الملخص
ObjectiveTo preliminarily explore the mechanism of Shufeng Tongluo prescription (SFTLP) in inhibiting airway inflammation in asthma mice by affecting the expression levels of eotaxin in the serum, CC type chemokine receptor 3 (CCR3), and extracellular signal-regulated kinase (ERK) phosphorylation in lung tissues. MethodSeventy C57BL/6 mice were randomly divided into a blank group, a model group, low-, medium-, and high-dose SFTLP groups (7.75, 15.5, 30 g·kg-1), a pertussis toxin (PTX) group, a CCR3 inhibitor (SB328437) group, a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) group, a p38 protein kinase antagonist inhibitor (SB203580) group, and an ERK inhibitor (PD98059) group. The asthma model was induced in mice by intraperitoneal injection of ovalbumin (OVA) and aluminum hydroxide [Al(OH)3] combined with OVA atomization (0.2 mL for all). After modeling, hematoxylin-eosin staining (HE staining) was used to observe the inflammatory infiltration of lung tissues in mice. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of eotaxin [CC chemokine ligand (CCL) 11 and CCL24) in each group. Western blot was used to detect the levels of ERK phosphorylation and CCR3 in lung tissues. ResultCompared with the blank group, the model group showed obvious bronchial constriction, lumen stenosis, damaged alveolar structure, massive inflammatory cell infiltration in lung tissues, mucous plug in the bronchus, edema in the submucosal tissues of the trachea, increased folds, increased serum levels of CCL11 and CCL24 (P<0.01), and increased expression of CCR3 protein in lung tissues (P<0.05). The ERK levels in lung tissues of the model group and the PTX group increased (P<0.05). The level of p-ERK in lung tissues of the model group and the low-dose SFTLP group increased (P<0.05). As revealed by pathological results, compared with the model group, the high-dose SFTLP group showed relieved lung lesions. The high-dose SFTLP group and the SB328437 group showed reduced CCL11 content (P<0.05). The low- and high-dose SFTLP group, the PTX group, the SB203580 group, the PD98059 group, and the SB328437 group showed decreased CCR3 protein expression in lung tissues (P<0.05). The high-dose SFTLP group and the PD98059 group showed reduced p-ERK level (P<0.05). The PD98059 group showed reduced ERK level (P<0.05). ConclusionSFTLP can inhibit airway inflammation in asthma, and the mechanism may be related to the inhibition of eosinophil activation by down-regulating CCR3 and CCL11 expression and ERK phosphorylation.
الملخص
<p><b>OBJECTIVE</b>To identify ubiquitinated proteins from complex human multiple myeloma (MM) U266 cells, a malignant disorder of differentiated human B cells.</p><p><b>METHODS</b>Employing a globally proteomic strategy combining of immunoprecipitation, LC-MS/MS and SCX-LC-MS analysis to identified ubiquitination sites, which were identified by detecting signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da).</p><p><b>RESULTS</b>In total, 52 ubiquitinated proteins containing 73 ubiquitination sites of which 14 and 59 sites contained LRGG-tag and GG-tag were identified, respectively.</p><p><b>CONCLUSION</b>Classification analysis by of the proteins identified in the study based on the PANTHER showed that they were associated with multiple functional groups. This suggested the involvement of many endogenous proteins in the ubiquitination in MM.</p>
الموضوعات
Humans , Cell Line, Tumor , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Physiology , Multiple Myeloma , Metabolism , Neoplasm Proteins , Genetics , Metabolism , Proteomics , Methods , Ubiquitinationالملخص
We cloned human U6 promoter from pAVU6 + 27 vector into pXSN to transcripe small RNA. Meanwhile, a shRNA targeting GDF-8 was cloned down-stream of the hU6 promoter to construct recombinant vector. Then the packing cell GP-293 was co-transfected the recombinant with pVSV-G to gernarate virus particle. Resistant C2C12 cell pools were screened using G418. Levels of mRNA and protein of GDF-8 were tested by Real-Time PCR and western blotting. Cell proliferation and cell cycle were analyzed using MTT and FACS. The expression of GDF-8 was dramatically decreased by the retrovirus-based system in C2C12 cells. Cells proliferated effectively after integrating the recombinant. The cells in G0/G1 phase decreased by 13.7%, while cells in S phase increased by 14.9%. In conclusion, the retrovirus-based RNAi could be used to stably silence GDF-8. It can be a powerful tool in curing muscle atrophy.