局部注射环孢素A对非肥胖糖尿病小鼠下颌下腺分泌功能及炎症的影响

doi:10.19723/j.issn.1671-167X.2021.04.022

摘要/Abstract

摘要：

目的: 探讨非肥胖糖尿病(non-obese diabetic, NOD)小鼠下颌下腺局部注射环孢素A(cyclosporine A, CsA)对腺体唾液分泌功能及炎症的影响。方法: 选用21只14周龄和18只21周龄雌性NOD小鼠,随机平均分为低剂量组、高剂量组和对照组。NOD小鼠下颌下腺局部注射CsA 1周后,检测刺激性唾液流率;取下颌下腺标本,制作石蜡切片,用苏木精-伊红(hematoxylin-eosin staining, HE)染色,显微镜下观察腺体淋巴细胞浸润程度;用徕卡图像分析系统计数淋巴细胞浸润灶的数量,计算灶性指数和淋巴细胞浸润灶的面积比;用实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qRT-PCR)检测下颌下腺中肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)、干扰素-γ(interferon-γ, IFN-γ)、白介素-4(interleukin-4, IL-4)、IL-13、IL-17F、IL22和IL-23a等炎症细胞因子的表达;用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, TUNEL)检测下颌下腺凋亡细胞;用全自动生化分析仪测量血清肌酐(serum creatinine,Scr)、尿素氮(blood urea nitrogen, BUN)、尿酸(uric acid, UA)、丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天冬氨酸氨基转移酶(aspartate aminotransferase,AST)、碱性磷酸酶(alkaline phosphatase, ALP)、白蛋白(albumin, ALB)和γ-谷氨酰基转移酶(γ-glutamyl transferase,GGT), 评估肝肾功能。结果: 下颌下腺局部注射CsA后,14周龄和21周龄NOD小鼠的刺激性唾液流率较同龄对照组明显增加(P<0.01或P<0.05);14周龄低剂量组NOD小鼠下颌下腺淋巴细胞浸润灶的灶性指数和面积比较同龄对照组显著减少(P<0.01);低剂量和高剂量组减轻炎症反应和改善唾液分泌功能的作用相似;下颌下腺整体炎症细胞因子表达水平无明显降低;低剂量和高剂量组下颌下腺凋亡细胞数和对照组相比有减少趋势,但差异无统计学意义;局部注射CsA对NOD小鼠肝肾功能无影响。结论: 局部应用CsA可减轻NOD小鼠的下颌下腺淋巴细胞浸润,并改善唾液分泌功能。

关键词: 舍格伦综合征, 环孢素A, 唾液腺, 下颌下腺, 非肥胖糖尿病小鼠

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

中图分类号:

R782.3

朱忆颖,闵赛南,俞光岩. 局部注射环孢素A对非肥胖糖尿病小鼠下颌下腺分泌功能及炎症的影响[J]. 北京大学学报（医学版）, 2021, 53(4): 750-757.

ZHU Yi-ying,MIN Sai-nan,YU Guang-yan. Effect of topical injection of cyclosporine A on saliva secretion and inflammation in the submandibular gland of non-obese diabetic mice[J]. Journal of Peking University (Health Sciences), 2021, 53(4): 750-757.

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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

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