The Effect And Mechanism Of Ubiquitin Conjugating Enzyme Rad6B Deficit On Skin Mitochondria

Objective: Rad6 B is an E2 ubiquitin conjugating enzyme(E2).Our previous research showed that depletion of Rad6 B in mice led to defects in repair of DNA damage,making neurons more prone to aging.Recently,we found that loss of Rad6 B can cause skin aging in mice,but its role in skin tissue and its mechanism are still unknown.This study explored the effect of Rad6 B loss on mitochondrial function in skin tissues with age and whether it can lead to skin aging.To study potential target genes of Rad6 B,and provide the basis for skin aging pathology and treatment.Methods: Gene knockout mice and littermate wild control mice were obtained through paired breeding.Grouped to 1 month old,3 month old,and 12 month old.Tissue sections were obtained using mice skin and embedding techniques.The thickness of skin tissue,the content of collagen fibers,and the number of subcutaneous lipid droplets were observed by HE,Masson,and Oil red-O staining;PCNA and PGC-1α respectively marked the proliferation of skin tissue and mitochondrial biosynthesis by Immunohistochemical dyeing;qRT-PCR assay was used to screen factors related to skin proliferation and metabolism;Western blotting experiments was performed to detect the expression of five complexes in the mitochondrial respiratory chain,mitochondrial fusion and fission,and aging-related proteins and explore the substrate of Rad6B;ATP content detection kit was used to detect the ATP content of mice skin tissue.We Extract primary fibroblasts from the skin,the membrane potential of mitochondria and the colocalization of complexes Ⅰ and Ⅳ on mitochondria were observed by mitochondrial red fluorescent probe and immunofluorescence ND1 and COX4 staining;DCFH-DA fluorescent probe was used to observe the level of intracellular reactive oxygen species ROS;β-galactosidase staining for detecting cell senescence;CCK-8 assay and RTCA assay for detecting primary skin fibroblast viability and proliferation;the mitochondrial morphology was observed by transmission electron microscopy;the oxygen consumption(OCR)and extracellular acidification rate(ECAR)of the primary skin fibroblast were measured by XF24 extracellular flux analyzer.Results: 1.HE,Masson,Oil Red O staining revealed that the skin phenotypes of WT and KO mice were significantly different with age,indicating that knocking out Rad6 B may cause physiological structure changes in mice skin;2.In western blot,qRT-PCR,immunohistochemical experiments,we noticed that the absence of Rad6 B affects the proliferation and metabolism of the skin,and different changes have occurred in each group,this difference was prominent in 1-month-old and 12-month-old groups in particularly;3.In western blot experiments,we detected that Rad6B-knockout reduced the activity of mitochondrial complex I and IV,mitochondrial dynamics was declining in old age,thus the function of mitochondria was impaired;4.Rad6B-knockout contribute to abnormal in mitochondrial membrane potential and structure,cell viability and proliferation,ROS level,and cell senescence in primary skin fibroblasts;5.energy metabolism changes in Rad6B-knockout primary skin fibroblasts with age,which was probably by reprogramming the metabolic network and thus compensated the lack of ATP production in primary skin fibroblasts of the old KO mice.Conclusion: Collectively,these findings indicate that the depletion of Rad6 B with age will lead to a decrease in collagen fiber content,skin cells proliferation,mitochondrial complexes,mitochondrial function and subsequent metabolic disorders,and accelerate skin aging in mice.Therefore,Rad6 B may be essential for mitochondrial function and maintain normal tissue metabolism.