BMSCs mediates endothelial cell autophagy by upregulating miR-155-5p to alleviate ventilator-induced lung injury.

In this study, we explored to detect the effects and mechanism of bone-marrow-derived mesenchymal stem cells (BMSCs) on ventilator-induced lung injury (VILI). We transplanted BMSCs in mice and then induced VILI using mechanical ventilation (MV) treatment. The pathological changes, the content of PaO2 and PaCO2 , wet/dry weight ratio (W/D) of the lung, levels of tumor necrosis factor-α and interleukin-6 in bronchoalveolar lavage fluid, and apoptosis were detected. The autophagy-associated factor p62, LC3, and Beclin-1 expression were analyzed by western blot. The quantitative polymerase chain reaction was applied to detect abnormally expressed microRNAs, including miR-155-5p. Subsequently, we overexpressed miR-155-5p in VILI mice to detect the effects of miR-155-5p on MV-induced lung injury. Then, we carried out bioinformatics analysis to verify the BMSCs-regulated miR-155-5p that target messenger RNA. It was observed that BMSCs transplantation mitigated the severity of VILI in mice. BMSCs transplantation reduced lung inflammation, strengthened the arterial oxygen partial pressure, and reduced apoptosis and the W/D of the lung. BMSCs promoted autophagy of pulmonary endothelial cells accompanied by decreased p62 and increased LC3 II/I and Beclin-1. BMSCs increased the levels of miR-155-5p in VILI mice. Overexpression of miR-155-5p alleviated lung injury in VILI mice following reduced apoptosis and increased autophagy. Finally, TAB2 was identified as a downstream target of miR-155-5p and regulated by miR-155-5p. BMSCs may protect lung tissues from MV-induced injury, inhibit lung inflammation, promote autophagy through upregulating of miR-155-5p.

Down-regulation of microRNA-155 suppressed Candida albicans induced acute lung injury by activating SOCS1 and inhibiting inflammation response.

Acute lung injury caused by Candida albicans could result in high mortality and morbidity. MicroRNA-155 (miR-155) and suppressor of cytokine signaling 1 (SOCS1) have been believed to play a key in the regulation of inflammatory response. Whether miR-155/SOCS1 axis could regulate the acute lung injury caused by C. albicans has not been reported. The acute lung injury animal model was established with acute infection of C. albicans. miR-155 inhibitor, miR-155 mimic, and sh-SOCS1 were constructed. The binding site between miR-155 and SOCS1 was identified with dual luciferase reporter assay. Knockdown of miR-155 markedly inhibited the germ tube formation of C. albicans. Knockdown of miR-155 significantly up-regulated the expression of SOCS1, and the binding site between miR-155 and SOCS1 was identified. Knockdown of miR-155 improved the acute lung injury, suppressed inflammatory factors and fungus loading through SOCS1. Knockdown of SOCS1 greatly reversed the influence of miR-155 inhibitor on the cell apoptosis in vitro. The improvement of acute lung injury caused by C. albicans, suppression of inflammatory response and C. albicans infection, and inhibitor of cell apoptosis were achieved by knocking down miR-155 through SOCS1. This research might provide a new thought for the prevention and treatment of acute lung injury caused by C. albicans through targeting miR-155/SOCS1 axis.

Inhibition of Candida albicans in vivo and in vitro by antimicrobial peptides chromogranin A-N12 through microRNA-155/suppressor of cytokine signaling 1 axis.

Antimicrobial peptides (AMPs) have proven to inhibit a variety of pathogens. Chromogranin A-N12 (CGA-N12) is a kind of AMP, and it is characterized by stable structure, high anti-Candida activity, and good safety. However, it remains unclear whether CGA-N12 could effectively inhibit the growth of Candida albicans (C. albicans). Colony forming assays were used to measure minimal inhibitory concentration (MIC), minimal fungicidal concentration (MFC), and time-kill curve. Disseminated C. albicans rabbit model was established to investigate the influence of CGA-N12 on histological damage. The protein and mRNA levels of suppressor of cytokine signaling 1 (SOCS1) after treatment were investigated. The MIC and MFC of CGA-N12 against C. albicans was 6 mg/mL. CGA-N12 considerably inhibited germ tube formation of C. albicans. The fungal load in the tissues and inflammatory factors in the serum were suppressed by CGA-N12. CGA-N12 significantly reduced the histological changes caused by C. albicans, and the protein and mRNA levels of SOCS1 were markedly inhibited. The inhibition effect of CGA-N12 on C. albicans and significant improvement of histological damage by CGA-N12 through microRNA-155/SOCS1 axis were proved in this study. This study proposes a novel therapeutic strategy for the treatment and prevention of C. albicans.Abbreviations: AMPs: Antimicrobial peptides; MIC: Minimal inhibitory concentration; MFC: Minimal fungicidal concentration; AIDS: Acquired immune deficiency syndrome; PBS: Phosphate buffer saline; FBS: Fetal bovine serum; ROS: Reactive oxygen species; CFU: Colony formation unit; CGA: Chromogranin A; SOCS1: Suppressor of cytokine signaling 1; SDA: Sabouraud Dextrose Agar; GRAVY: Grand average of hydropathicity; C. parapsilosis: Candida parapsilosis; C. albicans: Candida albicans.

Expression of chromogranin A-derived antifungal peptide CGA-N12 in Pichia pastoris.

The human chromogranin A-derived peptide CGA-N12, which is composed of 12 amino acid residues with the sequence ALQGAKERAHQQ, showed strong antifungal activity and the least hemolytic activity in previous studies. However, synthetic peptides are relatively expensive to produce. Recombinant expression of peptides in the host cells, such as bacteria or yeast, can fastly provide cost-efficient products of peptides. Here, we developed an innovative system to produce CGA-N12 peptides in the yeast Pichia pastoris GS115 using genetic engineering technology. In order to directly secret short CGA-N12 peptides into the culture media from GS115 cells and enhance its expression effect, the structure of the CGA-N12 coding sequence was designed to mimic that of native α-factor gene of Saccharomyces cerevisiae. Four long primer pairs with sticky end were used to synthesize CGA-N12 expression sequence which contains four copies of CGA-N12 flanked by a Lys-Arg pair and two Glu-Ala repeating units. Endogenous proteases Kex2 and Ste13 in Golgi apparatus recognize and excise Lys-Arg and Glu-Ala pair to release short CGA-N12 peptides from the tandem repeat sequences, respectively. The CGA-N12 peptides were successfully expressed in Pichia pastoris with a yield of up to 30 mg/L of yeast culture as determined using HPLC. Our study indicated that the strategy employed in this work may be a good way to express small-molecule peptides directly in the Pichia pastoris system.

Viroporin activity and membrane topology of classic swine fever virus p7 protein.

Viroporins are a group of viral proteins that participate in viral replication cycles, including modification of membrane permeability and promotion of viral release. Although biological data have been accumulated on viroporion-like proteins of other viruses belonging to family Flaviviridae, the viroporin activity and membrane topology of p7 protein from classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae, are largely unknown. In this study, sequence analysis of the primary structure of p7 polypeptide demonstrates that p7 contains two putative transmembrane regions connected by a short hydrophilic segment. Expression of p7 protein in Escherichia coli leads to the permeabilization of bacterial cells to small molecules. The p7 protein also enhances the permeability of mammalian cells, increasing the intracellular Ca(2+) concentration and the permeability of cells to the translation inhibitor Hygromycin B. This protein is an integral membrane protein and can form homo-oligomers. It mainly localizes to the ER at the early stage of the expression and can be transferred to the plasma membrane at the late stage of the expression. Detergent permeabilization assays confirmed that the p7 protein is a 2-pass transmembrane protein and its N and C termini are exposed to the ER lumen. Deletion analysis showed that amino acid residues 41-63 may be essential for the viroporin activity of the protein. Our studies demonstrate that CSFV p7 possesses properties commonly associated with viroporins, which could be a potential target for the development of a therapeutic intervention for classic swine fever virus infection.

Immunization of mice by hollow mesoporous silica nanoparticles as carriers of porcine circovirus type 2 ORF2 protein.

BACKGROUND: Porcine circovirus type 2 (PCV2) is a primary etiological agent of post-weaning multi-systemic wasting syndrome (PMWS), which is a disease of increasing importance to the pig industry worldwide. Hollow mesoporous silica nanoparticles (HMSNs) have gained increasing interest for use in vaccines. METHODS: To study the potential of HMSNs for use as a protein delivery system or vaccine carriers. HMSNs were synthesized by a sol-gel/emulsion(oil-in-water/ethanol) method, purified PCV2 GST-ORF2-E protein was loaded into HMSNs, and the resulting HMSN/protein mixture was injected into mice. The uptake and release profiles of protein by HMSNs in vitro were investigated. PCV2 GST-ORF2-E specific antibodies and secretion of IFN-γ were detected by enzyme-linked immunosorbent assays, spleen lymphocyte proliferation was measured by the MTS method, and the percentage of CD4+ and CD8+ were determined by flow cytometry. RESULTS: HMSNs were found to yield better binding capacities and delivery profiles of proteins; the specific immune response induced by PCV2 GST-ORF2-E was maintained for a relatively long period of time after immunization with the HMSN/protein complex. CONCLUSION: The findings suggest that HMSNs are good protein carriers and have high potential for use in future applications in therapeutic drug delivery.