iTRAQ-Based Proteomics Analysis Reveals the Effect of Rhubarb in Rats with Ischemic Stroke

Abstract

Background. Rhubarb, a traditional Chinese medicine, promotes viscera and remove blood stasis. Rhubarb is skilled in smoothening meridians, improving blood circulation which exhibits better efficacy on cerebral ischemic stroke. In this study, we aimed to analyze the underlying mechanisms of rhubarb which treated rats of middle cerebral artery occlusion (MCAO) model according to an iTRAQ-based proteomics and bioinformatics analysis. 30 rats were randomly allocated into three groups including sham group (SG), model group (MG), and rhubarb group (RG). Rhubarb group was given a gavage of rhubarb decoction at dose of 3 g/kg and the remaining groups were prepared with normal saline by gavage. Rats from MG and RG were induced into MCAO model. The effects of rhubarb were estimated by Modified Neurological Severity Score (mNSS) and cerebral infarct volume. The brain tissues were measured via the quantitative proteomic approach of iTRAQ coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, the bioinformatics analysis of overlapping differentially expression proteins (DEPs) was conducted by DAVID, KEGG, and Cytoscape. Specific selective DEPs were validated by Western blotting. Rats treated with rhubarb after MCAO showed a significant reduction on mNSS and cerebral infarct volume compared with MG. In MG versus SG and RG versus MG, we identified a total of 4578 proteins, of which 287 were DEPs. There were 76 overlapping DEPs between MG versus SG and RG versus MG. Through bioinformatics analysis, 14 associated pathways were searched including cGMP-PKG signaling pathway, tuberculosis, synaptic vesicle cycle, amyotrophic lateral sclerosis, long-term potentiation, and so on. 76 overlapping DEPs mainly involved synaptic vesicle cycling biological processes based on GO annotation. Further, the selective overlapping DEPs were verified at the protein level by using Western blotting. Our present study reveals that rhubarb highlights promising neuroprotective effect. Rhubarb exerts novel therapeutic action via modulating multiple proteins, targets, and pathways.

Proteomic Assessment of iTRAQ-Based NaoMaiTong in the Treatment of Ischemic Stroke in Rats

“…Longao [10] modified method was established to establish MCAO cerebral ischemia reperfusion rat model: rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg/kg). After complete anesthesia, the rats were fixed in the supine position on the operating table.…”

“…Frozen cerebra were quickly removed and sectioned into 5 mm slices over ice after reperfusion for 6 h [10]. Brain sections were removed and the remaining brain tissue was frozen in liquid nitrogen and stored at -80°C prior to proteomic analysis.…”

“…Brain sections were removed and the remaining brain tissue was frozen in liquid nitrogen and stored at -80°C prior to proteomic analysis. The anterior and posterior of the brain slice were incubated in Tetrazolium Chloride (2% TTC) at 37°C for 20 mins [10]. Images of the infarct were taken and infarct volume was measured using an image analysis system.…”

Neuroprotective Effects of Anthraquinones from Rhubarb in Central Nervous System Diseases

“…Meanwhile, Li j et al [37] reported that rhubarb aglycone reduced thrombolysis-caused brain microvascular basement membrane impairment through decreasing the level of IgG and increasing the level of type IV collagen (CoLIV) and laminin (LN) and then decreased the intracranial hemorrhage ratio and mortality in cerebral ischemia rats. What is more, Xiangping Lin et al [38] found 287 differentially expression proteins (DEPs) and 76 overlapping DEPs between model group (MG) versus sham group (SG) and rhubarb group (RG) versus MG with iTRAQ-based proteomics analysis. There were 14 DEPs related pathways including synaptic vesicle cycle, cGMP-PKG signaling pathway, amyotrophic lateral sclerosis, long-term potentiation, tuberculosis, and so on.…”

Proteomic-Based Approaches for the Study of Ischemic Stroke

“…Ferulic acid exerts a neuroprotective effect in cerebral ischemia through its anti-oxidant and anti-inflammation activity. Koh In addition, proteomics analysis has been used to light on the mechanisms underlying pleiotropic actions of rt-PA [41] and the neuroprotection of traditional Chinese medicines (baicalin [42], Ginkgo biloba extract (EGb761) [43], curcumin [44], Danhong injection [45], quercetin [46], rhubarb [47], hydroxysafflor yellow A [48], Xiao-Xu-Ming decoction [49]), asialoEPO [50], melatonin [51], estradiol [52], PACAP38 [53], tetrandrine [54], simvastatin [55], resveratrol [56], PPARα agonist [57], tetramethylpyrazine nitrone [58], and grape seed extract [59] against ischemic injury).…”