Glycine Protects against Hypoxic-Ischemic Brain Injury by Regulating Mitochondria-Mediated Autophagy via the AMPK Pathway

Abstract

Hypoxic-ischemic encephalopathy (HIE) is detrimental to newborns and is associated with high mortality and poor prognosis. Thus, the primary aim of the present study was to determine whether glycine could (1) attenuate HIE injury in rats and hypoxic stress in PC12 cells and (2) downregulate mitochondria-mediated autophagy dependent on the adenosine monophosphate- (AMP-) activated protein kinase (AMPK) pathway. Experiments conducted using an in vivo HIE animal model and in vitro hypoxic stress to PC12 cells revealed that intense autophagy associated with mitochondrial function occurred during in vivo HIE injury and in vitro hypoxic stress. However, glycine treatment effectively attenuated mitochondria-mediated autophagy. Additionally, after identifying alterations in proteins within the AMPK pathway in rats and PC12 cells following glycine treatment, cyclosporin A (CsA) and 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside (AICAR) were administered in these models and indicated that glycine protected against HIE and CoCl₂ injury by downregulating mitochondria-mediated autophagy that was dependent on the AMPK pathway. Overall, glycine attenuated hypoxic-ischemic injury in neurons via reductions in mitochondria-mediated autophagy through the AMPK pathway both in vitro and in vivo.

Figure 1

Glycine attenuated hypoxic-ischemic injury in the brains of neonatal rats. (a) TTC staining of sham rats, rats of HIE, and HIE rats with administration of glycine 200 mg/kg, 400 mg/kg, 600 mg/kg, 800 mg/kg, and 1000 mg/kg. (b) Calculation of infarct area shown by TTC staining (of the sham group). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001 versus the HIE group. n = 5. (c) Protein expression level of HIF-1α of sham rats, rats of HIE, and HIE rats with administration of glycine 200 mg/kg, 400 mg/kg, 600 mg/kg, 800 mg/kg, and 1000 mg/kg. (d) Analyses of HIF-1α (of GAPDH). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001 versus the HIE group. n = 3. (e) The brains were isolated from pups of the sham group, HIE group, and HIE with administration of the glycine group. Ratio of wet and dry is calculated in each group. n = 5. The brains (fixed with 4% paraformaldehyde) further showed dropsical areas and effectiveness of glycine administration. (f) HE staining of the cortex and hippocampus on the ipsilateral sides in rats of each group. (h) Nissl staining of the cortex on the ipsilateral sides in rats of each group is shown. (i) The analysis of cell number of neurons in the cortex on each group. ^∗∗∗ P < 0.001. n = 5. (j) Nissl staining of the hippocampus on the ipsilateral sides in rats of each group. (k) Analysis of cell number of neurons in the hippocampus on each group. ^∗∗∗ P < 0.001. n = 5. (l) Transmission electron microscope reveals status of mitochondria happened in tissues. (m) Transmission electron microscope discovers autophagosomes (red arrows) in tissues.

Figure 2

Glycine protected against hypoxic-ischemic injury in the brains via attenuation of mitochondrial-mediated autophagy. (a) Protein expression level of Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I on the cortex from ipsilateral sides of the sham group, HIE group, and HIE with administration of the glycine group (of GAPDH). (b) Analyses of Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I in each group. (c) Protein expression level of Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I on the hippocampus from ipsilateral sides of each group (of GAPDH). (e) Analyses of Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I in each group. (e) Protein expression level of Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I in each group from the cortex. (f) Analyses of Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I in each group. (g) Protein expression level of Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I in each group from the hippocampus. (h) Analyses of Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I in each group. (i) HE stainings of the cortex of four groups. (j) HE stainings of the hippocampus among four groups. (k) Nissl stainings in the cortex from four groups and neuron numbers were analyzed. ^∗∗∗ P < 0.001. (l) Nissl stainings in the hippocampus from four groups and neuron numbers were analyzed. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001. n = 5.

Figure 3

Glycine eliminated mitochondria-mediated autophagy via regulation of the AMPK pathway. (a) Protein expression level of p-AMPK_α in the cortex from ipsilateral sides of each group and analysis of p-AMPK_α (of AMPK_α), p-mTOR (of GAPDH). (b) Protein expression level of p-AMPK_α on the hippocampus from ipsilateral sides of each group and analysis. (c) Protein expressions of p-mTOR, p-AMPK_α, AMPK_α, Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I from the cortex. (d) Analyses of protein expressions above. (e) Protein expressions of p-mTOR, p-AMPK_α, AMPK_α, Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I from the cortex. (f) Analysis of protein expressions above. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001. n = 5.

Figure 4

Glycine improved prognosis of rats following hypoxic-ischemic injury. (a) Weights of rats of 7 days, 14 days, and 28 days. ^∗ P < 0.05 and ^∗∗∗ P < 0.001. ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001. (b) The brains isolated from 14-day-old rats of each group. Fresh brain tissue is shown at the first line and dehydrated brains tissue is shown at the second line. n = 3. (c) HE staining of the cortex and hippocampus from ipsilateral sides of each group. n = 3.

Figure 5

Glycine attenuated CoCl₂-induced cytotoxicity, cellular ROS, and apoptosis in PC12 cells. (a) PC12 cells treated with CoCl₂ on dose-dependent manner for 12 hours and 24 hours by CCK8. (b) PC12 cells were pretreated with different concentrations of glycine for 12 hours and 24 hours. Then, PC12 cells were treated with CoCl₂ (800–1000 μM) and cell viability was determined by CCK8. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001 versus the control group. ^## P < 0.01 and ^### P < 0.001 versus the CoCl₂ group. (c) ROS generation of the control group, CoCl₂ group, and CoCl₂ + glycine group was determined by flow cytometry assay of DCFH-DA. ^∗ P < 0.05. n = 3. (d) Apoptotic cells of each group were detected by TUNEL and DAPI staining. ^∗∗∗ P < 0.001. n = 3.

Figure 6

Glycine attenuated CoCl₂-induced mitochondrial ROS generation, restored the MPP, improved Mfn-2 protein levels, and alleviated autophagy in PC12 cells. (a) Mitochondrial ROS generation of each group was detected by MitoSOX and DAPI staining. (b) Mitochondrial membrane potential of each group was determined by TMRE and Hoechst staining. (c) Protein expression level of Mfn-2 from outer membrane of mitochondria was measured by immunofluorescence. (d) Autophagic vacuoles from each group were measured by MDC staining. ^∗∗ P < 0.01 and ^∗∗∗ P < 0.001. n = 3.

Figure 7

Glycine protected PC12 cells against CoCl₂-induced injury via regulation of mitochondria-mediated autophagy. (a) Protein expression level of Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I in PC12 cells of each group. (b) Analyses of Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I in each group. (c) After treatment of CsA, protein expression levels of Mfn-2, p62, parkin, PINK1, Bnip3, LC3 II, and LC3 I in PC12 cells of four groups were determined by Western blotting. (d) Analysis of all protein expressions. (e) Cell viability of four groups was detected by CCK8. (f) Apoptotic cells from each group were measured by TUNEL and DAPI staining. (g) Mitochondrial ROS generations were detected by immunofluorescence of MitoSOX and DAPI staining. (h) Mitochondrial membrane potential was detected in each group by TMRE and Hoechst staining. (i) The Mfn-2, protein of mitochondrial outer membrane, was used to measure function of mitochondria. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001. n = 3.

Figure 8

Glycine protected PC12 cells against CoCl₂ injury via regulation of AMPK-dependent mitochondria-mediated autophagy. (a) p-AMPK_α protein expression was detected by Western blotting in each group. (b) Analyses of p-AMPK_α protein (of AMPK_α) and p-mTOR (of GAPDH). (c) Agonist of the AMPK pathway, CsA, was used to overactivate proteins from the AMPK pathway. Protein expressions of four groups were detected by Western blotting. (d) Analysis of all protein expressions. (e) Cell viability in four groups was also measured by CCK8. (f) Autophagic vacuoles from each group were measured by MDC staining. (g) Apoptotic cells from each group were measured by TUNEL and DAPI staining. (h) Mitochondrial ROS generations were detected by immunofluorescence of MitoSOX and DAPI staining. (i) Mitochondrial membrane potential was detected in each group by TMRE and Hoechst staining. (j) Mfn-2, protein of mitochondrial outer membrane, was used to measure function of mitochondria. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001. n = 3.