Cell culture
Immortalized clones of human salivary gland acinar (NS-SV-AC) and ductal (NS-SV-DC) cells have been generated [27]. These cells were cultured at 37 °C in serum-free keratinocyte medium (SFKM; Gibco/Thermo Fisher Scientific, Grand Island, NY, USA) in an atmosphere containing 5 % CO2.
Animals
A total of 10 C57BL/6 and 20 MRL/MpJ/lpr/lpr (MRL/lpr) female mice were purchased from Japan SLC (Hamamatsu, Japan) and were used as the control and experimental groups, respectively. Mice were kept at a constant ambient temperature (22–24 °C) with a 12-h/12-h light/dark cycle and received a solid diet ad libitum in the animal facility of Tokushima University under specific pathogen-free conditions. The experimental protocol described below was approved by the ethics committee of Tokushima University (permit number 13015). The control groups included 6-week-old, 12-week-old, 20-week-old, and 24-week-old mice (n = 5 for each). The experimental groups included 12-week-old and 20-week-old mice (n = 5 for each).
LIPUS
LIPUS was applied by a modified version of the ST-Sonic clinical device (ITO Co., Tokyo, Japan). The modified system consisted of a 5.0-cm2 circular surface transducer and a cell culture plate. The ultrasound head had a mean beam nonuniformity value of 2.7 and an effective radiating area of 4.1 cm2. An ultrasound signal was transmitted at a frequency of 3 MHz in vitro and 1.5 MHz in vivo. It had a spatial average intensity value of 30 mW/cm2 and a pulse rate of 1:4 (2 ms on and 8 ms off). A six-well plate was maintained in vitro with its top above water level in a foam-fronted plastic sliding assembly containing an aperture of dimensions matching to the monolayer (Additional file 1: Figure S1). The distance between the transducer and the cells was approximately 1 mm. The cell cultures were treated with 20 min of a single ultrasound exposure. The tank water was maintained at 37 ± 0.5 °C. The LIPUS exposure assembly was maintained in a humidified atmosphere of 5 % CO2 at 37 °C during all experiments, and an electronic control panel activated an alert signal if the coupling gel or liquid was depleted. Control samples were treated in parallel, although LIPUS was not applied. The in vivo submandibular glands of the mice received 20 min of LIPUS per day for 14 days.
Cell proliferation
Cells were grown in 96-well microplates (2 × 104 cells/well) in SFKM. After the appropriate incubation period, the number of attached cells was counted using a Z1 COULTER COUNTER (Beckman Coulter, Fullerton, CA, USA). Moreover, the 2-(2-meth-oxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay was performed using Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan). The results were obtained at day 0 as a baseline reading. Subsequently, the culture medium was removed and the cells were stimulated with or without TNF-α (10 ng/ml). Four hours later, the cultured cells were exposed to LIPUS or sham exposure. After 1 day of culturing, the cell numbers were counted again. Cell proliferation was evaluated based on the ratio of treated cells to untreated control cells.
Immunofluorescence
Cells grown on a coverslip were treated with or without TNF-α (10 ng/ml) for 4 h, then received LIPUS or sham exposure. Twenty-four hours later, the cells were washed twice with phosphate-buffered saline (PBS), fixed in 4 % paraformaldehyde in PBS for 20 min, and then incubated for 1 h at 37 °C with goat antihuman AQP5 antibody (1:100, sc-28628; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After three washes with PBS containing 1 % bovine serum albumin, the cells were incubated for 1 h with Alexa Flour 488–conjugated secondary antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA) at room temperature (RT) in the dark. After the unbound antibodies were washed away, coverslips were mounted using fluorescence mounting medium (Dako, Glostrup, Denmark). Bound antibody was observed using a fluorescence microscope (BZ-9000; KEYENCE, Osaka, Japan), and the fluorescence intensity was quantified by using the BZ analyzer (KEYENCE).
Net fluid secretion rate measurements
The net fluid secretion rates for NS-SV-AC and NS-SV-DC cells treated with or without TNF-α (10 ng/ml), as well as those that additionally received LIPUS or sham exposure, were measured using a modified method described previously [28]. Briefly, 4 h after LIPUS or sham exposure, the liquid on the apical side was collected and its volume was measured using a calibrated pipette.
RNA isolation and real-time polymerase chain reaction analysis
Cultured NS-SV-AC and NS-SV-DC cells were treated with or without TNF-α (10 ng/ml) or interleukin (IL)-1β (1 ng/ml) and received LIPUS or sham exposure. After 4 h, total cellular RNA was extracted using NucleoSpin RNA (MACHEREY-NAGEL, Düren, Germany). First-strand cDNA was synthesized from total RNA (1000 ng) using a High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA). Using real-time PCR analysis with StepOnePlus (Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems), mRNA levels of AQP5, TNF-α, and A20 were examined. The following TaqMan probe mixtures were used: TaqMan gene expression assays; AQP5, Hs00387048_m1; TNF-α, Hs01113624_g1; A20, Hs00234713_m1; and β-actin, Hs01060665_g1 (Applied Biosystems). The cycling conditions included 20 s at 95 °C, 40 cycles of 1 s at 95 °C, and 20 s at 60 °C. Detection of β-actin was used as an internal control. Expression of AQP5, TNF-α, and A20 were calculated using the cycle threshold method.
Western blot analysis
Cultured in vitro NS-SV-AC and NS-SV-DC cells were treated with or without TNF-α (10 ng/ml) or IL-1β (1 ng/ml) and then received LIPUS or sham exposure. After 24 h, the cells were precipitated and lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Salivary glands were homogenized in vivo with T-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific). The samples were centrifuged, and the protein concentration of each supernatant was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific) and microplate reader (Corona Electric, Hitachinaka, Japan). SDS-PAGE was used to separate each 20-μg sample in vitro and each 40-μg sample in vivo, and the separated proteins were then transferred electrophoretically onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked for 1 h at RT with 0.1 % Tris-buffered saline with Tween 20 (TBS-T) containing 3 % skim milk, then incubated overnight at 4 °C with antihuman AQP5 antibody (1:500; Santa Cruz Biotechnology), anti-TNF-α antibody (1:500, catalog number 3707; Cell Signaling Technology), phosphorylated inhibitor of nuclear factor of κ light polypeptide gene enhancer in B cells, α subunit (phospho-IκBα) antibody (1:1000, catalog number 9246; Cell Signaling Technology), IκBα antibody (1:1000, catalog number 9242; Cell Signaling Technology), phospho-NF-κB p65 antibody (1:1000, catalog number 3033; Cell Signaling Technology), NF-κB p65 antibody (1:1000, catalog number 8242; Cell Signaling Technology), phosphorylated inhibitor of nuclear factor κB kinase subunit β (phospho-IKKβ) antibody (1:1000, catalog number 2697; Cell Signaling Technology), IKKβ antibody (1:1000, catalog number 2678; Cell Signaling Technology), interleukin 1 receptor-associated kinase 1 (IRAK1) antibody (1:1000, catalog number 4504; Cell Signaling Technology), or anti-β-actin antibody (1:1000, catalog number 4967; Cell Signaling Technology) in TBS-T. The membranes were washed three times with TBS-T for 15 min and then incubated for 1 h with the appropriate secondary antibodies conjugated to horseradish peroxidase (HRP). Bound antibodies were visualized using a Western blot detection system with LumiGLO reagent (Cell Signaling Technology) according to the manufacturer’s instructions. Protein bands were quantitated in vivo by densitometric analysis using image analysis software (CS Analyzer; ATTO, Tokyo, Japan).
Fluid secretion measurements
Control mice (aged 6, 12, 20, and 24 weeks old) and experimental mice (12 and 20 weeks old) had their salivary volumes measured following LIPUS treatment. A modified measurement method described previously was used [29]. Briefly, an intramuscular injection of pilocarpine (5 mg/kg) was administered without anesthesia. The total volume of saliva was then determined gravimetrically after a 20-minute collection period according to a method used in a Saxon test for the diagnosis of patients with SS [30].
Histology
After measuring fluid secretion, all salivary glands were resected, fixed with 4 % phosphate-buffered formaldehyde (pH 7.2), and prepared for histological examination. Formalin-fixed tissue sections (6 μm) were then subjected to hematoxylin and eosin staining, and three pathologists independently evaluated the histology without being informed of the condition of each mouse.
Histological grading was performed according to a previously proposed method [31]. Briefly, longitudinal sections of all glands were examined at × 150 magnification and scored for the degree of inflammatory infiltrate observed. Scoring ranged from 1 to 4 and was used to indicate that 1= 1–5 foci of mononuclear cells were observed among more than 20 cells; 2= more than 5 such foci were observed without significant parenchymal destruction; 3= multiple confluent loci were observed with moderate degeneration of parenchymal tissue; and 4= extensive infiltration of the glands with mononuclear cells and extensive parenchymal destruction were observed, respectively.
Immunohistochemical staining of AQP5
Sections of salivary glands were deparaffinized and rehydrated in a xylene-ethanol series. After the endogenous peroxidases in each section were blocked, the sections were incubated overnight with an antihuman AQP5 antibody (1:200 in an antibody solution buffer; Santa Cruz Biotechnology) at 4 °C. After the sections were washed three times with PBS, they were incubated with EnVision + Rabbit/HRP (Dako) as a secondary antibody. Immunoreactivity was detected using diaminobenzidine (Dako), and each section was counterstained with Mayer’s hematoxylin.
Statistical analysis
Mean and standard deviation values were calculated. Significant differences in experimental data were analyzed by one-way analysis of variance, followed by the Tukey–Kramer test and the Bonferroni–Dunn test as a post hoc test to examine mean differences at the 5 % level of significance.