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

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

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

Endotoxic responses to bacterial lipopolysaccharide (LPS) are triggered by Toll-like receptor 4 (TLR4) and involve the production of inflammatory mediators, including interleukin-6 (IL-6), by macrophages. The detailed mechanism of IL-6 production by macrophages in response to LPS has remained unclear, however. We now show that LPS induces IL-6 synthesis in mouse peritoneal macrophages via the leukotriene B4 receptor BLT2. Our results suggest that TLR4-MyD88 signaling functions upstream of BLT2 and that the generation of reactive oxygen species (ROS) by NADPH oxidase 1 (Nox1) and consequent activation of the transcription factor nuclear factor (NF)-kappaB function downstream of BLT2 in this response. These results suggest that a TLR4-MyD88-BLT2-Nox1-ROS-NF-kappaB pathway contributes to the synthesis of IL-6 in LPS-stimulated mouse macrophages.
Animals ; Cell Line ; Interleukin-6/*biosynthesis ; Leukotriene B4/metabolism ; Ligands ; Lipopolysaccharides/immunology ; Macrophages/immunology/*metabolism ; Macrophages, Peritoneal/immunology/metabolism ; Mice ; Myeloid Differentiation Factor 88/*metabolism ; NADH, NADPH Oxidoreductases/metabolism ; NF-kappa B/metabolism ; Reactive Oxygen Species/metabolism ; Receptors, Leukotriene B4/*metabolism ; Signal Transduction

This study was aimed at exploring the expression pattern of P2X family receptors (P2XR) in peritoneal macrophages and their relationship with the activation states of macrophages in Notch1-induced mouse T-ALL model. After establishment of the leukemia model, F4/80(+) peritoneal macrophages, F4/80(+)CD206(+) M2-like and F4/80(+)CD206(-) M1-like peritoneal macrophages were sorted by flow cytometry based on F4/80 and CD206 surface markers. The expression of P2XR in each cell population was detected by real time RT-PCR. The results showed that macrophages,M1-like and M2-like macrophages moderately expressed P2XR except for P2X5R. The expression of P2XR varied with the development of leukemia. The expression of P2X1R and P2X7R in peritoneal macrophages increased steadily; the expression of P2X2R and P2X3R decreased at late stage of leukemia;the expression of P2X4R slightly decreased at intermediate stage;the expression of P2X6R kept unchanged. At intermediate stage of leukemia, the expression of P2XR in M1-like and M2-like peritoneal macrophages varied. M1-like macrophages expressed higher level of P2X1R than M2-like macrophages, whereas M2-like macrophages expressed higher level of P2X7R than M1-like macrophages, which suggested that the expression of P2XR were related to the activation states. It is concluded that the expression of P2XR in peritoneal macrophages from leukemia mice is related to the progression of leukemia and the activation states of macrophages, which lay a foundation for further studying the role of macrophages in the development of leukemia.
Animals ; Cytokines ; metabolism ; Disease Models, Animal ; Macrophages, Peritoneal ; metabolism ; Mice ; Mice, Inbred C57BL ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; Receptor, Notch1 ; metabolism ; Receptors, Purinergic P2X ; metabolism ; Signal Transduction

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 study the effects and immunoregulation mechanism of the traditional Mongolian medicine Wuweifengshi capsule on adjuvant arthritis (AA).

METHODWister rats were divided into several groups: normal group, AA model group, Wuweifengshi capsule groups (with low, moderate, high dose of 0.2, 0.4, 0.8 g x kg(-1) x d(-1) respectively), and Zhonglun-5 group (original dose of 1.68 g x kg(-1) x d(-1)). The edema degree, the level of IL-1beta, TNF-alpha, PGE2, NO and MDA and the activity of SOD in serum were detected. Through cell culture, the effects of the medicine on AA rat's splenic cell's multiplication capacity were studied. The influence of celiac macrophage cell culture fluid of AA rats' on C57BL/6J mice thymic cell multiplication capacity under the medicine was evaluated.

RESULTWuweifengshi capsule showed an inhibiting function on the level of IL-1beta, TNF-alpha, PGE2, NO and increased the activity of SOD in serum, but showed no significant influence on MDA. It also inhibited the AA rat's splenic cell's multiplication capacity and the influence of celiac macrophage cell culture fluid of AA rat's on C57BL/6J mice thymic cell multiplication capacity.

CONCLUSIONThe anti-AA effect of Wuweifengshi capsule is possibly due to its inhibition of relevant cytokines and its adjustment of corresponding enzyme's activity and immunization organ's cell multiplication capacity.

Animals ; Arthritis, Experimental ; drug therapy ; immunology ; metabolism ; pathology ; Capsules ; Dehydroascorbic Acid ; analogs & derivatives ; blood ; Dinoprostone ; metabolism ; Disease Models, Animal ; Edema ; drug therapy ; Female ; Interleukin-1beta ; metabolism ; Lymphocytes ; immunology ; metabolism ; Macrophages, Peritoneal ; metabolism ; Male ; Medicine, Mongolian Traditional ; Mice ; Nitric Oxide ; metabolism ; Rats ; Spleen ; cytology ; metabolism ; Superoxide Dismutase ; blood ; Tumor Necrosis Factor-alpha ; 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

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