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. 2014 Aug 12;9(8):e104924.
doi: 10.1371/journal.pone.0104924. eCollection 2014.

Role of Nrf2/ARE pathway in protective effect of electroacupuncture against endotoxic shock-induced acute lung injury in rabbits

Jian-bo Yu  1 , Jia Shi  1 , Li-rong Gong  1 , Shu-an Dong  1 , Yan Xu  1 , Yuan Zhang  1 , Xin-shun Cao  1 , Li-li Wu  1
Affiliations

Role of Nrf2/ARE pathway in protective effect of electroacupuncture against endotoxic shock-induced acute lung injury in rabbits

Jian-bo Yu et al. PLoS One. .

Abstract

NF-E2 related factor 2 (Nrf2) is a major transcription factor and acts as a key regulator of antioxidant genes to exogenous stimulations. The aim of current study was to determine whether Nrf2/ARE pathway is involved in the protective effect of electroacupuncture on the injured lung in a rabbit model of endotoxic shock. A dose of lipopolysaccharide (LPS) 5 mg/kg was administered intravenously to replicate the model of acute lung injury induced by endotoxic shock. Electroacupuncture pretreatment was handled bilaterally at Zusanli and Feishu acupoints for five consecutive days while sham electroacupuncture punctured at non-acupoints. Fourty anesthetized New England male rabbits were randomized into normal control group (group C), LPS group (group L), electroacupuncture + LPS group (group EL) and sham electroacupuncture + LPS (group SEL). At 6 h after LPS administration, the animals were sacrificed and the blood samples were collected for biochemical measurements. The lungs were removed for calculation of wet-to-dry weight ratios (W/D), histopathologic examination, determination of heme oxygenase (HO)-1 protein and mRNA, Nrf2 total and nucleoprotein, as well as Nrf2 mRNA expression, and evaluation of the intracellular distribution of Nrf2 nucleoprotein. LPS caused extensive morphologic lung damage, which was lessened by electroacupuncture treatment. Besides, lung W/D ratios were significantly decreased, the level of malondialdehyde was inhibited, plasma levels of TNF-α and interleukin-6 were decreased, while the activities of superoxide dismutase, glutathione peroxidase and catalase were enhanced in the electroacupucnture treated animals. In addition, electroacupuncture stimulation distinctly increased the expressions of HO-1 and Nrf2 protein including Nrf2 total protein and nucleoprotein as well as mRNA in lung tissue, while these effects were blunted in the sham electroacupuncture group. We concluded that electroacupuncture treatment at ST36 and BL13 effectively attenuates lung injury in a rabbit model of endotoxic shock through activation of Nrf2/ARE pathway and following up-regulation of HO-1 expression.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Microphotographs of representative lung section stained with hematoxylin and eosin (original magnification×400).
A. The normal structure of lung from the control group (Group C); B. Severe alveolar edema, hemorrhage, the infiltration of leukocytes and thickened alveolar septum were observed in LPS group (Group L); C. Slight attenuation of the lung injury were displayed in treatment with electroacupuncture (Group EL); D. No improvement of the lung pathology were reflected in sham electroacupuncture stimulation (Group SEL). Black arrows: hemorrhage and infiltration of leukocytes in alveolar space; Red arrows: fracture of alveolar septum; Blue arrows: thickened alveolar septum. Scale bars: 50 µm.
Figure 2
Figure 2. Real-time PCR analysis of HO-1 mRNA (A) and Nrf2 mRNA (B) expressions in the lung tissue of four groups.
Data were presented as mean±SD. “C” presents group C, “L” presents group LPS, “EL” presents group electroacupuncture + LPS and “SEL” presents group sham electroacupuncture + LPS. The relative expressions of HO-1 mRNA and Nrf2 mRNA in group EL were higher than that in group C and group L (P<0.05), while no significant differences were found between group SEL and group L in terms of the above mentioned mRNA expressions(P>0.05). Ten control, LPS, EL and SEL experiments were performed for each group.
Figure 3
Figure 3. Western blot analysis of HO-1 protein (A), Nrf2 nucleoprotein (B) and Nrf2 total protein (C) relative expressions in lung tissue of four groups.
Data were presented as mean±SD. “C” presents group C, “L” presents group LPS, “EL” presents group electroacupuncture + LPS and “SEL” presents group sham electroacupuncture + LPS. The expressions of HO-1 protein, Nrf2 nucleoprotein and total protein in group EL were higher compared with group C and group L (P<0.05). While sham electroacupuncture treatment exhibited the similar expressions of the above mentioned proteins to that of group L (P>0.05). Ten control, LPS, EL and SEL experiments were performed for each group. *P<0.05 versus control group; +P<0.05 versus LPS group.
Figure 4
Figure 4. Immunofluorescence assays of nuclear localization of Nrf2 protein using fluorescence microscope (original magnification×400).
“A” showed the pictures of immunofluorescence staining, while “B” presented the distribution ratios of Nrf2 nucleoprotein to the number of nuclei in unit area of five fields among four groups. Green standed for Nrf2-FITC stained sections, while blue standed for images of DAPI stained nuclei. It was confirmed that Nrf2 increasingly translocated from cytoplasm into the nucleus by electroacupuncture protocols (P<0.05) rather than sham electroacupuncture stimulation (P>0.05). Data were representative of three independent experiments. Values were mean ± SD, and ten control, LPS, EL and SEL experiments were performed for each group.
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This research was supported by grants No. 81372096 from the National Natural Science Foundation of China, Beijing, China, and grant 12ZCZDSY03300 from the Key Project of Tianjin Science and Technology Support, Tianjin, China. The funders had no role in study design, data collection and analysis, decision to publish,or preparation of the manuscript.

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