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, 2020, 4909103
eCollection

Direct Peritoneal Resuscitation With Pyruvate Protects the Spinal Cord and Induces Autophagy via Regulating PHD2 in a Rat Model of Spinal Cord Ischemia-Reperfusion Injury

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Direct Peritoneal Resuscitation With Pyruvate Protects the Spinal Cord and Induces Autophagy via Regulating PHD2 in a Rat Model of Spinal Cord Ischemia-Reperfusion Injury

Ying Xiong et al. Oxid Med Cell Longev.

Abstract

Direct peritoneal resuscitation with pyruvate (Pyr-PDS) has emerged as an interesting candidate to alleviate injury in diverse organs, while the potential mechanism has yet to be fully elucidated. To explore the effect of autophagy in the spinal cord ischemia-reperfusion (SCIR) injury and the underlying mechanism, we established a model of SCIR in vivo and in vitro. In vivo, male SD rats underwent aortic occlusion for 60 min and then followed by intraperitoneally infused with 20 mL of pyruvate or normal saline for 30 min, and the spinal cords were removed for analysis after 48 h of reperfusion. The functional and morphological results showed that Pyr-PDS alleviated SCIR injury; meanwhile, the expression of autophagy-related genes and transmission electron microscopy displayed autophagy was activated by SCIR injury, and Pyr-PDS treatment could further upregulate the degree of autophagy which plays a protective part in the SCIR injury, while there is no significant difference after treatment with saline. In addition, SCIR injury inhibited expression of PHD2, which results to activate its downstream HIF-1α/BNIP3 pathway to promote autophagy. In the Pyr-PDS, the results revealed PHD2 was further inhibited compared to the SCIR group, which could further activate the HIF-1α/BNIP3 signaling pathway. Additionally, oxygen-glucose deprivation and reoxygenation were applied to SH-SY5Y cells to mimic anoxic conditions in vitro, and the expression of autophagy-related genes, PHD2, and its downstream HIF-1α/BNIP3 pathway showed the same trend as the results in vivo. Besides, IOX2, a specific inhibitor of PHD2 was also treated to SH-SY5Y cells during reoxygenation, in which the result is as same as the pyruvate group. Then, we observed the expression of autophagy-related genes and the HIF-1α signal pathway in the process of reoxygenation; the results showed that as the reoxygenation goes, the expression of the HIF-1α signal pathway and degree of autophagy came to decrease gradually, while treated with pyruvate could maintain autophagy high and stable through keeping PHD2 at a lower level during reoxygenation, and the latter was observed downregulated during reoxygenation process from 0 to 24 hours in a time-effect way. The above results indicated that direct peritoneal resuscitation with pyruvate showed effective protection to ischemia-reperfusion of the spinal cord through activating autophagy via acting on PHD2 and its downstream HIF-1α/BNIP3 pathway.

Conflict of interest statement

The authors declared no conflicts of interest.

Figures

Figure 1
Figure 1
PHDs could hydroxylate HIF-1α under normoxia, and the latter is discerned by the von Hippel-Lindau (VHL) tumor suppressor protein and then degraded by the proteasome. Under hypoxic conditions, HIF-1α is stabilized due to inactivation of PHDs, and then BNIP3 is upregulated as the direct target of HIF-1α. Upregulation of BNIP3 will bind to Rheb which would inhibit mTOR signaling and competing with Beclin-1-Bcl-2 and Beclin-1-Bcl-xl complexes allowing the release of Beclin-1. Autophagy would be enhanced via both sides, eventually, reducing inhibition of autophagy and enhancing activation of autophagy. Meanwhile, under hypoxia, accumulation of HIF-1α can induce the transcription of PHD2, in contrast, to ensure swift removal of HIF-1α after reoxygenation.
Figure 2
Figure 2
Pyr-PDS showed protective effect after SCIR. (a) Pyr-PDS improved BBB score after SCIR injury at 1 h, 6 h, 12 h, 24 h, 36 h, and 48 h post injury. (b) Serum creatinine showed SCIR has no influence to kidney function. Data are shown as the mean ± S.E.M., n = 18.
Figure 3
Figure 3
Direct peritoneal resuscitation with pyruvate alleviates SCIR injury and Nissl staining. (a–d) Representative section for hematoxylin-eosin staining (HE) staining at 48 hours after injury. Scale bar 200 μm for magnification 20x. (e–h) Nissl staining to display the survival neurons. Scale bar 200 μm for magnification 20x. (i, j) Spinal cord injury histological scores and statistics of the Nissl staining results. Data are shown as mean ± S.E.M.; p < 0.05, ∗∗p < 0.01 vs. control, n = 6.
Figure 4
Figure 4
Pyr-PDS increased expression of mRNA and protein related to autophagy in the spinal cord. The mRNA expression levels of p62 (a), Beclin-1 (b), and LC3 (c) were determined by qPCR. The expression levels of autophagy-related protein were detected by Western blot (d), and summarized data of protein expression was exhibited (e). Data are shown as the mean ± S.E.M.; p < 0.05, ∗∗p < 0.01vs. control.
Figure 5
Figure 5
SCIR could activate autophagy compared to the sham group, and Pyr-PDS could further increase degree of autophagy. Transmission electron microscopy (TEM) exhibited fragmented mitochondria and autophagosomes with double membranes which were marked with black arrowheads. (a–c) Scale bar = 2 μm; (d–f) Scale bar = 500 nm.
Figure 6
Figure 6
SCIR increased mRNA expression of the HIF-1α signal pathway, which could be further upregulated after Pyr-PDS treatment. (a–d) mRNA expression of HIF-1α, BNIP3, Rheb, and mTOR. (e) mRNA expression of PHD. (f) Protein expression of PHD2 and summarized data of protein expression. Data are shown as the mean ± S.E.M.; p < 0.05, ∗∗p < 0.01 vs. control.
Figure 7
Figure 7
Pyruvate/IOX2 could attenuate injury induced by OGD/R in SH-SY5Y cells. (a) The result of CCK-8 showed 1000 μM of pyruvate was cytotoxic in comparison to others. (b) The result of CCK-8 showed 500 μM of IOX2 was cytotoxic in comparison to others. (c) Both pyruvate and IOX2 play a protective role of SH-SY5Y treated by OGD/R according to the result of CCK-8. (e–h) Flow cytometry displays the apoptosis rate is increased in the OGD/R group, while downregulated by pyruvate/IOX2, and summarized data of flow cytometry was exhibited (d). Data are shown as the mean ± S.E.M.; p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001vs. control.
Figure 8
Figure 8
SH-SY5Y cells treated with OGD/R higher expressed autophagy-related proteins, HIF-1α and PHD2 (a), which could be further increased by pyruvate/IOX2 treatment. Summarized data of protein expression was also exhibited (b–f). Data are shown as the mean ± S.E.M.; p < 0.05, ∗∗p < 0.01 vs. control.
Figure 9
Figure 9
Reperfusion could reverse the increase expression of autophagy-related genes (a–d) and the HIF-1α signal pathway (e–h) in mRNA level in SH-SY5Y cells compared with the OGD group, while pyruvate could reverse the decline of the HIF-1α pathway and autophagy-related mRNA expression during reperfusion. Data are shown as the mean ± S.E.M.; p < 0.05, ∗∗p < 0.01 vs. control.
Figure 10
Figure 10
We detected the expression of PHD2 in both mRNA and protein levels of SH-SY5Y cells, as displayed in (a) the expression of PHD2 is decreased in SH-SY5Y cells treated with OGD; the expression of PHD2 was further downregulated in the SH-SY5Y cells in the group of pyruvate, while as shown in (b), the results exhibited the expression of PHD2 of SH-SY5Y cells treated with OGD is increased with the time of reoxygenation. Statistical significance was considered difference with p < 0.05, ∗∗p < 0.01 vs. control.

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