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Research Article
Zhongmin Chen
Chongqing University of Technology
Corresponding Author
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Hydrogen (H2), a new type of medical gas molecule, which has significant preventive effect on numerous diseases and its anti-inflammatory properties has been proven in previous studies. However, the mechanisms of H2 anti-inflammatory activity in signal transduction pathway or protein level regulation are inadequately inexplicit. In the current study, the effect of H2 on LPS-induced inflammation in RAW 264.7 cells were assessed and its molecular mechanisms were clarified. The in vitro model of inflammation was induced by lipopolysaccharide (LPS) in RAW264.7 cells. Cell viability was evaluated by MTT assay. Protein expression of inflammatory mediators were analyzed by ELISA and Western blot. mRNA levels were detected by RT-qPCR. In addition, RNA sequencing (RNA-seq) was conducted to explore the molecular targets of H2 anti-inflammatory. According to the findings, H2 reversed LPS-induced variety in NO levels and TNF-a production as well as IL-6, IL-10 proteins and related mRNA levels in macrophages. RNA-seq newly discovered that H2 acted on inflammatory signaling molecule protein kinase C 8 (PKC8) and heterodimer activator protein-1 (AP-1). The WB analysis was then used to determine the key proteins in the inflammatory signaling pathway involved in PKC8 and AP-1, which found that H2 inhibited the phosphorylation of key proteins in the NF-kB and MAPKs pathways, thereby the expression of mRNA and inflammatory mediators were affected. The findings of this study show that H2 may serve as a promising anti-inflammatory gas in mitigating inflammatory conditions.
Keywords
Hydrogen, Anti-inflammatory property, NF-κB pathway, MAPKs pathway, RNA sequencing
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No competing interests reported.
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This preprint is under consideration at Scientific Reports. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Zhongmin Chen
Chongqing University of Technology
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 12 Nov, 2021
No community comments so far
Editor invited
On 10 Nov, 2021
Submission checks complete
On 10 Nov, 2021
First submitted
On 31 Oct, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Biotechnology and Bioengineering
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Hydrogen (H2), a new type of medical gas molecule, which has significant preventive effect on numerous diseases and its anti-inflammatory properties has been proven in previous studies. However, the mechanisms of H2 anti-inflammatory activity in signal transduction pathway or protein level regulation are inadequately inexplicit. In the current study, the effect of H2 on LPS-induced inflammation in RAW 264.7 cells were assessed and its molecular mechanisms were clarified. The in vitro model of inflammation was induced by lipopolysaccharide (LPS) in RAW264.7 cells. Cell viability was evaluated by MTT assay. Protein expression of inflammatory mediators were analyzed by ELISA and Western blot. mRNA levels were detected by RT-qPCR. In addition, RNA sequencing (RNA-seq) was conducted to explore the molecular targets of H2 anti-inflammatory. According to the findings, H2 reversed LPS-induced variety in NO levels and TNF-a production as well as IL-6, IL-10 proteins and related mRNA levels in macrophages. RNA-seq newly discovered that H2 acted on inflammatory signaling molecule protein kinase C 8 (PKC8) and heterodimer activator protein-1 (AP-1). The WB analysis was then used to determine the key proteins in the inflammatory signaling pathway involved in PKC8 and AP-1, which found that H2 inhibited the phosphorylation of key proteins in the NF-kB and MAPKs pathways, thereby the expression of mRNA and inflammatory mediators were affected. The findings of this study show that H2 may serve as a promising anti-inflammatory gas in mitigating inflammatory conditions.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
No competing interests reported.
This is a list of supplementary files associated with this preprint. Click to download.
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