DataSheet1_BNTA alleviates inflammatory osteolysis by the SOD mediated anti-oxidation and anti-inflammation effect on inhibiting osteoclastogenesis.zip

Abnormal activation and overproliferation of osteoclast in inflammatory bone diseases lead to osteolysis and bone mass loss. Although current pharmacological treatments have made extensive advances, limitations still exist. N-[2-bromo-4-(phenylsulfonyl)-3-thienyl]-2-chlorobenzamide (BNTA) is an artificially synthesized molecule compound that has antioxidant and anti-inflammatory properties. In this study, we presented that BNTA can suppress intracellular ROS levels through increasing ROS scavenging enzymes SOD1 and SOD2, subsequently attenuating the MARK signaling pathway and the transcription of NFATc1, leading to the inhibition of osteoclast formation and osteolytic resorption. Moreover, the results also showed an obvious restrained effect of BNTA on RANKL-stimulated proinflammatory cytokines, which indirectly mediated osteoclastogenesis. In line with the in vitro results, BNTA protected LPS-induced severe bone loss in vivo by enhancing scavenging enzymes, reducing proinflammatory cytokines, and decreasing osteoclast formation. Taken together, all of the results demonstrate that BNTA effectively represses oxidation, regulates inflammatory activity, and inhibits osteolytic bone resorption, and it may be a potential and exploitable drug to prevent inflammatory osteolytic bone diseases.</p

Histological grading scale by Masuda et al [31].

<p>Histological grading scale based on four categories of degenerative changes. A normal disc is defined as 4 points. A higher score implies more severe degeneration in nucleus pulposus and annulus fibrosus.</p

Sox9-transduced BMSCs in C/Gp gels.

<p>(A) Scanning electron microscopy observation of the C/Gp gels. (B) The C/Gp was liquid at room temperature and gelled at 37°C. (C) Live/dead assay of the BMSCs encapsulated in C/Gp gels after 3 days of culture. (D) The MTT assay was used to assess the BMSC proliferation in C/Gp gels over 14 days. (E) The gene expression analysis of the BMSCs in C/Gp gels after 21 days of culture. #<i>P</i><0.01. Scale bar = 25 μm.</p

Safranin O staining of sections from the 5 groups at 6 (A–J) and 12 weeks (K–T) post-transplantation.

<p>Scale bar = 200 μm.</p

Sox9-transduced BMSCs in monolayer.

<p>(A) P3 BMSCs exhibited a uniform fibroblast-like shape, and fluorescence microscopy showed Sox9 transduction efficiency at days 3, 7 and 14. (B) Sox9 expression was evaluated by western blot at days 3, 7, 10 and 14 after transduction. (C) Real-time PCR for the expressions of <i>Sox9</i>, <i>Aggrecan</i>, <i>Col II</i>, <i>Col I</i> and <i>Col X</i> was performed at day 21 after transduction using <i>GAPDH</i> as a housekeeping gene. All results represent the mean±SD of triplicate samples. *<i>P</i><0.05, #<i>P</i><0.01. Scale bar = 200 μm.</p

Haematoxylin and eosin staining of sections from the 5 groups at 6 (A–J) and 12 weeks (K–T) post-transplantation.

<p>Scale bar = 500 μm.</p

Imaging studies.

<p>(A) T2-weighted images of the treated discs (outlined by the red box) and adjacent control discs at 6 and 12 weeks post-transplantation. (B) The mean T2 of NP at 6 and 12 weeks post-transplantation. *P<0.05, #P<0.01 vs. the Sox9 group. Coronal and sagittal CT reconstruction images for the GFP (C) and Sox9 (D) groups.</p

Macroscopic views of the representative discs at 12 weeks post-transplantation:

<p>: (A) normal control group; (B) degeneration group; (C) C/Gp group; (D) GFP group and (E) Sox9 group.</p

Type II collagen immunohistological staining and histological scores.

<p>Representative sections from the 5 groups at 6 (A–E) and 12 weeks (F–J) post-transplantation. (K) Quantitative analysis of the type II collagen staining using mean density. (L) Histological scores of discs from the 5 groups at 6 and 12 weeks post-treatment. *<i>P</i><0.05, #<i>P</i><0.01 vs. the Sox9 group. Scale bar = 200 μm.</p

Sequence of primers.

<p>Sequence of primers.</p