Xerostomia is known as a subjective complaint of dry mouth and is caused by a reduction in salivary secretion and by changes in the composition of saliva related to the decline in the function of salivary glands [1
]. There are several factors which lead to xerostomia, including systemic disease such as diabetic mellitus, autoimmune diseases, and renal failure [1
], as well as the use of certain medications, such as antiallergy agents, antianxiety agents, anticholinergic agents, antidepressants, calcium channel blockers and anorexiants [4
]. Biological aging also induces xerostomia [5
]. Furthermore, xerostomia leads to infectious conditions, such as periodontal disease and caries dentium, in the oral cavity [11
]. These oral infections aggravate diabetes [12
] and increase the risk of atherosclerosis and cardiovascular disease [13
]. Thus, xerostomia lies at the root of degenerative illness.
Salivary glands are subdivided into three major glands (the parotid, submandibular and sublingual glands) and numerous minor glands. These glands are innervated by autonomic nerves [14
]. The cholinergic parasympathetic nerve innervates all salivary glands and the parasympathetic nerve derived from the superior salivary nucleus innervates the submandibular glands (SMGs) and sublingual glands (SLGs), while parotid glands are innervated by the nerve originated from the inferior salivary nucleus. Minor salivary glands, in turn, are innervated by the parasympathetic fibers of the buccal branch of the mandibular nerve, whereas adrenergic sympathetic innervation of SMGs and parotid glands comes from the thoracic segment of spinal cord. The SLGs and minor salivary glands receive a sparse sympathetic nerve fiber. In parasympathetic and/or sympathetic nerves, non-adrenergic and non-cholinergic neurotransmitters, such as substance P, vasoactive intestinal peptide (VIP), neuropeptide Y, neurokinin A, and cholecystokinin (CCK), regulate nerve activity [15
We previously reported that the administration of whey from the milk of Jersey cattle mitigates age-dependent atrophy and functional decline of salivary glands accompanied with changes in gene expression [17
]. The major components of whey proteins are of β-lactoglobulin, α-lactalbumin, bovine serum albumin, and immunoglobulin, representing 50%, 20%, 10% and 10% of the whey fraction, respectively [18
]. Although β-lactoglobulin A and B are the most prevalent variants, the β-lactoglobulin C variant is observed in Jersey cattle. There is one amino acid difference between the B variant (Gln59
) and C variant (His59
]. In the present study, we investigated the effect of Leu57
(LHK) and His59
(HK) peptides on the gene expression profile, age-dependent atrophy and dysfunction of rat salivary glands.
2. Experimental Section
2.1. Experimental Animals and Collection of Saliva
Twenty-week-old male Wistar rats were purchased from SLC, Inc. (Shizuoka, Japan) and given standard laboratory chow (MF; Oriental Yeast, Tokyo, Japan) and water ad libitum. At the age of 80 weeks, rats were divided into the following four experimental groups (n = 3 per group): (I) water-treated control group, (II) LLHK-treated group, (III) LHK-treated group, and (IV) HK-treated group. They were provided water, LLHK (1 mg/mL) (Peptide Institute, Inc. Osaka, Japan), LHK (0.75 mg/mL) (Peptide Institute, Inc. Osaka, Japan), or HK (0.6 mg/mL) (Bachem AG, Bubendorf, Switzerland) ad libitum for 4 weeks. Five-week-old male Wistar rats were also purchased from SLC, Inc. and given a standard laboratory chow and water ad libitum until 16 weeks old. The animals were housed in an environmentally controlled room (22 ± 2 °C) with a 12-h light/dark cycle in accordance with the guidelines of Tokushima University Animal Experiment Committee. Whole saliva was collected from rats during the first 5–15 min after intraperitoneal administration of cevimeline hydrochloride (10.0 mg/kg) (Wako, Pure Chemical. Ind., Osaka, Japan) by a micropipette and placed in Eppendorf tubes kept on ice. Before and after the collection of saliva, the weight of each tube was measured. Salivary glands were rapidly harvested from rats euthanized by spinal dislocation under inhalation anesthesia. All animal procedures were approved by Tokushima University Animal Experiment Committee (TA14120 approved on 1 October 2014, T29-76 approved on 15 November 2017, and T29-124 approved on 1 April 2018) and the Ethics Committee of Animal Experiments in Suntory (APRV000644 approved on 21 May 2018).
2.2. Histological Analysis
Salivary glands for light microscopy examination were fixed by immersion in 10% phosphate-buffered formalin and then processed for paraffin sectioning. Routinely, 5-µm sections were cut and stained with hematoxylin and eosin.
2.3. Isolation of Total RNA
The total RNA from SLGs of 3 control (water-treated 84-week-old) rats and 3 experimental (LHK-treated 84-week-old) rats was isolated using an RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and QIAcube (Qiagen GmbH). RNA purity was quantitated by an absorbance ratio of 260/280 nm using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The integrity of the RNA was assessed by Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). The total RNA samples which had a ratio more than 2.0 and an integrity greater than 9.0 were used in this study.
2.4. DNA Microarray Experiments
Cyanine-3 (Cy3)-labeled cRNA was generated from 0.15 µg total RNA by Low Input Quick Amp Labeling Kit, One-Color (Agilent) and then purified by RNeasy column (QIAGEN, Valencia, CA, USA). cRNA yield and labeling efficiency were validated by ND-1000 spectrophotometer. Then, 0.6 µg of Cy3-cRNA (more than 6 pmol Cy3/µg of cRNA) was fragmented for 30 min at 60 °C in a reaction mixture of 25 µL containing fragmentation buffer (Agilent) and blocking reagent (Agilent). After fragmentation, 25 µL of hybridization buffer was added to the sample and then hybridized to SurePrint GE Unrestricted Microarrays (Agilent, G2519F) for 17 h at 65°C in a rotating hybridization oven. The array was washed twice in GE Wash Buffer (Agilent) and immediately dried. After washing on the DNA Microarray Scanner (Agilent, G2505C), using one color scan setting for 8 × 60 k array slides, the slides were scanned (Scan Area: 61 × 21.6 mm, Scan resolution: 5 µm, dye channel: green, PMT: 100%). This array allowed us to analyze 30,584 Entrez known and unknown genes.
2.5. Analysis of Microarray Data
The scanned images were preprocessed by Feature Extraction Software 10.7.1.1 (Agilent) and then the data were imported into GeneSpring GX software version 14.9 (Agilent). After normalizing the array data, the restriction lists and functional classifications were summed in the comparison of data from 3 different experiments. The signal levels of the genes which were upregulated or downregulated by at least 2.0-fold were further analyzed. The expression change was taken as informative when the p value was <0.05.
2.6. Measurement of α-Amylase Activity in Parotid Glands and Saliva
The α-amylase activity in parotid gland homogenate and saliva was measured as described by Bernfeld using amylose (Sigma Chemical Co., St. Louis, MO, USA) as the substrate [21
]. The incubation mixture consisted of 1% soluble amylose in 0.02 M phosphate buffer (pH 6.9) containing 0.0067 M NaCl. In order to measure α-amylase activity, NaCl was added. After incubation at 20 °C for 3 min, the reaction was stopped by the addition of 1% dinitrosalicylic acid. The tube was heated for 5 min in boiling water. After cooling, absorbance was measured at 540 nm. Amylase activity was expressed as units per mg protein of parotid gland or ml of saliva, where 1 U amylase was defined as the quantity of enzyme that generated 1 mg of maltose in 1 min at 20 °C.
2.7. Statistical Analysis
Data are expressed as the mean value ± standard error. To test for statistically significant differences between two groups, a paired Student’s t test was used for biochemical tests. In microarray analysis, a moderated t test was used. A p value of less than 0.05 was considered to be statistically significant.
This is the first report showing that the administration of LLHK, LHK, and HK prevented and reversed age-dependent atrophy and functional decline of the salivary glands via changes in gene expression. LLHK, LHK, and HK are fragments of β-lactoglobulin C from Jersey cattle. Beta-lactoglobulin, a major whey protein, is an important source of biologically active peptides [24
]. These peptides have been reported to play many crucial physiological roles and have anti-hypertensive, anti-oxidation, antimicrobial, anti-carcinogenic, pathogen adhesion, immunomodulating, and opioid activities, and they can decrease body cholesterol levels. Although many fragments from β-lactoglobulin have been characterized, there have been no investigations of LLHK, LHK, and HK to our best knowledge.
Saliva has important functions, such as in mastication, oral microbial defense, gustation, lubrication, speech, deglutition, digestion, mineralization of teeth, and the protection of mucosal tissues [25
]. These essential functions of saliva are strictly connected to its volume and composition when secreted from salivary glands [26
]. During aging, salivary volume decreases and the composition of saliva changes [2
]. The administration of LLHK, LHK, and HK upregulated major salivary protein genes, such as proline-rich proteins (Prpmp5
, Prp2, Prb1, Prp15
), cystatins (Cst5
, Cyss, Vegp2
), amylases (Amy1a, Amy2a3
), and lysozyme (Lyzl1
), suggesting that these peptides Change to “15”.reversed the age-dependent decline of salivary function.
Parotid glands and SMGs are innervated by cholinergic and adrenergic nerves. In addition to these nerves, the VIP and His-Met (HM) immunoreactive nerve, which is a non-adrenergic and non-cholinergic nerve, is in close proximity to the acini and ducts of parotid glands and SMGs [15
]. HM is known to have the same precursor as VIP. It is possible that LLHK, LHK, and HK have an effect on non-adrenergic and non-cholinergic nerves in salivary glands. Immunocytochemical investigations have demonstrated the presence of a number of neuropeptides surrounding the inferior salivary nucleus [27
]. Although it is uncertain that the HM immunoreactive nerve remains in existence in the inferior salivary nucleus, the oral administration of LLHK, LHK, and HK may influence central and/or peripheral non-adrenergic and non-cholinergic nerves.
Acinar cell atrophy, cytoplasmic vacuolization, and lymphocyte infiltration are detected in the SMGs of aged mice [8
]. Meanwhile, in humans, the aging effects marked by a progressive decrease in glandular volume and acini volume accompanied by an increase in the volume of ducts, stroma, and adipose tissues are observed in SLGs [28
]. The transcriptional changes by LHK-administration occurred within 1 week, but it took several weeks for the restoration of morphological decline (unshown data). In this study, we showed that the 1-month administration of LLHK, LHK, and HK peptides derived from β-lactoglobulin C prevented and/or reversed the age-dependent atrophy of SLGs.
Gly-His-Lys (GHK) is a peptide isolated from human serum [29
] that has various biological functions, including in liver growth, angiogenesis, and nerve outgrowth, as well as anti-oxidation, anti-inflammatory, anti-cancer, anti-pain, and cell protective and regenerative actions [30
]. GHK has also been shown to act on the expression of genes relevant to nervous system functioning and cognitive decline [31
]. GHK has a high affinity for ionic copper, and studies have reported that, upon the formation of the GHK-Cu complex, these functions are advanced [32
]. Furthermore, it has been reported that GHK changes the gene expression pattern in lungs via the activation of transforming growth factor beta (TGF-β) [33
LHK treatment induced the expression of the Tcfap2β
gene in salivary glands. As reported previously, whey consumption induces Tcfap2β
]. The AP-2 family of transcription factors consists of five members, AP-2α, 2β, 2γ, 2δ
, and 2ε
, each encoded by a separate gene [34
], and is known to have tissue-specific expression to regulate the expression of other genes [23
]. The transcription factor AP-2 promotes cartilage and skeletal development and reduces apoptotic cell death in renal epithelial cells [35
]. LLHK, LHK, and HK, which are derived from β-lactoglobulin C, may induce salivary gland-specific genes via the transcription factor AP-2β.
In this study, we investigated the effects of LLHK, LHK, and HK on salivary glands. As the structure of LHK and GHK are very similar, LLHK, LHK, and HK may also have multiple functions.