Alignment of the sequences of CsBPI homologues.

<p>Dots denote gaps introduced for maximum matching. Numbers in brackets indicate overall sequence identities between CsBPI and the compared sequences. The consensus residues are in black; the residues that are ≥75% identical among the aligned sequences are in blue. The GenBank accession numbers of the aligned sequences are as follows: <i>Oplegnathus fasciatus</i>, BAM21037.1; <i>Larimichthys crocea</i>, KKF08800.1; <i>Larimichthys crocea</i>, ABO32254.1; <i>Salmo salar</i>, NP_001135199.1; <i>Takifugu rubripes</i>, XP_003976395.2; <i>Osmerus mordax</i>, ACO08980.1; <i>Astyanax mexicanus</i>, XP_007237045.1; <i>Plecoglossus altivelis altivelis</i>, BAG49475.1.</p

Tissue specific expression of <i>CsBPI</i>.

<p><i>CsBPI</i> expression in the intestine, muscle, brain, blood, heart, liver, kidney, gill, and spleen of tongue sole was determined by quantitative real time RT-PCR. For convenience of comparison, the expression level in intestine was set as 1. Vertical bars represent means ± SEM (N = 3). N, the number of times the experiment was performed.</p

Effect of rCsBPI on bacterial and viral infection in tongue sole.

<p>Tongue sole were infected with Pseudomonas fluorescens (A and B) or megalocytivirus (C and D) in the presence or absence (control) of rCsBPI or rTrx. At various time points post-infection, pathogen loads in the kidney and spleen of the fish were determined. Values are shown as means ± SEM (N = 3). N, the number of times the experiment was performed. **<i>P</i> < 0.01, *<i>P</i> < 0.05.</p

<i>CsBPI</i> expression in response to bacterial and viral infection.

<p>Tongue sole were infected with <i>Pseudomonas fluorescens</i> (A and B), megalocytivirus (C and D), or PBS (control), and <i>CsBPI</i> expression in kidney (A and C) and spleen (B and D) was determined by quantitative real time RT-PCR at various time points. For convenience of comparison, the expression level of the control fish was set as 1. Values are shown as means ± SEM (N = 3). N, the number of times the experiment was performed. **<i>P</i> < 0.01.</p

Effect of rCsBPI on the expression of immune genes in tongue sole.

<p>Tongue sole were administered with rCsBPI, rTrx, or PBS (control), and expression of the immune genes in head kidney was determined by quantitative real time RT-PCR. In each case, the expression level of the control fish was set as 1. Values are shown as means ± SEM (N = 3). N, the number of times the experiment was performed. **<i>P</i> < 0.01, *<i>P</i> < 0.05.</p

Effect of rCsBPI on phagocytosis.

<p>Tongue sole peripheral blood lymphocytes were incubated with FITC-labeled <i>Pseudomonas</i> <i>fluorescens</i> in the absence (B) or presence of rCsBPI (C) or rTrx (D); the control cells (A) were incubated without bacteria. The cells were then analyzed by fluorescence activated cell sorting. M1 represents the cellular population with enhanced fluorescence as a result of bacterial uptake. Values are shown as means ± SEM (N = 3). N, the number of times the experiment was performed.</p

Bactericidal activity of rCsBPI.

<p><i>Edwardsiella tarda</i>, <i>Pseudomonas fluorescence</i>, <i>Streptococcus iniae</i>, and Staphylococcus aureus were incubated with or without (control) rCsBPI or rTrx for different hours. After incubation, the number of survived bacterial cells (shown as Colony Forming Unit, CFU) was determined by plate count. Values are shown as means ± SEM (N = 3). N, the number of times the experiment was performed. **<i>P</i> < 0.01, *<i>P</i> < 0.05.</p

Malé levodistributivní kvazigrupy

<p><b>Effect of rCsBPI on bacterial membrane permeability (A) and cellular structure (B).</b> (A) <i>Pseudomonas</i> <i>fluorescens</i> was incubated with or without (control) rCsBPI or rTrx for different hours. After incubation, the cells were stained with PI solution, and PI-positive cells were determined. Values are shown as means ± SEM (N = 3). N, the number of times the experiment was performed. (B) P. fluorescens was treated with rCsBPI for 2 h (B3) and 4 h (B4) or treated with rTrx for 4 h (B2); the control cells (B1) were untreated. The cells were then subjected to microscopy. Bar, 500 nm. **<i>P</i> < 0.01.</p

Tongxinluo Protects against Pressure Overload–Induced Heart Failure in Mice Involving VEGF/Akt/eNOS Pathway Activation

<div><p>Background</p><p>It has been demonstrated that Tongxinluo (TXL), a traditional Chinese medicine compound, improves ischemic heart disease in animal models via vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). The present study aimed to investigate whether TXL protects against pressure overload–induced heart failure in mice and explore the possible mechanism of action.</p><p>Methods and Results</p><p>Transverse aortic constriction (TAC) surgery was performed in mice to induce heart failure. Cardiac function was evaluated by echocardiography. Myocardial pathology was detected using hematoxylin and eosin or Masson trichrome staining. We investigated cardiomyocyte ultrastructure using transmission electron microscopy. Angiogenesis and oxidative stress levels were determined using CD31 and 8-hydroxydeoxyguanosine immunostaining and malondialdehyde assay, respectively. Fetal gene expression was measured using real-time PCR. Protein expression of VEGF, phosphorylated (p)-VEGF receptor 2 (VEGFR2), p–phosphatidylinositol 3-kinase (PI3K), p-Akt, p-eNOS, heme oxygenase-1 (HO-1), and NADPH oxidase 4 (Nox4) were measured with western blotting. Twelve-week low- and high-dose TXL treatment following TAC improved cardiac systolic and diastolic function and ameliorated left ventricular hypertrophy, fibrosis, and myocardial ultrastructure derangement. Importantly, TXL increased myocardial capillary density significantly and attenuated oxidative stress injury in failing hearts. Moreover, TXL upregulated cardiac nitrite content and the protein expression of VEGF, p-VEGFR2, p-PI3K, p-Akt, p-eNOS, and HO-1, but decreased Nox4 expression in mouse heart following TAC.</p><p>Conclusion</p><p>Our findings indicate that TXL protects against pressure overload–induced heart failure in mice. Activation of the VEGF/Akt/eNOS signaling pathway might be involved in TXL improvement of the failing heart.</p></div

TXL reduces cardiac fibrosis and ameliorates myocardial ultrastructure derangement after TAC.

<p>(<b>A</b>) Masson trichrome–stained sections of left ventricles. Scale bar, 50 µm. (<b>B</b>) Quantification of cardiac fibrosis area from Masson trichrome–stained sections (n = 5 per group). (<b>C</b>) Transmission electron micrographs of cardiomyocytes the respective treatment groups. Scale bar, 2 µm. Data are mean ± SEM. **<i>P</i><0.01, ***<i>P</i><0.001. NS, not significant.</p