Given the potential of natural products (NPs) for various aspects of human well-being, it is important to involve a variety of stakeholders and disciplines in the investigation of their health-related properties. For this reason, IUPHAR has initiated a project aimed at establishing a science-based registry of NPs and their pharmacological activities, with a view to providing a better understanding of their potential influences on human health. From a pharmacological perspective, it is important to evaluate the effects of selected NPs on important determinants of health such as, e.g. immune response and inflammation (among others). IUPHAR calls for a concerted effort and asks stakeholders to contribute to this project.

There is controversy about a causal role of serotonin (5-HT) in depression, some arguing that there is no proof for impaired brain 5-HT function in depressed patients. Major depressive disorder comes with multiple endophenotypes; not surprisingly classical antidepressants (tricyclics, MAO inhibitors, SSRIs, SNRIs) are not universally effective. Most antidepressants target the 5-HT system, partially if not exclusively, but treatment-resistant depression (TRD) remains a major issue. The most recent and heavily investigated class of potential rapid acting antidepressant, anxiolytic, and/or anti PTSD drugs, namely psychedelics (psilocybin, LSD, DMT, ayahuasca, etc..) or entactogens (MDMA, ibogaine), all target the 5-HT system, at least in part. Phase II / III clinical trials support psychedelics- and/or MDMA-assisted psychotherapy as a new class of rapid acting treatments for GAD, MDD, TRD, PTSD, and other disorders. Psilocybin and MDMA have FDA breakthrough status for TRD/MDD and PTSD, respectively, whereas LSD just received FDA breakthrough status for GAD. All psychedelics act as 5-HT2A receptor agonists, although LSD, DMT, psilocybin may also target other 5-HT and/or dopamine receptors. Psychedelics produce rapid onset and long-lasting antidepressant effects after one or two administrations. They all promote synaptogenesis and synaptic plasticity. Neuroinflammation plays a major role in anxiety, depression, PTSD. Interestingly, psychedelic-induced 5-HT2A receptor agonism has profound anti-(neuro)inflammatory effects. Altogether, the 5-HT system plays an essential, but not unique role in MDD and related disorders. MDD, TRD and PTSD may be considered as biochemical, neurological and immune conditions, given the emerging role of neuroplasticity and neuroinflammation, which until recently, have been overlooked.

The total iridoid glycoside extract of Lamiophlomis rotata (IGLR) has been proven to be the main pharmacological ingredient for wound healing; however, it is still unclear, which compounds in the extract are responsible for this effect. We developed and validated a mass spectrometry imaging (MSI) method to reveal the relationship between phytochemical components and the pharmacological efficacy in IGLR. Shanzhiside methyl (SM) ester, 8-O-acetylshanzhiside methyl ester (ASM), and Phlorigidoside C (PhC) have been identified as quality markers in IGLR to accelerate wound healing by MSI method. As per MSI data with UMAP manifold analysis, these compounds overlapped in the same class with endogenous metabolites (lactate, citrate and succinate). In addition, UPLC-Q/TOF-MSn coupled with multivariate analyses for skin tissues in the normal and wound groups further confirmed the results. Furthermore, MSI data from livers and kidneys revealed that SM, ASM, and PhC absorption significantly increased, whereas their elimination rates remarkably decreased in the injured group. Collectively, the overall data demonstrated that SM, ASM, and PhC selectively increased in the new granulation tissue and that the bioavailability of these three quality markers was remarkably improved in the wound group after continuous gavage for 7 days. This protocol can be applied to reveal the relationship between phytochemical components and the pharmacological efficacy of other iridoid glycoside extracts.

Abstract Background and Purpose: Activation of Nrf2 holds great promise for treating major depressive disorder (MDD). Trilobatin (TLB) is a naturally occurring food additive conferring robust neuroprotection with Nrf2 activation potency. This study was designed to explore whether TLB is able to overcome MDD, and the role of Nrf2 in the antidepressant effect of TLB. Experimental Approach: Mice and primary hippocampal astrocytes challenged with lipopolysaccharide (LPS) were used to decipher the effects of TLB on MDD. Nrf2-deficient mice were treated with TLB and fecal microbiota transplantation (FMT) to validate the potential targets of ICS Ⅱ on ALI. Key results: TLB ameliorated depressive-like behavior in LPS-induced MDD mouse model. Single-cell RNA sequencing analysis of the hippocampus revealed that astrocytes exhibited distinct MDD-related clustering with increased Nrf2 in the antidepressant effect of TLB. TLB directly bound to Nrf2 and increased Nrf2-antioxidant response element (ARE) binding activity, which ultimately restored mitochondrial function, reduced oxidative stress and neuroinflammation. TLB improved intestinal microbiota dysbiosis and attenuated intestinal barrier through increasing expressions of the tight junction proteins. Fecal microbiota transplantation from TLB-treated mice also ameliorated depression-like behavior after LPS insult. Furthermore, by using genetically modified Nrf2-knockout mice, we validated that the antidepressant effect of TLB were Nrf2/ARE signaling dependent, suppressing astrocytes activation and gut microbiota dysbiosis. These favorable effects of TLB were abrogated in Nrf2 deficiency mice. Conclusions and implications: Our results reveal a new-found pharmacological property of TLB: serves as a novel and naturally-occurring Nrf2 activator to conquer MDD through modulating microbiota-gut-brain axis.

Background and Purpose Botulinum Neurotoxin type A1 (BoNT/A) is one of the most potent neurotoxins known. At the same time, it is also one of the safest therapeutic agents used for the treatment of several human disorders and in aesthetic medicine. Notwithstanding great effectiveness, strategies to accelerate the onset and prolong BoNT/A action would significantly ameliorate its pharmacological effects with beneficial repercussions on clinical use. Experimental Approach Here, we combined BoNT/A with two fast-acting inhibitors of excitation-contraction coupling (ECCI), either the µ-conotoxin CnIIIC or dantrolene, and tested the effect of their co-injection on a model of hind-limb paralysis in rodents using behavioral, biochemical, imaging and electrophysiology assays. Key Results We report that the BoNT/A-ECCI combination accelerated the onset of muscle relaxation. Surprisingly, it also potentiated the peak effect and extended the duration of the three BoNT/A commercial preparations onabotulinumtoxinA, abobotulinumtoxinA and incobotulinumtoxinA. We found that ECCI co-injection increased the number of BoNT/A molecules entering motoneuron terminals, which induced a faster and larger cleavage of SNAP-25 during the onset and peak phases, and prolonged the attenuation of nerve-muscle neurotransmission during the recovery phase. We estimate that ECCI co-injection yields a three-fold potentiation in BoNT/A pharmacological activity. Conclusions and Implications Overall, our results show that the pharmacological activity of BoNT/A can be combined and synergized with other bioactive molecules and uncover a novel strategy to enhance BoNT/A neuromuscular effects without altering the neurotoxin moiety or intrinsic activity, thus maintaining its exceptional safety profile.

Background and Purpose: Lycorine is an alkaloid that was in the bulb of the genus Lycoris. It has properties of anti-inflammatory. This study aimed to investigate the molecular mechanism by which lycorine can reduce acute lung injury (ALI). Experimental Approach: ALI model was established by intranasal injection of lipopolysaccharide (LPS). In vitro, A549 cells were treated with LPS and pretreated with lycorine for 1 hour. Key Results: The results showed that lycorine reduced histopathological changes in lung, myeloperoxidase (MPO) activity, and the production of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in mice. Lycorine dose-dependently inhibited the production of TNF-α, IL-1β, and IL-6. It also inhibited the transmission of TLR4/NF-κB passway in LPS-stimulated A549 cells. Lycorine increased cholesterol efflux through the activated LXRα-ABCA1/ABCG pathway. Lycorine has a good binding ability with LXRα. After adding the LXRα inhibitor, the anti-inflammatory effect of lycorine was eliminated. Conclusion and Implications: Lycorine can reduce ALI that was induced by lipopolysaccharide. The anti-inflammatory mechanism of lycorine is related to the up-regulation of the LXRα-ABCA1/ABCG pathway, which inhibits TLR4-mediated inflammation by increasing cholesterol efflux and reducing TLR4 transport to lipid rafts.

Background and Purpose: Treatment of osteoarthritis (OA) pain often yields unsatisfactory results, making the search for new pain-relieving options essential. Vortioxetine, an antidepressant with a unique mechanism of action, has recently shown analgesic properties. We aimed to investigate its effects in the OA model and gain insight into the underlying molecular mechanisms. Duloxetine (a second-line drug for pain relief in OA) was studied as a reference. Experimental Approach: In the monoiodoacetate (MIA)-induced model of knee OA in rats of both sexes, pain-related behaviour was assessed in weight-bearing and von Frey tests. Antidepressants were administered orally once daily for 28 days. Gene expression of pain-related mediators (Ngf, Il-1β, Tnf-α, Bdnf, and Tac1 encoding substance P) and oxidative stress parameters were determined after completion of the treatment/behavioural testing protocol. Key results: Vortioxetine dose-dependently reduced weight-bearing asymmetry and mechanical allodynia of the paw ipsilateral to the MIA-injected knee. Duloxetine was also effective. Vortioxetine reduced the increased Ngf mRNA expression in the MIA-injected knees to the level observed in the sham-injected counterparts. It also reduced oxidative stress parameters in the affected knees, more effectively in females than in males. Duloxetine showed no effect on locally increased Ngf mRNA expression and oxidative stress. Both antidepressants decreased mRNA expression of pain-related mediators in the lumbar L3-L5 ipsilateral DRGs and spinal cords, which were upregulated in MIA-injected rats. This effect was male-specific. Conclusion and Implications: Vortioxetine may be effective against chronic pain in OA. This effect appears to be mediated, at least in part, by normalization of NGF expression in the affected joint.

Background and Purpose Quetiapine is a second-generation atypical antipsychotic drug that has been commonly prescribed for the treatment of schizophrenia, major depressive disorder (depression), and other psychological disorders. Targeted inhibition of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels, which generate Ih, may provide effective resistance against schizophrenia and depression. We investigated if HCN channels could contribute to the therapeutic effect of quetiapine, and its major active metabolite norquetiapine. Experimental Approach Two-electrode voltage clamp recordings were used to assess the effects of quetiapine and norquetiapine on currents from wild-type and mutant HCN1 and HCN2 expressed in Xenopus laevis oocytes. Key Results Norquetiapine, but not quetiapine nor 7-hydroxy quetiapine, has an inhibitory effect on HCN1 channels. Norquetiapine selectively inhibited HCN1 currents by shifting the voltage-dependence of activation to more hyperpolarized potentials in a concentration-dependent manner with an IC50 of 13.9 ± 0.8 μM for HCN1 and slowing channel opening, without changing the kinetics of closing. Inhibition by norquetiapine primarily occurs from in the closed state. Norquetiapine inhibition is not sensitive to the external potassium concentration, and therefore, likely does not block the pore. Norquetiapine inhibition also does not dependent on the cyclic-nucleotide binding domain. Norquetiapine had no effect on HCN2 channels. Conclusions and Implications HCN channels are key targets of norquetiapine, the primary active metabolite of quetiapine. These data help to explain the therapeutic mechanisms by which quetiapine aids in the treatment of anxiety, major depressive disorder, bipolar disorder, and schizophrenia, and may represent a novel structure for future drug design of HCN inhibitors.

Background and purpose: The impact of inflammatory bowel disease (IBD) on drug disposition is a critical concern, yet whether this impact is circadian time-dependent remains unexplored. We aim to discern the temporal effects of colitis on drug disposition and toxicity. Experimental Approach: The RNA-sequencing was used to screen colitis-affecting genes. The liver microsomal incubation and the pharmacokinetic study were used to analyze the enzymatic activity of key enzymes. Dual luciferase assays and chromatin immunoprecipitation (ChIP) were employed to elucidate regulatory relationships among molecules. Key Results: CYP1A2 and CYP2E1, pivotal in theophylline metabolism, exhibit reduced hepatic expression at Zeitgeber time (ZT)8 in colitis mice, a difference diminishing by ZT20. Moreover, theophylline metabolism is suppressed, and hepato-cardiac toxicities are markedly elevated in colitis mice at ZT8, while remaining unchanged at ZT20. The circadian clock component BMAL1 positively regulates CYP1A2 and CYP2E1 in the liver of colitis mice. A combination of assays, integrating liver-specific Bmal1 knockout mice and targeted activation of BMAL1 have unveiled that the dysregulation observed in Cyp1a2 and Cyp2e1 during colitis can be attributed to the perturbation in BMAL1 functionality in the liver. Luciferase reporter and ChIP assays collectively substantiate the role of BMAL1 in the modulation of Cyp1a2 and Cyp2e1 transcription through its binding affinity to E-box-like sites. Conclusion and implication: Our findings establish a strong link between IBD and chronopharmacology, shedding light on how IBD affects drug disposition and toxicity over time. Our research establishes a theoretical foundation for chronopharmacological approaches In the treatment of IBD.

Rationale: Fentanyl remains the primary cause of fatal overdoses, and its co-use with methamphetamine (METH) is a growing concern. The optical isomers of METH, dextromethamphetamine (d-METH) and levomethamphetamine (l-METH), differ substantially in dose expression and thus may differentially contribute to the racemate’s bidirectional effects. Furthermore, it is unknown which of METH’s monoamine (MA) receptor mechanisms mediate these respiratory effects. Thus, systematic evaluation of monoamine receptor selective agents may identify treatment targets for OIRD. Methods: The two optical isomers of METH, d-METH and l-METH, were tested in adult male mice to determine their effects on basal and fentanyl-depressed minute volume (MVb; i.e., respiratory frequency x tidal volum) using whole-body plethysmography. Next, six selective agonists at MA receptors involved in METH’s activity [phenylephrine (PNE; α1), clonidine (CLON; α2), SKF-82958 (SKF; D1), quinpirole (QPR; D2), 8-OH-DPAT (8-OH; 5HT1A), and DOI (5HT2)] were singly tested on basal MVb, and then in combination with fentanyl. Results: d-METH elevated MVb and l-METH decreased MVb. Under fentanyl-depressed conditions, the bidirectional effects of racemic METH were recreated by d-METH while l-METH significantly exacerbated OIRD at 1.0 and 3.0 mg/kg. MVb was dose-dependently increased by PNE and SKF and decreased by CLON and QPR. Neither 8-OH nor DOI altered basal MVb. Under fentanyl-depressed conditions, SKF transiently elevated MVb, while PNE more persistently increased it, while DOI transiently increased MVb, and 8-OH decreased MVb. Conclusions: d-METH and l-METH differentially contribute to the bidirectional respiratory modulation observed with the racemate and selective activation of MA receptors altered basal respiration and OIRD.

Background and Purpose: Traumatic brain injury (TBI) imposes life-long physical, psychological, and financial burdens on affected individuals. The current study investigated the effects of chronic nicotine exposure via E-cigarette (E-cig) on TBI-associated behavioral and biochemical changes. Experimental Approach: Adult C57/BL6J male mice were subjected to controlled cortical impact (CCI) followed by daily exposure to E-cigarette (E-Cig) vapor for six weeks. The effects of chronic nicotine exposure on sensorimotor functions, locomotion, and sociability were evaluated by nesting, open field, and social approach, respectively. Immunoblots were performed to assess changes of mature brain-derived neurotrophic factor (mBDNF) and associated downstream signaling proteins (p-Akt and p-Erk). Histological analyses of the cortex were performed to evaluate the effects of chronic nicotine exposure on Microglia-mediated neuroinflammation. Key Results: Post-injury chronic nicotine exposure significantly improved nesting performance in CCI mice. Histology analysis revealed that chronic nicotine exposure increased the survival of cortical neurons in the perilesion cortex. Immunoblots of cortical tissue revealed that chronic nicotine exposure significantly upregulated mBDNF expression, P-Erk, and p-Akt in the perilesion cortical tissue of CCI mice. Additional IF microscopy revealed elevated mBDNF and p-Akt expression was predominantly localized in cortical neurons of CCI mice. Furthermore, immunolabeling of Iba1 showed that chronic nicotine exposure attenuates microglia-mediated chronic neuroinflammation in the perilesional cortex of CCI mice. Conclusions and Implications: Post-injury chronic nicotine exposure via vaping facilitates sensorimotor function recovery by upregulating neuroprotective mBDNF/TrkB/Akt/Erk signaling. Results from this study support the neuroprotective properties of nicotine, further investigation is needed due to its highly addictive nature.

Background and purpose: Bardoxolone methyl (2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester, CDDO-Me) is a potent activator of nuclear factor erythroid 2-related factor (Nrf)2, which induces anti-oxidative-associated genes. CDDO-Me is known to exert protective effects against chronic inflammatory diseases in the kidney and lungs. However, its pharmacological effects on non-alcoholic steatohepatitis (NASH) caused by fat accumulation remain unknown. In this study, we examined the hepatoprotective effects of CDDO-Me in a diet-induced NASH mouse model, and elucidated its pharmacological mechanisms using RNA-seq analysis. Experimental approach: CDDO-Me was orally administered to mice fed a choline-deficient, L-amino acid-defined, high-fat diet, and histological, biochemical, and transcriptome analyses were performed on the livers of mice that developed NASH. Key results: CDDO-Me administration induced the expression of antioxidant genes and cholesterol transporters downstream of Nrf2 and significantly improved the symptoms of NASH. Whole-transcriptome analysis revealed that CDDO-Me inhibited the inflammatory pathway that leads to phagocyte recruitment, in addition to activating the Nrf2-dependent pathway. Among inflammatory pathways, CC chemokine ligand (CCL)3 and CCL4 in the downstream of NF-B, which are associated with the recruitment of macrophages expressing CC chemokine receptor (CCR)1 and CCR5, were released into blood in NASH mice. In contrast, CDDO-Me directly inhibited the expression of CCL3-CCR1 and CCL4-CCR5 in macrophages. Conclusions and Implications: Overall, this study revealed the potent hepatoprotective effect of CDDO-Me in a NASH mouse model, and demonstrated that its pharmacological effects were closely associated with the reduction of macrophage infiltration through CCL3-CCR1 and CCL4-CCR5 inhibition, in addition to Nrf2-mediated hepatoprotective effects.

Background and Purpose: Oligomeric Aβ1-42 (oAβ42) exhibits agonist-like action at human α7- and α7β2-nicotinic receptors (collectively, α7*-nAChR). Aβ1-42 and an N-terminal Aβ peptide fragment (N-Aβ fragment: Aβ1-15/16) have been shown to modulate presynaptic Ca2+ and enhance hippocampus-based synaptic plasticity via α7* nAChR. Both the N-Aβ fragment and its essential core sequence, the N-Aβcore hexapeptide (Aβ10-15), protect against Aβ-associated synapto- and neurotoxicity, also involving nAChR. Here, we investigated how oAβ42, the N-Aβ fragment and N-Aβcore regulate the functional activity of α7*-nAChRs. Experimental approach: Single-channel patch clamp recordings measured the impact of ACh, oAβ42, the N-Aβ fragment, and the N-Aβcore on the function of concatenated, human α7- and α7β2-containing nAChR expressed in nAChR-null SH-EP1 cells. Molecular dynamics simulations identified potential sites of interaction between the N-Aβ fragment and the orthosteric α7*-nAChR binding interfaces. Key Results: Relative to the effects of ACh alone, oAβ42 preferentially enhanced α7β2-nAChR open probability and open-dwell times. Co-application with the N-Aβcore neutralized these effects. Further, we demonstrate that the N-Aβ fragment alone, or in combination with ACh or oAβ42, resulted in selective enhancement of α7-nAChR single-channel open probability and open-dwell times (compared to ACh or oAβ42). Conclusions and Implications: Our findings show the functional diversity of Aβ peptides in regulating α7*-nAChR function, with implications for a wide range of nAChR-mediated functions in AD. Single-channel recordings of the differential effects of oAβ42, N-Aβ fragment and/or N-Aβcore on α7*-nAChR isoform function revealed the complexities of their interactions with α7*-nAChR, with new insights into the neuroprotective actions of these N-Aβ-derived peptides.

Abstract Background and Purpose Pneumoconiosis, especially silicosis has emerged as a prominent occupational disease with remarkable global implications with no definitive cure available. While pirfenidone and nintedanib have been approved in treating idiopathic pulmonary fibrosis, their potential efficacy as anti-fibrotic agents in advanced silicosis warrants further investigation. Thus, we aimed to assess the individual and combined effects of pirfenidone and nintedanib in treating advanced silicosis mice and further elucidate the underlying mechanisms involved in their therapeutic actions. Experimental Approach We administrated monotherapy or combination therapy of pirfenidone and nintedanib with low and high doses in silicosis mouse models established after 6 weeks and then evaluated lung function, inflammatory responses, and fibrotic status. Moreover, we employed transcriptomic and metabolomic analyses to unravel the mechanisms underlying different therapeutic strategies. Key Results Both pirfenidone and nintedanib were demonstrated to be effective for advanced silicosis, with superior outcomes when used in combination. Transcriptomic and metabolomic analyses revealed that pirfenidone and nintedanib primarily exerted their therapeutic effects through modulation of immune responses, signaling cascades, circadian rhythm, and metabolic processes of substances including lipid, amino acids, nucleotides, and carbohydrates. Conclusion and Implications In conclusion, pirfenidone and nintedanib, either administered individually or in combination, exhibit remarkable potential in advanced silicosis mouse models. Further, combined therapy outperformed monotherapy even at a half dose. These therapeutic benefits are attributed to their influence on diverse signaling pathways and metabolic processes. Keywords: silicosis, pulmonary fibrosis, pirfenidone, nintedanib, multi-omics.

Title: Orphan peptide and G protein-coupled receptor signalling in alcohol use disorderRoberta Goncalves Anversa1,2*, Xavier J Maddern1,2*, Andrew J Lawrence1,2and Leigh C Walker1,2*Contributed equally1Florey Institute of Neuroscience and Mental Health, VIC 3052 Australia2Florey Department of Neuroscience and Mental Health, University of Melbourne, VIC 3052 Australia

Abstract Background and Purpose Aging is usually accompanied by mitochondrial dysfunction, reduced energy levels, and cell death in the brain and other tissues. Mitochondria play a crucial role in maintaining cellular energy through oxidative phosphorylation (OXPHOS). However, OXPHOS is impaired as mitochondrial oxygen supply decreases with age. We explored whether pharmacologically increased oxygen diffusion by crocetin can restore OXPHOS and help delay aging of brain and other vital organs. Experimental Approach Stress-free chronic treatment of aged C57BL/6J mice with crocetin followed by an analysis of behavior, hippocampi whole transcriptome, and key energy metabolites by LCMS was performed. Key Results The aged mice treated with crocetin for four months displayed significantly improved memory behavior, neuromuscular coordination, and ATP and NAD+ levels in the brain and other vital organs, leading to an increased median life span. The transcriptomic analysis of hippocampi from crocetin-treated mice revealed that enhanced brain energy level was caused by the upregulation of genes linked to OXPHOS, and their expression was close to the expression in young mice. The chronic treatment of aged astrocytes also showed improved mitochondrial membrane potential and energy state of the cells. Conclusion and Implications Our data suggest that restoring the OXPHOS and the normal energy state of the cell can delay aging and enhance longevity. Therefore, molecules like crocetin should further be explored to treat age-related diseases.

Background and Purpose: The classical theory of receptor action has been used for decades as a powerful tool to estimate molecular determinants of ligand-induced receptor activation (i.e. affinity and efficacy) from experimentally observable biological responses. However, it is also a well-recognized fact that the receptor-binding and activation mechanisms, and the parameters thereof, described in the classical theory contradict with the modern view of receptor activation based on allosteric principles. Experimental Approach: We used mathematical analysis, along with some numerical simulations, to answer the key question as to what extent the classical theory is compatible -if at all- with the modern understanding of receptor activation. Key Results: Here, we showed conclusively that 1) receptor activation equations based on allosteric principles contain the logic of the classical theory in disguise, and therefore, 2) estimates of “intrinsic efficacy” () obtained by means of classical techniques (i.e. null methods or fitting the operational model to concentration-response data) are equivalent to the allosteric coupling factors that represent the molecular efficacy of ligands. Conclusion and Implications: Thus, we conclude that despite the right criticisms it has received so far, the classical theory may continue to be useful in estimating ligand efficacy from experimental data, if used properly. Here, we also provide rigorous criteria for the proper use of the theory. These findings not only have implications on ligand classification, but also resolve some long lasting discussions in the field of bias agonism in GPCR, which requires reasonable estimates of relative ligand efficacies at different signalling pathways.

Background and Purpose: Abnormal activation of the NLRP3 inflammasome in macrophages is closely associated with Ulcerative colitis (UC), and targeting the NLRP3 inflammasome has been proposed as a potential therapeutic approach, but the underlying mechanism by which it regulates intestinal inflammation remains unclear. Anemoside B4 (AB4) has anti-inflammatory activity, but whether it alleviates UC by inhibiting the activation of NLRP3 inflammasome remains unclear. More importantly, the molecular targets of AB4 remain unknown. Experimental Approach: We explored the role of AB4 in the development of dextran sodium sulfate (DSS)-induced colitis in wild-type (WT) mice and its effect on NLRP3 inflammasome. We isolated intestinal macrophages and epithelial cells, and validated them in DSS-induced NLRP3-deficient (NLRP3-/-) mice. The target and molecular mechanism of AB4 were identified in LPS-induced macrophages in vitro and DSS-induced macrophage-specific CD1d depletion (CD1d-/-) mice in vivo. Key Results: This study showed that AB4 had a strong anti-inflammatory effect DSS-induced colitis in WT mice, whereas the protective effects were lost in NLRP3-/- mice. AB4 inhibited the activation of NLRP3 inflammasome in colonic macrophages without affecting intestinal epithelial cells. Mechanistically, AB4 might target CD1d thus reducing the AKT-STAT1-PRDX1-NF-κB signaling pathway, eventually inhibiting the activation of NLRP3 inflammasome. Macrophage-specific CD1d depletion had been shown to reverse the protective effect of AB4. Conclusions and Implications: Our data showed that AB4 attenuated DSS-induced colitis by inhibiting CD1d-dependent NLRP3 inflammasome activation in macrophages. Therefore, as a natural product with high safety index, AB4 might be considered a promising candidate drug for the treatment of colitis.