Exploring the attenuation mechanisms of <i>Dalbergia odorifera</i> leaves extract on cerebral ischemia-reperfusion based on weighted gene co-expression network analysis

Background: The attenuation function of Dalbergia odorifera leaves on cerebral ischemia-reperfusion (I/R) is little known. The candidate targets for the Chinese herb were extracted from brain tissues through the high-affinity chromatography. The molecular mechanism of D. odorifera leaves on cerebral I/R was investigated. Methods: Serial affinity chromatography based on D. odorifera leaves extract (DLE) affinity matrices were applied to find specific binding proteins in the brain tissues implemented on C57BL/6 mice by intraluminal middle cerebral artery occlusion for 1 h and reperfusion for 24 h. Specific binding proteins were subjected to mass-spectrometry to search for the differentially expressed proteins between control and DLE-affinity matrices. The hub genes were screened based on weighted gene co-expression network analysis (WGCNA). Then, predictive biology and potential experimental verification were performed for the candidate genes. The protective role of DLE in blood-brain barrier damage in cerebral I/R mice was evaluated by the leakage of Evans blue, western blotting, immunohistochemistry, and immunofluorescent staining. Results: 952 differentially expressed proteins were classified into seven modules based on WGCNA under soft threshold 6. Based on WGCNA, AKT1, PIK3CA, NOS3, SMAD3, SMAD1, IL6, MAPK1, TGFBR2, TGFBR1, MAPK3, IGF1R, LRG1, mTOR, ROCK1, TGFB1, IL1B, SMAD2, and SMAD5 18 candidate hub proteins were involved in turquoise module. TGF-β, MAPK, focal adhesion, and adherens junction signaling pathway were associated with candidate hub proteins. Gene ontology analysis demonstrated that candidate hub proteins were related to the TGF-β receptor signaling pathway, common-partner SMAD protein phosphorylation, etc. DLE could significantly reduce the leakage of Evans blue in mice with cerebral I/R, while attenuating the expression of occludin, claudin-5, and zonula occludens-1. Western blotting demonstrated that regulation of TGF-β/SMAD signaling pathway played an essential role in the protective effect of DLE. Conclusion: Thus, a number of candidate hub proteins were identified based on DLE affinity chromatography through WGCNA. DLE could attenuate the dysfunction of blood-brain barrier in the TGF-β/SMAD signaling pathway induced by cerebral I/R.