Summary： This study aimed to examine the effect of the 24 N-terminal amino acids （N24） ofp55PIK, 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-ct）, 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-r,B p65） in HaCaT cells. The expression of the NF-kB inhibitor alpha （Iv, B-a） protein in LPS-stimulated HaCaT cells after the EP intervention was measured by Western blotting. The resillts showed that EP treatment increased TNF-a secretion from HaCaT cells. EP at certain concentrations could effectively inhibit the LPS-stimulated release of TNF-a, IL-6 and IL-8 from HaCaT cells. The ELISA assay demonstrated that the concentrations of TNF-a, 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.
This study examined the synergetic effect of class ⅠA Phosphoinositide 3-kinases catalytic subunit p110β knockdown in conjunction with oxaliplatin treatment on colon cancer cells. Down-regulation of p110β by siRNA interference and oxaliplatin treatment were applied in colon cancer cell lines HT29, SW620 and HCT116. MTT assay was used to measure the inhibitory effect of p110β knockdown on the proliferation of colon cancer cell lines. SubG1 assay and Annexin-Ⅴ FITC/PI double-labeling cytometry were applied to detect cell apoptosis. And cell cycle was evaluated by using PI staining and flow cytometry. The expression of caspase 3, cleaved PARP, p-Akt, T-Akt and p110β was determined by western blotting. The results suggested that down-regulation of p110β expression by siRNA obviously reduced cell number via accumulation in G0-G1 phase of the cell cycle in the absence of notablely increased apoptosis in colon cancer cell lines HT29 and SW620 (S phase arrest in HCT116). Moreover, inhibition of p110β expression increased oxaliplatin-induced cell apoptosis and cell cycle arrest in HT29, HCT116 and SW620 cell lines. In addition, increases of cleaved caspase-3 and cleaved PARP induced by oxaliplatin treatment were determined by immunoblotting in p110β knockdown group compared with normal control group and wildtype group. It is concluded that down-regulated expression of p110β could inhibit colon cancer cells proliferation and result in increased chemosensitivity of colorectal cancer cells to oxaliplatin through augmentation of oxaliplatin-induced cell apoptosis and cell cycle arrest.
5-aminosalicylic acid（5-ASA） is drug of choice for the treatment of ulcerative colitis（UC）. In this study, the efficacy of topical versus oral 5-ASA for the treatment of UC was examined as well as the action mechanism of this medication. A flexible tube was inserted into the rat cecum to establish a topical administration model of 2,4,6-trinitrobenzene sulfonic acid（TNBS）-induced UC. A total of 60 rats were divided into sham operation group（receiving an enema of 0.9% saline solution instead of the TNBS solution via the tube）, model group, topical 5-ASA group, oral Etiasa group（a release agent of mesalazine used as positive control） and oral 5-ASA group（n=12 each）. Different treatments were administered 1 day after UC induction. The normal saline（2 mL） was instilled twice a day through the tube in the sham operation group and model group. 5-ASA was given via the tube in the topical 5-ASA group（7.5 g/L, twice per day, 100 mg/kg）, and rats in the oral Etiasa group and oral 5-ASA group intragastrically received Etiasa（7.5 g/L, twice per day, 100 mg/kg） and 5-ASA（7.5 g/L, twice per day, 100 mg/kg）, respectively. The body weight was recorded every day. After 7 days of treatment, blood samples were drawn from the heart to harvest the sera. Colonic tissues were separated and prepared for pathological and related molecular biological examinations. The concentrations of 5-ASA were detected at different time points in the colonic tissues, feces and sera in different groups by using the high pressure liquid chromatography（HPLC）. The results showed that the symptoms of acute UC, including bloody diarrhea and weight loss, were significantly improved in topical 5-ASA-treated rats. The colonic mucosal damage, both macroscopical and histological, was significantly relieved and the myeloperoxidase activity was markedly decreased in rats topically treated with 5-ASA compared with those treated with oral 5-ASA or Etiasa. The mRNA and protein expression of IL-1β, IL-6, and TNF-α was down-
