Background

Oxidative stress is involved in the development of many inflammatory, metabolic and aging diseases.

Objective

In this study we investigated, the protective effects of H₂ on RAW 264.7 macrophage cell against LPS-and H₂O₂-induced oxidative stress by the inhibition of MAPK pathway and also activate the Nrf2 pathway.

Results

Our results showed H₂ increased the macrophage cell proliferation and generated ROS and NO against LPS stimulation to exert an active immune response. Similarly, H₂ protected the macrophage cell from H₂O₂-induced oxidative stress. H₂ reduced the LPS-and H₂O₂-induced inflammatory cytokine production and intracellular calcium influxes. H₂ inhibited the LPS-and H₂O₂-induced phosphorylation of MAPK pathway and its downstream signaling molecules. Furthermore, H₂ protected the macrophage cell from mitochondrial apoptosis. H₂ increased Nrf2 protein expression indicating its strong anti-oxidative effects against oxidative stress.

Conclusion

Collectively, our results indicate the strong antioxidant role of H₂ against LPS-and H₂O₂-induced oxidative stress on macrophage cells by activating the Nrf2 pathway and inhibiting the MAPK-signaling pathway.

Graphic Abstract

Our results clearly showed that LPS increased the cellular ROS by recognizing the TLR4 and H₂O₂ rapidly increased the cellular (1) and mitochondrial (2) oxidative stress. Excessive ROS/NO molecules cause intracellular calcium influxes (3) As a results imbalance the cellular membrane homeostasis and activate the stress response MAPK signaling pathway with its downstream signaling protein and mitochondrial caspase protein (4) that collapse the anti-oxidant mechanisms and induced the inflammatory cytokine secretion; leads to cell apoptosis (5) Whether H₂ reduced the cellular and mitochondrial oxidative stress, intracellular calcium influxes and inhibits the stress response MAPK, caspase cell signaling pathway through the activation of Nrf2/ARE signaling pathway (6) Consequently, increased the antioxidant enzymes and reduced the inflammatory cytokine that influences the macrophage cell proliferation (7) to protect the cell from apoptosis. The different effects of ROS and H₂ used in this study are indicated in red and green, red arrow depicts ROS effects in the cell, green arrow depicts H₂ effects.

中文翻译：

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分子氢通过激活Nrf2和抑制MAPK信号通路来保护氧化应激诱导的RAW 264.7巨噬细胞

背景

氧化应激参与许多炎症，代谢和衰老疾病的发展。

目的

在这项研究中，我们研究了H ₂对RAW 264.7巨噬细胞对LPS和H ₂ O ₂诱导的氧化应激的保护作用，其机制是通过抑制MAPK途径并激活Nrf2途径。

结果

我们的结果表明，H ₂增加了巨噬细胞的增殖，并产生了针对LPS刺激的ROS和NO，从而产生了主动的免疫反应。类似地，H ₂保护巨噬细胞免受H ₂ O ₂诱导的氧化应激。H ₂减少了LPS和H ₂ O ₂诱导的炎症细胞因子的产生以及细胞内钙的涌入。H ₂抑制LPS和H ₂ O ₂诱导的MAPK途径及其下游信号分子的磷酸化。此外，H ₂保护巨噬细胞免受线粒体凋亡。高₂ Nrf2蛋白表达增加，表明其对氧化应激具有很强的抗氧化作用。

结论

总体而言，我们的结果表明H ₂通过激活Nrf2途径和抑制MAPK信号传导途径，对LPS和H ₂ O ₂诱导的巨噬细胞氧化应激具有很强的抗氧化作用。

图形摘要

我们的结果清楚地表明，LPS通过识别TLR4和H ₂ O ₂迅速增加了细胞（1）和线粒体（2）的氧化应激而增加了细胞ROS 。过量的ROS / NO分子引起细胞内钙内流（3），结果使细胞膜稳态失衡，并通过其下游信号蛋白和线粒体胱天蛋白酶（4）激活应激反应MAPK信号通路，从而破坏了抗氧化机制并诱导炎性细胞因子分泌；导致细胞凋亡（5）H ₂是否通过激活Nrf2 / ARE信号通路降低了细胞和线粒体的氧化应激，细胞内钙的流入并抑制了应激反应MAPK，胱天蛋白酶细胞的信号通路（6）因此，增加了抗氧化酶并减少了影响巨噬细胞的炎性细胞因子增殖（7）保护细胞免于凋亡。本研究中使用的ROS和H ₂的不同作用以红色和绿色表示，红色箭头表示细胞中ROS的作用，绿色箭头表示H _2的作用。