Comparison of <i>in vitro</i> anti-influenza activities of RO5464466 and RO5487624.

a<p>: All numbers (EC₅₀, CC₅₀, and IC₅₀) are means of three independent experiments and shown in micromolar concentrations;</p><p>Some data from A/Weiss/43 have been shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029120#pone.0029120-Tang1" target="_blank">[29]</a>.</p>b<p>: MDCK cells were used;</p>c<p>: + means nearly 100% protection of BHA from trypsin degradation in the presence of compounds (10 µM).</p

<i>In vivo</i> efficacy of RO5487624.

<p>40 LD₅₀ of A/FM/1/47 (H1N1) were used for viral challenge in all groups by intranasal inoculation. The first dosage of all treatments was given either 1 h before (A) or 3 h after (B) virus challenge (see details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029120#s2" target="_blank">Materials and Methods</a> section). Mice in untreated control group were orally administrated with PBS twice a day for 7 days. Animals (n = 10 in each group) was monitored for 14 days starting from virus challenge. Data was analyzed with Chi-square for mortality rate, * <i>P</i><0.05; ** <i>P</i><0.001, and with Kaplan-Meier for mean survival day, # <i>P</i><0.05; ## <i>P</i><0.001, respectively.</p

SDS-PAGE of trypsin sensitivity assay showing RO5464466 protected BHA from trypsin digestion in pH-dependent and dose-dependent manner.

<p>(A) 6 µg BHA was incubated with compounds of different concentrations for 15 minute at 31°C prior to acidification to pH 5.0 with 0.25 M citrate (pH 4.2). The mixture was neutralized to a final pH of 7.5 and treated with 2 µg of trypsin for 30 minutes at 37°C. The extent of trypsin cleavage on BHA was analyzed on a 10% SDS-PAGE gel. MW was shown on the right in thousands. Untreated BHA, trypsin alone, BHA trypsin digestion without a prior acidification step were used as controls and included in this figure. (B) Dose-dependent protection of BHA by RO5464466. (C) pH-dependent protection of BHA by RO5464466. After incubated with 10 µM of RO5464466, BHA was acidified to different final pH shown on the top of the gel before neutralization and trypsin digestion. As a negative control, DMSO-treated BHA was adjusted to two pH values (5.0 and 5.2) before neutralization and trypsin digestion. (D) RO5464466 protected BHA from trypsin digestion not by directly inhibiting trypsin enzymatic activity. RO5464466 was added into the reaction either before or after acidification of BHA.</p

Pharmacokinetic analysis of RO5487624.

<p>Mean plasma concentration-time profiles of RO5487624 after an oral dose (30 mg/kg, ◊, 100 mg/kg □, and 200 mg/kg, Δ) to CD-1 mice. Vertical lines are standard deviation of each group (n = 3). Concentration corresponding to RO5487624's EC₉₀ after protein binding adjustment is marked by a dotted line.</p

RO5464466 inhibited HA-mediated hemolysis of chicken erythrocytes.

<p>After mixing RO5464466 with virus-containing allantoic fluid, suspension of freshly prepared chicken erythrocytes was added. The mixture was then acidified with different pH from 4.85 to 6. The suspension was incubated at 37°C for 30 minutes. The final concentration of RO5464466 was 10 µM. After a brief spin, supernatants containing released hemoglobin were transferred to a second plate for measuring OD₅₄₀. As a control, suspensions of erythrocytes alone were acidified (pH control) similarly to measure low pH-caused hemolysis. In the DMSO control group, every step was the same as described in RO5464466 group except the testing compound was omitted. All conditions were tested in duplicated wells. *, results significantly different (RO5464466 treated samples vs. DMSO controls) by Student's t test (<i>P</i><0.05).</p

RO5464466 blocked the production of progeny virus and inhibited an established influenza virus infection.

<p>(A) Decrease of progeny virus production in cells treated with RO5464466 and other reference compounds. 12-well plates were seeded with MDCK cells at a density of 1.5×10⁵ cells per well. On the next day, serially diluted compounds and 150 pfu virus were added per well. 48 hours later, treatment was stopped and viral titer in cell monolayer was measured in TCID₅₀. Virus yield was shown in a log scale, a mean of two wells. (B) Dynamics of virus yield reduction in the presence of inhibitors. In this experiment, all treatments began at the same time when virus was added into culture wells but were stopped at different time points of 24, 48, and 72 hours post-infection, respectively. (C) Inhibition of plaque formation by RO5464466. Details of seeding MDCK cells into 6-well plates and infecting monolayers with influenza A/Weiss/43 (H1N1) were described in the plaque reduction assay in the section of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029120#s2" target="_blank">Materials and Methods</a>. Instead of using 3 ml of agarose-containing overlay for each well, 2.5 ml of overlay was used. At each time point (0, 12, 24, 48 hours post-infection), 2.5 ml of culture medium that contained compound of 2-fold final concentration was added on the top of the overlay. At 72 hours post-infection, cell monolayers were fixed and stained to show plaques. Representative data of three independent experiments was shown.</p

Salvianolic Acid A, a Novel Matrix Metalloproteinase-9 Inhibitor, Prevents Cardiac Remodeling in Spontaneously Hypertensive Rats

<div><p>Cardiac fibrosis is a deleterious consequence of hypertension which may further advance to heart failure and increased matrix metalloproteinase-9 (MMP-9) contributes to the underlying mechanism. Therefore, new therapeutic strategies to attenuate the effects of MMP-9 are urgently needed. In the present study, we characterize salvianolic acid A (SalA) as a novel MMP-9 inhibitor at molecular, cellular and animal level. We expressed a truncated form of MMP-9 which contains only the catalytic domain (MMP-9 CD), and used this active protein for enzymatic kinetic analysis and Biacore detection. Data generated from these assays indicated that SalA functioned as the strongest competitive inhibitor of MMP-9 among 7 phenolic acids from <i>Salvia miltiorrhiza</i>. In neonatal cardiac fibroblast, SalA inhibited fibroblast migration, blocked myofibroblast transformation, inhibited secretion of intercellular adhesion molecule (ICAM), interleukin-6 (IL-6) and soluble vascular cell adhesion molecule-1 (sVCAM-1) as well as collagen induced by MMP-9 CD. Functional effects of SalA inhibition on MMP-9 was further confirmed in cultured cardiac H9c2 cell overexpressing MMP-9 <i>in vitro</i> and in heart of spontaneously hypertensive rats (SHR) <i>in vivo</i>. Moreover, SalA treatment in SHR resulted in decreased heart fibrosis and attenuated heart hypertrophy. These results indicated that SalA is a novel inhibitor of MMP-9, thus playing an inhibitory role in hypertensive fibrosis. Further studies to develop SalA and its analogues for their potential clinical application of cardioprotection are warranted.</p> </div

SalA inhibited myofibroblast transformation induced by MMP-9 CD.

<p>Fluorescence immunohistochemistry, using a specific α-SMA first antibody following by a second antibody conjugated with FITC, was performed to demonstrate myofibroblast transformation induced by MMP-9. Nuclei were stained with DAPI and images are shown at 400x. MMP-9/SalA(1) or MMP-9/SalA(10) denoted 1 µmol/L SalA or 10 µmol/L SalA was used to detect the inhibitory effect of SalA on MMP-9.</p

Effects of SalA on basic cardiovascular characteristics of SHR.

<p>All the values are expressed as mean ± S.E. Heart weight divided by body weight (HW/BW), lung weight divided by body weight (LW/BW) and kidney weight divided by body weight (KW/BW) were also calculated. #<i>p</i><0.05 versus SalA (0 mg/kg). n = 20 per each group. Results are expressed as mean±S.E.</p

SalA inhibited the secretion of cytokines and collagen induced by MMP-9 CD.

<p>(A) MMP-9 CD stimulated the secretion of IL-6, ICAM, sVCAM-1, TNF-α and SalA reversed the effects of MMP-9 CD partially. (B) SalA inhibited collagen secretion induced by MMP-9 CD. MMP-9/SalA(1) or MMP-9/SalA(10) denoted 1 µmol/L SalA or 10 µmol/L SalA was used to detect the inhibitory effects of SalA on MMP-9. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 versus Con; #<i>p</i><0.05, ### <i>p</i><0.001 versus MMP-9.</p