti-neuroinflammation and anti-oxidative effects, which serves as a potential therapeutic agent for the treatment of neural inflammation-associated CNS diseases.

Abstract Neuroinflammation and oxidative stress play crucial roles in many neurological diseases of the central nervous system. Targeting key proteins in inflammatory signaling may provide a new therapy for neuroinflammation. Hecubine is an active monoterpene indole alkaloid found in Ervatamia officinalis and the majority of its biological activities have not yet been explored. In the present study, we investigated the effects and mechanism of Hecubine on LPS-mediated neuroinflammation in vivo and in vitro for the first time. The results demonstrated that Hecubine reduced LPS-stimulated inflammatory cytokines overexpression, activated TREM2 expression, as well as suppressed the levels of TLR4-, MyD88-, and NF-κB-related proteins in BV2 microglia cells. Hecubine also exhibited an antioxidative effect, as evidenced by the reduction of ROS production and activation of the Nrf2/HO-1 pathway. Further drug target identification revealed that TREM2 is a primary interacting target of Hecubine. Knockdown of TREM2 mRNA expression significantly abolished Hecubine-induced anti-inflammatory and antioxidative effects via the upregulation of TLR4 signaling and the downregulation of Nrf2 pathway proteins. In vivo, after injection of LPS into the brain of zebrafish larvae, Hecubine administration obviously rescued behavioral deficits, inhibited the expression of pro-inflammatory cytokines, and prevented oxidative stress by activating TREM2. Taken together, Hecubine directly targets TREM2 to reduce neuroinflammation and oxidative stress and serves as a potential therapeutic agent for the treatment of neural inflammation-associated CNS diseases.

Background: Accumulating evidence suggest that behavioral sensitization is involved in the process of drug addiction. Zebrafish are sensitive to a variety of addictive drugs and are thus suitable for the study of behavioral sensitization. However, in contrast to mature rodent models of behavioral sensitization, how this phenomenon manifests in aquatic organisms, especially zebrafish, is largely unknown. In this study, we developed a morphineinduced behavioral sensitization adult zebrafish model and performed a preliminary investigation of the underlying mechanisms. Methods: Behavioral sensitization was established in zebrafish by observing their behavior after treatment and challenge with morphine. The effect of morphine was evaluated by a behavioral locomotor test. Different doses of morphine and withdrawal times were used to evaluate the establishment of the behavioral sensitization model. Results: Hyperlocomotion was induced after administration of morphine in adult zebrafish. After withdrawing the drug for a period, challenge with low-dose morphine evoked behavioral sensitization in zebrafish acutely pretreated with morphine. Low-dose morphine failed to induce behavioral sensitization in zebrafish if the withdrawal time was less than 5 days or more than 7 days. Morphine induced behavioral sensitization in zebrafish may involve dopaminergic, glutamatergic and opioid systems. Conclusion: A single low-dose of morphine could induce behavioral sensitization in zebrafish acutely pre-treated with morphine, and this phenomenon was highly correlated with drug dose and withdrawal time. These findings suggest that zebrafish is a suitable model for the study of behavioral sensitization.