The Phenolic Fraction of Mentha haplocalyx and Its Constituent Linarin Ameliorate Inflammatory Response through Inactivation of NF-κB and MAPKs in Lipopolysaccharide-Induced RAW264.7 Cells.

Mentha haplocalyx has been widely used for its flavoring and medicinal properties and as a traditional Chinese medicine with its anti-inflammation properties. The present study was designed to investigate the anti-inflammatory effects and potential molecular mechanisms of the phenolic fraction of M. haplocalyx (MHP) and its constituent linarin in lipopolysaccharide (LPS)-induced RAW264.7 cells. The high-performance liquid chromatography coupled with linear ion trap-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap MS) was used to analyze the chemical composition of MHP. Using the enzyme-linked immunosorbent assay (ELISA) and quantitative realtime polymerase chain reaction (qRT-PCR), the expression of pro-inflammatory meditators and cytokines was measured at the transcriptional and translational levels. Western blot analysis was used to further investigate changes in the nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and Akt signaling pathways. Fourteen phenolic constituents were identified from MHP based on the data of the mass spectrometry (MS)/MS analysis. MHP and linarin decreased the production of NO, tumor necrosis factor-α (TNF-α), interlenkin-1ß (IL-1ß), and IL-6. The messenger ribonucleic acid (mRNA) expression levels of inducible NO synthase (iNOS), TNF-α, IL-1ß, and IL-6 were also suppressed by MHP and linarin. Further investigation showed that MHP and linarin down-regulated LPS-induced phosphorylation content of NF-κB p65, inhibitor kappa B α (IκBα), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. However, MHP and linarin showed no inhibitory effect on the phosphorylated Akt. These results suggested that MHP and linarin exerted a potent inhibitory effect on pro-inflammatory meditator and cytokines production via the inactivation of NF-κB and MAPKs, and they may serve as potential modulatory agents for the prevention and treatment of inflammatory diseases.

Screening differential miRNAs responsible for permeability increase in HUVECs infected with influenza A virus.

Severe influenza infections are featured by acute lung injury, a syndrome of increased pulmonary microvascular permeability. A growing number of evidences have shown that influenza A virus induces cytoskeletal rearrangement and permeability increase in endothelial cells. Although miRNA's involvement in the regulation of influenza virus infection and endothelial cell (EC) function has been well documented, little is known about the miRNA profiles in influenza-infected endothelial cells. Using human umbilical vein endothelial cells (HUVECs) as cell models, the present study aims to explore the differential miRNAs in influenza virus-infected ECs and analyze their target genes involved in EC permeability regulation. As the results showed, permeability increased and F-actin cytoskeleton reorganized after HUVECs infected with influenza A virus (CA07 or PR8) at 30 MOI. MicroRNA microarray revealed a multitude of miRNAs differentially expressed in HUVECs after influenza virus infection. Through target gene prediction, we found that a series of miRNAs were involved in PKC, Rho/ROCK, HRas/Raf/MEK/ERK, and Ca2+/CaM pathways associated with permeability regulation, and most of these miRNAs were down-regulated after flu infection. It has been reported that PKC, Rho/ROCK, HRas/Raf/MEK/ERK, and Ca2+/CaM pathways are activated by flu infection and play important roles in permeability regulation. Therefore, the cumulative effects of these down-regulated miRNAs which synergistically enhanced activation of PKC, Rho/ROCK, Ras/Raf/MEK/ERK, and Ca2+/CaM pathways, can eventually lead to actin rearrangement and hyperpermeability in flu-infected HUVECs.

[The transfection of lentiviral siRNA vectors targeting ezrin, radixin and moesin facilitates influenza virus replication in primary pulmonary microvascular endothelial cells].

OBJECTIVE: To construct lentivirus siRNA vectors targeting ezrin (E), radixin (R) and moesin (M), transfect rat primary pulmonary microvascular endothelial cells (PMVECs) with the vectors and observe the effects of the transfection on E, R, M gene expressions and influenza virus replication. METHODS: According to the principles of siRNA synthesis, siRNA sequences targeting E, R and M genes were synthesized and cloned into lentiviral plasmid GV248 to construct lentiviral siRNA vectors Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA, respectively. Random Lenti-control-siRNA was also constructed targeting nonsense sequences. Rat PMVECs were cultured by peripheral lung tissue-sticking method. Rat PMVECs were transfected with Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA, respectively. The levels of ezrin, radixin and moesin mRNA and proteins were detected by real-time quantitative PCR and Western blotting, the distributions of ERM proteins were detected using confocal laser scanning microscope; After lentivirus-transfected PMVECs were infected with influenza virus, the effects of lentiviral vectors on influenza virus replication were measured by real-time quantitative PCR. RESULTS: Lenti-E-siRNA, Lenti-R-siRNA, Lenti-M-siRNA and Lenti-control-siRNA were successfully constructed according to sequencing identification of recombinant plasmids. The infection efficacy of lentiviral siRNA vectors in rat PMVECs was above 80% at a multiplicity of infection (MOI) of 10. The transcription and expressions of ezrin, radixin and moesin in rat PMVECs were significantly inhibited by Lenti-E-siRNA, Lenti-R-siRNA, Lenti-M-siRNA respectively. Lentivirus control (containing no target genes) and Lenti-control-siRNA inhibited influenza virus replication in influenza virus-infected PMVECs, while Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA increased the levels of influenza virus mRNA in the infected PMVECs. CONCLUSION: Lentiviral siRNA vectors targeting E, R and M genes were constructed successfully and could inhibit E, R and M gene expressions specifically and effectively. Lentiviral vectors could inhibit influenza virus replication whereas lentiviral siRNA vectors targeting E, R and M facilitated influenza virus replication.

p38MAPK, Rho/ROCK and PKC pathways are involved in influenza-induced cytoskeletal rearrangement and hyperpermeability in PMVEC via phosphorylating ERM.

Severe influenza infections are featured by acute lung injury, a syndrome of pulmonary microvascular leak. A growing number of evidences have shown that the pulmonary microvascular endothelial cells (PMVEC) are critical target of influenza virus, promoting microvascular leak. It is reported that there are multiple mechanisms by which influenza virus could elicit increased pulmonary endothelial permeability, in both direct and indirect manners. Ezrin/radixin/moesin family proteins, the linkers between plasma membrane and actin cytoskeleton, have been reported to be involved in cell adhesion, motility and may modulate endothelial permeability. Studies have also shown that ERM is phosphorylated in response to various stimuli via p38MAPK, Rho/ROCK or PKC pathways. However, it is unclear that whether influenza infection could induce ERM phosphorylation and its relocalization. In the present study, we have found that there are cytoskeletal reorganization and permeability increases in the course of influenza virus infection, accompanied by upregulated levels of p-ERM. p-ERM's aggregation along the periphery of PMVEC upon influenza virus infection was detected via confocal microscopy. Furthermore, we sought to determine the role of p38MAPK, Rho/ROCK and PKC pathways in ERM phosphorylation as well as their involvement in influenza virus-induced endothelial malfunction. The activation of p38MAPK, Rho/ROCK and PKC pathways upon influenza virus stimulation were observed, as evidenced by the evaluation of phosphorylated p38 (p-p38), phosphorylated MKK (p-MKK) in p38MAPK pathway, ROCK1 in Rho/ROCK pathway and phosphorylated PKC (p-PKC) in PKC pathway. We also showed that virus-induced ERM phosphorylation was reduced by using p38MAPK inhibitor, SB203580 (20 µM), Rho/ROCK inhibitor, Y27632 (20 µM), PKC inhibitor, LY317615 (10 µM). Additionally, influenza virus-induced F-actin reorganization and hyperpermeability were attenuated by pretreatment with SB203580, Y27632 and LY317615. Taken together, we provide the first evidence that p38MAPK, Rho/ROCK and PKC are involved in influenza-induced cytoskeletal changes and permeability increases in PMVEC via phosphorylating ERM.