To Investigate The Protective Effects And Mechanisms Of Phenylephrine On Irradiated Rat Submandibular Gland In Early Phase

Background: Radiotherapy for head and neck cancer inevitably leads to salivaryinjury, resulting in diminished life quality. Because of salivary glands’ exquisiteradiosensitivity and hypofunction in recovery, no effective modalities are available yet.Therefore, it is important to investigate the mechanisms of irradiation injury, and findpractical and effective approaches in order to augment patients’ life quality afterradiotherapy. It is reported that phenylephrine can significantly reduce the irradiatedparotid gland damage in rat, however, the mechanism of that remains unclear.Nicotinamide phosphoribosyltransferase (Nampt) is recently reported that it couldstrengthen cell’s resistance to oxidative stress. However, its role in radiogenicsubmandibular gland injury and its relation with phenylephrine remain unknown yet.Objective: In this study, we investigated the the expression and distribution ofnicotinamide phosphoribosyl transferase (Nampt) in rat salivary glands, studied theeffects of phenylephrine on irradiated rat submandibular gland and Nampt in the glandin early phase.Part Ⅰ: Expression and localization of nicotinamide phosphoribosyltransferasein salivary glands of ratMethods: Parotid, submandibular and sublingual glands were removed from sixhealthy wistar rats. Immunohistochemical assay was applied to detect the distribution ofNampt in parotid gland, submandibular gland and major sublingual gland of rat. Theexpressions of Nampt mRNA and protein were investigated by reversetranscription-polymerase chain reaction (RT-PCR) and Western Blot in parotid gland, submandibular gland and major sublingual gland of rat.Results: Nampt was widely distributed in parotid gland, submandibular gland andmajor sublingual gland of rat, mainly localized in the cytoplasm and nucleus of bothserous acinar cells and ductal cells. The results of RT-PCR and Western Blot showedthat Nampt mRNA and protein were highest expressed in rat parotid gland, followed bysubmandibular gland and major sublingual gland.Conclusions: Nampt mRNA and protein were expressed in parotid gland,submandibular gland and major sublingual gland of rat, and may be involved inunderlying mechanisms of physiology and pathological physiology in salivary gland.Part Ⅱ: Effects of Phenylephrine on Irradiated Submandibular Gland and on theExpression of Nicotinamide Phosphoribosyltransferase in the Glandin Early PhaseMethods: Eighteen healthy wistar rats were randomly divided into three groups:(1)the control group (without irradiation);(2) the simply irradiation group (only irradiationwithout phenylephrine treatment);(3) the phenylephrine treatment group (Phenylephine(5mg/kg) was injected intraperitoneally twice per day). X-ray radiation of20Gy wasdelivered to rat submandibular gland region on a medical linear accelerator underanesthesia. The submandibular glands were removed on the post-radiation day7underregular anesthetization. The morphological changes in submandibular gland wereobserved by light microscope. Immunohistochemistry was used to detect theexpressions of PCNA and Nampt in rat submandibular gland. Apoptosis was quantifiedusing the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nickend labeling method (TUNEL). Western blot was utilized to detect the proteinexpressions of Nampt in submandibular gland.Results:1. The morphological changes of marked cell shrinkage, pyknotic nuclei andvacuolization were observed in acinar cells under light microscope after radiation,compared with the control. Cell atrophy decreased dramatically in phenylephrinetreatment group.2. Results from immunohistochemistry demonstrated that the expression ofPCNA was increased by74.86%(P <0.01) in the simple irradiated group comparedwith the control group, and was increased by162.18%（P <0.01）in the irradiated group with phenylephrine treatment. The numbers of PCNA positive cells under400×magnification in control, simple irradiated and phenylephrine treatment groups were17.7±2.27,30.95±3.66and81.15±5.63respectively.Results fromimmunohistochemistry illustrated that the expression of Nampt was significantlydecreased in the simple irradiated group, and was markedly increased in the irradiatedgroup with phenylephrine treatment.3. Results from TUNEL demonstrated that there were lots of TUNEL positivecells in the irradiated group, and only few TUNEL positive cells were seen inphenylephrine treatment group. The number of positive TUNEL cells under400×magnification in the control, simply irradiation, and phenylephrine treatment groupswere41.22±8.02,220.33±37.92, and65.78±9.80, respectively.4. Results from western blot demonstrated that the protein expression of Namptwas down-regulated by44.85%（P <0.05） in the simple irradiated group comparedwith the control group, and was up-regulated by86.63%（P <0.05）in phenylephrinetreatment group compared with the simple irradiated group.Conclusions:1. Our results showed that phenylephrine could reduce the apoptosis and improvethe proliferation in rat submandibular gland after irradiation. Therefore, thecytoprotective effect of phenylephrine on the irradiated salivary gland may be a novelclinical strategy for preventing the irradiated injury during the early stage afterradiotherapeutic treament of head and neck malignant tumor.2. Phenylephrine could promote the expression of Nampt in irradiated ratsubmandibular gland after radiation in early phase, Nampt is hopefully to be the newtarget for the clinical treatment of irradiated salivary gland injury.