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 mu g/mL of for 4 h. Changes in the protein and mRNA levels of tumor necrosis factor-alpha (TNF-alpha), 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-kappa B p65) in HaCaT cells. The expression of the NF-kappa B inhibitor alpha (I kappa B-alpha) protein in LPS-stimulated HaCaT cells after the EP intervention was measured by Western blotting. The results showed that EP treatment increased TNF-alpha secretion from HaCaT cells. EP at certain concentrations could effectively inhibit the LPS-stimulated release of TNF-alpha, IL-6 and IL-8 from HaCaT cells. The ELISA assay demonstrated that the concentrations of TNF-alpha, 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-kappa B p65 protein was primarily expressed in the cytoplasm of non-stimulated HaCaT cells. After LPS stimulation, NF-kappa B p65 was translocated into the nucleus, and the nuclear expression of this protein increased. The nuclear NF-kappa B p65 protein expression was inhibited after the addition of EP. Western blotting showed that I kappa B-alpha expression began to decrease 30 min after LPS stimulation and declined to a trough 4 h later. I kappa B-alpha expression began to gradually recover 16 h after LPS stimulation but remained at a lower-than-normal level at 24 h. Greater I kappa B-alpha 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-alpha, IL-6, and IL-8, which involves the inhibition of the hydrolysis of I kappa B-alpha and thereby blockage of the nuclear translocation of NF-kappa B p65.