miR-124-Antagonist-Loaded Liposomal Nanoparticles Negatively Regulate the Toll-Like Receptor (TLR)-Signaling Pathway in Alveolar Epithelial Cells in Pulmonary Tuberculosis

Abstract

miR-124 is intensively expressed in the alveolar epithelial cells of pulmonary tuberculosis. This study focused on exploring the negative regulation of miR-124-antagonist-loaded liposomal nanoparticles on the Toll-like receptor (TLR)-signal transduction pathway in the alveolar epithelial cells from pulmonary tuberculosis, aiming to provide theoretical evidence for the treatment of pulmonary tuberculosis. The purchased alveolar epithelial cells were grouped into Blank group, Empty-vector group, Bacillus Calmette-Guerin (BCG) group, Nanoparticle+MiR-124 Antagonist group, MiR-124 Antagonist group, and MiR-124 Agonist group. The liposomal nanoparticles were identified. The following aspects were investigated: mRNA level of miR-124, mRNA and protein levels of Myeloid differentiation factor 88 (MyD 88), Toll-like receptor the 6 (TLR 6) and their downstream molecules Nuclear Factor-κB (NF-κB) and Tumor necrosis factor TNF receptor-associated factor 6 (TRAF 6) secretion level of cytokines (NF-κB, IL-8, IL-1α, TNF-α and IL-6), as well as the regulatory link between miR-124-antagonists with TLR6 and MyD88. The liposomal nanoparticles were uniform in size, with an average particle size of (35.25±10.58) nm and an average Zeta potential of (−48.55±10.27) mV. The miR-124 level was the strongest in the MiR-124 Agonist group, while being the lowest in the Blank group. The miR-124 level was relatively higher in the BCG group and Empty-vector group, while being significantly reduced in the Nanoparticle+MiR-124 Antagonist group, which was higher than the Blank group. The miR-124 level in the MiR-124 Antagonist group was higher than that in the Nanoparticle+MiR-124 Antagonist group (P <0.05). The mRNA and protein levels of MyD88, TLR6, NF-κB and TRAF6 were the highest in the MiR-124 Agonist group, while being the lowest in the Blank group. The transcription and translation levels of TRAF6, TLR6, NF-κB and MyD88 were relatively higher in the BCG group and Empty-vector group, while being significantly reduced in the Nanoparticle+ MiR-124 Antagonist group, which were higher than in the Blank group. The transcription and translation levels of TRAF6, TLR6, NF-κB and MyD88 were in the MiR-124 Antagonist group were higher than that in the Nanoparticle+MiR-124 Antagonist group (P <0.05). The secretion levels of inflammatory factors (NF-κB, IL-8, IL-1α, TNF-α and IL-6) were the highest in the MiR-124 Agonist group, while being the lowest in the Blank group. The levels of these inflammatory factors were relatively higher in the BCG group and Empty-vector group, while being significantly reduced in the Nanoparticle+MiR-124 Antagonist group, which were elevated compared to that in the Blank group. The secretion quantities of these inflammatory factors in the MiR-124 Antagonist group were higher than that in the Nanoparticle+MiR-124 Antagonist group (P <0.05).Dual luciferase experiments indicated that miR-124-antagonists may retard TLR6 and MyD88 to affect the immune response of pulmonary alveolar epithelial cells in pulmonary tuberculosis. The fluorescence intensity of mutant plasmid was significantly stronger than that of wild-type plasmid (P < 0.05). In the alveolar epithelial cells from pulmonary tuberculosis, the miR-124-antagonistloaded liposomal nanoparticles can significantly reduce the expression of TLR6 and MyD88, and their downstream molecules (NF-κB and TRAF6), leading to the reduced secretion of the inflammatory factors. As a result, the inflammatory response of lung tissue was alleviated, while the immune function was restored. This regulation was achieved by the miR-124-antagonist-loaded liposomal nanoparticles via negatively regulating the TLR6/MyD88 pathways.