Role of apolipoprotein O in autophagy via the p38 mitogen-activated protein kinase signaling pathway in myocardial infarction

Abstract Objectives To explore the role and possible mechanisms of action of apolipoprotein O (APOO) in autophagy in Myocardial Infarction (MI) in vivo and in vitro. Methods Differential gene expression and single Gene Set Enrichment Analysis (GSEA) were used to evaluate MI-related candidate genes. Animal and cell MI models were established. Sh-APOO, si-APOO, and SB203580 were used to inhibit the expression of APOO or p38MAPK. Western blot and qRT-PCR were used to analyze the expression levels of the target protein or mRNA. Apoptosis was observed using the TUNEL assay. The plasma concentrations of CK-MB and cTn-I in humans and mice were determined. Results In the GSE23294 dataset, APOO mRNA was highly expressed in the left ventricle of mice with MI; GSEA revealed that APOO was positively correlated with p38MAPK, autophagy, and apoptosis. The plasma concentration of APOO in patients with MI was significantly higher than that in healthy subjects. The expression of APOO, Beclin-1, LC3, and Bax in mouse and AC16 cell MI models increased, while the level of Bcl-2 decreased. After silencing the APOO gene, the expression of APOO was downregulated; meanwhile, changes in autophagy, apoptosis and myocardial cell injury were reversed in vivo and in vitro. Furthermore, autophagy was alleviated after AC16 cells were treated with SB203580. Conclusions The increased APOO expression in mouse and cell MI models may activate autophagy and apoptosis by regulating the p38MAPK signaling pathway, thus aggravating the myocardial injury. HIGHLIGHTS APOO was highly expressed in the left ventricle of mice with myocardial infarction. Increasing of APOO may activate autophagy and apoptosis in myocardial infarction. The regulation of APOO in autophagy and apoptosis was regulated by p38MAPK signaling pathway.

Construction and expression of recombinant fusion protein of thioredoxin-ApoO / 中南大学学报(医学版)

Objective To construct human apolipoprotein O (apolipoprotein O, ApoO) expression vector and obtain recombinant fusion protein thioredoxin (Trx)-ApoO by pET prokaryotic expression system. Methods The ApoO gene fragment from the human liver cDNA library was amplified by PCR. The resulting product was cloned into pET-32a(+) vector and sequenced. The confirmed cDNA was cloned into plasmid E.coli DH10B and then transformed into E.coli BL 21 (DE3) where it was induced to express protein by isopropyl β-D-1-thiogalactopyranoside (IPTG).The fusion protein was purified by Ni-NTA resin. Results The ApoO gene was cloned by PCR and a 519 bp DNA fragment was shown on the agarose electrophoresis. The cloned gene was sequenced and demonstrated to have the same sequence as that of human ApoO gene in GenBank which justified a successful construction of recombinant plasmid. ApoO cDNA gene fragment was induced by IPTG, and a 34 kD recombinant fusion protein Trx-ApoO was tested on sodium dodecyl sulfate polyacrylamide (SDS-PAGE). Conclusion Human ApoO gene is successfully cloned and its recombinant fusion protein Trx-ApoO is expressed.