Sjogren's syndrome (SS) is a systemic autoimmune disease that tends to occur in middle-aged and older women [1]. The main manifestation of SS is dryness caused by dysfunction of exocrine glands, such as salivary glands and lacrimal glands. Subsequently, it can cause symptoms such as kidney damage, joint pain, muscle weakness, and other symptoms, which seriously affect the normal life of patients [2]. The pathogenesis of SS is currently unknown, but it may be related to inflammatory responses, immune abnormalities, and genetic factors [3]. Salivary gland epithelial cells (SGECs) are the main source of salivary secretion [4,5]. When SS occurs, the death of SGECs caused by the interaction of these factors is one of the key factors of salivary gland secretory dysfunction [5]. In order to further treat and alleviate the symptoms of SS, it is particularly important to explore the mode and mechanism of SGECs' secretory function.

There are four main death modes in SS SGECs: apoptosis, autophagy, pyroptosis, and ferroptosis, all of which affect the secretion function of SGECs to varying degrees [6]. Among them, ferroptosis has been shown to play a crucial role in the occurrence and development of SS [7]. Ferroptosis is a programmed form of cell death. The main mechanism is the accumulation of large amounts of lipid peroxides in the cell, which subsequently catalyze unsaturated fatty acids on the cell membrane, which in turn leads to membrane rupture and cell death [8]. In addition, when ferroptosis occurs, mitochondrial shrinkage, loss of mitochondrial crests, cytoplasmic concentration, and so on occur in the cell [9]. Glutathione peroxidase 4 (GPX4) is a natural ferroptosis inhibitor that uses glutathione (GSH) to reduce lipid peroxides to non-toxic lipid alcohols, thereby protecting cells from toxic damage from lipid peroxides [10]. In our previous study, we reported the downregulation of GPX4 in SGECs during SS [7]. However, the role of GPX4 in the secretory function of SGECs and the underlying mechanisms are still unknown.

Aquaporin-5 (AQP5) is an important protein involved in salivary secretion in SGECs. AQP5 is mainly located on the cell membrane and plays an important role in controlling the efflux of intracellular saliva [11]. At the time of SS occurrence, the expression of AQP5 in SGECs is significantly reduced, which in turn leads to decreased salivary secretion [12]. In addition, when the SS mouse model was treated with AQP5 gene therapy, the salivary secretion function of the mice was significantly improved [13]. Levels of GPX4 and AQP5 are downregulated in SGECs during SS [7]. However, the regulatory role of GPX4 in AQP5 expression in SGECs has not been investigated.

The signal transducer and activator of transcription 4 (STAT4) is a key regulatory transcription that can bind to DNA fragments to affect various gene expressions [14]. STAT4 is involved in various autoimmune diseases, including SS, rheumatoid arthritis, systemic lupus erythematosus, etc [15,16]. Among them, STAT4 is closely related to SS susceptibility and also affects the inflammatory response in SS, but the specific mechanism is unknown [17,18]. However, the role of STAT4 on AQP5 transcription regulation has not been reported.

In this study, bioinformatics analysis of GSE databases from SS patients revealed a strong positive correlation between the downregulated GPX4 and AQP5. STAT4 was the key transcription regulatory factor with a strong negative correlation with GPX4 and AQP5 in SS SGECs. Based on these data, we hypothesized that GPX4 regulates AQP5 expression in SGECs and salivary secretion via STAT4. Both GPX4 and AQP5 were downregulated in the salivary glands of SS patients and the SS mice model. GPX4 overexpression upregulated and GPX4 knockdown downregulated AQP5 expression and water secretion by SGECs. The level of lipid ROS and STAT4 phosphorylation and nuclear translocation were upregulated in the SS salivary gland and SS SGECs. A series of in vitro and in vivo studies unraveled that GPX4 regulates lipid ROS which further dictates STAT4 phosphorylation and nuclear translocation to regulate AQP5 transcription in SGECs and salivary secretion. In summary, our results indicated a vital role of the downregulated GPX4 in salivary secretion via the lipid ROS/pSTAT4/AQP5 axis, unraveling novel therapeutic targets to recuperate the salivary secretion in SS patients.