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Artesunate attenuated progression of atherosclerosis lesion formation alone or combined with rosuvastatin through inhibition of pro-inflammatory cytokines and pro-inflammatory chemokines.

ABSTRACT

Backgrounds: Inflammation plays an important role in all stages of atherosclerosis, but little is known about the therapeutic effects of quenching inflammation in atherosclerotic lesions formation.

Purpose: Herein, the effect of artesunate, a derivant from artemisinin from the traditional Chinese herb sweet wormwood, could attenuate the progression of atherosclerosis lesion formation alone or combined with rosuvastatin in Western-type diet (WD) fed [ApoE.sup.-/-] mice, and explored its possible mechanisms.

Methods: The methods such as ELISA for plasma lipids and cytokines analyses, qRT-PCR and western blot for mRNA and protein expressions, and MTT assay for human umbilical vein endothelial cells (HUVECs) viability were used for in vivo and in vitro experiments.

Results: Artesunate could attenuate the progression of atherosclerosis lesion formation alone or combined with rosuvastatin in WD fed [ApoE.sup.-/-] mice without changes in food uptake, body weight and plasma lipids level, but with a significant reduction of pro-inflammatory cytokine, such as TNF-[alpha] and IL-6. Furthermore, artesunate could down-regulate the pro-inflammatory chemokines such as 1L-8 and MCP-1 in aorta of mice. Besides, artesunate didn't influence IL-8 and MCP-1 secretion in HUVECs up-regulated by TNF-[alpha], but inhibited IL-8 and MCP-1 secretion up-regulated by LPS.

Conclusion: AS attenuated progression of atherosclerosis lesion formation alone or combined with rosuvastatin through anti-inflammatory effect, resulting in down-regulation of TNF-[alpha] and IL-6, and further down-regulating IL-8 and MCP-1 expressions in aorta of WD fed [ApoE.sup.-/-] mice. Rosuvastatin combined with artesunate could more effectively attenuate the progression of atherosclerosis lesions than when treated by one of them, demonstrating that lipid-lowering agents combined with anti-inflammatory agents could provide the greater benefit for cardiovascular disease patients. Artesunate is worth further investigating as a candidate drug for the treatment of atherosclerosis.

Keywords:

Atherosclerosis

Artesunate

Inflammation

TNF-[alpha]

MCP-1

Introduction

Atherosclerosis is a chronic vascular disease which involves the progressive occlusion of blood vessels. It is characterized by sub-endothelial accumulation of inflammatory cells and lipids, which collectively contribute to occlusive disease or to less occlusive plaques at high risk for disruption (Ross R, 1999; Joana V and Oliver S, 2015).

Hypercholesterolemia is widely recognized as an important risk factor for atherosclerosis development. Animal studies have shown that reduced hypercholesterolemia strongly decreases the number of lesion macrophage foam cells within a few days. Later in time, a reduction of plaque size is observed (Kapourchali FR, et al, 2014). In humans, non-invasive imaging confirmed that plasma cholesterol-lowering alone can promote atherosclerosis regression (Bruckert E.et al, 2014). Statins, widely used for the treatment of atherosclerosis, are a group of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors based on their lipid-lowering properties via blocking cholesterol biosynthesis.

It is also confirmed that atherosclerotic plaque development is an inflammation-driven condition (Buttari B, et al. 2015). The host inflammatory response and resultant cellular and soluble mediator mobilization is critical in innate immune responses and crucial for host defense against infections. However, in the face of unrelenting persistent inflammation, the initiation, progression, and degenerative features of chronic diseases like native atherosclerosis or the vasculopathy of autoimmune disorders like rheumatoid arthritis may appear (Galkina E and Ley K, 2009; Tall AR, et al, 2015). One of the key early events in the pathogenesis of atherosclerosis is inflammation-triggered endothelial activation that leads to the attraction and adhesion of monocytes to the endothelium followed by their transmigration into the subendothelial space. This process is primarily mediated by several intracellular signaling events, leading to the elevated expression of a number of pro-inflammatory cytokines such as tumor necrosis factor-[alpha] (TNF-[alpha]), interleukin-6 (1L-6) and several pro-inflammatory chemokines such as interleukin-8 (IL-8) and monocyte chemoattractant rotein-1 (MCP-1) (Ramji DP and Davies TS, 2015; Drechsler M, et al. 2015).

In recent years, there has been growing interest on the anti-inflammatory effects of natural components existed in commonly used traditional herbs. Artemisinin is an active ingredient in sweet wormwood, a Traditional Chinese Medicine (Tu Y, 2011). Artemisinin and its derivates such as artesunate (AS), hydroartemisinin, artemether and arteether have been clinically used to treat malaria. AS is a water-soluble hemisuccinate derivative of dihydroartemisinin, and also possesses anti-cancer, anti-angiogenesis and anti-inflammatory activities in human rheumatoid arthritis fibroblast-like synoviocytes (Lai HC, et al, 2014; Wang Z, et al, 2012; Wang HY, et al, 2014.) In our previous experiments, we found AS could protect sepsis model mice challenged with a heat-killed E. coli and staphylococcus aureus via reduction of inflammation (Jiang W, et al, 2011; Li B, et al, 2010). On the other hand, the modulate atherosclerosis effects of AS have been reported previously such as via inhibition of type I IFN in systemic lupus erythematosus patients (Gu F, et al, 2014) or inhibition of STAT1 (Feng XB, et al, 2015).

As mentioned above, unrelenting persistent inflammation may result in the initiation and progression of atherosclerosis. Therefore, was considered whether it could attenuate the progression of atherosclerosis lesion formation through its anti-inflammatory effect. With these considerations in mind, we undertook the current study to investigate the effects of alone or combined with rosuvastatin in WD fed [ApoE.sup.-/-] mice, and its possible mechanisms.

Materials and methods

Materials

Injectable artesunate (AS) was purchased from Guilin Nanyao Ltd (Guangxi, China), no endotoxin was detected. Rosuvastatin calcium (Rosuvastatin) was purchased from AstraZeneca UK limited. Oil red stain and Movat's stain were purchased from Senbeijia Biological Inc (Nanjing, China). Enhanced chemiluminescence reagents were purchased from Pierce, Inc. (Thermo Fisher Scientific Inc., Rockford, IL, USA).

Mice ELISA kits for plasma lipids were purchased from Senbeijia Biological Inc (Nanjing, China). Mouse ELISA kits for TNF-[alpha] and IL-6 and human ELISA kits for MCP-1 and IL-8 were purchased from Boster Ltd (Wuhan, China). Avian myeloblastosis virus (AMV) reverse transcriptase and T4 polynucleotide kinase were purchased from Promega (Madison, WI, USA). Quantitative real-time PCR (qRT-PCR) Master Mix was purchased from ToYoBo Ltd (Osaka, Japan). Total Protein Extraction Kit was purchased from BestBio Ltd (Shanghai, China). Antibodies for western blot were all purchased from Santa Cruz Biotechnology, Inc (CA, US).

All primers were synthesized by Invitrogene Ltd (Shanghai, China). Cell culture medium and fetal bovine plasma were obtained from Invitrogen (Life Technologies Corporation, Carlsbad, CA, USA).

Mice

[ApoE.sup.-/-] mice lack the gene encoding apolipoprotein E (ApoE) and then spontaneously develops hypercholesterolemia, and atherosclerotic lesions similar to those found in human (Zhang S, 1992). Herein, C57BL/6J [ApoE.sup.-/-] mice were purchased from the Model Animal Research Center MARC at Nanjing University (body weight 16-20 g, 4-6 weeks old). All procedures were approved by the committee on Ethics of Animal Experiment of The Third Military University of Medicine, in accordance with the Guide for the Care and Use of Laboratory Animals.

Mice were housed individually in wire-bottomed cages in a temperature controlled room (22 [+ or -] 0.8 [degrees]C) with a 12 h light-dark cycle and a relative humidity of 55% [+ or -] 10%. All mice were fed with an atherogenic WD containing 21% calorie from fat and 0.2% cholesterol. Mice had an ad libitum access to food and water.

Mice treatment

Mice were randomly divided into six groups (6 mice/group): control group, rosuvastatin treatment group (as a positive control, 10mg/kg/day via intragastric administration), three treatment groups (1.5, 5 and 15 mg/kg/day, intramuscular injection), and combination group (5 mg/kg/day AS combined with 10 mg/kg/day rosuvastatin). The food intake, body weight were measured every day. Six months later, after an overnight abrosia, mice were anesthetized with an intraperitoneal injection of pentobarbital, plasma samples were collected from the orbital vein.

Herein, the doses of AS and rosuvastatin were converted from the clinical dosage regimens. 60 mg of AS is proposed to treat malaria in adult patients according to the drug conversion principle applied in pharmacology studies, conversion the dose of AS used from adult to mouse, 60 mg of AS in adults is the equivalence dose of about 15 mg/kg/day in mice. Therefore, 1.5, 5 and 15 mg/kg/day of AS were used in the present experiment. Similarly, the dose of rosuvastatin is also converted from clinical dose. Commonly, the dose of 10-20 mg/60kg/day is proposed for rosuvastatin to treat atherosclerosis in adult. Therefore, the equivalence dose is about 10-20 mg/kg/day of rosuvastatin in mouse, 10 mg/kg/day of rosuvastatin was used in the present experiment.

Tissue preparation

The aortas were immediately frozen in liquid nitrogen for RNA isolation and Western blot analysis. For RNA isolation, thoracic aortas were additionally perfused with RNA Later to prevent RNA degradation. Frozen samples of aortas were crashed on liquid nitrogen and total RNA was prepared in accordance with the manufacturer's instruction.

The plaque area determination

The aortas of three mice of each group were opened longitudinally, stained with Oil Red O, and pinned out on a white surface. The percentage of the plaque area stained by Oil Red 0 and the total luminal surface area was determined by Image Pro Plus analysis software (Version 6.0). Lesions were reported as percentage of the plaque area consisting of aortas.

The other three aortas of each group were used to pathological section since they were the area of greatest plaque formation (Coleman et al 2006). Samples used to pathological section were fixed in 4% paraformaldehyde and embedded in paraffin wax according to standard procedures. Serial sections were stained by Oil Red 0 or Movat's stain.

Plasma lipids and cytokines analyses

A whole blood sample (n = 6 for each group) were held for 30 min at room temperature to allow clotting. The sample was centrifuged at 3000 g for 10 min at 4[degrees]C. The plasma was transferred into separate tubes without disturbing blood clots and stored at -80[degrees]C. Plasma lipids were measured at the end of the study using mice ELISA kits for total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), respectively. For TNF-[alpha], IL-6 assays, the plasmas were tested using respective ELISA kits according to the manufacturer's guidelines.

Protein expressions analysis

Aortas were homogenized in modified Krebs buffer. Western blot analysis of IL-8 and MCP-1 was performed by a conventional method and detected by ECL chemiluminescence according to the manufacturer's instructions.

Cytotoxicity assay

Cytotoxicity was determined using the methylthiazolyltetrazolium assay as previously reported. The absorbance was measured at 490 nm by a microplate reader (Jiang W, et al, 2011).

Cytokines release assays

Human Umbilical Vein Endothelial Cells (HUVECs, kindly provided by professor Xiaohong Chen form The Third Military Medical University) were cultured at 37[degrees]C under humidified conditions of 95% air and 5% C[O.sub.2] in M199 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all from Gibco-BRL, Grand Island, NY, USA). HUVECs (5.0 x [10.sup.5] cells/ml, 2 ml or 0.5 ml) were incubated in 6-well or 24-well plates until confluent, and the washed twice and incubated with plasma-free medium for 12 h before different concentrations of AS (1.25, 2.5, 5 and 10 [micro]g/ml) were added. After incubation for 12 h, cells in 24-well were stimulated with TNF-[alpha] (10 [micro]g/ml) (R&D Systems; Minneapolis, MN) or LPS (0.1 [micro]g/ml, from E. coli 055: B5) (Sigma; St. Louis, MO, USA) for 24 h. After incubation, the supernatants were collected for MCP-1 and IL-8 assays.

mRNA expressions analysis

Total RNA was extracted from 1.0 x [10.sup.6] cells and reverse-transcribed to cDNA according to manufacturer's instructions qRT-PCR was performed using SYBR Green and the ABI PRISM 7500 Sequence Detection System (Applied Biosystems). cDNA was amplified by PCR with specific primers for MCP-1 and IL-8: primers for MCP-1 were 5'-GCATCCACGTGTTGGCTCA-3' (Forward) and 5-'CTCCAGCCTACTCATTGGGATCA-3' (Reverse). Primers for IL-8 were 5'-ACCACACTGCGCCAACACAGAAAT-3' (Forward) and 5'-TCCAGACAGAGCTCTCTTCCATCAGA-3' (Reverse). Primers for GAPDH were purchased from ABI.

Statistical analysis

Statistical analyses were performed using the SPSS 10.0 package. All data were presented as means [+ or -]SD of three to five independent experiments. The differences among groups were analyzed by One-way ANOVA and Student's-t test. Values of P < 0.05 were considered to be statistically significant.

Results

AS attenuates progression of atherosclerosis in WD-fed [ApoE.sup.-/-] mice

In order to make sure whether AS alone or combined with rosuvastatin could attenuate the progression of atherosclerosis lesion formation of mice, the effects of AS alone or combined with rosuvastatin in WD-fed [ApoE.sup.-/-] mice were observed. Our results showed mice treated with AS and rosuvastatin didn't result in any statistically significant differences in body weight and food intake (Fig. 1) (P > 0.05). However, Oil Red O-positive areas of enface aorta were significantly reduced in mice treated with AS or rosuvastatin, especially in the combination group. The plaque area rate (compared to total aortic area) reduced from 24.8% [+ or -] 5.5% (control group) to 9.3% [+ or -] 2.3% (rosuvastatin), 16.1% [+ or -] 2.2% (1.5 mg/kg/day AS), 12.8% [+ or -] 2.5% (5 mg/kg/day AS), 8.6% [+ or -] 1.7% (15 mg/kg/day AS); furthermore, the effect of AS combined with rosuvastatin was much better than rosuvastatin alone or AS alone (P<0.05) by Chi-square test, the plaque area rate was 5.1% [+ or -] 1.6% (Figs. 2A, 2B and 2C). Above results demonstrated that AS alone and combined with rosuvastatin could significantly attenuate the progression of atherosclerosis in WD-fed [ApoE.sup.-/-] mice.

AS doesn't affect the plasma lipid levels in WD-fed [apoE.sup.-/-] mice

Hyperlipidaemia is widely recognized as the most important risk factor for atherosclerosis development; therefore the plasma lipids levels were detected. Our results showed the plasma lipids levels in mice of control or AS groups were relatively high, and there were no difference of plasma TC, TG, HDL-C and LDL-C levels between two groups (P>0.05). But they decreased in rosuvastatin alone or combination groups although there were no difference between rosuvastatin alone and combination groups (P> 0.05). Above results demonstrated AS didn't influence the plasma lipids level in [ApoE.sup.-/-] mice and its anti-atherogenic effects were little related to plasma lipids levels (Table 1).

AS reduces plasma TNF-[alpha] and IL-6 levels in [apoe.sup.-/-] mice

It is also well accepted that inflammation plays an important role in all stages of the atherosclerotic process, and AS have antiinflammation effect proved by our previous experiments (Jiang W, et al, 2011; Li B, et al, 2010). Therefore, the plasma proinflammation cytokines levels were detected. Our results showed that plasma TNF-[alpha] level significantly decreased in [ApoE.sup.-/-] mice treated by AS in a dose-dependent manner: the TNF-[alpha] level respectively reduced from 238.7 [+ or -]35.3 pg/ml (control group) to 195.8 [+ or -]10.9 pg/ml (AS, 1.5 mg/kg/day), 169.8 [+ or -]18.0 pg/ml (AS, 5 mg/kg/day), and 98.1 [+ or -] 8.2 pg/ml (AS, 1.5 mg/kg/day). Besides, TNF-[alpha] level also decreased from 238.7 [+ or -]35.3 pg/ml in control group to 194.6 [+ or -]10.0 pg/ml in rosuvastatin group. Furthermore, when the TNF-[alpha] level reduced to 75.4 [+ or -] 11.9 pg/ml in combination group; the reduction degree was much higher than mice treated by one of them (Fig. 3). The IL-6 level changes were similar to TNF-[alpha] (Fig. 3).

AS reduces both MCP-1 and IL-8 mRNA and protein expressions of aorta

As we know, pro-inflammatory chemokines, such as IL-8 and MCP-1, are essential for monocyte rolling, firm adhesion to endothelial cells and their subsequent transmigration into vascular tissue. To gain insights into the molecular mechanism of antiatherogenic effect of AS, mRNA and proteins expressions of MCP-1 and IL-8 of aorta were examined by qRT-PCR (Fig. 4A) and Western blot analysis (Fig. 4B). As the result shown, AS and rosuvastatin could significantly attenuate the up-regulation of both MCP-1 and IL-8 mRNA expression in aorta in a dose dependent manner separately. And when the mice were treated with combination, the reduction degrees were much higher than when treated by only one of them.

AS doesn't inhibit the expression of MCP-1 and IL-8 of HUVECs treated with TNF-[alpha] but inhibit the expression of MCP-1 and IL-8 of HUVECs treated with LPS

Firstly, the cytotoxic effects of AS on the HUVECs were investigated using MTT assay. AS (2.5 - 100 [micro]g/ml) treatment for 24 h didn't induce any cellular toxicity in the HUVECs (Fig. 5). Hence, the concentrations of AS used in our in vitro experiments were less than 20 [micro]g/ml. Above results suggested that AS induced inhibition of cytokine release was not related to its cellular toxicity.

In vivo experiment, the progression of atherosclerosis was found to be attenuated by AS through inhibition of pro-inflammatory cytokine such as TNF-[alpha] and IL-6, and pro-inflammatory chemokines such as MCP-1 and IL-8. Then we wanted to make sure whether the reduction of chemokines was a direct effect of AS or not. Therefore, the influence of AS on the expressions of MCP-1 and IL-8 of HUVECs stimulated by TNF-[alpha] was observed. The results showed there were no differences among groups with different concentrations of AS treatment, which inferred that the expression of MCP-1 and IL-8 of HUVECs stimulated by TNF-[alpha] couldn't be inhibited by AS (Figs. 6A, 6B). However, AS significantly attenuate the up-regulation of both MCP-1 and IL-8 expression of HUVECs stimulated by LPS in a dose-dependent manner separately (Fig. 7).

Discussion

To the best of our knowledge, this is the report demonstrating that AS could attenuate the progression of atherosclerosis lesion formation alone or combined with rosuvastatin through inhibition of pro-inflammatory cytokines and pro-inflammatory chemokines in Western-type diet (WD) fed ApoE-/- mice.

Atherosclerosis is a main event and fundament of many cardiovascular diseases; it is the second cause of death in elder people. It is well know that atherosclerosis is a chronic inflammatory disease, which is characterized by the accumulation of lipids and fibrous elements that result from interactions between vascular cells and inflammatory cells (Joana V and Oliver S, 2015). While it has been shown that plasma cholesterol-lowering alone can attenuated progression of atherosclerosis lesion formation, the effect of quenching inflammation on atherosclerosis lesion formation has received as yet little attention (Kapourchali FR, et al, 2014).

Artemisinin is an active ingredient in the Chinese herb sweet wormwood; AS is the most widely used member of this family. In previous study, it has reported that AS alone decreased the plasma cholesterol and triglyceride and reduced the area of aortic root lesions in rabbits fed a Western-type diet (Wang YL, et al, 2013). Another report pointed that AS might influence CVD risk by reduced serum levels of total cholesterol, HDL-cholesterol concentrations (Adebayo JO, et al, 2011). Herein, AS alone or combined with rosuvastatin could attenuate the progression of atherosclerosis lesion formation of mice but had little effect on plasma lipids levels in our experiment. Although the results from our lab was different from those from other lab, AS did attenuate the progression of atherosclerosis.

The accumulation of plasma lipids, especially low-density lipoproteins (LDLs), in the walls of arteries is thought to be the most important factor in atherosclerosis progress, which in turn induces the migration of mononuclear cells and development of inflammation (Badimon L, et al, 2012). Therefore, the influence of AS on plasma lipids changes in WD fed [ApoE.sup.-/-] mice was investigated. Herein, AS was found to attenuate the progression of atherosclerosis lesion formation alone or combined with rosuvastatin, but there was no difference of plasma TG, TC, LDL and HDL levels between mice with and without AS treatment groups, demonstrating AS didn't attenuate the progression of atherosclerosis via reduction of the plasma lipids in [ApoE.sup.-/-] mice.

As we know, the earliest phase of atherosclerosis progression involves vascular endothelial cell dysfunction, and the exact cause of endothelial dysfunction leading to loss of barrier function remains unknown, but it is known that pro-inflammatory stimuli adversely affect endothelial function (Campbell LA and Rosenfeld ME, 2015). TNF-[alpha] and IL-6 are key cytokines in the inflammatory cascade, are proatherosderotic factors that triggers vascular inflammation and subsequent atherosclerosis development. Consistently, human studies have demonstrated that TNF-[alpha] is remarkably elevated in the plasma and arteries of subjects with vascular complications and that anti-TNF-[alpha] therapy improved aortic stiffness and carotid intima media thickness in patients with inflammatory arthropathies (Rossi D, et al. 2015; Nanau RM and Neuman MG, 2014). Furthermore, TNF-[alpha] and IL-6 can trigger several intracellular signaling events that ultimately up-regulate the expression of chemokines in endothelial cells, such as MCP-1 and IL-8, which are important in the onset and progress of inflammation. Many studies have reported the critical role of chemokines in the initiation of complex inflammatory process and pathogenesis of atherosclerosis, increase of MCP-1 and IL-8 might result in the dysfunction of endothelial cells, and leads to loss of barrier function that promotes inflammatory cell uptake and lipid accumulation (Deng B, et al. 2015; Shin WS, et al, 2002). Herein, AS was shown to reduce pro-inflammatory cytokines such as TNF-[alpha] and IL-6, meanwhile, MCP-1 and IL-8 expressions was reduced in mice aorta. It was inferred that AS could attenuate the progression of atherosclerosis lesion formation alone or combined with rosuvastatin consistency by decreasing of pro-inflammatory cytokines releases, resulting in decreasing of chemokines and eventually leading to decreased recruit monocyte into the arterial wall and improved vascular endothelial cell function.

Lipopolysaccharide (LPS) plays very important role for the atherosclerosis; it triggers the release of inflammatory cytokines that accelerate its initiation and progression (Jiang W., et al, 2011). Previously, AS had been reported to exert a protective effect on LPS-induced HUVEC activation and injury, which might be related to the inhibition of TNF-[alpha] mRNA expression and a decrease in the secretion of IL-1[beta], IL-6 and IL-8 from TNF-[alpha]--stimulated rheumatoid arthritis or fibroblast like synoviocytes (Mirshafiey A, et al, 2006; Wang Z, et al, 2012; Wang HY, et al, 2014.). Herein, the effects of AS to attenuate LPS-induced IL-8 and MCP-1 production were investigated in HUVECs. As the results showed, AS reduced LPS-induced MCP-1 and IL-8 expressions but didn't reduce TNF-[alpha]-induced MCP-1 and IL-8 expressions in HUVECs, demonstrating AS-reduced MCP-1 and IL-8 expressions in endothelial cells was the outcome via inhibiting LPS-induced release of chemokines.

Statins are a group of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that was used in atherosclerosis treatment because of their capacity to block cholesterol biosynthesis and modulate inflammatory reactions (Keating GM and Robinson DM, 2008). Therefore, rosuvastatin that impacts both two important mechanisms of atherosclerosis progression was used as a positive control. Though statins in general are well tolerated, myopathy and acute renal events have been a significant concern with the use of high-potency statin drugs, in particular simvastatin and rosuvastatin (Toth PP., et al, 2014; DiNicolantonio JJ, et al, 2013). Since these adverse effects are frequently dose related, there remains an overt need to identify agents such as anti-inflammatory agents. When they are combined with statins, two kinds of agents can provide better benefit for cardiovascular disease patients with the least added risk. Therefore, rosuvastatin was also used as a combination drug with AS. Herein, the results showed rosuvastatin combined with AS could more effectively attenuate the progression of atherosclerosis lesions than when treated by one of them, demonstrating that lipid-lowering agents combined with anti-inflammatory agents could provide the greater benefit for cardiovascular disease patients.

Conclusion

In conclusion, AS attenuated progression of atherosclerosis lesion formation alone or combined with rosuvastatin through antiinflammatory effect, resulting in down-regulation of TNF-[alpha] and IL-6, and further down-regulating IL-8 and MCP-1 expressions in aorta of WD fed [ApoE.sup.-/-] mice. Rosuvastatin combined with AS could more effectively attenuate the progression of atherosclerosis lesions than when treated by one of them, demonstrating that lipid-lowering agents combined with anti-inflammatory agents could provide the greater benefit for cardiovascular disease patients. Artesunate is worth further investigating as a candidate drug for the treatment of atherosclerosis.

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

ARTICLE INFO

Article history:

Received 17 September 2015

Revised 31 May 2016

Accepted 3 June 2016

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Weiwei Jiang (a, 1), Yanyan Cen (a, 1), Yi Song (a), Pan Li (a), Rongxin Qin (a), Chao Liu (a), Yibo Zhao (a), Jiang Zheng (b), Hong Zhou (a), *

(a) Department of Pharmacology, College of Medicine, The Third Military Medical University, Chongqing 400038, P. R. China

(b) Medical Research Center, Southwestern Hospital, The Third Military Medical University, Chongqing 400038, P. R. China

Abbreviations: ApoE, apolipoprotein e; WD, western-type diet; TNF-[alpha], tumor necrosis factor-[alpha]; IL-6, interleukin-6; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; ECs, endothelial cells; HUVECs, human umbilical vein endothelial cells; TC, total cholesterol; TG, triglyceride; LDL, low density lipoprotein; HDL, high density lipoprotein; LPS, Lipopolysaccharide.

* Corresponding author; Department of Pharmacology, College of Medicine, The Third Military Medical University, Gaotanyan Street 30, Shapingba District, Chongqing, P. R. China.. Tel./fax: +86 23 6875 2366.

E-mail address: zhouh64@163.com (H. Zhou).

(1) They equally contribute to this work.

http://dx.doi.org/10.1016/j.phymed.2016.06.004

Table 1
Artesunate didn't influence the plasma lipid levels (mmol/L) in
WD-fed [ApoE.sup.-/-] mice.

Groups                        TC

Control                       16.7 [+ or -] 1.9
Rosuvastatin (10 mg/kg/day)   13.1 [+ or -] 1.5 *
Artesunate (1.5 mg/kg/day)    16.6 [+ or -] 1.3
Artesunate (5 mg/kg/day)      17.6 [+ or -] 1.3
Artesunate (15 mg/kg/day)     16.9 [+ or -] 1.3
Combination                   13.3 [+ or -] 1.0 **

Groups                        TG

Control                       1.3 [+ or -] 0.2
Rosuvastatin (10 mg/kg/day)   1.0 [+ or -] 0.1 **
Artesunate (1.5 mg/kg/day)    1.2 [+ or -] 0.1
Artesunate (5 mg/kg/day)      1.2 [+ or -] 0.1
Artesunate (15 mg/kg/day)     1.2 [+ or -] 0.1
Combination                   1.0 [+ or -] 0.2 **

Groups                        LDL                    HDL

Control                       14.8 [+ or -] 1.8      5.8 [+ or -] 2.1
Rosuvastatin (10 mg/kg/day)   11.5 [+ or -] 1.5 **   5.6 [+ or -] 1.3 *
Artesunate (1.5 mg/kg/day)    15.2 [+ or -] 1.6      6.0 [+ or -] 0.8
Artesunate (5 mg/kg/day)      163 [+ or -] 0.7       5.7 [+ or -] 0.4
Artesunate (15 mg/kg/day)     15.2 [+ or -] 1.1      5.4 [+ or -] 0.5
Combination                   12.4 [+ or -] 1.6 *    5.2 [+ or -] 0.3 *

* P < 0.05 vs control, ** P < 0.01 vs control.


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Author:Jiang, Weiwei; Cen, Yanyan; Song, Yi; Li, Pan; Qin, Rongxin; Liu, Chao; Zhao, Yibo; Zheng, Jiang; Zh
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Oct 15, 2016
Words:5204
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