| Objective: Radiotherapy is one of the usual means of head and neck malignancies treatment. However, salivary glands are partially or totally included in the radiation field and caused irreversible xerostomia. Aquaporin(AQP) water transport proteins exist throughout nature and play a fundamental role in transmembrane water movements in many plant and animal tissues. At present, 13 mammalian aquaporin isoform have been identified. Water movement through membrane of acinar cells into lumen in salivary gland is facilitated by aquaporins (AQPs).The molecular pathways through which salivary gland secrete water are not yet explained. The molecular mechanism of radiation-induced xerostomia remain unclear and require further investigation. The present study was focused on elucidation of AQPs expression, AQP5 mRNA and protein localization in rat submandibular gland. Further studies were carried to observe the functional effects of irradiation on rat submandibular gland and to test the hypothesis that AQP5 mRNA and protein expression level were down-regulated after irradiation in rat salivary gland, which may be one of the molecular mechanism of radiation-induced xerostomia. The protective effects of danshen and the proliferation and apoptosis of rat submandibular gland cells were observed after in vivo local irradiation.Materials and methods:. We used reverse transcription-polymerase chain reaction(RT-PCR) to examine the expression of various aquaporin isoform (AQP 1-9 mRNA) in rat submandibular gland. In situ hybridization and immunohistochemistry study with paraformaldehyde-fixed , paraffin-embedded submandibular glands sections at 5μm thickness were used to elaborate the cellular localization of AQP5 mRNA and protein and AQP1 protein. Rats were locally irradiated with a single dose of 15 Gy X-ray. Saliva were collected from rat submandibular glands by intraorally cannulated with polyethylene tube (PE10) at 3 and 30 days postirradiation. A tracheostomy was performed and saliva was collected into preweighted tubes on ice for 20 minutes after subcutaneous injection with 2mg/kg pilocarpine. Rat submandibular glands were removed rapidly and weighted at the same time point. The volume was determined gravimetrically and saliva flow rate is recorded as flow per minute and also normalized to the submandibular gland weight. Protein concentrations of saliva were measured by Bio-Rad protein assay on a spectrophotometric apparatus set at 595nm. Osmolality of saliva were determined by freezing point depression with cryoscopic osmosmeter (Osmomat 030 ).Sections at 5μm thickness were stained with conventional hematoxylin and eosin (H&E) to evaluate the radiation-induced morphological changes in irradiated rat submandibular gland. We analyzed messenger RNA (mRNA) and protein expression of AQP5?in the submandibular glands of rats 3?and 30 days after irradiation. The levels AQP5 in irradiated rat submandibular glands were assessed by semi-quantitative RT-PCR and Westernblot. Further in situ hybridization and immunohistochemistry studies were carried to observe the localization and alteration of AQP5 after irradiation. To evaluate the protective effects of danshen from radiation, rats were intraperitoneally injected with 0.5ml danshen solution. The rat of control was simultaneously administered with same dose of saline. The function and the expression level of AQP5 were assessed as above. BrdU and TUNEL assay were used to observe the proliferation proliferate activity and apoptosis of rat submandibular gland cells at 0h,1d,3d,6d,10d,15d,20d,30d postirradiation.Results: The result of RT-PCR demonstrated that mRNA for AQP5 and AQP8 are expressed at significant level in normal rat submandibular gland. The In situ hybridization revealed a very strong signal over the secretory lobules but not over excretory ducts and stromal septae in submandibular glands. Immunostaining for AQP5 was detected on the apical membrane and intercellular secretory canaliculi of acinar cells in rat submandibular gland. No apparent staining was obse...
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