OBJECTIVE: To explore the mechanism by which estradiol modulates the immunophenotype of macrophages through the endoplasmic reticulum stress pathway. METHODS: Peritoneal macrophages isolated from C57 mice were cultured in the presence of 60 ng/mL interferon-γ (IFN-γ) followed by treatment with estradiol (1.0 nmol/L) alone, estradiol with estrogen receptor antagonist (Acolbifene, 4 nmol/L), estradiol with IRE1α inhibitor (4 μ 8 C), or estradiol with IRE1α agonist. After the treatments, the expression levels of MHC-Ⅱ, iNOS and endoplasmic reticulum stress marker proteins IRE1α, eIF2α and ATF6 in the macrophages were detected with Western blotting, and the mRNA levels of TGF-β, IL-6, IL-10 and TNF-α were detected with RT-PCR. RESULTS: Estrogen treatment of the macrophages significantly decreased the expressions of M1-related proteins MHC-Ⅱ (P=0.021) and iNOS (P < 0.001) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.004), increased the mRNA expression of TGF-β (P=0.002) and IL-10 (P=0.008), and up-regulated the protein expressions of IRE1α (P < 0.001) and its downstream transcription factor XBP-1 (P < 0.001). Addition of the estrogen inhibitor obviously blocked the effect of estrogen. Compared with estrogen treatment alone, combined treatment of the macrophages with estrogen and the IRE1α inhibitor 4 μ 8 C significantly up-regulated the protein expressions of MHC-Ⅱ (P=0.002) and iNOS (P=0.003) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.024), and obviously down-regulated the mRNA expression of TGF-β (P < 0.001) and IL-10 (P < 0.001); these changes were not observed in cells treated with estrogen and the IRE1α agonist. CONCLUSION Estrogen can inhibit the differentiation of murine macrophages into a pro-inflammatory phenotype by up-regulating the IRE1α-XBP-1 signaling axis, thereby producing an inhibitory effect on inflammatory response.
Animals ; Cell Differentiation/drug effects* ; Endoribonucleases/metabolism* ; Estradiol/pharmacology* ; Estrogens/metabolism* ; Interleukin-10 ; Interleukin-6/metabolism* ; Macrophages, Peritoneal/metabolism* ; Mice ; Phenotype ; Protein Serine-Threonine Kinases/metabolism* ; RNA, Messenger/metabolism* ; Signal Transduction/drug effects* ; Transforming Growth Factor beta/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Up-Regulation/drug effects* ; X-Box Binding Protein 1/metabolism*

OBJECTIVE: : To investigate the effect of polysaccharides (LBPs) on TLR/NF-κB independent pathway and serum tumor necrosis factor (TNF-α) level in diabetic MyD88-knockout mice. METHODS: : Diabetes was induced by feeding high-fat/high-sugar diet and injection of low-dose streptozotocin in MyD88-knockout mice. The diabetic mice were randomly divided into model group, positive control group and LBPs group. The expressions of TRAM, TRIF, TRAF6, RIP1 and TNF-α mRNA and proteins in mouse peritoneal macrophages were detected by real-time RT-PCR and Western blotting after LBPs treatment for 3 month. Serum TNF-α was determined with ELISA kit. RESULTS: : Real time RT-PCR showed that compared with model group, the relative expressions of and mRNA in macrophages of LBPs group were significantly decreased and expression of was significantly increased (all <0.05). Expression of TRAM, TRIF, TRAF6, RIP1 and TNF-α proteins as well as serum TNF-α level had no significant difference between LBPs group and model group (all >0.05). CONCLUSIONS : LBPs may not inhibit serum TNF-α level through TLR/NF-κB independent pathway.
Animals ; Diabetes Mellitus, Experimental ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation ; drug effects ; Macrophages, Peritoneal ; drug effects ; Mice ; Mice, Knockout ; Myeloid Differentiation Factor 88 ; genetics ; NF-kappa B ; genetics ; Random Allocation ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; blood ; metabolism

ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI/CLA-1) are the key proteins in reverse cholesterol transport (RCT). The high expression of ABCA1 and SR-BI/CLA-1 can decrease the danger of atherosclerosis. The purpose of the study is to find ABCA1 and CLA-1up-regulators for treating atherosclerosis by using cell-based high throughput screening models. Among 20 000 compounds screened, E0869 [1-(3, 4-dimethylphenyl)-1-oxopropan-2-yll4-((methylsulfonyl)methyl)benzoate] was found as the positive hit. The up-regulated activities of E0869 in ABCAl1-LUC and bCA-l1-LUC HepG2 cell were 160% and 175%, respectively. The EC50 values of E0869 in ABCAl1-LUC and CLA-l1-LUC HepG2 cell were 3.79 and 1.42 pμol- x ,(-1) respectively. E0869 could upregulate the mRNA and protein levels of ABCA1, SR-BI/CLA-1 and ABCGJ1genes in HepG2 and RAW264.7 cells by Real-Time Quantitative PCR and Western blotting analysis, but could not influence the expression of FAS, SREBP-l1 and CD36. Foam cell assay showed that E0869 could inhibit lipids accumulation in mouse peritoneal macrophages RAW264.7. Cholesterol efflux assay showed that E0869 could induce HDL-mediated cholesterol efflux in mouse peritoneal macrophages RAW264.7. In conclusion, E0869 could up-regulate ABCA1 and CLA-1 activity, and had good anti-atherosclerotic activity in vitro.
ATP Binding Cassette Transporter 1 ; metabolism ; Animals ; Atherosclerosis ; drug therapy ; Biological Transport ; Cholesterol ; Hep G2 Cells ; High-Throughput Screening Assays ; Humans ; Macrophages, Peritoneal ; drug effects ; Mice ; RNA, Messenger ; Scavenger Receptors, Class B ; metabolism ; Up-Regulation

This work was mainly studied the effects of the four alkaloids from Coptidis Rhizoma on the mouse peritoneal macrophages in vitro and preliminarily discussed the regulating mechanisms. The effect of alkaloids from Coptidis Rhizoma on the vitality of macrophages was measured by the MTT assay. The effect of alkaloids on the phagocytosis of macrophages was determined by neutral red trial and respiratory burst activity was tested by NBT. The expressions of respiratory-burst-associated genes influenced by alkaloids were detected by qRT-PCR. The conformation change of membrane protein in macrophages by the impact of alkaloids was studied by fluorospectro-photometer. Results showed that the four alkaloids from Coptidis Rhizoma could increase the phagocytosis of macrophages in different level and berberine had the best effect. Berberine, coptisine and palmatine had up-regulation effects on respiratory burst activity of mouse peritoneal macrophages stimulated by PMA and regulatory activity on the mRNA expression of PKC, p40phox or p47phox, whereas the epiberberine had no significant influence on respiratory burst. Moreover, alkaloids from Coptidis Rhizoma could change the conformation of membrane protein and the berberine showed the strongest activity. The results suggested that the four alkaloids from Coptidis Rhizoma might activate macrophages through changing the conformation of membrane protein of macrophages and then enhanced the phagocytosis and respiratory burst activity of macrophages. Furthermore, the regulatory mechanism of alkaloids on the respiratory burst activity of macrophages may be also related to the expression level of PKC, p40phox and p47phox.
Alkaloids ; pharmacology ; Animals ; Cells, Cultured ; Coptis ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gene Expression ; drug effects ; Macrophages, Peritoneal ; drug effects ; Mice ; Phosphoproteins ; genetics ; metabolism ; Protein Kinase C ; genetics ; metabolism ; Rhizome ; chemistry

OBJECTIVETo investigate effect of Chaiqin Chengqi Decoction (, CQCQD) on changes of neuronal acetylcholine receptor alpha 7 (nAChRα7) of peritoneal macrophages in acute pancreatitis (AP).

METHODSEighteen Kunming mice were equally randomized into the control group, AP group and CQCQD treatment group. AP was induced by two intraperitoneal injections of 4 g/kg L-arginine at 1 h apart, while control mice received saline injections. At 72 h after the first injection of L-arginine, mice in the treatment group were intragastrically administered 0.1 mL/10 g CQCQD every 2 h for 3 times, whilst mice in the other two groups received the same amount of saline feeding. Mice were sacrificed by cervical dislocation 2 h after the last feeding of either CQCQD or saline. Peritoneal macrophages were collected for determination of nAChRα7 mRNA and protein expression. Serum was collected for detection of interleukin-6 (IL-6), IL-10 and acetylcholine (ACh) levels, and pancreas was for histopathology analysis.

RESULTSThe CQCQD treatment significantly ameliorated the severity of AP as evidenced by reducing the pancreatic histopathology score (4.5±0.5 vs. 6.2±1.7, P<0.05) and the serum IL-6 levels (1228.3±419.2 pg/mL vs. 1589.6±337.3 pg/mL, P<0.05). The mRNA and protein expression of nAChRα7 of the peritoneal macrophages in the AP group were similar to the control group (P>0.05), but were significantly up-regulated after the CQCQD treatment (P<0.05). The serum ACh levels in the AP group were significantly lower than those in the control group (3.1±0.6 μg/mL vs 4.8±0.7 μg/mL P<0.05), but were significantly increased after the CQCQD treatment (5.6±1.5 μg/mL vs 3.1±0.6 μg/mL, P<0.05).

CONCLUSIONCQCQD is protective against L-arginine-induced AP through mechanisms involving nAChRα7 of peritoneal macrophages.

Acetylcholine ; pharmacology ; Acute Disease ; Animals ; Blotting, Western ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Interleukin-10 ; blood ; Interleukin-6 ; blood ; Macrophages, Peritoneal ; drug effects ; metabolism ; pathology ; Mice ; Neurons ; drug effects ; metabolism ; Pancreas ; drug effects ; pathology ; Pancreatitis ; blood ; drug therapy ; pathology ; RNA, Messenger ; genetics ; metabolism ; alpha7 Nicotinic Acetylcholine Receptor ; genetics ; metabolism

OBJECTIVETo analyze the difference of chemical compounds of frankincense-myrrh before and after their compatibility, and evaluate the effect of differentiated compounds on NO generated by LPS-induced peritoneal macrophage cells in rats, in order to discuss synergetic material basis of frankincense-myrrh compatibility from the prospective of change in chemical constituents.

METHODUPLC-Q-TOF-MS/MS combined technology was used to analyze the chemical components of frankincense-myrrh before and after their compatibility. MarkerLynx 4. 1 statistical software was used to analyze differentiated compounds before and after their compatibility.

RESULTThe results of PCA showed that there were significant differences in the combined extracts of frankincense-myrrh and the chromatogram of their combined liquid, suggesting significant differences in their chemical compounds before and after their compatibility; after their compatibility, the dissolution of pentacyclic triterpenoid (alpha-boswellic acid, beta-boswellic acid) and tetracyclic triterpenoid (elemonic acid, 3-acetoxy-16-hydroxy-dammar-24-ene, 3-hydroxytirucalla-8,24-dien-21-oic acid or 3-hydroxytirucalla-7,24-dien-21-oic acid) increased notably, while the dissolution of both yclic sesquiterpenes and macrocyclic diterpenoids decreased. According to the evaluation on in vitro activity, 2-methoxy-8, 12-epoxy-germa-1 (10), 7, 11-triene-6-ketone, 2-methoxy-5-acetoxyl-furan-germa-1 (10)-alkene-6-ketone and 3-carbonyl Euphorbia kansui-8, 24-diene-21-carboxylic acid notably inhibited NO generated by LPS-induced peritoneal macrophage cells in rats.

CONCLUSIONThese findings provide scientific basis and reference for studies on anti-inflammatory material basis of frankincense-myrrh compatibility.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; chemistry ; isolation & purification ; Boswellia ; chemistry ; Chromatography, Liquid ; Commiphora ; chemistry ; Drug Synergism ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Lipopolysaccharides ; pharmacology ; Macrophages, Peritoneal ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Molecular Structure ; Nitric Oxide ; metabolism ; Tandem Mass Spectrometry ; Terpenes ; chemistry ; isolation & purification ; Triterpenes ; chemistry ; isolation & purification

OBJECTIVETo study the toxic mechanism of toxic raphides from Pinellia ternata.

METHODMouse peritoneal macrophage in vitro culture model was adopted to study dose-dependent and time-dependent curves of toxic raphides, with TNF-alpha, IL-1beta and IL-6 in supernatant as indexes. Scanning electron microscopy was used to observe the changes in surface morphology of raphides-treated macrophages. Macrophages-neutrophils co-cultured the transport model to study the effect of toxic raphides' stimulation of macrophages on neutrophils migration.

RESULTToxic raphides' stimulation of macrophages could cause the increase in the levels of TNF-alpha, IL-1beta and IL-6 released, and showed dose dependence and time dependence. Scanning electron microscopy showed that toxic raphides were swallowed by macrophages, with notable cell membrane creases, increase in the number of pseudopods and decrease in integrity of cell membranes, and could significantly induce migration of neutrophils.

CONCLUSIONThe inflammatory process induced by toxic raphides is mainly mediated by macrophages. The toxic mechanism of toxic raphides from P. ternata is that toxic raphides penetrate into tissues to activate resident macrophages, release phagocytic and inflammatory cytokines, and cause migration of neutrophils, which finally results in acute inflammatory response.

Animals ; Drugs, Chinese Herbal ; toxicity ; Inflammation Mediators ; toxicity ; Interleukin-1beta ; immunology ; Interleukin-6 ; immunology ; Macrophages, Peritoneal ; drug effects ; immunology ; Male ; Mice ; Mice, Inbred ICR ; Pinellia ; chemistry ; Tumor Necrosis Factor-alpha ; immunology

Inflammasome is a large protein complex activated upon cellular stress or microbial infection, which triggers maturation of pro-inflammatory cytokines interleukin-1β and interleukin-18 through caspase-1 activation. Nod-like receptor family protein 3 (NLRP3) is the most characterized inflammasome activated by various stimuli. However, the mechanism of its activation is unclear and its exact cellular localization is still unknown. We examined the potential co-localization of NLRP3 inflammasome with mitochondria and seven other organelles under adenosine triphosphate, nigericin or monosodium urate stimulation in mouse peritoneal macrophages using confocal microscopy approach. Our results revealed that the activated endogenous apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome forms in the cytoplasm and co-localizes with NLRP3 and caspase-1, but not with any of the organelles screened. This study indicates that the ASC pyroptosome universally localizes within the cytoplasm rather than with any specific organelles.
Adenosine Triphosphate ; pharmacology ; Animals ; Apoptosis Regulatory Proteins ; CARD Signaling Adaptor Proteins ; Carrier Proteins ; analysis ; metabolism ; Caspase 1 ; analysis ; metabolism ; Cytoplasm ; metabolism ; Cytoskeletal Proteins ; analysis ; metabolism ; Inflammasomes ; analysis ; metabolism ; Macrophages, Peritoneal ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Microscopy, Confocal ; Mitochondria ; metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein ; Nigericin ; pharmacology ; Uric Acid ; pharmacology

ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I (apoA-I), and plays a key role in the initial steps of the whole process of reverse cholesterol transport (RCT). Upregulation of ABCA1 is beneficial for atherosclerosis (AS) prevention and/or therapy, which indicated that ABCA1 was a target for anti-AS drug development. In the previous study, a high-throughput screening method was established using ABCA1p-LUC HepG2 cell line to find the upregulators of ABCA1. In the present study, compound 2030421B was found using this method, with EC50 of 0.50 microg x mL(-1). The compound was further identified as an upregulator of ABCA1 expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analysis. Studies also showed that the 2030421B could induce apoA-I-mediated cholesterol efflux and inhibit lipids uptake into mouse peritoneal macrophages RAW264.7.
ATP Binding Cassette Transporter 1 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; Animals ; Anticholesteremic Agents ; administration & dosage ; chemistry ; pharmacology ; Apolipoprotein A-I ; metabolism ; Benzaldehydes ; administration & dosage ; chemistry ; pharmacology ; Biological Transport ; Cells, Cultured ; Cholesterol ; secretion ; Dose-Response Relationship, Drug ; Hep G2 Cells ; High-Throughput Screening Assays ; Humans ; Lipid Metabolism ; Lipids ; analysis ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Molecular Structure ; RNA, Messenger ; Up-Regulation ; drug effects

OBJECTIVETo observe the effect of andrographolide on the activation of mitogen-activated protein kinases (MAPKs) and expression of nuclear factor-κB (NF-κB) in macrophage foam cells.

METHODSThe mouse peritoneal macrophages were cultured in the media in the presence of oxidized low-density lipoprotein (ox-LDL), ox-LDL+andrographolide, or neither (control). The phosphorylation of MAPK molecules (p38MAPK, JNK, ERK1/2) and the expressions of NK-κB p65 were examined by Western blot.

RESULTSAs compared with cells in the control group, the expressions of phospho-p38 and NF-κB p65 were increased in the cells cultured with either ox-LDL or ox-LDL+andrographolide (P<0.01), but attenuated significantly in the presence of ox-LDL+ andrographolide when compared with ox-LDL (P<0.05). The phospho-JNK increased in the presence of either ox-LDL or ox-LDL+andrographolide when compared with control cells (P<0.01), but no significant difference existed between ox-LDL and ox-LDL+andrographolide (P>0.05). The expression of phospho-ERK1/2 was increased in the presence of ox-LDL compared with the control cells (P<0.01), but no significant differences existed between the cells cultured in the presence of ox-LDL+andrographolide and the control medium (P>0.05).

CONCLUSIONSAndrographolide could inhibit the activation of ERK1/2, p38MAPK and NK-κB induced by ox-LDL in macrophage foam cells, which might be one of its mechanisms in preventing atherosclerosis.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Atherosclerosis ; immunology ; metabolism ; prevention & control ; Cells, Cultured ; Diterpenes ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Foam Cells ; cytology ; drug effects ; enzymology ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lipoproteins, LDL ; metabolism ; MAP Kinase Signaling System ; drug effects ; immunology ; Macrophages, Peritoneal ; cytology ; drug effects ; enzymology ; Mice ; Mice, Inbred Strains ; NF-kappa B ; metabolism ; Vasculitis ; drug therapy ; immunology ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism