Differences in responses to the intracellular macrophage environment between Mycobacterium bovis BCG vaccine strains Moreau and Pasteur

BACKGROUND The Bacille Calmette-Guérin (BCG) vaccine comprises a family of strains with variable protective efficacy against pulmonary tuberculosis (TB) and leprosy, partly due to genetic differences between strains. OBJECTIVES Previous data highlighting differences between the genomes and proteomic profiles of BCG strains Moreau and Pasteur led us to evaluate their behaviour in the macrophage microenvironment, capable of stimulating molecular responses that can impact the protective effect of the vaccine. METHODS Strain infectivity, viability, co-localisation with acidified vesicles, macrophage secretion of IL-1 and MCP-1 and lipid droplet biogenesis were evaluated after infection. FINDINGS We found that BCG Moreau is internalised more efficiently, with significantly better intracellular survival up to 96 h p.i., whereas more BCG Pasteur bacilli were found co-localised in acidified vesicles up to 6 h p.i. IL-1β and MCP-1 secretion and lipid droplet biogenesis by infected macrophages were more prominent in response to BCG Pasteur. MAIN CONCLUSION Overall, our results show that, compared to Pasteur, BCG Moreau has increased fitness and better endurance in the harsh intracellular environment, also regulating anti-microbial responses (lower IL-1b and MCP-1). These findings contribute to the understanding of the physiology of BCG Moreau and Pasteur in response to the intraphagosomal environment in a THP-1 macrophage model.

Comparative of Forensic DNA Identification Using Cell Lysis Method and Magnetic Beads Method / 法医学杂志

OBJECTIVES@#To compare the effects of cell lysis method and magnetic beads method in forensic DNA identification and to explore these two methods in forensic DNA identification.@*METHODS@#The genome DNA of THP-1 cells in different quantities was extracted by the cell lysis method and magnetic beads method, and the DNA content was quantified by real-time quantitative PCR. The cell lysis method and magnetic beads method were used to type the STR of human blood with different dilution ratios.@*RESULTS@#When the numbers of THP-1 cell were 100, 400 and 800, the DNA content extracted by cell lysis method were (1.219±0.334), (5.081±0.335), (9.332±0.318) ng, respectively; and the DNA content extracted by magnetic beads method were (1.020±0.281), (3.634±0.482), (7.896±0.759) ng, respectively. When the numbers of THP-1 cells were 400 and 800, the DNA content extracted by the cell lysis method was higher than that by the magnetic beads method. The sensitivity of cell lysis method and magnetic beads method was similar in STR typing of human blood at different dilution ratios. Complete STR typing could be obtained at 100, 300 and 500-fold dilutions of blood samples, but could not be detected at 700-fold dilution. STR typing of undiluted human blood could not be detected by cell lysis method.@*CONCLUSIONS@#The cell lysis method is easy to operate and can retain template DNA to the maximum extend. It is expected to be suitable for trace blood evidence tests.

Effect of IgG dimer concentration on the binding ability of IgG Fc fragment in IVIG to THP-1 cell surface receptors / 中国输血杂志

【Objective】 To investigate the effect of immunoglobulin G (IgG) dimer concentration of intravenous immunoglobulin (IVIG) on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Methods】 Firstly, protein purification and high performance liquid chromatography (HPLC) were used to prepare different concentrations of IgG dimers. After that, IgG dimer was added to IVIG to prepare IVIG containing different concentrations of IgG dimer. Finally, based on the method established in our laboratory, we analyzed the effect of IgG dimer concentration in IVIG on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Results】 When the concentration of IgG dimer in IVIG was 1.11%-10.30%, its binding ability to Fc receptors on the surface of THP-1 cell was 97.67%-135.33%, and this binding ability was positively correlated with the concentration of IgG dimer. When the IgG dimer concentration exceeded 13.22%, the binding ability had no correlation with the IgG dimer concentration. 【Conclusion】 A certain concentration of IgG dimer can promote the binding ability of the IgG Fc fragment in IVIG to receptors on the surface of THP-1 cells, which needs further verification from animal experiments and clinical data.

Effect of Trichomonas vaginalis macrophage migration inhibitory factor on THP-1 macrophages / 中国血吸虫病防治杂志

Objective To investigate the effect of Trichomonas vaginalis macrophage migration inhibitory factor (TvMIF) on THP-1 macrophages.. Methods Recombinant TvMIF protein was prokaryotic expressed and purified, and endotoxin was removed after identification. Following exposure to TvMIF at concentrations of 0, 1, 5, 10, 50 and 100 ng/mL, the cytotoxicity of the recombinant TvMIF protein to THP-1 macrophages was tested using cell counting kit (CCK)-8 assay, and the apoptosis of THP-1 macrophages and reactive oxygen species (ROS) were detected using flow cytometry. The relative expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), caspase-1, interleukin-1β (IL-1β) and IL-18 genes was quantified using real-time fluorescent quantitative PCR (qPCR) assay, and the expression of caspase-1, NLRP3, gasdermin D (GSDMD), gasdermin D N-terminal (GSDMD-NT) and pro-IL-1β proteins were determined using Western blotting assay. Results Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) displayed successful expression and purification of the recombinant TvMIF protein with a molecular weight of 15.5 kDa, and the endotoxin activity assay showed the successful removal of endotoxin in the recombinant TvMIF protein (endotoxin concentration < 0.1 EU/mL), which was feasible for the subsequent studies on protein functions. Flow cytometry revealed that the recombinant TvMIF protein at a concentration of 10 ng/mL and less promoted the apoptosis of THP-1 macrophages, and the highest apoptotic rate of THP-1 macrophages was seen following exposure to the recombinant TvMIF protein at a concentration of 5 ng/mL, while the recombinant TvMIF protein at concentrations of 50 and100 ng/mL inhibited the apoptosis of THP-1 macrophages. Exposure to the recombinant TvMIF protein at a concentration 1 ng/mL resulted in increased ROS levels in THP-1 macrophages. qPCR assay quantified significantly elevated caspase-1, NLRP3, IL-18 and IL-1β expression in THP-1 macrophages 8 hours post-treatment with the recombinant TvMIF protein at a concentration 1 ng/mL, and Western blotting determined increased caspase-1, NLRP3, pro-IL-1β, GSDMD and GSDMD-NT protein expression in THP-1 macrophages following exposure to the recombinant TvMIF protein at a concentration 1 ng/mL. Pretreatment with MCC950 significantly reduced GSDMD and GSDMD-NT protein expression. Conclusions High-concentration recombinant TvMIF protein inhibits macrophage apoptosis, while low-concentration recombinant TvMIF protein activates NLRP3 inflammasome and promotes macrophage pyroptosis.

Proteomic characterization of four subtypes of M2 macrophages derived from human THP-1 cells / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Macrophages are widely distributed immune cells that contribute to tissue homeostasis. Human THP-1 cells have been widely used in various macrophage-associated studies, especially those involving pro-inflammatory M1 and anti-inflammatory M2 phenotypes. However, the molecular characterization of four M2 subtypes (M2a, M2b, M2c, and M2d) derived from THP-1 has not been fully investigated. In this study, we systematically analyzed the protein expression profiles of human THP-1-derived macrophages (M0, M1, M2a, M2b, M2c, and M2d) using quantitative proteomics approaches. The commonly and specially regulated proteins of the four M2 subtypes and their potential biological functions were further investigated. The results showed that M2a and M2b, and M2c and M2d have very similar protein expression profiles. These data could serve as an important resource for studies of macrophages using THP-1 cells, and provide a reference to distinguish different M2 subtypes in macrophage-associated diseases for subsequent clinical research.

Phytochemical composition and biological activities of extracts from ten species of the family Melastomataceae Juss / Composição fitoquímica e atividades biológicas de extratos de dez espécies da família Melastomataceae Juss

Plants possess a renewable source of metabolites with enormous chemical structural diversity, which may have potential therapeutic relevance. Furthermore, this chemical diversity favors the possibility of finding new and different chemical constituents with antimicrobial, antioxidant and anti-tumor activities. This work analyzed preliminary phytochemical profiles and evaluated the antimicrobial, antioxidant and cytotoxic activities of hexane extracts of leaves of ten species of the family Melastomataceae. Phytochemical screening was performed using staining methods while total phenols and flavonoids were quantified by spectrophotometry. Antimicrobial activity was evaluated using the disk diffusion method. Antioxidant activity was determined by the 2,2-diphenyl-1-picrylhydrazil (DPPH) method. Toxicity was recorded using the lethality test with Artemia salina Leach (1819). Cytotoxic activity of the extracts was assessed in vitro with acute monocytic leukemia cells (THP-1). Phytochemical analysis detected the presence of tannins, terpenes, steroids, polyphenols and flavonoids and the absence of alkaloids. Clidemia capitellata (Bonpl.) D. Don had the greatest amount of polyphenols (205.95 mg/g ± 4.14) while Clidemia hirta (L.) D. Don had the highest content of total flavonoids (143.99 mg/g ± 4.18). The hexane extracts did not show antimicrobial activity nor toxicity against Artemia salina. The extract of Tibouchina francavillana Cogn. was the most active in sequestering the DPPH radical. The extracts showed cytotoxicity in THP-1 cells with the appearance of apoptotic bodies and cell death. The extracts of Miconia amoena, Clidemia sericea and Clidemia capitellata are non-toxic against Artemia salina and induce the formation of apoptotic bodies and cell death of the THP-1 lineage.

Os vegetais apresentam uma fonte renovável de metabólitos com enorme diversidade química estrutural, os quais podem apresentar potencial relevante na terapêutica, aumentando as possibilidades de encontrar novos e diferentes constituintes químicos com atividades antimicrobiana, antioxidante e antitumoral. Este trabalho analisou o perfil fitoquímico preliminar e as atividades antimicrobiana, antioxidante, citotóxica dos extratos em hexano das folhas de dez espécies da família Melastomataceae. A triagem fitoquímica foi executada utilizando métodos de coloração e quantificação de fenóis e flavonoides totais por espectrofotometria. A atividade antimicrobiana foi realizada pelo método de difusão em disco. A atividade antioxidante foi determinada pelo método 2,2-difenil1-picrilhidrazila (DPPH). A toxicidade foi registrada utilizando o ensaio de letalidade com Artemia salina Leach (1819). A atividade citotóxica dos extratos foi realizada in vitro com células leucêmicas monocítica aguda (THP-1). A análise fitoquímica detectou a presença de taninos, terpenos, esteroides, polifenóis, flavonoides e ausência de alcaloides. A maior quantificação de polifenóis foi da Clidemia capitellata (Bonpl.) D. Don (205,95 mg/g ± 4,14) e o extrato de Clidemia hirta (L.) D. Don apresentou maior teor de flavonoides totais (143,99 mg/g ± 4,18). Os extratos hexânicos não demostraram atividade antimicrobiana e nem toxicidade frente à Artemia salina. O extrato de Tibouchina francavillana Cogn. foi o mais ativo no sequestro do radical DPPH. Os extratos apresentaram citotoxicidade em células THP-1, com visualização de corpos apoptóticos e morte celular. Os extratos de Miconia amoena, Clidemia sericea e Clidemia capitellata são atóxicos contra Artemia salina e induzem a formação de corpos apoptóticos e morte celular da linhagem THP-1.

Effect of IgG fragments in IVIG(pH4)on phagocytosis of sensitized erythrocytes by macrophage / 中国输血杂志

【Objective】 To research the effect of the Fc, Fab and F(ab′)2 fragments of immunoglobulin G, the main components of Human Immunoglobulin(pH4) for Intravenous Injection(IVIG), on the phagocytic function of macrophages derived from THP-1 cells. 【Methods】 First of all, IVIG was digested with papain and pepsin to obtain Fc, Fab and F(ab′)2, and these components were then identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Afterwards, propylene glycol monomethyl ether acetate (PMA) was used to induce THP-1 cells to differentiate into M0 macrophages. Finally, the sensitized erythrocytes were labeled with carboxy fluorescein succinimidyl ester (CFSE), and the effect of the above components on the phagocytic ability of M0 macrophages to engulf sensitized erythrocytes was detected by flow cytometry. 【Results】 The identification results of SDS-PAGE showed that the prepared IgG fragments met the requirements of subsequent experiments. Flow cytometry performs showed that the phagocytosis model of M0 macrophages had been successfully established. When the concentration of Fc increased from 0.1μg/ mL to 10μg/ mL, the phagocytosis rate of erythrocytes sensitized by M0 macrophages decreased from (24.21±0.58) % to (12.27±0.19) %. When the concentration of IVIG protein increased from 0.1 μg/ml to 10 μg/ml, the phagocytosis rate decreased from (20.57±0.39) % to (0.20±0.03) %. Meanwhile, at the same protein concentration (10 μg/ml), the inhibitory effect of Fc on phagocytosis was only half that of IVIG. In addition, Fab, F(ab′)2, and human serum albumin could not inhibit phagocytosis of M0 macrophages. 【Conclusion】 IVIG can effectively inhibit the phagocytosis of THP-1 derived M0 macrophages, which is mainly dependent on the Fc, but not related to the Fab of IgG and F (ab′)2.

Effect of miltirone on proliferation and apoptosis of leukemia THP-1 cells / 中国中药杂志

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.

Phytochemical composition and biological activities of extracts from ten species of the family Melastomataceae Juss / Composição fitoquímica e atividades biológicas de extratos de dez espécies da família Melastomataceae Juss

Plants possess a renewable source of metabolites with enormous chemical structural diversity, which may have potential therapeutic relevance. Furthermore, this chemical diversity favors the possibility of finding new and different chemical constituents with antimicrobial, antioxidant and anti-tumor activities. This work analyzed preliminary phytochemical profiles and evaluated the antimicrobial, antioxidant and cytotoxic activities of hexane extracts of leaves of ten species of the family Melastomataceae. Phytochemical screening was performed using staining methods while total phenols and flavonoids were quantified by spectrophotometry. Antimicrobial activity was evaluated using the disk diffusion method. Antioxidant activity was determined by the 2,2-diphenyl-1-picrylhydrazil (DPPH) method. Toxicity was recorded using the lethality test with Artemia salina Leach (1819). Cytotoxic activity of the extracts was assessed in vitro with acute monocytic leukemia cells (THP-1). Phytochemical analysis detected the presence of tannins, terpenes, steroids, polyphenols and flavonoids and the absence of alkaloids. Clidemia capitellata (Bonpl.) D. Don had the greatest amount of polyphenols (205.95 mg/g ± 4.14) while Clidemia hirta (L.) D. Don had the highest content of total flavonoids (143.99 mg/g ± 4.18). The hexane extracts did not show antimicrobial activity nor toxicity against Artemia salina. The extract of Tibouchina francavillana Cogn. was the most active in sequestering the DPPH radical. The extracts showed cytotoxicity in THP-1 cells with the appearance of apoptotic bodies and cell death. The extracts of Miconia amoena, Clidemia sericea and Clidemia capitellata are non-toxic against Artemia salina and induce the formation of apoptotic bodies and cell death of the THP-1 lineage.

Os vegetais apresentam uma fonte renovável de metabólitos com enorme diversidade química estrutural, os quais podem apresentar potencial relevante na terapêutica, aumentando as possibilidades de encontrar novos e diferentes constituintes químicos com atividades antimicrobiana, antioxidante e antitumoral. Este trabalho analisou o perfil fitoquímico preliminar e as atividades antimicrobiana, antioxidante, citotóxica dos extratos em hexano das folhas de dez espécies da família Melastomataceae. A triagem fitoquímica foi executada utilizando métodos de coloração e quantificação de fenóis e flavonoides totais por espectrofotometria. A atividade antimicrobiana foi realizada pelo método de difusão em disco. A atividade antioxidante foi determinada pelo método 2,2-difenil1-picrilhidrazila (DPPH). A toxicidade foi registrada utilizando o ensaio de letalidade com Artemia salina Leach (1819). A atividade citotóxica dos extratos foi realizada in vitro com células leucêmicas monocítica aguda (THP-1). A análise fitoquímica detectou a presença de taninos, terpenos, esteroides, polifenóis, flavonoides e ausência de alcaloides. A maior quantificação de polifenóis foi da Clidemia capitellata (Bonpl.) D. Don (205,95 mg/g ± 4,14) e o extrato de Clidemia hirta (L.) D. Don apresentou maior teor de flavonoides totais (143,99 mg/g ± 4,18). Os extratos hexânicos não demostraram atividade antimicrobiana e nem toxicidade frente à Artemia salina. O extrato de Tibouchina francavillana Cogn. foi o mais ativo no sequestro do radical DPPH. Os extratos apresentaram citotoxicidade em células THP-1, com visualização de corpos apoptóticos e morte celular. Os extratos de Miconia amoena, Clidemia sericea e Clidemia capitellata são atóxicos contra Artemia salina e induzem a formação de corpos apoptóticos e morte celular da linhagem THP-1.

Phytochemical composition and biological activities of extracts from ten species of the family Melastomataceae Juss

Abstract Plants possess a renewable source of metabolites with enormous chemical structural diversity, which may have potential therapeutic relevance. Furthermore, this chemical diversity favors the possibility of finding new and different chemical constituents with antimicrobial, antioxidant and anti-tumor activities. This work analyzed preliminary phytochemical profiles and evaluated the antimicrobial, antioxidant and cytotoxic activities of hexane extracts of leaves of ten species of the family Melastomataceae. Phytochemical screening was performed using staining methods while total phenols and flavonoids were quantified by spectrophotometry. Antimicrobial activity was evaluated using the disk diffusion method. Antioxidant activity was determined by the 2,2-diphenyl-1-picrylhydrazil (DPPH) method. Toxicity was recorded using the lethality test with Artemia salina Leach (1819). Cytotoxic activity of the extracts was assessed in vitro with acute monocytic leukemia cells (THP-1). Phytochemical analysis detected the presence of tannins, terpenes, steroids, polyphenols and flavonoids and the absence of alkaloids. Clidemia capitellata (Bonpl.) D. Don had the greatest amount of polyphenols (205.95 mg/g ± 4.14) while Clidemia hirta (L.) D. Don had the highest content of total flavonoids (143.99 mg/g ± 4.18). The hexane extracts did not show antimicrobial activity nor toxicity against Artemia salina. The extract of Tibouchina francavillana Cogn. was the most active in sequestering the DPPH radical. The extracts showed cytotoxicity in THP-1 cells with the appearance of apoptotic bodies and cell death. The extracts of Miconia amoena, Clidemia sericea and Clidemia capitellata are non-toxic against Artemia salina and induce the formation of apoptotic bodies and cell death of the THP-1 lineage.

Resumo Os vegetais apresentam uma fonte renovável de metabólitos com enorme diversidade química estrutural, os quais podem apresentar potencial relevante na terapêutica, aumentando as possibilidades de encontrar novos e diferentes constituintes químicos com atividades antimicrobiana, antioxidante e antitumoral. Este trabalho analisou o perfil fitoquímico preliminar e as atividades antimicrobiana, antioxidante, citotóxica dos extratos em hexano das folhas de dez espécies da família Melastomataceae. A triagem fitoquímica foi executada utilizando métodos de coloração e quantificação de fenóis e flavonoides totais por espectrofotometria. A atividade antimicrobiana foi realizada pelo método de difusão em disco. A atividade antioxidante foi determinada pelo método 2,2-difenil-1-picrilhidrazila (DPPH). A toxicidade foi registrada utilizando o ensaio de letalidade com Artemia salina Leach (1819). A atividade citotóxica dos extratos foi realizada in vitro com células leucêmicas monocítica aguda (THP-1). A análise fitoquímica detectou a presença de taninos, terpenos, esteroides, polifenóis, flavonoides e ausência de alcaloides. A maior quantificação de polifenóis foi da Clidemia capitellata (Bonpl.) D. Don (205,95 mg/g ± 4,14) e o extrato de Clidemia hirta (L.) D. Don apresentou maior teor de flavonoides totais (143,99 mg/g ± 4,18). Os extratos hexânicos não demostraram atividade antimicrobiana e nem toxicidade frente à Artemia salina. O extrato de Tibouchina francavillana Cogn. foi o mais ativo no sequestro do radical DPPH. Os extratos apresentaram citotoxicidade em células THP-1, com visualização de corpos apoptóticos e morte celular. Os extratos de Miconia amoena, Clidemia sericea e Clidemia capitellata são atóxicos contra Artemia salina e induzem a formação de corpos apoptóticos e morte celular da linhagem THP-1.

Effect of polydatin on the proliferation and apoptosis of THP-1 cells and the mechanism / 中国当代儿科杂志

OBJECTIVES@#To explore the effect of polydatin on the proliferation and apoptosis of acute monocytic leukemia cell line THP-1 and the possible mechanism.@*METHODS@#After THP-1 cells were treated with polydatin at gradient concentrations for 24 hours and 48 hours, their proliferation was determined by CCK-8 assay, and half maximal inhibitory concentration (IC50) was calculated. Logarithmically growing THP-1 cells were divided into two groups, a polydatin treatment group (treated with IC50 of polydatin) and a blank control group (treated without polydatin solution), and incubated for 48 hours. Cell apoptosis and cell cycle were measured by flow cytometry. The expression levels of PI3K, AKT, p-AKT, mTOR, p-mTOR, p70 S6K, and p-p70 S6K proteins were measured by Western blotting.@*RESULTS@#After treatment with polydatin, the proliferation of THP-1 cells was strongly inhibited, and the IC50 at 48 hours was 1 800 μmol/L. After treatment with 1 800 μmol/L polydatin solution for 48 hours, the apoptosis rate of THP-1 cells increased significantly compared with the blank control group (P<0.05). The cell cycle was arrested in the G0/G1 and S phases, with a significantly increased proportion of cells in the G0/G1 phase and a significantly decreased proportion of cells in the S phase, as compared with the blank control group (P<0.05). The expression levels of PI3K, AKT, p-AKT, mTOR, p-mTOR, p70 S6K, and p-p70 S6K proteins decreased significantly compared with the blank control group (P<0.05).@*CONCLUSIONS@#Polydatin can effectively inhibit the proliferation, block the cell cycle, and induce the apoptosis of THP-1 cells, which may be related to inhibition of the PI3K/AKT/mTOR signaling pathway.

Limonium duriusculum (de Girard) Kuntze Exhibits Anti-inflammatory Effect Via NF-κ B Pathway Modulation

HIGHLIGHTS L. duriusculum n-BuOH extract reduces inflammatory responses both in vitro and in vivo. L. duriusculum n-BuOH extract inhibits NF-κB-dependent transcriptional responses. L. duriusculum n-BuOH extract decreases the expression of TNF-α and IL-6 genes.

Abstract Limonium duriusculum is used in folk medicine to treat inflammatory disorders and has gained attention due to its richness in apigenin. The present investigation was performed to evaluate and confirm its anti-inflammatory properties, in cell lines and animal models. The potential anti-inflammatory properties of n-butanol (n-BuOH) extract of L. duriusculum (BEL) and isolated apigenins were examined on NF-κB transcriptional activity in TNFα- or LPS-stimulated cells, and on in vivo acute inflammatory models (carrageenan induced paw edema and peritonitis). BEL treatment was able to inhibit the activity of an NF-κB reporter gene in HCT116 cells both in the absence and in the presence of exogenous TNFα, used as NF-κB pathway inducer. This anti-inflammatory effect was even more potent compared to Apigenin (APG1) and was confirmed using monocyte-derived THP-1 cells treated with LPS to stimulate NF-κB-dependent transcription of IL-6 and TNFα mRNAs. Apigenin7-O-β-(6''-methylglucuronide) (APG2) was instead inactive both in HCT116 and THP-1 cells. BEL (oral, 200 mg/kg) led to paw swelling inhibition, vascular permeability and peritoneal leukocyte and PN migration diminution. Apigenins (intraperitoneal, APG1, APG2: 20 mg/kg) also evoked a significant anti-edema effect, early vascular permeability and leukocyte influx reduction. Collectively, this study demonstrates for the first time the effectiveness of L. duriusculum to inhibit NF-κB-dependent transcriptional responses in HCT116 and THP-1 cells. In vivo studies also established that L. duriusculum possesses a potential anti-inflammatory effect, confirm its traditional, empirical use, that could be attributed to its richness in apigenin.

Enterovirus 71 can induce autophagy and apoptosis of THP-1 macrophages / 南方医科大学学报

OBJECTIVE@#To investigate enterovirus 71 (EV71)-induced of autophagy, apoptosis and the related signaling pathways in THP-1 macrophages.@*METHODS@#THP-1 macrophages were infected with EV71 at the multiplicity of infection (MOI) of 0.1 for 2, 8 or 16 h, and the cell proliferation and toxicity were analyzed using CCK-8 kit. The intracellular viral nucleic acid in THP-1 macrophages were detected by fluorescence quantitative PCR, and the ultrastructural changes of the cells were observed using transmission electron microscopy. Cell apoptosis induced by EV71 infection was detected using Hoechst 33342 staining and AnnexinV/PI double staining. Western blotting was performed for analysis of changes in autophagy and apoptosis of the cells and in the expressions of the related proteins. The effect of EV71 infection on apoptosis of THP-1 macrophages incubated with 3-MA and Ac-DEVD-CHO inhibitor for 2 h was assessed using Western blotting.@*RESULTS@#EV71 infection significantly lowered the cell survival rate of THP-1 macrophages at 2, 8 h and 16 h after the infection ( < 0.05). The total copy number of viral nucleic acid in THP-1 macrophages incubated with EV71 increased significantly and progressively over time ( < 0.01). Intracellular autophagosomes and virions could be seen in EV71-infected THP-1 macrophages. The total apoptotic rate of the infected cell also increased significantly over time ( < 0.01). EV71 infection significantly increased LC3 conversion (LC3-Ⅱ/ LC3-I) and the expression of cleaved caspase 3 protein and decreased the protein expressions of p62, Bcl-2 and caspase-3 ( < 0.01) without causing obvious changes in cleaved caspase-8 (>0.05). 3-MA significantly inhibited the EV71-induced autophagy of THP-1 macrophages and reduced LC3 conversion (LC3-Ⅱ/LC3-I) and p62 protein expression at 8 h after EV71 infection ( < 0.01). Compared with DMSO, Ac-DEVD-CHO significantly inhibited EV71-induced apoptosis of THP-1 macrophages (15.5% 7.7%, < 0.01).@*CONCLUSIONS@#EV71 not only can infect and replicate in THP-1 macrophages, but also induces autophagy and cell apoptosis possibly by activating LC3/p62 autophagy pathway and caspase apoptosis pathway.

Essential Oil from Alpinia zerumbet Rhizome Inhibits Macrophage-derived Foam Cell Formation Via Modulating Expression of CD36 and ABCA1 / 中国实验方剂学杂志

Objective::To clarify the inhibitory effect of essential oil from Alpinia zerumbet rhizome (EOFAZ) on oxidized low-density lipoprotein (ox-LDL)-induced transformation of macrophage into foam cell and explore its possible mechanism. Method::THP-1 monocyte was incubated with 100 μg·L-1 phorbol myristate acetate (PMA) to grow into macrophage, experiment was divided into 4 groups as follows, control group, model group (80 mg·L-1 ox-LDL), EOFAZ at low dose (80 mg·L-1 ox-LDL+ 4 μg·L-1 EOFAZ)and EOFAZ at high dose (80 g·L-1 ox-LDL+ 20 μg·L-1 EOFAZ). Mathye thiazolye telrazliurn (MTT) method was employed to examine the influence of EOFAZ on macrophage viability. Western blot was used to analyze the expression level of cluster of differentiation 36(CD36) and ATP-binding cassette transporter A1(ABCA1) protein in macrophage. Enzyme-linked immunosorbent assay (ELISA) was used to detect cholesteryl ester contents in macrophage. Oil red O staining was applied to determine the accumulation of lipids in macrophage. Result::EOFAZ showed non-toxic effect on macrophage. Compared to control group, macrophage in model group displayed higher level of cholesteryl ester and lipid droplet(P<0.01), as well as significant increasing of CD36 expression (P<0.01), but no effect on ABCA1 expression. EOFAZ notably reduced the contents of lipids and cholesteryl ester(P<0.01), down-regulated expression of CD36 and up-regulated expression of ABCA1 in macrophage in comparison with the model group(P<0.01), indicating that EOFAZ inhibited transformation of macrophage into foam cell. Conclusion::EOFAZ could inhibit ox-LDL-induced transformation of macrophage into foam cell, the underlying mechanism may involves its ability to increase CD36 expression and decrease ABCA1 expression in macrophage.

Effect of Arsenic Trioxide Combined with Dihydroartemisinin on THP-1 Cells Proliferation and Apoptosis / 中国实验方剂学杂志

Objective:To investigate the effects of arsenic trioxide combined with dihydroartemisinin on proliferation, cell cycle, and apoptosis of THP-1 cells, and explore the mechanism. Method:The thiazolyl blue （MTT） method was applied to detect the effect of different concentrations of arsenic trioxide, dihydroartemisinin and arsenic trioxide combined with dihydroartemisinin on the proliferation of THP-1 cells. Annexin V/propidium iodide（PI）assay was used to detect the change of THP-1 cell cycle and apoptosis．Western blot was performed to assess the expression of cysteine protease-3（Caspase-3）, cleaved Caspase-3, B-lymphocytoma-2（Bcl-2） and Bcl-2 associated X protein （Bax）. The changes of cell morphology were observed under high intension microscope. Result:Compared with blank group, arsenic trioxide and dihydroartemisinin both exhibited obvious antiproliferative effect on the human acute monocytic leukemia cell line THP-1 in time-dose dependence （P<0.01）. After 48 h, compared with the same dose of arsenic trioxide or that of dihydroartemisinin alone, the inhibition effect of 1 µmol·L-1 arsenic trioxide combined with 2 µmol·L-1 dihydroartemisinin on proliferation of THP-1 cells was significantly stronger （P<0.01）. Compared with the control group, arsenic trioxide combined with dihydroartemisinin significantly arrested the cells in G1 phase （P<0.01）, induced the downregulation of Caspase-3 and Bcl-2 （P<0.01） and upregulation of cleaved Caspase-3 significantly（P<0.05）. Conclusion:Arsenic trioxide combined with dihydroartemisinin can significantly inhibit the proliferation and induce apoptosis of THP-1 cells. The possible mechanism may be related to arrest the cells in G1 phase, reduce the expression of Caspase-3 and Bcl-2, increase the expression of cleaved Caspase-3.

Polarization of human acute monocytic leukemia THP-1 cells-derived macprophages induced by Nippostrongylus brasiliensis proteins in vitro / 中国血吸虫病防治杂志

Objective To investigate the polarization of human acute monocytic leukemia THP-1 cells-derived macrophages induced by Nippostrongylus brasiliensis proteins in vitro, so as to provide insights into the elucidation of the mechanisms underlying host immune responses to hookworm infections. Methods The in-vitro culture of N. brasiliensis was established and maintained in the laboratory, and the third- (L3) and fifth-stage larvae (L5) were collected under a sterile condition for preparation of L3 and L5 proteins. The in-vitro culture of THP-1 cells was established, stimulated with 500 ng/mL PMA to yield M0 macrophages that were adherent to the plate wall. The LPS + IFN-γ group, IL-4 + IL-13 group, L3 protein group and L5 protein group were given stimulation with 500 ng/mL LPS plus 100 ng/mL IFN-γ, IL-4 and IL-13 (both 100 ng/mL), L3 protein (5 mg/mL) and L5 protein (5 mg/mL), respectively, while the negative control group was given no stimulation. The cell morphology was observed using microscopy, the mRNA expression of M1/M2 macrophages-specific genes was quantified using a quantitative real-time PCR (qPCR) assay, and the surface markers of M1/M2 macrophages were detected using flow cytometry, while the levels of cytokines secreted by M1/M2 macrophages were measured using enzyme-linked immunosorbent assay (ELISA) following stimulations, so as to examine the polarization of THP-1-derived macrophages induced by N. brasiliensis proteins in vitro. Results Following stimulation with PMA, THP-1 cells appeared wall-adherent M0 macrophages, and polarized to typical M1 macrophages following stimulation with LPS + IFN-γ, and typical M2 macrophages following stimulation with IL-4 + IL-13, IL-3 protein or L5 protein. There was a significant difference in the proportion of M1 macrophages among the negative control group, the LPS + IFN-γ group, the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (χ2 = 3 721.00, P < 0.001), with the highest proportion detected in the LPS + IFN-γ group, and there was also a significant difference in the proportion of M2 macrophages among groups (χ2 = 105.43, P < 0.001). There were significant differences among groups in terms of the mRNA expression of CCL2 (F = 191.95, P < 0.001), TNF-α (F = 129.95, P < 0.001), IL-12b (F = 82.89, P < 0.001), PPARγ (F = 11.30, P < 0.001), IL-10 (F = 9.51, P < 0.001) and Mrc1 genes (F = 12.35, P < 0.001). In addition, there were significant differences in the proportion of positive CD86 and CD206 expression among groups (χ2 = 24 004.33 and 832.50, P < 0.001). Higher IL-1β and TNF-α levels were measured in the LPS + IFN-γ group than in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (P < 0.001), and greater TGF-β1 and IL-10 levels were seen in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group than in the negative control group and the LPS + IFN-γ group (P < 0.05). Conclusions Both L3 and L5 proteins of N. brasiliensis may induce the polarization of THP-1-derived macrophages to M2 type in vitro.

Acetoacetate extract of pleione bulbocodioides (franch. ) rolfe induces apoptosis of human leukemia thp-1cells through a mitochondria-regulated intrinsic apoptotic pathway / Extrato de acetoacetato de Pleione bulbocodioides (Franch. ) Rolfe induz a apoptose de células THP-1 de leucemia humana através de uma via apoptótica intrínseca regulada por mitocôndrias

The tubers of three orchidaceous plants, includingPleione bulbocodioides (Franch.) Rolfe, have been used as 'Shan-Ci-Gu' in traditional Chinese medicine for the treatment of bacterial infections and cancers for thousands of years. In this study, the effects of an acetoacetate (EtOAc) extract of P. bulbocodioides on the cell viability and apoptosis of THP-1 (human acute monocytic leukemia cell line) cells and its interaction with possible apoptotic pathways were investigated. THP-1 cells were treated with the EtOAc extract of P. bulbocodioides at different concentrations. The results showed that THP-1 cell viability was significantly inhibited by the EtOAc extract ofP. bulbocodioides with an IC50 of 51.37±2.68 µ g/ mL at 24 h. The examination of cytotoxic effects on healthy cells showed that the EtOAc extract of P. bulbocodioidesdid not show any effect on healthy Vero cells. Selectivity indexes were greater than 15.57, suggesting that the EtOAc extract of P. bulbocodioides had selective toxicity against THP-1 cells. The results of annexin V-FITC/PI and DAPI staining showed that the EtOAc extract of P. bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased in the treatment groups compared with that in the control group (P<0.05). The distribution of cells in the G2 phase of the cell cycle increased along with typical cell apoptosis-induced morphological changes. The levels of the pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 increased with increasing concentration of acetoacetate extract of P. bulbocodioides, while the anti-apoptosis protein Bcl-2 was downregulated. Cyt c and AIF, which are characteristic proteins of the mitochondria-regulated intrinsic apoptosis pathway, also increased in the cytosol with increasing concentrations of the EtOAc extract of P. bulbocodioides. These results showed that the EtOAc extract of P. bulbocodioidessignificantly inhibits cell viability and induces cell apoptosis in the human leukemia cell line THP-1 through a mitochondria-regulated intrinsic apoptotic pathway

Os tubérculos de três plantas orquidáceas, incluindo Pleione bulbocodioides (Franch.) Rolfe, têm sido usados como "Shan-Ci-Gu" na medicina tradicional chinesa para o tratamento de infecções bacterianas e cânceres por milhares de anos. Neste estudo, os efeitos de um extrato de acetoacetato (EtOAc) de P. bulbocodioides na viabilidade celular e apoptose de células THP-1 (linhagem celular de leucemia monocítica aguda humana) e sua interação com possíveis vias apoptóticas foram investigados. As células THP-1 foram tratadas com o extrato EtOAc de P. bulbocodioides em diferentes concentrações. Os resultados mostraram que a viabilidade das células THP-1 foi significativamente inibida pelo extrato EtOAc de P. bulbocodioides com IC50 de 51,37 ± 2,68 µ g/mL às 24 h. O exame dos efeitos citotóxicos em células saudáveis mostrou que oextrato de EtOAc de P. bulbocodioides não mostrou nenhum efeito sobre células Vero saudáveis. Os índices de seletividade foram maiores que 15,57, sugerindo que o extrato de EtOAc de P. bulbocodioides teve toxicidade seletiva contra as células THP-1. Os resultados da coloração da anexina V-FITC/PI e DAPI mostraram que o extrato de EtOAc de P. bulbocodioides induziu a apoptose celular de maneira dose-dependente. A taxa de apoptose foi aumentada nos grupos de tratamento em comparação com o grupo controle (P <0,05). A distribuição de células na fase G2 do ciclo celular aumentou juntamente com alterações morfológicas típicas induzidas pela apoptose celular. Os níveis das proteínas pró-apoptóticas Bax, PARP clivada e caspase-3 clivada aumentaram com o aumento da concentração do extrato acetoacetato de P. bulbocodioides, enquanto a proteína anti-apoptose Bcl-2 foi regulada negativamente. Cyt c e AIF, que são proteínas características da via de apoptose intrínseca regulada por mitocôndrias, também aumentaram no citosol com concentrações crescentes do extrato de EtOAc de P. bulbocodioides. Estes resultados mostraram que o extrato de EtOAc de P. bulbocodioides inibe significativamente a viabilidade celular e induz a apoptose na linha celular de leucemia humana THP-1 através de uma via apoptótica intrínseca regulada por mitocôndrias.

Bioactive A-ring rearranged limonoids from the root barks of

Screening active natural products, rapid identification, and accurate isolation are of great important for modern natural lead compounds discovery. We hereby reported the isolation of seven new neotecleanin-type limonoids (-), seven new limonoids with 5-oxatricyclo[5.4.0.11., 4.]hendecane ring system (-), and two new precursors (-) together with four known limonoids (-) from the root barks of . Their structures, including their absolute configurations, were elucidated based on analyses of HR-ESI-MS, 1D/2D NMR, ECD spectrum calculations and single-crystal X-ray diffraction techniques. Compounds , , , , , , showed significant anti-inflammatory activities in LPS-induced RAW 264.7 cell line, BV2 microglial cells, and -stimulated THP-1 human monocytic cells. Walrobsin M () exhibited anti-inflammatory activity with IC value of 7.96±0.36 μmol/L, and down-regulated phosphorylation levels of ERK and p38 in a dose-dependent manner.

Establishment and application of STING agonist in-vitro screening model / 药学学报

Tumor immunotherapy is a critical field in the development of anticancer drugs. The research of stimulator of interferon genes (STING) agonist provides a new idea for immunotherapy. Innate immune response can effectively be induced by nucleic acids in mammalian cytoplasm. In recent years, a large number of studies have confirmed that the cGAS-cGAMP-STING signaling pathway plays a key role in cytoplasmic DNA recognition and immune defense activation. The dysfunction of cGAS-cGAMP-STING is closely related to the tumorigenesis, and is a potent target for drug development. In this study, based on THP-1 dual cells which are stably expressing cGAS-STING pathway and THP-1 KO-STING cells which are stably depleted STING, a screening method for STING agonists was established by detection of luciferase. Furthermore, the accuracy and sensitivity of the model were verified using positive compound, providing a simple, efficient and accurate screening platform for high-throughput screening of STING agonists.

Study on expression, role and mechanism of leukocyte adhesion molecule CD44 in pulmonary tuberculosis patients / 中国免疫学杂志

Objective: To study the expression of CD44 in pulmonary tuberculosis patients and explore the possible mechanisms. Methods: 236 patients suspected with pulmonary tuberculosis were selected and divided into pulmonary tuberculosis group ( n= 152) and non-pulmonary tuberculosis group ( n= 84), and 100 healthy people were randomly selected as healthy control group. The expression of CD44 was evaluated by qRT-PCR and ELISA in peripheral blood and pleural effusion from different patients. The CD44 levels at pre and post-treatment time points were determined by ELISA. The inducing factors of increased CD44 and the potentialroles played by CD44 in the pathogenesis of TB were also evaluated. We elucidated whether CD44 detection could combine with T-spot. TB to diagnose pulmonary tuberculosis rapidly and accurately. Results: The expression of CD44 in pulmonary tuberculosis patients was higher compared with the non-pulmonary tuberculosis patients and healthy people, and would be down-regulated after treatment for 3, 6 and 9 months. Besides, CD44 could not remove H37 Ra by the CFU assay, and could promote the expression of CCL-2, indicating that CCD4 promote the mobility the THP-1 via induction of CCL-2. Besides, TNF-α neutralizing antibody, added into the macrophages, could inhibit the expression of CD44, and functional TNF-α induced the expression of TNF-α. Conclusion: CD44 is highly expressed in pulmonary tuberculosis patients, which may be due to the high expression of TNF-α in pulmonary tuberculosis patients, stimulating macrophages to produce CD44, and it will provide a basis for clinical diagnosis of pulmonary tuberculosis.