1.CRT promoted c-FLIP expression via NF-κB pathway in rheumatoid arthritis fibroblast-like synoviocytes
Ya-Chong JIAO ; Shan-Shan HUANG ; Chun-You WAN ; Yi-Xin LIU ; Yang WANG ; Ying-Yu BAI ; Jun MA ; Fang ZHENG
Tianjin Medical Journal 2018;46(2):113-117
Objective To investigate the molecular mechanisms of upregulated expression of cellular Fas-associated death domain-like interleukin-1 beta converting enzyme inhibitory protein(c-FLIP)by calreticulin(CRT)in patients with rheumatoid arthritis (RA). Methods The semi-quantitative analysis and localization of c-FLIP in RA and osteoarthritis (OA)synovium were detected by immunohistochemistry.The fibroblast-like synoviocytes(FLS)were isolated by enzymatic digestion of synovial tissue specimens obtained from RA and OA patients,and cultured as an in vitro experiment model.The expressions of c-FLIP in RA and OA synovial fibroblasts were detected by immunofluorescence and Western blot assay. Whether CRT influenced c-FLIP expression and its molecular mechanism were explored by Western blot assay. Results The high expression of c-FLIP was found in RA synovium, mainly in the lining and sublining areas of FLS and vascular endothelial cells detected by immunohistochemistry.Meanwhile,weak staining of c-FLIP was observed in OA synovium.The expression of c-FLIP was significantly higher in RA synovium than that of OA synovium(t=11.717,P<0.001).Results of immunofluorescence and Western blot assay showed that c-FLIP was mainly located in cytoplasm, and which was higher expressed in FLS of RA than that of OA. The increased c-FLIP expression and phosphorylation of NF-κB were detected after being co-incubated with exogenous CRT (0, 10, 50, 100 μg/L), in dose-dependent manner. The effect of CRT upregulating c-FLIP expression was blocked by NF-κB inhibitor BAY 11-7082.Conclusion CRT can increase c-FLIP expression at least partly through NF-κB pathway in RA,which may provide therapeutic target for the treatment of RA.
2.Effect of eicosapentaenoic acid on mRNA expression of tight junction protein ZO-1 in intestinal epithelial cells after Escherichia coli LF82 infection.
Li-Jun HAO ; Yan LIN ; Wei ZHANG ; Jiao TIAN ; Ya WANG ; Peng-De CHEN ; Chong-Kang HU ; Ling-Chao ZENG ; Jie YANG ; Bao-Xi WANG ; Xun JIANG
Chinese Journal of Contemporary Pediatrics 2017;19(6):693-698
OBJECTIVETo investigate the change in the expression of tight junction protein ZO-1 in intestinal epithelial cells (Caco-2 cells) and the protective effect of eicosapentaenoic acid (EPA) after adherent-invasive Escherichia coli (E.coli) LF82 infection.
METHODSThe Caco-2 cell line was used to establish an in vitro model of tight junction of intestinal epithelial cells. Caco-2 cells were divided into EPA treatment groups (0, 25, 50, 100, and 200 μmol/L EPA) and EPA (0, 25, 50, 100, and 200 μmol/L EPA)+E.coli LF82 treatment (0, 6, and 12 hours) groups. A microscope was used to observe the morphological characteristics of the cells. MTT assay was used to determine the cell growth curve. The activity of alkaline phosphatase (ALP) at both sides of the cell membrane was compared to evaluate the Caco-2 cell model. MTT assay and flow cytometry were used to investigate the effects of different concentrations of EPA on the survival rate and apoptosis rate of Caco-2 cells. RT-qPCR was used to measure the mRNA expression of ZO-1 in Caco-2 cells after EPA and/or E.coli LF82 treatment. ELISA was used to measure the change in the level of tumor necrosis factor-α (TNF-α) in culture supernatant.
RESULTSAfter EPA treatment (25 and 50 μmol/L), the proliferation of Caco-2 cells was induced in a dose-dependent manner. The survival rates of the cells were significantly higher than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant inhibitory effect on the proliferation of Caco-2 cells in a dose-dependent manner. The survival rates of the cells were significantly lower than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant increase in cell apoptosis rate compared with the control group (P<0.05). The 6- and 12-hour E.coli LF82 treatment groups had decreasing mRNA expression of ZO-1 in Caco-2 cells over the time of treatment and had significantly lower mRNA expression of ZO-1 than the untreated group (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed an increase in the mRNA expression of ZO-1 with the increasing concentration of EPA, as well as significantly higher mRNA expression of ZO-1 than the Caco-2 cells treated with E.coli LF82 alone (P<0.05). The Caco-2 cells treated with E.coli LF82 alone for 6 or 12 hours had increasing secretion of TNF-α over the time of treatment and had significantly higher secretion than the untreated Caco-2 cells (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed a reduction in the secretion of TNF-α with the increasing concentration of EPA and had significantly lower secretion than the Caco-2 cells treated with E.coli LF82 alone (P<0.05).
CONCLUSIONSEPA can effectively prevent the destruction of tight junction of intestinal epithelial cells induced by E.coli LF82 infection and inhibit the secretion of inflammatory factors. Therefore, it has a certain protective effect on intestinal mucosal barrier.
Apoptosis ; drug effects ; Caco-2 Cells ; Eicosapentaenoic Acid ; pharmacology ; Escherichia coli ; pathogenicity ; Humans ; Intestinal Mucosa ; metabolism ; microbiology ; RNA, Messenger ; analysis ; Tight Junctions ; drug effects ; Tumor Necrosis Factor-alpha ; secretion ; Zonula Occludens-1 Protein ; genetics
3.Acquirement and evaluation of murine ventricular extracellular matrix.
Yu-Dong JIANG ; Wen-Si LI ; Chong YU ; Lu WANG ; Xiao-Xi SUN ; Jiao-Ya XI
Acta Physiologica Sinica 2014;66(6):709-717
Cardiac extracellular matrix (ECM), generated from the process of decellularization, has been widely considered as an ideal source of biological scaffolds. However, current ECM preparations are generally difficult to be applied to generate cardiac tissue. Our research was aimed to improve decellularization protocols to prepare cardiac ECM slices. Adult murine ventricular tissues were embedded in low melting agarose and cut into 300 μm slices, and then were divided randomly into three groups: normal cardiac tissue, SDS treated group (0.1% SDS) and SDS+Triton X-100 treated group (0.1% SDS+0.5% Triton X-100). Total RNA content and protein content quantification, HE staining and immunostaining were used to evaluate the removal of cell components and preservation of vital ECM components. Furthermore, murine embryonic stem cell-derived cardiomyocytes (mES-CMs) and mouse embryonic fibroblasts (MEFs) were co-cultured with ECM slices to evaluate biocompatibility. The relative residual RNA and protein contents of ECM slices significantly decreased after decellularization. HE staining showed that SDS+Triton X-100 treatment better destroyed cellular structure and removed nuclei of ECM slices, compared with SDS treatment. Immunostaining showed that collagen IV and laminin were better preserved and presented better similarity to original cardiac tissue in ECM slices acquired by SDS+Triton X-100 treatment. However, collagen IV and laminin were significantly decreased and arranged disorderly in SDS treated group. We observed effective survival (≥ 12 days) of MEFs and mES-CMs on ECM slices acquired by SDS+Triton X-100 treatment, and signs of integration, whereas those signs were not found in SDS treated group. We concluded that, compared with traditional SDS method, new combined protocol (SDS+Triton X-100) generated ECM slices with better component and structural preservation, as well as better biocompatibility.
Animals
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Extracellular Matrix
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chemistry
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Heart Ventricles
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cytology
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Mice
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Octoxynol
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Sodium Dodecyl Sulfate
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Tissue Engineering
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methods
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Tissue Scaffolds
4.Mechanism of Proliferation and Apoptosis of Acute Promyelocytic Leukemia Cell Line NB4 Induced by TPA.
Pan ZHAO ; Chong ZHANG ; Xue-Mei DONG ; Lu-Wei YAN ; Le-Yuan MI ; Ya-Jiao LI ; Jia-Chao KANG ; Jing WANG
Journal of Experimental Hematology 2023;31(5):1296-1302
OBJECTIVE:
To investigate the effect of phorbol-12-myristate-13-ace-tate (TPA) on the proliferation and apoptosis of acute promyelocytic leukemia cell line NB4 and its molecular mechanism.
METHODS:
The effect of different concentrations of TPA on the proliferation of NB4 cells at different time points was detected by CCK-8 assay. The morphological changes of NB4 cells were observed by Wright-Giemsa staining. The cell cycle and apoptosis of NB4 cells after TPA treatment were detected by flow cytometry. The mRNA expressions of NB4 cells after TPA treatment were analyzed by high-throughput microarray analysis and real-time quantitative PCR. Western blot was used to detect the protein expression of CDKN1A, CDKN1B, CCND1, MYC, Bax, Bcl-2, c-Caspase 3, c-Caspase 9, PIK3R6, AKT and p-AKT.
RESULTS:
Compared with the control group, TPA could inhibit the proliferation of NB4 cells, induce the cells to become mature granulocyte-monocyte differentiation, and also induce cell G1 phase arrest and apoptosis. Differentially expressed mRNAs were significantly enriched in PI3K/AKT pathway. TPA treatment could increase the mRNA levels of CCND1, CCNA1, and CDKN1A, while decrease the mRNA level of MYC. It could also up-regulate the protein levels of CDKN1A, CDKN1B, CCND1, Bax, c-Caspase 3, c-Caspase 9, and PIK3R6, while down-regulate MYC, Bcl-2, and p-AKT in NB4 cells.
CONCLUSION
TPA induces NB4 cell cycle arrest in G1 phase and promotes its apoptosis by regulating PIK3/AKT signaling pathway.
Humans
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Leukemia, Promyelocytic, Acute
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Caspase 3/metabolism*
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Caspase 9/pharmacology*
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Phosphatidylinositol 3-Kinases/metabolism*
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Proto-Oncogene Proteins c-akt/metabolism*
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bcl-2-Associated X Protein/metabolism*
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Cell Line, Tumor
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Cell Division
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Apoptosis
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RNA, Messenger
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Cell Proliferation