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. 2020 Sep;22(3):1969-1975.
doi: 10.3892/mmr.2020.11290. Epub 2020 Jun 30.

Butorphanol protects PC12 cells against OGD/R-induced inflammation and apoptosis

Zijing Yang  1 , Li Wang  1 , Yingjun Hu  1 , Feixiang Wang  1
Affiliations

Butorphanol protects PC12 cells against OGD/R-induced inflammation and apoptosis

Zijing Yang et al. Mol Med Rep. 2020 Sep.

Abstract

The aim of the present study was to examine the effects of butorphanol on neural injury in an oxygen glucose deprivation/reoxygenation (OGD/R) model using PC12 cells, and to investigate whether mitochondrial apoptosis was involved in these effects. To establish the OGD/R model, PC12 cells were cultured under hypoxia and low glucose conditions. Expression levels of inflammatory cytokines were evaluated by detecting the levels of tumor necrosis factor‑α, interleukin (IL)‑1β, IL‑6 and monocyte chemoattractant protein‑1. Oxidative stress was evaluated by measuring the levels of reactive oxygen species, lactate dehydrogenase activity and myeloperoxidase concentration. Apoptosis, protein expression and cell viability were determined by flow cytometry, western blotting and by using a Cell Counting Kit‑8, respectively. Compared with the control group, cell viability, expression of inflammatory factors and oxidative stress were all decreased in the OGD/R group. All the above changes could be mitigated by treatment with butorphanol. In addition, butorphanol treatment resulted in a significant upregulation of Bax, and downregulation of Bcl‑2, activated caspase‑3, caspase‑9 and poly ADP‑ribose polymerase, increased the expression of X‑linked inhibitor of apoptosis protein and enhanced ATP activity. To conclude, these results suggested that the protective effects of butorphanol are associated with the inhibition of OGD/R‑induced inflammation and apoptosis injury, and may be partially associated with the inhibition of mitochondrial apoptosis.

Keywords: butorphanol; oxygen glucose deprivation/reoxygenation; neural injury; mitochondrial apoptosis; oxidative stress.

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Figures

Figure 1.
Figure 1.
Butorphanol reduces cell viability following OGD/R injury. (A) The safe dose of butorphanol on PC12 cells was detected by CCK-8. *P<0.05 vs. 0 µM. (B) Effect of butorphanol (1–4 µM) on the viability of PC12 cells after OGD/R injury. Data are expressed as the mean ± standard deviation (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. Control; #P<0.05, ##P<0.01 and ###P<0.001 vs. OGD/R. OGD/R, oxygen glucose deprivation/reoxygenation.
Figure 2.
Figure 2.
Effect of butorphanol on expression of inflammatory factors following OGD/R injury. *P<0.05, **P<0.01 and ***P<0.001 vs. control; #P<0.05, ##P<0.01 and ###P<0.001 vs. OGD/R. TNF-α, tumor necrosis factor-α; IL-, interleukin; MCP-1, monocyte chemoattractant protein-1; OGD/R, oxygen glucose deprivation/reoxygenation.
Figure 3.
Figure 3.
Effect of butorphanol on oxidative stress in OGD/R injury. *P<0.05, **P<0.01 and ***P<0.001 vs. control; ##P<0.01 and ###P<0.001 vs. OGD/R. ROS, reactive oxygen species; LDH, lactate dehydrogenase; MPO, myeloperoxidase; OGD/R, oxygen glucose deprivation/reoxygenation.
Figure 4.
Figure 4.
Effect of butorphanol on apoptosis of PC12 cells following OGD/R injury. (A and B) Apoptosis of PC12 cells in each group. (C) Relative protein levels of Bax and Bcl-2 in each group, β-actin was used as the control. *P<0.05, **P<0.01 and ***P<0.001 vs. control; #P<0.05, ##P<0.01 and ###P<0.001 vs. OGD/R. OGD/R, oxygen glucose deprivation/reoxygenation.
Figure 5.
Figure 5.
Effects of butorphanol on expression of mitochondrial-mediated apoptosis associated proteins and ATP activity. (A) Relative protein levels of XIAP, cleaved-PARP, caspase-3, caspase-9, cleaved caspase-3 and cleaved caspase-9 in each group, β-actin was used as the control. (B) ATP activity of PC12 cells was determined by a detection kit. (C) XIAP protein expression was examined by an immunofluorescence assay. *P<0.05, **P<0.01 and ***P<0.001 vs. control; #P<0.05, ##P<0.01 and ###P<0.001 vs. OGD/R. OGD/R, oxygen glucose deprivation/reoxygenation; XIAP, X-linked inhibitor of apoptosis protein; PARP, poly ADP-ribose polymerase.
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