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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Pharmacol. | doi: 10.3389/fphar.2019.00979

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

 Guang-Ming Yang1, Ke Yan2, Peng Wang1, Jun-Li Zhang1,  Zi-Hao Pan1 and  Yang Pan1*
  • 1Nanjing University of Chinese Medicine, China
  • 2School of Pharmacy, Nanjing University of Chinese Medicine, China

Smooth muscle (SM) contraction is one of the important physiological functions of 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 model group, the phosphorylation of RLC in 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. ITRAQ (isobaric Tags for Relative and Absolute Quantification)-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 DAPs 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.

Keywords: vascular smooth muscle relaxation, Neoliensinine, Nelumbo nucifera GAERTN, regulatory light chain phosphorylation, iTRAQ

Received: 19 Feb 2019; Accepted: 31 Jul 2019.

Copyright: © 2019 Yang, Yan, Wang, Zhang, Pan and Pan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Yang Pan, Nanjing University of Chinese Medicine, Nanjing, China, ypan@njucm.edu.cn