**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 TNFα, 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 peroxynitrite. 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 nucifera* possess strong antioxidant activity and suppress restenosis in a rat model [12]. As liensinine is one of the major alkaloids presents in *Nelumbo nucifera,* 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 IC50 of 1.8 μg/mL (Figure 2a) and significantly inhibiting serum lipid peroxidation with 30 and 40 μg/mL concentrations of liensinine (Figure 2b). The DPPH antioxidant activity of liensinine was even better than another major alkaloid, neferine, with IC50 of 10.665 μg/mL (17.01 μM) [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), TNFα, 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-α 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 prostaglandins through the COX-2 pathway, and these prostaglandins are known to be mitogenic, simulating cell proliferation [38]. Our results showed promising activity of liensinine to suppress NO release from LPS-induced RAW264.7. The 25% reduction of NO by liensinine at a concentration of 20 μg/mL (Figure 6b) was comparable to 20 μM of neferine [33]. Furthermore, liensinine also notably decreased the protein expression of COX-2 and iNOS. The trend of iNOS and COX-2 inhibition shown by liensinine was similar to that shown by glucosamine (a commercially available anti-inflammatory drug) at a concentration of 2.5 to 10 mM in LPS-induced RAW264.7 cells [39].

IL-6 is a well characterized inflammatory mediator and it is released by VSMC after the induction of potent stimulants such as TNF-α [40] and angiotensin II [41]. IL-6 is crucial in vascular remodelling and it, along with ox-LDL, is a potential prognostic marker in predicting cerebral vascular and cardiovascular disorders [42]. In our ELISA result (Figure 5), liensinine suppressed the TNF-α induced IL-6 level in VSMC by approximately 50% at 30 μg/mL concentration. During atherosclerosis, the vascular lumen is narrowed by a fibrous cap composed of (among many other things) VSMC and extracellular matrix. Various growth factors and cytokines produced by endothelial cells and inflammatory cells contribute to the proliferation and migration of VSMC leading to fibrous cap formation [29,43,44]. Cytokines like PDGF can induce proliferation of VSMC to a significantly high level, whereas TNF-α is known to stimulate the migration of VSMC from tunica media to the site of a lesion by increasing MMP-9 expression. MMP-9 is a key gelatinolytic enzyme responsible for the degradation of the elastic lamina barrier of the extra cellular matrix [45,46]. In our previous publications, we have shown that PDGF and TNF-α promote the proliferation and MMP-9-dependent migration of VSMC [11,12,14]. In our result, liensinine inhibited the PDGF-BB induced proliferation/growth of VSMC, revealing its potent anti-proliferating activity. At a 30 μg/mL concentration of liensinine, the proliferation of VSMC was almost completely inhibited to the level of the control (without PDGF) (Figure 3). The potent anti-proliferative activity of liensinine at a concentration of 30 μg/mL is comparable to 50 μM of epigallocatechin-3-O-gallate [47] and 20mM of carnosine [48]. Likewise, liensinine at a concentration of 10, 20 and 30 μg/mL significantly attenuated the expression of MMP-9 induced by TNF-α (Figure 4). We speculate that the notable inhibition of MMP-9 enzymatic activity by *Nelumbo nucifera* leaf extract in our previous study, at a concentration of 250 μg/mL, was in part exerted by liensinine [23]. Taken together, our results provide the mechanistic pathway to attenuate the progression of atherogenesis via controlling vascular inflammation by liensinine possibly by targeting VSMC proliferation, MMP-9 expression and inflammatory mediators released by macrophages (Figure 7).
