Zhe Xue; Yuyan Guo; Fangyun Wang; Qinping Yang; Qiuhong Chen; Tingting Lin; Shunhe Lin
Abstract
Background: miR-196b-5p was found to be significantly reduced in endometriosis, but its function and the mechanisms involved remained unclear.Objective: To explore the effect of miR-196b-5p on manipulating macrophage phenotype and the underlying mechanisms in endometriosisMethods: The endometriosis mice ...
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Background: miR-196b-5p was found to be significantly reduced in endometriosis, but its function and the mechanisms involved remained unclear.Objective: To explore the effect of miR-196b-5p on manipulating macrophage phenotype and the underlying mechanisms in endometriosisMethods: The endometriosis mice and End1/E6E7 cells were used for in vivo and in vitro experiments, respectively. QRT-PCR was used to detect miR-196b-5p, suppressor of cytokine signaling 1 (SOCS1), high mobility group AT-Hook 1 (HMGA1), and CCL2 expressions. Western blot was used to detect SOCS1 and HMGA1 protein levels while luciferase reporter assay was performed to determine the interaction between miR-196b-5p and SOCS1/ HMGA1. ELISA was used to measure CCL2, IL-10, and IL-6 levels and immunohistochemical staining and flow cytometry were used to examine CD86 and CD206 expressions.Results: Significantly reduced levels of miR-196b-5p, and increased levels of SOCS1, HMGA1, and CCL2 were observed in the ectopic endometrium of mice with endometriosis. The miR-196b-5p mimic significantly reduced the lesion size, increased M1 macrophages, and decreased M2 macrophages in the ectopic endometrium of mice with endometriosis. End1/E6E7 cells transfected with miR196b-5p mimic significantly increased M1 macrophages, decreased M2 macrophages and reduced the migration in PMA-treated THP1 cells. Conversely, transfection with a miR-196b-5p inhibitor led to the opposite outcomes. miR-196b-5p targeted SOCS1/HMGA1, and miR-196b-5p inhibitor significantly up-regulated CCL2 and IL-10, and down-regulated IL-6 levels in End1/E6E7 cells. These effects were markedly reversed by si-SOCS1/si-HMGA1.Conclusion: miR-196b-5p elevates M1 macrophages and decreases M2 macrophages in endometriosis, possibly by targeting SOCS1/ HMGA1. This research may provide a novel insight into the pathological mechanisms of endometriosis.
Lidan Luo; Yuan Fang; Qi Yuan; Jinmao Liao; Zheng Zhang
Abstract
Background: Pyroptosis is a programmed cell death related to caspase-1, accompanied by the secretion of pro-inflammatory cytokines. Objectives: To explore the effects of LPS on the P2X7R/NLRP3 pathway in macrophages, and hepatocytes pyroptosis in mice. Methods: LPS was used to establish an animal ...
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Background: Pyroptosis is a programmed cell death related to caspase-1, accompanied by the secretion of pro-inflammatory cytokines. Objectives: To explore the effects of LPS on the P2X7R/NLRP3 pathway in macrophages, and hepatocytes pyroptosis in mice. Methods: LPS was used to establish an animal model of the acute liver injury. The macrophage RAW264.7 was induced by LPS to establish a cell model. The P2X7R inhibitor A438079 and agonist BZATP were added. RAW264.7 was co-cultured with AML-12 cells. Pyroptosis and the ratio of CD11b+CD86+/CD11b+CD206+ were analyzed by flow cytometry. ELISA, WB, and qRT-PCR were applied to analyze factors involved in the P2X7R/NLRP3 pathway. Results: LPS induced liver damage in mice, promoted cell pyroptosis and increased the levels of IL-18, IL-1β, ALT, AST, and TBIL. P2X7R, GSDMD, and GSDMD-N expressions also increased in the LPS group. LPS induced macrophage activation in vivo. NLRP3, ASC, P2X7R, and caspase-1 expressions in vitro promoted. ELISA confirmed that the IL-1β and IL-18 levels repressed in the BZATP (P2X7R agonist) group, while the trend was opposite in the A438079 (P2X7R inhibitor) group. LPS activated the P2X7R/NLRP3 pathway in macrophages. After RAW264.7 was co-cultured with AML-12 cells, the pyroptosis of AML-12 cells promoted but the proliferation decreased in the BZATP group. GSDMD and GSDMD-N expressions promoted in the BZATP group, while the trend was opposite in the A438079 group. Conclusion: LPS activated macrophages via P2X7R activation of NLRP3 and induced hepatocyte pyroptosis, which provided novel potential targets for the liver injury treatment.
Cheah Wen Yapp; Amal Widaad Mohaimin; Adi Idris
Volume 14, Issue 3 , September 2017, , Pages 250-256
Abstract
Background: Cytosolic double-stranded RNA (dsRNA) is an important ‘molecular signature’ for the detection of intracellular viral infections. Although intracellular dsRNA is a known potent inducer of apoptosis, the optimal time and dose for the onset of dsRNA-mediated apoptosis have not been ...
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Background: Cytosolic double-stranded RNA (dsRNA) is an important ‘molecular signature’ for the detection of intracellular viral infections. Although intracellular dsRNA is a known potent inducer of apoptosis, the optimal time and dose for the onset of dsRNA-mediated apoptosis have not been studied in detail. Objective: To perform an accurate temporal assessment of the cell death responses in dsRNA-dependent cytotoxicity. Methods: Poly I:C (PIC), a synthetic dsRNA molecule was delivered intracellularly into J774.1 and RAW264.7 murine macrophages via electroporation. Cell viability was measured using the MTT assay and apoptosis was determined by sub-G0/G1 DNA content using flow cytometry. Results: Loss of cell viability was seen as early as 3h post-electroporation of macrophages. A significant increase in the sub-G0/G1 DNA content consistent with apoptosis was observed in PIC-electroporated macrophages as early as 3h post electroporation. Conclusion: Intracellular PIC delivery induces rapid macrophage cell death.
Ziba Ghasemi; Babak Farrokhi; Farah Miraghasi; Ardalan Ejaz Ahmad; Nariman Mosaffa
Volume 3, Issue 3 , September 2006, , Pages 121-126
Abstract
Background: Polysaccharides have long been used as immune-modulators in various pathologic conditions including inflammation and solid malignancies. Objective: To evaluate the effects of Zymosan and Betaglucan on cytotoxic reactions in an effectortarget conjugate system. Methods: Blood was obtained from ...
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Background: Polysaccharides have long been used as immune-modulators in various pathologic conditions including inflammation and solid malignancies. Objective: To evaluate the effects of Zymosan and Betaglucan on cytotoxic reactions in an effectortarget conjugate system. Methods: Blood was obtained from 20 healthy subjects; purified mononuclear leukocytes (monocytes and lymphocytes) were extracted and cultured as effector cells by a cytotoxic method. Both adherent and non-adherent cells interacted with the K562 myeloid cell line. The effector-target (E:T) ratio was 1:1, 1:10, and 1:20. To evaluate stimulatory effects of Betaglucan and Zymosan on cytotoxic reactions, samples were divided into case and control groups based on the presence or absence of Betaglucan and Zymosan. MTT assay and sFas ligand (sFasL) concentrations were used to assess the increased killing capacity of effector cells. Results: Our results revealed that Zymosan and Betaglucan can induce cytotoxic responses in macrophages and lymphocytes (P<0.05). The best result was achieved with E:T ratio of 1:1. Both macrophages and lymphocytes produced sFasL following stimulation by Zymosan and Betaglucan, however, the level of production was not statistically significant (P>0.05). Conclusion: Zymosan and Betaglucan can be used as enhancers of the killing capacity of the immune cells; therefore, Betaglucan and Zymosan can be applied as systemic stimulators of the immune response in inflammation and chronic infection.
