key: cord-0068180-tjdxx1cs
authors: Errachid, Abdelmounaim; Nohawica, Michal; Wyganowska-Swiatkowska, Marzena
title: A comprehensive review of the influence of Epigallocatechin gallate on Sjögren's syndrome associated molecular regulators of exocytosis (Review)
date: 2021-09-21
journal: Biomed Rep
DOI: 10.3892/br.2021.1471
sha: df5b8864c2279b3712fae156fee30f8371e80ab8
doc_id: 68180
cord_uid: tjdxx1cs

Sjögren's syndrome (SS) is an autoimmune disorder that affects the salivary glands, leading to reduced secretory functions and oral and ocular dryness. The salivary glands are composed of acinar cells that are responsible for the secretion and production of secretory granules, which contain salivary components, such as amylase, mucins and immunoglobulins. This secretion process involves secretory vesicle trafficking, docking, priming and membrane fusion. A failure during any of the steps in exocytosis in the salivary glands results in the altered secretion of saliva. Soluble N-ethylmaleimide-sensitive-factor attachment protein receptors, actin, tight junctions and aquaporin 5 all serve an important role in the trafficking regulation of secretory vesicles in the secretion of saliva via exocytosis. Alterations in the expression and distribution of these selected proteins leads to salivary gland dysfunction, including SS. Several studies have demonstrated that green tea polyphenols, most notably Epigallocatechin gallate (EGCG), possess both anti-inflammatory and anti-apoptotic properties in normal human cells. Molecular, cellular and animal studies have indicated that EGCG can provide protective effects against autoimmune and inflammatory reactions in salivary glands in diseases such as SS. The aim of the present article is to provide a comprehensive and up-to-date review on the possible therapeutic interactions between EGCG and the selected molecular mechanisms associated with SS.

The quantity and composition of the daily salivary production of the major (submandibular, sublingual and parotid) and minor salivary glands is important for oral health. In a healthy individual the salivary glands produce 0.5-1.5 l saliva per a day, which is composed of 99.5% water 0.3% protein and 0.2% organic and inorganic substances (1) (2) (3) . The salivary glands are composed of acinar cells, which are responsible for the secretion and production of secretory granules (3) (4) (5) . These granules contain amylase, mucins and immunoglobulins, which are essential for the maintenance of a stable oral environment, lubrication, digestion and in the immunity of the oral mucosa (6) .

There are two types of acinar cells, mucinous and serous acinar cells, and each plays a different role in the proper functioning of the salivary glands. Mucinous acinar cells are characterized by an accumulation of a large number of mucinous granules in the apical cytoplasmic region (7) . These cells are responsible for the production of mucins, which are essential for lubrication and oral health (8) . Serous acinar cells also accumulate secretory granules, but are responsible for the secretion of digestive amylase α, which aids primary food digestion (7, 8) .

Sjögren's syndrome (SS) is an autoimmune disease characterized by a dysfunction of the salivary glands. The primary symptom is dryness of the mouth and eyes due to impaired salivary and lacrimal gland function (9, 10) . It has been reported that several factors may be responsible for this, such

as acinar cell atrophy and apoptosis (9, 11) , glandular denervation (12) , inhibition of cytokine neurotransmitter release (13) , Acetylcholine (ACh) depletion and increased secretion of cholinesterase (14) , presence of anti-muscarinic autoantibodies blocking muscarinic M3 receptors (14) , decreased nitric oxide production [which in turn can disrupt calcium release induced by altered levels of cyclic adenosine diphosphate-ribose (15) ], altered calcium tunneling (16) , and aberrant aquaporin (AQP)5 expression and localization (17) . Tea and its polyphenols, a product of the dried leaves of Camellia sinensis, have been suggested to possess several pharmacological properties (18) . The green tea polyphenols (GTPs) are useful in delaying or managing SS-like autoimmune disorders that can affect multiple tissues or organs (19, 20) . The prevalence of SS seems to be higher in the US population compared to the Japanese and Chinese population (21, 22) . It was reported that apoptosis, cytotoxicity and autoantigen expression (23) , as well as oxidative stress (24) , all of which are involved in SS pathogenesis and the primary cellular mechanisms underlying its development, are inhibited by GTPs (19, 25, 26) .

The major GTP, Epigallocatechin-3-gallate (EGCG), inhibits autoantigen expression in normal human keratinocytes and immortalized normal human salivary acinar cells (26) . Another study demonstrated that EGCG can protect normal human salivary acinar cells from tumor necrosis factor-α (TNF-α)-induced cytotoxicity, and the phosphorylation of p38 mitogen-activated protein kinase (MAPK) serves a major role in this protection (27) .

EGCG undergoes modification by gut microbes following oral administration (28) . Multiple resultant gut soluble metabolites have been reported to possess pharmacological properties (29) . EGCG reaches systemic circulation at low concentrations, and excretion is complete within several hours (30) . Identification of the true active component of EGCG, and conversely the true effect of EGCG on the body, is a contentious topic (31) . Nonetheless, whether the activity of EGCG is carried out by the unaltered molecule, its microbial metabolites, or its cellular metabolites, in the framework of SS and the selected proteins, this activity may be beneficial.

The dysregulation of secretory granules in SS by selected families of molecules, including soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs), AQP5, actin, and tight junctions, are comprehensively discussed. Furthermore, the possible mechanism of protection by GTPs against the dysregulation of these molecules is reviewed.

PRISMA guidelines were used to plan this comprehensive review (32) . Fig. 1 shows a flowchart summarizing the results of the search strategy carried out for this review. The review was split in to two bouts of data gathering, the molecular action of selected molecules in SS covered by search key 1, and the effect of EGCG on selected molecules covered by search key 2. Key 1: Sjogren AND salivary gland AND (aquaporin OR SNARE OR actin OR tight junction); and Key 2: EGCG AND (aquaporin OR SNARE OR actin OR tight junction).

The global inclusion criteria were: Published after 1998, the full text available, the article was written in the English language and it described non-trivial involvement of selected molecules. The PubMed, Science Direct and Cochrane databases were searched, and Medical Subject Headings Terms used where permitted by the search engine. Abstracts were screened for the selection criteria, and the chosen full text articles were screened again. Applicable results are summarized in Tables I-IV , and a pictorial representation is shown in Figs. 2 and 3.

Physiological roles of SNAREs. SNARE proteins have been shown to serve a major role in the regulation of exocytosis (33) . Vesicle-associated membrane proteins (VAMPs) are SNAREs that play a crucial role in the regulation of secretory vesicle trafficking (34) . Indeed, these vesicles are transported from the trans-Golgi network to the plasma membrane via the cytoskeleton (35) . Once these vesicles reach the plasma membrane, interactions between Ras-related protein (RAB), mammalian uncoordinated-18 (MUNC18) and SNARE proteins promote the formation of trans-SNARE (t-SNARE) (36) . During fusion between the plasma membrane and the secretory vesicles, a small opening is created at the point of contact between the two membranes (vesicles and plasma membrane), which gradually increases in size until exocytosis (37, 38) .

In the salivary gland, mucous acinar cells contain a large number of mucin granules aggregated in the apical cytoplasm (39, 40) . Several studies have shown that the exocytosis of mucins required precise localization of SNAREs and other components (41) . In acini cells, the secretory pathway synthesizes, processes and exocytoses mucins (42) . Several families of proteins, such as SNAREs, RAB and MUNC-18-bound proteins, are involved in the assembly of the membrane fusion complex (42) (43) (44) . The localization of each unit of this molecular machinery is essential for exocytosis (43) . VAMP8 in patients with SS. Studies of endosomal compartments, notably VAMP8 (42) , have indicated that it is an important component in exocytosis (44) . In SS, the expression and localization of the VAMP8 protein in acinar cells has been shown to be associated with dysfunction of the salivary labial glands. In healthy individuals, the localization of VAMP8 is cytoplasmic in the apical region of acinar cells. In contrast, in patients with SS, it was observed that the expression of this protein is decreased and localized throughout the entire cytoplasm. This aberration in VAMP8 distribution is considered to be related to ectopic mucin secretion (45) .

Other studies in VAMP8-deficient (VAMP8-/-) mice (normal at birth) have shown that the absence of interaction between VAMP8 with t-SNARE, Syntaxin (STX)4, and soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)23 induces abnormal accumulation of secretory granules, and increased production of amylases and carbonic anhydrase VI in acinar cells of the salivary glands (46, 47) . This suggests that the expression and distribution of SNARE proteins serve an essential role in the secretion of salivary gland proteins.

STX in patients with SS. Studies of STX in rat parotid glands have shown that the localization of STX4 is abundantly expressed across the entire plasma membrane, whereas STX2 and STX3 are expressed only at the apical level of the plasma membrane (48, 49) . In contrast, in the submandibular glands of humans, STX4 is expressed in the apical and basolateral plasma membranes, and STX2 is localized in both the apical plasma membrane and the cytoplasmic vesicles (50) .

In the labial salivary glands in patients with primary SS, the pattern of expression and/or localization of STX3 and STX4 exhibits several differences compared with healthy individuals. The localization of STX4 does not change compared with healthy individuals, whereas its expression is decreased in patients with SS. In contrast, whereas STX3 is normally localized only at the cytoplasmic level in the apical region of acinar cells, its expression was drastically increased in the entire cytoplasm in patients (45) . It has been reported that increased STX3 expression in patients is related to the fusion of secretory granules that were previously described as large pleomorphic granules (51) .

STX4-VAMP8 and STX3-SNAP23 complexes are overabundant in acinar cells of the salivary labial glands in patients with SS (45) . It is interesting to note that the interactions between STX4 and VAMP8 are observed only in the basolateral membrane of patients, suggesting a major role of this complex in secretory vesicle trafficking dysfunction and exocytosis (45) . Additionally, it has been reported that the co-localization of STX4 and RAB3D in mature secretory vesicles in the subapical region is altered from the apical to basolateral plasma membrane in patients with SS. This perturbation of SNARE complexes results in altered interactions, distribution and co-localization, which in-turn induce alterations in the distribution of secretory mucins in patients with SS (45, 51, 52) .

Altered expression and localization of SNARE complexes with aberrant mucin exocytosis in the basal region could induce mucin accumulation in the extracellular matrix. It has also been suggested that the STX3/SNAP23/VAMP8 complex may serve a major role in homotypic granule-granule fusion in Labial Salivary Gland (LSG) acinar cells in patients with SS, as described in the pancreas (44) .

SNAPs in patients with SS. SNAP23 belongs to the SNAP25 (also known as Q-SNARE) family and is anchored and Table I . Known distribution of protein involved in the mucosecretory mechanism between the salivary acini and ducts, in healthy individuals and patients with SS.

Aquaporins localized in the plasma membrane (53) . Whilst SNAP25 is expressed in the plasma membrane of neuronal and endocrine cells, SNAP23 which is a non-neuronal homologue of SNAP25, is specifically expressed in the apical and basolateral membranes of human submandibular glands (50) . In contrast, in rat parotid glands, SNAP23 has been detected in the apical plasma membrane and intracellular membranes, and forms complexes with STX3, STX4, VAMP2 and VAMP3 (49) . Several differences have been noted in the comparison of the expression and localization of SNAP23 in acinar cells of the labial salivary glands between healthy individuals and patients with SS. Whilst the expression of SNAP23 was detected throughout the plasma membrane in healthy individuals, its expression was absent in the apical plasma membrane and decreased in the lateral plasma membrane in patients with SS. In contrast, no changes were detected in the basal plasma membrane (45).

Physiological roles of AQP5. In human salivary glands, it is hypothesized that AQP5 is the only AQP involved in salivary secretion (54) . In the human parotid, submandibular, sublingual and labial glands, AQP5 labelling was localized to the apical membrane of acinar cells, and its expression was not detectable in duct cells (55, 56) . Proper expression and subcellular localization of AQP5 are required to maintain homeostasis (56, 57) . Parasympathetic innervation is essential for the expression and distribution of AQP5 in the salivary gland (58, 59) . Acetylcholine (ACh) acts on M3 muscarinic acetylcholine receptors to induce translocation of AQP5 in the rat parotid glands (60) , whereas in the rat submandibular gland, cholinergic denervation reduces AQP5 expression (61) .

AQP5 in patients with SS. Studies have shown defective function of AQP5 in patients with SS (56, 62) . Abnormal localization of AQP5 has been reported in patients with SS compared with patients without SS but with xerostomia. AQP5 was shown to be expressed at both the apical and basolateral membranes in the acinar cells of minor salivary glands in patients with SS compared to healthy individuals, in whom AQP5 was restricted to the apical membrane of acinar cells (56) .

Analysis of the saliva of transgenic mice lacking AQP5 showed that it is viscous and hypertonic (63) . Indirect immunofluorescence tests performed in patients with SS detected the anti-AQP5 Immunoglobulin G (IgG), which may explain the low rate of resting salivary flow (64) . The same authors demonstrated in another study that these anti-AQP5 antibodies found in patients with SS recognize different epitopes of AQP5, suggesting their role in salivary gland dysfunction (65) . Treatments of SS AQP5 expression with plant extracts, such as Dendrobium candidum, has been shown to yield positive results when crude materials or the isolated phenolic active compound chrystoxine (66,67).

Physiological role of actin. Cytoskeletal components, such as actin filaments, play a major role in the proliferation and differentiation of cells (68) . They are also involved in salivary gland protein secretion (69) . It has been reported that stimuli which promote salivation can regulate F-actin activity within acinar cells of the salivary glands. Indeed, when salivatory stimuli are absent, F-actin, which is located under the plasma membrane and separates the secretory granules from the luminal membrane, prevents the secretory granules from reaching their exocytotic destination in the human parotid and submandibular gland (70) .

Following stimulation, the actin-cytoskeleton is rearranged and disassembled, consequently allowing secretory granules to reach their destination for exocytosis (71) . F-actins are not only involved in the regulation of the secretion granules trafficking, but also regulate the formation of these vesicles and their movement to the cell membrane (72) . Similarly, it has been reported that depolymerization of F-actin in the rat submandibular gland prevents amylase release and exocytosis (73, 74) .

It has been suggested in other studies that proteins, such as cofilin, a protein necessary for the depolymerization of actin and for controlling the renewal and branching of microfilaments, may play a role in secretory vesicle trafficking (75, 76) . In adrenal chromaffin cells, cofilin is indispensable in the reorganization of the cortical actin cytoskeleton, which is necessary to allow the movement of secretory granules not yet attached to the plasma membrane (75). (38) . cytoplasm (labial salivary protein level is decreased and is Fusion of secretory glands) (38) .

localized throughout the entire granules (44) . STX4: Expressed in the apical and cytoplasm (labial salivary glands) (38) . basolateral plasma membranes STX3: Expression is increased and (submandibular glands) (38) .

localized throughout the entire STX3: Expressed in the apical cytoplasm and the basolateral plasma region of acinar cells (labial salivary membrane in patients (labial salivary glands) (38) . SNAP23: Expressed glands) (38) . in the apical membrane and in the STX4: Expression is decreased and basolateral plasma membrane localized at the basal plasma membrane (submandibular glands, labial (labial salivary glands) (38) . salivary glands) (38, 43) .

SNAP23: Expression is absent in the apical plasma membrane and decreased in the lateral plasma membrane (labial salivary glands) (38) . Tight

Claudins: Expressed in the apical Claudin-1, 4 and 5: Expression id Alteration of paracellular junctions plasma membrane (92) . Occludin:

increased (92, 106) . permeability in the salivary Expressed in the apical plasma Claudin-3, 4 and 5: Localized in the gland (92, 105) . Presence membrane (90, 91) .

basal plasma membrane (92, 105, 106) . of exosomes containing ZO-1: Cytosolic expression (90, 91) Occludin: Expression is decreased (92) . autoantigens from salivary ZO-1: Expression is decreased (92) . gland epithelial cell lines (99) . Actin F-actin: Located under the plasma Expression of (cofilin-1, α-enolase and Presence of anti-Moesin (82). membrane (parotid and subman RGI2) increased (81) . The expression of anti-cofilin-1, dibular gland) (75) .

Decrease in actin-α1/ α2 levels (104). α-enolase and RGI2 Actin-α1/α2: Present in myoepithe increased (81) . lial cells around the acini (104). AQP, aquaporin; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; VAMP, vesicle-associated membrane proteins; STX, syntaxin; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; ZO-1, Zonula occludens-1; RGI2, respiratory growth induced protein 2. Several molecules have been reported to play a major role in this regulation.

Recently, a study on moesin, a structural protein involved in cytoskeletal organization and signaling pathways, showed the presence of anti-moesin antibodies by ELISA and western blotting in patients with SS (77).

Gland tissue samples from patients with primary-SS and primary SS/mucosal associated lymphoid tissue (MALT) lymphoma exhibited significantly upregulated expression of cofilin 1, α-enolase and Rho GDP-dissociation inhibitor 2 compared to non-SS controls. ELISA tests that were used to detect autoantibodies against these proteins showed that three autoantibodies were upregulated in patients with primary-SS/MALT lymphoma compared with patients with primary-SS and the healthy controls, and that patients with primary-SS also had higher levels compared with the healthy controls (76) . This suggests that these autoantibodies may affect the functional role of F-actin and may be indirectly involved in altered secretory vesicle trafficking and exocytosis dysfunction. However, these results require further research to understand the molecular mechanism of the immune reaction against these proteins in vivo.

Physiological role of tight junctions. Tight junctions are protein complexes that are localized in the plasma membrane, such as claudin and occludin, or in the cytosol, such as zonula occludens 1 (ZO1). The functional role of tight junctions is to facilitate the transcellular epithelial flow of ions and water (78, 79) .

Comparison of the expression and localization of claudin-1, claudin-3, claudin-4, ZO1 and occludin in labial salivary gland between patients with SS and healthy individuals showed several differences. Whereas the expression of claudin-1 and claudin-4 was significantly increased in patients with SS, the levels of ZO1 and occludin were decreased compared to the healthy individuals. In contrast, no changes were observed in the expression of claudin-3, although a relocation of claudin-3 and claudin-4 was observed in these patients, which may alter the transcellular permeability of the salivary gland (80) .

It has been shown in patients with SS that TNF and interferon (IFN) levels are elevated, resulting in the alteration of tight junctions and the dysfunction of the salivary epithelium (41, 81) . In these patients, the alteration of tight junctions induced by increased TNF and IFN may cause cell alterations, increasing paracellular permeability (82), upregulated expression of claudin-1 and claudin-4, redistribution of claudin-3 and claudin-4 from the apical to the basal plasma membrane, and a strong negative regulation of occludin and ZO1 expression (80) . The molecular mechanisms proposed for TNF and IFN action on tight junctions (83, 84) are endocytosis of occluding (85), claudin-1 and junctional adhesion molecule 1 by activation of Ras homologue family member A (RhoA)/RhoA kinase (83) , and promotion of accumulation of myosin II-dependent vesicles in the apical region and reorganization of the cytoskeleton (86) . Other mechanisms have also been suggested, such as an indirect effect of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), either by modulating ZO1 mRNA translation or ZO1 protein degradation via the proteasome (80) .

It has been reported that exosomes that are released by epithelial cells in salivary glands contain autoantigens [anti-SS-related antigen A autoantibodies (SSA), SS type B antigen (SSB) and Anti-Smith], which may induce an immune response in these glands (87) . Furthermore, a study of the microvilli that are localized in the apical zone of the acinar cells of labial salivary gland from patients with SS has shown that they are disorganized, possibly due to alterations of the tight junctions and modifications of the actin cytoskeleton (88) .

A recent study on AQP-null mice suggested that AQP5 and tight junctions are functionally related (89) . Indeed, this previous study demonstrated that alterations of the tight junctions were also detected in these mice. Conversely, water transport was only partially altered (51, 89) , suggesting that alteration of AQP5 allows water to pass through other routes.

As previously mentioned, AQP5 in patients with SS is detected at both the apical and basolateral plasma membranes of acinar cells, whereas normally these proteins are localized only to the apical membrane (89, 90) . This indicates that the alteration in AQP5 distribution may be related to the loss of integrity of tight junction proteins. All proteins and their distribution in healthy individuals and patients with SS are described in Tables II and III 

EGCG in autoimmune disorders. EGCG, the ester of epigallocatechin and gallic acid, is the most abundant catechin in green tea. Its potential effects on human health and disease have been widely examined (91, 92) . Targets for EGCG include phosphoinositide 3-kinase/protein kinase B, Janus kinase/signal transducer and activator of transcription proteins, MAPK, as well as proteases, such as metalloproteinases and urokinase (93) .

Autoantigen expression and apoptosis are the most common factors that lead to salivary gland damage (19, 27) . It has been shown that the administration of EGCG in drinking water in non-obese diabetic mice had a protective effect against autoimmune reactions in the salivary glands, as well as inhibiting apoptosis and cell proliferation (27) .

Inhibition of apoptosis (94), a cellular process in which cells undergo controlled cell death, may be achieved through dampening of proteolytic polymerases. This activity is dissimilar to the pro-apoptotic results seen in cancer studies, and this difference in behavior could be due to EGCG binding to sugars (95) , such as ADP-ribose, is necessary for the intrinsic metabolic apoptotic pathway (96) inhibiting their ability to bind to Poly-(ADP-ribose) polymerases and carry out apoptosis in the salivary glands. Additionally, EGCG itself can be an agonist of receptor mediated apoptosis, particularly in cell lines were expression of the TNF superfamily of receptors are upregulated (97) . EGCG also inhibits the cellular process of proliferation (98) . Both apoptosis and cellular proliferation require intracellular remodeling, especially of components such as actin filaments (99, 100) . Inhibition of both of these processes may result in a suspended state of the tissue in cell cycle arrest (101) .

Other studies have shown that green tea extract can also reduce the levels of autoantibodies in animal serum and that EGCG affects TNF-α levels by preventing cytotoxicity in salivary gland cells ex vivo (102, 103) . EGCG can exert an anti-inflammatory effect by inhibiting interleukin-1β, suppressing dendritic cell maturation and reducing T cell activation (104) . A summary of EGCG interactions with the selected proteins is shown in Table IV .

EGCG and the SS autoimmune response. The study of the expression of autoantigens genes [SSA, SSB, fodrin, centromere protein C, golgin-67, coilin and poly (ADP-ribose) polymerase] in normal human epidermal keratinocytes and immortalized salivary gland acinar cells has shown that exposure to EGCG inhibits the expression of these genes (26) , which may explain the low levels of autoantibodies and salivary lymphocyte infiltration following GTP administration in an accepted mouse model of SS [Non-Obese Diabetic (NOD) mice] and in a mouse model of lupus erythematosus [Murphy Roths Large (MRL) mice] (25) .

An autoimmune sialadenitis model of MRL-Fas-lpr mice demonstrated that AQP5 expression is reduced in the salivary glands damaged by apoptosis of cells, and that EGCG was able to restore AQP5 expression and improve the functionality of the glands. The molecular mechanism of EGCG action has been suggested to involve inactivating both the NF-κB and the N-terminal c-Jun kinase, as well as preserving the activity of protein kinase A (105).

Anti-inflammatory activity of EGCG. EGCG affects the submandibular gland in a NOD animal by reducing the size of the SS foci (25) . By using a MTT viability assay on the human salivary gland cell line NS-SV-AC, it was shown that EGCG could ameliorate the effects of TNF-α produced by inflammatory cells, via the attenuation of the cytotoxic effect at the target acinar cells. Similarly, the phosphorylation of p38 was shown to be induced by EGCG via modulation of the MAPK signal transduction pathways, suggesting the presence of another mechanism by which GTPs can attenuate SS pathogenesis (27) . p38 is a key modulator of inflammation, regulating TNF-α and interleukin mediator secretion (106) .

In a dextran sulphate sodium mouse model of colitis, EGCG has been found to be effective at reducing the severity of inflammation and symptoms both preventatively and therapeutically (107) .

SNAREs and EGCG. EGCG, but not its metabolites, has been found to marginally increase the lysosomal proteolysis and autophagy (108) (109) (110) . Lysosomal function is dependent on cathepsins and inhibition of Cathepsin S has been found to induce autophagy through reactive oxygen species-mediated phosphatidylinositol 3-kinase and c-Jun N-terminal kinase signaling pathways (111) . Similarly, Cathepsin S activity was found to be elevated in the lacrimal glands of the SS murine model (112) .

AQPs and EGCG. EGCG significantly inhibits the expression levels of AQP4 in a traumatic brain injury model (113) , as well as a rat spinal injury model (114) , reducing the associated oedema. EGCG prevention of vasogenic oedema associated with status epilepticus was also mediated through regulation of AQP4 expression, however this time via its upregulation (115) .

AQP mediated moisture retention is increased with EGCG; however, whilst moisturizing cream containing the additive was found to cause cellular retention by increasing AQP3 mRNA expression (116) , a murine model of EGCG induced liver failure found that the associated cellular turgidity was concomitant to inhibition of AQP2 (117) . Cellular apoptosis stimulated by EGCG in ovarian cancer was also associated with inhibition of AQP5 production (118) , whereas a murine model of sialadenitis exhibited upregulated secretin levels due to the EGCG-mediated increase in AQP5 expression (105) .

Actin and EGCG. Extracellular expression of α-enolase was inhibited in a kidney model of calcium oxalate monohydrate (COM) crystal binding, resulting in improved outcomes and reduced extracellular crystallization (119) . Furthermore, EGCG was found to maintain and protect the apical microvillar structure and F-actin organization of tubular epithelial cells in a COM induced injury model (120) .

Whilst EGCG can prevent IFN-γ mediated disorganization and inhibition of moesin binding (121) , at high enough concentrations, it can begin to disrupt F-actin organization, which is associated with reduced rates of proliferation (119) .

Otherwise, commonly in the literature EGCG is found to have an anti-fibrotic effect, associated with an inhibition of α-smooth muscle actin expression (122) (123) (124) .

Tight junctions and EGCG. Reports have shown that whilst EGCG is unable to easily cross the intestinal or blood-brain barrier, there is some mechanism involving tight junctions, which, in vivo allows it (125,126). On a molecular level, EGCG was associated with stimulated secretion of tight junction proteins zonula occludens-1 (ZO-1) and occluding (127) (128) (129) in response to bacterial and TNF-α stimulation.

The protective role of EGCG against inflammation stimulated by cytokine release extends to inhibition of tight junction dysfunction induced by IFN-γ (130, 131) . These findings have been replicated in a colitis mouse model, showing a significant increase in expression of occludin, claudin-1 and ZO-1, whilst inhibiting claudin-4, all of which were associated with EGCG mediated inhibition of IL-6 and 12, as well as IFN-γ (107).

Selecting publications on EGCG with a narrow pharmacokinetic focus is challenging. Epidemiological and longitudinal studies on the health benefits in large populations are far outnumbered by cellular, histological and pharmacological investigations. This problem is further exacerbated by the complex interactions EGCG can have, which depend on its route of administration, the gut microflora and its cellular metabolism, which appears to be organ specific. Nonetheless, some signs of structured analysis of EGCG are appearing in the literature, allowing a comprehensive look at its role in salivary physiology.

It is striking to observe the action of the autoimmune response on AQP5, actin and tight junction directly or indirectly. It seems that the alteration of the localization and expression of AQP5, SNAREs, actin and tight junctions can lead to salivary gland dysfunction in SS (Table I; Fig. 1 ). An attractive hypothesis is that SNAREs act as a regulator of the distribution and movement of all these components through the cell, and the autoimmune reaction is the result of the alterations of these proteins. However, as mentioned above GTPs, such as EGCG, have multiple protective effects for autoimmune disease. They maintain total serum autoantibody production at moderate levels due to an inhibitory effect on antigen expression (26) and suppress the increase in TNF-α-induced apoptotic activity in human salivary gland acinar cells in vitro (27) . However, the mechanisms for such multi-level protection by polyphenols are still poorly understood and warrant further investigation of this unique phytochemical with regard to its potential human benefits. The apical plasma membrane water channel AQP5 plays an important role in transporting water across the apical surface of the salivary gland epithelia (58, 59) .

Multiple studies have demonstrated the presence of anti-AQP5 IgG antibodies in patients with primary-SS (132, 133) . Others have demonstrated that EGCG (592 µg/mouse in drinking water, for 57 days) was able to restore AQP5 expression levels and improve gland functionality (105) . These findings suggest a central role of AQP5 in SS, without excluding the importance of SNAREs (134, 135) , actin (136) and tight junctions (51, 89) , in the regulation of AQP trafficking, activity and distribution. It is also necessary to underline the involvement of these molecules in the regulation of secretory vesicles and exocytosis. Alterations of the molecules involved in the regulation of AQP5 trafficking and activity may lead to dysfunction in secretion of vesicles. However, the direct link between the alterations of the trafficking activity of AQP5, the presence of the anti-AQP5 antibodies and the impact on the secretory granule trafficking dysfunction requires further investigation.

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Metabolic and colonic microbiota transformation may enhance the bioactivities of dietary polyphenols

Function of green tea catechins in the brain: Epigallocatechin gallate and its metabolites

New insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3-gallate

The PRISMA 2020 statement: An updated guideline for reporting systematic reviews

SNAREs and traffic

A targeting signal in VAMP regulating transport to synaptic vesicles

Transport from the ER through the Golgi Apparatus

Vesicle tethering complexes in membrane traffic

SNARE-mediated membrane fusion

The multifaceted role of SNARE proteins in membrane fusion

Drooling in Parkinson's Disease: A review

Microvascular autologous submandibular gland transfer in severe cases of keratoconjunctivitis sicca

Disruption of tight junction structure in salivary glands from Sjögren's syndrome patients is linked to proinflammatory cytokine exposure

Endobrevin, a novel synaptobrevin/VAMP-like protein preferentially associated with the early endosome

Core proteins of the secretory machinery

VAMP8 is the v-SNARE that mediates basolateral exocytosis in a mouse model of alcoholic pancreatitis

Aberrant localization of fusion receptors involved in regulated exocytosis in salivary glands of Sjögren's syndrome patients is linked to ectopic mucin secretion

VAMP8/endobrevin as a general vesicular SNARE for regulated exocytosis of the exocrine system

A role of VAMP8/endobrevin in regulated exocytosis of pancreatic acinar cells

Interaction of SNARE proteins in rat parotid acinar cells

Intracellular localisation of SNARE proteins in rat parotid acinar cells: SNARE complexes on the apical plasma membrane

The human submandibular gland: Immunohistochemical analysis of SNAREs and cytoskeletal proteins

Enhanced degradation of proteins of the basal lamina and stroma by matrix metalloproteinases from the salivary glands of Sjögren's syndrome patients: Correlation with reduced structural integrity of acini and ducts

Interferon-γ-induced unfolded protein response in conjunctival goblet cells as a cause of mucin deficiency in Sjögren syndrome

Identification of SNAP-47, a novel Qbc-SNARE with ubiquitous expression

Aquaporin expression in developing human teeth and selected orofacial tissues

Identification and localization of aquaporin water channels in human salivary glands

Abnormal distribution of aquaporin-5 water channel protein in salivary glands from Sjögren's syndrome patients

Salivary acinar cells from aquaporin 5-deficient mice have decreased membrane water permeability and altered cell volume regulation

Aquaporin-5 water channel in lipid rafts of rat parotid glands

Water channels and zymogen granules in salivary glands

Acetylcholine acts on M3 muscarinic receptors and induces the translocation of aquaporin5 water channel via cytosolic Ca2+ elevation in rat parotid glands

Effects of phenylephrine on transplanted submandibular gland

Defective cellular trafficking of lacrimal gland aquaporin-5 in Sjögren's syndrome

Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels

Detection of autoantibodies against aquaporin-5 in the sera of patients with primary Sjögren's syndrome

Functional Epitopes for Anti-Aquaporin 5 Antibodies in Sjögren Syndrome

Dendrobium candidum extract increases the expression of aquaporin-5 in labial glands from patients with Sjögren's syndrome

Dendrobium officinale polysaccharides ameliorate the abnormality of aquaporin 5, pro-inflammatory cytokines and inhibit apoptosis in the experimental Sjögren's syndrome mice

Modulation of mesenchymal stem cell actin organization on conventional microcarriers for proliferation and differentiation in stirred bioreactors

Presence of cytoskeleton proteins in parotid glands and their roles during secretion

Cytoskeletal regulation of human salivary secretion studied by high resolution electron microscopy and confocal laser microscopy

cAMP and Ca(2+)-mediated secretion in parotid acinar cells is associated with reversible changes in the organization of the cytoskeleton

The subapical actin cytoskeleton regulates secretion and membrane retrieval in pancreatic acinar cells

Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells

Differences in the regulatory mechanism of amylase release by rat parotid and submandibular glands

Cofilin activation during Ca(2+)-triggered secretion from adrenal chromaffin cells

Investigation of three potential autoantibodies in Sjogren's syndrome and associated MALT lymphoma

Identification of moesin as a novel autoantigen in patients with Sjögren's syndrome

Molecular physiology and pathophysiology of tight junctions I. Tight junction structure and function: Lessons from mutant animals and proteins

Effect of polyunsaturated fatty acids on tight junctions in a model of the human intestinal epithelium under normal and inflammatory conditions

Acute regulation of tight junction ion selectivity in human airway epithelia

Cytokine mRNA expression in salivary gland biopsies of Sjögren's syndrome

Cytokine mRNA expression in labial salivary glands and cytokine secretion in parotid saliva in Sjögren's syndrome

Proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma alter tight junction structure and function in the rat parotid gland Par-C10 cell line

Interferon-gamma decreases barrier function in T84 cells by reducing ZO-1 levels and disrupting apical actin

TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation

Expression from the human occludin promoter is affected by tumor necrosis factor alpha and interferon gamma

Mechanism of IFN-gamma-induced endocytosis of tight junction proteins: Myosin II-dependent vacuolarization of the apical plasma membrane

The role of epithelial cells in the pathogenesis of Sjögren's syndrome

Interaction between transcellular and paracellular water transport pathways through Aquaporin 5 and the tight junction complex

Expression of aquaporin 3 and 5 as a potential marker for distinguishing dry mouth from Sjögren's syndrome

Pharmacokinetics and safety of green tea polyphenols after multiple-dose administration of epigallocatechin gallate and polyphenon E in healthy individuals

Plant-derived anti-inflammatory compounds: Hopes and disappointments regarding the translation of preclinical knowledge into clinical progress

Can EGCG alleviate symptoms of down syndrome by altering proteolytic activity?

Epigallocatechin-3-gallate inhibits H 2 O 2 -induced apoptosis in mouse vascular smooth muscle cells via 67kD laminin receptor

Influence of herbal medicines on HMGB1 release, SARS-CoV-2 viral attachment, acute respiratory failure, and sepsis. A literature review

ADP-ribose) polymerase in DNA damage-response pathway: Implications for radiation oncology

Epigallocatechin-3-gallate induces the apoptosis of hepatocellular carcinoma LM6 cells but not non-cancerous liver cells

Epigallocatechin-3-gallate induces apoptosis and cell cycle arrest in HTLV-1-positive and -negative leukemia cells

Shaping up to divide: Coordinating actin and microtubule cytoskeletal remodelling during mitosis

The actin cytoskeleton as a sensor and mediator of apoptosis

The green tea catechin epigallocatechin gallate induces cell cycle arrest and shows potential synergism with cisplatin in biliary tract cancer cells

Human salivary tea catechin levels and catechin esterase activities: Implication in human cancer prevention studies

Epigallocatechin-3-gallate, a green tea-derived polyphenol, inhibits IL-1 beta-dependent proinflammatory signal transduction in cultured respiratory epithelial cells

Epigallocatechin-3-gallate, constituent of green tea, suppresses the LPS-induced phenotypic and functional maturation of murine dendritic cells through inhibition of mitogen-activated protein kinases and NF-kappaB

Epigallocatechin gallate stimulates the neuroreactive salivary secretomotor system in autoimmune sialadenitis of MRL-Fas(lpr) mice via activation of cAMP-dependent protein kinase A and inactivation of nuclear factor κB

The biology of p38 kinase: A central role in inflammation

P-145 oral administration of EGCG, a green tea polyphenol, both suppresses and rescues mice from DSS-induced colitis

Isorhamnetin, a 3'-methoxylated flavonol, enhances the lysosomal proteolysis in J774.1 murine macrophages in a TFEB-independent manner

Epigallocatechin-3-Gallate (EGCG) promotes autophagy-dependent survival via influencing the balance of mTOR-AMPK pathways upon endoplasmic reticulum stress

The role of Epigallocatechin-3-Gallate in autophagy and endoplasmic reticulum stress (ERS)-induced apoptosis of human diseases

Inhibition of cathepsin S induces autophagy and apoptosis in human glioblastoma cell lines through ROS-mediated PI3K/AKT/mTOR/p70S6K and JNK signaling pathways

Tear cathepsin S as a candidate biomarker for Sjögren's syndrome

Epigallocatechin-3-Gallate (EGCG) attenuates traumatic brain injury by inhibition of edema formation and oxidative stress

Anti-edema effect of epigallocatechin gallate on spinal cord injury in rats

Epigallocatechin-3-Gallate and PEDF 335 peptide, 67LR activators, attenuate vasogenic edema, and astroglial degeneration following status epilepticus

Identification of compounds in red wine that effectively upregulate aquaporin-3 as a potential mechanism of enhancement of skin moisturizing

A mouse model of subacute liver failure with ascites induced by step-wise increased doses of (-)-epigallocatechin-3-gallate

Inhibitory effect of Epigallocatechin gallate on ovarian cancer cell proliferation associated with aquaporin 5 expression

Epigallocatechin-3-gallate reduces the proliferation of benign prostatic hyperplasia cells via regulation of focal adhesions

Microvillar injury in renal tubular epithelial cells induced by calcium oxalate crystal and the protective role of epigallocatechin-3-gallate

Interferon-gammainduced STAT1-mediated membrane retention of NHE1 and associated proteins ezrin, radixin and moesin in HT-29 cells

Effects of epigallocatechin gallate on diethyldithiocarbamate-induced pancreatic fibrosis in rats

Epigallocatechin-3-gallate, a green-tea polyphenol, suppresses Rho signaling in TWNT-4 human hepatic stellate cells

Dual-drug loaded nanoparticles of Epigallocatechin-3-gallate (EGCG)/Ascorbic acid enhance therapeutic efficacy of EGCG in a APPswe/PS1dE9 Alzheimer's disease mice model

Transepithelial transport of theasinensins through Caco-2 cell monolayers and their absorption in Sprague-Dawley rats after oral administration

Tea polyphenols protect gingival keratinocytes against TNF-α-induced tight junction barrier dysfunction and attenuate the inflammatory response of monocytes/macrophages

Epigallocatechin gallate inhibits endotoxin-induced expression of inflammatory cytokines in human cerebral microvascular endothelial cells

Green tea polyphenols enhance gingival keratinocyte integrity and protect against invasion by Porphyromonas gingivalis

Green tea polyphenol (-)-epigallocatechin gallate blocks epithelial barrier dysfunction provoked by IFN-gamma but not by IL-4

Role of flavonoids in intestinal tight junction regulation

Salivary mucins: Protective functions in relation to their diversity

Reduced sulfation of muc5b is linked to xerostomia in patients with Sjögren syndrome

Pre-and post-synaptic effects of botulinum toxin A on submandibular glands

Selective regulation of maize plasma membrane aquaporin trafficking and activity by the SNARE SYP121

Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking

Subcellular distribution of aquaporin 5 in salivary glands in primary Sjögren's syndrome

Atrophy of myoepithelial cells in parotid glands of diabetic mice; detection using skeletal muscle actin, a novel marker

Claudin-3 is required for modulation of paracellular permeability by TNF-α through ERK1/2/slug signaling axis in submandibular gland

Disruption of endothelial barrier function is linked with hyposecretion and lymphocytic infiltration in salivary glands of Sjögren's syndrome

The authors declare that they have no competing interests.