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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.