**1. Introduction**

Atherosclerosis is defined as a chronic vascular inflammatory disorder that progresses with the lipid oxidation due to hypercholesteremia, diabetes mellitus, hypertension and various other disorders [1]. Oxidized lipids induce the secretion of various cytokines and recruit macrophages and T-lymphocytes at the site of a lesion [2]. Further, accelerated vascular smooth muscle cell (VSMC) migration and proliferation contribute to atherosclerotic plaque development [3,4]. It is also stimulated by oxidative stress, which produces different inflammatory cytokines; tumor necrosis factor-A(TNF-A), interleukin-6 (IL-6) and growth factor such as platelet-derived growth factor-BB (PDGF-BB). According to the previous study, treatment of IL-6 to C57Bl/6 mice increased fatty streak cores by approximately five times as revealed by oil red o staining of aortic sinus serial section, and increased the release of inflammatory cytokine, IL-1β and TNF-Ain the plasma [5]. Moreover, TNF-Aand PDGF-BB are already reported to stimulate the migration of human aortic VSMC from media to the intima of blood vessels [6]. These migrated cells are extensively proliferated under the influence of inducing agents like PDGF, TNF-Aand lipopolysaccharide (LPS) in

**Citation:** Jun, M.Y.; Karki, R.; Paudel, K.R.; Panth, N.; Devkota, H.P.; Kim, D.-W. Liensinine Prevents Vascular Inflammation by Attenuating Inflammatory Mediators and Modulating VSMC Function. *Appl. Sci.* **2021**, *11*, 386. https://doi.org/10.3390/app11010386

Received: 13 December 2020 Accepted: 30 December 2020 Published: 3 January 2021

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the intimal layer of arteries forming atheroma [7–9]. Mitogen-activated protein kinases (MAPKs), a family of serine-threonine kinases, regulate cell adhesion, migration and proliferation on human aortic VSMC in response to external stimuli including TNF-A [10] and PDGF-BB [11,12]. The role of matrix metalloproteinase (MMP) is well known due to their function for disrupting matrices composed of gelatin or elastin, which could permit human aortic VSMC migration by destroying the elastic lamina present between the intima and media [13,14]. Vascular inflammation is a result of toxic insult by the mediators released by the macrophage. Initially, monocytes normally circulating in the blood vessel are migrated to tunica media due to endothelial dysfunction. At this site, they engulf the oxidized lowdensity lipoprotein (ox-LDL) and become activated after changing the morphology from macrophage to foam cell, as recognized by the accumulation of fatty streaks on lipid laden molecules [15,16]. Activated macrophages release inflammatory mediators like nitric oxide (NO) via the inducible nitric oxidase pathway, and prostaglandins via cyclooxygenase pathway. Collectively, these endogenous inflammatory agents trigger the formation of a necrotic core at the site of an atherosclerosis lesion [17,18].

Liensinine is a bisbenzylisoquinoline alkaloid found in various part of the lotus (*Nelumbo nucifera* Gaertn.) including seeds (Figure 1). Liensinine and other bisbenzylisoquinoline alkaloids present in lotuses are reported as potent anticancer, anti-inflammatory, antioxidant, cardiovascular protective and neuroprotective agents [19–21]. Traditionally, the seed embryo of the lotus has been used as medicine in China for cardiovascular diseases, nervous disorders and sleeplessness [22]. Previously, we reported the anti-atherosclerotic activity of *Nelumbo nucifera* leaf extract and its alkaloid rich fraction through inhibition of neointimal hyperplasia in rats and inhibiting VCMC proliferation and migration [12,23]. In the current study, we aim to determine the similarly beneficial activity of the liensinine against vascular inflammation through anti-inflammatory, anti-proliferative, anti-migratory and anti-oxidative activities.

**Figure 1.** The flower and seeds of *Nelumbo nucifera* and the chemical structure of liensinine.

### **2. Materials and Methods**
