scholarly journals Identification of a host collagen inducing factor from the excretory secretory proteins ofTrichinella spiralisusing immunoscreening

2018 ◽  
Author(s):  
Mi Kyung Park ◽  
Hae-Jin Kim ◽  
Min Kyoung Cho ◽  
Shin Ae Kang ◽  
So Young Park ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Serine Proteases ◽  
Collagen Synthesis ◽  
Trichinella Spiralis ◽  
Expression System ◽  
Molecular Characteristics ◽  
Secretory Proteins ◽  
Collagen Gene ◽  
Smad Signaling

AbstractBackgroundIn a previous study, we found thatTrichinella spiralisexcretory and secretory proteins (ES-P) most likely activate collagen synthesis via TGF-β/Smad signaling, and this event could influence collagen capsule formation.Methodology/Principal FindingsIn order to identify the specific collagen inducing factor, ES-P was fractionated by a Superdex 200 10/300 GL column. We obtained three large fractions, F1, F2, and F3, but only F3 had collagen gene inducing ability. After immunoscreening, 10 collagen inducing factor candidates were identified. Among them, TS 15-1 and TS 15-2 were identical to the putative trypsin ofT. spiralis. The deduced TS 15-1 (M.W. = 72 kDa) had two conserved catalytic motifs, an N-terminal Tryp_SPc domain (TS 15- 1n) and a C-terminal Tryp_SPc domain (TS 15-1c). To determine their collagen inducing ability, recombinant proteins (rTS 15-1n and rTS 15-1c) were produced using the pET-28a expression system. TS 15-1 is highly expressed during the muscle larval stage and has strong antigenicity. We determined that rTS 15-1c could elevate collagen I via activation of the TGF-β1 signaling pathwayin vitroandin vivo.Conclusion/SignificanceIn conclusion, we identified a host collagen inducing factor fromT. spiralisES-P using immunoscreening and demonstrated its molecular characteristics and functions.Author SummaryTrichinella spiraliscan make collagen capsules in host muscle cells during its life cycle, which encapsulates muscle stage larvae. Many investigators have tried to reveal the complex mechanism behind this collagen capsule architecture, and it has been suggested that several serine proteases in excretory-secretory proteins of the parasite are potential collagen capsule inducing factors. In addition, collagen synthesis is activated through the TGF-β/Smad signaling pathway and these events are closely related with protease activated receptor 2 which was activated by various serine proteases. In this study, we isolated and characterized a collagen gene expression inducer fromT. spiralisES-P using immunoscreening and investigated the candidate protein for its usefulness as a wound healing therapeutic agent.

scholarly journals Periplaneta Americana Extract may Attenuate 2,4,6-Trinitrobenzenesulfonic Acid-Induced Intestinal Fibrosis by Inhibiting TGF-β/Smad Signaling Pathway

2021 ◽  
Author(s):  
Jing Liu ◽  
Pin Lv ◽  
Xiang Rao ◽  
Jiajia Wang
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Periplaneta Americana ◽  
Collagen I ◽  
Smad Signaling ◽  
Intestinal Fibrosis ◽  
Smad Signaling Pathway

Abstract PurposeIntestinal fibrosis is an incurable digestive disease accompanied by stricture formation, and it has an increasing incidence in recent years. Periplaneta americana is one of the medicinal insects with a long history. There are few reports on the effect of intestinal fibrosis. This study aims to evaluate the inhibitory effect of PA treatment on intestinal fibrosis. MethodsTNBS was used to establish intestinal fibrosis model by enema in BALB/c mice. The mice were treated with PA (50, 100, 200 mg/kg body weight) and 5-aminosalicylic acid (5-ASA) (40mg/kg) by gavage once a day for 6 weeks. At the end of the last week, the mice were sacrificed. Colon samples were collected for H&E and Masson staining. The mRNA and protein expression of α-smooth muscle actin (α-SMA), collagen I and the transforming growth factor-β (TGF-β) / Smad signaling pathway were conducted by real-time PCR and western blot analysis. In vitro, TGF-β1 was used to induce intestinal fibrosis at human colon fibroblasts (CCD-18Co). And using real-time PCR and western blot methods to detect the expression of α-SMA and collagen I. ResultsPA inhibited the expression of α-SMA and collagen I in vivo and in vitro. But the difference was that PA inhibited the TGF-β/Smad signaling pathway in vivo, and the same results had not been obtained in vitro. Conclusion: PA may attenuate intestinal fibrosis by inhibiting TGF-β/Smad signaling pathway, but more experiments were needed to prove it in vitro. ConclusionsPA has potential pharmacological effects in inhibiting intestinal fibrosis, and the TGF-β/Smad signaling pathway seemed promising.

scholarly journals Modulation of Calretinin Expression in Human Mesothelioma Cells Reveals the Implication of the FAK and Wnt Signaling Pathways in Conferring Chemoresistance towards Cisplatin

2019 ◽  
Vol 20 (21) ◽  
pp. 5391 ◽  
Author(s):  
Wörthmüller ◽  
Salicio ◽  
Oberson ◽  
Blum ◽  
Schwaller
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Expression System ◽  
Single Agent ◽  
Wnt Signaling Pathway ◽  
Tumor Formation ◽  
Mesenchymal Transition

Malignant mesothelioma (MM) is an aggressive asbestos-linked neoplasm, characterized by dysregulation of signaling pathways. Due to intrinsic or acquired chemoresistance, MM treatment options remain limited. Calretinin is a Ca2+-binding protein expressed during MM tumorigenesis that activates the FAK signaling pathway, promoting invasion and epithelial-to-mesenchymal transition. Constitutive calretinin downregulation decreases MM cells’ growth and survival, and impairs tumor formation in vivo. In order to evaluate early molecular events occurring during calretinin downregulation, we generated a tightly controlled IPTG-inducible expression system to modulate calretinin levels in vitro. Calretinin downregulation significantly reduced viability and proliferation of MM cells, attenuated FAK signaling and reduced the invasive phenotype of surviving cells. Importantly, surviving cells showed a higher resistance to cisplatin due to increased Wnt signaling. This resistance was abrogated by the Wnt signaling pathway inhibitor 3289-8625. In various MM cell lines and regardless of calretinin expression levels, blocking of FAK signaling activated the Wnt signaling pathway and vice versa. Thus, blocking both pathways had the strongest impact on MM cell proliferation and survival. Chemoresistance mechanisms in MM cells have resulted in a failure of single-agent therapies. Targeting of multiple components of key signaling pathways, including Wnt signaling, might be the future method-of-choice to treat MM.

The protective effects of bone morphogenetic protein-7 against epithelial injury and matrix metalloproteases upregulation induced by silica in vitro

2016 ◽  
Vol 36 (9) ◽  
pp. 892-900 ◽  
Author(s):  
D Liang ◽  
G An ◽  
Z Zhu ◽  
Y Wang ◽  
G Yang ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Signaling Pathway ◽  
Western Blotting ◽  
Neutralizing Antibody ◽  
Collagen I ◽  
Bone Morphogenetic Protein 7 ◽  
Smad Signaling ◽  
Morphogenetic Protein ◽  
Smad Signaling Pathway

Objective: We investigate the effects of bone morphogenetic protein-7 (BMP-7) on models with silica-induced and macrophage-mediated fibrosis and its possible mechanisms in vitro. Methods: Rat alveolar II epithelial (RLE-6TN) cells were incubated with the supernatant of mouse macrophage-like cells (RAW264.7) and treated with 0, 25, 50, and 100 μg/mL silica. Using Western blotting, the epithelial markers (surfactant proteins-C and E-cadherin) and the mesenchymal markers (fibronectin (FN) and viminten (Vim)) were detected. After neutralizing the BMP-7, the progress of fibrosis was assessed by the content of hydroxyproline (Hyp) and collagen I, III protein levels as well as the Smad signaling pathway proteins, including phosphorylated Smad1/5(P-Smad1/5) and phosphorylated Smad2/3(P-Smad2/3). Collagen I was also identified by immunofluorescence and pretreated with SB-431542, LDN-193189, or anti-BMP-7-neutralizing antibody. In addition, the levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 were detected using Western blotting. Results: The model of RLE-6TN cells was established successfully, the expressions of Vim, FN, MMP-2, and MMP-9 were upregulated, while the concentration of silica is increased. Neutralizing BMP-7 stimulated the decrease of P-Smad1/5 and the increase of P-Smad2/3, as well as the collagen I, collagen III, FN, and Hyp via Smad signaling pathway. Furthermore, pretreated with LDN-193189 or anti-BMP-7-neutralizing antibody, the expression of collagen I was increased, yet it was decreased with SB-431542 intervention. Conclusion: The activated BMP/Smad and suppressed transforming growth factor-β/Smad pathways could suppress silica-induced fibrosis via a MMP-dependent mechanism. BMP-7 is expected to be the optimized strategy of delaying the interstitial changes.

scholarly journals Proteomic Analysis ofTrichinella spiralisMuscle Larval Excretory-Secretory Proteins Recognized by Early Infection Sera

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Li Wang ◽  
Zhong Quan Wang ◽  
Dan Dan Hu ◽  
Jing Cui
Keyword(s):  
Molecular Weight ◽  
Serine Proteases ◽  
Trichinella Spiralis ◽  
Early Stage ◽  
False Negative ◽  
Hypothetical Protein ◽  
Secretory Proteins ◽  
Dnase Ii ◽  
Negative Results ◽  
Protective Antibodies

Although the excretory-secretory (ES) proteins ofTrichinella spiralismuscle larvae are the most commonly used diagnostic antigens for trichinellosis, their main disadvantage is the false negative results during the early stage of infection and cross-reaction of their main components (43, 45, 49, and 53 kDa) with sera of patients with other helminthiasis. The aim of this study was to identify early specific diagnostic antigens inT. spiralisES proteins with 30–40 kDa. The ES proteins were analyzed by two-dimensional electrophoresis (2-DE), and a total of approximately 150 proteins spots were detected with isoelectric point (pI) varying from 4 to 7 and molecular weight from 14 to 66 kDa. When probed with sera from infected mice at 18 days postinfection, ten protein spots with molecular weight of 30–40 kDa were recognized and identified by MALDI-TOF/TOF-MS. All of ten spots were successfully identified and characterized to correlate with five different proteins, including two potential serine proteases, one antigen targeted by protective antibodies, one deoxyribonuclease (DNase) II, and one conserved hypothetical protein. These proteins might be the early specific diagnostic antigens for trichinellosis.

scholarly journals MiRNA-21 Regulates Bronchial Epithelial Cell Proliferation by Activating Tgfβ1/Smad Signaling Pathway and Its Correlation with Asthma Severity in Children

2021 ◽  
Author(s):  
Yang Kang ◽  
Minghui Bai ◽  
Liling Deng ◽  
Linbo Fan ◽  
Xing Wang
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Bronchial Epithelial Cell ◽  
Control Group ◽  
Healthy Children ◽  
Bronchial Epithelial ◽  
Smad Signaling ◽  
Smad Signaling Pathway ◽  
Tgf Β1

Background: This research was designed to probe into the role of miRNA-21 in the pathogenesis of childhood asthma and its correlation with the severity. Methods: Fifty-four children with bronchial asthma admitted to the Third Affiliated Hospital of Qiqihar Medical University from Jun 2018 to Dec 2019 were included. Forty nine healthy children underwent physical examination at this time period were also enrolled. The miR-21 expression in peripheral blood serum was analyzed by qRT-PCR. The relationship between the expression and severity of asthma in children was explored by Spearman correlation analysis and ROC curve. Bronchial epithelial cell lines were cultured in vitro and divided into blank control group, negative control group and miR-21 inhibition and activation group. The changes of cell proliferation after treatment were detected by CCK-8 test in different groups. The expression of TGF-β1/Smad signaling pathway protein in cells was assessed by Western blot (WB). Results: Compared with that of healthy children, the miR-21 expression in peripheral blood serum of asthmatic children was higher (P<0.001). MiR-21 expression was positively correlated with the severity of illness (r=0.853, P<0.001). The results of cell experiments in vitro signified that miR-21 can promote the proliferation of bronchial epithelial cells, and may be involved in regulating the expression of TGF-β1/ Smad3 signaling pathway, thus affecting cell proliferation. Conclusion: miRNA-21 regulates the proliferation of bronchial epithelial cells by activating TGFβ1/Smad signaling pathway. And it is positively correlated with the severity of asthma in children.

scholarly journals HuangQi Decoction Ameliorates Renal Fibrosis via TGF-β/Smad Signaling Pathway In Vivo and In Vitro

2016 ◽  
Vol 38 (5) ◽  
pp. 1761-1774 ◽  
Author(s):  
Jie Zhao ◽  
Li Wang ◽  
Ai-li Cao ◽  
Ming-Qian Jiang ◽  
Xia Chen ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Ureteral Obstruction ◽  
Interstitial Fibrosis ◽  
Unilateral Ureteral Obstruction ◽  
Type I ◽  
Renal Interstitial Fibrosis ◽  
Smad Signaling ◽  
Smad Signaling Pathway ◽  
Tgf Β1

Objective: Traditional Chinese Medicine compound HuangQi decoction is widely used in clinical treatment of chronic kidney disease, but its role on renal interstitial fibrosis and the underlying mechanism remains unclear. The aim of this study is to investigate the effect of HuangQi decoction on renal interstitial fibrosis and its association with the TGF-β/Smad signaling pathway Methods: A total of 120 C57/BL mice were randomly divided into six groups: sham group, sham plus high-dose HuangQi decoction (1.08g/kg) group, unilateral ureteral obstruction (UUO) model group, and UUO model plus low to high doses of HuangQi decoction (0.12g/kg, 0.36g/kg and 1.08g/kg respectively) groups. Animals were sacrificed 14 days after the administration and ipsilateral kidney tissue was sampled for pathologic examinations. Immunohistochemistry, PCR and western blot were used to detect the expressions of related molecules in the TGF-β/Smad signaling pathway. TGF-β1 was used in in vitro experiments to induce human kidney proximal tubule epithelial cells (HK2). Results: HuangQi decoction improved ipsilateral kidney fibrosis in UUO mice and downregulated the expressions of TGF-β1, TβRI, TβRII, Smad4, Smad2/3, P-Smad2/3, α-SMA, collagen type I, III and IV in a dose-dependent manner while upregulated the expression of Smad7 in the same fashion. Similar results were found in in vitro studies. Conclusion: The protective effect of HuangQi decoction for unilateral ureteral obstruction kidney damage in mice was mediated by downregulating the TGF-β/Smad signaling pathway.

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