title: Effects of the 24 N-terminal amino acids of p55PIK on endotoxinstimulated release of inflammatory cytokines by HaCaT cells.
abstract: This study aimed to examine the effect of the 24 N-terminal amino acids (N24) of p55PIK, a regulatory subunit of phosphatidylinositol 3-kinase (PI3K), on the endotoxin lipopolysaccharide (LPS)-stimulated release of the cytokines (CKs) by HaCaT cells. The fusion protein, trans-acting activator of transcription (TAT)-N24 (an experimental peptide, EP) containing the N24 of PI3K-p55PIK, was constructed, and TAT-N24 fusion peptide was expressed and identified in BL21 E·coli. HaCaT cells (a human keratinocyte cell line) was cultured and stimulated by LPS at 100 ng/mL for 1, 2, 4, 8, 16 or 24 h, or by LPS at 10, 100 ng/mL, 1, 10 or 100 μg/mL of for 4 h. Changes in the protein and mRNA levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) released by HaCaT cells following EP intervention were determined by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Immunofluorescence confocal laser scanning microscopy was utilized to detect the protein expression and translocation of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB p65) in HaCaT cells. The expression of the NF-κB inhibitor alpha (IκB-α) protein in LPS-stimulated HaCaT cells after the EP intervention was measured by Western blotting. The results showed that EP treatment increased TNF-α secretion from HaCaT cells. EP at certain concentrations could effectively inhibit the LPS-stimulated release of TNF-α, IL-6 and IL-8 from HaCaT cells. The ELISA assay demonstrated that the concentrations of TNF-α, IL-6 and IL-8 in the supernatants of LPS-stimulated cells were reduced from 208.06±30.18, 86.4±9.78 and 260.59±54.05 pg/mL to 121.78±22.26, 53.18±7.36 and 125.08±35.17 pg/mL, respectively, in the supernatants of cells treated by LPS and EP combined. Real-time PCR also revealed that the expression of the three pro-inflammatory CKs was significantly decreased after EP intervention. Immunofluorescence confocal laser scanning microscopy showed that NF-κB p65 protein was primarily expressed in the cytoplasm of non-stimulated HaCaT cells. After LPS stimulation, NF-κB p65 was translocated into the nucleus, and the nuclear expression of this protein increased. The nuclear NF-κB p65 protein expression was inhibited after the addition of EP. Western blotting showed that IκB-α expression began to decrease 30 min after LPS stimulation and declined to a trough 4 h later. IκB-α expression began to gradually recover 16 h after LPS stimulation but remained at a lower-than-normal level at 24 h. Greater IκB-α expression was found in cells treated with LPS and EP combined than those treated with LPS alone. It was concluded that EP can effectively inhibit the LPS-stimulated expression of TNF-α, IL-6, and IL-8, which involves the inhibition of the hydrolysis of IκB-α and thereby blockage of the nuclear translocation of NF-κB p65.
authors.affiliation: Department of Breast Surgery, Henan Provincial People's Hospital, The People's Hospital of Zhengzhou University, Zhengzhou, 450003, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Lv
authors.affiliation: Department of Breast Surgery, Henan Provincial People's Hospital, The People's Hospital of Zhengzhou University, Zhengzhou, 450003, China.
authors.forename: Wei
authors.initials: W
authors.lastname: You
authors.affiliation: Department of Breast Surgery, Henan Provincial People's Hospital, The People's Hospital of Zhengzhou University, Zhengzhou, 450003, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Department of Molecular Medical Center, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors.forename: Jun-Bo
authors.initials: JB
authors.lastname: Hu
authors.affiliation: Department of Breast Surgery, Henan Provincial People's Hospital, The People's Hospital of Zhengzhou University, Zhengzhou, 450003, China. zhangbin841919@gmail.com.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. wangjhxh@163.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D016207:Cytokines; D004731:Endotoxins; D019869:Phosphatidylinositol 3-Kinases; C523442:PIK3R3 protein, human
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