Label-free quantitative proteomic analysis of serum exosomes in mice with thoracic aortic aneurysm.

OBJECTIVE: Thoracic aortic aneurysm (TAA) is a cardiovascular disease with high morbidity and mortality. However, the causes and mechanisms of TAA are not fully understood. Serum exosomes from mice with TAA were used to explore the markers associated with this disease. METHODS: C57BL/6 mice were divided into three groups and given ordinary drinking water, ordinary drinking water plus a saline osmotic pump, or drinking water containing ß-aminopropionitrile (BAPN) (1 g/kg/d) plus an angiotensin II (Ang II) (1 µg/kg/min) osmotic pump. Haematoxylin and eosin staining of thoracic aortic tissues was performed. The basic characteristics of exosomes were analysed. Differentially expressed proteins (DEPs) were identified by LC‒MS/MS. Protein‒protein networks and enrichment analysis were used to explore possible molecular mechanisms. RESULTS: The present study elucidated the protein expression profile of serum exosomes in mice with TAA induced by BAPN combined with Ang II. In this work, the expression of a total of 196 proteins was significantly dysregulated in serum exosomes of mice with TAA, with 122 proteins significantly upregulated and 74 proteins markedly downregulated. Notably, Haptoglobin (Hp) and Serum amyloid p-component (Sap) identified based on the PPI network were significantly upregulated and have been strongly linked to cardiovascular disease. Interestingly, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the upregulated and downregulated proteins were involved in the complement and coagulation cascade pathways. CONCLUSIONS: This study showed that the identified DEPs have potential as biomarkers for the diagnosis of TAA and provided a more comprehensive understanding of the pathophysiological mechanisms of TAA.

[CpG ODN synergistically promotes proliferation and migration of mouse RAW264.7 macrophages induced by lipopolysaccharide].

Objective To investigate the effects of CpG oligodeoxynucleotide (CpG ODN) on the proliferation and migration of macrophages induced by lipopolysaccharide (LPS) and its mechanism. Methods In vitro inflammatory cell model was established by 1 mg/L LPS added into the culture medium of mouse RAW264.7 macrophages. CCK-8 assay was performed to assess the effect of CpG ODN (500 nmol/L) on the proliferation of macrophages induced by LPS; TranswellTM assay was used to measure the effect of CpG ODN (500 nmol/L) on the migration of macrophages induced by LPS. Western blot analysis was performed to detect the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK), c-Jun N-terminal kinase (JNK), extracellular regulated protein kinases (ERK) and nuclear factor kappa-B subunits 65 (NF-κBp65). The specific inhibitors for p38MAPK (SB203580), JNK (SP600125), ERK (PD98059) and NF-κBp65 (BAY11-7082) were used to further explore the possible mechanism underlying the effects of CpG ODN. Real-time PCR was used to detect the role of CpG ODN in LPS-induced transcription of COX2 and MCP-1 in macrophages. Results CpG ODN synergistically enhanced the proliferation and migration of LPS-stimulated macrophages and promoted the transcription of COX2 and MCP-1. It also selectively enhanced the phosphorylation of JNK and ERK proteins in MAPK signaling pathway. To blockage JNK and ERK signaling pathways with its specific inhibitors dramatically inhibited the effects of CpG ODN. Conclusion CpG ODN can synergistically promote the proliferation and migration of LPS-stimulated macrophages through JNK and ERK pathway as well as the transcription of COX2 and MCP-1.

CpG-Oligodeoxynucleotides Alleviate Tert-Butyl Hydroperoxide-Induced Macrophage Apoptosis by Regulating Mitochondrial Function and Suppressing ROS Production.

Oxidative stress and mitochondrial dysfunction are related to disease pathogenesis. Oligodeoxynucleotide containing CpG motifs (CpG ODN) demonstrate possibilities for immunotherapy applications. The aim of the present work is to explore the underlying mechanism of the cytoprotective function of CpG ODN by employing the oxidative stress modulation in immune cells. We used the imaging flow cytometry to demonstrate that tert-butyl hydroperoxide (t-BHP) induces mitochondrial-mediated apoptosis and ROS production in RAW264.7 cells. After pretreatment with CpG ODN, the percentage of apoptotic cells and ROS production was both markedly reduced. The decrease in mitochondrial membrane potential (MMP) induced by t-BHP was partially reversed by CpG ODN. The t-BHP induced upregulation of the expression of apoptosis-related proteins (cleaved-caspase 3, cleaved-caspase 9, cleaved-PARP, and bax) was notably decreased in the presence of CpG ODN. Furthermore, we found that CpG ODN enhanced phosphorylation of ERK1/2 and Akt to inhibit ROS production. In conclusion, the protective effect of CpG ODN in mitigation of t-BHP-induced apoptosis is dependent on the reduction of ROS.