The Role And Mechanism Of Transcription Factor NFE2L2/NRF2 In Enhanced Differentiation Of Osteoclasts And Osteoporosis Caused By Cadmium Exposure

Objective:Osteoporosis is a common systemic metabolic bone disease.In recent years,the incidence rate of osteoporosis has increased year by year with the aggravation of population aging,and its damage to the quality of life of patients has become increasingly prominent.Among the many cells involved in the occurrence and development of osteoporosis,osteoclasts,as the core cells that play the role of bone absorption,have received extensive attention.In recent years,the role of environmental pollution exposure in osteoporosis has become increasingly clear,especially the damage of cadmium,which takes kidney and bone as direct target organs,to bone homeostasis.Environmental cadmium exposure has become a clear risk factor for bone loss and osteoporosis.Existing studies have shown that cadmium exposure can promote the differentiation of osteoclasts by increasing the level of ROS in cells,leading to increased bone resorption and destruction of bone microstructure.The transcription factor Nfe2l2 is an important transcription factor that regulates the homeostasis of cell oxidative balance and resists oxidative damage.Clear evidence shows that it plays an important role in osteoclast differentiation and cadmium-induced renal injury.However,the role and specific mechanism of NFE2L2 in cadmium-induced osteoporosis are unclear.In order to clarify the role of NFE2L2 in cadmium-induced osteoclast differentiation enhancement,this study used Nfe2l2 knockout mice to give cadmium exposure to clarify the role of NFE2L2 in cadmium-induced osteoporosis,and used mouse bone marrow primary cells,Nfe2l2 silenced and overexpressed RAW264.7 cell line to explore the role and molecular mechanism of NFE2L2 in osteoclast differentiation enhancement under cadmium exposure.Method:Using wild type,Nfe2l2 knockout C57BL/6 mice and their controls,a model of cadmium exposure in drinking water was established.Wild-type mice were randomly divided into Control,50 ppm and 100 ppm groups according to body weight.Nfe2l2knockout mice and their controls were randomly divided into Control and 100 ppm groups.Control group was given distilled water.All mice were given cadmium water at 12 weeks until the end of the experiment.The bone and plasma of mice were collected for Micro CT,pathology,mechanics and plasma detection.Nfe2l2 was used to knock out murine myeloid cells and Nfe2l2 silenced and overexpressed RAW264.7cell line,and cadmium treatment was given to induce differentiation,and the cell differentiation ability was tested.The Nfe2l2 silenced RAW264.7 cell line was treated with cadmium to detect the expression of NFE2L2 and related antioxidant genes.On this basis,antioxidant and various types of ROS inhibitors were used to analyze ROS level and production mode,and evaluate the differentiation ability of cells after intervention.Result:1.Cadmium exposure led to bone loss in mice:Compared with the control group,the amount of drinking water in Wild-type mice decreased,and there was no difference in body weight and diet results;In the 100 ppm group,the cancellous bone of the femur of mice was significantly reduced,and the bone volume,bone trabecular thickness and bone trabecular number were decreased.2.The loss of bone mass caused by cadmium exposure was aggravated by the loss of Nfe2l2:In the 100 ppm Nfe2l2^-/-group,the femoral cancellous mass was significantly reduced,the bone volume,the number of bone trabeculae and the area of cortex were reduced,and the maximum load and fracture load were reduced.3.Nfe2l2 deficiency promoted the increase of osteoclast activity induced by cadmium exposure:The number of osteoclasts in Nfe2l2^-/-mice in the control group was significantly increased compared with that in the Wild-type mice,and the number of osteoclasts in Nfe2l2^-/-mice in the 100 ppm group was further increased compared with that in the wild-type mice;In the 100 ppm group,Nfe2l2^-/-mouse osteoclast surface/bone surface and osteoclast number/bone circumference increased,and the bone resorption markers CTX1 and TRAP5b in plasma increased.4.Nfe2l2 inhibits the enhanced differentiation of osteoclasts induced by cadmium exposure:Compared with the control,Nfe2l2^-/-mouse bone marrow primary cells and Nfe2l2 silenced RAW264.7 cell line increased the number of TRAP-positive cells under the conditions of induced differentiation,and the expression level of the m RNA of Cathepsin K,ATP6V0D2 and H⁺-atpase,the indicators of osteoclast function and fusion,increased the expression level of NFATc1 protein,and the difference was further amplified after cadmium treatment;Compared with the control,the number of TRAP-positive cells in the RAW264.7 cell line overexpressed with Nfe2l2 was reduced under the condition of induced differentiation,the m RNA expression level of Cathepsin K,ATP6V0D2 and H⁺- atpase,the expression level of NFATc1 protein was decreased,and the difference after cadmium treatment was smaller than that of the cells silenced with Nfe2l2.5.Cadmium exposure led to the increase of intracellular antioxidant enzymes and ROS levels during differentiation:Cadmium exposure led to the increase of m RNA and protein levels of antioxidant enzymes and phase II detoxification enzymes in cells,and the expression levels of antioxidant enzymes and phase II detoxification enzymes in Nfe2l2 silenced cells were lower than those in control cells under basic and cadmium exposure conditions;The ROS probe DCFH-DA coincides with the mitochondrial marker.Fluorescence quantitative analysis and flow cytometry showed that the ROS level in cells increased with the increase of differentiation time,and the ROS level increased more significantly after cadmium treatment.6.Antioxidants and ROS inhibitors reduce the intracellular ROS level and inhibit osteoclast differentiation under cadmium treatment:NAC,DPI and Mito Q effectively reduce the increase of ROS level caused by cadmium exposure,reduce the increase of osteoclast differentiation caused by cadmium exposure,and inhibit the increase of Cathepsin K,ATP6V0D2,H⁺-atpase m RNA and NFATc1 protein level.Conclusion:Cadmium exposure in drinking water can cause bone loss in mice.After Nfe2l2deletion,mice are more sensitive to the bone loss caused by cadmium exposure.The important reason for this effect is the increase of osteoclast activity.Cadmium treatment led to the increase of ROS production in the cytoplasm and mitochondria of osteoclasts and promoted the differentiation of osteoclasts.NFE2L2 reduces the intracellular ROS level by regulating the expression of intracellular antioxidant enzymes,thus inhibiting the enhanced differentiation of osteoclasts induced by cadmium exposure...