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

Abstract

Background. NaoMaiTong (NMT) is widely used in the treatment of cerebral ischemia but the molecular details of its beneficial effects remain poorly characterized. Materials and Methods. In this study, we used iTRAQ using 2D LC-MS/MS technology to investigate the cellular mechanisms governing the protective effects of NMT. The transient middle cerebral artery occlusion (MCAO) rat model was established and evaluated. The degree of cerebral ischemia was assessed through scoring for nerve injury symptoms and through the assessment of the areas of cerebral infarction. Brain tissues were subjected to analysis by iTRAQ. High-pH HPLC and RSLC-MS/MS analysis were performed to detect differentially expressed proteins (DEPs) between the treatment groups (Sham, MCAO, and NMT). Bioinformatics were employed for data analysis and DEPs were validated by western blot. Results. The results showed that NMT offers protection to the neurological damage caused by MCAO and was found to reduce the areas of cerebral infarction. We detected 3216 DEPs via mass spectrometry. Of these proteins, 21 displayed altered expression following NMT intervention. These included DEPs involved in translation, cell cycle regulation, cellular nitrogen metabolism, and stress responses. Pathway analysis revealed seven key DEPs that were enriched in ribosomal synthesis pathways, tight junction formation, and regulation of the actin cytoskeleton. According to protein-protein interaction analysis, RPL17, Tuba, and Rac1 were affected by NMT treatment, which was validated by western blot analysis. Discussion. We therefore identify new pharmacodynamic mechanisms of NMT for the prevention and treatment of ischemic stroke. These DEPs reveal new targets to prevent ischemic stroke induced neuronal damage.

Integrated Analysis of the Cecal Microbiome and Plasma Metabolomics to Explore NaoMaiTong and Its Potential Role in Changing the Intestinal Flora and Their Metabolites in Ischemic Stroke

“…The sham, nimodipine, and NMT groups were treated intragastrically with either autoclaved water, with nimodipine (12.34mg/1 Kg dissolved in water), or NMT extractum (0.96 g/Kg, approximately equal to 3.085 g/Kg botanical drugs) daily for 7 days following surgery, respectively. According to the clinical dose, the dose of NMT was determined by the previous dose-effect relationship study ( Chen et al, 2012 ; Yang et al, 2017 ; Li et al, 2019 ). The weight of each rat was recorded daily.…”

“…NaoMaiTong (NMT), an effective treatment used clinically in IS for many years ( Li et al, 2019 ; Zhang W. et al, 2020 ), is a multi-botanical drug preparation consisting of emodin, aloe-emodin, 3′-Methyoxy puerarin, Ligustilide, Senkyunolide, Ginsenoside Rg1 etc. ( Fan et al, 2017 ).…”

Proteomic investigations of acute ischemic stroke in animal models: a narrative review

“…The results showed that disruption of brain–blood barrier integrity, inflammation, and dysregulated energy homeostasis contributed to ischemia-induced brain injury, and Buyang Huanwu Decoction treatment reversed levels of most inflammation and neurodegeneration-associated proteins including A2m, Serpinb5, Ces1c, Hspa1a, Jup, and Nptxr in the cerebrospinal fluid. [51] Li et al [52] identified 3216 proteins associated with MCAO in mice using iTRAQ-based proteomics, of which 21 were differentially expressed following NaoMaiTong treatment. Enrichment analysis indicated that NaoMaiTong treatment might exert its neuroprotective effects through modulation of three pathways: ribosome function (Rpl26, Rpl17, Rpl39, and Rps13), tight junctions, and regulation of actin cytoskeleton (Tuba, Wasl, and Rac1).…”

“…The expression changes in Rpl17, Tuba, and Rac1 were validated using western blot assay. [52] Proteomics results showed that the therapeutic effects of rhubarb, another Chinese traditional medicine, might result from modulation of various pathways such as cGMP-PKG signaling and the synaptic vesicle cycle via restoration of the expression levels of multiple proteins including Mapk1, Syn1, and Calm1 in MCAO rats. [53] Furthermore, Eftud2, mTOR, Rab11, Ppp2r5e, Hk1, and Eno2, which are associated with innate immune regulation, mTOR signaling, membrane trafficking, cell growth, and HIF-1 signaling pathways, were identified as key hub proteins underlying the effects of Hydroxysafflor Yellow A against ischemia-induced injury in rats using proteomic analysis.…”

“…Many biomarker candidates were reported in multiple studies, including A2m, Ahsg, Alb, Apoa1, Apoa4, cdc42, Ctsd, Gfap, Hpx, Mug1, Rhoa, S100b, Tf, and Ttr. [28,33,34,36,[41][42][43][44][45][49][50][51][52][53][54][55][56] Considering the complex physiological changes induced by ischemia, a panel of proteins might be the most effective AIS biomarker.…”