Atrial natriuretic peptide (ANP) activity deficiency contributes to salt-sensitive hypertension in humans and mice. However, the role of ileal microbiota in salt sensitivity in ANP deficiency-related cardiac injury has not been investigated yet. This study used ANP^−/− mice to analyze the role of the salt-sensitive ileal microbiome on cardiac injury. ANP^−/− mice showed an increase in blood pressure (BP), the heart weight/body weight (HW/BW) ratio, and cardiac hypertrophy compared with wild-type (WT) mice. ANP deficiency did not impact the histological structure but reduced occludin expression in the ileum. Antibiotics significantly relieved BP and cardiac hypertrophy in ANP^−/− mice. A high-salt diet (HSD) increased BP, the HW/BW ratio, and cardiac hypertrophy/fibrosis in WT and ANP^−/− mice, and an HSD treatment in ANP^−/− mice exacerbated these cardiac parameters. The HSD markedly decreased muscularis layer thickening, villus length, and numbers of Paneth and goblet cells in the ileum of WT and ANP^−/− mice. Furthermore, the HSD increased the level of TLR4 and IL-1β in ANP^−/− mice ileum compared with WT mice. Antibiotics reduced the HW/BW ratio, cardiac hypertrophy/fibrosis, and the level of TLR4 and IL-1β in the ileum, and rescued the muscularis layer thickening, villus length, and numbers of Paneth and goblet cells in the ileum of HSD-ANP^−/− mice. Importantly, ANP deficiency induced the colonization of Burkholderiales bacterium YL45, Lactobacillus johnsonii, and Lactobacillus reuteri in the ileum on the NSD diet, which was only observed in HSD-induced WT mice but not in WT mice on the NSD. Besides, the HSD significantly enhanced the sum of the percentage of the colonization of Burkholderiales bacterium YL45, Lactobacillus johnsonii, and Lactobacillus reuteri in the ileum of ANP^−/− mice. Ileal microbiota transfer (IMT) from ANP^−/− mice to healthy C57BL/6J mice drove Lactobacillus johnsonii and Lactobacillus reuteri colonization in the ileum, which manifested an increase in BP, the HW/BW ratio, cardiac hypertrophy, and ileal pathology compared with IMT from WT mice. The HSD in C57BL/6J mice with IMT from ANP^−/− mice drove the colonization of Burkholderiales bacterium YL45, Lactobacillus johnsonii, and Lactobacillus reuteri in the ileum and further exacerbated the cardiac and ileal pathology. Our results suggest that salt-sensitive ileal microbiota is probably related to ANP deficiency-induced cardiac injury.

In situ nucleic acid visualization techniques are important tools and methods for studying the functions of nucleic acid molecules (DNA and RNA) and are widely used in various fields, including the spatial localization of gene expression, the influence of nucleic acid molecules on biological processes, and pathological diagnosis. On the basis of their mechanisms, these techniques can be categorized into direct detection methods, such as fluorescence in situ hybridization signal amplification-based techniques, such as Hybridization Chain Reaction (HCR), rolling circle amplification (RCA) and RNAscope; and combined coding and high-throughput imaging approaches, such as sequential Fluorescence In Situ Hybridization (seqFISH). In this review, we have established a comprehensive classification framework based on the fundamental principles of probe design and the mechanisms of signal amplification. We conducted an in-depth comparison of the strengths and limitations of in situ visualization methodologies in terms of spatial resolution, target throughput, and clinical applications. Furthermore, we explored potential major challenges in the development of in situ nucleic acid visualization technologies and proposed potential optimization strategies to address them. Finally, we looked ahead to the future directions for the expansion and development of in situ nucleic acid visualization technologies and analyzed their unique value in clinical translation.