**4. Discussion**

*H. sabdari*ff*a* Linne (*Malvaceae*), an attractive plant believed to be native to African countries, is cultivated in both Southern and Eastern Taiwan [26]. The calyces of the plant are typically used in foods and beverages, such as jam, jellies, and teas [15,26]. Previous studies have shown that various extracts of calyces of *H. sabdari*ff*a* L., including *H. sabdari*ff*a* aqueous extracts (HSEs), *H. sabdari*ff*a* anthocyanins (HAs), and its polyphenol-rich extracts (HPEs), have been reported to exhibit a wide variety of activities against hypertension, inflammation, liver disorders, diabetes, cancer, atherosclerosis, and other metabolic syndromes [26]. While the focus has been on the calyx, the leaves of this plant are also consumed as a leafy vegetable in many countries [15]. The *Hibiscus* leaf has been also reported to exert many biologic e ffects, including antioxidant [13,15], anti-hyperlipidemic [13,14], anti-cancer [17,18], anti-atherosclerotic [11,16], and anti-inflammatory [15] activities, as shown in Table S1. Our recent studies have indicated that HLP, a methanol extract of *Hibiscus*leaf, is rich in polyphenols [16], including ECG and other polyphenols (Cat, EA, Que, and FA; Table S2). In the literature, ECG-enriched HLP exhibited to inhibit ox-LDL uptake and lipid-laden foam cell formation, promoted cholesterol e fflux [16], and reduced ox-LDL-mediated endothelial cell injury and apoptosis [11], so HLP was expected to have potential as an anti-atherogenic agent. ECG, one of the major tea catechins, plays an important protective role in the cardiovascular system, and has been reported to possess anti-atherogenic properties in in vitro and in vivo studies [27]. It has been shown that the anti-atherosclerotic e ffect of Cat is associated with their antioxidant, anti-hypertensive, hypolipidaemic, and anti-mutagenic effects [28]. These Cat have been indicated to suppress the LDL oxidation and the foam cell formation in in vivo atherosclerotic lesions [27], and MMP-2 activities in cell culture supernatant of pulmonary VSMC [29]. Previous studies have indicated that EA, a polyphenolic compound present in berries, scavenged free radicals and improved lipid peroxidation [30]. According to the past and present works, the findings cooperatively show the anti-atherosclerotic activities of HLP may be contributed by their biological properties of these polyphenolic components.

In the comparison of components between both extracts, polyphenolic extracts from flowers (HPE) and leaves (HLP) of *H. sabdari*ff*a* L., the total flavonoid content of HPE and HLP was estimated to near to 20% and 75%, respectively, via Jia method [16,22]. The results show the polyphenolic extract of *Hibiscus* leaves exhibited an about 3.8-fold content of flavonoids compared to that of its flowers. In addition to the above, HLP seems to possess stronger protective properties from VSMC dysfunction than HPE [22], including the inhibitory e ffects on MMP expression and cell migration (Figures 2 and 4). Huang et al. demonstrated that HPE at the doses of 0.01 and 0.10 mg/mL reduced high glucose-stimulated cell migration about 30% and 80%, respectively, by measuring the wound-healing assay [22]. In this study, above the dose of HLP at 0.01 mg/mL could completely reverse the TNFα-increased proportion of cell migration (Figure 4a). These data sugges<sup>t</sup> that HLP could exert the anti-migratory e ffect at lower doses than HPE.

Atherosclerosis is a multistep and chronic inflammatory process that involves interactions between various soluble mediators, endothelial cells, monocytes, and VSMCs. Monocyte-derived growth factors and cytokines further a ffect the vascular wall by stimulating VSMC migration and proliferation [31]. The inflammatory cytokine TNFα has been shown to play a vital role in the disruption of the vascular circulation, and its increased expression induces the production of ROS, resulting in endothelial cell injury and VSMC dysfunction [32]. The blockade of TNFα has been demonstrated to improve cardiovascular morbidity and mortality in chronic inflammatory disease [33]. The model of TNFα-stimulated VSMCs has been applied to mimic the VSMC dysfunction during atherosclerotic development [31]. Therefore, the atheroprotective e ffects of HLP were investigated in a model of VSMCs exposed to TNFα in vitro. The extract, at a concentration in a range of 0.01–0.10 mg/mL, possessed inhibitory e ffects on VSMC migration, as evidenced by the results of the decreased activities and expressions of MMPs, the levels of key migratory proteins, and the wound-healing and invasive abilities of VSMCs (Figures 1d and 2, Figures 3 and 4). The e ffect of the higher concentrations (>0.10 mg/mL) showed that it repressed cell growth and DNA synthesis, as well

as enhanced cell apoptosis (Figure 5). Similarly, Won et al. reported that the mechanisms of catechins action against cardiovascular diseases include the inhibition of VSMC proliferation [34]. Further studies have also indicated that many polyphenols prevented atherosclerosis through inhibiting proliferation and/or inducing apoptosis of VSMC [35]. The findings of this study reveal, for the first time, the protective effects of HLP on this model, examined in each test, provide bifunctional results of HLP. Therefore, it is convincing that HLP could potentially be used in the treatment of atherosclerosis.

As mentioned above, the migration of VSMCs from medial to intima contributes to the formation of atherosclerosis [2]. MMPs, a family of proteinases, promote ECM degradation, which, in turn, facilitates cell migration. Activated Akt induced AP-1, which is required for the production of MMPs [36]. However, upon TNF-α stimulation, the mechanism(s) mediating MMP activation and the MMP-regulated downstream signals have ye<sup>t</sup> to be clarified. TNF-α promoted VSMC chemotaxis via Akt and MAPK activation, as reported by Chan et al. [31]. The Akt antagonist PTEN was also shown to be involved in VSMC migration [37]. Consistent with previous reports, this study confirmed that it is therefore possible that HLP inhibits TNF-α-stimulated MMP-9 activation by downregulating Akt/AP-1 pathway, and subsequently prevents VSMC migration. Furthermore, previous studies have indicated that oxidative stress modulated by ROS promotes VSMC dysfunction developing atherosclerosis and also induces MMP-mediated ECM remodeling and cell cycle progression [6,7,38]. In this study, TNF-α increased the production of intracellular ROS, especially of the H2O2 level, and this might influence the overall signaling pathways of VSMC migration/proliferation. In this regard, HLP attenuated ROS generation against TNF-α stimulation (Figure 7), and it might contribute to MMP inhibition and cell cycle regulation by HLP. Further investigations are needed to clarify this issue. Consistent with previous reports and our past results, as shown in Table S4, this study confirmed that HLP possesses strong antioxidant ability in inhibiting the VSMC dysfunction and atherosclerotic development.

Next, to study the mechanism(s) of HLP-inhibited VSMC proliferation, the regulation of cell cycle arrest was examined. VSMCs begin to divide in response to mitogens and enter the S phase upon vascular injury [9]. Cdks, working in conjunction with their activating subunits (cyclins), provide the driving force for cell cycle transitions [34]. The kinase activities of these cyclin/cdk complexes are regulated by cdi, including Ink4 proteins (p16, p18, and p19) and Cip/Kip proteins (p21, p27, and p57), and their upstream factor, the gatekeeper of mammalian cell cycle, p53 [10,39]. As expected, HLP treatments induced a G0/G1 phase growth arrest by inducing the cki-mediated cell cycle regulation (Figure 6). In addition, PCNA expression is increased in unstable atherosclerotic plaque [40]; moreover, PCNA is highly expressed in human VSMCs [41]. Consistent with previous reports, our studies showed that TNF-α significantly reduced A7r5 cell growth and increased PCNA expression (Figure 6b). Further data demonstrate a marked and dose-dependent reduction in neointimal expression of PCNA after fed with HLP in HFD-treated rabbits (Figure 8e), indicating that HLP ameliorates atherosclerosis by reducing PCNA expression, both in vitro and in vivo.
