The Protective Effect Of Bafilomycin A1 Against Cobalt Nanoparticle-induced Adverse Biological Reactions In Vitro And In Vivo

Part I Experimental study of Bafilomycin A1 inhibition on murine macrophage cytotoxic and pro-inflammatory cytokine release induced by Co NPsObjective: To study the protective effect of Bafilomycin A1 on the resultant cytotoxicity of mouse macrophages and inflammatory responses induced by cobalt nanoparticles(Co NPs).Methods: 1. RAW264.7 cells were treated with different concentrations of Co NPs, Co2+ and Bafilomycin A1 cells,and the viabilities were evaluated by MTT; 2. RAW264.7 cells treated with different concentrations of Co NPs, Co2+ and Bafilomycin A1 cells were divided into four groups: control group, Co NPs group, Co NPs+ low concentration Bafilomycin A1 group, Co NPs + high concentration Bafilomycin A1 group. Cell viability was detected by MTT, which was used to assessed the Co NPs cytotoxicity inhibition of Bafilomycin A1; 3. Cell numbers were counted by DAPI staining; 4. Cell ultrastructural changes in each group were detected by scanning electron microscope; 5. Expression of inflammatory cytokines IL-6, TNF-α and IL-1β were analyzed by ELISA assay. Results: 1. The toxic effects of Co NPs and Co2+on RAW264.7 cells appeared time, concentration-dependent; 1.1 There were no cytotoxicity of Bafilomycin A1 on RAW264.7 cells; 1.2 The onset of cytotoxic concentrations of Co NPs and Co2+ were approximately 50, 400 μM; 2. With the increase of Bafilomycin A1 concentration, the cytotoxic was gradually weakened(P <0.05); 3.The number of RAW264.7 cells increased when the concentrations of Bafilomycin A1 role of added under fluorescent microscopy; 4. SEM showed much more cell pseudopod and cell spreading area in blank control group; Cell pseudopods almost disappeared in Co NPs group and the cells were significantly reduced in size; With the increase of Bafilomycin A1 concentration, cell pseudopodia gradually increased and cell spreading area were similar to the control group; 5. Inflammatory cytokines gradually decreased(P <0.05) with the increase of concentration of Bafilomycin A1. Conclusion: 1. The toxic effect of Co NPs on RAW264.7 cells is stronger than Co2+; There is no cytotoxicity of Bafilomycin A1; 2. Bafilomycin A1 possess protective effect on cytotoxicity and inflammation caused Co NPs.Part II Experimental study of Bafilomycin A1 inhibition on osteoclast differentiation of murine macrophage induced by cobalt nanoparticlesObjective: To explore the influence of Bafilomycin A1 on osteoclast differentiation induced by cobalt nanoparticles. Methods: 1. Inhibition of osteoclast differentiation induced Co NPs were evaluated by cell morphological changes, TRAP staining on 5th day. 2.2 Real-time PCR were used to assess the Inhibition of osteoclast differentiation induced Co NPs on 6th day. Results: 1. On 5th day, the number of irregular cells and its size increased, which take on large circular, oval, etc. in shape; The number and size of multinucleated cells also increased, the same with nucleus number. Few multinucleated cells scattered in the control group, far less than Co NPs group. With the increase of Bafilomycin A1 concentration, the number of multinucleated cells gradually decreased and the cell size became smaller. 2. TRAP staining showed much more positive TRAP staining cells appeared in Co NPs group. With increase Bafilomycin A1 concentrations, the number of TRAP-positive staining osteoclasts decreased and polynuclear cell becomes smaller. 3. The gene expression of TRAP, RANK and Cath-K detected by Real-time PCR presented the similar trend change with above experiments. Conclusion: 1. The mouse monocyte- macrophage RAW 264.7 cells line could be induced into osteoclasts; 2. Co NPs could accelerate osteoclasts differentiation of macrophage induced by RANKL; 3. Bafilomycin A1 can inhibit Co NPs-induced acceleration osteoclasts differentiation, and the process of inhibition take in a dose-dependent manner.Part III To study the mechanism of biological effects protection of Bafilomycin A1 on adverse effects caused by Co NPs in mouse macrophageObjective: To study the effect of p H on Co NPs dissolution through the inhibition of Bafilomycin A1 on intracellular Co NPs dissolution, cell ultrastructure and oxidative stress levels changes; To study the mechanism of biological effects protection of Bafilomycin A1 on adverse effects caused by Co NPs in vivo. Methods:(1) Measuring the level of Co2+ released by Co NPs in different p H medium by ICP-MS;(2)Evaluating the inhibition of Bafilomycin A1 on intracellular Co NPs dissolution by ICP-MS and EDX;(3) Observing the changes of ultrastructure by TEM;(4)Evaluating the inhibition of intracellular oxidative stress by Bafilomycin A1 from the detection of GSH/GSSG, SOD, CAT, GPx level. Results:(1) Ion release showed time and p H dependence and were less than the onset of Co2+ cytotoxic concentration at each time point;(2) Intracellular Co NPs release showed time dependence, and intracellular Co NPs release decreased with the increase of concentration of Bafilomycin A1(P<0.05);(3)The number of follicles and lysosomes increased dramatically in Co NPs group with swallowed Co NPs by lysosomal and dissapeared organelles; When increased the concentration of Bafilomycin A1, the number of follicular and lysosomal gradually decreased, and the disappeared organelles reappeared;(4)The level of GSH, SOD, GPx, CAT levels were significantly lower in Co NPs group and rise with increased Bafilomycin A1 concentration. Conclusion: Co NPs showed very low solubility in the extracellular environment; Low p H environment resulted in the release of more dissolved cobalt ions, destroyed the cellular antioxidant defense mechanisms and resulted in oxidative stress, which is the basic reason of cell toxicity, inflammation and osteoclast differentiation and so on caused by Co NPs.Part IV Experimental study of Bafilomycin A1 inhibition on acute inflammation and osteolysis around the skull induced by Co NPsObjective: To investigate the effects of Bafilomycin A1 on acute inflammation and osteolysis around the skull induced by Co NPs. Methods:(1) The model of acute inflammation and osteolysis around the skull was established, tissue inflammation and inflammatory cell were were analyzed by histological stain;(2) Measurement of bone tissue pathology staining and metrology;(3) Micro-CT analysis of bone tissue in each group;(4) measurement of proinflammatory cytokines in each group around the skull. Results:(1) The result of tissue pathology staining and inflammatory cell count analysis showed the highest intensive inflammatory cells in Co NPs group. The number of inflammatory cells decreased(p <0.05) when the concentration of Bafilomycin A1 increased.(2) Pathology staining presented the obvious skull osteolysis along with inflammatory cell infiltration. When increasing the concentrations of Bafilomycin A1, the scope of bone resorption, dissolving area and the number of inflammatory cells decreased.(3) Micro-CT analysis showed significant bone resorption in Co NPs group. The extent of bone resorption decreased along with increasing concentrations of Bafilomycin A1; and so it is the BMD and BMC results(P <0.05).(4)TNF-α, IL-1β, and IL-6 protein expression was significantly increased in Co NPs group(P <0.05), and decreased with increasing doses of Bafilomycin A1 which presented in a dose-dependent manner(P <0.05). Conclusion: Co NPs could induce inflammation, secrete TNF-α, IL-1β and IL-6 and other inflammatory cytokines involved in regulation of osteoclast and result in bone resorption. Bafilomycin A1 inhibitsinflammation and bone resorption around the skull induced by Co NPs in a dose-response manner.