Patients with BPPV treated at Affiliated Hospital of Inner Mongolia Medical University (Hohhot, China) were enrolled from April 2017 to December 2018. Participants in the control group had no prior history of vertigo onset. For inclusion in the experimental group, patients were required to have met the diagnostic criteria for BPPV (20). Predisposing factors (position or head position change), vertigo characteristics upon onset and positive physical examination (Dix-Hallpike or Roll test) were used to confirm diagnosis. In total, 48 patients were enrolled in the experimental group in strict accordance with the inclusion and exclusion criteria, and 48 controls were recruited. The patients comprised 30 females and 18 males (ratio, 1.67:1.00) aged 50–80 years (mean age, 64.65±1.24 years). The majority (86%) of patients were evaluated within 1 week of symptom onset. The controls comprised 28 females and 20 males (ratio, 1.40:1.00) aged 50–80 years (mean age, 63.25±1.33 years).

Primary exclusion criteria for both the experimental and control groups included: i) Head trauma or ear surgery history; ii) prior medication such as calcium, VD, hormone and associated drug substitution therapy; iii) ear disorders, such as vestibular neuronitis, sudden deafness, otitis media or Meniere's disease; iv) other central nervous system diseases such as transient ischemic attack of posterior circulation, cerebral infarction, cerebral hemorrhage or migraine; v) relevant endocrine diseases such as hypothalamic or pituitary space-occupying lesions; vi) severe renal insufficiency; vii) long-term anxiety or aversion to noisy environments; and viii) unwillingness to cooperate. Written informed consent was obtained from all participants for the use of their blood samples. The present study was approved by the Ethics Committee of the Affiliated Hospital of Inner Mongolia Medical University.

Before treatment, the bone mineral density of patients was measured via dual-energy x-ray absorptiometry using a Hologic Discovery dual-energy x-ray absorptiometer (Hologic, Inc.). Bone quality change was determined by T value: T≥-1.0, normal bone mineral density; −2.5<T<-1.0, osteopenia; and T≤-2.5, osteoporosis.

The subjects were food-fasted for 12 h and liquid-fasted for 8 h before the test. Plasma 1,25(OH)₂D₃ levels were measured using an electrochemiluminescence kit (Elecsys Vitamin D total) according to the manufacturer's directions (Roche Diagnostics). This assay has a sensitivity of 0.83 ng/ml and a measurement range of 0.83–322.50 ng/ml.

A total of 5 ml peripheral venous blood was collected from all subjects and anticoagulated using citric acid. Lymphocyte separation medium (cat. no. Corning LSM 25-072-CV; Corning, Inc.) was used to separate lymphocytes from peripheral blood, and total RNA was extracted using TRIzol^® reagent (cat. no. 15596026, Invitrogen; Thermo Fisher Scientific, Inc.). Total mRNA extraction (PolyATtract^® mRNA Isolation Systems; cat. no. Z5210), first chain synthesis and PCR fluorescent quantitation kits (GoTaq^® qPCR Master Mix; cat. no. A6001) were purchased from Promega Corporation and used according to the manufacturer's protocols. RT-qPCR was performed using the following thermocycling conditions: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 5 sec at 95°C, 10 sec at 60°C and 30 sec at 72°C. The 2^−ΔΔCq method was used to calculate the relative gene expression levels (21). The housekeeping gene β-actin was used as an internal reference. The relative amount of the target gene was obtained by dividing the mean copy number of the target gene by the mean copy number of reference gene. The primer sequences were as follows: Otoconin-90 (OC90) forward, 5′-AGTGGTTTGGATGGTGCCAA-3′ and reverse, 5′-GCACCATCATTTCCACGAGC-3′; VDR forward, 5′-CAGGCTATCATTACGGAGTC-3′ and reverse, 5′-CTGGCATTTGTTTCTGTTCT-3′; NAPDH oxidase 3 (NOX3) forward, 5′-TTTTGGGTTCAACACTGGCT-3′ and reverse, 5′-GTCTAATTGCCTCCTCCACG-3′; and β-actin forward, 5′-TAGTTGCGTTACACCCTTTCTTG-3′ and reverse, 5′-TGCTGTCACCTTCACCGTTC-3′.

Protein expression levels of OC90 and NOX3 were detected in each serum sample using ELISA kits (cat. nos. CSB-EL016255HU and CSB-E17448h-1, respectively; both Cusabio Technology LLC.) according to the manufacturer's instructions. The calibration curve was plotted according to the concentration and optical density value of the standard to calculate the concentration of test sample.

A total of 12 VDR gene knockout and 12 knock-in female C57BL/6JGpt mice (age, 6–8 weeks; weight, ~20 g) were purchased from GemPharmatech Co., Ltd. All mice were raised in specific pathogen free conditions with 12 h light/dark cycle and free access to food and water at 21–24°C and 40–67% humidity. After 3 days, one group of knockout (n=6) and knock-in mice (n=6) was sacrificed to harvest inner ear tissue for subsequent experiments. Another group of knockout mice (n=6) was injected with 1,25(OH)₂D₃ 100 ng/day via the caudal vein daily for 1 week. A total of 12 control wild-type C57BL/6JGpt mice (GemPharmatech Co., Ltd.) were injected with an equivalent dose of normal saline daily for 1 week. The present study was approved by the Animal Ethics Committee of Affiliated Hospital of Inner Mongolia Medical University (approval no. QZ2017023). All experiments were conducted in accordance with the Code for the Care and Use of Animals for Scientific Purposes (22) and the principles of replacing, refining, and reducing.

Bilateral temporal bones of mice were separated, and the muscle and other adherent soft tissues were removed manually. Inner ear samples were ground in liquid nitrogen and placed in PBS buffer (cat. no. C0221A; Beyotime Institute of Biotechnology) containing 1% protease inhibitor (cat. no. 78438; Invitrogen; Thermo Fisher Scientific, Inc.), then placed on ice for 10 min. The solution was centrifuged at 4°C at 12,000 × g for 10 min, and the supernatant was reserved. Following quantification using the BCA Protein Assay kit (cat. no. P0012S, Beyotime Institute of Biotechnology), and electrophoresis (40 µg protein/lane separated via 10% SDS-PAGE), proteins were transferred onto a PVDF membrane. Then, the membrane was blocked for 1 h with 5% skimmed milk at 25°C. After washing, the membrane was incubated at 4°C overnight with the following prediluted primary antibodies: Anti-transferrin (1:1,000; 77 kDa; cat. no. ab109503; Abcam), anti-GAPDH (1:2,000; 36 kDa; cat. no. ab181602; Abcam), anti-VDR (1:1,000; 48 kDa; ab109234; Abcam), anti-NOX3 (1:1,000; 65 kDa; cat. no. ab254572; Abcam) and anti-OC90 (1:1,000; 51 kDa; cat. no. PA5-71564; Invitrogen; Thermo Fisher Scientific, Inc.). The membrane was then incubated with horseradish peroxidase-conjugated secondary antibody (1:1,000; cat. no. A0208; Beyotime Institute of Biotechnology) without agitation for 60 min at 20–25°C. Signals were visualized using ECL reagents (EMD Millipore) and detected using an Amersham™ Imager 680 (Cytiva). ImageJ software (1.8.0; National Institutes of Health) was used for densitometry.

Data are presented as the mean ± SD of three independent repeats. SPSS 19.0 software (IBM Corp.) was used for statistical analysis. Normally distributed data were analyzed via independent sample t-tests; non-parametric Wilcoxon rank-sum tests were used to analyze data with non-normal distributions. Receiver operating characteristic (ROC) curve analysis was performed to reveal the potential diagnostic value of VDR expression levels for BPPV. Differences among multiple groups were detected via ANOVA (one-way) with post hoc Tukey's test. χ² test was used for categorical data. Pearson's correlation analysis was used to assess the correlation between VDR mRNA and 1,25(OH)2D3 levels. P<0.05 was considered to indicate a statistically significant difference.

Data was collected from patients with BPPV diagnosed in Affiliated Hospital of Inner Mongolia Medical University. There was no difference in the age, sex ratio, BMI, or diabetes and hypertension rate between the patient and control groups (Table I). However, cases of decreased bone density (particularly osteoporosis) in patients with BPPV were significantly higher than in the control group (Table I). As shown in Table II, BPPV most commonly involved the posterior (n=25, 52.1%), horizontal (n=16, 33.3%) and anterior canals (n=7, 14.6%). The proportion of patients with canalolithiasis (n=38) was 79.2% and that of cupulolithiasis (n=10) was 20.8%.

The level of 1,25(OH)₂D₃ was measured in patients with BPPV; results indicated that plasma VD levels in patients with BPPV were significantly decreased compared with the control group (Fig. 1A). Serum VD expression levels in patients with canalolithiasis and cupulolithiasis exhibited no significant difference (Fig. S1). As demonstrated via RT-qPCR, serum mRNA expression levels of otolith-associated proteins OC90 and NOX3 were notably reduced in patients with BPPV compared with in controls (Fig. 1B and C). ELISA indicated that protein levels of OC90 were lower in the serum of patients with BPPV compared with in the control group; this was supported by western blot analysis (Fig. 1D and E). Finally, NOX3 protein levels were also significantly lower in the serum of patients with BPPV compared with in the controls, as revealed via both ELISA and western blotting (Fig. 1F and G). The expression levels of 1,25(OH)₂D₃ and otolith-associated proteins were lower in patients with BPPV than in controls. This suggested that the expression levels of VD regulated OC90 and NOX3 expression, and were associated with the occurrence and development of BPPV.

As VD exerts its biological role primarily via interacting with VDR, a potential association between VDR expression levels and BPPV was investigated. RT-qPCR was performed to analyze VDR expression levels in the serum of patients with BPPV: Compared with the control, VDR expression was notably decreased (Fig. 2A), and its expression was positively correlated with that of 1,25(OH)₂D₃ (r=0.3292; P=0.0223; Fig. 2B). Furthermore, ROC curve analysis reported an area under the curve of 0.877 and cutoff value of 1.545, suggesting that VDR was a candidate diagnostic marker of BPPV (Fig. 2C). The mRNA and protein expression levels of VDR were also detected and shown to be significantly lower in patients with BPPV (Fig. 2D and E).

In order to verify the effects of VDR on the expression levels of OC90, VDR^−/− mice were purchased, and inner ear tissue was harvested for the detection of OC90 expression levels. VDR expression levels were first verified in knockout mice: mRNA and protein levels of VDR were significantly decreased in VDR^−/− mice compared with wild-type mice (Fig. 3A and B). Subsequent RT-qPCR experiments using inner ear tissue found that mRNA levels of OC90 were significantly lower in VDR^−/− mice compared with those in wild-type mice (Fig. 3C); western blot analysis showed the same trend for OC90 protein expression (Fig. 3D). Similarly, NOX3 mRNA and protein expression levels were significantly downregulated in VDR^−/− mice compared with in wild-type mice (Fig. 3E and F). This suggested that there was an association between VDR and both OC90 and NOX3 expression levels. In order to investigate whether VD acted via VDR to regulate expression levels of OC90 and NOX3, activated VD or saline was injected into the caudal vein of VDR^−/− mice (wild-type injected with saline, VDR^−/− injected with saline and VDR^−/− injected with activated VD), and inner ear tissue was collected from each group 1 week later. Injection of VD did not reverse the inhibiting effect of the VDR^−/− on otolith protein formation (Fig. 3G-J). This indicated that VD required VDR to exert its biological function.

VDR overexpression mice were constructed to further verify the role of VDR in regulating OC90 and NOX3 expression. The model was successful, indicated by the detection of VDR mRNA and protein (Fig. 4A and B). RT-qPCR and western blotting were used to detect mRNA and protein levels of OC90 and NOX3 in inner ear tissue. mRNA and protein expression levels of both OC90 and NOX3 were significantly increased in VDR overexpression mice compared with in wild-type mice (Fig. 4C-F). Thus, VDR was important for expression of the otolith-associated proteins OC90 and NOX3.