Table 1.
Primers for the analysis of target genes by qRT-PCR.
Fig 1.
Alterations in osteogenic function of MC3T3-E1 cells during TNF-α-triggered inflammation.
(A-E) Gene expression levels of Runx2, ALP, OPG and RANKL, as well as ALP activity, respectively, were determined in cell lysates. (F) Expression of ALP was directly examined in a 24-well plate. (G) Cellular activity was determined by the CCK-8 method. N = 5–7 cell lines/group. P< 0.05 versus the vehicle-treated group.
Fig 2.
Changes in mitochondrial function and morphology of MC3T3-E1 cells during inflammation.
(A) Representative images of Mitosox red staining. (B) Level of mitochondrial ROS was assessed by Mitosox red staining intensity. (C) Representative images of TMRM staining. (D) Level of mitochondrial membrane potential was assessed by TMRM staining intensity. (E) ATP production was detected by an ATP assay kit. (F) Representative images of Mitotracker Red staining. Middle panels show magnified images corresponding to the indicated bilateral images. (G) Quantification of average mitochondrial length. (H) Quantification of immunoreactive bands of Drp1. Representative immunoblots are shown in the lower panel. Image intensity was quantified using NIH Image J software. (Scale bar = 10 μm). N = 5–7 cell lines/group. P< 0.05 versus the vehicle-treated group.
Fig 3.
Effect of NAC treatment on the inflammatory response induced by TNF-α.
The cells were treated with 1 mM NAC for 24 h. (A) Representative images of Mitosox red staining. (B) Level of mitochondrial ROS. (C) Representative images of Mitotracker Red staining. Lower panels show magnified images corresponding to the indicated images. (D) Quantification of average mitochondrial length. (E) Quantification of immunoreactive bands of Drp1. Representative immunoblots are shown in the lower panel. (F) Representative images of TMRM staining. (G) Level of mitochondrial membrane potential. (H) Level of ATP production. (I-L) Gene expression levels of osteogenic markers, ALP activity, ALP expression level and cellular activity, respectively. Image intensity was quantified using NIH Image J software. (Scale bar = 10 μm). N = 5–7 cell lines/group. P< 0.05 versus the vehicle-treated group and NAC-added group.
Fig 4.
Effect of Mdivi-1 treatment on the inflammatory response induced by TNF-α.
The cells were treated with 10 μM Mdivi-1 for 24 h. (A) Representative images of Mitotracker Red staining. Lower panels show magnified images corresponding to the indicated images. (B) Quantification of average mitochondrial length. (C) Representative images of Mitotracker Red staining. Lower panels show magnified images corresponding to the indicated images. (D) Quantification of average mitochondrial length. (E) Representative images of TMRM staining. (F) Level of mitochondrial membrane potential. (G) Level of ATP production. (H-K) Gene expression levels of osteogenic markers, ALP activity, ALP expression level and cellular activity, respectively. Image intensity was quantified using NIH Image J software. (Scale bar = 10 μm). N = 5–7 cell lines/group. P< 0.05 versus the vehicle-treated group and NAC-added group. (L) Working hypothesis: ROS-induced oxidative stress resulting from TNF-α exposure triggers an increase in Drp1 expression leading to mitochondrial dynamic imbalance, resulting in mitochondrial dysfunction and subsequent osteogenic dysfunction.