Objectives: This study aims to investigate the effects of ionizing radiation on the secretion of the paracellular pathway in rat submandibular glands (SMGs) and reveal the changes in the tight junction (TJ) protein claudin-4.
Methods: A total of 24 Wistar rats were randomly divided into control and irradiation groups. The irradiation groups were further divided into 1, 4, and 12 weeks groups after irradiation. One-time 20 Gy irradiation was given to the SMG area on the experimental side of the irradiation group. At 1, 4, and 12 weeks after irradiation, the secretion of SMGs was measured using the Schirmer's test. The pathological changes in the gland tissues were observed under light microscopy after hematoxylin⁃eosin (HE) staining. The changes in the TJ ultrastructure were observed under transmission electron microscopy. The immunofluorescence staining and Western blot were used to detect the expression levels of muscarinic acetylcholine M3 receptor, aquaporin 5 (AQP5), and claudin-4 protein.
Results: At 1, 4, and 12 weeks after irradiation, the secretion of SMGs in the irradiation group was significantly decreased and lower than that in the control group (P<0.01). At 1 week, the interstitial edema was observed in SMG tissues. Nuclear pyknosis, decreased number of acinar cells, and small focal necrosis with inflammatory infiltration were also observed over time. However, these changes were most evident at 12 weeks after irradiation. In the irradiation group, the TJ ultrastructure of glands at different times appeared to be fuzzy, collapsed, and had decreased electron density. Moreover, the width of TJs was remarkably decreased (P<0.01). The expression levels of M3 and AQP5 were decreased in a time-dependent manner, and the fluorescence intensity was significantly reduced after irradiation. However, the expression levels and fluorescence intensity of claudin-4 were enhanced in different degrees.
Conclusions: The changes in the TJ structure, the upregulation of the claudin-4 expression, and the damage in the paracellular pathway were involved in the hyposecretion of SMGs after irradiation.
目的: 探讨电离辐射引起的紧密连接（TJ）蛋白claudin-4的改变及其对大鼠下颌下腺旁细胞分泌功能的影响。方法: 将24只Wistar大鼠随机分为对照组和照射组，照射组又分为照射后1、4、12周组，照射组一次性给予20 Gy射线局部照射实验侧下颌下腺区。照射后1、4及12周，用Schirmer实验检测分泌量；苏木精-伊红（HE）染色观察腺体组织病理变化；透射电镜观察TJ超微结构；免疫荧光染色和蛋白质印迹（Western blot）检测毒蕈碱型乙酰胆碱受体（M受体）亚型M3、水通道蛋白5（AQP5）及claudin-4的蛋白表达。结果: 电离辐射后1、4、12周，腺体分泌量逐渐降低（P<0.01）；1周时腺体组织以间质水肿为主，随时间进行出现核固缩，腺泡细胞数目减少，小灶性腺体坏死伴炎症浸润，12周时最明显；照射组腺体不同时期TJ超微结构表现为模糊、崩塌、电子密度降低，TJ宽度显著减少（P<0.01）；M3及AQP5表达随时间依赖性下调，荧光强度在照射后明显减弱，而claudin-4表达及荧光强度则表现为不同程度增强。结论: 下颌下腺照射后TJ结构改变，claudin-4表达上调，旁细胞水分泌途径损伤参与了下颌下腺电离辐射后分泌低下。.
Keywords: claudin-4; ionizing radiation; paracellular pathway; submandibular gland; tight junction.