## 4 Discussion  
In our study, we have shown that liensinine inhibits the key features of vascular inflammation mediated by altered VSMC function due to PDGF and TNF-\(\alpha\), and macrophage function by LPS (Figure 7). During vascular inflammation, toxic insults to the blood vessel wall are mediated by oxidative stress, lipid peroxidation, and inflammation mediators released by VSMC and activated macrophage facilitates atherosclerosis progression [29; 30]. Under stressful conditions, our bodies generate free radicals such as superoxide anion, which in turn convert NO to peroxpiritrite. Peroxynitrite facilitates the oxidative modification of cholesterol to produce enormous quantities of lipid peroxidation byproducts [18]. Blood/serum lipids such as low-density lipoprotein (LDL) are involved in the progression/pathogenesis of numerous diseases including atherosclerosis. Oxidized-LDL upregulates the scavenger receptors on macrophages followed by the increased engulfment of ox-LDL and conversion of macrophages to foam cells characterized by accumulation of fatty streaks [31; 32]. Therefore, pharmacological intervention inhibiting serum lipid peroxidation can slow down the process of vascular inflammation. We have previously shown that alkaloid rich fractions of _Nelumbo uncifera_ possess strong antioxidant activity and suppress restenosis in a rat model [12]. As liensinine is one of the major alkaloids presents in _Nelumbo uncifera_, we sought to investigate if an antioxidant effect is exerted by liensinine. In our antioxidant activity assay, liensinine showed potent activity as revealed by scavenging the DPPH free radical with IC\({}_{50}\) of 1.8 \(\upmu\)g/mL (Figure 2a) and significantly inhibiting serum lipid peroxidation with 30 and 40 \(\upmu\)g/mL concentrations of liensinine (Figure 2b). The DPPH antioxidant activity of liensinine was even better than another major alkaloid, neferine, with IC\({}_{50}\) of 10.665 \(\upmu\)g/mL (17.01 \(\upmu\)M) [33]. Figure 7: The mechanism of action of liensinine to inhibit vascular inflammation: During the progression of vascular inflammation, low density lipoprotein (LDL) is oxidized to ox-LDL by free radicals and these ox-LDL are engulfed/phagocytized by macrophages. The macrophage converts its phenotype to activated foam cells distinguished by the accumulation of fatty streaks of ox-LDL. These activated macrophages release numbers of inflammation mediators such as nitric oxide (NO), TNF-\(\alpha\), inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2). Similarly, the intact vascular smooth cells (VSMC) in the tunica media are activated by cytokines such as TNF-\(\alpha\) and growth factor such as PDGF. VSMCs release cytokines such as IL-6 and initiate proliferation mediated by PDGF and migration mediated by matrix metalloproteinase-9 (MMP-9) enzymatic activity at the site of a lesion. Collectively, this process leads to vascular inflammation flowed by atherosclerosis. In physiological systems, atherogenesis is initiated in major arteries after endothelial dysfunction triggered by oxidative stress, leading to significant changes in the permeability of the vascular intimal layer and resulting in transportation of ox-LDL to the vascular inner layer [34]. After endothelial cells are activated by atherogenic risk factors such as ox-LDL, they overexpress cell adhesion molecules such as intercellular adhesion molecules and vascular cell adhesion molecules to attract circulating cells including monocytes and leukocytes. The transported ox-LDL are engulfed by scavenger receptors of macrophages/monocytes [35]. Macrophages are activated after phagocytosis of ox-LDL and in turn overexpress iNOS and COX-2 [17; 36]. It is well established that high iNOS level corresponds with a massive release of NO from macrophages and subsequent inflammatory response [37]. Similarly, LDL also stimulates the production of various prostaglandandins through the COX-2 pathway, and these prostaglandandins are known to be mitogenic, simulating cell proliferation [38].