The intestinal barrier, comprised of a mucus layer (chemical barrier), cell layer (mechanical barrier), and lamina propria (immune barrier), helps maintain biological homeostasis by separating the luminal contents from the interstitium [1]. Intestinal defects are associated with various diseases, such as inflammatory bowel disease (IBD), irritable bowel syndrome, and celiac disease, as well as systemic diseases, such as human immunodeficiency virus, graft versus host disease, and autism [2]. Standardized diagnostic methods or therapies that target the intestinal barrier do not exist. Therefore, identifying molecular biomarkers to diagnose diseases associated with increased intestinal permeability quickly and accurately is imperative.

The mucus layer is the gastrointestinal tract's first line of defense, protecting epithelial cells from contact with and stimulation from intestinal bacteria, their metabolites, or food antigens [3]. Intestinal goblet cells of the epithelial layer synthesize mucin2, the best-characterized secreted mucin in the intestine. Mucin2, which forms in the ribosome and endoplasmic reticulum, is highly polymerized and stored in the vesicles of cells until secretory signals are released after glycosylation in the Golgi apparatus [4]. In general, mucin2 is slowly released into the extracellular space, where it hydrates and depolymerizes to form a dense gel structure that acts as a barrier [5]. Endocytosis, autophagy, and inflammasome formation in goblet cells are necessary for the synthesis and secretion of mucus. Abnormalities in these processes lead to disordered mucin synthesis and mucin2 secretion [6]. Impaired autophagy in intestinal epithelial cells deteriorates the gut microenvironment [7], indicating that cellular autophagy plays an important role in maintaining the intestinal barrier.

Autophagy is a composite molecular pathway that delivers intracellular components to the lysosomal compartment for degradation and recycling with key roles in metabolic stress [8], neurodegenerative disease [9], cancers [10], immunity [11]. Autophagy is also essential for barrier maintenance. For example, in lipopolysaccharide (LPS)-induced lung injury, autophagy protects the endothelial barrier by enhancing zonula occluden-1 expression [12]. Additionally, autophagy regulates claudin 5, providing therapeutic functions to the blood-brain barrier [13]. Other evidence suggests that autophagy regulates mucin secretion, which affects the intestinal barrier and inflammation [14], and autophagy signaling fortifies the intestinal mucus layer [15]. Similarly, autophagy deficiency in Drosophila caused by autophagy-related gene (ATG) deficiencies increased intestinal barrier permeability [16]. Therefore, autophagy may be essential for epithelial barrier repair.

Thymosin β4 (Tβ4) is a widely distributed peptide with high concentrations in the thymus, spleen, brain, liver, kidney, and gastrointestinal tract [17,18]. Tβ4 plays a vital role in several physiological processes, namely, promoting angiogenesis, enhancing endothelial progenitor cell viability, triggering cell proliferation and migration, and forming intracellular capillary-like structures [19]. Tβ4 also improves inflammation, has cardioprotective effects, and promotes skin wound healing, with promising applications for treating dry eye diseases, but how Tβ4 affects the intestinal barrier remains unknown. Therefore, this study evaluated the underlying regulatory mechanisms and effects of Tβ4 in vivo and in vitro to clarify its effects on the intestinal barrier.