The Role Of Nrf2 In Bone Metabolism And The Mechanisms Of Regulation In Osteoclast Differentiation

Objective: Bone is the body’s calcified connective tissue,which is constantly renewed and remodeled to maintain a high degree of dynamic balance.Normal bone metabolism plays an important role in maintaining mineral homeostasis and the skeletal structural integrity in the body.Bone resorption mediated by osteoclasts and bone formation mediated by osteoblasts are two core processes in maintaining bone homeostasis,which are regulated by a variety of factors,including cytokines,hormones and mechanical stimulation.The breakdown of the balance of bone metabolism will cause the disorder of bone microstructure as the main manifestation of the disease.For example,osteoclast mediated bone resorption leads to pathologic osteoporosis.The high rate of disability and mortality caused by osteoporosis seriously affects the quality of life of individuals,and brings great social and financial burden.So,it is of very important implications to explore the pathogenesis of osteoporosis in order to guide the clinical prevention and treatment of osteoporosis.Recent studies have shown that oxidative stress causes damage to osteoclasts and osteoblasts.The dysfunction is one of the causes of osteoporosis.Oxidative stress is mainly caused by the imbalance between oxygen free radicals(especially ROS)and antioxidant processing capacity.Nuclear factor erythroid-derived 2-like 2(NFE2L2,Nrf2)is an important transcription factor regulating adaptive antioxidant responses and is closely related to bone metabolism.However,the effects of Nrf2 on bone metabolism are multiple and uncertain.Nrf2 plays an important role in osteoclast differentiation,and Nrf2 can inhibit osteoclast differentiation by reducing intracellular ROS levels through upregulation of antioxidant enzyme expression.Preliminary results from the section have also shown that Nrf2 deficiency can lead to increased sensitivity to foreign stimuli and greater susceptibility to osteoporosis in mice.On the other hand,animal experiments have shown that deletion of Nrf2 in bone tissue would lead to a decrease in bone mass manifested as low bone density.Previous results also showed that Nrf2 knockdown in mice would result in reduced bone strength in the femur and conus and load-induced bone formation would be significantly reduced compared to wild controls.Therefore,in this study,the ovarian removal mouse model,aging mouse model and rotational exercise mouse model were established by using systemic Nrf2 knockout mice,aiming to clarify the role of Nrf2 in bone metabolism,and to study the role and mechanism of Nrf2 in osteoclast differentiation through in vivo cellular assays,aimed at providing new targets for clinical prevention and cure of osteoporosis and theoretical basis.Methods:1.Estrogen deficient osteoporosis model: Systemic Nrf2 knockout(Nrf2-KO)mice and co-litter wild-type(Nrf2-WT)mice were used.From birth to 12 weeks of age,12 to15 female mice were random for each genotype into two groups: the other group was the castration model group(OVX)and the other group was the Sham operation control group(Sham).After surgery,the body weight of the mice was detected weekly,and the basic indexes such as food intake and water intake,body composition analysis were monitored.After 28 weeks of modeling,fasting blood glucose level of mice was measured,all organs were collected for weighing,and blood from mice eyeball was collected under carbon dioxide anesthesia for the determination of plasma bone resorptive markers Trap-5B and CTX1 and plasma bone formation marker P1 NP.Prepare the right femur for micro-CT scan analysis.2.Aged osteoporosis model: Nrf2-KO mice and Nrf2-WT female mice were used,with 12-15 mice in each group.After feeding to 24 weeks,6-8 mice from each group were randomly selected to finish modeling.As the control group(Cont),the eye blood of mice was collected under carbon dioxide anesthesia for the determination of plasma bone resorptive markers TRAP-5B and CTX1.The left femur was collected for histopathological TRAP staining,and the right femur was analyzed by micro-CT scan.The remaining mice were fed until modeling was completed at 65 weeks of age.As the Aged group,the relevant experiment was conducted,and the detection method was the same as the control group.3.In vitro cell experiments were used to study the molecular mechanism of Nrf2 in the process of osteoclast differentiation: Primary bone marrow mesenchymal stem cells from male Nrf2-KO and Nrf2-WT mice were used as experimental materials,and RANKLinduced differentiation was performed to detect ROS levels,osteoclast differentiation ability and downstream gene expression.NRF2 gene was stably silenced by lentivirus in rat osteoclast cell line RAW 264.7.The silenced group(NRF2-KD)and the control group(Scramble)were differentiated by NAC and DPI,respectively.ROS level,localization,osteoclast differentiation ability and downstream gene expression were detected.Two different genotypes of RAW264.7 were used for lentivirus stable silencing of c-FOS gene and specific inhibitor treatment,and osteoclast differentiation ability of NRF2-KD and Scramble groups was detected.4.Mice forced rotate Exercise model: Female Nrf2-KO and Nrf2-WT mice were selected,12-15 of each genotype,and randomly divided into two groups at 16 weeks of age,one group was Exercise building group and the other was relatively Sedentary group.Mice in the Exercise group were given forced rotation Exercise for 60 minutes per day at a speed of 10 m /min.The mice in the Sedentary group were not treated in the cage during the period.The body weight and body composition of mice were monitored weekly during the exercise experiment.Modeling was completed after 12 weeks.The fasting blood glucose level of mice was measured,and all organs were collected for weighing.The blood of mice eyeballs was collected under carbon dioxide anesthesia for the determination of plasma bone resorptive markers Trap-5B and CTX1 and plasma bone formation marker P1 NP.Micro-ct scan of the right femur and HE staining of the other femur were performed.5.Forced rotation movement model after OVX: Female Nrf2-KO and Nrf2-WT mice were selected,with 14-18 mice in each group.All mice were OVX at 12 weeks of age.Four weeks after surgery,mice of all genotypes were randomly divided into two groups,7-9 mice in each group.One group was OVX-exercise group,which was given forced rotational Exercise,and the other group was OVX-sedentary group,which was not given any treatment.The exercise time was 60 min per day and the speed was 10 m/min.The body weight and body composition of mice were detected during experiments weekly.After 12 weeks of modeling,fasting glucose levels were measured,and plasma bone resorptive markers TRAP-5B and CTX1 and plasma bone formation marker P1 NP were detected.The right femur was scanned with micro-CT and the other femur was stained with HE.Results:1.Nrf2-deficient mice showed increased sensitivity to osteoporosis due to estrogen deficiency or aging.Bone mass decreased in the OVX group and the aged group,and the serious of osteoporosis increased after global Nrf2 deletion.Micro-CT scanning analysis and specific pathological staining were performed on femur of Nrf2-KO and Nrf2-WT mice,respectively,and it was found that all the mice with different genotypes showed osteoporosis after OVX surgery,and the Nrf2-KO group displayed more obvious osteoporosis.Data analysis of cancellous trabecular bone showed that compared with the sham group,bone volume(BV/TV)and bone trabecular number(Tb.N)decreased in the OVX group,and the decrease was more obvious in the Nrf2-KO group than in the Nrf2-WT group,and the difference was statistically significant(P < 0.05).In the modeling experiment of senile osteoporosis,compared with the control group,the bone volume(BV/TV)of aged Nrf2-KO group was decreased,and the difference was statistically significant(P < 0.05).The number of trabecular bone(Tb.N)decreased,while the trabecular space(Tb.Sp)increased,but there was no statistical significance.TRAP pathological staining analysis showed that the number of osteoclasts in Nrf2-KO group was significantly increased compared with Nrf2-WT group.Plasma TRAP-5B and CTX1 determination showed that Nrf2-KO group in the elderly mice had significantly higher Nrf2-WT than that in the elderly mice,and the difference was statistically significant(P <0.05).2.Nrf2 deletion promotes osteoclast differentiation by increasing intracellular and mitochondrial ROS levels and activating c-Fos.TRAP staining of Nrf2 deletion representing osteoclast differentiation ability was increased in BMMs and RAW 264.7cells,as well as expression levels of Cathepsin K,H+-ATPase,Atp6v0d2 and NFATC1 of osteoclast differentiation related genes.The difference was statistically significant(P <0.05).However,Nrf2 overexpression in RAW 264.7 cells decreased TRAP staining and expression of osteoclast differentiation related genes.ROS was detected by flow cytometry after RAW 264.7 was treated with RANKL,and ROS levels were significantly increased after Nrf2 silencing.Mito-Sox and Mito-Red staining showed that ROS is mainly present in the cytoplasm at 15 min,and could be detected in cytoplasm and mitochondria for 24-48 h.In NRF2-KD and Scramble cells treated with three ROS inhibitors of different mechanisms,TRAP staining and expression levels of Cathepsin K,H+-ATPase,Atp6v0d2 and NFATC1 genes related to osteoclast differentiation were decreased.Decreased TRAP staining and Cathepsin K,H+-ATPase and Atp6v0d2 were also found in NRF2-KD and Scramble cells treated with C-FOS silencing and inhibitors,with statistically significant differences(P < 0.05).3.Loss of Nrf2 reduced the sensitivity of mice to exercise-induced bone mass increase,but had no effect on bone mass increase after emasculation.Micro-ct analysis of femur after 16 weeks of passive exercise showed: Bone volume(BV/TV)and bone trabecular thickness(Tb.Th)were increased in the exercise group.After Nrf2 was knocked out,compared with the Nrf2-WT group,the rate of bone mass increase after exercise was lower than that before exercise,and the difference was statistically significant(P < 0.05).Serological detection of bone formation marker P1 NP showed an increase after exercise,and the increase was not significant in Nrf2-KO group compared with Nrf2-WT group.However,in the exercise group after castration surgery,both Nrf2-KO group and Nrf2-WT group could increase bone mass compared with the relatively static group after castration surgery.In Nrf2-KO group or Nrf2-WT group,BV/TV and Tb.Th showed less.Tb.Sp and Tb.N showed an increase,and the difference was not statistically significant.Conclusions:1.Both ovarian removal and aging can induce osteoporosis,systemic Nrf2 knockout mice are more sensitive to decreased bone mass caused by estrogen loss and aging,mainly showing increased osteoclast and enhanced osteoclast function.These results suggest that Nrf2 plays a vital role in the development of osteoporosis.2.Loss of Nrf2 can enhance osteoclast differentiation.The levels of ROS in the cytoplasm were increased 15-30 min after osteoclast differentiation,while ROS levels in the cytoplasm and mitochondria were increased 24-48 h after osteoclast differentiation.ROS inhibitors blocked the enhancement of osteoclast differentiation caused by Nrf2 deletion,while c-FOS silencing and inhibitor treatment also reduced osteoclast differentiation.We concluded that loss of Nrf2 promotes osteoclast differentiation by increasing intracellular and mitochondrial ROS levels and c-FOS expression.3.Exercise can increase the bone mass of femur in mice,which may have a relationship to the enhanced differentiation ability of osteoblasts.The sensitivity of mice to exercise-induced increase in bone mass was decreased after systemic Nrf2 deletion.Reexercise also increased bone mass in mice after castration surgery,but Nrf2 deletion had no effect on exercise-induced bone mass.