A Mechanism Study Of Dark Plum Spray To Treat Radiation-induced Xerostomia By Promoting Salivary Gland Cells Autophagy

Objective:We set up the rat model of radiation-induced salivary gland damage to observe weight and salivary function on radiation-induced salivary gland after using dark plum spray.Submandibular glands were collected to analyze pathological morphologic change and to detect the expression levels of autophagy-related genes to explore the function,structure and molecular mechanism in submandibular gland cells after radiation-induced damage,laying the foundation for further investigating the molecular mechanism and clinical application in treating radiation-induced xerostomia.Methods:A total of 140 Wistar rats,6～7 weeks and weighs about 170～210 grams,approved by Animal Ethics Committee,were randomly divided into the Control Group(CG),Untreated Group(UG),Negative Control Group(NCG),Positive Control Group(PCG),Experimental Group(EG).CG were fed normally without any processing.UG,NCG,PCG,EG were given a one-time 18 Gy irradiation locally on the submandibular glands.NCG,PCG,EG were respectively given normal saline oral spray,pilocarpine oral spray and dark plum oral spray three times a day after irradiation.CG and UG were still fed normally.The 1st,7th,14th,28th day after irradiation,weight were measured,saliva were collected and salivary flow rate calculated to explore salivary function from all the groups(7 rats of each group).After death,submandibular glands were draw out to analyze pathological morphologic change by HE staining,explore the expression levels of autophagy-related genes Beclinl and Atg5 by Reverse Transcription-Polymerase Chain Reaction and Western blot.Results:(1)The body weight difference in CG was significantly higher than the other groups(P<0.05)in the 7th,14th,28th day after irradiation;the body weight difference in EG was significantly higher than UG,(P<0.05)but no statistically significant than PCG in the 14th,28th day after irradiation.(2)The salivary flow rate in all the irradiation groups was significantly less than CG(P<0.05)in the 1st day after irradiation;the salivary flow rate in UG was significantly less than EG,CG and PCG the salivary flow rate in EG was no statistically significant than CG and PCG in the 7th day after irradiation.(3)Morphological change of submandibular gland cells was observed by H-E staining.The submandibular gland cells of EG,UG,NCG,and PCG became subtle atrophy,the size of gland cells became smaller,some cells were necrosis,obviously nuclear pyknosis and inflammatory cell infiltration,cell compartment broadened in the 1st day after irradiation;Compared with EG,UG and PCG were repaired in the 7th and 14th day after irradiation;UG and NCG still have serious damage but the structure of EG were obvious and rules,no shrink gland,necrosis or inflammatory cell infiltration in the 28th day after irradiation.It showed that the damage submandibular gland cells have been effectively repaired.(4)The autophagy-related genes Beclin1 and Atg5 both expressed in all the groups.RT-PCR showed that the expressions of Beclinl in EG,UG,PCG was higher than CG,there was no statistically significant between EG and UG of the expressions of Beclinl in the 1st,7th,14th day after irradiation.The expressions of autophagy-related genes Atg5 in EG,UG,NCG and PCG were significantly higher than CG in the 1st,7th day after irradiation.(5)Western blot showed that the expressions of Beclinl in CG was lower than EG,UG,NCG and PCG in the 1st,7th day after irradiation;the expressions of Atg5 in the UG,NCG,PCG and EG were higher than CG in the 1st,7th,14th day after irradiation but there was no statistically significant between EG and CG of the expressions of Atg5 in the 1st and 7th day after irradiation.Conclusions:(1)Dark plum spray can improve rat’s appetite after salivary gland radiation-induced damage.Especially in the second to fourth weeks after irradiation,the body weight of salivary gland radiation-induced damaged rats were increased.(2)Dark plum spray can effectively repaired the submandibular gland cells with radiation-induced damage.However,as the present study datum we failed to find the connection between dark plum spray and autophagy after salivary gland irradiation.(3)The autophagic activity of submandibular gland cells may have connection with the early course of radiation-induced injury.