Nitrogen-doped Carbon Dots Suppress Osteoclastic Ostelysis Via Downregulating Reactive Oxygen Species

Research background and purpose: Over formation and activation of osteoclasts can lead to a variety of osteolytic diseases,such as osteoporosis,Peget’s disease,inflammation-induced bone destruction,and osteolysis with tumors.Bone homeostasis is a dynamic process.There are two main roles involved in the regulation of bone homeostasis: osteoblasts derived from bone marrow mesenchymal progenitor cells mediate bone formation and osteoclasts derived from hematopoietic stem cells mediate bone resorption.Nowadays,the clinical drugs for osteoclast osteolysis are basically not targeted drugs,and there are many uncertain side effects,so they are not widely used,so it is an urgent problem to develop drugs with little side effects of osteoclast osteolysis.In our previous study,nitrogen-doped carbon dots(N-CDs)were hydrolyzed by chitosan and cross-linked with unsaturated acrylamide or carboxylic acid and then carbonized.It is a kind of fluorescent nano-imaging material,they have good biocompatibility.It has been reported that nitrogen-doped carbon dots can effectively down-regulate reactive oxygen species(ROS),and protect against oxidative stress by eliminating endogenous / exogenous ROS,ROS is an significant second messenger involved in the regulation of osteoclast development,differentiation and bone resorption.In this study,we will explain the molecular mechanism of nitrogen-doped carbon dots on the regulation of ROS and osteoclasts from three aspects: the mechanism of nitrogen-doped carbon dots down-regulating ROS,the formation of osteoclasts and the function of bone resorption,and the establishment of an animal model of osteolysis to explore the salvage effect of nitrogen-doped carbon spots on bone loss caused by abnormal osteoclasts.The research results of this topic can provide a theoretical basis for the clinical transformation of nanomaterials and the research and development of new nanomaterials.Experimental methods: High yield nitrogen-doped carbon dots(N-CDs)was synthesized by strengthening carbon-carbon double bond,the proportion of chitosan with the highest scavenging rate of active oxygen was determined,and the structure of nitrogen-doped carbon point was characterized.In vitro experiment:(bone marrow macrophages BMMs),was extracted from bone marrow mononuclear macrophages of mouse tibia.ROS kit was used to detect the clearance rate of reactive oxygen species of N-CDs and the clearance rate of DPPH.Then the expression of antioxidant enzyme protein and gene of osteoclasts treated with N-CDs were detected by western blot and RT-PCR.Then the effect of N-CDs on the proliferation of BMMs was determined by cck8 kit,and the effect of N-CDs on osteoclast differentiation was further explored by tartaric acid staining,and the effect of osteoclast specific gene expression was quantitatively analyzed by RT-PCR technique,and then the effect of N-CDs on osteoclast skeleton and bone resorption function was detected.Finally,western blot and nuclear experiments were used to explore the molecular mechanism of N-CDs on osteoclasts,and ALP experiments were used to study the effects of N-CDs on osteoblasts.In vivo experiment: the model of local osteolysis of calvaria induced by LPS in mice: 8-week-old male C57 mice were randomly divided into three groups: blank control group,LPS group and LPS+N-CDs group.A week later,the samples of mice were collected,Micro-CT scanning and tissue analysis of bone mass changes,HE staining,TRAP staining and CTSK,RUNX2,osteocalcin immunohistochemistry to observe the histological morphology of mice.Local osteolysis model of tibia induced by breast cancer in nude mice: 8-week-old female nude mice were randomly divided into three groups: blank control group,tumor group and tumor+N-CDs group.Four weeks later,the samples of nude mice were collected,Micro-CT scanning and tissue analysis of bone mass changes,HE staining and TRAP staining to observe the histological morphology of nude mice.Results: In this study,based on ROS as an important second messenger to activate osteoclast differentiation and bone resorption,we first synthesized nitrogen-doped carbon dots with high yield and strong oxygen free radical scavenging rate,and then verified that N-CDs could inhibit the production of reactive oxygen species in cells.Then we proved that N-CDs enhanced the expression of antioxidant enzyme proteins and antioxidant genes in vitro.It was also found that N-CDs inhibited osteoclast differentiation and bone resorption function,and inhibited the expression of maturation marker genes of osteoclasts.We found that N-CDs effectively eliminated RANKL-induced ROS generation,thus weakening the activation of NF-κB and MAPK signal cascades downstream of ROS.In vivo,N-CDs can effectively protect the osteolysis of calvaria induced by LPS in mice and tibia osteolysis induced by breast cancer in nude mice.In summary,based on the good biocompatibility of N-CDs and the ability to effectively clear ROS,as well as inhibiting the activation of osteoclasts,nanomaterials have been provided for the clinical treatment of osteolytic diseases.Conclusion: N-CDs effectively eliminates RANKL-induced ROS formation and weakens the activation of NF-κB and MAPK signal pathways downstream of ROS,thus inhibiting the formation and activation of osteoclasts.N-CDs can protect mice from skull bone destruction induced by lipopolysaccharide(LPS)and tibial osteolysis induced by breast cancer cells.