Effect Of Cadmium On Chicken Cartilage Development And Chondrocyte Apoptosis

Cadmium(Cd)is a heavy metal pollutant in the environment and has toxic effects on the body.Studies have shown that bone is one of the main target organs of Cd,and long-term Cd exposure can result in many bone diseases.The proliferation,differentiation and apoptosis of chondrocytes play an important role in this.Avian chondrocyte abnormalities can lead to a variety of bone diseases,such as Tibial Dyschondroplasa,rickets,osteoarthritis,joint degeneration,etc.The cartilage growth plate of avian has more abundant chondrocytes and deeper blood vessel invasion.Therefore,the differentiation of chondrocytes is very important in bone diseases.However,there are few reports on the effects of Cd on chicken cartilage development,chondrocyte proliferation,differentiation and apoptosis.This study used chicken embryo cartilage and primary chondrocyte as subjects to explore the efrects of Cd on chicken cartilage maturation and chondrocyte apoptosis.1.Effect of Cd on the development of chicken embryo cartilageIn order to investigate the effect of Cd on the development of chicken embryo cartilage,chicken embryo yolk sac was inoculated with different concentrations of Cd(0,0.05,0.1,0.2 mg/mL)in day 8 of incubation.After 8 days,the development of cartilage growth plate was observed by paraffin section and embryo staining.The structure of articular cartilage was observed by scanning electron microscopy.The expression of cartilage-related gene Acan,Col2al,Coll0al,Sox9,Mmp-13,Rankl and Opg were detected by qRT-PCR.The results showed that Cd could reduce the secretion of aggrecan and make condensate nucleus;the collagen fibers on the cartilage surface of the treated group became loose and disordered.Compared with the control group,0.05 mg/mL Cd significantly increased the expression levels of Acan,Sox9,and Col2al(P?O.05),which is characteristic transcription factors in early differentiation of cartilage,but the expression levels in late differentiation of cartilage,Mmp-13 and CollOal significant(P?0.05)or extremely significant(P<0.01)decreased with dose-dependent manner.It is indicated that low concentration of Cd can promote early differentiation of cartilage,but high concentration of Cd can inhibit the growth and development of cartilage.2.Effect of Cd on proliferation and hypertrophy of chicken chondrocytesTo investigate the effect of Cd on proliferation and hypertrophy of chicken chondrocytes,.chicken chondrocytes cultured in vitro were treated with different concentrations of Cd(0,0.2,2,5,10,20 uM)for 2,4,6 days.CCK-8 was used to detect the level of cell proliferation,Alcian blue stain to authenticate chondrocytes,and the expression of chondrocyte-associated gene Acan,Col2al,Col10al,Sox9,Mmp-13,Rankl and Opg were detected by qRT-PCR.The results showed that compared with the control,expression of mRNA in the early stage of chondrocyte differentiation,Acan,Col2al and Sox9 was significantly increased(P?0.01)after treatment with 0.2 ?M Cd in 2 days.With the increase of Cd concentration,the expression was decreased.The expression of mRNA in the late stage of chondrocyte differentiation,CollOal and Mmp-13 of chondrocyte treated by Cd was significant(P<0.05)or significantly decreased(P<0.01).After 6 days of treatment with 2,5,10 ?M Cd,the expression levels of Rankl and Opg were significantly higher than control(P?0.01).It indicates that low concentration of Cd can promote chondrocyte proliferation at an early stage,but Cd inhibits the differentiation and maturation of chondrocytes as time goes by,and may affect PRANKL/OPG pathway.3.Effect of Cd on apoptosis of chicken chondrocytesIn order to investigate the effect of Cd on chondrocyte apoptosis,chicken chondrocytes cultured in vitro were treated with different concentrations of Cd(0,0.2,2,5,10,20 ?M)for 12 h,CCK-8 was used to detect the activity of chondrocyte,Annexin V-FITC/PI was used to detect chondrocyte apoptosis,JC-1 was used to detect mitochondrial membrane potential,nuclear was stained with DAPI to observe the morphological changes,Scanning electron microscopy was used to observe the surface ultrastructure of chondrocytes,and Western Blot was used to detect the expression of Cleaved caspase-3.The results showed that,5?M Cd could significantly reduce(P<0.05)the activity and apoptosis rate of chondrocyte compared with the control,and make damage to cell surface structure.2 ?M Cd could significantly decrease(P<0.05)the level of mitochondrial membrane potential in chondrocytes and make damage to nucleus.Meanwhile,the expression level of Cleaved caspase-3 was significantly increased(P<0.05)compared with the control.It indicated that Cd can induce apoptosis of chicken chondrocytes.