Emodin Inhibits Homocysteine-Induced C-Reactive Protein Generation in Vascular Smooth Muscle Cells by Regulating PPARγ Expression and ROS-ERK1/2/p38 Signal Pathway

Atherosclerosis is an inflammatory disease. As an inflammatory molecule, C-reactive protein (CRP) plays a direct role in atherogenesis. It is known that the elevated plasma homocysteine (Hcy) level is an independent risk factor for atherosclerosis. We previously reported that Hcy produces a pro-inflammatory effect by inducing CRP expression in vascular smooth muscle cells (VSMCs). In the present study, we observed effect of emodin on Hcy-induced CRP expression in rat VSMCs and molecular mechanisms. The in vitro results showed that pretreatment of VSMCs with emodin inhibited Hcy-induced mRNA and protein expression of CRP in a concentration-dependent manner. The in vivo experiments displayed that emodin not only inhibited CRP expression in the vessel walls in mRNA and protein levels, but also reduced the circulating CRP level in hyperhomocysteinemic rats. Further study revealed that emodin diminished Hcy-stimulated generation of reactive oxygen species (ROS), attenuated Hcy-activated phosphorylation of ERK1/2 and p38, and upregulated Hcy-inhibited expression of peroxisome proliferator-activated receptor gamma (PPARγ) in VSMCs. These demonstrate that emodin is able to inhibit Hcy-induced CRP generation in VSMCs, which is related to interfering with ROS-ERK1/2/p38 signal pathway and upregulating PPARγ expression. The present study provides new evidence for the anti-inflammatory and anti-atherosclerotic effects of emodin.


Introduction
Cardiovascular diseases resulting from atherosclerosis are the leading cause of mortality and morbidity all over the world. In traditional views, atherosclerosis is considered as a progressive narrowing of the artery lumen due to hyperlipidemia. Nevertheless, epidemiological analysis reveals that atherosclerosis still occurs in patients with normal or low cholesterol level [1]. In the recent years, a growing number of evidence from laboratory and clinical studies show that inflammation plays a pivotal role in all phases of atherogenesis, and therefore support that atherosclerosis is a chronic inflammatory disease [2,3].
Among the various inflammatory molecules, C-reactive protein (CRP) is a representative inflammatory cytokine. CRP is not only considered as the most reproducible marker to predict cardiovascular risk, but also accepted as an independent risk factor for cardiovascular events [4]. Moreover, an increasing number of data suggest that CRP directly participates in the initiation and progression of atherosclerosis through multiple activities in the inflammatory response [5].
Elevated plasma homocysteine (Hcy) level is an independent risk factor for atherosclerosis [6,7], and hyperhomocysteinemia is estimated to account for 10% of cardiovascular events [8]. Data from cell culture, animal experiments and clinical investigations suggest that Hcy appears to promote atherosclerosis formation by the pleiotropic effects including pro-inflammation [9]. Our previous research confirmed that Hcy is able to stimulate CRP expression in rat vascular smooth muscle cells (VSMCs) via reactive oxygen species (ROS) and mitogen activated protein kinase (MAPK) signal pathway [10]. Moreover, the locally generated CRP is anticipated to intensify the inflammatory process in the vessel wall and thus contribute to atherogenesis [11].
Emodin, 1, 3, 8-trihydroxy-6-methylanthraquinone, is a natural active ingredient found in many Chinese herbs, such as Rheum officinale, Rheum palmatum and Polygonam cuspidatum. In addition to its traditional usage as a laxative, emodin exhibits various biological activities including anti-cancer, anti-bacterial, anti-oxidant, and anti-inflammatory effects [12]. Meng et al. report that emodin inhibits TNF-α-induced expressions of MMP-2 and MMP-9 in VSMCs to produce an anti-inflammatory effect [13]. Despite the report, there is no direct evidence to prove that emodin exerts an anti-inflammatory effect through inhibition of CRP production in vascular cells. Therefore, the present study observed the inhibitory effect of emodin on Hcy-stimulated CRP expression in VSMCs, and probed the roles of ROS-MAPK signal pathway and peroxisome proliferator-activated receptor gamma (PPARγ) in the effect so as to provide a new evidence for its anti-inflammatory and anti-atherosclerotic effects.

Culture of rat aortic smooth muscle cells
VSMCs derived from the thoracic aorta of male Sprague-Dawley (SD) rats according to Hadrava's method [14] were cultured in DMEM supplemented with 100 U/ml penicillin, 100 U/ml streptomycin and 10% fetal bovine serum (FBS)in the condition of 5% CO 2 and 95% air at 37°C. The cells between passages 3 and 8 were used in the experiments. Before the experiments, the cells was starved for 24 h in DMEM containing 0.1% FBS. All the experimental procedures were approved by the Institutional Animal Care Committee of Xi'an Jiaotong University.

Assessment of the cell viability
The viability of VSMCs was examined by the MTT method [10]. VSMCs seeded in a 96-well plate at a concentration of 10 4 cells/well were incubated for 24 h with 0.1, 1, 10, 100 μM emodin or DMEM for control. Then, 20 μl MTT (5 mg/ml) was added to each well for 4 h's further incubation at 37°C. After that, the culture medium was removed and the formazan crystal was completely dissolved by 150 μl DMSO. Finally, formazan absorbance was assessed at 490 nm with an ELISA-reader (Thermo, NH, USA)

Measurement of ROS
H 2 DCF-DA fluorescent probes were used to detect the intracellular ROS in VSMCs [15]. VSMCs incubated with the different concentrations of emodin (0.1, 1, 10 μM) for 24 h were exposed to Hcy (100 μM) for 40 min after preloaded with H 2 DCF-DA (10 μM) for 30 min at 37°C. The fluorescence was detected by a fluorescence microscope (Olympus BX51, Japan) at the excitation wavelength of 488 nm and the emission wavelength of 525 nm.

Methionine diet-induced hyperhomocysteinemic model
Male SD rats aged 8 weeks from Experimental Animal Center of Xi'an Jiaotong University School of Medicine were divided into control group, hyperhomocysteinemic model group and two emodin-treated groups. Rats in control groups were maintained with normal diet, while rats in the model group and emodin-treated groups were fed with diet contained 2% methionine for 4 weeks [16]. Meanwhile, emodin was orally administrated to rats in emodin-treated groups at the dose of 40 or 80 mg/kg per day for 4 weeks. At the end of the experiment, blood samples were collected via the abdominal aorta, and serum was separated for detecting Hcy and CRP levels. Then, the thoracic aorta of rats was removed for analyzing mRNA expression of CRP with RT-PCR, and detecting protein expressions of CRP, PPARγ, ERK1/2, p38, p-ERK1/2 and p-p38 by Western blot. Hcy concentration was examined by a commercial available kit based on the enzyme cycling method (Beijing Strong Biotechnologies, Beijing, China), and CRP levels were assayed by ELISA.

Statistical analysis
All values were shown as mean ± S.E.M. Statistical significance between groups was assessed by one-way ANOVA followed by Tukey's test. P < 0.05 was considered statistically significant.

Effect of emodin on the viability of VSMCs
In order to choose the proper concentrations of emodin used for the in vitro study, effect of emodin on the viability of VSMCs was observed.   inhibited Hcy-induced mRNA and protein accumulation of CRP in VSMCs in a concentration-dependent manner (P < 0.05 vs. Hcy alone). Inhibition of CRP was 34.9%, 53.3%, 70.7% for mRNA expression, and 11.9%, 38.5%, 71.8% for protein expression, respectively. In addition, emodin or DMSO (solvent control) alone did not significantly change the basal CRP expression in VSMCs.

Effect of emodin on Hcy-stimulated intracellular ROS generation in VSMCs
As shown in Fig 2C and 2D, there was a basal DCF fluorescence in control VSMCs, which represented ROS generation. Exposure of the cells to 100 μM Hcy for 40 min resulted in a significant raise of DCF fluorescence with 8.8 times over control (P < 0.05 vs. control). After preincubation of the cells with emodin at 0.1, 1 and 10 μM, Hcy-stimulated ROS generation was markedly diminished (P < 0.05 vs. Hcy alone), and inhibitory rate was 28.9%, 81.4% and 60.9% respectively.

Effects of emodin on Hcy-regulated MAPK phosphorylation and PPARγ expression in VSMCs
Our previous study found that Hcy is capable of activating ERK1/2 and p38, which mediate Hcy-induced CRP expression in VSMCs [10]. In the present study, effect of emodin on Hcyinduced activation of ERK1/2 and p38 was assessed by Western blot analysis. The results in Fig 3A and 3B illustrated that Hcy facilitated phosphorylation of ERK1/2 and p38 in VSMCs (P < 0.05 vs. control), while emodin ameliorated Hcy-activated phosphorylation of ERK1/2 and p38 (P < 0.05 vs. Hcy alone).
The result in Fig 3C revealed that incubation of the cells with 100 μM Hcy for 12 h reduced PPARγ expression (P < 0.05 vs. control). Compared with Hcy alone, pretreatment of the cells with emodin for 24 h antagonized the inhibition of PPARγ expression by Hcy in a concentration-dependent manner (P < 0.05 vs. Hcy alone).

Effects of emodin on serum Hcy and CRP in hyperhomocysteinemic rats
As seen from Fig 4A and 4B, serum Hcy and CRP levels were significantly increased after feeding of the high methionine diet to rats for 4 weeks (P < 0.05 vs. control), whereas simultaneous administration of emodin to hyperhomocysteinemic rats decreased serum CRP concentration (P <0.05 vs. model) and but, did not change serum Hcy level.
Effect of emodin on CRP expression in the thoracic aorta of hyperhomocysteinemic rats

Effects of emodin on MAPK phosphorylation and PPARγ expression in the thoracic aorta of hyperhomocysteinemic rats
The results from the in vivo experiment showed that ERK1/2 and p38 phosphorylation was increased, and PPARγ protein expression was decreased in the thoracic aorta of hyperhomocysteinemic rats (P < 0.05 vs. control). Compared with model rats, p38 phosphorylation, but not ERK1/2, was decreased, and PPARγ expression was increased in the thoracic aorta of emodin-treated rats (Fig 5, P < 0.05 vs. model group).

Discussion
It has been widely accepted that atherosclerosis is a chronic inflammatory disease in the vessel wall, and anti-inflammatory therapy is a new strategy for prevention and treatment of atherosclerosis [17]. As one of the common risk factors for cardiovascular diseases, hyperhomocysteinemia is associated with formation of atherosclerosis. We previously reported that Hcy produces a pro-inflammatory effect on VSMCs by inducing CRP expression [10]. It is known that CRP participates in initiation and progression of atherosclerosis through modulating the activities and expressions of multiple factors implicated in atherogenesis [5]. In the present study, we observed the effect of emodin on Hcy-induced CRP production in protein and mRNA levels in VSMCs. The results showed that pretreatment of the cells with emodin inhibited Hcy-induced mRNA and protein expression of CRP in a concentration-dependent manner in vitro. The results from the in vivo experiment revealed that emodin not only inhibited CRP expression in the vessel wall, but also reduced the circulating CRP level in hyperhomocysteinemic rats. These confirm that emodin is able to inhibit CRP production in VSMCs and may thus alleviate the vascular inflammation. The in vivo experiment also exhibited that emodin did not significantly decrease serum Hcy level of hyperhomocysteinemic rats, suggesting that the inhibitory effect of emodin on Hcy-induced CRP expression is not resulted from interfering with Hcy metabolism.
It is reported that ROS mediate Hcy-induced CRP expression in VSMCs [10], and emodin exerts an anti-oxidant property [18,19]. The further study displayed that emodin diminished Hcy-stimulated ROS generation in VSMCs. This implicates that emodin reduces Hcy-induced CRP production possibly via inhibition of ROS generation elicited by Hcy in VSMCs.
MAPK signaling plays a pivotal role in the inflammatory process of atherosclerosis. Hcy is capable of activating MAPK [20], which is crucial for Hcy-induced CRP production in VSMCs [10]. The present results showed that emodin attenuated Hcy-activated phosphorylation of ERK1/2 and p38 in VSMCs in vitro, which is consistent with Zhu's study conducted in TGF-β1-stimulated NRK-49F cells [21]. However, emodin only inhibited p38 phosphorylation, and did not significantly affect ERK phosphorylation in the thoracic aorta of hyperhomocysteinemic rats in vivo. The mechanism underlying the difference is unclear and merits further investigation. Existing evidence indicates that ROS serve as an upstream event of MAPK signaling, and Hcy activates ERK1/2 and p38 through stimulated ROS production in VSMCs [10]. In combination of the present results with known reports, it is inferred that emodin inhibits Hcyactivated phosphorylation of ERK1/2 and p38 via reducing ROS generation in VSMCs.
Activation of NF-κB is responsible for the expression of many inflammatory cytokines including CRP. We previously demonstrated that NF-κB plays a pivotal role in Hcy-induced CRP expression in VSMCs [10]. Furthermore, it has been demonstrated that emodin is able to inhibit the activation of NF-κB [22,23]. Therefore, it is concluded that emodin may decrease CRP expression via inhibiting Hcy-stimulated NF-κB activation in combination of our previous results with reports from others.
PPARγ is a nuclear hormone receptor, and plays a beneficial role in the regulation of vascular inflammation [24,25]. PPARγ agonists have been demonstrated to exert the protective effects against Hcy-mediated atherogenesis [26]. Zhou et al. report that emodin induces PPARγ expression in atherosclerotic plaque and promotes the plaque stability in fat-fed apolipoprotein E-deficient mice [27]. However, whether emodin regulates PPARγ expression in VSMCs remains unknown. Therefore, we observed the effect of emodin on PPARγ expression in VSMCs. The results found that emodin upregulated Hcy-inhibited PPARγ expression in VSMCs in vivo and in vitro, implicating that emodin also exerts an anti-inflammatory effect by PPARγ. However, more experiments are needed to clarify how emodin upregulates PPARγ expression and the crosstalk between PPARγ and ROS-MAPK signal pathway.

Conclusion
The present study demonstrates that emodin is able to inhibit Hcy-induced CRP generation in VSMCs, which is related to interfering with ROS-ERK1/2/p38 signal pathway and upregulating PPARγ expression. These provide new evidence for the anti-inflammatory and anti-atherosclerotic effects of emodin.