Combined proteomic and metabolomic analyses of cerebrospinal fluid from mice with ischemic stroke reveals the effects of a Buyang Huanwu decoction in neurodegenerative disease.

Ischemic stroke is one of the most common causes of death worldwide and is a major cause of acquired disability in adults. However, there is still a need for an effective drug for its treatment. Buyang Huanwu decoction (BHD), a traditional Chinese medicine (TCM) prescription, has long been used clinically to aid neurological recovery after stroke. To establish potential clinical indicators of BHD efficacy in stroke treatment and prognosis, we conducted a combined proteomic and metabolomic analysis of cerebrospinal fluid (CSF) samples in a mouse stroke model. CSF samples were obtained from male mice with acute ischemic stroke induced by middle cerebral ischemic/reperfusion (CI/R) injury, some of which were then treated with BHD. Label-free quantitative proteomics was conducted using nano-LC-MS/MS on an LTQ Orbitrap mass and metabolomic analysis was performed using nanoprobe NMR and UHPLC-QTOF-MS. The results showed that several proteins and metabolites were present at significantly different concentrations in the CSF samples from mice with CI/R alone and those treated with BHD. These belonged to pathways related to energy demand, inflammatory signaling, cytoskeletal regulation, Wnt signaling, and neuroprotection against neurodegenerative diseases. In conclusion, our in silico data suggest that BHD treatment is not only protective but can also ameliorate defects in pathways affected by neurological disorders. These data shed light on the mechanism whereby BHD may be effective in the treatment and prevention of stroke-related neurodegenerative disease

Multiplex Brain Proteomic Analysis Revealed the Molecular Therapeutic Effects of Buyang Huanwu Decoction on Cerebral Ischemic Stroke Mice

<div><p>Stroke is the second-leading cause of death worldwide, and tissue plasminogen activator (TPA) is the only drug used for a limited group of stroke patients in the acute phase. Buyang Huanwu Decoction (BHD), a traditional Chinese medicine prescription, has long been used for improving neurological functional recovery in stroke. In this study, we characterized the therapeutic effect of TPA and BHD in a cerebral ischemia/reperfusion (CIR) injury mouse model using multiplex proteomics approach. After the iTRAQ-based proteomics analysis, 1310 proteins were identified from the mouse brain with <1% false discovery rate. Among them, 877 quantitative proteins, 10.26% (90/877), 1.71% (15/877), and 2.62% (23/877) of the proteins was significantly changed in the CIR, BHD treatment, and TPA treatment, respectively. Functional categorization analysis showed that BHD treatment preserved the integrity of the blood–brain barrier (BBB) (Alb, Fga, and Trf), suppressed excitotoxicity (Grm5, Gnai, and Gdi), and enhanced energy metabolism (Bdh), thereby revealing its multiple effects on ischemic stroke mice. Moreover, the neurogenesis marker doublecortin was upregulated, and the activity of glycogen synthase kinase 3 (GSK-3) and Tau was inhibited, which represented the neuroprotective effects. However, TPA treatment deteriorated BBB breakdown. This study highlights the potential of BHD in clinical applications for ischemic stroke.</p></div

Effects of BHD on neurological deficits and brain function in mice after cerebral ischemic/reperfusion (CIR) injury.

<p>(A) Typical animal-tracking profiles within 3 min for the evaluation of neurological deficits. (B) Representative micro-PET analysis of brain function (glucose metabolism) in live mice at 24 h after stroke. Animal groups include sham, vehicle-treated animals (ischemic stroke, CIR), BHD-treated animals (CIR+BHD; 1.0 g/kg, p.o., twice daily), and TPA-treated animals (CIR+TPA; 10 mg/kg, i.v. at day one), with treatment administered 2 h after ischemic stroke. The experiment was repeated at least 3–5 times, with similar results. #<i>p</i> < 0.05 compared with the sham group; *<i>p</i> < 0.05 compared with the CIR group.</p

Neuroprotective effects of BHD on the GSK-3 pathway.

<p>(A) BHD treatment significantly increased the phosphorylation level of PKCs, Akt, CaMKII and GSK-3; thus, inhibiting Tau phosphorylation yielded neuroprotective activity. (B) Quantitative analysis of the phosphorylated proteins was mentioned above. The values represent the ratio of the treatment group compared with the sham group, using α-tubulin as a loading reference. Data are presented as the mean ± S.D. Each protein expression with triplicates was statistically analyzed using Student <i>t</i>-test. *<i>p</i> < 0.05 compared with CIR/Sham.</p

Validation of the selected proteins by western blotting.

<p>(A) The levels of upregulated proteins (Cntn1, Actn1, Mag, Aplp1 and Gap43) and downregulated protein (Bdh) affected by CIR were recovered to the basal situation in the BHD-treated group (compared with the sham group). (B) Quantitative analysis of the selected proteins mentioned above. The values represent the ratio of the treatment group compared with the sham group, using α-tubulin as a loading reference. (C) The histogram indicates the ratio of the proteins selected in the iTRAQ experiment. (D) The levels of upregulated proteins (Alb and Gdi1) and downregulated proteins (Grm5 and Gfap) affected by CIR were reversed in the BHD-treated group (compared with the sham group). (E) Quantitative analysis of the selected proteins mentioned above. The values represent the ratio of the treatment group compared with the sham group, using β-actin as a loading reference. Data are presented as the mean ± S.D. Each protein expression with triplicates was statistically analyzed using Student <i>t</i>-test. *<i>p</i> < 0.05 compared with CIR/Sham.</p

Gene-ontology-based functional categorization of proteins that were significantly changed (fold change > 1.3) by CIR/sham, CIR+TPA/sham and CIR+BHD/sham treatments.

<p>(A) Biological Process, (B) Cellular Component, and (C) Molecular Function.</p

Significant changes in the protein ratios in CIR/sham, CIR+TPA/sham and CIR+BHD/sham groups according to the changes of at least 1.3-fold (<i>p</i>-value < 0.05).

<p>Significant changes in the protein ratios in CIR/sham, CIR+TPA/sham and CIR+BHD/sham groups according to the changes of at least 1.3-fold (<i>p</i>-value < 0.05).</p

Summary of the overall findings of the iTRAQ-based proteomics analysis.

<p>BHD treatment significantly preserved the integrity of the BBB, suppressed glutamate excitotoxicity, and improved the energy metabolism. Mechanistically, BHD enhanced kinase activity (Akt, PKC, and CaMKII), thereby inhibiting GSK-3 and Tau activity, which suggests a neuroprotective effect.</p