The Role Of Autophagy In Maintaining Homeostasis Of Aged Salivary Gland

Objectives:Cytokeratin-14 positive(KRT14+)progenitor cells play a vital role in the development of salivary gland.Part of KRT14+progenitor cells still retained stemness in adult salivary glands.These progenitor cells can differentiate into ductal cells to maintain the homeostasis of the adult salivary gland,especially when destructions occurred.Autophagy is an evolutionally conserved biological process.It is orchestrated by the synergistic effect of a large number of autophagy-related proteins and intracellular membrane structure.Autophagy eliminates misfolded proteins and damaged organelles in an orderly and precise manner,thereby maintaining intracellular homeostasis.Autophagy is widely involved in cell differentiation and organ development,and also plays an important role in cells resisting internal and external stimuli.However,the study of autophagy in the maintenance of salivary gland homeostasis and damage repair dominated by KRT14+progenitor cells remains to be further investigate.Therefore,we established a conditional knockout mouse(cKO)which specifically deleted Atg5 in KRT 14+precursor cells.According to the analysis the phenotype of cKO salivary gland and its changes through aging process,we reveal the function and the possible mechanism of autophagy in KRT 14+progenitor cells which maintained the homeostasis of salivary gland.Methods:(a)To generate the cKO(Krt14-Cre;Atg5f/f)mice,we crossed Krt14-Cre;Atg5f/+mice with the Atg5f/f mice.(b)To investigate the effect of aging to the autophagy deficient KRT 14+progenitor cells,we choose 3 and 8 month old mice for futher investigation.(c)To analyze histology of salivary glands,we measured morphological alternations of each group by hematoxylin and eosin(H&E)staining.(d)Immunohistochemical staining was performed to detect the expression of proteins possibly related to morphological and physiological differences within salivary glands.ATG5,LC3,p62,and Ubiquitin were detected to measure the actication of autophagy.Ki67 and Caspase3 were stained as the markers of cell proliferation and apoptosis.The subpopulation of cells in salivary gland were identified by the expression of AQP5 and KRT14.Pyroptosis was measured by detecting the expression of NLRP3,Caspase1,and IL-1?.(e)We further located different sub-tissue expression pattern of proteins using double-or triple-labeling immunofluorescence staining.The functional proteins(ATG5,Caspase3,Capasel,Ki67)were colocalized with the cellular markers of the salivary gland(KRT14,AQP5).(f)To investigate salivary physiology,we measured the flow rate of saliva in each group,then further detected the buffering capacity and the concentration of calcium.Results:(a)Compared with the control group,cKO mice had no obvious changes in the appearance of body and salivary gland.(b)The specific knockout of Atg5 in KRT 14+precursor cells inhibited the activation of autophagy in ductal cells.(c)Among the 3-month-old mice,the SMGs of cKO mice had no obvious difference in histological morphology and salivary gland function,but there was apoptosis activation in the SMGs of cKO mice.(d)Among 8-month-old mice,the SMGs phenotype of mice in the cKO group showed significant age related changes.Compared with the control group,the results showed hypertrophy of ductal cells and accumulation of intracellular ubiquitin-positive puncta in the SMGs of cKO mice.Within the cKO salivary gland,the apoptosis was activated in ductal cells and the pyroptosis could be detected in interductal regions;Function of the salivary gland was also impaired which were revealed by the decreased secretion of saliva,weakened buffering capacity,decreased calcium ion content.However,the control group showed no changes in the functions of salivary gland.Conclusion:Our results demonstrated that the deletion of Atg5 in KRT 14+progenitor cells inhibits autophagy and induces an age-dependent dysfunction,rather than the developmental defects of mSMGs.The underlying mechism of this age dependent dysfunction might be that the autophagy-deficient KRT 14+progenitor cells could differentiate into ductal cells to maintain salivary gland homeostasis through aging,while newly differentiated ductal cells had dysfunction due to autophagy deficiency,and at the same time,apoptosis and pyroptosis in salivary glands were activated simultaneously.