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ObjectiveTo investigate the effects of protein phosphatase 2A (PP2A) inhibitors on the viability of pancreatic cancer cell line PANC-1 and its mechanism.MethodsPANC-1 cells were treated with PP2A inhibitors Cantharidin or Okadiac acid.The activity degree of NF-κB pathway was tested by Western blot.NF-κB pathway was blocked from all sectors by PP2Acα plamid transfection,NF-κB inhibition of protein kinase α (IKKα) and NF-κB inhibitor α (IκBα) dominant negative mutant and p65 interfering plasmid.Cell viability was determined by MTT.ResultsPP2A inhibitors could induce phosphorylation of IKKα,further phosphorylation of IκBα and degradation and followed by the release of p65 into nucleus.When PP2Acα,IKKα dominant negative mutant and IκBα dominant negative mutant were overexpressed,or p65 was interfered,the inhibition rate of Cantharidin on cell viability decreased (31.85±13.37) ％,(23.48±8.98)％,(22.63±5.81)％ and (20.88±3.24)％respectively,and the inhibition rate of Okadiac acid on cell viability decreased (40.17 ± 11.65)％,(27.34±14.28)％,(24.85±3.39)％ and (27.08±3.81)％ respectively.ConclusionsPP2Ainhibitors play a role in preventing pancreatic cancer through PP2Acα/IKKα/IκBα/p65 pathway.
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Objective To explore the dynamic expressions of interleukin-1 (IL-1)receptor associated kinase (IRAK)-M and IRAK-4 in rats with or without endotoxin tolerance (ETT)in acute liver failure (ALF).Methods Sixty-six male SD rats were divided into three groups:ALF group,ETT group and control group.The rats in ETT group received daily lipopolysaccharide (LPS)intraperitoneal injection for 5 days,while the rats in ALF group received daily injection with same volume of 0.75% NaCl solution.Both ETT group and ALF group received intraperitoneal injection with D-galactosamine (D-GalN)and LPS at 24 hours after the 5th injection of LPS or NaCl solution.At 2,6,12,24 and 48 h after D-GalN and LPS injection,the serum levels of tumor necrosis factor-α (TNF-α)and interleukin-6 (IL-6)were detected by enzyme-linked immunosorbent assay (ELISA).The liver pathologic changes were observed with HE staining by microscope.The mRNA expressions of IRAK-4,IRAK-M and NF-κB (p65)were detected by reverse transcriptase polymerase chain reaction (RT-PCR).The pairwise comparison between groups was done by lease significant difference (LSD)and Dunnet's t test.Results The liver pathologic changes in ETT group were much milder than those of ALF group.In ALF group,IRAK-4 mRNA/β-actin absorbance ratios at 2,6,12,24and 48 h after D-GalN and LPS challenge were 0.711 ±0.074,0.904±0.118,1.012 ±0.098,1.534±0.279 and 1.451±0.290,respectively,while the IRAK-M mRNA/β-actin absorbance ratios were 0.496±0.018,0.516±0.089,0.503±0.023,0.503±0.057 and 0.469±0.142,respectively.In ETT group,the IRAK-4 mRNA/β-actin absorbance ratios at 2,6,12,24 and 48 h after D-GalN and LPS challenge were 0.619±0.083,0.587±0.033,0.623±0.034,0.720±0.044 and 0.654±0.041,respectively,while the IRA'K-M mRNA/β-actin absorbance ratios were 0.929 ± 0.064,1.111±0.138,1.113±0.027,1.891±0.315 and 1.710±0.303,respectively.The IRAK-M mRNA expression level was significantly increased and IRAK-4 mRNA expression level was relatively decreased in ETT group compared to ALF group.The differences were all statistically significant at 2,6,12,24 and 48 h after D-GalN and LPS challenge (F= 17.305,54.921,121.031,67.607,55.279,respectively; F=19.506,43.777,110.823,302.681,202.822,respectively; F=172.003,59.519,987.055,68.463,96.601,respectively; all P＜0.05).Conclusion LPS pretreatment may induce ETT in rat model by downregulating the expression of IRAK-4 and upregulating the expression of IRAK-M.
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Objective To investigate the effect of corticotrophin-releasing factor (CRF) on the regulation of Toll-like receptor 4/NF-κB signaling pathway expression in human intestinal epithelial cell line HT-29. Methods HT-29 cells were divided into four groups, normal control group, LPS group (LPS 20 μg/ml stimulated for 24 h), CRF group (CRF 20 ng/ml stimulated 24 h) and CRF+ LPS group (CRF incubated for 12 h then changed to LPS for another 12 h). After stimulation, the expression of TLR4 mRNA of each group was examined by reverse transcriptase polymerase chain reaction (RT-PCR). Total cell protein were extracted and the expression of TLR4 and NFκB p65 at protein level were detected by western blotting.Cell culture supernatant was collected and the secretion of interleukin-8 was detected by enzyme-linked immunoasorbent assay (ELISA). Results The expression of TLR4 in LPS group at mRNA and protein level were 0.31±0.04 and 0.48±0.17,there was no significant difference compared with normal control group (0.28±0.02 and 0.45±0.12,t＝0.216 and 0.712 , P＞0.05 ). In CRF group which were 1.05±0.06 and1. 08±0.21, significantly higher than normal control group (t＝3.721 and 3.802, P＜0.05). In CRF+LPS group which were 1.68±0.05 and 1.81±0. 18,significantly higher than CRF group (t＝4. 816 and 3. 918, P＜0.05).The results of NF-κB p65 expression at protein level and interleukin-8 expression of cell culture supernatant were consistent with the results of TLR4 expression at mRNA and protein level.Conclusion CRF not only activate TLR4/NF-κB signaling pathway in human intestinal epithelial cell,but enhance the reaction of intestinal epithelial cell to LPS as well, which resulting in increased interleukin-8 secretion.
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Objective To study the signal transduction mechanism of nicotine induced periodontal ligament fibroblasts (PDLFs) apoptosis. Methods This study used 1 μg/ml, 10μg/ml and 100μg/ml nicotine to intervene PDLFs cells for 24h separately. NF-κB, p53, I-κB and Caspase3 expression were detected. Results After Nicotine was done on PDLFs cells for 24h, the transcription of p53, and Caspase3,and the translation of Caspase 3 protein were increased, while NF-κB was decreased. At the same time, the transcription of NF-κB decreased gradually with the concentration of nicotine increased ( r = 0. 707, F =33. 705, P ＜0. 01 ), nevertheless, I-κB was reversed ( r =0. 964, F =374. 883, P ＜0. 01 ). p53 expression was increased gradually with the concentration of nicotine increased ( r =0. 957, F = 153. 377, P ＜0. 01).Both Caspase3 mRNA (r =0.935, F =318.371, P ＜0.01) and protein (r =0.677, F =8. 459, P ＜ 0. 05 )increased gradually. Conclusion Nicotine induced PDLFs apoptosis was mediated through NF-κB and p53 pathway.