Inflammatory bowel disease (IBD) is characterized by chronic relapsing intestinal inflammation and closely associated with persistent inflammatory responses and impairment of the mucosal barrier integrity. In this work, we demonstrated that Th17/Treg immune imbalance drove inflammation and pyroptosis in IBD, as revealed by single-cell RNA sequencing, and pyruvate kinase M2 (PKM2) and ubiquitin-like with PHD and RING finger domains 1‌ (Uhrf1) displayed a negative correlation in IBD clinic samples. Target fishing analysis revealed that eupatolide (EPT) covalently bound to C165 of PKM2 with a dissociation constant (Kd) of 106 nM, corroborated by follow-up chemical biology assays. We gained a deeper understanding of the mechanistic by which EPT interfered PKM2 function, specifically by promoting its interaction with Uhrf1, enhancing the K48-linked ubiquitylation and degradation of PKM2 to block its nuclear translocation. Critically, PKM2 silencing mitigated dextran sodium sulfate (DSS)-driven inflammation and pyroptosis across in vitro and in vivo models, with EPT showing no further efficacy upon PKM2 knockdown in IBD mice. This work uncovered Uhrf1-dependent PKM2 ubiquitination/degradation as a previously unrecognized therapeutic axis for IBD, while positioning EPT as a molecular glue capable of targeting the Uhrf1-PKM2 complex.

Background : Acute respiratory distress syndrome (ARDS) is an acute respiratory failure syndrome arising from non-cardiogenic pulmonary edema, characterized by limited therapeutic options and a poor prognosis, underscoring an urgent need for effective treatments. Reduning injection (RDN) is a traditional Chinese medicine formulation widely used in clinical practice for treating respiratory tract infections. However, its specific role and underlying mechanism in ARDS remain to be fully elucidated. Purpose : This study aimed to examine the multi-target protective effects of RDN against ARDS and elucidate its underlying mechanisms. Study design and methods : A lipopolysaccharide (LPS)-induced ARDS model was established to evaluate the therapeutic potential of RDN. A multifaceted experimental approach, encompassing flow cytometry, immunofluorescence, the integration target fishing technique of thermal-solvent (TS) proteomics and affinity chromatography (AC), Western blot, real-time PCR, immunohistochemistry, scRNA-seq analysis, molecular docking, and molecular dynamics, was adopted to uncover the therapeutic mechanism of RDN in ARDS. Results : RDN alleviated the progression of ARDS by improving alveolar barrier function and reducing histopathological lung damage. The underlying mechanisms involved the inhibition of the MAPK, NF‑κB, and NLRP3 pathways, which led to the decreasing of release for key inflammatory cytokines (IL‑6, IL‑1β, and TNF‑α). It also attenuated pulmonary infiltration of macrophages and neutrophils and modulated the Treg/Th17 balance, thereby maintaining pulmonary immune homeostasis. Furthermore, employing the target fishing technique, we identified folliculin (Flcn), integrin alpha-V (Itgav), and phospholipase C-β3 (Plcb3) as direct intracellular targets of RDN, which was further supported by various chemical biological methods. LC‑MS/MS analysis revealed that components of RDN entered the blood and lungs, among which isochlorogenic acid A (IAA) and genipin 1-gentiobioside (GG) were identified as bioactive components that directly bound to Flcn, Itgav, and Plcb3, respectively. Conclusion : RDN alleviated LPS-induced ARDS by modulating inflammatory and immune responses via multi-target regulation of Flcn, Itgav, and Plcb3, thereby inhibiting the NLRP3, NF-κB, and MAPK pathways.

Sepsis is a systemic inflammatory response syndrome triggered by an uncontrolled host response to infection, which causes acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Baicalein (Bai) is one of the major active components of Scutellariae Radix for treating pulmonary diseases. However, its specific underlying molecular mechanism is unknown for treating sepsis-mediated ALI. Herein, we found that Bai inhibited the nuclear factor kappa B (NF-κB) pathway to suppress inflammation and oxidative stress in vitro and in vivo, alleviating the course of lung injury. Affinity chromatography revealed that Bai directly bound to eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) through hydrogen bond interaction with Q108 and D110, exhibiting a dissociation constant (K d) of 336 nM. Mechanistically, Bai blocked the interaction between eEF1A1 and peroxiredoxin 4 (Prdx4) to inhibit ring finger protein 14 (RNF14)-mediated ubiquitin-dependent degradation of Prdx4 based on APEX2 and Co-IP experiments. Moreover, eEF1A1 knockdown could repress inflammation, whereas its overexpression exerted an opposite effect in the in vitro experiments. Notably, Bai did not display any additional protective effect in LPS-mediated eEF1A1 knockdown cells, while its effect was weakened in LPS-mediated eEF1A1 overexpression cells. In addition, our study revealed that the course of sepsis-mediated lung injury was alleviated in vivo by eEF1A1 knockdown, and no extra effects were exhibited by Bai in sepsis-mediated eEF1A1 knockdown mice. These findings collectively suggested that targeting eEF1A1 represented a promising therapeutic strategy against sepsis-associated lung injury through dual anti-inflammatory and antioxidant mechanisms, particularly via Bai's role as a direct blocker disrupting eEF1A1-Prdx4 interactions.

SIGNIFICANCE:Cellular proteostasis is essential for cellular proteome integrity, which is exquisitely sensitive to the redox environment. Heat shock proteins (HSPs) are the central chaperones that sense and adapt to these redox fluctuations. Emerging evidence demonstrates dysregulation of cellular HSPs-modulated redox-proteostasis in protein aggregation diseases, including cancers, senescence, neurodegenerative diseases, limb-girdle muscular dystrophy type D1, and β-thalassemia, making HSPs promising therapeutic targets in disease treatment. RECENT ADVANCES:Redox post-translational modifications (PTMs) serve as master switchboards to dynamically modulate the structure and chaperone function of HSPs. Redox PTMs allow HSPs to participate in protein synthesis and folding, conformational maintenance, and degradation, thereby maintaining cellular proteostasis. Beyond their chaperone functions, HSPs also play critical roles in organelle-specific stress responses, such as mitochondrial unfolded protein response, endoplasmic reticulum (ER) stress, and unfolded protein response. CRITICAL ISSUES:Despite the well-known contributions of HSPs to redox-proteostasis, the double-edged functions of HSPs in protein aggregation diseases remain unclear. The main issues covered in this review include the regulation of HSPs by redox PTMs, the important role of HSPs in proteostasis and organelle-specific stress responses, dual modulation of HSPs in protein aggregation diseases, and pharmacological agents targeting HSPs.Further Directions:The functional diversity of HSPs in redox-proteostasis makes them promising therapeutic targets in disease treatment. Further studies should focus on exploiting agents that precisely target cysteine residues modifications on HSPs with good blood-brain barrier (BBB) penetration and low toxicity. Antioxid. Redox Signal. 45, 78-112.

Antioxidant research has recently become a popular topic. Medicinal plants are important sources of novel active compounds. Diarylheptanoids, a typical family of secondary plant metabolites, are of great interest owing to their extensive spectrum of biological activities. They possess a unique 1,7-diphenylmethane structural skeleton. Thus, this review summarizes the natural linear or macrocyclic diarylheptanoids with antioxidant activity in the last two decades. In addition, the relationships between the structural characteristics of natural diarylheptanoids and their antioxidant capacity were also discussed. All the available data highlight the potential of natural diarylheptanoids as novel antioxidants.

Phagocytosis is a fundamental mechanism used by the body to resist pathogens and restore physiological homeostasis. Herein, to identify small molecules with anti-inflammatory properties via phagocytosis inhibition, we constructed a library of natural products and evaluated their ability to modulate phagocytosis in RAW264.7 macrophages. Berberine (BBR) is the major constituent of traditional Chinese medicine Coptidis Rhizoma that is recorded in Chinese Pharmacopoeia with the effect of clearing heat-toxin, and is used in the therapeutic management of various inflammatory diseases. BBR was found to inhibit phagocytosis and significantly alleviate inflammation via suppressing interleukin-1α (IL-1α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α), according to real-time quantitative polymerase chain reaction (RT-qPCR) analyses, and phosphorylated-p65 (p-p65), iNOS, and cyclooxygenase-2 (COX-2), according to western blot analyses. BBR inhibited the expression of F-actin, a key protein in phagosome formation. Notably, BBR exerted its phagocytosis effects through targeting phosphoinositide 3-kinase (PI3K), thereby activating the small GTPase-Cdc42 (CDC42), Wiskott-Aldrich syndrome protein (WASP), and actin-related protein 2/3 complex subunit 2 (Arp2/3). BBR attenuated LPS-mediated inflammation through promoting macrophage phagocytosis. We determined that BBR targets the toll-like receptor 4 (TLR4)-PI3K-CDC42 pathway, thereby inhibiting the nuclear factor-kappa B (NF-κB) pathway, and consequently regulating phagocytosis and the inflammatory response. Our findings suggest that BBR might serve as a candidate for the development of phagocytic inhibitors.

Pantothenate kinases (PANKs), which regulate the first and rate-limiting step of coenzyme A (CoA) biosynthesis, have emerged as therapeutic targets for various human diseases. PANKs family consists of PANK1, PANK2, PANK3, and PANK4. We summarized the research progress of the PANK family in the last two decades. Notably, PANKs play critical roles in diverse pathophysiological mechanisms underlying human diseases, including pantothenate kinase-associated neurodegeneration (PKAN), propionic acidemia (PA), lipid metabolic disorders, Parkinson's disease (PD), glioma, clear cell renal cell carcinoma (ccRCC), and insulin resistance (IR). Nevertheless, the precise relationship between PANKs and human diseases remained ambiguous. Therefore, this review provides a comprehensive summary of the structural properties, molecular mechanisms, and relevant modulators that govern PANK activity. In conclusion, targeting PANKs as regulators of CoA biosynthesis and disease pathogenesis contributes to connecting biochemistry with pharmacotherapy.

In continuation of research aimed at identifying anti-inflammatory agents from natural sesquiterpenoids, an activity-guided fractionation approach utilizing lipopolysaccharide (LPS)-mediated RAW264.7 cells was employed to investigate chemical constituents from Inula Britannica (I. britannica). Seven novel sesquiterpenoid dimers inulabritanoids A-G (1-7) and two novel sesquiterpenoid monomers inulabritanoids H (8) and I (9) were isolated from I. britannica together with eighteen known compounds (10-27). The structural elucidation was accomplished through comprehensive analysis of 1D and 2D nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HR-MS), and electronic circular dichroism (ECD) spectra, complemented by quantum chemical calculations. Compounds 1, 2, 12, 16, 19, and 26 demonstrated inhibitory effects on NO production, with IC50 values of 3.65, 5.48, 3.29, 6.91, 3.12, and 5.67 μmol·L-1, respectively. Mechanistic studies revealed that compound 1 inhibited IκB kinase β (IKKβ) phosphorylation, thereby blocking nuclear factor κB (NF-κB) nuclear translocation, and activated the kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signal pathway, leading to decreased expression of NADPH oxidase 2 (NOX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), IL-1β, and IL-1α and increased expression of NAD(P)H: quinone oxidoreductase 1 (NQO-1) and heme oxygenase-1 (HO-1), thus exhibiting anti-inflammatory effects in vitro. These results indicate that dimeric sesquiterpenoids may serve as promising candidates for anti-inflammatory drug development.

Neuroinflammation is a complex immunological phenomenon characterized by a dysregulated inflammatory response in the central nervous system (CNS) that can be triggered by various pathological injuries, such as toxins, which are involved in Parkinson’s and Alzheimer’s diseases (PD and AD), therefore, suppressing neuroinflammation serves as an effective treatment for CNS diseases. Herein, we found that natural soluble epoxide hydrolase (sEH) inhibitor 1- O -acetyl-4 R ,6 S -britannilactone (AB) regulated mitogen-activated protein kinase (MAPK) and AMP-activated protein kinase (AMPK) pathways to suppress the microglial activation by regulating inflammation and autophagy in vitro and in vivo, contributing to the improvement of lipopolysaccharide (LPS)-mediated neuroinflammation. Protein microarray analysis indicated that AB could selectively target PDZ binding kinase (PBK) through covalently binding to C70, exhibiting a dissociation constant (Kd) of 0.62 μM, which was corroborated by subsequent chemical biology experiments. We gained a deeper understanding of the mechanistic by which AB interfered PBK function, specifically by disrupting its interaction with tumor necrosis factor alpha-induced protein-8 like-2 (TIPE2), blocking the serine 3 (S3) phosphorylation-mediated ubiquitylation and degradation of TIPE2. Additionally, our study revealed that PBK genetic deletion alleviated the course of LPS-mediated neuroinflammation in vitro and in vivo, and AB did not exhibit any extra effects in LPS-mediated PBK -/- mice. These findings first offered broader prospects for treating neuroinflammation by targeting PBK to repress inflammation and activate autophagy, suggesting that AB had the potential to serve as a direct inhibitor in the PBK–TIPE2 interaction.

Inflammatory Bowel Disease (IBD), is a chronic illness characterized by severe abdominal pain, diarrhea, and weight loss, seriously diminishing patients' quality of life. Andrographolide (AND), a natural diterpenoid from Andrographis paniculata, and its sulfated metabolite, andrographolide sodium bisulfite (ASB), have showed potential anti-inflammatory effects. However, their mechanism in IBD remains elusive. This study investigated the impact of AND and its sulfated derivative ASB, on inflammatory responses in IBD. Our findings revealed that AND and ASB significantly reduced disease activity index (DAI) scores and enhanced intestinal barrier function in dextran sodium sulfate (DSS)-induced mice, thereby ameliorating the course of IBD. Furthermore, AND and ASB inhibited both the mitogen-activated protein kinase (MAPK) and NLRP3 pathways to reduce the release of inflammatory cytokines IL-6 and TNF-α. This mechanism was accompanied by a restoration of immune balance through the modulation of T-helper 17 (Th17) and regulatory T (Treg) cells. The ability of AND and ASB to mitigate chronic inflammation and maintain immune equilibrium presented a promising therapeutic approach for IBD management. These findings suggested that AND and ASB might provide novel therapeutic approaches for IBD, thereby warranting further investigation into their clinical efficacy for disease treatment and maintenance of remission.

Ethnopharmacological relevanceBerberine (BBR) is the main active component from Coptidis rhizome, a well-known Chinese herbal medicine used for metabolic diseases, especially diabetes for thousands of years. BBR has been reported to cure various metabolic disorders, such as nonalcoholic fatty liver disease (NAFLD). However, the direct proteomic targets and underlying molecular mechanism of BBR against NAFLD remain less understood.Aim of the studyTo investigate the direct target and corresponding molecular mechanism of BBR on NAFLD is the aim of the current study.Materials and methodsHigh-fat diet (HFD)-fed mice and oleic acid (OA) stimulated HepG2 cells were utilized to verify the beneficial impacts of BBR on glycolipid metabolism profiles. The click chemistry in proteomics, DARTS, CETSA, SPR and fluorescence co-localization analysis were conducted to identify the targets of BBR for NAFLD. RNA-seq and shRNA/siRNA were used to investigate the downstream pathways of the target.ResultsBBR improved hepatic steatosis, ameliorated insulin resistance, and reduced TG levels in the NAFLD models. Importantly, Aldo-keto reductase 1B10 (AKR1B10) was first proved as the target of BBR for NAFLD. The gene expression of AKR1B10 increased significantly in the NAFLD patients’ liver tissue. We further demonstrated that HFD and OA increased AKR1B10 expression in the C57BL/6 mice’s liver and HepG2 cells, respectively, whereas BBR decreased the expression and activities of AKR1B10. Moreover, the knockdown of AKR1B10 by applying shRNA/siRNA profoundly impacted the beneficial effects on the pathogenesis of NAFLD by BBR. Meanwhile, the changes in various proteins (ACC1, CPT-1, GLUT2, etc.) are responsible for hepatic lipogenesis, fatty acid oxidation, glucose uptake, etc. by BBR were reversed by the knockdown of AKR1B10. Additionally, RNA-seq was used to identify the downstream pathway of AKR1B10 by examining the gene expression of liver tissues from HFD-fed mice. Our findings revealed that BBR markedly increased the protein levels of PPARα while downregulating the expression of PPARγ. However, various proteins of PPAR signaling pathways remained unaffected post the knockdown of AKR1B10.ConclusionsBBR alleviated NAFLD via mediating PPAR signaling pathways through targeting AKR1B10. This study proved that AKR1B10 is a novel target of BBR for NAFLD treatment and helps to find new targets for the treatment of NAFLD by using active natural compounds isolated from traditional herbal medicines as the probe.

Berberrubine (BRB), belonging to the benzylisoquinoline alkaloid, is a main metabolite of berberine in vivo. BRB was previously proven to undergo metabolic activation mediated by P450s. In this study, the chemical interactions between BRB and CYP2D6 enzyme were investigated. First, a variety of P450s participated in the metabolism of berberine transformed to BRB, but CYP2D6 was the most involved enzyme. A time-, concentration-, and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent inhibition of CYP2D6 was caused by BRB. The inhibitory effect of BRB on CYP2D6 was irreversible. The maximum reaction rate constants of inactivation (k inact) and half-maximal inactivation (K I) of BRB on CYP2D6 were 0.0410 min-1 and 3.798 mu M, respectively. Metoprolol, a classic substrate of CYP2D6, attenuated CYP2D6 from inactivation by BRB. Glutathione (GSH) and catalase/superoxide dismutase failed to protect against the inactivation of CYP2D6 caused by BRB. Three cys-based adducts derived from the reaction of electrophilic metabolites of BRB with CYP2D6 were detected by ultra performance liquid chromatography-mass spectrometry (UPLC-MS)/MS. The reactive metabolites derived from BRB might be responsible for the inactivation of CYP2D6. In summary, BRB was characterized as a mechanism-based inactivator of CYP2D6.

BackgroundChemotherapeutic agents including cisplatin, gemcitabine, and pemetrexed, significantly enhance the efficacy of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC) by increasing PD-L1 expression and potentiating T cell cytotoxicity. However, the low response rate and adverse effects limit the application of chemotherapy/ICI combinations in patients.MethodsWe screened for medicinal herbs that could perturb PD-L1 expression and enhance T cell cytotoxicity in the presence of anti-PD-L1 antibody, and investigated the underlying mechanisms.ResultsWe found that the aqueous extracts of Centipeda minima (CM) significantly enhanced the cancer cell-killing activity and granzyme B expression level of CD8+ T cells, in the presence of anti-PD-L1 antibody. Both CM and its active component 6-O-angeloylplenolin (6-OAP) upregulated PD-L1 expression by suppressing GSK-3β-β-TRCP-mediated ubiquitination and degradation. CM and 6-OAP significantly enhanced ICI-induced reduction of tumor burden and prolongation of overall survival of mice bearing NSCLC cells, accompanied by upregulation of PD-L1 and increase of CD8+ T cell infiltration. CM also exhibited anti-NSCLC activity in cells and in a patient-derived xenograft mouse model.ConclusionsThese data demonstrated that the induced expression of PD-L1 and enhancement of CD8+ T cell cytotoxicity underlay the beneficial effects of 6-OAP-rich CM in NSCLCs, providing a clinically available and safe medicinal herb for combined use with ICIs to treat this deadly disease.

Introduction Triple-negative breast cancer (TNBC) has a high mortality rate and limited treatment options. Tetrahydrocurcumin (THC), a major metabolite of curcumin, has potential antitumor activities. However, the antitumor effects and mechanism of THC in TNBC remain elusive. Objectives To investigate the mechanism of THC in combating TNBC by targeting TRIP13 to disrupt the interaction of the TRIP13/USP7/c-FLIP complex and mediate c-FLIP ubiquitination both in vitro and in vivo. Methods Apoptosis was measured by TUNEL and flow cytometry. Click chemistry-based target fishing, CETSA, DARTS, and SPR were used to identify direct target of THC. Protein interactions was examined using co-immunoprecipitation. The role of USP7 in THC-mediated c-FLIP ubiquitination was evaluated by in vitro deubiquitination assay. Human breast cancer clinical samples were employed to assess the expression of c-FLIP, TRIP13, and USP7. The impact of THC on USP7/TRIP13/c-FLIP was analyzed using co-immunoprecipitation, confocal microscopy, molecular docking and dynamics simulations. Results THC effectively inhibits TNBC cell proliferation and tumor growth in vitro and in vivo without significant toxicity. Mechanistically, THC induces extrinsic apoptosis in TNBC primarily by promoting degradation of c-FLIP, a key negative regulator in the apoptotic pathway. Furthermore, utilizing click chemistry-based target fishing, we identified TRIP13, a component of the highly conserved AAA ATPase family, as a direct target of THC in combating TNBC. Interestingly, contrary to previous drug-target studies, the knockdown of TRIP13 further amplified the antitumor effects of THC. After in-depth investigation, it was revealed that TRIP13 forms a trimeric complex with USP7 and c-FLIP in TNBC cells. THC specifically targets TRIP13 to disrupt the interaction of TRIP13/USP7/c-FLIP, leading to the ubiquitination of c-FLIP, ultimately inducing extrinsic apoptosis. Conclusions These findings offer new insights into the novel molecular mechanisms of anti-TNBC effects of THC and present a promising targeted therapeutic strategy for TNBC.

Sesquiterpene dimers are mainly found in the Asteraceae family. However, conflicting reports on the structures of these compounds can be found in the literature. Herein, we describe ten sesquiterpene dimers isolated from the flowers of Inula japonica, including configurational revisions of japonicone H (1-1), japonicone D (2-1), inulanolide A (4-1), japonicone X (5-1), and inulanolide F (5-2) to compounds 1, 2, 4, and 5, respectively. Five new related metabolites (3 and 6-9) are also described. Application of GIAO NMR/DP4+ analyses and ECD/OR calculations enabled us to revise the absolute configurations of an additional 13 sesquiterpene dimers isolated from plants of the genus Inula. Compounds 1, 2, 4, and 6 exhibited inhibition of nitric oxide production in lipopolysaccharide activated RAW264.7 macrophages with IC50 values of 4.07-10.00 mu M.

The application of photocatalyzed Minisci-type reactions in LSF accelerates the discovery of drug candidates in a green way.

Schisandra chinensis, a traditional Chinese medicine, has been widely applied in China to treat diabetes and its complications. The aim of this study was to discover the active compounds and explain related molecular mechanism contributing to the anti-diabetic effect of Schisandra chinensis. Herein, the therapeutic effects of Schisandra chinensis extracts on type 2 diabetes mellitus (T2DM) were firstly confirmed in vivo. Subsequently, various lignans were isolated from Schisandra chinensis and tested for hypoglycemic activity in palmitic acid-induced insulin-resistant HepG2 (IR-HepG2) cells. Among these lignans, R-biar-(7S,8R)-6,7,8,9-tetrahydro-1,2,3,12,13,14-hexamethoxy-7,8-dimethyl-7-dibenzo [a, c] cyclooctenol (compound 2) and Gomisin A (compound 4) were identified significantly increased the glucose consumption in IR-HepG2 cells. Meanwhile, compounds 2 and 4 activated the insulin receptor substrate-1 (IRS-1)/phosphoinositide 3-kinase (PI3K)/Ak strain transforming (AKT) pathway, which regulates glucose transporter 2 (GLUT2) and glucose-6-phosphatase (G6Pase), essential for gluconeogenesis and glucose uptake. These compounds also inhibited the nuclear factor-κB (NF-κB) signaling pathway, reducing interleukin-6 (IL-6) levels. Importantly, the hypoglycemic effects of compounds 2 and 4 were diminished after Toll-like receptor 4 (TLR4) knockdown. Cellular thermal shift assays confirmed increased TLR4 protein stability upon treatment with these compounds, indicating direct binding to TLR4. Furthermore, TLR4 knockdown reversed the effects of compounds 2 and 4 on the NF-κB and IRS-1/PI3K/AKT pathways. Taken together, compounds 2 and 4 alleviate IR by targeting TLR4, thereby modulating the NF-κB and IRS-1/PI3K/AKT pathways. These findings suggest that compounds 2 and 4 could be developed as therapeutic agents for T2DM.

The immune system serves as a role of diseases, such as Parkinson's disease, and acute lung injury. An immunoregulatory activity-directed separation depended on phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Ion)-mediated Jurkat leukemic T cells was used for studying chemical constituents from Inula britannica L. in depth. Five previously undescribed aromatic sesquiterpenoid dimers inulabritanoids J-N (1-5) and a previously undescribed germacrane-type sesquiterpenoid britanicafanin F (6) were afforded from I. britannica as well as eight known sesquiterpenoids (7-14). Their structures were elucidated through 1D and 2D NMR, HRMS, and ECD spectra along with quantum chemical calculations. Immunomodulatory effects of compounds 1-14 were assayed in PMA plus Ion-mediated Jurkat cells, and indicated that compounds 8, 9, and 13 displayed significantly inhibitory effects toward IL-2 and IFN-γ. Further investigation of mechanism of action revealed that compound 13 inhibited phosphorylations of p38, ERK, and JNK to suppress c-Jun and c-Fos expressions, resulting in blocking the nuclear translocation of AP-1 (a complex of c-Jun and c-Fos) to regulate mRNA expressions of IL-2 and IFN-γ. Molecular docking analysis demonstrated that compound 13 could enter into the cavity of p38, ERK, and JNK, and from hydrogen bond interactions with Gly33, Lys53 Ser154, and Asp168 for p38, Lys54, Glu71, Ser153, and Asp167 for ERK, and Met149 and Asn152 for JNK, which supported the abovementioned results. These findings suggested that sesquiterpenoids from the genus Inula served as immunomodulators for treating diseases involved in immune and inflammatory responses.