Metabolomic analysis and pharmacological validation of the cerebral protective effect of 3,4‑dihydroxybenzaldehyde on cerebral ischemia‑reperfusion injury

Abstract

3,4‑Dihydroxybenzaldehyde (DBD), one of the active components of Gastrodia elata, has a cerebral protective effect and can effectively combat cerebral ischemia/reperfusion (I/R) injury in rats. However, the metabolite profiles and underlying mechanisms associated with DBD remain unclear. To explore the level of energy metabolism and pharmacological targets in brain tissue following DBD treatment of stroke. The right middle cerebral artery of the rats was occluded for 2 h and reperfused for 24 h to simulate brain I/R injury. Pharmacological results showed that DBD reduced cerebral infarct volume, improved neurological function and increased adenosine triphosphate (ATP) content. Mitochondria are the primary sites for ATP generation and cellular energy supply and decreasing mitochondrial dysfunction can alleviate the energy expenditure of ischemic stroke. Through further experiments, it was found that mitochondrial damage was recovered following DBD treatment, which was manifested by the improvement of mitochondrial morphology under an electron microscope and the reduction of oxidative stress damage. The metabolomics of middle cerebral artery occlusion/reperfusion (MCAO/R) rat brain tissue was studied by the liquid chromatography‑tandem mass spectrometry metabolomics method. Significantly different metabolites were screened and the pathways involved included amino sugar and nucleotide sugar metabolism and pentose phosphate pathway. Finally, the present study performed targeted metabolic profiling and validated potential therapeutic targets. Uridine diphosphate N‑acetylglucosamine (UDP‑GlcNAc) levels were decreased in the MCAO/R group but significantly increased in the DBD group. TdT‑mediated dUTP nick end labeling (TUNEL) staining, hematoxylin and eosin staining and western blotting showed that brain cell apoptosis was inhibited and neuronal morphology improved. Among them, the regulatory enzyme O‑GlcNAc transferase (OGT) of UDP‑GlcNAc appeared to be significantly increased and neuronal apoptosis was inhibited following DBD treatment, which was verified by western blotting. Therefore, by analyzing mitochondrial dysfunction following I/R and the characterization of potential markers in mitochondrial energy metabolism, it was shown that OGT could inhibit neuronal apoptosis as a potential therapeutic target for brain I/R injury.

Keywords: 3; 4‑dihydroxybenzaldehyde; O‑GlcNAc transferase; ischemic stroke; metabolomics; mitochondrial dysfunction; uridine diphosphate N‑acetylglucosamine.

Figure 1.

DBD has a protective effect on cerebral I/R injury. (A) Chemical structure of DBD. (B) Representative images of brain sections stained with TTC to visualize infarcts. Red is normal tissue and white is infarcted. (C) Quantitative analysis of infarct size. (D) Neurological deficit scores in different groups. (E) ATP content of brain tissue. Oxidative stress-related indicators (F) ROS level and (G) MDA content. All data are presented as the mean ± standard error of the mean, n=6. ^##P<0.01 vs. Sham group; *P<0.05, **P<0.01 vs. Model group. DBD, 3,4-Dihydroxybenzaldehyde; I/R, ischemia/reperfusion; TTC, triphenyl tetrazolium chloride; ATP, adenosine triphosphate; ROS, reactive oxygen species; MDA, malondialdehyde.

Figure 2.

DBD has a protective effect on mitochondrial brain damage. (A) Electron microscope observation of the structure and morphology of mitochondria in different groups of brain tissues; the right image is the enlarged image in the left panels. Red arrows indicated typical changes in mitochondrial morphology. Magnification, ×400; scale bar=500 nm. The activity of mitochondrial respiratory chain enzyme complexes (B) I, (C) II, (D) III, and (E) IV in each group. (F) The mitochondrial swelling method determined the degree of mPTP opening and the higher the mitochondrial swelling value, the greater the opening degree of mPTP. (G) MMP levels, a decrease in MMP is an early signal of apoptosis. (H) mitochondrial Cyt-c content, an essential protein in apoptosis and an important mediator in the mitochondrial respiratory chain can reflect apoptosis. All data are presented as the mean ± standard error of the mean, n=6. Scale bar=500 nm ^#P<0.05, ^##P<0.01 vs. Sham group; *P<0.05, **P<0.01 vs. Model group. DBD, 3,4-Dihydroxybenzaldehyde; mPTP, mitochondrial permeability transition pore; Cyt-c, cytochrome c.

Figure 3.

CV distribution map in each group of samples. Red lines represent QC samples, green represent Sham samples, blue represents Model samples and purple represents Treat samples. The reference lines perpendicular to the X-axis correspond to CV values of 0.3 and 0.5. The two parallel to the X-axis corresponds to 75 and 85% of the total number of substances. CV, the coefficient of variation.

Figure 4.

PCA of metabolomic changes in each group and cluster heat map of metabolites were performed based on LC-MS/MS system. (A) In the PCA diagram of each group, each point represents a sample and color represents the samples in the same group. Green: Sham group, Red: Model group, Purple: Treat group. The Sham and Model groups were clearly divided, indicating that the metabolites in the samples following MCAO/R changed significantly. (B) Sample overall clustering heat map, where the clustering line on the left is the metabolite clustering line and the upper one is the sample clustering line. Scale is the expression level after normalization (the redder the color, the higher the expression level). PCA, principal component analysis; MCAO/R, middle cerebral artery occlusion/reperfusion.

Figure 5.

The enrichment analysis of differential metabolites and metabolic pathways following MCAO/R analysis by OPLS-DA. (A) OPLS-DA model validation plot, usually Q2, is higher than 0.9 and the model is best when P<0.05. (B) OPLS-DA score plot, representing the gap between Model group and Treat group. Green: Model group, Red: Treat group. (C) Volcano plot of differential metabolites, the greater the absolute value of the abscissa, the greater the fold difference in expression between the Model group and the Treat group. The greater the ordinate value, the more significant the differential expression. Green dots: Downregulated metabolites, red: upregulated metabolites and visualized in (D) Differential metabolite score plot (VIP≥1). (E) DA score plot of differential metabolic pathways. The dots are distributed on the left side of the central axis. The longer the line segment, the more the pathway's overall expression tends to be up-regulated; the larger the dots, the more metabolites and vice versa. The color reflects the size of the P-value; red, the smaller the P-value. MCAO/R, middle cerebral artery occlusion/reperfusion; OPLS-DA, orthogonal partial least squares discriminant analysis; VIP, variable importance in projection; DA, discriminant analysis.

Figure 6.

Graph of the central metabolic network with significant changes following MCAO/R. Compared with the Model group, elevated metabolites in the Treat group are indicated in red, decreased metabolites in green, and detected but not significant metabolites in blue. MCAO/R, middle cerebral artery occlusion/reperfusion; TCA, tricarboxylic acid cycle.

Figure 7.

Effects of DBD on UDP-GlcNAc and OGT in MCAO/R rats. (A) Quantitative results of the metabolite UDP-GlcNAc in each group. (B) Representative western blotting and quantitative analysis of OGT expression. All data are presented as the mean ± standard error of the mean, n=3. ^##P<0.01 vs. Sham group; *P<0.05, **P<0.01 vs. Model group. DBD, 3,4-Dihydroxybenzaldehyde; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine; OGT, O-GlcNAc transferase; MCAO/R, middle cerebral artery occlusion/reperfusion.

Figure 8.

DBD attenuates MCAO/R-induced apoptosis in rat brain cells. (A) Hematoxylin and eosin staining of rat brain tissue. Note the cellular structure and arrangement of nerve fibers in brain tissue. (B) TUNEL (red) staining in TUNEL staining, nuclei were stained with DAPI (blue) and detected under a fluorescence microscope. (C) Quantitative analysis of apoptotic cells in brain tissue. (D) Representative western blot bands of apoptosis-related proteins are displayed. (E, F) Western blot analysis of Bax and Bcl-2 expression in brain tissue. (G and H) Western blot analysis of Caspase-3 and cleaved-Caspase-3 expression in brain tissue. All data are presented as the mean ± standard error of the mean, n=3. Magnifications are ×400; scale bar=50 µm ^##P<0.01 vs. Sham group; **P<0.01 vs. Model group. DBD, 3,4-Dihydroxybenzaldehyde; MCAO/R, middle cerebral artery occlusion/reperfusion; TUNEL, TdT-mediated dUTP nick end labeling.