Neuroinflammation, mediated by microglia and astrocytes, is an abnormal immune reaction in central nervous system (CNS) disorders. Stimulation of TRPV1 has been found to enhance microglial activation, resulting in a pro-inflammatory response. Natural anthraquinones such as physcion and rhein are commonly found in rhubarb, a medicinal plant recognized for its dual role in culinary and therapeutic applications. The therapeutic potential and mechanisms of these anthraquinones remain largely unexplored. This research aims to examine how anthraquinones protect against neuroinflammation and delineate the underlying mechanisms in lipopolysaccharide (LPS)-mediated cellular and zebrafish models. Among the representative anthraquinone analogs, physcion and rhein showed potent functional inhibitory activity against the TRPV1 channel. The production of nitric oxide (NO) and secretion of pro-inflammatory factors triggered by LPS were significantly reduced in BV-2 cells through regulation of iNOS, IL-6, IL-1β, and TNF-α mRNA expression. Moreover, physcion and rhein inhibited calcium influx and exerted anti-neuroinflammatory effects, which were closely associated with the suppression of Ca2+/CAMKK2/AKT and the PI3K/AKT-mediated NF-κB activation pathways. Furthermore, physcion and rhein reduced LPS-driven neutrophil recruitment to the brain and ameliorated locomotor deficits in zebrafish larvae, with the restoration of IL-1β, IL-6, and TNF-α transcript levels to baseline. In conclusion, natural-derived anthraquinones from rhubarb, physcion and rhein, acted as functional inhibitors of TRPV1-mediated calcium dynamics and significantly reduced LPS-mediated neuroinflammation in microglial cells and zebrafish larvae, suggesting promise as therapeutics for neurological disorders.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Potassium voltage-gated channels are potential targets for the treatment of PD. The aim of this study is to identify novel potassium ion channel blockers for the treatment of PD through transcriptomic analysis of the coral species Galaxea fascicularis. After annotation by four different databases, four peptides were selected that showed characteristics of potassium ion channel blockers. These four peptides were subjected to multiple sequence alignment and phylogenetic analysis. These four peptides were identified as of Kunitz-type peptides, are known as potassium ion channel blockers. The structures of the peptides were modeled and subjected to molecular dynamics (MD) simulation to verify their stability, which indicated that the peptide GfKuz1 showed the highest potency to block KV1.3 (potassium voltage-gated channel subfamily A member 3) among the reference peptides. The MD simulation of the peptide-protein complexes showed that GfKuz1 interacted with KV1.3, and was more compact and stable than the other potassium ion channel. The blocking effect was confirmed by a potassium ion bioassay. Furthermore, GfKuz1 showed no toxicity to PC-12 cells or zebrafish at concentrations up to 100 mu M. In addition, GfKuz1 increased the PC-12 cell viability that was reduced by 6-hydroxydopamine hydrochloride, and also down-regulated the level of reactive oxygen species and activated the Nrf2 pathway. In summary, GfKuz1 reversed PD symptoms and is a potential peptide drug prototype for PD treatment.

Pain serves as a vital protective mechanism triggered by tissue damage. While NSAIDs and opioids offer relief, their prolonged usage is hindered by adverse effects. Developing analgesics with fewer side effects is crucial for effective pain treatment. The TRPV1 channel is a key target for pain relief, with its inhibitors effectively reducing hyperalgesia in animals. This research utilized virtual screening to identify TRPV1-selective natural compounds for potent analgesic properties. The physcion exhibited the notable affinity for TRPV1 compared to the compounds examined. After conducting molecular dynamics simulations, physcion emerged as the compound demonstrating the highest binding affinity towards TRPV1, a finding corroborated by calcium imaging, which validated its inhibitory impact. Furthermore, physcion mitigated the stretch number in the acetic acid-induced stretching model, prolonged the latency period in the hot water tail-flick and hot plate assays, and heightened the pain withdrawal threshold lowered by complete Freund’s adjuvant (CFA). Notably, physcion exerted a marked effect in ameliorating bone cancer-induced pain in the hot plate and von Frey tests. Additionally, physcion diminished the levels of inflammatory cytokines and the mRNA expression of both inflammatory and calcium-related genes in the CFA-induced murine model. Furthermore, physcion downregulated the expression of inflammatory genes induced by tumor necrosis factor-α (TNF-α) in RAW264.7 cells. The underlying mechanism potentially involves the suppression of the NF-κB and MAPK signaling cascades. Our investigation underscores the potential of physcion as a promising candidate for analgesic therapy.

Stony corals (Scleractinia) harbor abundant toxins as part of their survival strategy, and these molecules also provide novel lead candidates for drug development. In this study, we present the first comprehensive investigation of toxin profiles of the understudied Goniopora columna through transcriptome analysis, which has expanded the biochemical diversity of marine organism derived toxins. Then, a novel Kunitz-like polypeptide, named GcKuz1, which exhibits therapeutic potential for thrombosis was selected for functional studies and mechanistic investigation. In vitro experiments revealed that GcKuz1, at 2–16 μM, delayed plasma recalcification time and activated partial thromboplastin time without affecting prothrombin time. In murine models, GcKuz1, at 1–4 mg/kg dosage, significantly inhibited the FeCl3-induced carotid artery thrombosis and the carrageenan-induced mouse tail thrombosis. Moreover, GcKuz1 effectively attenuated thrombo-inflammation and cerebral tissue destruction, as well as restored blood–brain barrier integrity, in the transient middle cerebral artery occlusion model. Through enzyme kinetics assays and surface plasmon resonance (SPR) verification, GcKuz1 was shown to strongly inhibit the enzyme activity of plasma kallikrein (PKa) and FXIIa, two key factors involved in the contact-kinin pathway, via direct interaction, thereby exerting anticoagulation effects without impairing hemostasis. Notably, safety evaluation highlights the low toxicity, minimal hemolytic activity and reduced bleeding risk of GcKuz1, which underline its clinical availability. In conclusion, as a novel coral-derived protease inhibitor of FXIIa and PK, GcKuz1 offers potential therapeutic benefits in the treatment thrombosis-related cardiovascular diseases treatment by suppressing inflammation and preventing thrombus formation.

BACKGROUND: Elastic fibers (EF) disorganization contributes to increased tissue stiffness and impaired lung function in pulmonary fibrosis (PF). However, the complex structural features of EF are difficult to capture using conventional histology. Moreover, the molecular mechanisms governing EF homeostasis during fibrosis remain poorly understood. METHODS: A high-dimensional Elastic Fiber Algorithm (EFA) was developed to digitally quantify EF structural features in PF. Gene variation-rate analysis was performed to identify candidate regulators of EF homeostasis. Fibroblast-specific and pathological fibroblast-specific MFAP5 knockout mice were generated to assess the role of MFAP5 in PF rodent models. Bulk RNA sequencing was employed to investigate MFAP5-mediated fibroblast activation and signaling pathways. RESULTS: EFA revealed profound EF disorganization in fibrotic lungs, capturing various architectural alterations. Gene variation-rate analysis identified MFAP5 as a candidate regulator of EF homeostasis, with upregulated expression in PF patients and mouse models. Fibroblast-specific deletion of MFAP5 significantly attenuated fibrosis, restored EF architecture, reduced collagen deposition, and improved pulmonary function in PF mouse models. MFAP5-positive fibroblasts displayed a dynamic shift toward pathological states during PF progression. Mechanistically, MFAP5 promoted fibroblast activation and ECM production via αvβ3 integrin-mediated TGFβ signaling. CONCLUSIONS: MFAP5 is a key orchestrator of EF remodeling and fibroblast activation in PF. Targeting MFAP5 restores EF homeostasis, reduces fibrotic severity, and represents a potential therapeutic strategy for PF. ### Competing Interest Statement The authors have declared no competing interest.

Bone cancer pain (BCP) is one of the most common types of chronic pain in cancer patients, with a prevalence of up to 75%. However, the pathological mechanism and therapeutic approaches are limited. Here, we demonstrated that Na+/K+-ATPase α1 (NKAα1) is a critical regulator of nociception through interaction with purinergic P2X3 receptor (P2X3R) in the dorsal root ganglion (DRG). Conditional knockout of NKAα1 in transient receptor potential vanilloid 1-positive (TRPV1+) neurons led to an increase in P2X3R-dependent Ca2+ influx and neuronal hyperexcitability and also promoted pain hypersensitivity in BCP model mice. In addition, NKAα1 knockout in TRPV1+ neurons further enhanced C-C motif chemokine ligand 5 release, thereby exacerbating spinal glial cell activation and pain hypersensitivity in BCP mice. DR5-12D, a monoclonal antibody to stabilize the expression of NKAα1, markedly inhibited the hyperexcitability of DRG nociceptors and ameliorated pain hypersensitivity in BCP mice. Overall, NKAα1 modulates P2X3R-dependent Ca2+ influx and the excitability of DRG nociceptors, thereby providing valuable theoretical guidance for the treatment of BCP.

BACKGROUND Pulmonary hypertension (PH) is characterized by vascular remodeling without effective treatments. The initiation and progression of PH are heavily influenced by the dysfunction of pulmonary artery endothelial cells. Thioredoxin domain containing 5 (TXNDC5), a member of the protein disulfide isomerases (PDI) family, catalyzes the formation and rearrangement of disulfide bonds in proteins, crucial for endothelial cell function and maintaining vascular homeostasis in cardiovascular diseases, yet its role in PH remains unclear. METHODS Label-free proteomics was used to reveal the PDI protein expression profile in hypoxic PH patients. Multiplex immunofluorescence and Western blot analyses were performed to investigate the pathological role of TXNDC5. Mice with adeno-associated virus-mediated lung endothelial TXNDC5 overexpression, global knockout, and endothelial ablation of TXNDC5 were used to study the role of TXNDC5 in rodent PH models. In vitro experiments with cultured pulmonary arterial endothelial cells and bioinformatics assays were used to elucidate the underlying mechanisms. Pharmacological inhibition and endothelial-targeted TXNDC5 gene therapy were evaluated for their effects in PH. RESULTS TXNDC5 was significantly upregulated in the lungs of both PH patients and rodent models, with prominent expression in the endothelial layer of remodeled distal pulmonary arteries. Overexpression of TXNDC5 exacerbated Sugen5416/hypoxia (SuHx)-induced pulmonary artery remodeling, increased right ventricular systolic pressure, and right ventricular hypertrophy. Global or endothelial-specific deficiency of TXNDC5 exerted protective efficacy against the SuHx challenge. RNA sequencing and protein-protein interaction analysis revealed TXNDC5 as a potential regulator of extracellular matrix (ECM) homeostasis, with biglycan (BGN) identified as a critical downstream effector. TXNDC5 required BGN to regulate the ubiquitination of HIF-1α/2α, resulting in its abnormal accumulation to drive PH development. E64FC26, a TXNDC5 inhibitor, and endothelial-targeted TXNDC5 gene therapy exhibited therapeutic efficacy in SuHx-induced PH rats. CONCLUSIONS Our study reveals that TXNDC5 is a main modulator to regulate ECM homeostasis and may serve as a promising target for the treatment of PH. Graphic Abstract The role of TXNDC5 in regulating ECM homeostasis during PH. In the healthy state, BGN folding is properly maintained and HIF-1α/2α is rapidly degraded. During PH, TXNDC5 is upregulated, leading to excessive accumulation of BGN and impaired ubiquitination-mediated degradation of HIF-1α/2α, resulting in an imbalance in ECM homeostasis and contributing to vascular remodeling. Pharmacological or genetic inhibition of TXNDC5 restores BGN folding capacity and facilitates HIF-1α/2α degradation, thereby reestablishing ECM homeostasis and attenuating PH progression. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Key Program of National Natural Science Foundation of China Shenzhen Science Fund for Distinguished Young Scholars, RCJC20210706091946002 Shenzhen Science and Technology Program, GJHZ20240218111401002 National Natural Science Foundation of China, 82241021 [1]: pending:yes

Neuroinflammation, defined as the inflammatory response in the brain or spinal cord, plays a pivotal role in multiple neurodegenerative conditions including Parkinson's and Alzheimer's diseases. Transient receptor potential cation channel subfamily V member 1 (TRPV1), widely expressed in microglia, influences their function by regulating the production of these immune-modulating molecules. To identify peptides with anti-neuroinflammatory properties targeting TRPV1, a set of seven Kunitz-type peptides named DrKuz1 to 7 was discovered in Dipsastraea rotumana. Through molecular docking and dynamic simulations, it was shown that DrKuz1 interacted with key residues crucial for TRPV1 activation. Functional assays confirmed that DrKuz1 induced calcium influx in HEK293 cells overexpressing hTRPV1. Furthermore, DrKuz1 demonstrated its anti-inflammatory properties by reducing the levels of nitric oxide (NO), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) activated by lipopolysaccharides (LPS) in mouse microglial cells (BV-2). Moreover, DrKuz1 restored the LPS-activated inflammatory gene expression and abnormal locomotory behavior in zebrafish larvae. This anti-inflammatory effect of DrKuz1 has been found to involve modulation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in LPS-treated BV-2 cells. Therefore, DrKuz1 emerges as a promising tool for investigating TRPV1 function and as a potential therapeutic candidate for neuroinflammation.

BACKGROUND:Bruceine A (BA), a natural quassinoid compound derived from the fruit of Brucea javanica, has demonstrated diverse pharmacological activities. However, the treatment effectiveness of BA against ulcerative colitis (UC) and its underlying mechanisms of action are yet to be fully elucidated. PURPOSE:To explore the effects of BA against UC and uncover its underlying mechanisms, with a particular focus on its influence on inhibiting macrophage M1 polarization via the IL-17/NF-κB pathway. METHODS:A murine UC model was constructed using 3 % dextran sulfate sodium (DSS) and treated with BA. Colon length, histopathological damage, inflammatory mediators, and epithelial barrier integrity were assessed to evaluate BA's therapeutic efficacy. In vitro, cell viability and macrophage polarization biomarkers (e.g., iNOS and TNF-α) were quantified to evaluate the cytoprotective effects of BA. The potential mechanisms of BA in combating UC were investigated using network pharmacology, with direct targets validated through biophysical approaches. Furthermore, western blot analysis was employed to confirm BA's regulatory effect on the protein expression of the IL-17/NF-κB pathway in both UC mice and macrophages. RESULTS:BA treatment effectively restored colon length, attenuated histopathological inflammation, and repaired epithelial barrier via upregulating tight junction proteins (e.g., ZO-1 and Occludin) in UC mice. Although BA failed to enhance cell viability in DSS-injured IEC-6 cells, it potently inhibited M1 macrophage polarization in THP-1 cells, as evidenced by downregulated iNOS and TNF-α. Mechanistically, BA bound to heat shock protein 90 (HSP90), leading to a significant reduction in the expression of IL-17/NF-κB pathway-related proteins, thereby suppressing the IL-17 pathway and the NF-κB cascade activation. Notably, overexpression of HSP90 abrogated BA's inhibition of the IL-17/NF-κB pathway and M1 macrophage polarization. CONCLUSIONS:BA effectively inhibits macrophage M1 polarization, thereby attenuating inflammation in UC by targeting HSP90 and subsequently suppressing the activation of IL-17 and NF-κB pathway. These results imply that BA is a promising therapeutic candidate for UC treatment.

Introduction:Pain is a complex phenomenon involving physiological and psychological responses to noxious stimuli. Long-term opioid or NSAID use leads to reduced efficacy and tolerance. Initially a thermosensitive receptor, TRPV1 is increasingly recognized as a target for analgesic intervention. Methods:Our investigation is focused on the exploration of novel TRPV1 antagonists derived from natural sources through computational screening methodologies, aiming to assess their efficacy as analgesic agents. Results:Among the compounds screened, a promising TRPV1 antagonist named pinocembrin-7-o-3-o-galloyl-4-6-hexahydroxydiphenoyl-beta-d-glucoside (PINO) has exhibited superior stability in its interaction with TRPV1 through virtual screening and molecular dynamics simulation. A dosage of 20 mg/kg of PINO had been shown to reduce the writhing response in acetic acid-induced mice, elevate the thermal pain threshold in the hot water tail-flick and hot plate assays, and concurrently increase the mechanical pain threshold in CFA-induced inflammatory pain models in mice. Moreover, in a murine Lewis lung carcinoma cell line LL-induced bone cancer pain model, PINO also effectively raised the thermal and mechanical pain thresholds in mice. Furthermore, PINO had been found to attenuate the production and gene expression of pro-inflammatory cytokines. The underlying mechanism was attributed to the suppression of NF-κB and MAPK signaling cascades. Discussion:This innovative compound represents a prospective avenue for the management of acute, chronic, and bone cancer pain, providing a viable alternative analgesic option for individuals suffering from such conditions.

Phellodendri Chinensis Cortex, a time-honored Chinese materia medica, has long been applied in the management of gout and other diseases. Hyperuricemia (HUA) is the vital contributor for the occurrence and progression of gout. Oxyberberine (OBB), an oxidized protoberberine alkaloid, is naturally present in Phellodendri Chinensis Cortex. The work made a pioneering endeavor to unravel the potential anti-HUA effect and mechanism of OBB. Hight fructose-induced experimental HUA in rats was employed. Results indicated that OBB significantly reduced the body weight and UA level, and concurrently mitigated hepatic injury, which achieved superior therapeutic effect to its prodrug berberine (BBR). OBB treatment remarkably alleviated inflammatory response and oxidative stress. Furthermore, OBB appreciably activated the gene expression of key enzymes involved in the pentose phosphate pathway (PPP), and inhibited the transcriptional or translational expression of key enzymes in de novo purine biosynthesis (DNPB), as well as modulated purine salvage pathway (PSP), collectively leading to a reduction in UA level. Molecular docking and dynamic simulation further confirmed the dynamic stability of OBB-target protein complexes. Meanwhile, OBB favorably modulated the fructose-induced gut dysbiosis, enhanced the abundance of probiotics and abated detrimental counterparts, which were associated with promoting UA catabolism and inhibiting inflammation response via correlation analysis. In summary, our findings in pioneering endeavor found that OBB efficaciously ameliorated fructose-elicited HUA. The mechanism was intimately associated with alleviating endogenous purine original biosynthetic and metabolic pathways and favorably harmonizing gut microflora homeostatic disequilibrium. This study has uncovered innovative mechanisms underlying OBB's therapeutic potential for HUA and bolstered the scientific basis for the historical use of Phellodendri Chinensis Cortex and BBR in the management of HUA.

Purpose To investigate the therapeutic effect and underlying mechanism of tetrahydrocurcumin (THC) on nonalcoholic steatohepatitis (NASH) induced by high-fat diet (HFD). Methods NASH rat model was established through long-term feeding HFD, and the steatosis cell model was stimulated via palmitate acid (PA). The therapeutic effect of THC was evaluated in terms of liver function, lipid metabolism, liver pathophysiology, inflammation and oxidative stress in vivo, and lipid accumulation in vitro. The alteration in lipophagy was identified by using western blot and immunofluorescence. mTORC1-TFEB signaling pathway was measured by qRT-PCR, western blot and protein-ligand docking. In addition, chloroquine and MHY1485 were further introduced to validate the effect of THC on lipophagy and mTORC1-TFEB signaling pathway, respectively. Results THC effectively improved hepatic steatosis, inflammation and oxidative stress in NASH rats, and reduced lipid accumulation in steatosis L02 cells and Hep G2 cells. THC promoted lipophagy with increasing LC3B-II as well as decreasing P62 expression via lysosomal biogenesis upregulation, which was greatly weakened after chloroquine intervention. mTORC1-TFEB is a critical pathway for regulating lysosome in autophagy, THC treatment induced TFEB nucleus translocation via inhibiting mTORC1 to upregulate lysosomal biogenesis. However, these effects were partly eliminated by mTORC1 activator MHY1485. Conclusion THC restored lipophagy to reduce lipid accumulation by regulating mTORC1-TFEB pathway in NASH rats and steatosis hepatocytes. These findings suggested that THC represents a therapeutic candidate for NASH treatment.

Ethnopharmacological relevance: Palmatine is a main bioactive alkaloid of Cortex Phellodendri, which has been commonly prescribed for the treatment of hyperuricemia (HUA) in China. The metabolites of palmatine were crucial to its prominent biological activity. 9-Hydroxy-8-oxypalmatine (9-OPAL) is a novel liver-mediated secondary oxymetabolite of palmatine. Aim of the study: The current study was to assess the efficacy of 9-OPAL, a novel liver-mediated secondary oxymetabolite of palmatine derived from Cortex Phellodendri, in experimental HUA mouse model and further explore its underlying mechanism. Materials and methods: An in vitro metabolic experiment with oxypalmatine was carried out using liver samples. We separated and identified a novel liver metabolite, and investigated its anti-HUA effect in mice. HUA mice were induced by potassium oxonate and hypoxanthine daily for one week. After 1 h of modeling, mice were orally administered with different doses of 9-OPAL (5, 10 and 20 mg/kg). The pathological changes of the kidneys were evaluated using hematoxylin-eosin staining (H&E). The acute toxicity of 9-OPAL was assessed. The effects of 9-OPAL on serum levels of uric acid (UA), adenosine deaminase (ADA), xanthine oxidase (XOD), creatinine (CRE), blood urea nitrogen (BUN) and inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA) or biochemical method. Furthermore, Western blot, quantitative real-time PCR (qRT-PCR) and molecular docking were used to investigate the effect of 9-OPAL on the expression of renal urate transporters and NLRP3 signaling pathway in HUA mice. Results: 9-OPAL had been discovered to be a novel liver-mediated oxymetabolite of palmatine for the first time. Treatment with 9-OPAL significantly reduced the UA, CRE as well as BUN levels, and also effectively attenuated abnormal renal histopathological deterioration with favorable safety profile. Besides, 9-OPAL significantly decreased the serum and hepatic activities of XOD and ADA, dramatically inhibited the up-regulation of UA transporter protein 1 (URAT1) and glucose transporter protein 9 (GLUT9), and reversed the down-regulation of organic anion transporter protein 1 (OAT1). Additionally, 9-OPAL effectively mitigated the renal inflammatory markers (TNF-alpha, IL-1 beta, IL-6 and IL-18), and downregulated the transcriptional and translational expressions of renal Nod-like receptor family pyrin domain containing 3 (NLRP3), caspase-1, apoptosis-associated speck-like (ASC) and IL-1(3 in HUA mice. Molecular docking results revealed 9-OPAL bound firmly with XOD, OAT1, GLUT9, URAT1, NLRP3, caspase-1, ASC and IL-1(3. Conclusions: 9-OPAL was found to be a novel liver-mediated secondary metabolite of palmatine with favorable safety profile. 9-OPAL had eminent anti-hyperuricemic and renal-protective effects, and the mechanisms might be intimately associated with repressing XOD activities, modulating renal urate transporter expression and suppressing the NLRP3 inflammasome activation. Our investigation might also provide further experimental evidence for the traditional application of Cortex Phellodendri in the treatment of HUA.

Dysfunction of the Na+/K+-ATPase (NKA) has been documented in various neurodegenerative diseases, yet the specific role of NKAα1 in Parkinson's disease (PD) remains incompletely understood. In this investigation, we utilized NKAα1 haploinsufficiency (NKAα1+/−) mice to probe the influence of NKAα1 on dopaminergic (DA) neurodegeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our findings reveal that NKAα1+/− mice displayed a heightened loss of DA neurons and more pronounced motor dysfunction compared to the control group when exposed to MPTP. Intriguingly, this phenomenon coincided with the activation of ferroptosis and impaired mitophagy both in vivo and in vitro. To scrutinize the role and underlying mechanism of NKAα1 in PD, we employed DR-Ab, an antibody targeting the DR-region of the NKA α subunit. Our study demonstrates that the administration of DR-Ab effectively reinstated the membrane abundance of NKAa1, thereby mitigating MPTP-induced DA neuron loss and subsequent improvement in behavioral deficit. Mechanistically, DR-Ab heightened the formation of the surface NKAα1/SLC7A11 complex, inhibiting SLC7A11-dependent ferroptosis. Moreover, DR-Ab disrupted the cytosolic interaction between NKAα1 and Parkin, facilitating the translocation of Parkin to mitochondria and enhancing the process of mitophagy. In conclusion, this study establishes NKAα1 as a key regulator of ferroptosis and mitophagy, identifying its DR-region as a promising therapeutic target for PD.

6-benzylaminopurine (6-BA), classified as a "plant hormone", is an important ingredient in production of "toxic bean sprouts". Although there is no direct evidence of adverse effects, its hazardous effects have received some attention and aroused furious debate between proponents and environmental regulators. In this study, potential adverse effects of 6-BA were investigated by exposing zebrafish in vivo to 0.2 - 25 mg 6-BA/L. Results indicated that, when exposure was limited to early-life stage (4-36 hpf), 20 mg 6-BA/L caused early hatching, abnormal spontaneous movement, and precocious hyperactivity in zebrafish embryos/larvae. While under a continuous exposure regime, 6-BA at 0.2 mg/L was able to cause hyperactive locomotion and transcription of genes related to neurogenesis (gnrh3 and nestin) and endocrine systems (cyp19a and fshb) in 5 dpf larvae. Quantification by use of LC/MS indicated bioaccumulation of 6-BA in zebrafish increased when exposed to 0.2 or 20 mg 6-BA/L. These results suggested that 6-BA could accumulate in aquatic organisms and disrupt neuro-endocrine systems. Accordingly, exposure to 0.2 mg 6-BA/L increased production of estradiol (E2) and consequently E2/T ratio in zebrafish larvae, which directly indicated 6-BA is estrogenic. In silico simulations demonstrated potential for binding of 6-BA to estrogen receptor alpha (ERa) and cytochrome P450 aromatase (CYP19A). Therefore, induction of estrogenic effects, via potential interactions with hormone receptors or disturbance of downstream transcription signaling, was possible mechanism underlying the toxicity of 6-BA. Taken together, these findings demonstrate endocrine disrupting properties of 6-BA, which suggest concerns about risks posed to endocrine systems.

Approximately 50 million people are suffering from epilepsy worldwide. Corals have been used for treating epilepsy in traditional Chinese medicine, but the mechanism of this treatment is unknown. In this study, we analyzed the transcriptome of the branching coral Acropora digitifera and obtained its Kyoto Encyclopedia of Genes and Genomes (KEGG), EuKaryotic Orthologous Groups (KOG) and Gene Ontology (GO) annotation. Combined with multiple sequence alignment and phylogenetic analysis, we discovered three polypeptides, we named them AdKuz1, AdKuz2 and AdKuz3, from A. digitifera that showed a close relationship to Kunitz-type peptides. Molecular docking and molecular dynamics simulation indicated that AdKuz1 to 3 could interact with GABA A receptor but AdKuz2–GABA A remained more stable than others. The biological experiments showed that AdKuz1 and AdKuz2 exhibited an anti-inflammatory effect by decreasing the aberrant level of nitric oxide (NO), IL-6, TNF-α and IL-1β induced by LPS in BV-2 cells. In addition, the pentylenetetrazol (PTZ)-induced epileptic effect on zebrafish was remarkably suppressed by AdKuz1 and AdKuz2. AdKuz2 particularly showed superior anti-epileptic effects compared to the other two peptides. Furthermore, AdKuz2 significantly decreased the expression of c-fos and npas4a , which were up-regulated by PTZ treatment. In addition, AdKuz2 reduced the synthesis of glutamate and enhanced the biosynthesis of gamma-aminobutyric acid (GABA). In conclusion, the results indicated that AdKuz2 may affect the synthesis of glutamate and GABA and enhance the activity of the GABA A receptor to inhibit the symptoms of epilepsy. We believe, AdKuz2 could be a promising anti-epileptic agent and its mechanism of action should be further investigated.

The roots of Glycine tabacina are used to treat rheumatoid arthritis (RA) and joint infection in folk medicine. Glytabastan B (GlyB), a newly reported coumestan isolated from this species, was found to significantly attenuate IL-1β-induced inflammation in SW982 human synovial cells at 3 and 6 μM, as evidenced by the decreased levels of pro-inflammatory mediators and matrix metalloproteinases (MMPs). GlyB also suppressed RANKL-induced osteoclastogenesis, decreased the expression of osteoclastogenic markers (NFATc1, CTSK, MMP-9) and osteoclast-mediated bone resorption. Further, GlyB administration (12.5 and 25 mg/kg) significantly inhibited inflammation, osteoclast formation and disease progression in collagen-induced arthritis (CIA) mice. Integration of network pharmacology, quantitative phosphoproteomic and experimental pharmacology results revealed that these beneficial actions were closely associated with the blockade of GlyB on the activation of MAPK, PI3K/AKT and their downstream signals including NF-κB and GSK3β/NFATc1. Drug affinity responsive target stability (DARTS) assay, cellular thermal shift (CETSA) assay and molecular docking analysis confirmed that there were direct interactions between GlyB and its target proteins ERK2, JNK1 and class Ⅰ PI3K catalytic subunit p110 (α, β, δ and γ), which significantly contributed to the inhibition of activation of MAPK and PI3K/AKT pathways. In conclusion, these results strongly suggest GlyB is a promising multiple-target candidate for the development of agents for the prevention and treatment of RA.

Berberine (BBR) has extremely low concentration and high tissue distribution. However, current pharmacokinetic studies predominantly focus on its concentration in plasma, which could hardly make a comprehensive understanding of its pharmacokinetic process. This study made a pioneering endeavor to explore the erythrocyte-hemoglobin (Hb) self-assembly system of BBR by exploring the interaction of BBR with erythrocyte and the combination of BBR with Hb. Results showed that BBR had a low bioavailability (C0 = 2.833 μg/mL via intravenous administration of 2.5 mg/kg BBR and Cmax = 0.260 μg/mL via oral administration of 400 mg/kg BBR). Besides, BBR achieved higher concentrations in erythrocytes than plasma, and the erythrocytes count and Hb content were significantly decreased after intravenous administration. Hemolysis rate indicated the BBR-erythrocyte system (with 2% erythrocytes) was relatively stable without hemolysis at the concentration of 1.00 mg/mL. And the maximum percentage of drug loading was 100% when the BBR-erythrocyte concentration was 0.185 μg/mL. Furthermore, incubation of BBR and erythrocytes resulted in internalization of the erythrocyte membrane and the formation of intracellular vacuoles. The thermodynamic parameters indicated that the binding process of bovine hemoglobin (BHB) and BBR was spontaneous. UV-vis absorption spectra, synchronous fluorescence, circular dichroism and Raman spectra collectively indicated that BBR showed strong binding affinity toward BHB and affected the molecular environment of residues like tryptophan and tyrosine in BHB, resulting in the conformational changes of its secondary and tertiary structure. Molecular docking indicated BBR interacted with Arg-141 residue of BHB via hydrogen bond with the bond length of 2.55 Å. The ΔG value of the BHB-BBR system was -31.79 kJ/mol. Molecular dynamics simulation indicated the root mean square derivation of BBR-BHB was <0.025 nm, suggestive of stable conformation. Cumulatively, there was an erythrocyte-Hb self-assembled drug delivery system after oral or intravenous administration of BBR, which conceivably gained novel insight into the discrepancy between the extremely low plasma concentration and relatively high tissue concentration of BBR.

The present study aimed to evaluate the neuroprotective effects and underlying mechanisms of pinocembrin-7-methylether (PME), a natural bioflavonoid, in 6-hydroxydopamine (6-OHDA)-induced models of Parkinson's disease in vivo and in vitro. First, we found that PME decreased apoptosis in 6-OHDA-intoxicated SH-SY5Y cells. PME also blocked several 6-OHDA-induced mitochondrial apoptotic cascades, including loss of mitochondrial membrane potential, caspase 3 and PARP activation, and a decrease in the Bcl-2/Bax ratio. Also, PME suppressed 6-OHDA-induced oxidative stress while increasing antioxidant enzymatic activity. Further investigations indicated that PME significantly enhanced nuclear accumulation of Nrf2, improved ARE promoter activity, and upregulated HO-1 and NQO1 expression levels. In addition, siRNA-mediated Nrf2 knockdown abolished PME-induced anti-oxidative and anti-apoptotic effects. Interestingly, we found that PME promoted phosphorylation of AKT and ERK, whereas pharmacological inhibition of AKT or ERK pathways diminished PME-induced Nrf2 activation and protective actions. Moreover, PME attenuated 6-OHDA-induced loss of dopaminergic neurons and ameliorated locomotor deficiency in zebrafish, supporting the neuroprotective actions of PME in vivo. In summary, we found that PME conferred neuroprotection against 6-OHDA-induced neurotoxicity in PD models in vivo and in vitro. Taken together, our findings suggest that activation of Nrf2/ARE/HO-1 signaling cascades contributes to PME-induced anti-oxidative and neuroprotective actions, which are at least partially mediated by AKT and ERK pathways.

PcActx peptide, identified from the transcriptome of zoantharian Palythoa caribaeorum, was clustered into the phylogeny of analgesic polypeptides from sea anemone Heteractis crispa (known as APHC peptides). APHC peptides were considered as inhibitors of transient receptor potential cation channel subfamily V member 1 (TRPV1). TRPV1 is a calcium-permeable channel expressed in epileptic brain areas, serving as a potential target for preventing epileptic seizures. Through in silico and in vitro analysis, PcActx peptide was shown to be a potential TRPV1 channel blocker. In vivo studies showed that the linear and oxidized PcActx peptides caused concentration-dependent increases in mortality of zebrafish larvae. However, monotreatment with PcActx peptides below the maximum tolerated doses (MTD) did not affect locomotor behavior. Moreover, PcActx peptides (both linear and oxidized forms) could effectively reverse pentylenetetrazol (PTZ)-induced seizure-related behavior in zebrafish larvae and prevent overexpression of c-fos and npas4a at the mRNA level. The excessive production of ROS induced by PTZ was markedly attenuated by both linear and oxidized PcActx peptides. It was also verified that the oxidized PcActx peptide was more effective than the linear one. In particular, oxidized PcActx peptide notably modulated the mRNA expression of genes involved in calcium signaling and γ-aminobutyric acid (GABA)ergic-glutamatergic signaling, including calb1, calb2, gabra1, grm1, gria1b, grin2b, gat1, slc1a2b, gad1b, and glsa. Taken together, PcActx peptide, as a novel neuroactive peptide, exhibits prominent anti-epileptic activity, probably through modulating calcium signaling and GABAergic-glutamatergic signaling, and is a promising candidate for epilepsy management.