Protopine Exerts Neuroprotective Effects on Neonatal Hypoxic-Ischemic Brain Damage in Rats via Activation of the AMPK/PGC1α Pathway

Abstract

Introduction: Neonatal hypoxic-ischemic encephalopathy (HIE), caused by perinatal asphyxia, is characterized by high morbidity and mortality, but there are still no effective therapeutic drugs. Mitochondrial biogenesis and apoptosis play key roles in the pathogenesis of HIE. Protopine (Pro), an isoquinoline alkaloid, has anti-apoptotic and neuro-protective effects. However, the protective roles of Pro on neonatal hypoxic-ischemic brain injury remain unclear.

Methods: In this study, we established a CoCl₂-induced PC12 cell model in vitro and a neonatal rat hypoxic-ischemic (HI) brain damage model in vivo to explore the neuro-protective effects of Pro and try to elucidate the potential mechanisms.

Results: Our results showed that Pro significantly reduced cerebral infarct volume, alleviated brain edema, inhibited glia activation, improved mitochondrial biogenesis, relieved neuron cell loss, decreased cell apoptosis and reactive oxygen species (ROS) after HI damage. In addition, Pro intervention upregulated the levels of p-AMPK/AMPK and PGC1α as well as the downstream mitochondrial biogenesis related factors, such as nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM), but the AMPK inhibitor compound c (CC) could significantly reverse these effects of Pro.

Discussion: Pro may exert neuroprotective effects on neonatal hypoxic-ischemic brain damage via activation of the AMPK/PGC1α pathway, suggesting that Pro may be a promising therapeutic candidate for HIE, and our study firstly demonstrate the neuro-protective roles of Pro in HIE models.

Keywords: apoptosis; mitochondrial biogenesis; neonatal hypoxic-ischemic brain damage; protopine; reactive oxygen species.

Figure 1

Pro treatment attenuated acute hypoxia-ischemia (HI) brain injury in neonatal rats. (A) The chemical structure of protopine. (B) The timeline of in vitro experiments. (C) The representative images of TTC staining of Coronal brain sections 24 h after HI brain injury (n = 4). Scale bar: 0.5 mm. (D) Calculation of the infarct volume depicted by TTC staining. All data are presented as mean ± SD. (n = 4). (E) Representative images of the brain from each group 24 h after HI brain injury (n = 4). Scale bar: 0.5 mm. (F) The ratio of wet and dry in each group (n = 5). The data are presented as mean ± SD.

Figure 2

Pro treatment inhibited white matter damage and microglial activation after hypoxic-ischemic (HI) brain injury in neonatal rats. (A) The protein levels of MAP and MBP expression by Western blot 7 days after HI brain injury. (B) Analyses of MAP and MBP (n = 4). (C) Representative immunofluorescence staining images of IBA-1 (green) and DAPI (blue) on the brain tissues. Scale bar = 10 μm. (D) The ratio of IBA-1 positive cells (of all cells) (n = 3). (E) Representative images of the brain from each group 7 days after HI injury (n = 5). Scale bar: 0.5 mm. (F) Ratio of the injured hemisphere to the contralateral hemisphere. The data are presented as mean ± SD.

Figure 3

Pro ameliorated the tissue structural damage and reduced the loss of neurons. (A) Representative image of Nissl staining 7 days after HI brain injury. Scale bar = 100 μm. (B) Representative image of HE staining in the cortex, hippocampal CA1, CA3, and DG region 7 days after HI brain injury. Scale bar = 100 μm. (C) Quantification analysis of neuron numbers in brain cortex, hippocampal CA1, CA3, and DG region (n = 4). The data are presented as mean ± SD.

Figure 4

Pro treatment suppressed apoptosis and oxidative stress caused by HI brain damage and exerted neuroprotection via the AMPK/PGC1α pathway to activate NRF1/TFAM in positive feedback manner. (A) Representative immunofluorescence staining images of TUNEL (green) and DAPI (blue) in brain cortex and CA1 24 h after HI brain injury. Scale bar = 10 μm. (B) Quantitative analysis of TUNEL-positive cells in Cortex and hippocampal CA3 region (of all cells) (n = 3). (C) The MDA level in brain tissues 24 h after HI brain injury (n = 3). (D) The CAT level in brain tissues 24 h after HI brain injury (n = 3). (E) The protein levels of BCL-2 and BAX expression by Western blotting in brain tissues. (F) Analyses of BCL-2 and BAX (normalized to β-actin) (n = 4). (G) The protein levels of AMPK, p-AMPK, PGC1α, NRF1 and TFAM expression by Western blotting in brain tissues. (H) Analyses of AMPK, p-AMPK, PGC1α, NRF1 and TFAM (normalized to β-actin) (n = 4). The data are presented as mean ± SD.

Figure 5

Pro attenuated CoCl₂-induced oxidative stress damage and apoptosis in PC12 cell. (A) The mitochondrial ROS generation of PC12 cell was detected by MitoSOX (red) and DAPI (blue) staining (n = 3). Scale bar: 10 μ m. (B) Mean fluorescence intensity of ROS in the mitochondria of PC12 cells after CoCl₂ injury (n = 3). (C) The CAT level in PC12 cells after CoCl₂ injury (n = 3). (D) The MDA level in PC12 cells after CoCl₂ injury (n = 3). (E) Quantitative analysis of Annexin V and PI positive cells (n = 3). The data are presented as mean ± SD. (F) Representative immunofluorescence staining images of Annexin V FITC (green), PI (red) and Hoechest (blue) in PC12 cells after CoCl₂ injury. Scale bar: 100 μm.

Figure 6

CC reversed the neuro-protective effect of Pro after CoCl₂-induced injury in PC12 cell. (A) The generation of mitochondrial ROS in each group was detected by MitoSOX (red) and DAPI (blue) staining of PC12 cells after CoCl₂ injury (n = 3). (B) Mean fluorescence intensity of ROS in the mitochondria of each group (n = 4). (C) Representative and pooled data showing ROS production using flow cytometry (n = 3). (D) Quantitative analysis of ROS generation in PC12 cells using flow of ROS (n = 3). (E) The protein levels of BCL-2 and BAX expression by Western blotting in PC12 cells after CoCl₂ injury (n = 4). (F) Analysis of BCL-2 and BAX levels (normalized to β-actin) (n = 4). (G) Apoptosis in each group was analyzed and quantified using Annexin V FITC and PI staining flow cytometry (n = 3). (H) Quantitative analysis of Annexin V FITC and PI staining flow cytometry (n = 3). The data are presented as the mean ± SD.

Figure 7

Pro exerted neuroprotection through activates AMPK/PGC1α pathway to regulate NRF1/TFAM transcription factor. (A) The protein levels of AMPK, p-AMPK, PGC1α, NRF1, and TFAM expression by Western blotting in PC12 cells after CoCl₂ injury (n = 4). (B) Analyses of AMPK, p-AMPK, PGC1α, NRF1 and TFAM (normalized to β-actin) (n = 4). (C) The representative immunofluorescence staining images of PGC1α (green) and DAPI (blue) in PC12 from each group (n = 4). Scale bar: 10 μm. (D) Quantitative analysis of Mean fluorescence intensity of PGC1α in PC12 cells (n = 4). (E) The representative immunofluorescence staining images of TFAM (red) and DAPI (blue) in PC12 from each group (n = 4). Scale bar: 10 μm. (F) Quantitative analysis of Mean fluorescence intensity of TFAM in PC12 cells (n = 4). The data are presented as mean ± SD.

Figure 8

Pro improves the mechanism of action of HIE by activating AMPK/PGC1α pathway to activate NRF1/TFAM in positive feedback manner to inhibit oxidative stress damage and apoptosis.