Novel potential neuroprotective targets for DengZhanXiXin injection in middle cerebral artery occlusion rats recommended by quantitative proteomics and simulated docking

Abstract

Stroke, which leads to death and disability in high proportions globally, is one of the most deleterious neurological diseases. Ischemic stroke (IS) is the major cause of disease attack and accounts for ~70% of all incident stroke cases in China. Up to now, only two therapies for IS were officially approved, which are intravenous administration of recombinant tissue-plasminogen activator (rt-PA) and endovascular mechanical thrombectomy to rapidly recanalize the occluded artery, which both recanalize the occluded artery rapidly to reduce disability, but are limited in a fixed time window. In this study, the therapeutic effect of a traditional Chinese medicine, DengZhanXiXin injection (DZXI), was evaluated on middle cerebral artery occlusion (MCAO) rats at the neurobehavioral and pathophysiological levels through neurological tests, neurohistological staining, proteomic assay, and biological information analysis. We found that DZXI significantly ameliorated the neurological deficit, prevented infarct volume evolution, and protected cortical neural cells from death in ischemia penumbra on MCAO rats. Furthermore, corresponding therapeutic molecular targets were investigated through proteomic analysis of ischemic hemispheres of MCAO rats. One hundred ninety-one differentially expressed proteins involved in response to metal ions, neurofilament bundle assembly, and modulation of chemical synaptic transmission were identified between the MCAO model and DZXI groups after 7 days. DZXI influenced the expression levels of proteins in 13 specific biological functions, with cell signaling and chemical synaptic transmission-associated proteins being most affected. Subsequent molecular docking analysis predicted binding potential between key target proteins and DZXI compounds. The results suggested that DZXI ameliorates neurological deficits by potentially affecting cellular signaling and chemical synaptic transmission physiological processes.

Keywords: DengZhanXiXin injection; MCAO; cell signaling; ischemic stroke; molecular docking.

Figure 1

(A) mNSS score of the rats in each MCAO and DZXI treatment group. Red, blue, and green lines represent the mean scores of MCAO, DZXI, and sham groups, respectively (n = 10 in each group). Error bars indicate SD. The time and group effects on mNSS score were analyzed by two-way ANOVA followed by Turkey's HSD test, *P < 0.05, DZXI vs. model. (B) TTC staining of rat brain serial coronal slices in sham, MCAO, and MCAO with DZXI treatment groups. TTC staining showed red healthy zones and pale infarcted regions. (C) The infarction ratio in different groups was plotted (n = 5 in each group). Error bars indicate SD. The group difference was analyzed with one-way ANOVA followed by Turkey's HSD test, *P < 0.05.

Figure 2

(A) The morphological change of cortical penumbra neural cells in different groups was examined by HE staining. The magnification of the upper images is smaller for observing the overall arrangement of cortical neural cells, while the magnification of the lower images is larger, allowing for a detailed observation of the structure and morphology of neural cells. The black arrows in the image indicate dead cells with swollen morphology, blurred structure, or nuclear condensation. The scale bar is displayed in the lower left corner of the image. (B) The ratios of dead neural cells vs. total neural cells in different groups were plotted (n = 5 in each group). Error bars indicate SD. The group difference was analyzed with one-way ANOVA followed by the Turkey's HSD test, ***P < 0.001.

Figure 3

(A) The DEPs between the sham group and MCAO model group identified in proteomic assays were clustered according to their expression levels. (B) The DEPs between the MCAO model group and DZXI group identified in proteomic assays were clustered according to their expression levels. The relative TMT intensities of each protein (rows) in each group (columns) were indicated on a colored scale in both (A, B), where red represents a high expression level and blue represents a low expression level. (C). The top 10 biological processes enriched in the DEPs between the sham group and the MCAO model group were enriched. (D) The top 5 biological processes in which the DEPs between the model group and DZXI group were enriched. Both results in (C, D) were acquired with GO analysis.

Figure 4

The interaction network of the 191 DEPs between the model and DZXI 7-day treatment groups (acquired with STRING). Colored nodes represent query proteins, edges represent protein-protein associations, and different color lines indicate the different interactions between proteins.

Figure 5

The interaction subnetwork of cellular signaling and chemical synaptic transmission associated proteins (acquired with STRING). Nodes in red represent proteins associated with cell signaling, and green represents chemical synaptic transmission. Pde1b, Calcium/calmodulin-dependent3′5′-cyclic nucleotide phosphodiesterase 1B; Pde1c, Calcium/calmodulin-dependent3′5′-cyclic nucleotide phosphodiesterase 1C; Pde10a, cAMP and cAMP-inhibited cGMP3′5′-cyclic phosphodiesterase 10A; Adcy5, Adenylate cyclase 5; Gnal, Guanine nucleotide-binding protein G(olf) subunit alpha; Gng7, Guanine nucleotide-binding protein subunit gamma; Nt5e, 5′-nucleotidase; Prkcg, Protein kinase C gamma type; Prkag2, Protein kinase AMP-activated non-catalytic subunit gamma 2; Gria1, Glutamate receptor 1; Grm2, Metabotropic glutamate receptor 2; Cnih2, Protein cornichon homolog 2; Gabra5, Gamma-aminobutyric acid receptor subunit alpha-5; Gabrg2, Gamma-aminobutyric acid receptor subunit gamma-2; Gabrb1, Gamma-aminobutyric acid receptor subunit beta-1.

Figure 6

3D structural visualization of molecular docking simulation between DZXI compounds and their potentially targeted proteins. The binding affinity, binding sites (local and enlarged in 3D), and interacting residues (in 2D) of three target-compound pairs are exhibited. (A) PDE10A and 3,4-O-dicaffeoylquinic acid; (B) PRKCG and 3,5-O-dicaffeoylquinic acid; (C) CAR3 and 4,5-O-dicaffeoylquinic acid.

Figure 7

Overview of possible mechanisms inferred from prior studies underlying the DZXI therapeutic effect on ischemic stroke.