1,25?OH?₂D₃ Deficiency Promotes Colon Inflammation Via Induction Of Senescence Associated Secretory Phenotype

Epidemiological studies showed that 1,25-Dihydroxyvitamin D[1,25(OH)2D3]insufficiency appears to be associated with aging and colon cancer while underlying biological mechanisms remain largely unknown.Inflammatory bowel disease is one of the risk factors for colon cancer.la(OH)ase encoded by Cyp27b1 gene,involves in the metabolism of 25(OH)D3 to 1,25(OH)2D3.Cyp27bl knock-out mouse(Cyp27b1-/-)which chatacterized by defficient 1,25(OH)2D3 production,is a good model to study the function of 1,25(OH)2D3.We investigated whether 1,25(OH)2D3 deficiency has an impact on the colon.We found that 1,25(OH)2D3 deficient mice fed a rescue diet(high calcium,phosphate,and lactose)from weaning until 10 months of age displayed significant colon inflammation and cellular senescence phenotypes.ROS level,DNA damage,and the production of senescence-associated inflammatory cytokines were also increased significantly in the colon of Cyp27b1-/-mice.Thus,the hypothesis we are putting forward is that 1,25(OH)2D3 deficiency leads to colonic inflammation through secretion of senescence-associated inflammatory cytokines by senescent cells.Specific researches are as follows.Biochemical tests proved that,high calcium and phosphorus diet(containing 2%calcaium,1.25%phosphorus and 20%lactose)could rescue the levels of serum calcium and phosphorus in 10-month old female Cyp27b1-/-(KO)mice to normal levels,while the serum 1,25(OH)2D3 level remains much lower than their wide type(WT)littermares,thus excluding the influence of serum calcium and phophorus in this study.In order to determine whether 1,25(OH)2D3 deficiency could induce colon inflammation,we compared body weight,colon length and colon histologic structure between WT and Cyp27b1-/-mice.The results showed that,the colon in KO mice displayed a more advanced inflammation phenotype,including Cyp27b1-/-mice were significantly lighter than WT mice at all time points examined.Full colon length was shorter and the ratio of colon length vs body weight was also decreased,the thinner mucosa with disordered structure including crypt damage,decrease of goblet cells and submucosal edema in Cyp27b1-/-mice,besides,the CD3,F4/80 positive cells were obviously more in KO mice,indicating 1,25(OH)2D3 deficiency could accelerate inflammation phenotype of colon.In order to determine whether 1,25(OH)2D3 deficiency-induced colon inflammation is associated with colon senescence.By using immunohistochemistry,western blotting,we detected the expression levels of p16,as well as the level of senescence associated ?-galactosidase(SA-?-Gal)expression.The results showed that,compared with WT mice,in the KO colon tissues,protein level of p16 were increased significantly,p16 positive cells were obviously more in KO mice,SA-?-Gal expression was increased,indicating 1,25(OH)2D3 deficiency could promote colon senescence.In order to determine whether 1,25(OH)2D3 deficiency-induced colon inflammation is associated with multiplication and apoptosis.By using immunohistochemistry,western blotting,we detected the protein involved in cell cycle and proliferation expression levels of CyclinE,CyclinD,PCNA.The results showed that,compared with WT mice,in the KO colon tissues,protein level of CyclinE,CyclinD,PCNA were decreased significantly,PCNA positive cells decreased obviously more in KO mice and TUNEL positive cells no significant change,indicating 1,25(OH)2D3 deficiency-induced colon inflammation is associated with suppressed multiplication and increased apoptosis.In order to determine whether 1,25(OH)2D3 deficiency-induced colon inflammation is associated with SASP.by using immunohistochemistry and Real time RT-PCR,we detected ptotein level of key transcription factor NF-?B which regulates SASP,as well as the expression levels of main SASP factors(including IL-1?,IL-1?,IL-6,IL-8,MMP3 and MMP13).The results showed that,compared with WT mice,in the KO colon tissues transcription factor NF-?B were increased significantly in KO colon,besides,both protein and mRNA levels of the SASP factors were increased significantly in KO colon,indicating 1,25(OH)2D3 deficiency could increase SASP in the colon.In order to determine whether 1,25(OH)2D3 deficiency-induced cellular senescence is associated with increased DNA damage,by using immunohistochemistry and western blotting,we detected DDR signal pathway molecules(including 8-OhdG,?-H2AX).The results showed that,compared with WT mice,in the KO colon tissues,the protein levels of the above DDR signal pathway molecules were increased significantly in KO colon,indicating 1,25(OH)2D3 deficiency could increased DNA damage.In order to determine whether 1,25(OH)2D3 deficiency-induced cellular senescence is associated with increased oxidative stress,by using biochemistry,immunohistochemistry,western blotting and real-time PCR,we detected oxidative indicators(including ROS,T-SOD and H2O2 levels).The results showed that,compared with WT mice,in the KO colon tissues,ROS and H2O2 levels were increased,while T-SOD levels decreased;indicating 1,25(OH)2D3 deficiency could increase oxidative stress in the colon.These results demonstrated that 1,25(OH)2D3 deficiency might promote colon inflammation,which might be due to the increase of oxidative-stress,DNA damage respose,and subsequent colon senescence as well as SASP.This study not only expound the mechanism underlying the involvement of 1,25(OH)2D3 deficiency in colon inflammation,but might provide theoretical and experimental basis for active vitamin D in the prevention and treatment of colon inflammation.