Objective: To investigate the effect of microRNA (miR-148b) targeting decoy receptor 3 (DcR3) on macrophage polarization in sepsis. Methods: Experimental study. From December 2019 to December 2022, serum microRNA expression was detected in 3 patients with sepsis and 3 healthy controls in the clinical laboratory of Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Phorbol 12-myristate 13-acetate (PMA) was used to induce the differentiation of human acute monocytic leukemia cells THP-1 into macrophages, and then lipopolysaccharide (LPS) was added to stimulate the establishment of a sepsis cell model, and the expression changes of miR-148b and DcR3 were detected by RT-PCR and Western blot. Overexpression of DcR3 was used to detect the expression levels of TNF-α, CD163 and IL-10 in macrophages stimulated by LPS (100 ng/ml). Overexpression of miR-148b was used to observe the changes of molecular markers of macrophage polarization. The targeting regulation effect of miR-148b on DcR3 was determined by dual-luciferase reporter assay. t test was used to analyze whether there were statistical differences among the groups. Results: The expression of miR-148b was down-regulated (P<0.05) and the expression of DcR3 was up-regulated (P<0.01) in THP-1 macrophages stimulated by LPS. Overexpression of DcR3 inhibited the expression of TNF-α (P<0.05) and promoted the expression of CD163 (P<0.01) and IL-10 (P<0.01). When miR-148b mimics was added, the opposite effect was observed. The dual-luciferase reporter assay confirmed that miR-148b targets and binds to DcR3, inhibiting its transcription and expression. The results of flow cytometry showed that DcR3 could reverse the promoting effect of miR-148b on the CD86/CD163 ratio of macrophages (P<0.05). Conclusion: miR-148b inhibits the expression of DcR3, thereby inhibiting M2 polarization in LPS-stimulated macrophage cells.
Humans ; Interleukin-10 ; Lipopolysaccharides/pharmacology* ; Macrophages ; MicroRNAs/genetics* ; Receptors, Tumor Necrosis Factor, Member 6b/metabolism* ; Tumor Necrosis Factor-alpha

Objective: To investigate the effect of microRNA (miR-148b) targeting decoy receptor 3 (DcR3) on macrophage polarization in sepsis. Methods: Experimental study. From December 2019 to December 2022, serum microRNA expression was detected in 3 patients with sepsis and 3 healthy controls in the clinical laboratory of Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Phorbol 12-myristate 13-acetate (PMA) was used to induce the differentiation of human acute monocytic leukemia cells THP-1 into macrophages, and then lipopolysaccharide (LPS) was added to stimulate the establishment of a sepsis cell model, and the expression changes of miR-148b and DcR3 were detected by RT-PCR and Western blot. Overexpression of DcR3 was used to detect the expression levels of TNF-α, CD163 and IL-10 in macrophages stimulated by LPS (100 ng/ml). Overexpression of miR-148b was used to observe the changes of molecular markers of macrophage polarization. The targeting regulation effect of miR-148b on DcR3 was determined by dual-luciferase reporter assay. t test was used to analyze whether there were statistical differences among the groups. Results: The expression of miR-148b was down-regulated (P<0.05) and the expression of DcR3 was up-regulated (P<0.01) in THP-1 macrophages stimulated by LPS. Overexpression of DcR3 inhibited the expression of TNF-α (P<0.05) and promoted the expression of CD163 (P<0.01) and IL-10 (P<0.01). When miR-148b mimics was added, the opposite effect was observed. The dual-luciferase reporter assay confirmed that miR-148b targets and binds to DcR3, inhibiting its transcription and expression. The results of flow cytometry showed that DcR3 could reverse the promoting effect of miR-148b on the CD86/CD163 ratio of macrophages (P<0.05). Conclusion: miR-148b inhibits the expression of DcR3, thereby inhibiting M2 polarization in LPS-stimulated macrophage cells.
Humans ; Interleukin-10 ; Lipopolysaccharides/pharmacology* ; Macrophages ; MicroRNAs/genetics* ; Receptors, Tumor Necrosis Factor, Member 6b/metabolism* ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate the effects of X ray on human colorectal cancer cells for their tumor necrosis factor receptor-p55 (TNFR-p55) expression and release of soluble soluble TNFR-p55 (sTNFR-p55) in vitro.

METHODSThe protein expression of TNFR-p55 in Lovo cells exposed to X-ray was detected using immunohistochemistry, and enzyme-linked immunosorbent assay was used to examine the levels of sTNFR-p55 in the supernatants of the cell culture. The cell apoptosis of the exposed cells was analyzed with flow cytometry, and the changes in cell morphology were observed microscopically.

RESULTSX-ray exposure of cells resulted in a strong increase in TNFR-p55 expression of (P<0.01) and LoVo cell apoptosis (P<0.05). The levels of sTNFR-p55 in the supernatant of cells with X-ray exposure was significantly lowered in comparison with the levels before exposure (P<0.01). Optical microscopy showed that the exposed LoVo cells shrank and became spherical with cytoplasmic condensation and nuclear pyknosis.

CONCLUSIONX-ray exposure can induce LoVo cell apoptosis by increasing TNFR-p55 expression on the cell membrane and inhibiting the release of sTNFR-p55 in the supernatants.

Apoptosis ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; metabolism ; Tumor Necrosis Factor Decoy Receptors ; genetics ; metabolism ; X-Rays

Animals ; Lung ; drug effects ; metabolism ; Perchlorates ; toxicity ; Quaternary Ammonium Compounds ; toxicity ; RNA, Messenger ; genetics ; Rabbits ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

No abstract available.
Female ; Gastric Mucosa/*metabolism ; Helicobacter Infections/*genetics ; Humans ; Male ; Peptic Ulcer/*genetics ; *Promoter Regions, Genetic ; Tumor Necrosis Factor-alpha/*genetics

Tumor necrosis factor (TNF) is a cytokine that is produced by immune cells in response to bacterial and viral stimuli and plays important roles in various inflammatory diseases. TNF is produced as a membrane-bound precursor, which is then cleaved to release soluble mature protein. We expressed murine pro-TNF in Saccharomyces cerevisiae and examined processing and cellular localization of the recombinant protein. Yeast cells were transformed with an expression construct carrying the pro-TNF gene under the control of alcohol dehydrogenase promoter. Immunoblotting analysis of cell homogenate revealed expression of 26 kD pro-TNF in transformed cells. Upon centrifugation, pro-TNF transformed cells fractionated into the membrane/particulate. In a clone that expresses a high level of pro-TNF, mature 17 kD TNF was detected in the culture medium, although the amount was far smaller than that of cell-associated pro-TNF. Flow cytometric analysis of yeast spheroplasts demonstrated the presence of TNF on the cell surface. Our results show that pro-TNF expressed in yeast mainly resides in the cellular membrane with an orientation similar to that of pro-TNF produced in mammalian cells. Our data suggest that the transformed yeast cells can be used for the genetic analysis of pro-TNF processing machinery in immune cells.
Animal ; Cell Line ; Cell Membrane/metabolism ; Flow Cytometry ; Immunoblotting ; Mice ; Plasmids ; Protein Precursors/metabolism* ; Protein Precursors/genetics ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae/genetics ; Transformation, Genetic ; Tumor Necrosis Factor/metabolism* ; Tumor Necrosis Factor/genetics

Tumor necrosis factor (TNF) -alpha induces pleiotropic cellular effects through a 55kDa, type 1 receptor (TNFR1) and a 75kDa type 2 receptor (TNFR2). Moreover, it participates in the pathogenesis of several CNS diseases, including demyelinating diseases. TNF- receptors are differentially expressed and are regulated in many cell types. However, data regarding the TNF-alpha receptor expression and regulation in human astrocytes is limited to date. We investigated TNF-alpha receptor expression, its regulation by cytokines, and its functional role in primary cultured human fetal astrocytes, which are the most abundant cellular population in the central nervous system and are known to be immunologically active. In this study, astrocytes were found to constitutively and predominantly transcribe, translate and shed TNFR1 rather than TNFR2, but TNFR2 expression was increased by adding TNF-alpha, IL-1, and IFN-gamma, but not by adding LPS. To determine the functional roles of TNFR1 and TNFR2 on TNF induction, we investigated NF-kappaB activation and TNF-alpha induction after neutralizing TNFR1 and TNFR2 by an antibody treatment. We found that NF-kappaB activation and TNF-alpha induction are blocked by TNFR1 neutralizing antibody treatments.
Receptors, Tumor Necrosis Factor, Type II/genetics/*metabolism/physiology ; Receptors, Tumor Necrosis Factor, Type I/genetics/*metabolism/physiology ; RNA, Messenger/metabolism ; NF-kappa B/metabolism ; Humans ; Gene Expression Regulation ; Fetus/cytology ; Cytokines/*pharmacology ; Cells, Cultured ; Astrocytes/drug effects/*metabolism

Tumor necrosis factor (TNF) is a key cytokine in immunology system and is related to many human diseases. In order to inhibit the activity of TNF, cDNA coding for soluble TNF receptor II (sTNFRII) and human IgG Fc were linked using a flexible hinge. This gene was expressed in E. coli as a chimeric protein and purified by metal chelate chromatography. The results show that the fusion protein exists in the physiological form as a dimer, has the ability to bind with TNF and inhibits the cytotoxicity of TNF on L929 cells. Contrasting to monomer sTNFRII, the chimeric protein has an improved bioactivity, and displays potential prospects for research and application.
Antigens, CD ; genetics ; isolation & purification ; metabolism ; Blotting, Western ; methods ; Chromatography, Liquid ; Cloning, Molecular ; Gene Expression ; Genetic Engineering ; Humans ; Immunoglobulin Fc Fragments ; genetics ; isolation & purification ; metabolism ; Immunoglobulin G ; genetics ; isolation & purification ; metabolism ; Receptors, Tumor Necrosis Factor ; genetics ; isolation & purification ; metabolism ; Receptors, Tumor Necrosis Factor, Type II ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Recombination, Genetic ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo explore the molecular biology mechanism of acupuncture on improving immune function damage induced by chemotherapy in cancer-bearing mice.

METHODSSeventy-two mice (36 mice in 3-day treatment and 5-day treatment, respectively) which were successfully made into cancer-bearing model were divided into a blank group, a model group, an acupuncture group and a moxibustion group by stratified randomization method, 9 mice in each one. Except for the mice in the blank group, the remaining mice were treated with intra-peritoneal injection of cyclophosphamide (CTX, 150 mg/kg), to establish the cancer-bearing mice of CTX. The mice in the blank group were treated with intraperitoneal injection of 0.9% NaCl (identical dose as other groups). After 4 h, the mice in the acupuncture group and moxibustion group were treated with acupuncture and moxibustion at "Dazhui" (GV 14), "Geshu" (BL 17), "Shenshu" (BL 23), "Zusanli" (ST 36), once a day. The mice in the blank group and model group were treated with immobilization and fixation during the same time. On the next day of the end of 3-day and 5-day treatment, the sample was collected. The ELISA method was applied to measure the contents of IL-12 and TNF-α in spleen of all the mice.

RESULTSAfter 3-day and 5-day treatment, compared with the blank group, the contents of IL-12 and TNF-α in spleen in the model group were reduced (all P < 0.05); compared with the model group, the contents of IL-12 and TNF-α in spleen in the acupuncture group and moxibustion group were increased (all P < 0.05), but the content of IL-12 and TNF-α in the acupuncture group was not different from that in the moxibustion group (both P > 0.05).

CONCLUSIONAcupuncture and moxibustion could effectively increase the contents of IL-12 and TNF-α in spleen of CTX cancer-bearing mice, which could relieve chemotherapy-induced immune function damage to improve immune function.

Acupuncture Therapy ; Animals ; Cyclophosphamide ; adverse effects ; Humans ; Interleukin-12 ; genetics ; metabolism ; Male ; Mice ; Moxibustion ; Neoplasms ; chemically induced ; genetics ; metabolism ; therapy ; Spleen ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Astrocytes/metabolism* ; Cell Hypoxia ; Cells, Cultured ; Cytokines ; Humans ; Lipopolysaccharides ; Membrane Proteins/genetics* ; Proto-Oncogene Proteins/genetics* ; Tumor Necrosis Factor-alpha