miR-146a-5p regulates autophagy and NLRP3 inflammasome activation in epithelial barrier damage in the in vitro cell model of ulcerative colitis through the RNF8/Notch1/mTORC1 pathway |
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| Institution: | 1. Department of Anorectal Surgery, The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210029, China;2. First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023, China;3. Department of Anorectal Surgery, Xuzhou City Hospital of TCM, Xuzhou, Jiangsu 221000, China;1. Department of Pathogenic Biology, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, China;2. Sichuan-Chongqing jointly-established Research Platform of Zoonosis, Chengdu, China;3. Chong Qing Animal Disease Prevention and Control Center, Chongqing, China;1. Disease Biology Laboratory, School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT), Deemed to be University, Bhubaneswar, Odisha, India;2. Department of Rheumatology, Kalinga Institute of Medical Sciences, Bhubaneswar, Odisha, India;3. Immuno-Genomics and Systems Biology Lab, Institute of Life Sciences, Bhubaneswar, Odisha, India;1. Innovative Institute of Chinese Medicine and Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan 250355, China;2. Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada;1. Laboratorio de Microbioma, División de Investigación, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México, Mexico;2. Programa de Inmunología Molecular Microbiana, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México, Mexico;3. Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, Av. Ciudad Universitaria 3000, C.P. 04510, Coyoacán, Ciudad de México, Mexico;1. Biomedicine Research Center, Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China;2. Department of Anesthesiology, The Third Xiangya Hospital of Central South University, Changsha, China;3. Department of Anesthesiology, The Second Xiangya Hospital of Central South University, Changsha, China;4. Department of Anesthesiology and Center for Shock, Trauma, and Anesthesiology Research, University of Maryland School of Medicine, Baltimore, MD, United States of America |
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| Abstract: | Ulcerative colitis (UC) is a chronic inflammatory disease affecting the colon that can be influenced by microRNAs (miRNAs). This study aims to investigate the impact of miR-146a-5p on lipopolysaccharide (LPS)-induced Caco-2/HT-29 cell autophagy and NLRP3 inflammasome activation and the underlying mechanism, with the aim of identifying potential therapeutic targets. We used LPS to establish Caco-2/HT-29 cell models and measured cell viability by CCK-8. The levels of miR-146a-5p, RNF8, markers of NLRP3 inflammasome activation and autophagy, proteins involved in the Notch1/mTORC1 pathway, and inflammatory factors were assessed by RT-qPCR, Western blot, and ELISA. Intestinal epithelial barrier function was evaluated by measuring transepithelial electrical resistance. Autophagic flux was measured using tandem fluorescent-labeled LC3. miR-146a-5p was highly-expressed in LPS-induced Caco-2/HT-29 cells, and autophagy flux was blocked at the autolysosomal stage after LPS induction. Inhibition of miR-146a-5p suppressed NLRP3 inflammasome activation, reduced intestinal epithelial barrier damage, and facilitated autophagy inhibition in LPS-induced Caco-2/HT-29 cells. The autophagy inhibitor NH4Cl partially nullified the inhibitory effects of miR-146a-5p inhibition on NLRP3 inflammation activation. miR-146a-5p targeted RNF8, and silencing RNF8 partly abrogated the action of miR-146a-5p inhibition on promoting autophagy and inhibiting NLRP3 inflammasome activation. miR-146a-5p inhibition suppressed the Notch1/mTORC1 pathway activation by upregulating RNF8. Inhibition of the Notch1/mTORC1 pathway partially nullified the function of silencing RNF8 on inhibiting autophagy and bolstering NLRP3 inflammasome activation. In conclusion, miR-146a-5p inhibition may be a potential therapeutic approach for UC, as it facilitates autophagy of LPS-stimulated Caco-2/HT-29 cells, inhibits NLRP3 inflammasome activation, and reduces intestinal epithelial barrier damage by upregulating RNF8 and suppressing the Notch1/mTORC1 pathway. |
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| Keywords: | Ulcerative colitis miR-146a-5p RNF8 Notch1 mTORC1 Autophagy NLRP3 inflammasome Caco-2 cells |
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