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Journal of Peking University (Health Sciences) ›› 2021, Vol. 53 ›› Issue (4): 750-757. doi: 10.19723/j.issn.1671-167X.2021.04.022

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Effect of topical injection of cyclosporine A on saliva secretion and inflammation in the submandibular gland of non-obese diabetic mice

ZHU Yi-ying,MIN Sai-nan,YU Guang-yan()   

  1. Department of Oral and Maxilloficial Surgery, Peking University School and Hospital of Stomatology & National Center of Stomatology &National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
  • Received:2021-04-20 Online:2021-08-18 Published:2021-08-25
  • Contact: Guang-yan YU E-mail:gyyu@263.net
  • Supported by:
    National Natural Science Foundation of China(81671005);National Natural Science Foundation of China(81974151)

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Abstract:

Objective: To investigate the effects of topical administration of cyclosporine A (CsA) on salivary secretion and inflammation of the submandibular glands in non-obese diabetic (NOD) mice. Methods: Female NOD mice, 21 aged 14 weeks and 18 aged 21 weeks were selected and randomly divided into low-dose group, high-dose group and control group on average. CsA was injected into submandibular glands. One week later the saliva stimulated by pilocarpine was collected and measured. The submandibular glands were collected to make paraffin sections. The lymphocyte infiltration in submandi-bular gland was observed by microscope after hematoxylin-eosin (HE) staining. The number of lymphocyte infiltration foci was counted to calculate the focus sore and the ratio of lymphocyte infiltration area to total gland area was figured up by Leica image analysis system. The expressions of inflammatory cytokines tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-13, IL-17F, IL22 and IL-23a in the submandibular glands of the NOD mice were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell apoptosis in the submandibular gland was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The levels of serum creatinine (Scr), blood urea nitrogen (BUN), uric acid (UA), alanine aminotransferase (ALT), aspertate aminotransferase (AST), alkaline phosphatase (ALP), albumin (ALB) and γ-glutamyl transferase (GGT) were measured by automatic biochemical analyzer to evaluate liver and kidney functions. Results: After topical injection of CsA in the submandibular gland, the stimulated salivary flow rate of the 14- and 21-week-old NOD mice significantly increased compared with the control group (P<0.01 or P<0.05), and the number and area of lymphocyte infiltration foci in the 14-week-old NOD mice low-dose group significantly decreased compared with the control group (P<0.01). Low and high dose of CsA had similar effects on reducing inflammation and improving salivary secretion. The overall level of inflammatory cytokines in the submandibular gland did not decrease significantly. The number of cell apoptosis of submandibular gland in the NOD mice treated with CsA decreased compared with the control group, but there was no statistically significant difference. Topical injection of CsA had no adverse effect on liver and kidney function in the NOD mice. Conclusion: Topical injection of CsA can reduce lymphocyte infiltration in submandibular gland of NOD mice and improve salivary secretion.

Key words: Sjögren’s syndrome, cyclosporine A, salivary gland, submandibular gland, non-obese diabetic mice

CLC Number: 

  • R782.3

Table 1

Primer sequences used for qRT-PCR"

Primer Forward primer (5'-3') Reverse primer (5'-3')
TNF-α CCTGTAGCCCACGTCGTAG GGGAGTAGACAAGGTACAACCC
IFN-γ CTCAAGTGGCATAGATGTGGAAG TGACCTCAAACTTGGCAATACTC
IL-4 GGTCTCAACCCCCAGCTAGT GCCGATGATCTCTCTCAAGTGAT
IL-13 CCTGGCTCTTGCTTGCCTT GGTCTTGTGTGATGTTGCTCA
IL-17F TGCTACTGTTGATGTTGGGAC AATGCCCTGGTTTTGGTTGAA
IL-22 ATGAGTTTTTCCCTTATGGGGAC GCTGGAAGTTGGACACCTCAA
IL-23a AACTCCTCCAGCCAAGGATCA TCTTGGAACGGAGAGGGGG
GAPDH CTTTGGCATTGTGGAAGGGCTC GCAGGGATGATGTTCTGGGCAG

Figure 1

Variation in the stimulated saliva flow rate of NOD mice * P<0.05, # P<0.01, vs. control group; 14 weeks (n=7), 21 weeks (n=6)."

Figure 2

Histopathological changes of the submandibular glands of 14 weeks and 21 weeks NOD mice A, HE staining of 14 weeks NOD mice; B, HE staining of 21 weeks NOD mice; C, FS of submandibular glands; D, the ratio index representing the ratio of the lymphocytic foci area to the total area of glandular tissue. Control, the control group; Low, the low dose group; High, the high dose group; FS, focus score. 14 weeks (n = 7), 21 weeks (n = 6), *P<0.05, # P<0.01, vs. control group."

Figure 3

Cell apoptosis in the submandibular gland of 14 weeks and 21 weeks NOD mice A, TUNEL and DAPI staining of 14 weeks NOD mice; B, TUNEL and DAPI staining of 21 weeks NOD mice; C, percentage of apoptotic cells. TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; DAPI, 4’, 6-diamidino-2-phenylindole. Control, the control group; Low, the low dose group; High, the high dose group. 14 weeks (n = 7), 21 weeks (n = 6), P > 0.05 vs. control group."

Figure 4

Levels of cytokines in the submandibular gland of 14 weeks and 21 weeks NOD mice A, 14 weeks (n = 7); B, 21 weeks (n = 6); * P < 0.05, # P < 0.01, vs. control group. TNF-α, tumor necrosis factor α; IFN-γ, interferon γ; IL-4, interleukin 4; IL-13, interleukin 13; IL-17F interleukin 17F; IL-22 interleukin 22; IL-23a, interleukin 23a."

Table 2

NOD mice liver and renal function"

Groups UA/(μmoL/L) BUN/(mmoL/L) Scr/(μmoL/L) ALT/(U/L) AST/(U/L) GGT/(U/L) ALP/(U/L) ALB/(g/L)
14 weeks NOD mice (n=7), $\bar{x}±s$
Control 116.00±6.13 9.56±0.48 53.16±5.01 73.17±4.64 96.09±5.06 2.25±0.41 1.94±0.66 30.55±0.77
Low 110.26±10.60 9.96±0.65 52.20±5.27 72.98±5.31 97.20±7.44 1.80±0.34 1.64±0.78 25.56±0.31
High 119.36±10.14 10.60±0.54 55.51±6.98 79.19±2.56 99.72±2.56 1.03±0.31 1.84±0.94 29.20±0.92
21 weeks NOD mice (n=6), $\bar{x}±s$
Control 148.68±14.15 10.69±0.35 62.03±12.71 99.31±10.2 124.95±16.85 0.96±0.26 1.66±1.04 30.80±0.66
Low 128.88±0.92 9.35±0.57 51.39±5.98 85.74±3.36 112.92±7.17 1.27±0.39 0.73±0.27 30.14±0.54
High 133.70±12.30 10.44±0.41 56.32±6.62 91.04±5.72 121.06±11.13 1.41±0.31 1.82±0.77 30.20±0.51
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