Fig. 3

NETs affects viability of osteoblasts in vitro. The alkaline phosphatase staining of the osteoblasts (× 200). a MSU crystals induces the formation of NETs in vitro (× 40). DAPI fluorescence intensity and filamentous structures were observed in the MSU group. b The concentration of DNA was significantly higher in the MSU group than the NC group, but after the addition of DNase treatment, the supernatant DNA concentration decreased. ***P < 0.001 as determined by t test. c NETs adhere to osteoblasts. Osteoblasts were covered by NETs (reddish brown). d The expression of ALP and OPG was decreased, and RANKL was increased which were tested by WB. After digesting the NETs in the supernatant or adding the inhibitor of NE, the expression of markers was restored. e The ALP and OPG mRNA were decreased, while the RANKL mRNA was increased. After digesting the NETs in the supernatant or adding the inhibitor of NE, the expression of markers was restored. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by t test. f The viability of osteoblasts was tested by CCK-8. After treating osteoblasts with supernatant containing NETs, the viability of osteoblasts decreased. After digesting the NETs in the supernatant or adding the inhibitor of NE, the viability was restored. g IHC showed the expression of NE increased significantly after stimulation