1,25（OH）₂D₃ Postpones Aging-associated Bone Loss By Inhibiting Oxidative Stress And Cellular Senescence

Active vitamin D has proven effective in the prevention and treatment of osteoporosis or aging-mediated bone loss commonly occurred in elderly people.However,whether its anti-osteoporosis effect is due to its role in postponing bone aging still remains unknown.To test whether 1,25(OH)2D3 deficiency alone can protect against aging-induced bone loss,we compared the bone phenotypes of 1α(OH)ase-/-mice fed a high calcium/phosphate(rescue)diet after weaning with age-and diet-matched wild-type mice.Serum 1,25(OH)2D3 was undetectable,and serum calcium,phosphate and PTH was normalized in 1α(OH)ase-/-mice with a rescue diet.Our μCT results showed that with a normal diet(ND),1α(OH)ase-/-mice age 2 months with secondary hyperparathyroidism exhibited an increase of trabecular bone volume and osteoid volume reflecting unmineralized bone matrix.Of note,1α(OH)ase-/-mice displayed a more dramatic decline of trabecular bone mass from 6 to 12 months of age than WT mice.Histological staining also confirmed that aged 1α(OH)ase-/-mice lost more trabecular bone than WT mice.Moreover,1α(OH)ase-/-mice age 6 months showed an increase in oxidative stress(ROS)and in the expression of p16,p19,p53,p21 and SASP compared to WT mice.These results demonstrate that the progressive bone loss induced by 1,25(OH)2D3 deficiency might be attributable to its induction of aging.Free radical species was identified as a principal mediator of aging.To test the involvement of oxidative stress in aging-associated bone loss induced by 1,25(OH)2D3 deficiency.1α(OH)ase-/-mice age 3 months and their control littermates were exposed to or not to X ray irradiation,and then lumbar vertebrae of each group of mice was removed for analysis.Results showed that there was a remarkable rise of ROS levels,p16,p53,p21,sirt1 and SASP showed in WT and 1α(OH)ase-/-mice exposed to X ray irradiation,however,1α(OH)ase-/-mice exhibited a more dramatic increase of these parameters than WT mice upon irradiation.These findings suggest that 1,25(OH)2D3 deficiency may accelerate aging-induced bone loss by the increase of oxidative stress.Now that 1,25(OH)2D3 deficiency caused the rise of oxidative stress and accelerated aging-related bone loss,we next asked whether 1,25(OH)2D3 can prevent aging-induced bone loss by inhibiting oxidative stress.To answer this question,1α(OH)ase-/-mice and their wild-type littermates after weaning were subcutaneously injected with vehicle or 1,25(OH)2D3(1μg/Kg)thrice weekly or were fed a rescue diet with or without supplements containing the antioxidant NAC(N-acetyl-L-cysteine,lmg/ml).The indicated groups of mice at the age of 6 months were sacrificed and bone phenotypes analyzed.Our results showed that NAC treatment showed similar effects to 1,25(OH)=D3 supplementation in rescuing the bone loss in 1α(OH)ase-/-mice.The trabecular bone volume was remarkably in NAC-or 1,25(OH)2D3-treated 1α(OH)ase-/-mice as compared to 1α(OH)ase-/-mice fed a rescue diet(Fig.3a,b,c).In addition,the ALP-positive area reflecting the osteoblastic activity was significantly higher in 1α(OH)ase-/-mice than in NAC-or 1,25(OH)2D3-treated 1α(OH)ase-/-mice.Furthermore,NAC-or 1,25(OH)2D3-treatment remarkably showed a reduction of the TRAP+ cells compared to 1α(OH)ase-/-mice.The quantitative RT-PCR revealed that the transcript levels of senescence-associated secretion phenotype(SASP)-related molecules in NAC-or 1,25(OH)2D3-treated 1α(OH)ase-/-mice was significantly lower than in 1α(OH)ase-/-mice.In addition,The expression of aging-related proteins in NAC-or 1,25(OH)2D3-treated 1α(OH)ase-/-mice,such as p16,p53 and p21 was significantly lower than that in 1α(OH)ase-/-mice.These results demonstrate that 1,25(OH)2D3 appears to protect against aging-related bone loss by inhibiting oxidative stress and SASP.As a principal hallmark of aging,the expression of p16 was increased in 1α(OH)ase-/-mice.To assess whether the ablation of p16 can rescue the aging-associated bone loss induced by 1,25(OH)2D3 deficiency by postponing skeletal aging,we generated compound mutant mice with the homozygous deletion of both p16 and 1α(OH)ase[1α(OH)ase-/-p16-/-],and compared the bone phenotypes of the indicated groups.Our findings revealed that 1α(OH)ase-/-and 1α(OH)ase-/-p16-/-mice showed normalized serum calcium and phosphate compared to WT mice.Additionally,the ablation of p16 significantly increased the body weight in 1α(OH)ase-/-mice in a time-dose manner.Of note,the median life span was extended from 31 weeks in 1α(OH)ase-/-mice to 65 weeks in 1α(OH)ase-/-p16-/-mice.Moreover,μCT results showed that 1α(OH)ase-/-p16-/-mice exhibited more trabecular bone mass than 1α(OH)ase-/-mice.Furthermore,the ablation of p16 significantly reduced the SASP molecules leading to aging-related bone loss in 1α(OH)ase-/-mice.The increased levels of SASP and β-gal in 1α(OH)ase-/-p16-/-mice were significantly lower than that in 1α(OH)ase-/-p16-/-mice.These findings suggest that 1,25(OH)2D3 seems to postpone aging-related bone loss by inhibiting p16-mediated cellular senescenceThere was a gradual loss of Sirtl,a longlife gene involved in combating oxidative stress with advancing age in WT mice.However,1α(OH)ase-/-mice with a rescue diet age 6 months displayed more dramatic loss of Sirt1.To explore the involvement of Sirtl in 1,25(OH)2D3 deficiency induced aging-related bone loss,we overexpressed Sirtl in mesenchymal stem cell in 1α(OH)ase-/-mice by generating compound mice with the homozygous deletion of 1α(OH)ase and the overexpression of Sirtl in mesenchymal stem cell[1α(OH)ase-/-Sirt1TG].Our results showed that aged 1α(OH)ase-/-Sirt1TG mice still exhibited higher levels of Sirtl that was significantly lowered in 1α(OH)ase-/-mice.Additionally,the overexpressed Sirtl in mesenchymal stem cell significantly increased the body weight of 1α(OH)ase-/-mice.Also,the aging-induced bone loss in 1α(OH)ase-/-mice was largely rescued in 1α(OH)ase-/-SirtlTG mice.μCT results showed that 1α(OH)ase-/-Sirt1TG mice exhibited more trabecular bone mass than 1α(OH)ase-/-mice.As a deacetylase,sirt1 was reported to deacetylate p53 involved in the response to oxidative stress-mediated DNA damage.We found that 1,25(OH)2D3 deficiency significantly increased the expression of acetylated p53 with the decline of Sirt1.Moreover,the elevated mRNA expression of SASP induced by 1,25(OH)2D3 deficiency was markedly rescued by the overexpressed Sirt1 in mesenchymal stem cell.These findings suggest that the aging-related bone loss induced by 1,25(OH)2D3 deficiency may due to the more dramatic loss of Sirtl,causing high level of oxidative stress and SASP.