Hydrogen Sulfide Inhibits Transforming Growth Factor-β1-Induced EMT via Wnt/Catenin Pathway.

Hydrogen sulfide (H2S) has anti-fibrotic potential in lung, kidney and other organs. The exogenous H2S is released from sodium hydrosulfide (NaHS) and can influence the renal fibrosis by blocking the differentiation of quiescent renal fibroblasts to myofibroblasts. But whether H2S affects renal epithelial-to-mesenchymal transition (EMT) and the underlying mechanisms remain unknown. Our study is aimed at investigating the in vitro effects of H2S on transforming growth factor-β1 (TGF-β1)-induced EMT in renal tubular epithelial cells (HK-2 cells) and the associated mechanisms. The induced EMT is assessed by Western blotting analysis on the expressions of α-SMA, E-cadherin and fibronectin. HK-2 cells were treated with NaHS before incubating with TGF-β1 to investigate its effect on EMT and the related molecular mechanism. Results demonstrated that NaHS decreased the expression of α-SMA and fibronectin, and increased the expression of E-cadherin. NaHS reduced the expression of TGF-β receptor type I (TβR I) and TGF-β receptor type II (TβR II). In addition, NaHS attenuated TGF-β1-induced increase of β-catenin expression and ERK phosphorylation. Moreover, it inhibited the TGF-β1-induced nuclear translocation of ββ-catenin. These effects of NaHS on fibronectin, E-cadherin and TβR I were abolished by the ERK inhibitor U0126 or β-catenin inhibitor XAV939, or β-catenin siRNA interference. We get the conclusion that NaHS attenuated TGF-β1-induced EMT in HK-2 cells through both ERK-dependent and β-catenin-dependent pathways

Fractal Design Boosts Extrusion-Based 3D Printing of Bone-Mimicking Radial-Gradient Scaffolds

Although extrusion-based three-dimensional (EB-3D) printing technique has been widely used in the complex fabrication of bone tissue-engineered scaffolds, a natural bone-like radial-gradient scaffold by this processing method is of huge challenge and still unmet. Inspired by a typical fractal structure of Koch snowflake, for the first time, a fractal-like porous scaffold with a controllable hierarchical gradient in the radial direction is presented via fractal design and then implemented by EB-3D printing. This radial-gradient structure successfully mimics the radially gradual decrease in porosity of natural bone from cancellous bone to cortical bone. First, we create a design-to-fabrication workflow with embedding the graded data on basis of fractal design into digital processing to instruct the extrusion process of fractal-like scaffolds. Further, by a combination of suitable extruded inks, a series of bone-mimicking scaffolds with a 3-iteration fractal-like structure are fabricated to demonstrate their superiority, including radial porosity, mechanical property, and permeability. This study showcases a robust strategy to overcome the limitations of conventional EB-3D printers for the design and fabrication of functionally graded scaffolds, showing great potential in bone tissue engineering

NaHS decreased the TGF-β1-stimulated overexpression of TGF-β receptor type II (TβR II) and TGF-β receptor type I (TβR I).

<p>Pretreatment by the several concentrations of NaHS is given to HK-2 cells for 12 hours and then TGF-β1 was given to the cells for the following 36 hours. (A) NaHS decreased both TβR I and TβR II overexpression stimulated by TGF-β1 in a concentration-dependent manner in HK-2 cells. (B) Graphical representation of the relative quantification for TβR I and TβR II. The relative values were calculated by the density of TβR I and TβR II vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. ^#P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.</p

Molecular cloning and functional analysis of a novel type of Bowman-Birk inhibitor gene family in rice

Bowman-Birk inhibitor (BBI) genes encode serine protease inhibitors that have repetitive cysteine-rich domains with reactive sites for the trypsin or chymotrypsin family. We have identified seven BBI genes from japonica rice (Oryza sativa subsp. japonica var Teqing). All of the genes identified were found in a single cluster on the southern end of the long arm of rice chromosome 1. Four of the seven BBI genes have two repetitive cysteine-rich domains, whereas one has a truncated domain with only one reactive site. We have also identified three novel BBI genes, each of which possesses three repetitive domains instead of two. In situ hybridization analyses indicated that the accumulation of rice BBI transcripts was differentially regulated in germinating embryos and also in the leaves, roots, and flower organs at later developmental stages. Different members of the rice BBI gene family displayed different expression patterns during rice seed germination, and wounding induced the expression of rice BBI transcripts. The three-domain BBIs had higher expression levels than the two-domain BBIs. It was also found that the mRNA of rice BBI genes was present in abundant amounts in scutellar epithelium and aleurone layer cells. RBBI3-1, one of the three-domain RBBI, exhibited in vitro trypsin-inhibiting activity but no chymotrypsin-inhibiting activity. Overexpression of RBBI2-3 in transgenic rice plants resulted in resistance to the fungal pathogen Pyricularia oryzae, indicating that proteinase inhibitors confer resistance against the fungal pathogen in vivo and that they might play a role in the defense system of the rice plant

NaHS attenuates the TGF-β1 induced EMT via Wnt/catenin pathway.

<p>(A) β-catenin knockdown efficiency by siRNA in HK-2 cells. The cells were transfected with β-catenin siRNA or control siRNA for 6 hours and the culture medium was replaced by fresh medium. Western blot assay was used to detect the expression level of non-phosphorylated β-catenin. (B) After transfected with β-catenin siRNA or control siRNA for 6 hours, HK-2 cells were incubated with NaHS (100μM) for 12 hours and then with TGF-β1 for the following 36 hours. The expression level of fibronectin, E-cadherin, TβR I and α-SMA were examined by western blot assay. (C) Graphical representation of the relative quantification for the protein level. The relative values were calculated by the density of targeted proteins vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. ^#P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.</p

NaHS attenuated TGF-β1-stimulated EMT in HK-2 cells.

<p>Pretreatment by the several concentrations of NaHS is given to HK-2 cells for 12 hours and then TGF-β1 was given to the cells for the following 36 hours. (A) The influence of 400μM NaHS on the morphological change of HK-2 cells (B) Western blot assay for fibronectin, E-cadherin and α-SMA expressions in HK-2 cells. (C) Graphical representation of the relative quantification for fibronectin, E-cadherin and α-SMA. The relative values were calculated by the density of fibronectin, E-cadherin and α-SMA vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. ^#P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.</p

NaHS increased the Cystathionine β-synthase (CBS) expression in HK-2 cells.

<p>Pretreatment by the several concentrations of NaHS is given to HK-2 cells for 12 hours and then TGF-β1 was given to the cells for the following 36 hours. (A) NaHS increased the CBS expression level in a concentration‑dependent manner in TGF-β1-stimulated HK-2 cells. (B) Graphical representation of the relative quantification for CBS. The relative values were calculated by the density of of CBS vs β-actin (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to β-actin. ^#P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.</p