ITRAQ-Based Proteomics Analysis Reveals the Effect of Neoliensinine on KCl-Induced Vascular Smooth Muscle Contraction by Inhibiting Regulatory Light Chain Phosphorylation

Abstract

Smooth muscle (SM) contraction is one of the important physiological functions of the human body, and SM abnormal contraction will induce many diseases. The phosphorylated regulatory light chains (p-RLC) play a decisive role in SM contraction, and dephosphorylation of p-RLC is an effective way to relax SM. Our previous study showed that the novel benzylisoquinoline alkaloid, neoliensinine (Neo), could relax microvascular SM contracted by KCl hyperpolarization. In this study, mesenteric capillaries isolated from 45 mice were divided into normal tension group (Control), 124 mM KCl induced contraction model group (Model), and KCl and Neo-treatment group (Drug). The dephosphorylation levels of RLC in the three groups were measured. Compared with the model group, the phosphorylation of RLC in the drug group was decreased dramatically as expected, suggesting that the relaxation effect of Neo was caused by downregulating p-RLC of microvessel SM. In order to fully understand its fundamental mechanism, our research focused on the identification of target proteins in mice with KCl-induced contractile mesenteric capillary. Isobaric tags for relative and absolute quantification (ITRAQ) tagging was carried out by nanospray liquid chromatography–tandem mass spectrometry. The results allowed the upregulation of 164 differential abundance proteins (DAPs) among the 3,474 protein abundance disturbances identified from the model/control samples. Further comparison showed that there were 16 DAP convergences associated with vascular SM contraction between the drug/model and the drug/control samples. Among them, two proteins with known function, PLCβ and RhoGEF12, were selected as target proteins of the relaxation effect of Neo. The two selective target DAPs were verified by Western blot at protein level. The results suggested that changes of the two proteins were consistent with that of the iTRAQ results. Our present work reveals that Neo relaxes vascular smooth muscle via inhibition of RLC phosphorylation, and PLCβ and RhoGEF12 may be potential biomarkers for evaluating the effects mediated by Neo.

Evaluating the Therapeutic Role of Selected Active Compounds in Plumula Nelumbinis on Pulmonary Hypertension via Network Pharmacology and Experimental Analysis

“…In addition to the hyperproliferation of PASMCs, hypoxia-induced contraction of PASMCs is also one of the crucial factors leading to PH 32 . Recently study found that neoliensinine, a novel benzylisoquinoline alkaloid extracted from Plumula Nelumbinis, has the effect of relaxing vascular smooth muscle cells 33 . The above study results show that the anti-PH effect of Plumula Nelumbinis may also be related to the inhibition of excessive contraction of PASMCs.…”

Enrichment analysis of differentially expressed genes in chronic heart failure

“…This study also found that the vascular smooth muscle contraction pathway may play an essential role in the occurrence and progression of CHF. The vascular smooth muscle contraction pathway mediates hypertension, portal hypertension, and vascular endothelial injury (17,18). These pathological changes increase the risk of CHF to a certain extent.…”

Alkaloids from lotus (Nelumbo nucifera): recent advances in biosynthesis, pharmacokinetics, bioactivity, safety, and industrial applications

“…, in vitro cell experiments demonstrated that alkaloid rich fractions of lotus and neferine have potent inhibitory effects on vascular smooth muscle cell (VSMC) proliferation and migration by abolishing PDGF-Rb activation, dephosphorylation of extracellular signal-regulated kinase (ERK)1/2, inactivation of JNK/P38 MAPK, and downregulation of fractalkine gene expression(Jun et al 2016;Li, Tong, et al 2010;Zheng et al 2014). Through iTRAQ proteomics analysis, blocking phospholipase Cβ (PLCβ) and 145 multidomain guanine nucleotide exchange factor 12 (RhoGEF12) expression by activation of G protein-coupled receptor (GPCR) signaling may be responsible for neoliensinine-dependent vascular smooth muscle relaxation(Yang,Yan et al 2019). The protective effect of neferine (10 μM) on H9c2 cardiomyoblasts was demonstrated by reduced doxorubicin-induced apoptosis mediated by the regulation of the NADPH oxidase/ROS-mediated NF-κB redox and insulin-like growth factor (IGF)/Nrf2 signalling pathways(Bharathi Priya et al 2018;Priya et al 2017).…”