The Protective Effect Of Vitamin D On Cadmium-induced Toxicity In Primary Chondrocytes From Chicken Embryo

Cadmium(Cd)is a common heavy metal pollutant in the environment.The metabolic disorders of vitamin D in the body are secondary to the toxic effect of Cd on renal tubules in the body,which causes the occurrence of cartilage diseases,spontaneous fractures,avian gout and other metabolic diseases.Avian cartilage diseases are difficult to cure,and chondrocytes are the only constituent cells of cartilage.Therefore,the study on the proliferation,differentiation and the physiological functions of chondrocytes are of great significance for understanding the pathogenesis,treatment and prevention of cadmum-induced cartilage diseases.1?,25-dihydroxyvitamin D3(1?,25-(OH)2D3)is the strongest bioactive form of vitamin D in the body.It can act directly on bone cells and participate in maintaining the balance of bone homeostasis.However,whether 1?,25-(OH)2D3 can improve the toxic effect of Cd on primary chondrocytes of chicken embryo needs further study.Therefore,the aim of this study is to establish a stable primary chondrocytes of chicken embryos in vitro culture system,and to study the effects of 1?,25-(OH)2D3 and Cd on chondrocyte phenotype,proliferation,differentiation,apoptosis and other physiological functions at the cellular level,further more investigate whether 1?,25-(OH)2D3 can alleviate the toxic effect of Cd on primary chondrocytes,so as to provide theoretical basis for the clinical prevention and treatment of 1?,25-(OH)2D3 on cadium-induced chondrogenic diseases in poultry.1.Isolation,culture and phenotypic identification of primary chondrocytes from chicken embryoTrypsin and collagenase type IV two-step digestion method was used to isolate chondrocytes from tibia joint cartilage of chicken embryo.Alcian blue staining was used to identify the expression of acidic glycosaminoglycan in chondrocytes,the morphology of chondrocytes was observed under an inverted microscope,the proliferation rate of chondrocytes was detected by CCK-8,and chondrocyte differentiation marker genes were detected by qRT-PCR.The results showed that the identified cells were strongly positive for the expression of acidic glycosaminoglycan.The differentiation phenotype was normal,showing a typical "paving stone" shape under inverted microscope,which in line with the characteristics of chondrocytes.The proliferation of chondrocytes increased significantly after 5 d in vitro culture.With the extension of in vitro culture time,the expression levels of Sox 9,COL2A1 and ACAN mRNA of early differentiation related marker genes of chondrocytes gradually decreased,while the expression levels of late differentiation related genes Mmp-9 and RANKL mRNA gradually increased.It is shown that the chondrocytes from chicken embryo could mature and differentiate normally in vitro.2.Effects of vitamin D on the proliferation,differentiation and physiological functions of primary chondrocytes from chicken embryoIn order to explore the effects of different concentrations of 1?,25-(OH)2D3(10-8,10-9,10-10 mol/L)on primary chondrocytes of chicken embryo,relevant indexes were detected by CCK-8,qRT-PCR,Western blot and immunofluorescence.The results showed that different concentrations of 1?,25-(OH)2D3 promoted the proliferation of chondrocytes,maintained the normal differentiation phenotype of chondrocytes in vitro,and promoted the expression of Sox 9 and ACAN mRNA of early differentiation related marker genes of chondrocytes,among which the concentration of la,25-(OH)2D3 was the most obvious in 10-8 mol/L.10-8 mol/L 1?,25-(OH)2D3 also significantly promoted the expression of COL2A1,a major component of the extracellular matrix in chondrocytes.It is shown that 1?,25-(OH)2D3 positively promoted the proliferation,differentiation and physiological functions of primary chondrocytes in chicken embryo.3.Toxic effects of cadmium on primary chondrocytes from chicken embryoIn order to investigate the toxic effects of Cd on primary chondrocytes of chicken embryo,cells were treated with different concentrations of Cd(0,0.5,1,2,4,6,8,10?mol/L).The relevant indicators were detected by CCK-8,qRT-PCR,Western blot,flow cytometry,alcian blue staining and other techniques.The results showed that Cd above 0.5?mol/L significantly promoted chondrocyte apoptosis.Cd above 1 ?mol/L significantly inhibited the proliferation of chondrocytes and the expression of Sox 9,ACAN mRNA of early differentiation related marker genes of chondrocytes,and inhibited the expression of COL2A1.Cd above 2 ?mol/L significantly inhibited the secretion of acidic glycosaminoglycan by chondrocytes and promoted the expression of Mmp-9.It is shown that Cd could inhibit the proliferation and early differentiation of primary chondrocytes in chicken embryo,and promote the apoptosis of chondrocytes and the degradation of extracellular matrix.4.The protective effect of vitamin D on cadmium-induced toxicity in primary chondrocytes from chicken embryoIn order to investigate the protective effect of 1?,25-(OH)2D3 on Cd induced primary chondrocytic injury in chicken embryos,chondrocytes were treated with 10-8 mol/L 1?,25-(OH)2D3 and 2 ?mol/L Cd.The changes of related indexes were detected by CCK-8,Western blot,flow cytometry,immunofluorescence,alcian blue staining and other techniques.The results showed that 1?,25-(OH)2D3 could alleviate the inhibition effect of Cd on the expression of COL2A1 and acidic glycosaminoglycan in chondrocytes,mitigate the proliferation toxicity of Cd on chondrocytes,and inhibit the pro-apoptotic effect of Cd.1?,25-(OH)2D3 could also reduce the over-expression of Mmp-9 upregulated by Cd,so as to alleviate the degradation of chondrocyte extracellular matrix.In summary,1?,25-(OH)2D3 played a protective role in Cd induced chicken chondrocyte toxic injury cultured in vitro.