A Novel Network Pharmacology Strategy Based on the Universal Effectiveness-Common Mechanism of Medical Herbs Uncovers Therapeutic Targets in Traumatic Brain Injury

Abstract

Purpose: Many herbs can promote neurological recovery following traumatic brain injury (TBI). There must lie a shared mechanism behind the common effectiveness. We aimed to explore the key therapeutic targets for TBI based on the common effectiveness of the medicinal plants.

Material and methods: The TBI-effective herbs were retrieved from the literature as imputes of network pharmacology. Then, the active ingredients in at least two herbs were screened out as common components. The hub targets of all active compounds were identified through Cytohubba. Next, AutoDock vina was used to rank the common compound-hub target interactions by molecular docking. A highly scored compound-target pair was selected for in vivo validation.

Results: We enrolled sixteen TBI-effective medicinal herbs and screened out twenty-one common compounds, such as luteolin. Ten hub targets were recognized according to the topology of the protein-protein interaction network of targets, including epidermal growth factor receptor (EGFR). Molecular docking analysis suggested that luteolin could bind strongly to the active pocket of EGFR. Administration of luteolin or the selective EGFR inhibitor AZD3759 to TBI mice promoted the recovery of body weight and neurological function, reduced astrocyte activation and EGFR expression, decreased chondroitin sulfate proteoglycans deposition, and upregulated GAP43 levels in the cortex. The effects were similar to those when treated with the selective EGFR inhibitor.

Conclusion: The common effectiveness-based, common target screening strategy suggests that inhibition of EGFR can be an effective therapy for TBI. This strategy can be applied to discover core targets and therapeutic compounds in other diseases.

Keywords: AZD3759; astrocyte; epidermal growth factor receptor; luteolin; medicinal plants; traditional Chinese medicine.

Figure 1

Flowchart of this research. The common compounds of the effective herbs and the compound targets in TBI are screened. The potential common compounds-core target interactions are assessed by molecular docking and in vivo experiments.

Figure 2

The herb-compound network of the collected effective herbs. Sixteen TBI-effective herbs consist of 230 compounds. Octagons represent medicinal plants. Circles represent candidate compounds. Blue circles represent 209 specific compounds, and Orange circles represent 21 common compounds. The size of the node is proportional to the value of degree.

Figure 3

The common compound-core target interactions. (A) Venn diagram shows 131 overlapped genes between herb targets and TBI-related genes. (B) The PPI network of the overlapping targets highlights the core targets of TBI, including EGFR. (C) The top 15 enriched Wiki pathway of the overlapped targets shows the importance of the AGE/RAGE pathway, and EGFR tyrosine kinase inhibitor resistance pathways in TBI. (D) The 3D and 2D interaction diagrams display the binding model of luteolin and EGFR.

Figure 4

The therapeutic effects of luteolin in treating CCI mice. (A) H&E staining and Nissl staining of the cerebral cortex (N=3). The dotted lines indicate the damaged region. (B) mNSS shows that AZD3759 and luteolin partially reverse the neurological deficits of the CCI mice on days 7 and 14. (C) Foot fault test suggests that AZD3759 and luteolin reduce the foot fault rate after CCI. (D) AZD3759 and luteolin alleviate the body weight loss after CCI. Data are expressed as Mean ± SEM, N = 6 (B-D), **P < 0.01, ****P < 0.0001, Scar bar = 100 μm. CCI: Controlled Cortical Impact model.

Figure 5

Luteolin and AZD03759 attenuate astrocyte activation after CCI. (A) Immunofluorescent staining of GFAP (green) and EGFR (red) shows luteolin and AZD03759 decrease astrocyte number and astrocytic EGFR expression. (B) Morphological analysis suggests luteolin and AZD03759 suppress astrocyte activation. (C) Quantification of both EGFR-positive and GFAP-positive cells. (D-F) Quantitative metrics of astrocyte activation. Data are expressed as Mean ± SEM, N = 3, **P < 0.01, ***P < 0.001, ****P < 0.0001, Scar bar = 50 μm. CCI: Controlled Cortical Impact model.

Figure 6

Luteolin reduced glial scar formation after CCI. (A) Immunofluorescent staining of GFAP (green) and Brevican (red, CSPGs marker). (B) Quantification of Brevican expression. (C) The maximum area of the glial scar. (D) The average glial scar area. Data are expressed as Mean ± SEM, N = 3, ****P < 0.0001, Scar bar = 50 μm. CCI: Controlled Cortical Impact model.

Figure 7

Luteolin promoted axonal regeneration after CCI. (A) Immunofluorescent staining of GFAP (green) and GAP43 (red). (B) Quantification of GAP43 expression. Data are expressed as Mean ± SEM, N = 3, ****P < 0.0001, Scar bar = 50 μm. CCI: Controlled Cortical Impact model.