A Mechanism Study Of Calcitonin Gene-related Peptide Promoting Proliferation And Differentiation Of Rat Osteoblast Via The Signaling Pathway Of NOD-like Receptor Protein 3-reactive Oxygen Species-interleukin 1?

Background:Bone is a multi-functional and complex organ,and playe a role in hematopoiesis,regulation and storage of crucial minerals,as well as protecting the life-sustaining organs.While bone is injured,acute inflammation develop immediately,follow congenital immunoregulation of body.It affected the healing of local bone injury under the co-effect of tissue and organ.When bone fracture occur,local hematoma emerged firstly,at the same time,risk signal molecules were released and macrophages were activated as soon as they arrive the acute inflammatory location.During this stage,proinflammatory cytokines,growth factors and chemotactic factors were generated;the mesenchymal stem cells,osteoblasts and fibroblasts were recruited.Bone formation and healing would be inhibited if the inflammatory response remained imbalance during the acute inflammatory stage.The osteoblasts not only play a role in ossificating,but also regulating the inflammatory response.Once the main proinflammatory cytokine interleukin-1?(IL-1?)exhibited overexpression or imbalanced expression,the period of inflammatory response would extend,then influence bone healing.NOD-like receptor protein 3(NACHT,LRR-and pyrin-domain(PYD)-containing protein 3,NLRP3)is an intracellular multiprotein complex with a relative molecular mass of 700000,and regulate the release of IL-1? directly.It was proved that the calcitonin gene-related peptide(CGRP)is secreted by peripheral sensory nerves and can regulate the inflammatory response of body and expression of IL-1?,besides promotion in repair and reconstruction of bone trauma.Reactive oxygen species(ROS),which locate in the cellular mitochondria,might overload Ca2+ by upregulating the intracellular flow of Ca2+,resulting in mitochondria dysfunction and activation of NLRP3.In a conclusion,this experiment was designed to investigate whether CGRP could regulate proliferation and differentiation of osteoblast through NLRP3-ROS-IL-1? signaling pathway,further to elucidate the mechanism that CGRP facilitated repair of bone trauma by regulation of inflammatory response.Follow this design,we studyed the gene and protein expression of NLRP3 and IL-1? in the osteoblasts,and measured intracellular contents of ROS and mitochondria after exogenous CGRP stimulated rat osteoblast.Method:1.Cell culture and identification: Cranium of 10 newborn(born within 24-48 hours)BABL/c mice were sheared in aseptic condition.The cells were extracted by twice-enzyme isolation method and cultivated in 5%CO2?37? incubator.Cell passages were made while cell density grew to 80%.The third-generation cells were identified by alkaline phosphase(ALP)and alizarin red staining,and was taken for the following experiment.2.Experiment grouping: They were inoculated into 8 pores on two six-pore plates with a density of 5×105 / pore,and were divided into four groups by a completely random design(two pores for each group).CGRP of different concentration were respectively added in them after 24 hours of culture,0 ng/ml in group A(control group),10 ng/ml in group B,30 ng/ml in group C,100 ng/ml in group D.Subsequent experiments were conducted after 3 days' culture.3.Detection of expression of NLRP3 and IL-1? in osteoblast: We extracted RNA and protein from the cells in each group,.Then expression of NLRP3 and IL-1? in osteoblasts were detected by RT-PCR,Western blot and ELISA4.Detection of ROS content by flow cytometry: Each group with cells treated by CGRP for 3 days,as well as a new blank group(without CGRP)in the same state were taken.The total fluorescence of intracellular DCF(2',7'-dichlorofluorescin)in each group was detected by flow cytometry to determine the level of ROS.5.Detection of the quantity of intracellular mitochondria by laser scanning confocal microscope: The 3rd-generation primary osteoblasts in good condition were inoculated into four laser confocal culture dishes with a density of 1×105 /pore.CGRP was added to different groups after24 hours' adhesion to wall,MitoSOX Red reagent was then added.The quantity of intracellular mitochondria was observed by the laser scanning confocal microscope6.Proliferation and differentiation of osteoblast after the treatment of CGRP:Proliferation: the proliferation of cells in each group at 0h,24 h,48h and 72 h detected by CCK-8 method.Differentiation: the differentiation of cells in each group detected by ALP and alizarin red staining.Result:1.Cell culture and identification: The cultured cells have the same characteristics of osteoblasts,by microscopic examination.They showed positive by ALP and alizarin red staining,Both two methods proved that the cultivated cells were osteoblasts.2.Detection of the expression of NLRP3 and IL-1? in osteoblasts: As the CGRP concentration increased,the level of mRNA and protein expression of NLRP3 and IL-1? decreased.3.Detection of ROS concentration and quantity of mitochondria in osteoblast: As the CGRP concentration increased,the intracellular ROS content reduced,and the quantity of mitochondria decreased.4.Proliferation and differentiation of osteoblast by CGRP: CGRP could enhance the proliferation and differentiation of the osteoblast significantly.Conclusions:1.CGRP may regulate inflammatory response by inhibiting activation of NLRP3 and consequent secretion of IL-1?.2.After the detection of content of intracellular ROS and mitochondria,it was found that CGRP could promote the autophagy of intracellular mitochondrial and reduce the production of ROS,thereby inhibit the activation of NLRP3.3.CGRP could promote the proliferation of and differentiation of osteoblast significantly.