Image4_2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside ameliorates bleomycin-induced pulmonary fibrosis via regulating pro-fibrotic signaling pathways.jpg

2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-Glucoside (THSG) is the main active ingredient extracted from Polygonum multiflorum Thunb. (PMT), which has been reported to possess extensive pharmacological properties. Nevertheless, the exact role of THSG in pulmonary fibrosis has not been demonstrated yet. The main purpose of this study was to investigate the protective effect of THSG against bleomycin (BLM)-induced lung fibrosis in a murine model, and explore the underlying mechanisms of THSG in transforming growth factor-beta 1 (TGF-β1)-induced fibrogenesis using MRC-5 human lung fibroblast cells. We found that THSG significantly attenuated lung injury by reducing fibrosis and extracellular matrix deposition. THSG treatment significantly downregulated the expression levels of TGF-β1, fibronectin, α-SMA, CTGF, and TGFBR2, however, upregulated the expression levels of antioxidants (SOD-1 and catalase) and LC3B in the lungs of BLM-treated mice. THSG treatment decreased the expression levels of fibronectin, α-SMA, and CTGF in TGF-β1-stimulated MRC-5 cells. Conversely, THSG increased the expression levels of SOD-1 and catalase. Furthermore, treatment of THSG profoundly reduced the TGF-β1-induced generation of reactive oxygen species (ROS). In addition, THSG restored TGF-β1-induced impaired autophagy, accompany by increasing the protein levels of LC3B-II and Beclin 1. Mechanism study indicated that THSG significantly reduced TGF-β1-induced increase of TGFBR2 expression and phosphorylation of Smad2/3, Akt, mTOR, and ERK1/2 in MRC-5 cells. These findings suggest that THSG may be considered as an anti-fibrotic drug for the treatment of pulmonary fibrosis.</p

Comparison of the h<i>FTSJ2</i> mRNA expression levels in two lung cancer sublines (CL1-0 and CL1-5).

<p>(<b>A</b>) Morphology of CL1-0 and CL1-5 cells. (<b>B</b>) Determination of the h<i>FTSJ2</i> mRNA expression levels in the CL1-0 and CL1-5 cells in triplicate. (<b>C</b>) Relative quantification of the h<i>FTSJ2</i> mRNA expression. <i>GAPDH</i> mRNA was used as an internal control. The values are equal to = the means±SE; n = 3; **<i>P</i><0.01 vs. the non-heat shock group.</p

Protein sequence alignment of <i>E. coli</i> RrmJ with its FTSJ2 orthologs in 7 different species.

<p>The α-helices and β-strands were based on the RrmJ protein structure (PDB code: 1EIZ). The stars and triangles indicate the K-D-K-E catalytic center and the SAM binding residues in RrmJ, respectively. The residues with identical and similar chemical properties are highlighted in black and gray, respectively.</p

Primer sequences for real-time and semi-quantitative RT-PCR.

<p>Primer sequences for real-time and semi-quantitative RT-PCR.</p

Bacterial autolysis triggered by vancomycin plays an important role in the enhancement of biofilm formation.

<p>Determination of the number of viable biofilm-bound bacteria in subcutaneous catheters removed from healthy/diabetic mice (A) infected with VRSA strain SJC1200 (also chloramphenicol resistance) upon chloramphenicol (Cm) treatment or (B) infected with <i>cidA</i> null mutant SJC1201 (autolysis deficient VRSA) upon vancomycin treatment. Six mice were used in each group.</p

Vancomycin enhances biofilm formation with glucose but reduces propensity to form biofilms without glucose.

<p>(A) A static biofilm assay was performed when VRSA cells (SJC1200) were cultured in BHI medium or medium supplemented with 0.5% (BHIg) or 1.5% (BHIhg) glucose, respectively, in the absence/presence of vancomycin (32 μg/ml). Adherent cells from representative triplicate assays in each condition were stained with safranin O and are shown on the bottom. (B) A time course of the number viable cells was performed when VRSA cells were cultured in BHI or BHIg in the absence (Van0) or presence of vancomycin (32 μg/ml; Van32). The results are presented as the means±sd of the log₁₀ CFU/ml from three separate experiments. (C) A time course of the static biofilm assay was performed when VRSA cells were cultured in different conditions as above. (D) The time course of glucose consumption. (E) A time course of the static biofilm assay was performed as above, but vancomycin was added immediately after the glucose was exhausted in BHI (at 6 h). * <i>P</i> < 0.05 and ** <i>P</i> < 0.005 in this figure and hereafter.</p

High Glucose Concentration Promotes Vancomycin-Enhanced Biofilm Formation of Vancomycin-Non-Susceptible <i>Staphylococcus aureus</i> in Diabetic Mice

<div><p>We previously demonstrated that vancomycin treatment increased acquisition of eDNA and enhanced biofilm formation of drug-resistant <i>Staphylococcus aureus</i> through a <i>cidA</i>-mediated autolysis mechanism. Recently we found that such enhancement became more significant under a higher glucose concentration <i>in vitro</i>. We propose that besides improper antibiotic treatment, increased glucose concentration environment in diabetic animals may further enhance biofilm formation of drug-resistant <i>S</i>. <i>aureus</i>. To address this question, the diabetic mouse model infected by vancomycin-resistant <i>S</i>. <i>aureus</i> (VRSA) was used under vancomycin treatment. The capacity to form biofilms was evaluated through a catheter-associated biofilm assay. A 10- and 1000-fold increase in biofilm-bound bacterial colony forming units was observed in samples from diabetic mice without and with vancomycin treatment, respectively, compared to healthy mice. By contrast, in the absence of glucose vancomycin reduced propensity to form biofilms <i>in vitro</i> through the increased production of proteases and DNases from VRSA. Our study highlights the potentially important role of increased glucose concentration in enhancing biofilm formation in vancomycin-treated diabetic mice infected by drug-resistant <i>S</i>. <i>aureus</i>.</p></div

Subcellular localization of human FTSJ2 in TE671 and HepG2 cells.

<p>(<b>A</b>) Schematic of the hFTSJ2 over-expression vector. (<b>B</b>) Over-expression of the hFTSJ2 protein in the TE671-h<i>FTSJ2</i> and HepG2-h<i>FTSJ2</i> stable clones. (<b>C</b>) Immunofluorescence staining with anti-hFTSJ2 (green), MitoTracker for mitochondria (red), and DAPI for nuclei (blue) in the TE671-h<i>FTSJ2</i> and HepG2-h<i>FTSJ2</i> cells. (<b>D</b>) Mitochondrial localization of the hFTSJ2 protein in non-transfected TE671 cells. VDAC and MEK-1 were used as the mitochondrial and cytosolic fraction controls, respectively, in the Western blot analysis.</p

Vancomycin enhances catheter-associated biofilm formation in diabetic mice infected with vancomycin-non-susceptible <i>S</i>. <i>aureus</i>.

<p>(A) Experimental control of catheter-associated biofilm formation assay in mice. Determination of the number of viable biofilm-bound bacteria in subcutaneous catheters removed from healthy/diabetic mice upon infection with VSSA strain ATCC 12598 in the presence of vancomycin treatment. (B) Subcutaneous catheters removed from healthy/diabetic mice upon infection with VRSA strain SJC1200 in the absence/presence of vancomycin treatment. Two representative catheters in each group are shown in the figure. (C) Catheter-associated biofilm materials were observed under SEM. Photos were taken at the indicated magnifications. (D) Determination of the number of viable biofilm-bound bacteria in the removed catheters, as above. (E) Determination of the number of viable biofilm-bound bacteria in the catheters removed from diabetic mice infected with VISA strain Mu50 following vancomycin treatment. (F) The correlation between the blood glucose concentration and catheter biofilm-forming capacity. The capacity was evaluated by determining the number of viable biofilm-bound bacteria in subcutaneous catheters. The number of mice used in each group is shown.</p

Investigation of the mechanisms underlying vancomycin-triggered biofilm degradation in the absence of glucose.

<p>(A) Time courses of PIA production by VRSA strain SJC1200 in different media with/without vancomycin treatment. (B) Evaluation of bacteria-secreted DNase activity by visualizing DNA degradation after incubating a DNA product (861 bp) with the supernatant removed from different VRSA culture conditions. 12 hr and 24 hr: supernatant from a 12-h and a 24-h culture system, respectively. M: a 100-bp DNA ladder marker. P and N: positive and negative controls, respectively. (C) Detection of changes in the transcription levels of <i>cidA</i> and <i>agrA</i> in VRSA by qRT-PCR upon different treatments. The fold change of each transcript was compared with vancomycin-untreated samples at T₀. (D) Detection of the bacteria-secreted protease activity by the evaluation of the biofilm degradation (suppression) potential using a static biofilm assay in the absence (Non-PI) or presence (PI) of a protease inhibitor cocktail.</p