| Cadmium,one of most toxic heavy metals,is widely distributed in the environment and enters human and animal bodies directly and indirectly and mainly accumulating in liver,kidney and bone.Its biological half-life can be as long as 10-30 years in the body,but the toxic mechanism of cadmium on bone metabolism is unclear.In recent years,the role of P2X7 receptor on bone metabolism has attracted extensive attention.The changes in the function of osteoblasts and osteoclasts were studied by pharmacological activation or inhibition of P2X7 receptor.These studies found that P2X7 receptor knockout mice did not show significant abnormal bone metabolism,but P2X7 receptor knockout in ovariectomized mice model of osteoporosis increased the symptoms of osteoporosis.However,the regulatory mechanism of P2X7 receptor on the function of osteoblasts and osteoclasts has not been clarified.This study is based on mouse bone marrow mesenchymal stem cells and bone marrow mononuclear macrophage model for in vitro study,combining with the experiments of mice exposed to different time exploration of cadmium effects on osteoblast and osteoclast differentiation,by Western blot,qRT-PCR,immunofluorescence,immunohistochemical and flow cytometry test technology,the detection of cell differentiation and apoptosis related indicators,from molecular level to reveal the mechanism of cadmium to mice bone injury,which will provide new theoretical basis for the prevention and treatment of cadmium poisoning and therapeutic targets.The main research contents are as follows:1.Effect of cadmium on differentiation of osteoblasts and osteoclastsUsing osteoblasts and osteoclast precursors as models,osteoblasts and osteoclasts differentiation related indexes were detected by Western blot,ALP staining and Alizarin red staining.After prolonged(72h)cadmium exposure,cadmium inhibited bone formation and mineralization of BMSCs,gene and protein expression levels of key osteogenic proteins COL1A1,OPN and OCN,and osteogenic regulator RUNX2 were significantly down-regulated(p<0.01).Long-term cadmium exposure also significantly inhibited osteoclast formation and bone resorption activity,and the expression levels of osteoclast differentiation and bone resorption key proteins,such as TRAP,CAII,CK and MMP-9 were significantly lower than those in the control group(P<0.01).The protein expression of NFATC1,a key factor regulating osteoclast differentiation,was also significantly decreased(P<0.01).After short-term(12h)cadmium exposure,it was found that cell survival significantly increased at the later stage of osteoblast and osteoclast differentiation(P<0.01).Flow cytometry showed that the apoptosis rate of cells was decreased(P<0.01).Western blot analysis of autophagy in osteoblasts and osteoclasts showed that short-term cadmium exposure significantly inhibited the accumulation of LC3II in osteoblasts and increased the expression of P62,while short-term cadmium exposure increased the accumulation of LC3II in osteoclasts and accelerated the degradation of P62(p<0.01).In order to further verify the effect of short-term cadmium exposure on osteoclast activity,the osteoclasts were treated with cadmium for 12 h at the late stage of osteoclast differentiation,and the results showed that the bone resorption activity of osteoclasts was increased(p<0.01),the structure of pseudopod was more obvious than that of the control group,and the expression of Integrin αvβ3,the key protein of osteoclast adhesion,weres significantly up-regulated(P<001),significantly increased phosphorylation levels of pseudopod key proteins Paxillin,Vinculin,PYK2,and SRC(p<001).In conclusion,long-term cadmium exposure inhibits the differentiation of osteoblasts and osteoclasts,while short-term cadmium exposure is more conducive to the survival of osteoblasts and osteoclasts.2.The role of P2X7 receptor in the inhibition of differentiation of osteoblasts and osteoclasts by cadmiumIn order to investigate the effect of P2X7 receptor on the inhibition of osteoblast and osteoclast differentiation by cadmium,P2X7 knockdown and over-expression cell models were established by adenovirus and lentivirus transfection cells in this study.The indicators related to osteoblast and osteoclast differentiation were detected by Western blot,ALP staining,Alizarin red staining and TRAP staining.The results showed that P2X7 receptor protein expression level was decreased in a dose-dependent manner after long-term cadmium exposure(P<0.01),the downstream PI3K/Akt signaling pathway was also significantly inhibited(P<0.01).P2X7 receptor knockdown significantly inhibited ALP activity and the formation of mineralized nodules in osteoblasts,and expression of osteogenic key proteins were significantly decreased(P<0.01).The number of osteoclasts and bone resorption activity also significantly decreased(P<0.01),the expression of bone resorption key protein was significantly down-regulated(P<0.01).Overexpression of P2X7 receptor significantly alleviated the inhibition of osteoblast and osteoclast differentiation caused by long-term cadmium exposure(P<0.01).However,after blocking P2X7/PI3K/Akt signal,long-term cadmium exposure significantly enhanced the inhibitory effect on osteoblast and osteoclast differentiation(P<0.01).In conclusion,the P2X7/PI3K/AKT signaling pathway plays an essential role in the inhibition of differentiation of osteoblasts and osteoclasts by cadmium.3.Study on the pathogenesis of osteoporosis induced by cadmium in miceIn order to further investigate the effect of cadmium on osteoporosis in mice,we took mice established cadmium poisoning model.Micro-CT,TRAP staining,ALP staining,histology and Western blot were used to detect osteoporosis-related indexes.The results showed that BMD,BV/TV,Tb.Th,Tb.N,Tb.Sp and SMI had no significant changes after short-term(4 months)cadmium exposure compared with the control group(P>0.05).Cadmium exposure did not cause histological changes,but a large number of osteoclasts appeared in the bone tissue after cadmium exposure,which was significantly higher than that in the control group(P<0.01),the number of osteoblasts decreased significantly(P<0.01),the expressions of osteogenic key proteins and apoptotic proteins in bone tissue were significantly diseased(P<0.01).BMD,BV/TV,Tb.Th and Tb.N were significantly lower than those in the control group(P<0.01).Tb.Sp and SMI values were significantly increased(P<0.01).While the bone tissue of the cadmium group was uniformly stained,no obvious pathological changes were observed in the bone tissue of the control group,a large number of local osteogenic tissues were replaced by cartilage tissue,no obvious inflammation was observed,and a large number of hematopoietic cells and multinucleated giant cells were observed in the bone marrow.The number of osteoclasts and osteoblasts were significantly decreased compared with the control group(P<0.01);The expression of osteogenesis and bone resorption key proteins were significantly diseased(P<0.01),and the expression of apoptotic protein was significantly increased(P<0.01).Both long-term and short-term cadmium exposure significantly inhibited the expression of P2X7/PI3K/AKT signaling related proteins(P<0.01).In conclusion,long-term cadmium exposure mice showed significant osteoporosis,while short-term exposure did not show symptoms of osteoporosis.In conclusion:①Long-term cadmium exposure induces obvious osteoporosis in mice and short-term exposure does not.②The inhibition of differentiation of osteoblasts and osteoclasts is the main reason of osteoporosis caused by long-term cadmium exposure,while the promotion of osteoclast activity is the early manifestation of osteoporosis induced by short-term cadmium exposure.③Short-term cadmium exposure inhibits the apoptosis of osteoblasts and osteoclasts and autophagy plays a protective role in this process.Long-term cadmium exposure promotes the increase of apoptotic in bone tissues.④ P2X7 receptor knockdown inhibits the differentiation of osteoblasts and osteoclasts,and overexpression of P2X7 receptor prevents the differentiation of osteoblasts and osteoclasts caused by cadmium. |
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