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Attenuation of the macrophage inflammatory activity by TiO₂ nanotubes via inhibition of MAPK and NF-κB pathways.

Neacsu P, Mazare A, Schmuki P, Cimpean A - Int J Nanomedicine (2015)

Bottom Line: Results showed that the Ti/TiO2 significantly reduce the expression levels of the phosphorylated forms of p38, ERK1/2, c-Jun NH2-terminal kinase (JNK), IKKβ, and IkB-α.Furthermore, a significant reduction in the p65 nuclear accumulation on the nanotubular surface was remarked.However, the selective inhibitor for JNK signaling pathway (SP600125) was effective in reducing tumor necrosis factor alpha release as well as monocyte chemotactic protein-1 and nitric oxide production.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Bucharest, Bucharest, Romania.

ABSTRACT
Biomaterial implantation in a living tissue triggers the activation of macrophages in inflammatory events, promoting the transcription of pro-inflammatory mediator genes. The initiation of macrophage inflammatory processes is mainly regulated by signaling proteins of mitogen-activated protein kinase (MAPK) and by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. We have previously shown that titania nanotubes modified Ti surfaces (Ti/TiO2) mitigate the immune response, compared with flat Ti surfaces; however, little is known regarding the underlying mechanism. Therefore, the aim of this study is to investigate the mechanism(s) by which this nanotopography attenuates the inflammatory activity of macrophages. Thus, we analyzed the effects of TiO2 nanotubes on the activation of MAPK and NF-κB signaling pathways in standard and lipopolysaccharide-evoked conditions. Results showed that the Ti/TiO2 significantly reduce the expression levels of the phosphorylated forms of p38, ERK1/2, c-Jun NH2-terminal kinase (JNK), IKKβ, and IkB-α. Furthermore, a significant reduction in the p65 nuclear accumulation on the nanotubular surface was remarked. Following, by using specific MAPK inhibitors, we observed that lipopolysaccharide-induced production of monocyte chemotactic protein-1 and nitric oxide was significantly inhibited on the Ti/TiO2 surface via p38 and ERK1/2, but not via JNK. However, the selective inhibitor for JNK signaling pathway (SP600125) was effective in reducing tumor necrosis factor alpha release as well as monocyte chemotactic protein-1 and nitric oxide production. Altogether, these data suggest that titania nanotubes can attenuate the macrophage inflammatory response via suppression of MAPK and NF-κB pathways providing a potential mechanism for their anti-inflammatory activity.

No MeSH data available.


Related in: MedlinePlus

Effects of selective inhibitors of p38 (SB202190), ERK1/2 (U0126), and JNK (SP600125) on the production of TNF-α and MCP-1.Notes: Cells were allowed to adhere on the samples for 18 hours, then were incubated with specific MAPK inhibitors for 1 hour and further treated with 1 µg·mL−1 LPS for 24 hours. The secreted pro-inflammatory factors were detected in cell culture media using ELISA assay. The data are expressed as mean ± SD. *P<0.05; **P<0.01; ***P<0.001.Abbreviations: cpTi, commercial pure titanium; ELISA, enzyme-linked immunosorbent assay; JNK, c-Jun NH2-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemotactic protein-1; TNF-α, tumor necrosis factor α; ERK, extracellular signal-regulated kinase; SD, standard deviation; vs, versus.
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f4-ijn-10-6455: Effects of selective inhibitors of p38 (SB202190), ERK1/2 (U0126), and JNK (SP600125) on the production of TNF-α and MCP-1.Notes: Cells were allowed to adhere on the samples for 18 hours, then were incubated with specific MAPK inhibitors for 1 hour and further treated with 1 µg·mL−1 LPS for 24 hours. The secreted pro-inflammatory factors were detected in cell culture media using ELISA assay. The data are expressed as mean ± SD. *P<0.05; **P<0.01; ***P<0.001.Abbreviations: cpTi, commercial pure titanium; ELISA, enzyme-linked immunosorbent assay; JNK, c-Jun NH2-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemotactic protein-1; TNF-α, tumor necrosis factor α; ERK, extracellular signal-regulated kinase; SD, standard deviation; vs, versus.

Mentions: To further determine whether the inhibition of MAPKs contributes to the attenuation of macrophage inflammatory activity by TiO2 nanotubes, we have investigated the effect of specific MAPK inhibitors on the secretion of TNF-α, MCP-1, and NO by RAW 264.7 macrophages. A pretreatment of the cells with specific inhibitors (ie, SB202190 – p38 inhibitor, U0126 – ERK inhibitor, and SP600125 – JNK inhibitor) led to a decrease in the release of all analyzed pro-inflammatory mediators, both in the presence and absence of LPS (Figures 4 and 5). As shown in Figure 4, pretreatment with the ERK1/2-specific inhibitor (U0126) led to a dramatically diminished cytokine and chemokine expression in both culture conditions. Interestingly, under pro-inflammatory culture conditions (+LPS), a significant reduction in the release of TNF-α and MCP-1 on the surface of Ti/TiO2 was observed when RAW 264.7 cells were treated with the p38 (SB202190) and ERK (U0126) inhibitors. The only exception is for the JNK inhibitor (SP600125), which was ineffective in blocking the LPS-induced MCP-1 release. The above results suggest that ERK1/2 and p38 MAPK may constitute critical components in nanotopography-dependent regulation of TNF-α and MCP-1 release by macrophages. On the contrary, inhibition of the JNK signaling pathway contributes to the inhibitory action of titania nanotubes on LPS-induced TNF-α release, but not on MCP-1.


Attenuation of the macrophage inflammatory activity by TiO₂ nanotubes via inhibition of MAPK and NF-κB pathways.

Neacsu P, Mazare A, Schmuki P, Cimpean A - Int J Nanomedicine (2015)

Effects of selective inhibitors of p38 (SB202190), ERK1/2 (U0126), and JNK (SP600125) on the production of TNF-α and MCP-1.Notes: Cells were allowed to adhere on the samples for 18 hours, then were incubated with specific MAPK inhibitors for 1 hour and further treated with 1 µg·mL−1 LPS for 24 hours. The secreted pro-inflammatory factors were detected in cell culture media using ELISA assay. The data are expressed as mean ± SD. *P<0.05; **P<0.01; ***P<0.001.Abbreviations: cpTi, commercial pure titanium; ELISA, enzyme-linked immunosorbent assay; JNK, c-Jun NH2-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemotactic protein-1; TNF-α, tumor necrosis factor α; ERK, extracellular signal-regulated kinase; SD, standard deviation; vs, versus.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608594&req=5

f4-ijn-10-6455: Effects of selective inhibitors of p38 (SB202190), ERK1/2 (U0126), and JNK (SP600125) on the production of TNF-α and MCP-1.Notes: Cells were allowed to adhere on the samples for 18 hours, then were incubated with specific MAPK inhibitors for 1 hour and further treated with 1 µg·mL−1 LPS for 24 hours. The secreted pro-inflammatory factors were detected in cell culture media using ELISA assay. The data are expressed as mean ± SD. *P<0.05; **P<0.01; ***P<0.001.Abbreviations: cpTi, commercial pure titanium; ELISA, enzyme-linked immunosorbent assay; JNK, c-Jun NH2-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemotactic protein-1; TNF-α, tumor necrosis factor α; ERK, extracellular signal-regulated kinase; SD, standard deviation; vs, versus.
Mentions: To further determine whether the inhibition of MAPKs contributes to the attenuation of macrophage inflammatory activity by TiO2 nanotubes, we have investigated the effect of specific MAPK inhibitors on the secretion of TNF-α, MCP-1, and NO by RAW 264.7 macrophages. A pretreatment of the cells with specific inhibitors (ie, SB202190 – p38 inhibitor, U0126 – ERK inhibitor, and SP600125 – JNK inhibitor) led to a decrease in the release of all analyzed pro-inflammatory mediators, both in the presence and absence of LPS (Figures 4 and 5). As shown in Figure 4, pretreatment with the ERK1/2-specific inhibitor (U0126) led to a dramatically diminished cytokine and chemokine expression in both culture conditions. Interestingly, under pro-inflammatory culture conditions (+LPS), a significant reduction in the release of TNF-α and MCP-1 on the surface of Ti/TiO2 was observed when RAW 264.7 cells were treated with the p38 (SB202190) and ERK (U0126) inhibitors. The only exception is for the JNK inhibitor (SP600125), which was ineffective in blocking the LPS-induced MCP-1 release. The above results suggest that ERK1/2 and p38 MAPK may constitute critical components in nanotopography-dependent regulation of TNF-α and MCP-1 release by macrophages. On the contrary, inhibition of the JNK signaling pathway contributes to the inhibitory action of titania nanotubes on LPS-induced TNF-α release, but not on MCP-1.

Bottom Line: Results showed that the Ti/TiO2 significantly reduce the expression levels of the phosphorylated forms of p38, ERK1/2, c-Jun NH2-terminal kinase (JNK), IKKβ, and IkB-α.Furthermore, a significant reduction in the p65 nuclear accumulation on the nanotubular surface was remarked.However, the selective inhibitor for JNK signaling pathway (SP600125) was effective in reducing tumor necrosis factor alpha release as well as monocyte chemotactic protein-1 and nitric oxide production.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Bucharest, Bucharest, Romania.

ABSTRACT
Biomaterial implantation in a living tissue triggers the activation of macrophages in inflammatory events, promoting the transcription of pro-inflammatory mediator genes. The initiation of macrophage inflammatory processes is mainly regulated by signaling proteins of mitogen-activated protein kinase (MAPK) and by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. We have previously shown that titania nanotubes modified Ti surfaces (Ti/TiO2) mitigate the immune response, compared with flat Ti surfaces; however, little is known regarding the underlying mechanism. Therefore, the aim of this study is to investigate the mechanism(s) by which this nanotopography attenuates the inflammatory activity of macrophages. Thus, we analyzed the effects of TiO2 nanotubes on the activation of MAPK and NF-κB signaling pathways in standard and lipopolysaccharide-evoked conditions. Results showed that the Ti/TiO2 significantly reduce the expression levels of the phosphorylated forms of p38, ERK1/2, c-Jun NH2-terminal kinase (JNK), IKKβ, and IkB-α. Furthermore, a significant reduction in the p65 nuclear accumulation on the nanotubular surface was remarked. Following, by using specific MAPK inhibitors, we observed that lipopolysaccharide-induced production of monocyte chemotactic protein-1 and nitric oxide was significantly inhibited on the Ti/TiO2 surface via p38 and ERK1/2, but not via JNK. However, the selective inhibitor for JNK signaling pathway (SP600125) was effective in reducing tumor necrosis factor alpha release as well as monocyte chemotactic protein-1 and nitric oxide production. Altogether, these data suggest that titania nanotubes can attenuate the macrophage inflammatory response via suppression of MAPK and NF-κB pathways providing a potential mechanism for their anti-inflammatory activity.

No MeSH data available.


Related in: MedlinePlus