Xu H, Shan X F, Cong X, Yang N Y, Wu L L, Yu G Y, Zhang Y, Cai Z G
Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, Beijing, China.
Department of Physiology and Pathophysiology, Peking University Health Science Centre and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing, China.
J Dent Res. 2015 Oct;94(10):1454-62. doi: 10.1177/0022034515590087. Epub 2015 Jun 15.
Intraglandular injection of botulinum toxin type A (BoNT/A) is an effective treatment for sialorrhea. Despite numerous experimental and clinical studies on inhibition of saliva section by BoNT/A, the proteolysis of synaptosomal-associated protein 25 (SNAP-25) following BoNT/A treatment has not yet been confirmed in the salivary gland after injection of BoNT/A. More important, it is not known whether BoNT/A exerts a direct effect in acinar cells. Here, we show that injection of BoNT/A into the rat submandibular gland (SMG) decreased salivary flow in a dose-dependent manner; the inhibitory effect lasted at least 4 wk, and salivary flow recovered to normal levels by 12 wk. During the inhibitory period, SMG neurons and synapses expressed lower levels of full-length SNAP-25, and cleavage of SNAP-25 was observed, as indicated by detection of reduced molecular weight SNAP-25 using Western blotting. In addition, the water channel aquaporin 5 (AQP5) was downregulated and abnormally distributed in rat SMG after injection of BoNT/A. The direct effects of BoNT/A on AQP5 expression and distribution were assessed in vitro to exclude the influence of BoNT/A-induced inhibitory neurotransmission. In stable GFP-AQP5-transfected SMG-C6 cells, treatment with BoNT/A reduced the cell surface protein level of AQP5 in a dose- and time-dependent manner without affecting total AQP5 protein expression. Cell surface biotinylation and immunofluorescence demonstrated translocation of AQP5 from the membrane to the cytoplasm, which was confirmed by decreased levels of AQP5 protein in the membrane fraction and increased levels in the cytoplasmic fraction, suggestive of AQP5 redistribution. Taken together, these results indicated that BoNT/A reversibly decreased saliva secretion in rat SMGs through not only the presynaptic SNAP-25 cleavage but also the postsynaptic AQP5 redistribution. These data provide the first evidence for a direct effect of BoNT/A on the salivary gland.
A型肉毒杆菌毒素（BoNT/A）腺体内注射是治疗流涎症的一种有效方法。尽管针对BoNT/A抑制唾液分泌开展了大量实验和临床研究，但在注射BoNT/A后，唾液腺中BoNT/A治疗后突触体相关蛋白25（SNAP-25）的蛋白水解情况尚未得到证实。更重要的是，尚不清楚BoNT/A是否对腺泡细胞有直接作用。在此，我们发现向大鼠下颌下腺（SMG）注射BoNT/A可使唾液分泌呈剂量依赖性减少；这种抑制作用至少持续4周，且唾液分泌在12周时恢复至正常水平。在抑制期，SMG神经元和突触中全长SNAP-25的表达水平降低，并且观察到SNAP-25的裂解，这通过蛋白质印迹法检测到分子量降低的SNAP-25得以表明。此外，注射BoNT/A后大鼠SMG中的水通道水通道蛋白5（AQP5）表达下调且分布异常。为排除BoNT/A诱导的抑制性神经传递的影响，在体外评估了BoNT/A对AQP5表达和分布的直接作用。在稳定转染绿色荧光蛋白-AQP5的SMG-C6细胞中，BoNT/A处理以剂量和时间依赖性方式降低了AQP5的细胞表面蛋白水平，而不影响AQP5总蛋白表达。细胞表面生物素化和免疫荧光显示AQP5从细胞膜转移至细胞质，这通过膜组分中AQP5蛋白水平降低和细胞质组分中AQP5蛋白水平升高得到证实，提示AQP5发生了重新分布。综上所述，这些结果表明，BoNT/A不仅通过突触前SNAP-25裂解，还通过突触后AQP5重新分布可逆地减少大鼠SMG中的唾液分泌。这些数据为BoNT/A对唾液腺的直接作用提供了首个证据。
