RESUMEN
Aberrant TGFß/Smad7 signaling has been reported to be an important mechanism underlying the pathogenesis of ulcerative colitis. Therefore, the present study aimed to investigate the effects of a number of potential anticolitis agents on intestinal epithelial permeability and the TGFß/Smad7 signaling pathway in an experimental model of colitis. A mouse model of colitis was first established before antiTNFα and 5aminosalicyclic acid (5ASA) were administered intraperitoneally and orally, respectively. Myeloperoxidase (MPO) activity, histological index (HI) of the colon and the disease activity index (DAI) scores were then detected in each mouse. Transmission electron microscopy (TEM), immunohistochemical and functional tests, including Evans blue (EB) and FITCdextran (FD4) staining, were used to evaluate intestinal mucosal permeability. The expression of epithelial phenotype markers Ecadherin, occludin, zona occludens (ZO1), TGFß and Smad7 were measured. In addition, epithelial myosin light chain kinase (MLCK) expression and activity were measured. AntiTNFα and 5ASA treatments was both found to effectively reduce the DAI score and HI, whilst decreasing colonic MPO activity, plasma levels of FD4 and EB permeation of the intestine. Furthermore, antiTNFα and 5ASA treatments decreased MLCK expression and activity, reduced the expression of Smad7 in the small intestine epithelium, but increased the expression of TGFß. In mice with colitis, TEM revealed partial epithelial injury in the ileum, where the number of intercellular tight junctions and the expression levels of Ecadherin, ZO1 and occludin were decreased, all of which were alleviated by antiTNFα and 5ASA treatment. In conclusion, antiTNFα and 5ASA both exerted protective effects on intestinal epithelial permeability in an experimental mouse model of colitis. The underlying mechanism may be mediated at least in part by the increase in TGFß expression and/or the reduction in Smad7 expression, which can inhibit epithelial MLCK activity and in turn reduce mucosal permeability during the pathogenesis of ulcerative colitis.
Asunto(s)
Colitis Ulcerosa/metabolismo , Proteína smad7/genética , Proteína smad7/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Animales , Cadherinas/metabolismo , Colitis Ulcerosa/inducido químicamente , Colon/patología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Femenino , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Mucosa Intestinal/ultraestructura , Masculino , Mesalamina/administración & dosificación , Ratones Endogámicos C57BL , Quinasa de Cadena Ligera de Miosina/metabolismo , Ocludina/metabolismo , Peroxidasa/efectos de los fármacos , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos , Uniones Estrechas/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.