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p38/AP-1 pathway in lipopolysaccharide-induced inflammatory responses is negatively modulated by electrical stimulation.

Jeong D, Lee J, Yi YS, Yang Y, Kim KW, Cho JY - Mediators Inflamm. (2013)

Bottom Line: Through immunoblotting and immunoprecipitation analyses, we found that p38/AP-1 could be the major inhibitory pathway in the NGACP-mediated anti-inflammatory response.Indeed, inhibition of p38 by SB203580 showed similar inhibitory activity of the production of TNF- α and PGE2 and the expression of TNF- α and COX-2 mRNA.These results suggest that ACP-induced nanocurrents alter signal transduction pathways that are involved in the inflammatory response and could therefore be utilized in the treatment of various inflammatory diseases such as arthritis and colitis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.

ABSTRACT
Electrical stimulation with a weak current has been demonstrated to modulate various cellular and physiological responses, including the differentiation of mesenchymal stem cells and acute or chronic physical pain. Thus, a variety of investigations regarding the physiological role of nano- or microlevel currents at the cellular level are actively proceeding in the field of alternative medicine. In this study, we focused on the anti-inflammatory activity of aluminum-copper patches (ACPs) under macrophage-mediated inflammatory conditions. ACPs generated nanolevel currents ranging from 30 to 55 nA in solution conditions. Interestingly, the nanocurrent-generating aluminum-copper patches (NGACPs) were able to suppress both lipopolysaccharide-(LPS-) and pam3CSK-induced inflammatory responses such as NO and PGE2 production in both RAW264.7 cells and peritoneal macrophages at the transcriptional level. Through immunoblotting and immunoprecipitation analyses, we found that p38/AP-1 could be the major inhibitory pathway in the NGACP-mediated anti-inflammatory response. Indeed, inhibition of p38 by SB203580 showed similar inhibitory activity of the production of TNF- α and PGE2 and the expression of TNF- α and COX-2 mRNA. These results suggest that ACP-induced nanocurrents alter signal transduction pathways that are involved in the inflammatory response and could therefore be utilized in the treatment of various inflammatory diseases such as arthritis and colitis.

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Effect of NGACP treatment on the expression of proinflammatory mRNA. ((a) and (b)) The mRNA levels of proinflammatory genes (COX-2, iNOS, TNF-α, IL-1β, IL-6, IFN-β, and IL-12) were determined using semiquantitative RT-PCR or real-time PCR. *P < 0.05 and **P < 0.01 compared to control.
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fig3: Effect of NGACP treatment on the expression of proinflammatory mRNA. ((a) and (b)) The mRNA levels of proinflammatory genes (COX-2, iNOS, TNF-α, IL-1β, IL-6, IFN-β, and IL-12) were determined using semiquantitative RT-PCR or real-time PCR. *P < 0.05 and **P < 0.01 compared to control.

Mentions: To confirm the inhibitory effect on NO and PGE2 production, we first assessed inhibition at the transcriptional level. The mRNA levels of various inflammatory genes such as COX-2, iNOS, TNF-α, IL-1β, IL-6, IFN-β, and IL-12 were measured under NGACP treatment conditions. As Figure 3(a) shows, expression of most of the inflammatory genes except IL-1β was suppressed by NGACP treatment. Furthermore, real-time RT-PCR analysis showed similar inhibitory patterns on the expression of iNOS and COX-2 (Figure 3(b)), which implied that transcriptional activation of LPS-induced inflammatory responses was targeted by NGACP treatment. Because transcriptional activation is regulated by the nuclear translocation of various transcription factors such as NF-κB, AP-1, CREB, and STAT-1 [35], the nuclear levels of these proteins were then examined. As Figure 4(a) confirms, NGACP treatment clearly blocked the nuclear translocation of c-Jun at 6 h, while the other transcription factors were also inhibited based on the results for lamin A/C. In addition, AP-1-mediated luciferase activity was also suppressed by NGACP (data not shown). Because c-Jun is a representative transcription factor that is regulated by ERK, JNK, and p38 [36], we examined whether the activation of these enzymes could be blocked by NGACP treatment through analysis of their phosphorylation levels. As Figure 4(b) reveals, the phosphorylation of p38 at 1 h was strongly inhibited by NGACP treatment. In particular, because phosphorylation of MKK3/6, an upstream enzyme that phosphorylates p38 [37], was not blocked (Figure 4(c)) and the protein level of phospho-p38 diphosphatase, MKP-1 [38], was not enhanced (Figure 4(d)), NGACP treatment seems to directly target MKK3/6, although a specific enzyme assay would be required to obtain better evidence. In agreement with this finding, signaling complex formation between p38 and MKK3 was also defective, as demonstrated through immunoprecipitation and immunoblotting analyses (Figure 4(e)). Therefore, these results strongly suggest that the MKK3/p38 pathway could be the major inhibitory target of NGACP treatment. Finally, we confirmed the functional significance of p38 in the inflammatory response by using a specific inhibitor, SB203580 (SB), on LPS-activated RAW264.7 cells. SB also strongly suppressed the expression of COX-2 and TNF-α mRNA, as assessed using RT- and real-time PCR (Figure 5(a)) and inhibited the release of TNF-α and PGE2 (Figure 5(b)), which is in agreement with previously reported results [39].


p38/AP-1 pathway in lipopolysaccharide-induced inflammatory responses is negatively modulated by electrical stimulation.

Jeong D, Lee J, Yi YS, Yang Y, Kim KW, Cho JY - Mediators Inflamm. (2013)

Effect of NGACP treatment on the expression of proinflammatory mRNA. ((a) and (b)) The mRNA levels of proinflammatory genes (COX-2, iNOS, TNF-α, IL-1β, IL-6, IFN-β, and IL-12) were determined using semiquantitative RT-PCR or real-time PCR. *P < 0.05 and **P < 0.01 compared to control.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649710&req=5

fig3: Effect of NGACP treatment on the expression of proinflammatory mRNA. ((a) and (b)) The mRNA levels of proinflammatory genes (COX-2, iNOS, TNF-α, IL-1β, IL-6, IFN-β, and IL-12) were determined using semiquantitative RT-PCR or real-time PCR. *P < 0.05 and **P < 0.01 compared to control.
Mentions: To confirm the inhibitory effect on NO and PGE2 production, we first assessed inhibition at the transcriptional level. The mRNA levels of various inflammatory genes such as COX-2, iNOS, TNF-α, IL-1β, IL-6, IFN-β, and IL-12 were measured under NGACP treatment conditions. As Figure 3(a) shows, expression of most of the inflammatory genes except IL-1β was suppressed by NGACP treatment. Furthermore, real-time RT-PCR analysis showed similar inhibitory patterns on the expression of iNOS and COX-2 (Figure 3(b)), which implied that transcriptional activation of LPS-induced inflammatory responses was targeted by NGACP treatment. Because transcriptional activation is regulated by the nuclear translocation of various transcription factors such as NF-κB, AP-1, CREB, and STAT-1 [35], the nuclear levels of these proteins were then examined. As Figure 4(a) confirms, NGACP treatment clearly blocked the nuclear translocation of c-Jun at 6 h, while the other transcription factors were also inhibited based on the results for lamin A/C. In addition, AP-1-mediated luciferase activity was also suppressed by NGACP (data not shown). Because c-Jun is a representative transcription factor that is regulated by ERK, JNK, and p38 [36], we examined whether the activation of these enzymes could be blocked by NGACP treatment through analysis of their phosphorylation levels. As Figure 4(b) reveals, the phosphorylation of p38 at 1 h was strongly inhibited by NGACP treatment. In particular, because phosphorylation of MKK3/6, an upstream enzyme that phosphorylates p38 [37], was not blocked (Figure 4(c)) and the protein level of phospho-p38 diphosphatase, MKP-1 [38], was not enhanced (Figure 4(d)), NGACP treatment seems to directly target MKK3/6, although a specific enzyme assay would be required to obtain better evidence. In agreement with this finding, signaling complex formation between p38 and MKK3 was also defective, as demonstrated through immunoprecipitation and immunoblotting analyses (Figure 4(e)). Therefore, these results strongly suggest that the MKK3/p38 pathway could be the major inhibitory target of NGACP treatment. Finally, we confirmed the functional significance of p38 in the inflammatory response by using a specific inhibitor, SB203580 (SB), on LPS-activated RAW264.7 cells. SB also strongly suppressed the expression of COX-2 and TNF-α mRNA, as assessed using RT- and real-time PCR (Figure 5(a)) and inhibited the release of TNF-α and PGE2 (Figure 5(b)), which is in agreement with previously reported results [39].

Bottom Line: Through immunoblotting and immunoprecipitation analyses, we found that p38/AP-1 could be the major inhibitory pathway in the NGACP-mediated anti-inflammatory response.Indeed, inhibition of p38 by SB203580 showed similar inhibitory activity of the production of TNF- α and PGE2 and the expression of TNF- α and COX-2 mRNA.These results suggest that ACP-induced nanocurrents alter signal transduction pathways that are involved in the inflammatory response and could therefore be utilized in the treatment of various inflammatory diseases such as arthritis and colitis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.

ABSTRACT
Electrical stimulation with a weak current has been demonstrated to modulate various cellular and physiological responses, including the differentiation of mesenchymal stem cells and acute or chronic physical pain. Thus, a variety of investigations regarding the physiological role of nano- or microlevel currents at the cellular level are actively proceeding in the field of alternative medicine. In this study, we focused on the anti-inflammatory activity of aluminum-copper patches (ACPs) under macrophage-mediated inflammatory conditions. ACPs generated nanolevel currents ranging from 30 to 55 nA in solution conditions. Interestingly, the nanocurrent-generating aluminum-copper patches (NGACPs) were able to suppress both lipopolysaccharide-(LPS-) and pam3CSK-induced inflammatory responses such as NO and PGE2 production in both RAW264.7 cells and peritoneal macrophages at the transcriptional level. Through immunoblotting and immunoprecipitation analyses, we found that p38/AP-1 could be the major inhibitory pathway in the NGACP-mediated anti-inflammatory response. Indeed, inhibition of p38 by SB203580 showed similar inhibitory activity of the production of TNF- α and PGE2 and the expression of TNF- α and COX-2 mRNA. These results suggest that ACP-induced nanocurrents alter signal transduction pathways that are involved in the inflammatory response and could therefore be utilized in the treatment of various inflammatory diseases such as arthritis and colitis.

Show MeSH
Related in: MedlinePlus