The Change of Nuclear Factor-κB of Inflammation Model in Lung and the Effect of Eucalyptus Globules Oil and α-pinene on Activation of Nuclear Factor-κB
|Keywords||Nuclear factor -kB Inflammation of the lungs Globulus oil α a spinach ene THP- cell lines IkBα Laser scanning confocal microscope Western blot electrophoresis analysis Double antibody sandwich enzyme-linked rabbit plague assay|
Preface: most of the diseases of the respiratory system and immune inflammatory response is closely related, such as acute and chronic lung injury, asthma, chronic obstructive pulmonary disease (COPD), morbidity and mortality increase day by day. Nuclear factor-κB (NF-κB) as a ubiquitous multifunctional transcription factor in intracellular signal transduction pathways convergence point, not only involved in mediated immune response, cellular stress, apoptosis and proliferation and other related regulation of gene expression, and in the regulation of asthma, lung injury, COPD and other lung diseases related inflammatory response genes play a key role. Many types of cytokines involved in the inflammatory disease of the lungs, and the extremely complex signaling networks, will become, if we can be at a higher level of the overall aspects of the inflammatory factor regulation, rather than on isolated points of the upstream signaling pathways and conquer Look for the new direction of the new anti-inflammatory drugs. We NF-κB as an anti-inflammatory targets to inhibit NF-κB pathological overactivation of goal to find a specific inhibitor of NF-κB activity, the key link in the regulation of the inflammatory response of lung inflammatory diseases intervention, there will be a broad and far-reaching application prospects. The general term of the volatile oil extracted from the blue eucalyptus leaves and camphor tree leaves Eucalyptus globulus leaf extract, also known as blue eucalyptus oil, eucalyptus oil, and blue eucalyptus oil containing 1.8-eucalyptus oil prime, α-pinene , the current study found that eucalyptus oil has anti-inflammatory role, but its anti-inflammatory molecular mechanism is still not clear, in view of NF-κB in lung immunity, inflammation importantly Zhejiang University, a master's degree thesis lung inflammation model lieutenant colonel factor d change and blue by oil and a \u0026 en-activation of its nuclear translocation bit, so we target as an anti-inflammatory effect of NF-h to study blue oil's anti-inflammatory mechanisms. Objective: To establish the lipopolysaccharide (LPS) LPS tracheal instillation of THP-l cells induced inflammation model, low-dose and long-term sulfur dioxide (SO) inhalation-induced rat model of chronic inflammation of the lungs, the observation of the state of activation of the NF-h, explore the Blue Press the oil and its main component. The spinach ene model NF. h nuclear translocation of activated. Methods: Using human blood monocyte cell line THP. l, SD rats as experimental subjects, and the establishment of lipopolysaccharide (LPS) THP. l cells induced inflammation model, a small dose LPSu00 pg) the tracheal instillation long-term SO. Inhalation-induced rat model of chronic lung inflammation in blue, and their respective oil and a. Was ene administration group. Determined using indirect immunofluorescence cytochemistry method combined with laser scanning confocal microscope (LSCM) THP-l cells, alveolar macrophages in the bronchoalveolar lavage fluid of rats (Pulmonary alveolar macrophages, PAMs) to fluorescein isothiocyanate (FITC ) green fluorescent labeled NF-h p65 subunit (NFth/p65) position; use under the conditions of low temperature and suppression of protease the hypotonic lysis membrane extraction cytoplasmic protein, followed by hypertonic lysis nuclear membrane extract nucleoprotein, Folin phenol quantitative protein , and then double antibody sandwich enzyme-linked immunosorbent assay (SandwichELISA) analysis the nucleus within NF-h/p65 level, intracytoplasmic I the Immunoblot electrophoresis (Western.blot),. The Ba level of change, and semi-quantitative analysis. Statistical processing and analysis using the statistical package SPSS 11 knife, statistical data to two d, significant test using one-way ANOVA. Results: 1. Immunofluorescence LSCM analysis showed: @ normal state THP1 cells, rat PAMS NF seven B/p65 fluorescent markers are mainly distributed in the cytoplasmic domain nuclear area, after LPS 30 min stimulated THP-l cells, small doses fluorescently labeled LPS tracheal instillation of 3 W rats PAMS NF-h/p65 concentrated in the nucleus, cytoplasmic domain uncommon. ② After Blue by oil or a of a spinach allyl pretreatment Tm-l cells, LPS 30 min stimulation, NF-h/p65 fluorescent marker is still mainly located in the cytoplasmic domain; tracheal instillation through a small dose of LPS, 3 W within blue oil administration in rats PAMS NF-h/p65 fluorescent labeling is still mainly located in the cytoplasmic domain. 2. The Western seven lot analysis: @ the normal THPl nuclear protein low levels of NF-loB/p65 expression, Zhejiang University master's degree thesis lung inflammation model lieutenant colonel because of a d change and blue oil and a spinach ene its nuclear translocation activated by LPS 30 min stimulated THP * nucleus NF-h/p65 expression was significantly higher (P <0 knife 5 normal rat lung tissue nuclear protein low levels of NF-oh/p65 expression and the tracheal instillation after small doses of LPS and long-term SO inhaled rat lungs nucleus NF-h/p65 expression was significantly increased. @ Blue by oil or a of a spinach ene door, 10,100 pg · L ', 30min) pretreatment inhibited LPS 30min stimulation induced THP nucleus within NF / p65 expressed at high levels, and with the the the Blue by oil or increase in the dose of a-spinach-ene, NF-h/p657k level relatively lower, by statistical analysis, showed a good dose-dependent relationship, which Blue oil 10 pg · L 'and 100 knock · L' group, the team won the ene 100 Hg · L 'group, a statistically significant difference (p <0 knife 5); small doses Lps tracheal instillation, 3w within continuous blue oil Q0, 100,300 mg kg ') administration of rat lung tissue nucleus NF overlooking reduce / p65 expression than the model group, and showed a dose-dependent effect long-term SOZ inhalation , SW sustained blue oil (10 mg · kg-') NF 4 / p65 expression in the nucleus of the administration of rat lung tissue than the model group decreased significantly. ③ o school ene door, 10,100 pg · L-' the 30mifl) the normal cultured the state THpl cell nuclei NF a h/p65 no obvious effect (HO knife 5) the school _AT_ a ene (, 10, 100 pg · L \LPS-induced Itha degradation within the cytoplasm, and by statistical analysis, a dose-dependent relationship, including a one spinach ene 100 Bu g · L-'group had statistically significant difference (P <0.05). 3. Sandwich ELISA analysis showed: SO. Inhalation model NF-h A. . . the nm value normal rats was significantly higher (P <0.05X blue oil 100 mg · kg 'clever group NFth/p65 the A * m values ??with the model group decreased significantly (P <0 knife 5). Conclusion: 1.LPS vitro model of proinflammatory LPS stimulation can cause activation of THP-1 cells of NF-IcB nuclear translocation?