OBJECTIVE: To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM). METHODS: Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA. RESULTS: Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells. CONCLUSIONS CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.
Animals ; Humans ; Extracellular Traps ; Neutrophils ; Interleukin-8/metabolism* ; Dermatophagoides farinae ; Interleukin-6/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Epithelial Cells ; Interferon-gamma/metabolism* ; Tetradecanoylphorbol Acetate/pharmacology*

Tigliane type macrocyclic diterpenoids with special structures and diverse bioactivities are mainly extracted from plants of Euphorbiaceae and Thymelaeaceae. According to the different functional groups, they can be classified into types of phorbol esters, C-4 deoxyphorbol esters, C-12 deoxyphorbol esters, C-16 or C-17 substituted phorbol esters and others. Most of them present promising antiviral activities and cytotoxic activities and are expected to be developed as candidates for anti-AIDS, anti-tuberculosis, and anti-tumor clinical trials, demonstrating great potential for the application in healthcare. This paper reviews 115 novel tigliane-type diterpenoids discovered since 2013 and summarize their chemical structures and bioactivities, aiming to lay a foundation for further development and utilization of these compounds and provide new ideas for the development of clinical drugs.
Phorbols ; Molecular Structure ; Diterpenes/chemistry* ; Antiviral Agents ; Phorbol Esters

Extracellular traps released by neutrophils (neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations (0, 0.1, 1, and 10 μmol/L) of all-trans retinoic acid (ATRA) to differentiate HL-60 cells for different days (1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells (dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 μmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together, these observations suggest that dHL-60 cells differentiated with 1 μmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.
Humans ; Extracellular Traps ; Tetradecanoylphorbol Acetate/pharmacology* ; Neutrophils ; HL-60 Cells ; Tretinoin/pharmacology*

Thapsigargin (TGN) is a potent and selective inhibitor of sarco-endoplasmic Ca²⁺-ATPase, leading to rapid elevation of cytoplasmic Ca2+ concentration. Previous reports have shown that TGN increases the production of various cytokines from macrophages and dendritic cells. Here, we examine the effects of TGN on murine T cells. Nanomolar concentrations of TGN are a significant inducer of IL-2 production with full activity at 50 nM. Micromolar concentrations of TGN, however, are inhibitory to IL-2 production and T cell proliferation. The IL-2 production-inducing activity of TGN is much more prominent when T cells are primed with concanavalin A or anti-CD3 mAb, and is due to the increase of cytoplasmic Ca²⁺ concentration. TGN at 50 nM does not affect interferon-gamma or IL-4 production from T cells. Thus, the present study shows that low nanomolar concentrations of TGN could be useful in potentiating IL-2 production from antigen-primed T cells.
Cell Proliferation ; Concanavalin A ; Cytokines ; Cytoplasm ; Dendritic Cells ; Interferon-gamma ; Interleukin-2* ; Interleukin-4 ; Macrophages ; T-Lymphocytes* ; Tetradecanoylphorbol Acetate ; Thapsigargin*

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Down-Regulation ; drug effects ; Female ; Guanine Nucleotide Exchange Factors ; antagonists & inhibitors ; genetics ; metabolism ; Humans ; Indoles ; pharmacology ; MCF-7 Cells ; Maleimides ; pharmacology ; Protein Isoforms ; genetics ; metabolism ; Protein Kinase C ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Receptor, ErbB-2 ; genetics ; metabolism ; Tetradecanoylphorbol Acetate ; toxicity

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.
Animals ; Antigens, Helminth ; isolation & purification ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Culture Media, Conditioned ; chemistry ; pharmacology ; Gene Expression Regulation ; Hedgehog Proteins ; agonists ; antagonists & inhibitors ; genetics ; immunology ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; parasitology ; prevention & control ; Macrophage Activation ; drug effects ; Macrophages ; cytology ; drug effects ; immunology ; Models, Biological ; Monocytes ; cytology ; drug effects ; metabolism ; Pentoxifylline ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; RNA, Messenger ; genetics ; immunology ; Schistosoma japonicum ; chemistry ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology ; Zinc Finger Protein GLI1 ; genetics ; immunology ; Zygote ; chemistry

BACKGROUND: The goal of this in vitro study was to investigate the effect of lipid emulsion on vasodilation caused by toxic doses of bupivacaine and mepivacaine during contraction induced by a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), in an isolated endothelium-denuded rat aorta. METHODS: The effects of lipid emulsion on the dose-response curves induced by bupivacaine or mepivacaine in an isolated aorta precontracted with PDBu were assessed. In addition, the effects of bupivacaine on the increased intracellular calcium concentration ([Ca²⁺]ᵢ) and contraction induced by PDBu were investigated using fura-2 loaded aortic strips. Further, the effects of bupivacaine, the PKC inhibitor GF109203X and lipid emulsion, alone or in combination, on PDBu-induced PKC and phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) was examined by western blotting. RESULTS: Lipid emulsion attenuated the vasodilation induced by bupivacaine, whereas it had no effect on that induced by mepivacaine. Lipid emulsion had no effect on PDBu-induced contraction. The magnitude of bupivacaine-induced vasodilation was higher than that of the bupivacaine-induced decrease in [Ca²⁺]ᵢ. PDBu promoted PKC and CPI-17 phosphorylation in aortic VSMCs. Bupivacaine and GF109203X attenuated PDBu-induced PKC and CPI-17 phosphorylation, whereas lipid emulsion attenuated bupivacaine-mediated inhibition of PDBu-induced PKC and CPI-17 phosphorylation. CONCLUSIONS: These results suggest that lipid emulsion attenuates the vasodilation induced by a toxic dose of bupivacaine via inhibition of bupivacaine-induced PKC and CPI-17 dephosphorylation. This lipid emulsion-mediated inhibition of vasodilation may be partly associated with the lipid solubility of local anesthetics.
Anesthetics, Local ; Animals ; Aorta ; Blotting, Western ; Bupivacaine* ; Calcium ; Fura-2 ; In Vitro Techniques ; Mepivacaine* ; Muscle, Smooth, Vascular ; Myosin-Light-Chain Phosphatase ; Phorbol 12,13-Dibutyrate ; Phosphorylation ; Protein Kinase C ; Rats ; Solubility ; Vasodilation*

BACKGROUND: The goal of this in vitro study was to investigate the effect of lipid emulsion on vasodilation caused by toxic doses of bupivacaine and mepivacaine during contraction induced by a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), in an isolated endothelium-denuded rat aorta. METHODS: The effects of lipid emulsion on the dose-response curves induced by bupivacaine or mepivacaine in an isolated aorta precontracted with PDBu were assessed. In addition, the effects of bupivacaine on the increased intracellular calcium concentration ([Ca²⁺]ᵢ) and contraction induced by PDBu were investigated using fura-2 loaded aortic strips. Further, the effects of bupivacaine, the PKC inhibitor GF109203X and lipid emulsion, alone or in combination, on PDBu-induced PKC and phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) was examined by western blotting. RESULTS: Lipid emulsion attenuated the vasodilation induced by bupivacaine, whereas it had no effect on that induced by mepivacaine. Lipid emulsion had no effect on PDBu-induced contraction. The magnitude of bupivacaine-induced vasodilation was higher than that of the bupivacaine-induced decrease in [Ca²⁺]ᵢ. PDBu promoted PKC and CPI-17 phosphorylation in aortic VSMCs. Bupivacaine and GF109203X attenuated PDBu-induced PKC and CPI-17 phosphorylation, whereas lipid emulsion attenuated bupivacaine-mediated inhibition of PDBu-induced PKC and CPI-17 phosphorylation. CONCLUSIONS: These results suggest that lipid emulsion attenuates the vasodilation induced by a toxic dose of bupivacaine via inhibition of bupivacaine-induced PKC and CPI-17 dephosphorylation. This lipid emulsion-mediated inhibition of vasodilation may be partly associated with the lipid solubility of local anesthetics.
Anesthetics, Local ; Animals ; Aorta ; Blotting, Western ; Bupivacaine* ; Calcium ; Fura-2 ; In Vitro Techniques ; Mepivacaine* ; Muscle, Smooth, Vascular ; Myosin-Light-Chain Phosphatase ; Phorbol 12,13-Dibutyrate ; Phosphorylation ; Protein Kinase C ; Rats ; Solubility ; Vasodilation*

The present study was designed to target fish for potential bioactive components contained in a Huang Lian Jie Du decoction (HLJDD) and identify the underlying mechanisms of action for the treatment of sepsis at the molecular level. he bioactive components database of HLJDD was constructed and the sepsis-associated targets were comprehensively investigated. The 3D structures of the PAFR and TXA2R proteins were established using the homology modelling (HM) method, and the molecular effects for sepsis treatment were analysed by comparing the bioactive components database and the sepsis targets using computational biology methods. The results of the screening were validated with biological testing against the human oral epidermal carcinoma cell line KB in vitro. We found that multiple bioactive compounds contained in the HLJDD interacted with multiple targets. We also predicted the promising compound leads for sepsis treatment, and the first 28 compounds were characterized. Several compounds, such as berberine, berberrubine and epiberberine, dose-dependently inhibited PGE2 production in human KB cells, and the effects were similar in the presence or absence of TPA. This study demonstrates a novel approach to identifying natural chemical compounds as new leads for the treatment of sepsis.
Anti-Inflammatory Agents, Non-Steroidal ; pharmacokinetics ; Berberine ; analogs & derivatives ; pharmacokinetics ; Dinoprostone ; biosynthesis ; Drugs, Chinese Herbal ; chemistry ; pharmacokinetics ; Humans ; KB Cells ; Platelet Membrane Glycoproteins ; drug effects ; Protein Transport ; Receptors, G-Protein-Coupled ; drug effects ; Receptors, Thromboxane A2, Prostaglandin H2 ; drug effects ; Sepsis ; drug therapy ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacokinetics