Anti-inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma.

Trichospira verticillata is an annual herb that belongs to the family Asteraceae. Trichospira verticillata extract (TVE) elicits anti-plasmodial activity; however, there has been no detailed report about its anti-inflammatory effects and molecular mechanisms. In addition, herbal plants exhibit anti-inflammatory effects by suppressing the NLRP3 inflammasome. Therefore, the primary goal of this study was to examine the effects of TVE on NLRP3 inflammasome activation by measuring interleukin-1ß (IL-1ß) secretion. We treated lipopolysaccharides (LPS)-primed J774A.1 and THP-1 cells with TVE, which attenuated NLRP3 inflammasome activation. Notably, TVE did not affect nuclear factor-kappa B (NF-κB) signalling or intracellular reactive oxygen species (ROS) production and potassium efflux, suggesting that it inactivates the NLRP3 inflammasome via other mechanisms. Moreover, TVE suppressed the formation of apoptosis-associated speck-like protein (ASC) speck and oligomerization. Immunoprecipitation data revealed that TVE reduced the binding of NLRP3 to NIMA-related kinase 7 (NEK7), resulting in reduced ASC oligomerization and speck formation. Moreover, TVE alleviated neutrophilic asthma (NA) symptoms in mice. This study demonstrates that TVE modulates the binding of NLPR3 to NEK7, thereby reporting novel insights into the mechanism by which TVE inhibits NLRP3 inflammasome. These findings suggest TVE as a potential therapeutic of NLRP3 inflammasome-mediated diseases, particularly NA.

Colletodiol derivatives of the endophytic fungus Trichocladium sp.

The OSMAC (one strain many compounds) concept is a cultivation-based approach to increase the diversity of secondary metabolites in microorganisms. In this study, we applied the OSMAC-approach to the endophytic fungus Trichocladium sp. by supplementation of the cultivation medium with 2.5% phenylalanine. This experiment yielded five new compounds, trichocladiol (1), trichocladic acid (2), colletodiolic acid (3), colletolactone (4) and colletolic acid (5), together with five previously described ones (6-10). The structures were elucidated via comprehensive spectroscopic measurements, and the absolute configurations of compound 1 was elucidated by using TDDFT-ECD calculations. For formation of compounds 3-5, a pathway based on colletodiol biosynthesis is proposed. Compound 6 exhibited strong antibacterial activity against methicillin-resistant Staphylococcus aureus with a minimal inhibitory concentration (MIC) of 0.78 µM as well as a strong cytotoxic effect against the human monocytic cell line THP1 with an IC50 of 0.7 µM. Compound 8 showed moderate antibacterial activity against Mycobacterium tuberculosis with a MIC of 25 µM and a weak cytotoxic effect against THP1 cells with an IC50 of 42 µM.

Flavonoid extracted from Epimedium attenuate cGAS-STING-mediated diseases by targeting the formation of functional STING signalosome.

Hyperactivation of the cyclic-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signalling pathway has been shown to be associated with the development of a variety of inflammatory diseases, and the discovery of an inhibitor of the cGAS-STING signalling pathway holds great promise in the therapeutic interventions. Epimedium flavonoid (EF), a major active ingredient isolated from the medicinal plant Epimedium, has been reported to have good anti-inflammatory activity, but its exact mechanism of action remains unclear. In the present study, we found that EF in mouse bone marrow-derived macrophages (BMDMs), THP-1 (Tohoku Hospital Pediatrics-1) as well as in human peripheral blood mononuclear cells (hPBMC) inhibited the activation of the cGAS-STING signalling pathway, which subsequently led to a decrease in the expression of type I interferon (IFN-ß, CXCL10 and ISG15) and pro-inflammatory cytokines (IL-6 and TNF-α). Mechanistically, EF does not affect STING oligomerization, but inhibits the formation of functional STING signalosome by attenuating the interaction of interferon regulatory factor 3 (IRF3) with STING and TANK-binding kinase 1 (TBK1). Importantly, in vivo experiments, EF has shown promising therapeutic effects on inflammatory diseases mediated by the cGAS-STING pathway, which include the agonist model induced by DMXAA stimulation, the autoimmune inflammatory disease model induced by three prime repair exonuclease 1 (Trex1) deficiency, and the non-alcoholic steatohepatitis (NASH) model induced by a pathogenic amino acid and choline deficiency diet (MCD). To summarize, our study suggests that EF is a potent potential inhibitor component of the cGAS-STING signalling pathway for the treatment of inflammatory diseases mediated by the cGAS-STING signalling pathway.

Trilobolide-6-O-isobutyrate from Sphagneticola trilobata acts by inducing oxidative stress, metabolic changes and apoptosis-like processes by caspase 3/7 activation of human lung cancer cell lines.

BACKGROUND: Lung cancer, a chronic and heterogeneous disease, is the leading cause of cancer-related death on a global scale. Presently, despite a variety of available treatments, their effectiveness is limited, often resulting in considerable toxicity and adverse effects. Additionally, the development of chemoresistance in cancer cells poses a challenge. Trilobolide-6-O-isobutyrate (TBB), a natural sesquiterpene lactone extracted from Sphagneticola trilobata, has exhibited antitumor effects. Its pharmacological properties in NSCLC lung cancer, however, have not been explored. PURPOSE: This study evaluated the impact of TBB on the A549 and NCI-H460 tumor cell lines in vitro, examining its antiproliferative properties and initial mechanisms of cell death. METHODS: TBB, obtained at 98 % purity from S. trilobata leaves, was characterized using chromatographic techniques. Subsequently, its impact on inhibiting tumor cell proliferation in vitro, TBB-induced cytotoxicity in LLC-MK2, THP-1, AMJ2-C11 cells, as well as its effects on sheep erythrocytes, and the underlying mechanisms of cell death, were assessed. RESULTS: In silico predictions have shown promising drug-likeness potential for TBB, indicating high oral bioavailability and intestinal absorption. Treatment of A549 and NCI-H460 human tumor cells with TBB demonstrated a direct impact, inducing significant morphological and structural alterations. TBB also reduced migratory capacity without causing toxicity at lower concentrations to LLC-MK2, THP-1 and AMJ2-C11 cell lines. This antiproliferative effect correlated with elevated oxidative stress, characterized by increased levels of ROS, superoxide anion radicals and NO, accompanied by a decrease in antioxidant markers: SOD and GSH. TBB-stress-induced led to changes in cell metabolism, fostering the accumulation of lipid droplets and autophagic vacuoles. Stress also resulted in compromised mitochondrial integrity, a crucial aspect of cellular function. Additionally, TBB prompted apoptosis-like cell death through activation of caspase 3/7 stressors. CONCLUSION: These findings underscore the potential of TBB as a promising candidate for future studies and suggest its viability as an additional component in the development of novel anticancer drugs prototypes.

Compound Danshen Dripping Pill effectively alleviates cGAS-STING-triggered diseases by disrupting STING-TBK1 interaction.

BACKGROUND: The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon (IFN) genes (STING) pathway is critical in the innate immune system and can be mobilized by cytosolic DNA. The various inflammatory and autoimmune diseases progression is highly correlated with aberrant cGAS-STING pathway activation. While some cGAS-STING pathway inhibitor were identified, there are no drugs that can be applied to the clinic. Compound Danshen Dripping Pill (CDDP) has been successfully used in clinic around the world, but the most common application is limited to cardiovascular disease. Therefore, the purpose of the present investigation was to examine whether CDDP inhibits the cGAS-STING pathway and could be used as a therapeutic agent for multiple cGAS-STING-triggered diseases. METHODS: BMDMs, THP1 cells or Trex1-/- BMDMs were stimulated with various cGAS-STING-agonists after pretreatment with CDDP to detect the function of CDDP on IFN-ß and ISGs productionn. Next, we detect the influence on IRF3 and P65 nuclear translocation, STING oligomerization and STING-TBK1-IRF3 complex formation of CDDP. Additionally, the DMXAA-mediated activation mice model of cGAS-STING pathway was used to study the effects of CDDP. Trex1-/- mice model and HFD-mediated obesity model were established to clarify the efficacy of CDDP on inflammatory and autoimmune diseases. RESULTS: CDDP efficacy suppressed the IRF3 phosphorylation or the generation of IFN-ß, ISGs, IL-6 and TNF-α. Mechanistically, CDDP did not influence the STING oligomerization and IRF3-TBK1 and STING-IRF3 interaction, but remarkably eliminated the STING-TBK1 interaction, ultimately blocking the downstream responses. In addition, we also clarified that CDDP could suppress cGAS-STING pathway activation triggered by DMXAA, in vivo. Consistently, CDDP could alleviate multi-organ inflammatory responses in Trex1-/- mice model and attenuate the inflammatory disorders, incleding obesity-induced insulin resistance. CONCLUSION: CDDP is a specifically cGAS-STING pathway inhibitor. Furthermore, we provide novel mechanism for CDDP and discovered a clinical agent for the therapy of cGAS-STING-triggered inflammatory and autoimmune diseases.

Dehydrocostus lactone alleviates irinotecan-induced intestinal mucositis by blocking TLR4/MD2 complex formation.

BACKGROUND: Irinotecan (CPT-11) is used as chemotherapeutic drug for treatment of colorectal cancer. However, without satisfactory treatments, its gastrointestinal toxicities such as diarrhea and intestinal inflammation severely restrained its clinical application. Roots of Aucklandia lappa Decne. are used as traditional Chinese medicine to relieve gastrointestinal dysfunction and dehydrocostus lactone (DHL) is one of its main active components. Nevertheless, the efficacy and mechanism of DHL against intestinal mucositis remains unclear. PURPOSE: The present study aimed to investigate the protective effects of DHL on CPT-11-induced intestinal mucositis and its underlying mechanisms. METHODS: The protective effect of DHL was investigated in CPT-11-induced mice and lipopolysaccharide (LPS)+CPT-11 induced THP-1 macrophages. Body weight, diarrhea score, survival rate, colon length, and histopathological changes in mice colon and jejunum were analyzed to evaluate the protective effect of DHL in vivo. And DHL on reducing inflammatory response and regulating TLR4/NF-κB/NLRP3 pathway in vivo and in vitro were explored. Moreover, DHL on the interaction between TLR4 and MD2 was investigated. And silencing TLR4 targeted by siRNA was performed to validate the mechanisms of DHL on regulating the inflammation. RESULTS: DHL prevented CPT-11-induced intestinal damage, represented by reducing weight loss, diarrhea score, mortality rate and the shortening of the colon. Histological analysis confirmed that DHL prevented intestinal epithelial injury and improved the intestinal barrier function in CPT-11 induced mice. Besides, DHL significantly downregulated the level of inflammatory cytokines by inhibiting TLR4/NF-κB/NLRP3 signaling pathway in CPT-11-induced mice and LPS+CPT-11-induced THP-1 macrophages. In addition, DHL blocked TLR4/MD2 complex formation. Molecular docking combined with SIP and DARTS assay showed that DHL could bind to TLR4/MD2 and occludes the hydrophobic pocket of MD2. Furthermore, Silencing TLR4 abrogated the effect of DHL on LPS+CPT-11 induced inflammatory response in THP-1 macrophages. Additionally, DHL ameliorate the CPT-11-induced intestinal mucositis without affecting the anti-tumor efficacy of CPT-11 in the tumor xenograft mice. CONCLUSION: This study found that DHL exhibited the anti-inflammatory effects in CPT-11-induced intestinal mucositis by inhibiting the formation of TLR4/MD2 complex and then regulation of NF-κB/NLRP3 signaling pathway. DHL is potentially served as a novel strategy of combined medication with CPT-11.

Astaxanthin targets IL-6 and alleviates the LPS-induced adverse inflammatory response of macrophages.

Numerous natural compounds are recognized for their anti-inflammatory properties attributed to antioxidant effects and the modulation of key inflammatory factors. Among them, astaxanthin (AST), a potent carotenoid antioxidant, remains relatively underexplored regarding its anti-inflammatory mechanisms and specific molecular targets. In this study, human monocytic leukemia cell-derived macrophages (THP-1) were selected as experimental cells, and lipopolysaccharides (LPS) served as inflammatory stimuli. Upon LPS treatment, the oxidative stress was significantly increased, accompanied by remarkable cellular damage. Moreover, LPSs escalated the expression of inflammation-related molecules. Our results demonstrate that AST intervention could effectively alleviate LPS-induced oxidative stress, facilitate cellular repair, and significantly attenuate inflammation. Further exploration of the anti-inflammatory mechanism revealed AST could substantially inhibit NF-κB translocation and activation, and mitigate inflammatory factor production by hindering NF-κB through the antioxidant mechanism. We further confirmed that AST exhibited protective effects against cell damage and reduced the injury from inflammatory cytokines by activating p53 and inhibiting STAT3. In addition, utilizing network pharmacology and in silico calculations based on molecular docking, molecular dynamics simulation, we identified interleukin-6 (IL-6) as a prominent core target of AST anti-inflammation, which was further validated by the RNA interference experiment. This IL-6 binding capacity actually enabled AST to curb the positive feedback loop of inflammatory factors, averting the onset of possible inflammatory storms. Therefore, this study offers a new possibility for the application and development of astaxanthin as a popular dietary supplement of anti-inflammatory or immunomodulatory function.

Naturally occurring small molecules with dual effect upon inflammatory signaling pathways and endoplasmic reticulum stress response.

The endoplasmic reticulum (ER) is determinant to maintain cellular proteostasis. Upon unresolved ER stress, this organelle activates the unfolded protein response (UPR). Sustained UPR activates is known to occur in inflammatory processes, deeming the ER a potential molecular target for the treatment of inflammation. This work characterizes the inflammatory/UPR-related molecular machinery modulated by an in-house library of natural products, aiming to pave the way for the development of new selective drugs that act upon the ER to counter inflammation-related chronic diseases. Starting from a library of 134 compounds of natural occurrence, mostly occurring in medicinal plants, nontoxic molecules were screened for their inhibitory capacity against LPS-induced nuclear factor kappa B (NF-κB) activation in a luciferase-based reporter gene assay. Since several natural products inhibited NF-κB expression in THP-1 macrophages, their effect on reactive oxygen species (ROS) production and inflammasome activation was assessed, as well as their transcriptional outcome regarding ER stress. The bioactivities of several natural products are described herein for the first time. We report the anti-inflammatory potential of guaiazulene and describe 5-deoxykaempferol as a novel inhibitor of inflammasome activation. Furthermore, we describe the dual potential of 5-deoxykaempferol, berberine, guaiazulene, luteolin-4'-O-glucoside, myricetin, quercetagetin and sennoside B to modulate inflammatory signaling ER stress. Our results show that natural products are promising molecules for the discovery and pharmaceutical development of chemical entities able to modulate the inflammatory response, as well as proteostasis and the UPR.

Cucurbitacin B modulates M2 macrophage differentiation and attenuates osteosarcoma progression via PI3K/AKT pathway.

Osteosarcoma is a common malignant bone tumour characterised by an aggressive metastatic potential. The tumour microenvironment, particularly the M2-polarised macrophages, is crucial for tumour progression. Cucurbitacin B (CuB), a triterpenoid derivative, is recognised for its anti-inflammatory and antitumour properties. This study investigates CuB and its effect on M2 macrophage differentiation and osteosarcoma progression, aiming to contribute to new treatment strategies. In vitro, THP-1 monocytes were stimulated with PMA, IL-13 and IL-4 to induce differentiation into M2 macrophages. Additionally, the influence of CuB on the proliferation, migration and invasion of osteosarcoma cells in the context of M2 macrophages was scrutinised. Crucial signalling pathways, especially the PI3K/AKT pathway, affected by CuB were identified and validated. In vivo, the osteosarcoma model was employed to gauge the effects of CuB on tumour weight, lung metastasis, angiogenesis, cell proliferation and M2 macrophage markers. The results showed that CuB inhibited M2 macrophage differentiation, leading to reduced proliferation, migration and invasion of osteosarcoma cells. CuB manifested an inhibitory effect on the PI3K/AKT pathway during the differentiation of M2 macrophages. In mouse models, CuB markedly reduced the tumour weight and the number of lung metastases. It also reduced the expression of angiogenesis and cell proliferation markers in tumour tissues, decreased the quantity of M2 macrophages and their associated markers and pathway proteins. In conclusion, CuB impedes osteosarcoma progression by inhibiting M2 macrophage differentiation via the PI3K/AKT pathway, presenting the potential for therapeutic advancements in osteosarcoma treatment.

Difference in the cellular response following THP-1 derived phagocytic monocyte cells exposure to commercial aluminum-based adjuvants and aluminum-containing vaccines.

BACKGROUND: Aluminum-based adjuvants (ABAs) enhance the immune response following vaccine injection. Their mechanisms of action are not fully understood, and their bio-persistency have been described associated with long-term adverse effects. METHODS: We evaluated and compared the cellular effects of the two main ABAs and whole vaccines on ATP production, ROS generation and cytokines production (IL-6 and IL-10), using THP-1 cells. RESULTS: ABAs altered the cell energy metabolism by increasing ROS production after 24 h and reducing ATP production after 48 h. In addition, both ABAs and whole vaccines induced different kinetics of IL-6 production, whereas only ABAs induced IL-10 secretion. CONCLUSION: This study showed clearly, for a first time, a difference in cellular response to the ABAs and whole vaccines which should be taken into consideration in future studies focusing on the effect of ABA in vaccines. Future studies on ABAs should also pay attention to mitochondrial function alterations following exposure to ABA-containing vaccines.

Glycyrrhetinic acid suppresses breast cancer metastasis by inhibiting M2-like macrophage polarization via activating JNK1/2 signaling.

BACKGROUND: Breast cancer metastasis is leading cause of cancer death among women worldwide. Tumor-associated macrophages (TAMs) have been considered as potential targets for treating breast cancer metastasis because they promote tumor growth and development. Glycyrrhetinic acid (GA) is one of the most important phytochemicals of licorice which has shown promising anti-cancer efficacies in pre-clinical trials. However, the regulatory effect of GA on the polarization of TAMs remains elusive. PURPOSE: To investigate the role of GA in regulating the polarization of M2 macrophages and inhibiting breast cancer metastasis, and to further explore its underlying mechanisms of action. STUDY DESIGN: IL-4 / IL-13-treated RAW 264.7 and THP-1 cells were used as the M2-polarized macrophages in vitro. A 4T1 mouse breast cancer model and the tail vein breast cancer metastasis model were applied to study the effect of GA on breast cancer growth and metastasis in vivo. RESULTS: In vitro studies showed that GA significantly inhibited IL-4 / IL 13-induced M2-like polarization in RAW 264.7 and THP-1 macrophages without affecting M1-like polarization. GA strongly decreased the expression of M2 macrophage markers CD206 and Arg-1, and reduced the levels of the pro-angiogenic molecules VEGF, MMP9, MMP2 and IL-10 in M2 macrophages. GA also increased the phosphorylation of JNK1/2 in M2 macrophages. Moreover, GA significantly suppressed M2 macrophage-induced cell proliferation and migration in 4T1 cancer cells and HUVECs. Interestingly, the inhibitory effects of GA on M2 macrophages were abolished by a JNK inhibitor. Animal studies showed that GA significantly suppressed tumor growth, angiogenesis, and lung metastasis in BALB/c mice bearing breast tumor. In tumor tissues, GA reduced the number of M2 macrophages but elevated the proportion of M1 macrophages, accompanied by activation of JNK signaling. Similar results were found in the tail vein breast cancer metastasis model. CONCLUSION: This study demonstrated for the first time that GA could effectively suppress breast cancer growth and metastasis by inhibiting macrophage M2 polarization via activating JNK1/2 signaling. These findings indicate that GA could be served as the lead compound for the future development of anti-breast cancer drug.

Anti-Inflammatory Effect of Garcinol Extracted from Garcinia dulcis via Modulating NF-κB Signaling Pathway.

Garcinia is a significant medicinal plant with many beneficial phytoconstituents, including garcinol. This study investigated the anti-inflammatory effect of garcinol isolated from Garcinia dulcis fruit in LPS-activated THP-1 and Raw 264.7 macrophages. The results demonstrated that the low concentration of garcinol did not alter cell viability. Furthermore, co-incubation of garcinol with LPS inhibited the production of pro-inflammatory cytokines, including TNF-α, IL-8, IL-6, IL-1ß, and pro-inflammatory mediators, including iNOS and COX-2 at the mRNA and protein expression levels. Garcinol also decreased the secretion of TNF-α, IL-6, IL-1ß, PGE2, and NO. Moreover, the anti-inflammatory effects involved an alteration in the NF-κB signaling pathway. Downregulation of pIKKα/ß, pIκBα, and pNF-κB was observed, hence reducing the translocation of pNF-κB from the cytosol into the nucleus, which subsequently decreased the production of pro-inflammatory molecules. Therefore, garcinol isolated from Garcinia dulcis is a potential candidate as an anti-inflammatory agent for inflammation-related disease treatment.

Vitamin K3 Suppresses Pyroptosis in THP-1 Cells through Inhibition of NF-κB and JNK Signaling Pathways.

Vitamin K, a necessary nutritional supplement for human, has been found to exhibit anti-inflammatory activity. In the present study, we investigated the effects of vitamin K family on lipopolysaccharide (LPS) plus nigericin induced pyroptosis and explored the underlying mechanism of its action in THP-1 monocytes. Results showed that vitamin K3 treatment significantly suppressed THP-1 pyroptosis, but not vitamin K1 or K2, as evidenced by increased cell viability, reduced cellular lactate dehydrogenase (LDH) release and improved cell morphology. Vitamin K3 inhibited NLRP3 expression, caspase-1 activation, GSDMD cleavage and interleukin (IL)-1ß secretion in pyrophoric THP-1 cells. In addition, vitamin K3 inhibited the pro-inflammatory signaling pathways including nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK). Vitamin K3 treatment also attenuated tissue damage and reduced serum LDH, IL-1ß and IL-6 levels in LPS-induced systemic inflammation of mice. The reduced myeloperoxidase (MPO) activityand F4/80 expression indicated that vitamin K3 effectively reduced the infiltration of neutrophils and macrophages. Moreover, NLRP3 expression in monocytes/macrophages were also decreased in vitamin K3-treatedmice after LPS challenge. These findings suggest that vitamin K3 potently alleviates systemic inflammation and organ injury via inhibition of pyroptosis in monocytes and may serve as a novel therapeutic strategy for patients with inflammatory diseases.

Antcin A, a phytosterol regulates SARS-CoV-2 spike protein-mediated metabolic alteration in THP-1 cells explored by the 1 H-NMR-based metabolomics approach.

The mechanism of SARS-CoV-2 spike protein-mediated perturbations of metabolic pathways and modulation of antcin A, a steroid-like compound isolated from Taiwanofungus camphoratus, are not studied. Here, we investigated the metabolic alteration by SARS-CoV-2 spike protein and the regulatory effect of antcin A on SARS-CoV-2 spike protein-induced metabolic changes in the Phorbol 12-myristate 13-acetate (PMA)-induced human monocytes (THP-1) using proton nuclear magnetic resonance (1 H-NMR) and MetaboAnalyst 5.0 software. The cytotoxic potential of SARS-CoV-2 spike protein, antcin A, and dexamethasone was assessed by MTT assay. The metabolomic perturbations and their relation to human coronaviruses' receptors were evaluated by qPCR. This study indicated that the altered metabolites mediated by SARS-CoV-2 protein, such as methionine, phosphoenolpyruvic acid, canadine, glutamine, ethanolamine, and phenylalanine, were significantly reversed by antcin A. In addition, antcin A significantly inhibited SARS-CoV-2 spike protein-mediated up-regulation of TLR-4 and ACE2 receptors, while GRP78 inhibition was not statistically significant. This is the first study to use 1 H-NMR to investigate SARS-CoV-2 spike protein-induced metabolomic changes in PMA-induced THP-1 cells. Antcin A significantly reversed metabolomic alters while dexamethasone failed to fix them. Therefore, we believe that antcin A could be a potential candidate for therapeutic agents for viral infections related to a metabolic abnormality.

Effect of 5-aminolevulinic Acid on Mitochondrial Activity.

BACKGROUND: Mitochondrial dysfunction is considered an important mechanism in the pathogenesis of various diseases. Therefore, mitochondria are currently being considered as subjects for targeted therapies, particularly, phototherapy using 5-aminolevulinic acid. This study aimed to investigate the activity of mitochondria in cells with different mutation loads. MATERIALS AND METHODS: The study was conducted using 11 cybrid lines obtained from the THP-1 cell line (a human monocytic leukemia cell line) and platelets of patients with different mitochondrial mutations. RESULTS: Our results illustrate that 5-aminolevulinic acid was metabolized equally in all cell lines, however, there was a significant decrease in mitochondrial potential, which differed among lines. CONCLUSIONS: The results of this study can be used to develop a personalized therapeutic approach based on different mitochondrial activities.

Short-Chain Fatty Acids Weaken Ox-LDL-Induced Cell Inflammatory Injury by Inhibiting the NLRP3/Caspase-1 Pathway and Affecting Cellular Metabolism in THP-1 Cells.

Short-chain fatty acids (SCFAs) are important anti-inflammatory metabolites of intestinal flora. Oxidized low-density lipoprotein (ox-LDL)-induced macrophage activation is critical for the formation of atherosclerosis plaque. However, the association between SCFAs and ox-LDL-induced macrophage activation with respect to the formation of atherosclerosis plaque has not yet been elucidated. The present study investigated whether SCFAs (sodium acetate, sodium propionate, and sodium butyrate) can affect ox-LDL-induced macrophage activation and potential signaling pathways via regulation of the expression of the NLRP3/Caspase-1 pathway. Using human monocyte-macrophage (THP-1) cells as a model system, it was observed that ox-LDL not only induced cell inflammatory injury but also activated the NLRP3/Caspase-1 pathway. The exogenous supplementation of three SCFAs could significantly inhibit cell inflammatory injury induced by ox-LDL. Moreover, three SCFAs decreased the expression of IL-1ß and TNF-α via the inactivation of the NLRP3/Caspase-1 pathway induced by ox-LDL. Furthermore, three SCFAs affected cellular metabolism in ox-LDL-induced macrophages, as detected by untargeted metabolomics analysis. The results of the present study indicated that three SCFAs inhibited ox-LDL-induced cell inflammatory injury by blocking the NLRP3/Caspase-1 pathway, thereby improving cellular metabolism. These findings may provide novel insights into the role of SCFA intervention in the progression of atherosclerotic plaque formation.

Pharmacokinetics and pharmacodynamics of bioactive compounds in Penyanqing preparation in THP-1 inflammatory cells induced by Lipopolysaccharide.

BACKGROUND: Penyanqing (PYQ), a traditional Chinese medicine (TCM), has a good clinical efficacy for the treatment of pelvic inflammatory disease (PID). Previously, researches on its anti-inflammatory effect and mechanism in vitro, in silico, and in vivo have been reported by our team. However, the interrelationship between the anti-inflammatory activity and the active compounds in PYQ are not clear. Here, the pharmacokinetics-pharmacodynamics (PK-PD) study was carried out for more proper clinical use. METHODS: The plasma concentrations of salvianolic acid B (SAB), protocatechualdehyde (PRO), paeoniflorin (PE), astilbin (AST), ferulic acid (FE), and chlorogenic acid (CH) in SD rats after PYQ administration were determined by a selective and rapid HPLC-MS/MS method. In addition, the PK-PD on cell model was used to explore the relationship between the plasma concentration and inflammatory biomarkers (TNF-α, IL-1ß). RESULTS: The results of this study showed that the six components could reach the peak blood concentration within 0.29 h, indicating the rapid absorption of it. The eliminations of AST, CH, FE, PE, and PRO were relatively fast due to their mean residence times (MRTs) within 3 h, while the elimination of SAB was slower (MRT 5.67 ± 0.66 h). Combined with a THP-1 cell model, there was a significant correlation between inflammatory factors and component plasma concentrations with correlation coefficients in the range of -0.9--0.746. Correspondingly, the drug-containing plasma obtained at 0.25 h point exhibited the best inhibition effect on production of IL-1ß and TNF-α in LPS-induced THP-1 cells. CONCLUSION: The six main components in PYQ could be quickly absorbed, and there was a potential good correlation between their pharmacokinetics and the pharmacodynamics of PYQ.

Centella asiatica Modulates Nrf-2 Antioxidant Mechanisms and Enhances Reactive Oxygen Species-Mediated Apoptotic Cell Death in Leukemic (THP-1) Cells.

Centella asiatica is commonly used in traditional medicine owing to its many therapeutic properties including but not limited to antioxidant and antitumor potential. This study examined the antioxidant and antiproliferative effects of its crude (C) and fractionated (C3) ethanolic leaf extracts in THP-1 cells. In THP-1 cells, C and C3 cytotoxicity was evaluated (WST-1 viability assay; 24 h; [0.2-3 mg/mL]) and half maximal inhibitory concentration was obtained. Malondialdehyde (MDA; spectrophotometry), mitochondrial depolarization (Δψm), intracellular reactive oxygen species (IROS; flow cytometry), glutathione (GSH), oxidized GSH (GSSG) concentrations, adenosine triphosphate (ATP) levels, caspase activities (luminometry) and DNA fragmentation (single cell gel electrophoresis assay) were evaluated. Protein expression and gene expression was quantified by Western blotting and quantitative polymerase chain reaction, respectively. THP-1 cell viability was dose-dependently reduced by C and C3. MDA, IROS, GSH, and Δψm were increased and ATP was decreased by C and C3 (P < .01). Antioxidant gene expression, Nrf-2 protein expression, and GSSG levels (P < .01) were increased by C, but were decreased by C3. C and C3 elevated caspase activity and DNA damage (P < .0001), whereas they decreased glutathione peroxidase and Bcl-2 protein expressions (P < .003). c-PARP protein expression and c-myc gene expression was decreased by C, whereas they were increased by C3 (P < .002). C3 reduced OGG-1 gene expression (P < .0003). Antioxidant responses were increased by C, whereas they were decreased by C3. Both C and C3 exerted antiproliferative effects in THP-1 cells by enhancing apoptosis. Of note, C3 more effectively induced apoptosis.

[Effect of miltirone on proliferation and apoptosis of leukemia THP-1 cells].

To investigate the toxicity and related mechanism of miltirone to human acute myeloid leukemia THP-1 cells. To be specific, the active components and targets of miltirone were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the target proteins were converted into standard gene names with UniProt. Acute leukemia-rela-ted target genes were screened from GeneCards and DisGeNET. Venn diagram was constructed with Venny 2.1 to yield the common targets of the disease and the drug. The protein-protein interaction(PPI) network was constructed by STRING and Cytoscape 3.8.2. THP-1 cells in the logarithmic growth phase were treated with dimethyl sulfoxide(DMSO), and 2.5, 5, 10, 15, and 20 µmol·L~(-1) miltirone for 24 h, respectively. The proliferation rate of cells was analyzed by carboxyfluorescein diacetate succinimidyl ester(CFSE), apoptosis rate by flow cytometry with Annexin V-PE/7 AAD staining, and cell morphology by acridine orange staining. Real-time quantitative PCR(qPCR) was employed to detect the mRNA levels of nuclear receptor coactivator 2(NCOA2), poly(ADP-ribose) polymerase-1(PARP1), B-cell lymphoma-2(Bcl-2)-associated X protein(Bax), Bcl-2, and cysteine aspartyl protease-3(caspase-3). The effect of miltirone on apoptosis was detected in presence of caspase inhibitor Z-VAD-FMK. A total of 26 targets of miltirone, 1 046 genes related to acute leukemia, and 6 common targets of the two were screened out. Flow cytometry result showed miltirone at 10 µmol·L~(-1) can inhibit proliferation and promote apoptosis of THP-1 cells. The typical manifestations of apoptosis, such as cell shrinkage, nuclear rupture, and chromatin agglomerate were displayed by acridine orange staining. The decreased mRNA levels of NCOA2 and PARP1 and increased Bax/Bcl-2 ratio and the activity of pro-apoptotic protein caspase-3 were observed. Z-VAD-FMK can attenuate the apoptosis-inducing effect of miltirone. This study indicates that miltirone can inhibit the proliferation and promote the apoptosis of THP-1 cells, by down-regulating NCOA2 and PARP1, raising Bax/Bcl-2 ratio, and activating caspase-3.

Treating leishmaniasis in Amazonia, part 2: Multi-target evaluation of widely used plants to understand medicinal practices.

ETHNOPHARMACOLOGICAL RELEVANCE: Leishmaniasis are widely distributed among tropical and subtropical countries, and remains a crucial health issue in Amazonia. Indigenous groups across Amazonia have developed abundant knowledge about medicinal plants related to this pathology. AIM OF THE STUDY: We intent to explore the weight of different pharmacological activities driving taxa selection for medicinal use in Amazonian communities. Our hypothesis is that specific activity against Leishmania parasites is only one factor along other (anti-inflammatory, wound healing, immunomodulating, antimicrobial) activities. MATERIALS AND METHODS: The twelve most widespread plant species used against leishmaniasis in Amazonia, according to their cultural and biogeographical importance determined through a wide bibliographical survey (475 use reports), were selected for this study. Plant extracts were prepared to mimic their traditional preparations. Antiparasitic activity was evaluated against promastigotes of reference and clinical New-World strains of Leishmania (L. guyanensis, L. braziliensis and L. amazonensis) and L. amazonensis intracellular amastigotes. We concurrently assessed the extracts immunomodulatory properties on PHA-stimulated human PBMCs and RAW264.7 cells, and on L. guyanensis antigens-stimulated PBMCs obtained from Leishmania-infected patients, as well as antifungal activity and wound healing properties (human keratinocyte migration assay) of the selected extracts. The cytotoxicity of the extracts against various cell lines (HFF1, THP-1, HepG2, PBMCs, RAW264.7 and HaCaT cells) was also considered. The biological activity pattern of the extracts was represented through PCA analysis, and a correlation matrix was calculated. RESULTS: Spondias mombin L. bark and Anacardium occidentale L. stem and leaves extracts displayed high anti-promatigotes activity, with IC50 ≤ 32 µg/mL against L. guyanensis promastigotes for S. mombin and IC50 of 67 and 47 µg/mL against L. braziliensis and L. guyanensis promastigotes, respectively, for A. occidentale. In addition to the antiparasitic effect, antifungal activity measured against C. albicans and T. rubrum (MIC in the 16-64 µg/mL range) was observed. However, in the case of Leishmania amastigotes, the most active species were Bixa orellana L. (seeds), Chelonantus alatus (Aubl.) Pulle (leaves), Jacaranda copaia (Aubl.) D. Don. (leaves) and Plantago major L. (leaves) with IC50 < 20 µg/mL and infection rates of 14-25% compared to the control. Concerning immunomodulatory activity, P. major and B. orellana were highlighted as the most potent species for the wider range of cytokines in all tested conditions despite overall contrasting results depending on the model. Most of the species led to moderate to low cytotoxic extracts except for C. alatus, which exhibited strong cytotoxic activity in almost all models. None of the tested extracts displayed wound healing properties. CONCLUSIONS: We highlighted pharmacologically active extracts either on the parasite or on associated pathophysiological aspects, thus supporting the hypothesis that antiparasitic activities are not the only biological factor useful for antileishmanial evaluation. This result should however be supplemented by in vivo studies, and attracts once again the attention on the importance of the choice of biological models for an ethnophamacologically consistent study. Moreover, plant cultural importance, ecological status and availability were discussed in relation with biological results, thus contributing to link ethnobotany, medical anthropology and biology.