1.Effects of adenovirus-mediated bFGF, IL-1Ra and IGF-1 gene transfer on human osteoarthritic chondrocytes and osteoarthritis in rabbits.
Biao CHEN ; Jun QIN ; Hui WANG ; Jacques MAGDALOU ; Liaobin CHEN
Experimental & Molecular Medicine 2010;42(10):684-695
The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.
Adenoviridae/*genetics
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Animals
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Chondrocytes/drug effects/*metabolism
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Collagen Type II/genetics/metabolism
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Fibroblast Growth Factor 2/*genetics
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Gene Therapy/methods
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Genetic Vectors/administration & dosage/*genetics
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Humans
;
Insulin-Like Growth Factor I/*genetics/metabolism
;
Interleukin 1 Receptor Antagonist Protein/*genetics/metabolism
;
Interleukin-1/genetics/metabolism
;
Matrix Metalloproteinase 13/genetics/metabolism
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Matrix Metalloproteinase 3/genetics/metabolism
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Osteoarthritis/*therapy
;
Rabbits
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Tissue Inhibitor of Metalloproteinase-1/genetics
;
Transfection
2.Saccharomyces boulardii Activates Expression of Peroxisome Proliferator-activated Receptor-gamma in HT-29 Cells.
Sang Kil LEE ; Hyo Jong KIM ; Sung Gil CHI ; Jae Young JANG ; Ki Deok NAM ; Nam Hoon KIM ; Kwang Ro JOO ; Seok Ho DONG ; Byung Ho KIM ; Young Woon CHANG ; Joung Il LEE ; Rin CHANG
The Korean Journal of Gastroenterology 2005;45(5):328-334
BACKGROUND/AIMS: Saccharomyces boulardii (S. boulardii) has been reported to be beneficial in the treatment of inflammatory bowel disease, however, little is known about its mechanism of action. Peroxisome proliferator- activated receptor-gamma (PPAR-gamma) is recently found to regulate inflammation in intestinal epithelial cells. We hypothesized that the anti-inflammatory effects of S. boulardii are mediated, in part, through PPAR-gamma. To test this hypothesis, we examined the ability of S. boulardii to modulate the expression of PPAR-gamma in human colon cells. METHODS: Effects of S. boulardii on survival and proliferation of HT-29 human colon cells were assessed by MTT and [3H]thymidine incorporation assays. PPAR-gamma expression was assessed by Western blot and RT-PCR. Induction of interleukin-8 (IL-8) expression by tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), or lipopolysaccharide (LPS) was assessed by RT-PCR. RESULTS: S. boulardii did not affect viability and proliferation of HT-29 cells. S. boulardii up-regulated PPAR-gamma expression at both mRNA and protein levels. Pretreatment of HT-29 cells with S. boulardii blocked PPAR-gamma down-regulation by TNF-alpha, IL-1beta, or LPS, whereas it ameliorated IL-8 response to these proinflammatory factors. CONCLUSIONS: S. boulardii stimulates PPAR-gamma expression and reduces response of human colon cells to proinflammatory cytokines.
Cell Proliferation
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Colon/*metabolism
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*Gene Expression
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HT29 Cells
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Humans
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Interleukin-1/metabolism
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Interleukin-8/metabolism
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Lipopolysaccharides/pharmacology
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PPAR gamma/genetics/*metabolism
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Saccharomyces/*physiology
3.Association of IL33/ST2 signal pathway gene polymorphisms with myocardial infarction in a Chinese Han population.
Jin-hua YANG ; Fang-qin WU ; Qiang WEN ; Wen-cai ZHANG ; Yang-e WANG ; Xin XIONG ; Yan-wen SU ; Long-xian CHENG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2015;35(1):16-20
This study investigated the relationship between IL-33/ST2 signal pathway gene polymorphisms and myocardial infarction (MI) in Han Chinese. A case-control association analysis was performed on a total of 490 MI patients (MI group) and 929 normal subjects (NC group). Sequenom Mass Array and Taqman genotyping technique were used to analyze the tag single nucleotide polymorphisms (SNPs) in the genes encoding IL-33, ST2, and IL-1RaP (rs11792633, rs1041973 and rs4624606). The results showed that the frequencies of rs4624606 genotypes AA, TT, AT were 0.031, 0.647, 0.322 in MI group and 0.026, 0.712, 0.263 in NC group, and the allele frequencies of A and T were 0.192, 0.808 in MI group and 0.157, 0.843 in NC group. There were significant differences in rs4624606 genotypes and allele frequencies between MI group and NC group (P<0.05). For rs11792633, the allele frequencies of C and T were 0.45, 0.55 in MI group and 0.454, 0.546 in NC group with no significant differences found between the two groups. Compared with genotype CC+TC, rs11792633 genotype TT had an increased risk of hypertension (P<0.05). However, there were no significant differences in the frequencies of rs11792633 genotypes between the two groups. No significant differences were noted in the frequencies of rs1041973 genotype and allele between the two groups. Logistic regression analysis showed that rs4624606 genotypes AT and AA+AT were both significantly associated with MI (AT: OR=1.325, P=0.029, 95% CI=1.03-1.705; AA+AT: OR=1.316, P=0.028, 95% CI=1.03-1.681) after factors such as age, gender, smoking, drinking, body mass index (BMI), triglyceride (TG) and cholesterol were adjusted. Those carrying rs4624606 genotype AT or AA+AT had an increased risk of MI. No associations were found between the polymorphisms of the other two loci with MI. It was concluded that, in the IL33/ST2 signal pathway, the A allele of rs4624606 polymorphism of IL-1RaP gene is a potential independent risk factor for MI, and the genotypes AA+AT and AT are associated with the incidence of MI.
China
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Ethnic Groups
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genetics
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Female
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Humans
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Interleukin-1 Receptor-Like 1 Protein
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Interleukin-33
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Interleukins
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genetics
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metabolism
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Male
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Myocardial Infarction
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genetics
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Receptors, Cell Surface
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genetics
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metabolism
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Signal Transduction
;
genetics
4.Effects of exogenous IL-10 on IL-6 and ICAM-1 expression in inflammatory gingival tissue.
Pishan YANG ; Xiangmin QI ; Shaohua GE ; Min ZHAO
West China Journal of Stomatology 2002;20(5):343-345
OBJECTIVEThis study aimed at investigating effects of exogenous interleukine-10 (IL-10) on IL-6 and intercellular adhesion molecular (ICAM-1) expression in inflamed gingival tissue.
METHODSThe expression of IL-6 and ICAM-1 was examined using immunohistochemical techniques. Inflammatory gingival tissue treated with IL-10 was taken as the experimental group and the same patient's inflammatory gingival tissue without treatment of IL-10 was included into the control group.
RESULTSIL-6 expression was found mainly in monocytes, macrophages, lymphocytes, endotheliocytes and fibroblasts. The expression of ICAM-1 was found mainly in epithelial cells, monocot-macrophages, lymphocytes, endotheliocytes and fibroblasts. The immunohistochemical optical density (IOD) of the expression of IL-6 and ICAM-1 was detected by using Image-Proplus software, and the results showed that the expression in the experimental group differed significantly from that in the control group.
CONCLUSIONThe exogenous IL-10 may down-regulate IL-6 and ICAM-1 expression in inflammatory gingival tissue.
Adult ; Down-Regulation ; Female ; Gingiva ; metabolism ; Gingivitis ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; biosynthesis ; genetics ; Interleukin-10 ; pharmacology ; Interleukin-6 ; biosynthesis ; genetics ; Male ; Middle Aged
5.Study on the up-regulation of B7 molecules expression and immunogenicity of acute leukemia cells induced by interleukin 7.
Chinese Journal of Hematology 2005;26(5):289-292
OBJECTIVETo investigate the effects of interleukin 7 (IL-7) on B7 molecules expression and immunogenicity of acute leukemia (AL) cells.
METHODSThe B7 molecules expression on fresh acute leukemia cells and on the IL-7 exposed leukemia cells was detected by FACScan cytometer. B7-1 and B7-2 mRNA in IL-7 treated HL-60 cells were detected by reverse transcription-PCR (RT-PCR). The stimulation of proliferation of allogeneic peripheral blood mononuclear cells (PBMNC) by IL-7 treated leukemia cells was detected by MTT method. The level of interferon-gamma (IFN-gamma) secreted by the stimulated PBMNC was determined using enzyme-linked immunosorbent assays (ELISA). The blocking experiments were performed using monoclonal antibodies against B7-1, B7-2 and W6/32.
RESULTSB7-1 was weakly expressed in three, whereas B7-2 did in only one of eleven AL patients. IL-7 significantly enhanced B7 molecules expression on AL cells in a time-dependent manner. Furthermore, IL-7 could induce higher expression of B7-1 and B7-2 mRNAs on HL-60 cells. IL-7 treated leukemia cells could stimulate PBMNC proliferation and promote their IFN-gamma production. Anti-B7-1 and anti W6/32 but not anti-B7-2 monoclonal antibodies significantly inhibited the stimulated PBMNC proliferation and IFN-gamma secretion.
CONCLUSIONFresh AL cells express low level of B7-1 and B7-2 molecules. IL-7 enhances the B7 molecules expression on AL cells. The IL-7-treated leukemia cells can significantly stimulate the proliferation of allogeneic PBMNC and induce their IFN gamma secretion.
B7-1 Antigen ; genetics ; metabolism ; B7-2 Antigen ; genetics ; metabolism ; Humans ; Interleukin-7 ; pharmacology ; Leukemia ; immunology ; metabolism ; RNA, Messenger ; genetics ; Tumor Cells, Cultured ; Up-Regulation ; drug effects
6.Expression of recombinant extracellular region of human interleukin-1 receptor type I in Pichia pastoris.
Wei-hui LÜ ; Jun-hua ZHUANG ; Wei-ye CHEN ; Zhan-feng ZHANG ; Xian-zhang HUANG
Journal of Southern Medical University 2010;30(8):1841-1843
OBJECTIVETo construct pPICZalphaA-soluble interleukin-1 receptor type I (sIL-1RI) recombinant expression vector containing the gene fragment encoding the extracellular domain of sIL-1RI for its expression in Pichia pastoris.
METHODSsIL-1RI gene was amplified by RT-PCR and inserted into the yeast expression vector pPICZalphaA by digestion ligation. The recombinant plasmid pPICZalphaA-sIL1RI was transformed into E.coli Stb13, and the positive clones were analyzed by PCR and DNA sequencing. The pPICZalphaA-sIL1RI recombinant plasmid was electroporated into GS115 cells and the transformants were analyzed by PCR. After phenotype identification, the recombinant strains were induced by methanol to express the target protein, which was analyzed by Western blotting of the cell extract and supernatant.
RESULTSThe recombinant plasmid pPICZalphaA-sIL-1RI was constructed successfully, and the results of Western blotting showed that yeast induced by methanol expressed a protein of about 39 kD.
CONCLUSIONsIL-1RI protein has been successfully expressed in P.pastoris expression system, which provides the basis for further study of sIL-1RI.
Escherichia coli ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Pichia ; metabolism ; Plasmids ; Receptors, Interleukin-1 Type I ; biosynthesis ; genetics
7.Effect of interleukin-1beta on the variation of adenylyl cyclase expression in rats with seizures induced by L-glutamate.
Zhen WANG ; Qingying LIU ; Changgeng ZHU
Journal of Huazhong University of Science and Technology (Medical Sciences) 2004;24(6):540-587
To explore the mechanism of interleukin-1beta (IL-1beta) in the onset of seizure and the effect of IL-1beta on the expression of adenylyl cyclase (AC) in rats with seizure induced by L-glutamate. Experimental rats were first injected with IL-1beta and then L-glutamate (a dose under the threshold) was injected into the right lateral ventricle. The rats were sacrificed 4 h after the onset of epileptic activity and examined for changes in behavior, immunohistochemistry and compared with those with seizure induced by L-glutamate alone. It was found that the expression of AC in hippocampal and neocortex of rats with seizure induced by IL-1beta and L-glutamate were stronger than that of control group (P<0.05), without significant difference found between the L-glutamate group and IL-1beta plus L-glutamate group in the expression of AC, the latent period and the severity of seizure. When IL-ra were given (i.c.v.) first, there was no epileptic activity and the expression of AC did not increase. There were no differences in the expression of AC of rats with IL-1ra and that of control rats. But when 2-methyl-2-(carboxycyclopropyl) glycine (MCCG) was given (i.c.v.) first, the strongest expression of AC, the shortest latent period and the the most serious seizure activities were observed. The results indicated that IL-1beta could facilitate the onset of epilepsy induced by L-glutamate through IL-1R, metabotropic glutamate receptors might work with IL-1R and the increased expression of AC might be involved in the process.
Adenylyl Cyclases
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biosynthesis
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genetics
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Animals
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Glutamic Acid
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Hippocampus
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metabolism
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Interleukin-1
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pharmacology
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Male
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Neocortex
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metabolism
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Rats
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Seizures
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chemically induced
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enzymology
8.NLRC4 plays a regulatory role in F. nucleatum-induced pyroptosis in macrophages.
Wei Yi JIANG ; Zi Long DENG ; Wang Hong ZHAO
Journal of Southern Medical University 2022;42(10):1560-1565
OBJECTIVE:
To explore the mechanism of F.nucleatum-induced pyroptosis in macrophages and the regulatory role of inflammasomes.
METHODS:
Lactate dehydrogenase (LDH) cytotoxicity assay and Hoechst 33342/PI double fluorescence staining were used to analyze cytolysis in F.nucleatum-infected macrophage RAW264.7 cells.The expressions of pyroptosis-related proteins caspase-1, GSDMD and IL-1β were determined using Western blotting.Inflammasome activation in the cells was analyzed by detecting the mRNA expressions of NLRP3, NLRC4, AIM2, and NLRP1 with qRT-PCR.RNA interference technique was used to knock down the key molecules involved in pyroptosis regulation in the macrophages, and the pyroptosis and necrosis rates of the cells following F.nucleatum infection were examined.
RESULTS:
The results of LDH cytotoxicity assay and double-fluorescence staining showed that F.nucleatum infection caused swelling and lytic cell death in RAW264.7 cells.F.nucleatum infection resulted in the activation of caspase-1 and GSDMD and upregulated IL-1β expression in a multiplicity of infection (MOI)-and time-dependent manner (P < 0.05).qRT-PCR revealed significantly increased expression of NLRC4 mRNA in the macrophages after F.nucleatum infection (P < 0.05).NLRC4 silencing by siRNA strongly inhibited the activation of caspase-1/GSDMD pathway and reduced cell death (P < 0.05) and IL-1β expression in F.nucleatum-infected cells.
CONCLUSION
NLRC4 inflammasome drives caspase-1/GSDMD-mediated pyroptosis and inflammatory signaling in F.nucleatum-infected macrophages, suggesting the potential of NLRC4 inflammasome as a therapeutic target for F.nucleatum infections.
Pyroptosis/genetics*
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Inflammasomes/metabolism*
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Caspase 1/metabolism*
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Interleukin-1beta/metabolism*
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Macrophages/metabolism*
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RNA, Messenger/metabolism*
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NLR Family, Pyrin Domain-Containing 3 Protein/metabolism*
9.Mechanism of total flavonoids of Rhododendra simsii in alleviating ischemic brain injury.
Chen-Chen JIANG ; Lei SHI ; Xin-Ya ZHAO ; Hui ZHANG ; Zi-Xu LI ; Jia-Jun LU ; Yu-Xiang HE ; Di CAO ; Hao-Ran HU ; Jun HAN
China Journal of Chinese Materia Medica 2023;48(2):455-464
This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.
Animals
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Male
;
Rats
;
Apoptosis
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Brain Ischemia/metabolism*
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Caspase 3
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Interleukin-1
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Interleukin-6
;
Rats, Sprague-Dawley
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Reperfusion Injury/metabolism*
;
Tumor Necrosis Factor-alpha/genetics*
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Flavonoids/pharmacology*
;
Rhododendron/chemistry*
10.Characteristics of mRNA level of interleukin 12 and intercellular adhesion molecule 1 and monocyte chemotactic protein 3 in human nasal epithelial cells.
Xuewei ZHU ; Dongdong ZHU ; Kewei SUN ; Zhen DONG
Journal of Clinical Otorhinolaryngology Head and Neck Surgery 2008;22(23):1068-1070
OBJECTIVE:
To investigate the mRNA level of IL-12, ICAM-1 and MCP-3 in human nasal epithelial cells.
METHOD:
Firstly, human primary nasal epithelial cells were cultured, and then 4 pairs of primers were designed for detecting mRNA level of IL-12, ICAM-1 and MCP-3. The 938 bp PCR products of GAPDH were used as internal standards. The mRNA expression levels of IL-12, ICAM-1 and MCP-3 in primary nasal epithelial cells was measured with semi-quantitative reverse transcription-polymerase chain reaction.
RESULT:
The round or irregular primary nasal epithelial cells were observed sticking to the bottom of cell culture plates under 400 times optical microscope. The expressions of IL-12 p35, ICAM-1 and MCP-3 mRNA were found in primary nasal epithelial cells while IL-12 p40 subunit was not detected.
CONCLUSION
IL-12 p35, ICAM-1 and MCP-3 mRNAs are expressed in primary nasal epithelial cells, whereas effective IL-12 with integrity is not present in nasal epithelial cells.
Cells, Cultured
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Chemokine CCL7
;
metabolism
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Epithelial Cells
;
metabolism
;
Humans
;
Intercellular Adhesion Molecule-1
;
metabolism
;
Interleukin-12 Subunit p35
;
metabolism
;
Nasal Mucosa
;
cytology
;
metabolism
;
RNA, Messenger
;
genetics