The intestinal epithelial barrier is crucial for separating mammals from the external environment, protecting against infection and inflammation. Its integrity relies on factors like secreted defenses (mucins, antimicrobial proteins, IgA), epithelial cell apoptosis/proliferation, and cell junctions (adherens and tight junctions). Barrier dysfunction leads to bacterial translocation and diseases like inflammatory bowel disease (IBD), chronic infections, diabetes, and autoimmune disorders. TMEM16A, a Ca²⁺-activated Cl⁻ channel, is expressed in intestinal epithelial cells and influences fluid transport. Its role in intestinal epithelium is debated, with some studies suggesting involvement in mucus secretion while others report no effect on anion transport and mucus homeostasis. TMEM16A also exhibits diverse roles in other diseases. This study aimed to clarify TMEM16A's expression and function in intestinal epithelial cells using an in vitro model of LPS-induced barrier dysfunction in IEC-6 cells to minimize confounding factors.

Existing literature highlights the importance of intestinal epithelial barrier function in maintaining health and preventing various diseases. Studies have shown that dysfunction of this barrier contributes significantly to inflammatory bowel disease and other systemic illnesses. While the role of chloride channels and currents in intestinal inflammation has been documented, the specific role of TMEM16A in this process remains unclear and controversial. Previous research on TMEM16A has focused on its involvement in various physiological processes and diseases, such as cancer, hypertension, and cystic fibrosis, but its function within the intestinal epithelium needs further investigation.

The study used rat intestinal epithelial IEC-6 cells. A barrier dysfunction model was established by exposing the cells to different concentrations of lipopolysaccharide (LPS). TMEM16A was knocked down and overexpressed using siRNA and adenovirus, respectively. The effects on cell apoptosis and tight junctions were assessed using quantitative real-time PCR, western blotting, immunofluorescence, flow cytometry, and transepithelial electrical resistance (TER) measurements. The role of NF-κB in LPS-induced TMEM16A expression was investigated using siRNA targeting NF-κB p65. In vivo studies were performed on mice with dextran sodium sulfate (DSS)-induced and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis to assess TMEM16A expression in inflamed intestines. Statistical analysis included one-way ANOVA, Student's t-tests, and Kruskal-Wallis rank sum tests.

LPS (0.1, 1, and 10 µg/mL) significantly increased TMEM16A mRNA and protein expression in IEC-6 cells and RAW264.7 cells. SiRNA targeting NF-κB p65 inhibited LPS-induced TMEM16A expression. Low-dose LPS decreased TER, which was further reduced by TMEM16A knockdown but enhanced by TMEM16A overexpression. Conversely, high-dose LPS-induced TER reduction was aggravated by TMEM16A knockdown and alleviated by TMEM16A overexpression. Low-dose LPS slightly increased IEC-6 cell proliferation, which was further enhanced by TMEM16A overexpression. High-dose LPS decreased IEC-6 cell proliferation, which was partially rescued by TMEM16A overexpression. TMEM16A affected tight junction proteins (MLCK) via ERK1/2 signaling pathways in low-dose LPS-treated cells and influenced apoptosis markers (cleaved caspase-3, Bax, Bcl-2) via ERK1/2 signaling in high-dose LPS-treated cells. In vivo studies showed decreased TMEM16A expression in DSS-colitis and variable expression in TNBS-colitis.

This study reveals a dual role for TMEM16A in LPS-induced intestinal epithelial barrier dysfunction. At low LPS concentrations, TMEM16A exacerbates barrier disruption primarily by affecting tight junctions through ERK1/MLCK signaling. However, at high LPS concentrations, its protective effects against apoptosis via ERK/Bcl-2/Bax signaling become dominant. These findings contrast with previous studies showing TMEM16A downregulation in DSS-induced colitis. The discrepancy might arise from differences in experimental models and the complex interplay of various factors in vivo. The study highlights that the effects of TMEM16A could be context-dependent, influenced by LPS concentration and the predominant mechanism of barrier dysfunction (tight junction dysregulation vs. apoptosis).

TMEM16A exhibits a dual role in LPS-induced intestinal epithelial barrier dysfunction in vitro, exacerbating barrier damage at low LPS doses through tight junction disruption but protecting against apoptosis at high LPS doses. Further in vivo research is needed to explore TMEM16A's role in various stages of inflammatory bowel disease.

The study is limited to an in vitro model using IEC-6 cells and may not fully reflect the complexity of in vivo conditions. The in vivo results show variability in TMEM16A expression in different colitis models, suggesting the need for further investigation to understand its role in different inflammatory conditions. The study focused on TMEM16A protein expression rather than channel activity, which may not capture the whole picture of its functional effects.