The development of antibody–drug conjugates (ADCs) has accelerated due to their tumor-targeting precision. However, challenges in optimizing the target-linker-payload combination often lead to attrition from safety or efficacy issues, highlighting the need for ADCs with an improved therapeutic index. This study presents a newly designed B7-H3-targeted ADC, BR112, and demonstrates its superior preclinical efficacy across multiple models. BR112 comprises a humanized anti-B7-H3 antibody (112–59) conjugated to the microtubule inhibitor MMAE via a Pyridazinedione-valine-citrulline-p-aminobenzyl (PD-VC-PAB) linker with a drug-to-antibody ratio (DAR) of 4.15. The binding affinity and internalization profile of 112–59 were evaluated across multiple cell lines. In vitro cytotoxicity and in vivo antitumor efficacy were assessed in multiple cancer cell lines and three xenograft models. Pharmacokinetics of BR112 were analyzed in cynomolgus monkeys. The antibody 112–59 exhibited high binding affinity for human B7-H3 (Kd = 5.28 nM), with potent cell-binding (EC50 ≈ 1 nM) and superior internalization across multiple cancer cell lines, exceeding the performance of antibodies used in clinical-stage comparator ADCs (MGC018 and DS-7300). Consequently, BR112 demonstrated potent cytotoxicity in vitro, with IC50 values ranging from femtomolar to nanomolar. In vivo, BR112 induced complete tumor regression in xenograft models (HCC1954, HCC1806, Calu-6) at doses of 3–10 mg/kg, outperforming both MGC018 and DS-7300. Pharmacokinetic studies in cynomolgus monkeys showed dose-proportional exposure and minimal MMAE release, confirming linker stability. This study positions BR112 as a promising therapeutic candidate with enhanced efficacy and a stable pharmacokinetic profile for the treatment of B7-H3-expressing solid tumors.

Background: The NLRP3 inflammasome drives pathological inflammation in various diseases. PINK1/Parkin-associated mitophagy serves as a critical negative regulator of NLRP3 activation, yet pharmacological enhancers remain scarce. Muscone, a natural macrocyclic ketone with blood–brain barrier permeability, exhibits potent anti-inflammatory properties; however, its mechanistic role within the NLRP3-mitophagy axis remains undefined. Methods: LPS/ATP-stimulated macrophages were employed to assess stage-specific effects of muscone on NLRP3 priming (NF-κB signaling, NLRP3, and pro-IL-1β expression) and activation (ASC oligomerization, ASC–pro-caspase 1 complex formation, and IL-1β secretion). RNA sequencing and bioinformatic analysis were performed for pathway enrichment. Mitophagy was characterized by MitoSOX Red staining for mt-ROS detection, electron microscopy, Western blotting of LC3B-II in isolated mitochondria and PINK1 and Parkin in whole-cell lysates, and live-cell mitochondria–lysosome tracking. In vivo protective efficacy was assessed in an LPS-induced endotoxemia mouse model. Results: Muscone dose-dependently suppressed both the priming and activation stages of the NLRP3 inflammasome, maximally reducing IL-1β secretion by ~60% at 50 μM. Mechanistically, muscone amplified PINK1/Parkin-associated mitophagy, scavenging excessive mt-ROS and attenuating NLRP3 activation. These effects were corroborated by RNA-seq and comprehensive functional assays. In vivo, muscone (30 mg/kg) significantly improved survival (3/8 mice alive at 98 h when all LPS controls had died; 2/8 survived to the 132-h endpoint), with concomitant enhancement of mitophagy markers in peritoneal macrophages. Conclusions: Muscone functions as a PINK1/Parkin-associated mitophagy enhancer that maintains mitochondrial quality control during NLRP3-driven inflammatory responses. Its unique macrocyclic structure and blood–brain barrier permeability provide a promising scaffold for developing therapeutics against inflammatory disorders associated with NLRP3 inflammasome activation.

Background/Objectives: Silicosis is a chronic disease caused by long-term exposure to high levels of silica dust, which leads to extensive nodular fibrosis in the lungs. The disease is currently a serious occupational health hazard globally. Xuanfei Baidu decoction (XFBD) is a mature Chinese herbal medicine in China that has shown anti-inflammatory and anti-fibrotic effects in mouse experiments, making it a promising candidate for addressing the persistent inflammation and fibrosis in silicosis. Methods: Silicosis was induced in male C57BL/6J mice using crystalline silica (CS). XFBD’s early anti-inflammatory role was verified in vitro in peritoneal macrophages (PMs) and in vivo in silicosis mice, while its late anti-collagen deposition and anti-fibrotic activities were further investigated. Results: In vitro, XFBD effectively inhibits the activation of the NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome in CS-induced lipopolysaccharide (LPS)-primed PMs, decreases the release of inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), and modulates the phenotypic transition of macrophages from the M2 to the M1 phenotype. In vivo studies further validated that XFBD significantly downregulates the expression of NLRP3 and Cleaved-Caspase-1 proteins in the lung tissues of mice afflicted with silicosis. Additionally, XFBD enhanced pulmonary function, inhibited collagen deposition and pulmonary fibrosis in silicosis mice, and reversed epithelial–mesenchymal transition (EMT) by regulating key EMT-related proteins to slow fibrosis. Conclusions: The beneficial effects of XFBD on CS-induced pulmonary fibrosis can be attributed to the induction of macrophage polarization-mediated anti-inflammatory responses during the early stage of fibrotic development, as well as its anti-collagen deposition and anti-fibrotic activities during the intermediate stage of fibrotic development. This study provides preclinical evidence supporting XFBD as a promising candidate for prevention or adjunctive therapy, and its multi-target, time-phase mechanism offers a novel rationale and theoretical foundation for the development of new strategies against silicosis.

The NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome is an intracellular protein complex containing a nucleotide-binding oligomerization domain, leucine-rich repeats, and a pyrin domain. It is a key regulator of inflammation in viral pneumonia (VP). Small-molecule inhibitors targeting various NLRP3 binding sites are advancing into early clinical trials, but their therapeutic utility is incompletely established. Xuanfei Baidu Formula (XF), clinically used for VP treatment, attenuates NLRP3 activation by hampering caspase-11 to impede polarization of pro-inflammatory macrophages in a model of lipopolysaccharide (LPS)-induced lung injury inmice. Herein, we demonstrate that XF attenuated influenza A virus (IAV)-induced lung inflammation as well as lung injury in immunocompetent (but not in macrophage-depleted) mice. RNA sequencing of sorted lung macrophages from IAV-infected mice revealed that XF inhibited activation of the NLRP3 inflammation and interleukin (IL)-1β production. Quantitative nuclear magnetic resonance of XF enabled us to develop XF-Comb1, a fixed-ratio combination of five bioactive compounds that recapitulated the bioactivity of XF in suppressing NLRP3 activation in macrophages in vitro and in vivo. Interestingly, XF-Comb1 inhibited assembly of the NLRP3 inflammasome through multi-site interactions with functional residues of NLRP3, apoptosis-associated speck-like protein containing caspase recruitment domain (ASC), and caspase-1. Taken together, this work advances the development of NLRP3 inhibitors by translating a complex herbal formula into defined bioactive compounds.

Activation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome and oxidative stress are key pathological hallmarks of inflammatory bowel disease (IBD) and represent novel targets for therapeutic intervention. The therapeutic potential of the naturally occurring flavonoid kaempferol (KAE) in IBD, along with its underlying molecular mechanisms, remains incompletely understood. We established dextran sulfate sodium (DSS)-induced colitis and lipopolysaccharide (LPS)-induced systemic inflammation mouse models. KAE was administered as an intervention. In vitro, peritoneal macrophages (PMs) were stimulated to activate the NLRP3 inflammasome. We comprehensively evaluated the effects of KAE on interleukin-1β (IL-1β) release, NLRP3 inflammasome assembly, and mitochondrial reactive oxygen species (mt-ROS) production. Additionally, we assessed its impact on the Keap1-Nrf2 pathway. Molecular docking and ubiquitin-dependent degradation assays were conducted to confirm Nrf2 as a direct target of KAE. KAE significantly attenuated colitis development, marked by reduced NLRP3 expression and enhanced Nrf2 activation. It inhibited both the priming and assembly phases of NLRP3 inflammasome activation. Notably, Nrf2 inhibition completely abolished KAE-mediated removal of mt-ROS and downstream suppression of NLRP3 inflammasome activation. Mechanistically, KAE prevented Nrf2 proteolysis by directly binding to Arg415 of Keap1. Our findings demonstrate that KAE acts as an mt-ROS scavenger to protect against inflammatory pyroptosis, offering novel mechanistic insights and a promising therapeutic strategy for colitis treatment.

Cytokine synergy induced by the activation of multiple Toll-like receptors (TLRs) may result in uncontrollable life-threatening inflammation, the so-called cytokine storm. Owing to the complexity of the crosstalk for individual TLR signaling pathways, analyses of transcriptional changes in addition to differentially expression genes (DEGs) are needed. In the present study, we aimed to create algorithms to obtain a profile of synergistic cytokine production and to evaluate the anti-cytokine synergistic activity of ginsenoside Rg3. RAW264.7 macrophages were activated by TLR2/3 dual ligands; Rg3 was used as an intervention. After interleukin (IL)-6 secretion was detected as a preliminary readout for cytokine synergy, RNA sequencing-based bioinformatic analysis was performed, followed by qPCR and western blotting verification. Specifically, nonadditive transcriptional responses (DIFs) were applied as a measure of synergistic genes, and an anti-synergy score was created as a measure of the antagonistic effect of Rg3. A mouse model of TLR2/3 costimulation was subsequently established to evaluate the anti-cytokine synergistic effect of Rg3 in vivo. The results show that Rg3 alleviates synergistic cytokine production both in vitro and in vivo. Nuclear factor kappa B (NF-κB) and interferon regulatory factor 3 (IRF3) are novel targets of Rg3 related to its anti-cytokine synergistic effects. Our strategy of nonadditive transcriptional change analysis will be helpful for performing high-throughput screening for drugs with anti-cytokine synergy activities.

ETHNOPHARMACOLOGICAL RELEVANCE:Aging is a time-dependent decline in physiological function that reduces quality of life. In traditional Chinese medicine (TCM), aging is viewed as "deficiency" complicated by blood stasis and qi stagnation. "Kang Shuai Lao Pian" (KSLP) is a Ming Dynasty herbal formula widely used to repenish qi, nourish yin, calm the heart, and tranquilize the mind. AIM OF THE STUDY:We aimed to evaluate the systemic anti-aging activity of KSLP and to determine its mode of action in naturally senescent mice. MATERIALS AND METHODS:Male 16-18-month-old mice received daily intragastric gavage of KSLP (450 mg/kg) for 8 weeks. Skin structure, adipose distribution, neurotransmitters, adipose-derived stem cells (ADSCs) morphology and differentiation, 16S rRNA gene sequencing and untargeted metabolomics analysis of cecal feces were assessed. RNA sequencing (RNA-Seq) was performed on senescent ADSCs; metabolic-microbe correlations were analyzed by Pearson correlation. RESULTS:KSLP restored skin structure and dermal collagen density, normalized dopamine, norepinephrine, acetylcholine, and 5-hydroxytryptamine levels, and improved ADSCs osteogenic/adipogenic capacity- changes mechanistically linked to PI3K-Akt and cytokine-cytokine receptor interaction signaling pathways. In gut, KSLP reversed age-associated shifts in Firmicutes, Epsilonbacteraeota, Ruminococcaceae, and Helicobacter and modulated 57 metabolites particularly within amino acid, fatty acid, and nucleotide metabolic pathways. Correlation analysis identified Ruminococcaceae_UCG_014, Candidatus_Saccharimonas, Helicobacter, Rikenellaceae_RC9, and Ileibacterium as keystone taxa linked to KSLP-responsive metabolites. CONCLUSION:KSLP systemically improves skin architecture, ADSCs function, gut-microbiome balance, and metabolic profiles in naturally aged mice, supporting its clinical potential against age-related decline. Bioactivity-guided identification of active compounds and in vivo mechanistic validation are warranted.

E’Jiao is a traditional Chinese medicine derived from donkey skin. E’Jiao is reported to suppress elevated bone remodelling in ovariectomised rats but its mechanism of action is not known. To bridge this research gap, the current study aims to investigate the effects of E’Jiao on skeletal mineralisation, osteocyte and WNT signalling inhibitors in ovariectomised rats. Female Sprague–Dawley rats (3 months old) were ovariectomised and supplemented with E’Jiao at 0.26 g/kg, 0.53 g/kg and 1.06 g/kg, or 1% calcium carbonate (w/v) in drinking water. The rats were euthanised after two months of supplementation and their bones were collected for Fourier-transform infrared spectroscopy, histomorphometry and protein analysis. Neither ovariectomy nor treatment affected the skeletal mineral/matrix ratio, osteocyte number, empty lacunar number, and Dickkopf-1 and sclerostin protein levels (p > 0.05). Rats treated with calcium carbonate had a higher Dickkopf-1 level than baseline (p = 0.002) and E’Jiao at 0.53 g/kg (p = 0.002). In conclusion, E’Jiao has no significant effect on skeletal mineralisation, osteocyte and WNT signalling inhibitors in ovariectomised rats. The skeletal effect of E’Jiao might not be mediated through osteocytes.

Martynoside (MAR), a bioactive component in several well-known tonic traditional Chinese herbs, exhibits pro-hematopoietic activity during 5-fluorouracil (5-FU) treatment. However, the molecular target and the mechanism of MAR are poorly understood. Here, by adopting the mRNA display with a library of even-distribution (md-LED) method, we systematically examined MAR-protein interactions in vitro and identified the ribosomal protein L27a (RPL27A) as a key cellular target of MAR. Structural and mutational analysis confirmed the specific interaction between MAR and the exon 4,5-encoded region of RPL27A. MAR attenuated 5-FU-induced cytotoxicity in bone marrow nucleated cells, increased RPL27A protein stability, and reduced the ubiquitination of RPL27A at lys92 (K92) and lys94 (K94). Disruption of MAR binding at key residues of RPL27A completely abolished the MAR-induced stabilization. Furthermore, by integrating label-free quantitative ubiquitination proteomics, transcriptomics, and ribosome function assays, we revealed that MAR restored RPL27A protein levels and thus rescued ribosome biogenesis impaired by 5-FU. Specifically, MAR increased mature ribosomal RNA (rRNA) abundance, prevented ribosomal protein degradation, facilitated ribosome assembly, and maintained nucleolar integrity. Collectively, our findings characterize the target of a component of Chinese medicine, reveal the importance of ribosome biogenesis in hematopoiesis, and open up a new direction for improving hematopoiesis by targeting RPL27A.

Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, China, National Research andDevelopment Center for Edible Fungus Processing Technology, Henan University, Kaifeng, China, Henan Province Food Engineering Technology Research Center, Kaifeng, China, Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China, Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara, Türkiye, CREA-Research Centre for Food and Nutrition, Rome, Italy

Background: Menopause is accompanied by increased oxidative stress, partly contributing to weight gain and bone marrow adiposity. Traditional Chinese medication, E'Jiao, has been demonstrated to reduce excessive bone remodelling during oestrogen deprivation, but its effects on body composition and bone marrow adiposity during menopause remain elusive. Objective: To determine the effects of E'Jiao on body composition, bone marrow adiposity and skeletal redox status in ovariectomised (OVX) rats. Methods: Seven groups of three-month-old female Sprague Dawley rats were established (n=6/group): baseline, sham, OVX control, OVX-treated with low, medium or high-dose E'Jiao (0.26, 0.53, 1.06 g/kg, p.o.) or calcium carbonate (1% in tap water, ad libitum). The supplementation was terminated after 8 weeks. Whole-body composition analysis was performed monthly using dual-energy X-ray absorptiometry. Analysis of bone-marrow adipocyte numbers and skeletal antioxidant activities were performed on the femur. Results: Increased total mass, lean mass, and bone marrow adipocyte number were observed in the OVX control versus the sham group. Low-dose E'Jiao supplementation counteracted these changes. Besides, E'Jiao at all doses increased skeletal catalase and superoxide dismutase activities but lowered glutathione levels in the OVX rats. Skeletal malondialdehyde level was not affected by ovariectomy but was lowered with E'Jiao supplementation. However, peroxisome proliferator-activated receptor gamma protein expression was not affected by ovariectomy or any treatment. Conclusion: E'Jiao, especially at the low dose, prevented body composition changes and bone marrow adiposity due to ovariectomy. These changes could be mediated by the antioxidant actions of E'Jiao. It has the potential to be used among postmenopausal women to avoid adiposity.

ABSTRACT Neuraminidase (NA) is a pivotal surface enzyme and a key therapeutic target in combating the influenza A virus. Its evolution can lead to potential zoonotic transmission, seasonal epidemics, and the emergence of drug-resistant mutants. To gain comprehensive insights into the mutational effects and drug resistance profiles of NA, we employed a high-throughput profiling system to quantify the replication capacity of NA mutants at the single-nucleotide level in mouse lung tissues. The fitness of NA mutants is generally correlated with natural mutation occurrence and is constrained by both the requirement to maintain protein stability and NA function. Leveraging this system, we profiled the drug resistance to the three most commonly used neuraminidase inhibitors (NAIs): zanamivir, oseltamivir, and peramivir. In addition to identifying previously reported drug resistance mutations, we validated novel mutants. Notably, we identified an allosteric mutation that confers resistance to all three drugs, which may affect drug binding by interfering with the tetramerization of NA. Moreover, the fitness cost associated with drug-resistant mutations may limit their widespread dissemination. In summary, we provided a parallel characterization of NA’s fitness and drug resistance landscape in an in vivo context, which may guide the rational selection of antiviral drugs for optimal therapeutic efficacy and second-generation NAI development. IMPORTANCE NA is a crucial surface antigen and drug target of influenza A virus. A comprehensive understanding of NA’s mutational effect and drug resistance profiles in vivo is essential for comprehending the evolutionary constraints and making informed choices regarding drug selection to combat resistance in clinical settings. In the current study, we established an efficient deep mutational screening system in mouse lung tissues and systematically evaluated the fitness effect and drug resistance to three neuraminidase inhibitors of NA single-nucleotide mutations. The fitness of NA mutants is generally correlated with a natural mutation in the database. The fitness of NA mutants is influenced by biophysical factors such as protein stability, complex formation, and the immune response triggered by viral infection. In addition to confirming previously reported drug-resistant mutations, novel mutations were identified. Interestingly, we identified an allosteric drug-resistance mutation that is not located within the drug-binding pocket but potentially affects drug binding by interfering with NA tetramerization. The dual assessments performed in this study provide a more accurate assessment of the evolutionary potential of drug-resistant mutations and offer guidance for the rational selection of antiviral drugs.

The current prevention options for postmenopausal osteoporosis are very limited. E'Jiao is a collagen-rich traditional Chinese medicine with the potential to prevent osteoporosis but more comprehensive investigations are lacking. This study aimed to investigate the skeletal protective effects of E'Jiao in a rat model of osteoporosis caused by ovariectomy. Female Sprague Dawley rats (n = 42) were randomly assigned into baseline, sham, ovariectomised (OVX) control, OVX-treated with low-dose (0.26 g/kg), medium dose (0.53 g/kg) and high dose E'Jiao (1.06 g/kg), as well as calcium carbonate (1% w/v) groups. Daily treatment through oral gavage was initiated 7 days after OVX. The rats were euthanised after eight weeks of treatment. Bone mineral density and content were measured at baseline, 1 and 2 months after treatment. Blood was collected for the measurement of bone remodelling markers. Femur and tibial bones were collected for histomorphometry and biomechanical strength analysis. Untreated OVX rats showed high bone remodelling marked by the increased bone formation and bone resorption markers, as well as increased mineralising surface/bone surface ratio. In addition, osteoclast surface and single-labelled surface were increased while mineral apposition rate was reduced in the untreated OVX rats. These changes were antagonised by E'Jiao at all doses. However, the structural, cellular and biomechanical parameters were not affected by ovariectomy and treatment. In conclusion, E'Jiao prevented high bone remodelling during oestrogen deficiency but a long-term study will be required to establish its effects on structural and biomechanical changes due to oestrogen deficiency.

Postmenopausal osteoporosis transpires due to excessive osteoclastic bone resorption and insufficient osteoblastic bone formation in the presence of oestrogen insufficiency. Kang Shuai Lao Pian (KSLP) is a red ginseng-based traditional Chinese medicine known for its anti-ageing properties. However, studies on its effect on bone loss are lacking. Thus, the current study examined the skeletal protective effects of KSLP in an ovariectomised rodent bone loss model. Three-month-old female Sprague Dawley rats (n=42) were randomised into baseline, sham and ovariectomised (OVX) groups. The OVX rats were supplemented with low- (KSLP-L; 0.15 g/kg), medium- (KSLP-M; 0.30 g/kg), high-dose KSLP (KSLP-H; 0.45 g/kg) or calcium carbonate (1% w/v). The daily supplementation of KSLP was performed via oral gavage for eight weeks. Gavage stress was stimulated in the ovariectomised control with distilled water. The rats were euthanised at the end of the study. Whole-body and femoral bone mineral content and density scans were performed at baseline and every four weeks. Blood samples were obtained for the determination of bone remodelling markers. Histomorphometry and biomechanical strength testing were performed on femurs and tibias. High bone remodelling typically due to oestrogen deficiency, indicated by the elevated bone formation and resorption markers, osteoclast surface, single-labelled surface and mineralising surface/bone surface ratio, was observed in the untreated OVX rats. Whole-body BMD adjusted to body weight and Young's modulus decreased significantly in the untreated OVX rats. High-dose KSLP supplementation counteracted these degenerative changes. In conclusion, KSLP improves bone health by normalising bone remodelling, thereby preventing bone loss and decreased bone strength caused by oestrogen deficiency. Its anti-osteoporosis effects should be validated in patients with postmenopausal osteoporosis.

Babaodan (BBD) is a traditional Chinese medicine (TCM) prescribed for various inflammatory diseases, including viral hepatitis and acute genitourinary tract infection. Like other TCMs, BBD is a multi-component formula whose chemical composition and mode of action are largely unknown. The current study identified the bioactive ingredients of BBD using ultrahigh-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) followed by mass spectrometry molecular networking analysis. Subsequently, network pharmacology analysis was performed to predict the potential targets and pathways regulated by BBD. Eventually, a panel of compounds was selected and examined for their anti-inflammatory effects using lipopolysaccharide-stimulated RAW264.7 cells. Eighty-six compounds, including saponins, bile acids, and fatty acids, were identified. Tumor necrosis factor-alpha was identified as a key molecule. Pathways in cancer, inflammatory bowel disease, and hepatitis were predicted to be the major regulatory pathways. The results from bioassays validated ginsenoside Rb1, ginsenoside Rd, deoxycholic acid, chenodeoxycholic acid, and taurochenodeoxycholic acid as novel bioactive ingredients in BBD with anti-inflammatory effects. In conclusion, our study explains the anti-inflammatory efficacy of BBD from both chemical and biological aspects, which provides a scientific basis for the clinical application of BBD in inflammation-related diseases.

Mitochondria sense both intracellular and extracellular stress, with the subsequence released mt-ROS resulted from the interruption of its membrane integrity being a direct activator for NLRP3 inflammasome activation. Regulating the morphology and function of mitochondria could be a strategy against uncontrolled inflammation. We have previously reported that physiological concentrations of bilirubin exhibit anti-inflammatory effect by inhibiting both NF-κB and inflammasome activation. In the current study, we investigated its anti-NLRP3 inflammasome effect per se by means of detecting releasing of IL-1β and TNF-α, the formation of ASC oligomers and ASC-specks, as well as pro-caspase-1 recruitment. Mechanistically, with respect to the antioxidant nature of bilirubin, we evaluated the effect of bilirubin on the releasing of mt-ROS from mitochondria. In addition, mitochondrial morphofunction mainly including morphology and membrane potential in contact living macrophages was analyzed by applying a newly developed multiplexed high-content mitochondrial imaging analysis system using live-cell microscopy. We revealed that bilirubin targets and stabilizes mitochondrial membrane during NLRP3 inflammasome activation; defined doses of bilirubin could be considered as a mitochondria targeted medication against inflammasome-related diseases.

ETHNOPHARMACOLOGICAL RELEVANCE:BBD is a well-known traditional Chinese medicine widely used in clinic to treat viral hepatitis, cholecystitis, angiocholitis and urinary tract infection. According to traditional medicinal theory, BBD exerts the effects of "clearing and humid heat, activating blood and removing toxicity, curing jaundice and relieving pain", the signs of which are recognized as common symptoms of inflammation during infectious diseases in modern medicine. AIM OF THE STUDY:To determine the therapeutic effect of BBD on bacterial endotoxin lipopolysaccharide (LPS) induced sepsis and to investigate the relevant pharmacological and molecular mechanisms of action whereby BBD mitigates inflammation. MATERIALS AND METHODS:In vivo, a mouse sepsis model was induced by intraperitoneally injection of LPS; the BBD were formulated as drug suspension for intragastric administration. The survival rate, secretion of pro-inflammatory cytokines of IL-1β and TNF-α, and multiple organ injury of lung, liver and spleen were examined. In vitro, peritoneal macrophages (PMs) were stimulated with LPS plus ATP for NLRP3 inflammasome activation; polar gradient extractions of BBD from ultrapure water (sample 1) followed by 70% ethanol (sample 2) were added as interventions. In addition to detect the secretion of IL-1β and TNF-α, the activation of NF-κB, ASC-speck formation and ASC oligomerization were examined by western blotting and immunofluorescent stainning. Eventually, the extractions of BBD were applied for UPLC-QTOF-MS analyses; refer to the identified chemicals, the bioactive compounds in BBD with anti-NLRP3 inflammasome activities were discussed. RESULTS:BBD improved the survival of sepsis mice accomplished with diminished inflammatory cytokines production and multiple organ injury. Mechanistically, BBD inhibited both the NF-κB pathway and the assembly of NLRP3 complex in PMs. There were 29 chemical compounds identified from sample 1 and 20 from sample 2. Both samples contained bile acids and saponins and sample 2 contained 2 extra chemicals in the category of bile acids. CONCLUSIONS:BBD presents therapeutic role of endotoxin induced sepsis by inhibiting NLRP3-medaited inflammasome activation, which supports its traditional use for the treatment of infectious diseases. The bile acids and saponins are most likely related to the anti-NLRP3 inflammasome activation effect of BBD.

Shenmai injection (SMI), a traditional Chinese medicine (TCM) injection prepared from Red ginseng and Ophiopogon japonicus, is widely used in clinics to treat chemotherapy-induced myelosuppression. Similar to other TCM injections, SMI contains a high amount of carbohydrates (fructose, sucrose, and maltose) in addition to the bioactive substances, specifically ginsenosides (Rg1, Re, and Rb1). To date, the role of these carbohydrates in the hematopoietic function of SMI remains unclear. We aimed to investigate the hematopoietic effects and potential mechanisms of SMI and its components, focusing on the carbohydrates present in SMI. First, we evaluated the hematopoietic effect of SMI on 5-fluorouracil (5-FU)-induced myelotoxicity in a tumor-bearing mouse model. Then we prepared mixtures of ginsenosides and carbohydrates according to their proportions in SMI and evaluated their hematopoietic function in mice with 5-FU-induced myelosuppression. Finally, hematopoiesis-related molecular networks were built based on RNA sequencing (RNA-seq) of the bone marrow stromal cells (BMSCs), and the potential mechanisms of carbohydrates and ginsenosides were evaluated. SMI attenuated 5-FU-induced myelotoxicity in tumor-bearing mice. Both ginsenosides and carbohydrates increased the bone marrow nucleated cell (BMNC) count and improved the bone marrow morphology in myelosuppressive mice; they promoted the proliferation of BMSCs derived from those myelosuppressive mice. Bioinformatics analyses revealed ECM-receptor interaction, Hippo signaling, and Wnt signaling are common pathways regulated by both ginsenosides and carbohydrates; Gstt1, Gstp2, Gsta4 and Oplah in Glutathione metabolism pathway and Cd19, Cd79a, and Cd79b in B cell receptor pathway are uniquely regulated genes related to carbohydrates but not ginsenosides. Carbohydrates may collaborate with ginsenosides and contribute to the hematopoietic function of SMI. Carbohydrates could be considered as a bioactive component in this TCM injection.

Martynoside (MAR) is a bioactive glycoside of Rehmannia glutinosa, a traditional Chinese herb frequently prescribed for treating chemotherapy-induced pancytopenia. Despite its clinical usage in China for thousands of years, the mechanism of MAR’s hematopoietic activity and its impact on chemotherapy-induced antitumor activity are still unclear. Here, we showed that MAR protected ex vivo bone marrow cells from 5-fluorouracil (5-FU)-induced cell death and inflammation response by down-regulating the TNF signaling pathway, in which II1b was the most regulatory gene. Besides, using mouse models with melanoma and colon cancer, we further demonstrated that MAR had protective effects against 5-FU-induced myelosuppression in mice without compromising its antitumor activity. Our results showed that MAR increased the number of bone marrow nucleated cells (BMNCs) and the percentage of leukocyte and granulocytic populations in 5-FU-induced myelosuppressive mice, accompanied by an increase in numbers of circulating white blood cells and platelets. The transcriptome profile of BMNCs further showed that the mode of action of MAR might be associated with the increased survival of BMNCs and the improvement of the bone marrow microenvironment. In summary, we revealed the potential molecular mechanism of MAR to counteract 5-FU-induced bone marrow cytotoxicity both ex vivo and in vivo, and highlighted its potential clinical usage in cancer patients experiencing chemotherapy-induced multi-lineage myelosuppression.

The type I interferon (IFN) pathway is a key component of innate immune response upon invasion of foreign pathogens. It is also under precise control to prevent excessive upregulation and undesired inflammation cascade. In the present study, we report that Riok3, an atypical kinase, negatively regulates retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) sensing-induced type I IFN signaling. Riok3 deficiency selectively inhibits RNA viral replication in vitro, resulting from an upregulated type I IFN pathway. Mice with myeloid-specific Riok3 knockout also show a more robust induction of type I IFN upon RNA virus infection and are more resistant to RNA virus-induced pathogenesis. Mechanistically, Riok3 recruits and interacts with the E3 ubiquitin ligase TRIM40, leading to the degradation of RIG-I and melanoma differentiation-associated gene-5 (MDA5) via K48- and K27-linked ubiquitination. Collectively, our data reveal the mechanism that Riok3 employs to be a negative regulator of antiviral innate immunity.