Targeted knockout of Mx in the DF-1 chicken fibroblast cell line impairs immune response against Newcastle disease virus.

Newcastle disease virus (NDV) is an RNA virus taking poultry as the host, and the Newcastle disease (ND) caused by NDV is one of the diseases with serious damage to the health of poultry. Mx encoding by myxovirus resistance gene, induced by type I interferon (IFN), has a wide range of antiviral and GTPase activities in human, mice, and other species via inhibition virus replication. However, the antiviral ability of chicken Mx is still a controversial issue. To explore the effect of chicken Mx post-NDV infection, Mx-knockout DF-1 cells were constructed via CRISPR/Cas9 gene editing system. The number of copies of NDV was detected by RT-qPCR, and the mRNA expression levels of IRF-7, IFN-α, IFN-ß, TNF-α, p21, p27, and Bak in DF-1 cells were analyzed after NDV infection. Compared with control cells, virus titers were much higher in Mx-knockout DF-1 cells post-NDV infection. The deficiency of Mx aggravated the cell pathological features post-NDV infection, and promoted the expression levels of IRF-7, IFN-α, IFN-ß, and pro-inflammatory cytokine TNF-α in host cells. In addition, cells with Mx deficiency could alleviate the harm from virus by enhancing the expression of p21, p27, and Bak, which related to cell proliferation apoptosis. In conclusion, Mx played an important role in antivirus invasion. In the absence of Mx, cells could alleviate the harm from virus infection via retarding cell proliferation and enhancing cell apoptosis.

Structural basis of α-scorpion toxin action on Nav channels.

Fast inactivation of voltage-gated sodium (Nav) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Nav channel alone and in complex with a lethal α-scorpion toxin, AaH2, by electron microscopy, both at 3.5-angstrom resolution. AaH2 wedges into voltage-sensing domain IV (VSD4) to impede fast activation by trapping a deactivated state in which gating charge interactions bridge to the acidic intracellular carboxyl-terminal domain. In the absence of AaH2, the S4 helix of VSD4 undergoes a ~13-angstrom translation to unlatch the intracellular fast-inactivation gating machinery. Highlighting the polypharmacology of α-scorpion toxins, AaH2 also targets an unanticipated receptor site on VSD1 and a pore glycan adjacent to VSD4. Overall, this work provides key insights into fast inactivation, electromechanical coupling, and pathogenic mutations in Nav channels.

Daikenchuto (Da-Jian-Zhong-Tang) ameliorates intestinal fibrosis by activating myofibroblast transient receptor potential ankyrin 1 channel.

AIM: To investigate the anti-fibrotic effects of the traditional oriental herbal medicine Daikenchuto (DKT) associated with transient receptor potential ankyrin 1 (TRPA1) channels in intestinal myofibroblasts. METHODS: Inflammatory and fibrotic changes were detected in a 2,4,6-trinitrobenzenesulfonic acid (TNBS) chronic colitis model of wild-type and TRPA1-knockout (TRPA1-KO) mice via pathological staining and immunoblotting analysis. Ca2+ imaging experiments examined the effects of DKT and its components/ingredients on intestinal myofibroblast (InMyoFib) cell TRPA1 channel function. Pro-fibrotic factors and transforming growth factor (TGF)-ß1-associated signaling were tested in an InMyoFib cell line by qPCR and immunoblotting experiments. Samples from non-stenotic and stenotic regions of the intestines of patients with Crohn's disease (CD) were used for pathological analysis. RESULTS: Chronic treatment with TNBS caused more severe inflammation and fibrotic changes in TRPA1-KO than in wild-type mice. A one-week enema administration of DKT reduced fibrotic lesions in wild-type but not in TRPA1-KO mice. The active ingredients of DKT, i.e., hydroxy α-sanshool and 6-shogaol, induced Ca2+ influxes in InMyoFib, and this was antagonized by co-treatment with a selective TRPA1 channel blocker, HC-030031. DKT counteracted TGF-ß1-induced expression of Type I collagen and α-smooth muscle actin (α-SMA), which were accompanied by a reduction in the phosphorylation of Smad-2 and p38-mitogen-activated protein kinase (p38-MAPK) and the expression of myocardin. Importantly, 24-h incubation with a DKT active component Japanese Pepper increased the mRNA and protein expression levels of TRPA1 in InMyoFibs, which in turn negatively regulated collagen synthesis. In the stenotic regions of the intestines of CD patients, TRPA1 expression was significantly enhanced. CONCLUSION: The effects of DKT on the expression and activation of the TRPA1 channel could be advantageous for suppressing intestinal fibrosis, and benefit inflammatory bowel disease treatment.

Activation of Myofibroblast TRPA1 by Steroids and Pirfenidone Ameliorates Fibrosis in Experimental Crohn's Disease.

BACKGROUND & AIMS: The transient receptor potential ankyrin 1 (TRPA1) channel is highly expressed in the intestinal lamina propria, but its contribution to gut physiology/pathophysiology is unclear. Here, we evaluated the function of myofibroblast TRPA1 channels in intestinal remodeling. METHODS: An intestinal myofibroblast cell line (InMyoFibs) was stimulated by transforming growth factor-ß1 to induce in vitro fibrosis. Trpa1 knockout mice were generated using the Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. A murine chronic colitis model was established by weekly intrarectal trinitrobenzene sulfonic acid (TNBS) administration. Samples from the intestines of Crohn's disease (CD) patients were used for pathologic staining and quantitative analyses. RESULTS: In InMyoFibs, TRPA1 showed the highest expression among TRP family members. In TNBS chronic colitis model mice, the extents of inflammation and fibrotic changes were more prominent in TRPA1-/- knockout than in wild-type mice. One-week enema administration of prednisolone suppressed fibrotic lesions in wild-type mice, but not in TRPA1 knockout mice. Steroids and pirfenidone induced Ca2+ influx in InMyoFibs, which was antagonized by the selective TRPA1 channel blocker HC-030031. Steroids and pirfenidone counteracted transforming growth factor-ß1-induced expression of heat shock protein 47, type 1 collagen, and α-smooth muscle actin, and reduced Smad-2 phosphorylation and myocardin expression in InMyoFibs. In stenotic intestinal regions of CD patients, TRPA1 expression was increased significantly. TRPA1/heat shock protein 47 double-positive cells accumulated in the stenotic intestinal regions of both CD patients and TNBS-treated mice. CONCLUSIONS: TRPA1, in addition to its anti-inflammatory actions, may protect against intestinal fibrosis, thus being a novel therapeutic target for highly incurable inflammatory/fibrotic disorders.

Significant contribution of TRPC6 channel-mediated Ca2+ influx to the pathogenesis of Crohn's disease fibrotic stenosis.

Intestinal fibrosis is an intractable complication of Crohn's disease (CD), and, when occurring excessively, causes severe intestinal obstruction that often necessitates surgical resection. The fibrosis is characterized by an imbalance in the turnover of extracellular matrix (ECM) components, where intestinal fibroblasts/myofibroblasts play active roles in ECM production, fibrogenesis and tissue remodeling, which eventually leads to the formation of stenotic lesions. There is however a great paucity of knowledge about how intestinal fibrosis initiates and progresses, which hampers the development of effective pharmacotherapies against CD. Recently, we explored the potential implications of transient receptor potential (TRP) channels in the pathogenesis of intestinal fibrosis, since they are known to act as cellular stress sensors/transducers affecting intracellular Ca2+ homeostasis/dynamics, and are involved in a broad spectrum of cell pathophysiology including inflammation and tissue remodeling. In this review, we will place a particular emphasis on the intestinal fibroblast/myofibroblast TRPC6 channel to discuss its modulatory effects on fibrotic responses and therapeutic potential for anti-fibrotic treatment against CD-related stenosis.

In Vivo Cannulation Methods for Cardiomyocytes Isolation from Heart Disease Models.

Isolation of high quality cardiomyocytes is critically important for achieving successful experiments in many cellular and molecular cardiology studies. Methods for isolating cardiomyocytes from the murine heart generally are time-sensitive and experience-dependent, and often fail to produce high quality cells. Major technical difficulties can be related to the surgical procedures needed to explant the heart and to cannulate the vessel to mount onto the Langendorff system before in vitro reperfusion can begin. During this period, transient hypoxia and ischemia may damage the heart, resulting in low yield and poor quality of cells, especially for heart disease models that have fragile cells. We have developed novel in vivo cannulation methods to minimize hypoxia and ischemia, and fine-tuned the entire protocol to produce high quality ventricular myocytes. The high cell quality has been confirmed using important structural and functional criteria such as morphology, t-tubule structure, action potential morphology, Ca2+ signaling, responsiveness to beta-adrenergic agonist, and ability to have robust contraction under mechanically loaded condition. Together these assessments show the preservation of the cardiac excitation-contraction machinery in cells isolated using this technique. The in vivo cannulation method enables consistent isolation of high-quality cardiomyocytes, even from heart disease models that were notoriously difficult for cell isolation using traditional methods.