| Objective:Phosphatidylinositol 3-kinase protein family (PI3Ks) is closely related with the immune response, the expression deletion of its regulatory subunits and catalytic subunits or the application of the corresponding PI3Ks inhibitors can have a significant impact on the immune response. Among them, PI3Ks regulatory subunit of IA could connect with the corresponding catalytic subunit to form dimer structure via its i-SH2 domain, and then to control kinase activity of the catalytic subunit. The regulation of PI3Ks subunit of IA on the immune response, particularly on the inflammatory response, is controversial, and this paper aimed to disclose the regulation of protein p55PIK on inflammatory response, and preliminary studied the regulation mechanism of protein p55PIK on inflammatory reaction.Methods:To construct adenovirus vector for protein p55PIK expression to express protein p55PIK highly in HaCat cells; siRNA specific to protein p55PIK was transfected THP1 cells after being induced by PMA, to decrease the expression of protein p55PIK. The expression of protein p55PIK was affected alone or combined with the stimulation with LPS again, and the transcription levels of corresponding proinflammatory cytokines, including TNF-α, IL-1βand IL-6, were detected by Q-PCR; and the protein levels of corresponding proinflammatory cytokines, including TNF-α, IL-1βand IL-6, were detected with ELISA; and the activations of NFκB signaling pathway, PI3K/AKT pathway, IKK pathway, and MAPK signaling pathway were detected by Western Blot.Results:Adenovirus vector for protein p55PIK expression was constructed, and siRNA specific to protein p55PIK was screened successfully. p55PIK was expressed highly in HaCat cells alone or combined with LPS stimulation again could significantly promote the transcription and translation of inflammatory cytokines (P<0.05), and could activate NFκB signaling pathway and PI3K/AKT pathway, but had significant effect on IKK signaling pathway and MAPK pathway; p55PIK was expressed lowly in THP1 cells alone or combined with LPS stimulation again could significantly inhibit the transcription and translation of inflammatory cytokines (P＜0.05), and could inhibit the activation of NFkB signaling pathway and PI3K/AKT pathway, but had no obvious effect on IKK signaling pathway and MAPK pathway.Conclusion:p55PIK can promote the transcription and translation of inflammatory cytokines in immune cells by activating NFκB signaling pathways. High expression of p55PIK can promote the transcription and translation of inflammatory cytokines induced by LPS and the activation of NFκB and PI3K/AKT signaling pathways. Low expression of p55PIK can inhibit the transcription and translation of inflammatory cytokines induced by LPS, as well as can reduce the activation of NFκB and PI3K/AKT signaling pathways. Objective:To preliminarily study the relation of protein p55PIK with the regulation of inflammation.Methods:Vector pcDNA3.1-p55PIK expressing protein p55PIK highly was constructed and NFκB RelA reporter gene vector was constructed. The effect of protein p55PIK on the transcriptional activity of NFκB RelA was observed by NFκB RelA reporter gene vector being transfected in Hela cells with low or high expression of protein p55PIK. Protein p55PIK was highly expressed in Hela cells by pcDNA3.1-p55PIK, and protein p55PIK was highly expressed in Hela cells by p55PIK adenovirus vector. Nuclear and cytoplasmic components in cells were separated by nuclei and cytoplasm separation kit, and the expression of protein p55PIK and the activation of NFκB and PI3K/AKT signaling pathways in nuclei and cytoplasm were detected by Western Blot. The possible proteins belonged to NFκB signaling pathway and combined with protein p55PIK in immunologicprocess were detected by co-IP assay.Results:Vector pcDNA3.1-p55PIK expressing protein p55PIK highly was constructed, and NFκB RelA reporter gene vector was constructed successfully. Protein p55PIK could promote the transcriptional activity of NFκB RelA, and could positively regulate the transcriptional activation of NFκB RelA induced by LPS. High expression of protein p55PIK in Hela cells could promote the degradation of cytoplasmic protein IKBa, and high expression of protein p55PIK in HaCat cells could promote the degradation of nuclear protein IKBa while promote the increase of the phosphorylation of cytoplasmic NFκB p65. Immune process such as pV, PMA treatment on cells may promote combination of protein p55PIK and NFκB signaling pathways protein NFκB p65.Conclusion:Protein p55PIK can promote the transcriptional activity of NFκB RelA,positively regulating inflammatory reaction. The possible interaction between protein p55PIK and NFκB signaling pathways protein NFκB p65 can be detected during immune process such as pV, PMA treating. Objective:The effects of pretreatment with penetrating fusion peptide IP55 on the production of corresponding proinflammatory cytokines in immune cells (monocyte-macrophage lineage and primary monocytes and macrophages) and the activation of related signaling pathways were observed. The effect of pretreatment with penetrating fusion peptide IP55 on the release of proinflammatory cytokines in immune cells induced by LPS was observed. To preliminarily explore the therapeutic effect of penetrating fusion peptide IP55 on inflammatory in vivo.Methods:Prokaryotic expression vector of penetrating fusion peptide IP55 was constructed, and penetrating fusion peptide IP55 purified with the technology of protein purification and endotoxin removal was to affect on monocyte-macrophage lineage and primary monocytes and macrophages, then the effect on the productions of correspondin cytokines as TNF-α, IL-6 and IL-1βwith the changes of corresponding signaling pathways were observed. The protective effect of preventive treatment with penetrating fusion peptide IP55 on acute liver injury induced by LPS was observed with animal experiment.Results:Prokaryotic expression vector pTAT-HA of penetrating fusion peptide IP55 was constructed successfully. In hematological tumor cell line Jurkat and solid tumor cell line Hela, IP55 could enter cells efficiently, and inhibit cell cycle progression of tumor cells, and inhibit intracellular DNA synthesis; in monocyte-macrophage lineage THP1, pretreatment with penetrating fusion peptide IP55 had no significant effect on the production of proinflammatory cytokines (P>0.05), without activating NFκB signaling pathway, but could significantly inhibit the production of cytokines as TNF-α, IL-6 and IL-1βin monocyte-macrophage lineage THP1 induced by LPS (P<0.05), and partially inhibit the activation of NFκB signaling pathway; in primary monocytes and macrophages, it could partially inhibit the generation of proinflammatory cytokine TNF-a in monocytes and macrophages THP1 induced by LPS (P<0.05), but the effect on the other pro-inflammatory factors had no statistical significance (P>0.05). In vivo, penetrating fusion peptide IP55 could partially inhibit nuclear translocation of NFκB p65 in liver cells induced by LPS, and reduce the mortality of mice caused by acute liver injury induced by LPS.Conclusion:penetrating fusion peptide IP55 can effectively entry the cells and inhibit cell cycle progression of tumor cells and inhibit DNA synthesis; pretreatment with penetrating fusion peptide IP55 can significantly inhibit the production of cytokines as TNF-α, IL-6 and IL-1βin monocyte-macrophage lineage induced by LPS; pretreatment with penetrating fusion peptide IP65 can partially inhibit the generation of proinflammatory cytokine TNF-a in monocytes and macrophages THP1 induced by LPS; in the mice acute liver injury model induced by LPS, pretreatment with penetrating fusion peptide IP55 could partially inhibit nuclear translocation of NFκB p65 in liver cells induced by LPS and reduce the mortality of mice caused by acute liver injury. |
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