The Role And Mechanism Of Active Vitamin D Deficiency In The Development Of Knee Osteoarthritis

Knee osteoarthritis（OA）is the most common joint disease in the world,but its exact pathogenic mechanism remains unclear.A recent meta-analysis showed that there is a significant correlation between low-vitamin D levels and knee gap narrowing and knee OA progression,but the mechanism of active vitamin D deficiency in the development of knee OA remains unclear.In order to study the role and mechanism of active vitamin D deficiency in the development of knee osteoarthritis,we conducted the following investigation for three aspects.In order to clarify the role of active vitamin D deficiency in the development of knee osteoarthritis,2-month-old wild-type（WT）and 1α（OH）ase-/-mice on a high-calcium-phosphate diet were performed a Sham or anterior cruciate ligament transection（ACLT）,the phenotypic differences were compared and analyzed in the knee joints of mice at 6 months of age using imaging,histopathology,and molecular biology methods.The results showed that diet supplemented with high calcium and phosphorus can correct hypocalcemia,hypophosphatemia and secondary hyperparathyroidism caused by active vitamin D deficiency;both active vitamin D deficiency and ACLT could cause knee joint cartilage loss,and reduce the content of cartilage matrix glycosaminoglycans,down-regulate the expression level of type II collagen gene and protein,up-regulate the expression level of X-type collagen gene and protein,and increase the OARSI score,manifesting as the phenotype of knee osteoarthritis,and the loss of active vitamin D could further aggravate ACLTinduced the phenotype of knee osteoarthritis;active vitamin D deficiency and ACLT both induce arthritic osteophyte formation,and the loss of active vitamin D can increase the formation of knee osteoarthritis.Loss of active vitamin D and ACLT could both increase the senescence and senescence-associated phenotype（SASP）of chondrocytes,and the loss of active vitamin D can further aggravate ACLT-induced chondrocyte senescence and increase of SASP secretion.Active vitamin D mediated by VDR promoted synthesis of chondrocyte matrix proteins and inhibited degradation of chondrocyte matrix proteins.These results suggest that the loss of active vitamin D can accelerate knee cartilage senescence and SASP,inhibit the synthesis of cartilage matrix proteins,promote the degradation of cartilage matrix proteins,accelerate the loss of cartilage in knee joints and the arthritic osteophyte formation,thereby promoting the development of knee osteoarthritis.To determine whether exogenous active vitamin D or antioxidant NAC supplementation can prevent the occurrence of knee osteoarthritis caused by the loss of active vitamin D,post-weaning WT mice were given a high-calcium phosphorus diet,1α（OH）ase-/-mice were given a high-calcium phosphorus diet or injected subcutaneously with 1,25（OH）2D3（1 μg/kg）,three times a week,or given combine a high-calcium phosphorus diet and drinking water to supplement the antioxidant NAC（1 mg/L）to 6 months of age,phenotypic differences of the knee joints of mice were compared and analyzed at 6 months of age using images,histology,histopathology,and molecular biology.The results showed that active vitamin D or NAC supplementation could correct the loss of cartilage in the knee joint,reduced the content of cartilage matrix glycosaminoglycans,decreased the expression of type II collagen gene and protein,and increased the expression level of type X collagen gene and protein,elevated OARSI score,increased osteophyte formation,cartilage cell senescence and SASP caused by active vitamin D deficiency.These results suggest that active vitamin D can inhibit the oxidative stress,cartilage cell senescence,and SASP,promote the synthesis of cartilage matrix proteins,inhibit the degradation of cartilage matrix proteins,prevent the loss of articular cartilage and osteophyte formation,and play a role in the prevention of active vitamin D deficiency induced development of knee osteoarthritis.To determine whether mesenchymal stem cell（MSC）overexpression of Sirt1 can prevent the occurrence of knee osteoarthritis caused by the loss of active vitamin D,we first examined the effect of endogenous active vitamin D depletion and exogenous vitamin D supplementation on Sirt1 protein expression levels.To verify whether the vitamin D receptor could bind to the Sirt1 promoter region using the method of chromosome co-immunoprecipitation.We also generated a Sirt1 transgenic 1α（OH）ase-/-mice（1α（OH）ase-/-Sir1Tg）mouse model that overexpress Sirt1 in 1α（OH）ase-/-MSCs,their phenotypic differences with 1α（OH）ase-/-,Sirt1 Tg and WT mouse knee joints were compared to analyzed used imaging,histopathology,and molecular biology.The results showed that active vitamin D can upregulate Sirt1 expression through VDRmediated transcription;Overexpression of Sirt1 in MSCs can correct cartilage loss in the knee joint,decreased the content of cartilage matrix glycosaminoglycans and the level of type II collagen gene and protein expression,increased expression of X-type collagen gene and protein,elevated OARSI score,increased osteophyte formation,cartilage cell senescence and SASP,upregulated aging-related genes p16,p19,p21,p53 expression levels,down-regulated antioxidant-related transcription factor Nrf2 protein expression,upregulated senescence-related acetylated p53,and inflammation-related acetylated p65 protein expression levels caused by active vitamin D deficiency.These results indicate that overexpression of Sirt1 in MSCs can inhibit the p53 and p65 acetylation,inactivate the p16 and p19-p53-p21 signaling pathways,inhibit chondrocyte senescence and SASP,promote the synthesis of cartilage matrix proteins,and inhibit the degradation of cartilage matrix proteins,prevent the loss of articular cartilage and the formation of osteophytes,thereby preventing the development of knee osteoarthritis caused by active vitamin D deficiency.This study not only revealed the mechanism of active vitamin D deficiency in the induction of knee OA,but also provided theoretical and experimental evidence for the clinical use of active vitamin D,antioxidants,and Sirt1 agonists for clinical prevention and treatment of knee OA.