Modulation of oxidized-LDL receptor-1 (LOX1) contributes to the antiatherosclerosis effect of oleanolic acid

The International Journal of Biochemistry & Cell Biology 69 (2015) 142–152

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Modulation of oxidized-LDL receptor-1 (LOX1) contributes to the antiatherosclerosis effect of oleanolic acid Qixiao Jiang a,1 , Daoyan Wang a,1 , Yantao Han a , Zhiwu Han b,∗∗ , Weizhen Zhong a , Chunbo Wang a,∗ a b

Department of Pharmacology, Qingdao University Medical College, 308 Ningxia Road, Qingdao 266071, Shandong, China The Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Qingdao 266003, Shandong, China

a r t i c l e

i n f o

Article history: Received 13 March 2015 Received in revised form 15 October 2015 Accepted 22 October 2015 Available online 25 October 2015 Keywords: Oleanolic acid Quails Antiatherosclerosis Oxidized low-density lipoprotein LOX-1

a b s t r a c t Oleanolic acid (OA) is a bioactive pentacyclic triterpenoid. The current work studied the effects and possible mechanisms of OA in atherosclerosis. Quails (Coturnix coturnix) were treated with high fat diet with or without OA. Atherosclerosis was assessed by examining lipid profile, antioxidant status and histology in serum and aorta. Human umbilical vein endothelial cells (HUVECs) were exposed to 200 ␮g/mL ox-LDL for 24 h, then cell viability was assessed with MTT assay; reactive oxygen species (ROS) was assessed with DCFDA staining. Expression levels of LOX-1, NADPH oxidase subunits, nrf2 and ho-1 were measured with real time PCR and western blotting. Furthermore, LOX-1 was silenced with lentivirus and the expression levels assessment was repeated. OA treatment improved the lipid profile and antioxidant status in quails fed with high fat diet. Histology showed decreased atherosclerosis in OA treated animals. Ox-LDL exposure decreased viability and induced ROS generation in HUVECs, and this progression was alleviated by OA pretreatment. Moreover, elevated expression of LOX-1, NADPH oxidase subunits, nrf2 and ho-1 were observed in ox-LDL exposed HUVECs. OA pretreatment prevented ox-LDL induced increase of LOX-1 and NADPH oxidase subunits expression, while further increased nrf2 and ho-1 expression. Silencing of LOX-1 abolished ox-LDL induced effects in cell viability, ROS generation and gene expression. OA could alleviate high fat diet induced atherosclerosis in quail and ox-LDL induced cytotoxicity in HUVECs; the potential mechanism involves modulation of LOX-1 activity, including inhibition of expression of NADPH oxidase subunits and increase of the expression of nrf2 and ho-1. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Atherosclerosis is a chronic disease, which involves oxidative stress, inflammation and thrombosis in large and medium-sized arteries (Badimon et al., 2009). Many risk factors have been identified for atherosclerosis, such as smoking and metabolic syndrome

Abbreviations: Ox-LDL, oxidized low density lipoprotein; HUVECs, human umbilical vein endothelial cells; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2-H-tetrazolium bromide; DCFDA, 2 ,7 -dichlorofluorescein diacetate; ROS, reactive oxygen species; OA, oleanolic acid; LOX-1, letin-like oxidized low-density lipoprotein receptor-1; NADPH oxidase, nicotinamide adenine dinucleotide phosphate-oxidase; Nrf2, nuclear factor (erythroid-derived 2)-like 2; Ho-1, heme oxygenase 1. ∗ Corresponding author. Tel.: +86 532 83780029. ∗∗ Corresponding author. Tel.: +86 532 82911848. E-mail addresses: [email protected] (Z. Han), [email protected] (C. Wang). 1 Qixiao Jiang and Daoyan Wang contributed equally to this work. http://dx.doi.org/10.1016/j.biocel.2015.10.023 1357-2725/© 2015 Elsevier Ltd. All rights reserved.

(Unverdorben et al., 2009; Bonora, 2006). Because of the prevalence of atherosclerosis, and its negative consequence on public health as well as economy, preventative measures, such as preventative agents are highly desirable. Natural products are now receiving increasing attention as a significant resource for preventive agents (Mulvihill and Huff, 2012; Del Turco and Basta, 2012). Oleanolic acid (OA), a pentacyclic triterpenoid (Fig. 1), exists in various plants and medical herbs in the form of free acid or aglycone all over the world (Sultana and Ata, 2008; Ovesna et al., 2004). Examples of plants rich in oleanolic acid include Fructus Ligustrum lucidum (Xia et al., 2011) and Forsythiae fructus (Lee et al., 2010). OA has been reported to possess various health benefits such as hypolipidemic effect, hepatoprotection and anti-tumor effects (Kim et al., 2005; Li et al., 2002). Its antioxidative and antiinflammatory effects were also reported in an in vitro study on PC12 cells (Tsai and Yin, 2008). In addition, the protective effect of OA on vascular has been identified: OA induces vasorelaxation and evokes endothelium-dependent release of nitrite oxide (NO) (RodriguezRodriguez et al., 2008). Buus et al. (2011) also associated OA with

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2. Material and methods 2.1. Reagents

Fig. 1. Oleaolic acid chemical structure. Demonstration of oleanolic acid chemical structure and molecular weight.

atherosclerosis prevention in vivo. Since OA is a promising potential atherosclerosis prevention agent, we utilized a high-fat diet induced atherosclerosis model in quail to directly assess OA’s antiatherosclerosis effect. Furthermore, the model of ox-LDL induced cytotoxicity in HUVECs was also employed to investigate the mechanism of OA’s action. Among the identified risk factors of atherosclerosis, oxidized low-density lipoprotein (ox-LDL) attracted considerable attentions. Ox-LDL has been demonstrated to be involved in multiple steps of the pathogenesis of atherosclerosis, including endothelial dysfunction, proliferation and migration of vascular cells and stimulating transformation of lipid-rich foam cells from macrophages or smooth muscle cells (Li and Mehta, 2000; Mehta et al., 2004; Kao et al., 2009). Several studies had reported that ox-LDL has direct cytotoxicity on endothelial cells, which is a mimic of endothelial dysfunction during the pathogenesis of atherosclerosis (Kume and Kita, 2004; Chen et al., 2004), thus the in vitro ox-LDL exposure to endothelial cells is considered as a mimic of atherosclerosis (Wang et al., 2013). Identified mechanism of toxicity for ox-LDL includes activation of LOX1, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and subsequent overproduction of reactive oxygen species (ROS) (Pirillo et al., 2013). In endothelial cells, LOX-1 is the main ox-LDL receptor (Mehta et al., 2006). It has been reported that LOX-1 is involved in ox-LDL induced cytotoxicity (Imanishi et al., 2002). Multiple in vivo studies also confirmed the involvement of LOX-1 in atherosclerosis pathogenesis (Mehta et al., 2007; Inoue et al., 2005). However, the exact role of LOX-1 in early atherosclerosis pathogenesis remains to be fully elucidated. In this study, we investigated the role of LOX-1 in an ox-LDL induced cytotoxicity model and the protective effect of OA. Moreover, LOX-1 silencing with lentivirus allowed us to elucidate the role of LOX-1 in ox-LDL induced cytotoxicity. Following ROS generation, multiple protein could provide compensation, such as nuclear factor (erythroid-derived 2)-like 2 (nrf2) and heme oxygenase-1 (ho-1). Nrf2 is a nuclear receptor, upon activation, it translocates to nucleus and regulate the expression of nrf2 target genes such as NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (ho-1), glutathione S transferases (GSTs) and glutamate-cysteine ligase (GCL) (Kansanen et al., 2013), providing protection against ROS. On the other hand, ho-1 is a stress-inducible enzyme, which plays vital roles in various physiological and pathophysiological states related to oxidative stress (Otterbein and Choi, 2000). Over the past decade, increasing evidence has indicated that the induction of ho-1 serves as a host defense mechanism against the formation of atherosclerotic lesion (Ishikawa et al., 2001). Considering the central role of ROS in atherosclerosis pathogenesis, we investigated the influence of ox-LDL exposure on nrf2, ho-1, and the effects of OA treatment. This study is the first to associate LOX-1 modulation to the antiatherosclerosis effect of OA. Our data provided evidence for the role of LOX-1 in ox-LDL mediated cytotoxicity and for using OA as an anti-atherosclerosis agent.

OA (catalog number 03920590), 2 ,7 -dichlorofluorescin diacetate (DCFDA) (catalog number D6883) and MTT (catalog number M5655) were purchased from Sigma Aldrich (St. Louis, MO, US). Simvastatin was purchased from Merck China (Hangzhou, China). Nitric oxide (NO, 20131121), malondialdehyde (MDA, 20131112), superoxide dismutase (SOD, 20140110), catalase (CAT, 20131215), glutathione (GSH, 20140112), nicotinamide adenine dinucleotide phosphate (NADPH, 20131215) and glutathione peroxidase (GSH-Px, 20131210) kits were purchased from Nianjing Jiancheng (Nianjing, China). The gavage solution of OA was prepared in 1% sodium carboxymethylcellulose. For treatment of cell culture, a stock solution was prepared in dimethyl sulfoxide (DMSO) and diluted with culture medium immediately prior to the experiment. The final DMSO concentration was 0.1% in the medium. DMEM/High glucose was purchased from HyClone (Logan, UT, US). Human Ox-LDL (catalog number YB-002) was purchased from Yiyuan biotech (Guangzhou, China). Monoclonal antibodies against LOX-1 (ab53202), gp91 (ab129068), nrf2 (ab89443) and ho-1 (ab12220) were purchased from Abcam (Cambridge, MA, US). Polyclonal antibodies against p67 (#3923) and p47 (#4301) were purchased from Cell signaling (Danvers, MA, US). Monoclonal antibody against ␤-Actin (CW0096) was purchased from CWBIO (Beijing, China). HRP-conjugated anti-mouse IgG antibody (#7076S) and anti-rabbit IgG antibody (#7074S) were purchased from Cell signaling (Danvers, MA, US). Lentivirus loaded with LOX1 shRNA sequence or scramble control sequence were purchased from Genechem (Shanhai, China). 2.2. Animal treatment and sample collection The animal treatment was adopted from He et al. (2005) with modifications. Briefly, male quails (Coturnix coturnix) at 3 weeks age were obtained from Lanke Poultry Breeding Center (Jimo, Qingdao, China) and kept for one week for environment adaption, then put on normal diet (grinded corn 62%, soybean meal 15%, wheat bran 6%, peanut meal 5%, corn protein powder 5%, fish powder 2%, bone powder 2% (powder prepared mainly from mammalian bone, may also include some connective tissue), sodium chloride 0.3%, with other minor vitamins and minerals supplement; energy from carbonhydrates: 70.18%; energy from protein: 21.53%; energy from fat: 8.3%) or high fat diet (normal diet plus 1% w/w cholesterol and 14% w/w lard; energy from carbonhydrates: 50.40%; energy from protein: 15.46%; energy from fat: 34.13%), along with treatments: high fat diet quails were subjected to vehicle (1% sodium carboxymethylcellulose), simvastatin 15 mg/kg or OA 25, 50 or 100 mg/kg via gavage daily. For each group, 20 quails were included, and at least 16 quails per group survived until end of experiment. The dose of simvastatin was calculated from previous reported effective dose in rodents (Lee et al., 2012). Blood samples were collected from right jugular veins at the beginning of treatment and after four and a half weeks treatment. After ten weeks of treatment, the animals were sacrificed, serum was collected and frozen at −80 degree Celsius until use, and the aorta was quickly dissected out, either fixed in 4% formaldehyde or used subject to aorta lipid profile assessment. All procedures were approved by the Qingdao University Institutional Animal Care and Use Committee. 2.3. Serum lipid profile, antioxidant assessment and aorta lipid content measurement Quail serums were subjected to automatic biochemical analyzer Beckman AU5400 (Brea, CA, US) for the lipid profile; commercial

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kits were used to assess NO, MDA, SOD, CAT, GSH, NADPH and GSHPx following manufacturer’s instructions. Aortas were chopped and homogenized in 1% 1,4-dioxane. Samples were then incubated in a 37 degree Celsius incubator shaker for 72 h. Then 1 mL supernatant from every sample were collected and dried in tubes. 50 ␮L isopropanol were added to each tube to dissolve the lipids. The samples were then subjected to the same analyzer for lipid content. 2.4. Histology of quail aorta After 24 h fixation in 4% formaldehyde, the aortas were embedded in paraffin, sectioned at 6 ␮m on a microtome (Leica RM2016, Wetzlar, Germany) and the sections were stained with hematoxylin and eosin (Beyotime, Jiangsu, China) following manufacturer’s protocol. Pictures were taken with an Olympus BX51 (Tokyo, Japan) and analyzed with ImageJ (NIH, US). The ratio of atherosclerotic area (areas with significant fibromuscular lesion and smooth muscle cell proliferation) to total aorta area was calculated as a quantification of atherosclerosis. For details please refer to supplementary data. 2.5. Cell culture and treatment Human umbilical vein endothelial cells (HUVECs) were kindly provided by China Ocean University. HUVECs were cultured in DMEM/High glucose medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were incubated at 37 degree Celsius with 5% CO2 in a humidified environment. Cells in logarithmic growth phase and fusion up to 50–60% were treated with ox-LDL 200 ␮g/mL for 24 h, with or without pretreatment with OA (5, 10 or 20 ␮M) or vitamin E 200 ␮M for 24 h. Negative control cells were kept along with treated group and incubated under identical condition. 2.6. LOX-1 siRNA lentivirus package Four short hairpin RNAs (shRNA-1, 5 -AGGTACCTGTGC ATATATA-3 , shRNA-2,5 -GAATCTGAATCTCCAAGAA-3 , shRNA3,5 -CCTCCTAACACAAGAGCAA-3 , and shRNA-4,5 -GGTCTTCAGT TTCTTTACT-3 ) targeting the LOX-1 ORF (Genbank no. NM 001172632) and a non-targeting RNA sequence (shRNA5 -TTCTCCGAACGTGTCACGT-3 ) serving as a scramble control (Scr-shRNA), delivered by lentiviral vectors (hU6-MCS-CMVEGFP), were constructed. In preliminary experiments, shRNA-2 (5 -GAATCTGAATCTCCAAGAA-3 ) was found to be the most effective silencing LOX-1 expression, thus was chosen for subsequent experiments. Please refer to supplementary data for the confirmation of LOX-1 silencing. 2.7. Lentiviral transfection HUVECs were grown to 70–80% confluence and infected with lentivirus/LOX-1-shRNA or lentivirus/Scr-shRNA at multiplicity of infection (MOI) of 10. The infection efficiency was confirmed with fluorescence microscopy for EGFP protein three to five days posttransfection (Please refer to supplementary data). After five days incubation post-transfection, cells were harvested for real-time PCR or western blotting. 2.8. Determination of cytotoxicity HUVECs were cultured in 96-well plates at density of 5x103 cell/well and the cell viability was measured with the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay following manufacturer’s instructions. GSH concentration

and SOD activity was measured with commercial GSH and SOD assay kits according to the instruction provided by manufacturers. 2.9. Assessment of ROS in HUVECs Intracellular ROS level was measured with the fluorescent probe DCFDA. Cells at 50–60% confluence were treated as described previously, then washed for three times with medium. Cells were then loaded with DCFH-DA at a concentration of 10 ␮M for 20 min, washed for three times in medium, and then observed with a fluorescence microscope (Olympus BX-UCB, Tokyo, Japan) at excitation wavelength 488 nm and emission wavelength 530 nm. Three random views were selected for each sample, pictures were taken and quantified with ImageJ (NIH, US). Fluorescence intensity as an assessment of intracellular ROS. 2.10. Isolation of mRNA and real-time PCR Total RNA was extracted from cells with TRIzol Reagent (Invitrogen, Grand Island, NY, US) and reverse-transcribed with a Prime Script RT-PCR Kit (Takara BIO, Dalian, China) following manufacturer’s instructions. LOX-1 was amplified using the sense primer 5 -AAGCTTCACAGGAGTCAGAAAACG-3 and antisense primer 5 CAGATTCTGGTGGTGAAGTTCCA-3 . GAPDH was amplified using the sense primer 5 -GGATTTGGTCGTATTGGG-3 and antisense primer 5 -GGAAGATGGTGATGGGATT-3 . Real-time quantitative RT-PCR was performed with SYBR premix Ex TaqTM II (Takara Bio, Japan, RR820A) with a Light Cycler 480 (Roche Diagnostics, Mannheim, Germany). The reaction was initiated by heating at 95 degree Celsius for 30 s, then 95 degree Celsius, 5 s, 60 degree Celsius, 30 s for 40 cycles. Two delta delta Ct method was used to quantify gene expression as fold change to control group. 2.11. Western blotting After treatment, cells were harvested and lysed by radio immunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, MO, US) supplemented with protease inhibitor PMSF (Sigma, St. Louis, MO, US). Western blotting was performed as described previously (Wang et al., 2014). 2.12. Design and statistical analysis In animal study, quails were randomly assigned into each treatment group by drawing lots. Statistical analysis was performed with SPSS 17.0. All data were expressed as mean ± standard derivation. All the data were subjected to Levene’s test to confirm normal distribution, then the statistical significance was determined by one-way analysis of variance (ANOVA). When ANOVA detected statistical significance, the level of differences among groups was analyzed by least significant difference (LSD) test. Statistical significance was determined when P-values were less than 0.05. 3. Results 3.1. Effects of OA on serum lipid profile of high fat diet fed quails No significant differences were detected on serum lipid profile among groups at the beginning of treatment. High fat diet significantly deteriorated the serum and aorta lipid profile in quails, increasing total cholesterol, tryacylglycerol and LDL, while decreasing HDL after four and a half week and ten week treatment. Positive control drug simvastatin, and OA both reversed the changes in lipid profile (Table 1).

Q. Jiang et al. / The International Journal of Biochemistry & Cell Biology 69 (2015) 142–152 Table 1 Serum lipid profile of high fat diet-feed quails.

Table 2 Antioxidant effect of OA in high fat diet-feed quails.

Lipid profile before treatment (mM) Treatment

Total cholesterol

Control High fat diet Simvastatin OA 25 mg/kg OA 50 mg/kg OA 100 mg/kg

5.54 5.58 5.63 5.54 5.62 5.49

± ± ± ± ± ±

0.83 1.08 0.85 0.94 1.01 0.95

Triacylglycerol 1.15 1.11 1.08 1.09 1.07 1.06

± ± ± ± ± ±

0.15 0.17 0.22 0.21 0.18 0.11

LDL

HDL

1.42 1.39 1.46 1.40 1.39 1.42

± ± ± ± ± ±

0.46 0.51 0.57 0.35 0.47 0.28

3.44 3.45 3.49 3.44 3.57 3.42

± ± ± ± ± ±

0.70 1.11 0.72 0.85 0.90 0.80

Lipid profile after four and a half weeks high fat diet (mM) Treatment

Total cholesterol Triacylglycerol LDL

Control High fat diet Simvastatin OA 25 mg/kg OA 50 mg/kg OA 100 mg/kg

5.54 7.79 5.55 6.40 6.00 5.39

± ± ± ± ± ±

0.68 0.66a 0.59b 0.38b 0.43b 0.45bcd

1.26 1.77 1.18 1.38 1.36 1.06

± ± ± ± ± ±

0.39 0.31a 0.23b 0.39b 0.42b 0.20bc

1.23 1.95 1.35 1.71 1.55 1.33

HDL ± ± ± ± ± ±

0.19 0.27a 0.18b 0.20b 0.35b 0.23bcd

4.86 2.56 4.44 2.91 3.43 3.85

± ± ± ± ± ±

0.58 0.50a 0.89b 0.44 0.40bc 0.26bc

Lipid profile after ten weeks high fat diet (mM) Treatment

Total cholesterol Triacylglycerol LDL

Control High fat diet Simvastatin OA 25 mg/kg OA 50 mg/kg OA 100 mg/kg

5.88 8.18 5.55 6.40 5.80 5.49

± ± ± ± ± ±

0.57 0.57a 0.53b 0.42b 0.41bc 0.37bc

1.20 1.85 1.15 1.48 1.16 1.04

± ± ± ± ± ±

0.28 0.34a 0.21b 0.44b 0.18bc 0.21bc

145

1.19 2.01 1.35 1.75 1.55 1.36

HDL ± ± ± ± ± ±

0.17 0.23a 0.18b 0.12b 0.35bc 0.17bc

5.16 2.63 4.44 3.31 3.53 4.05

± ± ± ± ± ±

0.69 0.52a 0.89b 0.36b 0.35b 0.31bcd

Male quails (three week old at the beginning of experiments, four week old at the beginning of treatments) were subjected to high fat diet (1% cholesterol and 14% pork oil, w/w) for a total of ten weeks. Venous blood were collected from right jugular veins at the beginning of treatment and after four and a half weeks treatment. After ten weeks, quails were sacrificed and blood were collected. Serums were subjected to automatic biochemistry analyzer Beckman AU5400 (Brea, CA, US) for total cholesterol, triacylglycerol, low density lipoprotein (LDL) and high density lipoprotein (HDL). Data are shown as mean ± standard derivation, N = 10. a Significantly different from control group (P < 0.05). b Significantly different from high fat diet group (P < 0.05). c Significantly different from OA 25 mg/kg group (P < 0.05). d Significnatly different from OA 50 mg/kg group (P < 0.05).

3.2. Effects of OA on antioxidant status of high fat diet fed quails High fat diet significantly decreased the antioxidant capacity in quails, significantly increasing MDA level, while decreasing GSH and NADPH level. SOD, CAT and GSH-px activities were decreased as well. These changes were reversed by simvastatin or OA (Table 2). 3.3. Effects of OA on aorta morphology and lipid profile High fat-feed quails exhibited significantly increase of atherosclerotic area in aorta compared to control group, while OA and simvastatin significantly decreased atherosclerotic area in high fat diet quails compared to high fat diet only group (Fig. 2A and B). Consistently, total cholesterol, triacylglycerol and LDL were significantly increased in aorta lipid following high fat diet, which was reversed by simvastatin or OA (Fig. 2C–E), 3.4. Effects of OA on ox-LDL-induced cytotoxicity and ROS generationin HUVECs In HUVECs, ox-LDL exposure significantly decreased cell viability, while pretreatment with OA or vitamin E significantly alleviated the decrease (Fig. 3A). Similarly, ox-LDL exposure significantly increased ROS generation in HUVECs, which was also counteracted by pretreatment with OA or vitamin E (Fig. 3B).

Control High fat diet Simvastatin OA 25 mg/kg OA 50 mg/kg OA 100 mg/kg

Control High fat diet Simvastatin OA 25 mg/kg OA 50 mg/kg OA 100 mg/kg

NO(␮M)

MDA(nM)

SOD(U/mL)

34.19 ± 8.52 29.48 ± 7.47a 35.88 ± 6.57b 36.82 ± 8.68b 34.92 ± 9.63b 37.60 ± 9.15b

9.15 ± 2.57 12.26 ± 3.42a 7.23 ± 1.84b 9.41 ± 4.09b 7.98 ± 3.51b 6.64 ± 2.03b

213.35 ± 58.56 186.92 ± 47.86a 230.23 ± 51.98b 210.86 ± 58.66b 223.56 ± 68.59b 231.36 ± 65.32b

CAT (U/mL)

GSH (mg/L)

NADPH (␮M)

GSH-Px (U)

1.98 ± 0.53 1.60 ± 0.46a 2.17 ± 0.66b 1.82 ± 0.58 2.02 ± 0.58b 2.16 ± 0.62b

3.36 ± 0.79 2.87 ± 0.81a 3.49 ± 0.95b 3.35 ± 1.07b 3.48 ± 0.81b 3.54 ± 1.03b

3.50 ± 1.05 2.80 ± 0.90a 3.80 ± 1.00b 3.90 ± 1.40b 4.25 ± 1.50b 4.50 ± 1.25b

706.33 ± 183.12 612.79 ± 178.93a 836.42 ± 163.54b 780.66 ± 179.36b 823.59 ± 186.19b 831.62 ± 169.75b

Male quails (three week old at the beginning of experiments, four week old at the beginning of treatments) were subjected to high fat diet (1% cholesterol and 14% pork oil) for a total of ten weeks. After ten weeks, quails were sacrificed and blood were collected. Nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione peroxidase (GSH-Px) were measured with commercial kits following manufacturer’s protocols. Data are expressed as mean ± standard derivation, N = 10. a Significantly different from control group (P < 0.05). b Significantly different from high fat diet group (P < 0.05).

3.5. Time course changes of LOX-1, NADPH oxidase subunits and ho-1 in ox-LDL treated HUVECs Western blotting for LOX-1 and NADPH oxidase subunits (gp91hox, p47hox and p67hox) indicated that their levels were affected by ox-LDL treatment over time. Double-peak expression patterns were observed for LOX-1 and ho-1: their expression levels peaked at 3 h post ox-LDL exposure and returned to near-normal levels, then peaked again 24 h post ox-LDL exposure (Fig. 4A and B). In contrast, all the NADPH oxidase subunits exhibited significant increase in expression levels starting from 3 h post ox-LDL exposure, the expression level stayed at the induced levels until 12 h, and further increased at 24 h post ox-LDL exposure (Fig. 4A and C).

3.6. Pretreatment with OA alleviated ox-LDL induced LOX-1 up-regulation Real-time RT-PCR and western blotting were used to assess the effects of pretreatment with OA on ox-LDL induced LOX-1 upregulation. Consistent with time-course experiment results, ox-LDL exposure significantly increased LOX-1 expression, while pretreatment with vitamin E or OA effectively alleviated the increase. The alleviation induced by OA is dose dependent (Fig. 5A and C).

3.7. Pretreatment with OA inhibited the expression of the subunits of NADPH oxidase in HUVECs Western blotting was used to assess the protein levels of subunits of NADPH oxidase, including gp91phox, p67phox and p47phox. Similar to the case of LOX-1, following 24 h exposure to ox-LDL, the expression of all the subunits significantly increased, while pretreatment with OA for 24 h led to a dose-dependent reduction in the protein expressions of gp91phox, p67phox and p47phox (Fig. 5D and E).

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Fig. 2. Assessment of atherosclerosis in high fat diet fed quails and the effects of oleanolic acid. Male quails (three weeks old at the beginning of experiment, four weeks old at the beginning of treatments) were fed with high fat diet (1% cholesterol and 14% lard, w/w) for ten weeks. Quails were then sacrificed, aorta were collected for histology and lipid profile. For histology, aorta were fixed and embedded in paraffin, sectioned transversally on a microtome (Leica RM2016) at 6 ␮m and stained with hematoxlin and eosin. For artery lipid profile, lipids were extracted from homogenized aorta tissue with 1,4-dioxane and the lipid contents were measured using automatic biochemical analyzer Beckman AU5400 (Brea, CA, US). (A) Representative pictures of aorta sections stained with hematoxylin and eosin. They are control group, high fat diet group, simvastatin group, OA 25 mg/kg group, OA 50 mg/kg group and OA 100 mg/kg group. Arrow in the high fat diet group indicates atherosclerotic area. Scale bars represent 92.48 ␮m. (B) Quantification of the ratio of the atherosclerotic area to total aorta area (N = 3). (C) Quantification of the total cholesterol content in aorta (N = 10). (D) Quantification of the triacylglycerol content in aorta (N = 10). (E) Quantification of the LDL content in aorta (N = 10). (HF) high fat diet group; Sim: simvastatin group. (a) Statistically different from control group (P < 0.05). (b) Statistically different from high fat diet group (P < 0.05). (c) Statistically different from OA 25 mg/kg group (P < 0.05). (d): Statistically different from OA 50 mg/kg group (P < 0.05).

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Fig. 3. Cytotoxicity and ROS generation assessment in ox-LDL exposed HUVECs. HUVECs in logarithmic growth phase with cells fusion up to 50–60% were exposed to 200 ␮g/mL ox-LDL for 24 h, with or without pretreatment of vitamin E 200 ␮M or OA 5, 10, 20 ␮M for 24 h. Cells were then subjected to MTT assay for cell viability and DCFDA staining for ROS generation assessment. (A) Cell viability normalized to control group (N = 6 from six independent experiments). (B) ROS generation assessment (N = 3 from three independent experiments). (a) Statistically different from control group (P < 0.05). (b) Statistically different from ox-LDL group (P < 0.05). (c) Statistically different from OA 5 ␮M group (P < 0.05). (d) Statistically different from OA 10 ␮M group (P < 0.05).

3.8. Pretreatment with OA enhanced the expression of ho-1 and nrf2 in ox-LDL-exposed HUVECs Western blotting was used to assess the expression of ho-1 and nrf2 in ox-LDL-exposed HUVECs. 24-h exposure of ox-LDL to HUVECs significantly increased both ho-1 and nrf2 expression, while pretreatment with vitamin E or OA further enhanced expression of ho-1 and nrf2 (Fig. 5F and G). 3.9. Knockdown LOX-1 expression in HUVECs with lentivirus-based RNA silencing For confirmation of LOX-1 silencing with lentivirus, please refer to supplementary data. Western blotting for LOX-1 on HUVECs with or without LOX-1 knockdown revealed that ox-LDL exposure still increases LOX-1 expression in these LOX-1 knockdown HUVECs, but the level was significantly lower relative to control cells (Fig. 6). 3.10. LOX-1 knockdown inhibited ox-LDL induced cytotoxicity and ROS generation Knockdown of LOX-1 in HUVECs effectively inhibited ox-LDL induced cytotoxicity as determined with MTT assay. Exposure of ox-LDL to cells for 24 h resulted in a remarkable decrease in viability relative to control cells, while knockdown of LOX-1 effectively prevented the decrease (Fig. 7A). Meanwhile, DCFDA staining revealed that ROS generation was affected in a similar manner (Fig. 7B). 3.11. LOX-1 knockdown inhibited ox-LDL induced expression of NADPH oxidase subunits

Fig. 4. Time dependent change in western blotting for LOX1, gp91phox, p47phox, p67phox and ho-1. HUVECs in logarithmic growth phase with cells fusion up to 50–60% were exposed to 200 ␮g/mL ox-LDL for 0, 3, 6, 12 or 24 h. At desired time points, cells were harvested and subjected to western blotting. (A). Representative blot pictures for LOX-1, gp91phox, p47phox, p67phox and ho-1. (B) Quantification of LOX-1 and ho-1 expression levels by western blotting (N = 3 from three independent experiments). (C) Quantification of gp91phox, p47phox and p67phox expression levels by western blotting (N = 3 from three independent experiments). (a) Statistically different from 0 h time point (P < 0.05). (b) Statistically different from 3 h time point (P < 0.05).

Knockdown of LOX-1 in HUVECs significantly reduced ox-LDL induced expression of NADPH oxidase (gp91phox, p67phox and p47phox), while scramble control had no effect (Fig. 8). 3.12. LOX-1 knockdown inhibited ox-LDL induced expression of nrf2 and ho-1 Knockdown of LOX-1 in HUVECs significantly reduced ox-LDL induced expression of nrf2 and ho-1, while scramble control had no effect (Fig. 9).

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Fig. 5. Western blotting for LOX1, gp91phox, p47phox, p67phox, nrf2 and ho-1 expression in OA treated HUVECs. HUVECs in logarithmic growth phase with cells fusion up to 50–60% were exposed to 200 ␮g/mL ox-LDL for 24 h, with or without pretreatment of vitamin E 200 ␮M or OA 5, 10, 20 ␮M for 24 h. Cells were then harvested and subjected to real-time PCR or western blotting. (A) Quantification of LOX-1 expression level by Quantitative real-time PCR in treated HUVECs (N = 3 from three independent experiments). (B) Representative western blotting picture for LOX-1 in treated HUVECs. (C) Quantification of LOX-1 expression level by western blotting (N = 3 from three independent experiments). (D) Representative western blotting results for gp91phox, p47phox and p67phox in treated HUVECs. (E) Quantification for gp91phox, p47phox and p67phox western blotting (N = 3 from three independent experiments). (F) Representative western blotting picture for nrf2 and ho-1 in treated HUVECs. (G) Quantification of nrf2 and ho-1 expression levels by western blotting (N = 3 from three independent experiments). (a) Statistically different from control group (P < 0.05). (b) Statistically different from ox-LDL group (P < 0.05). (c) Statistically different from OA 5 ␮M group (P < 0.05). (d) Statistically different from OA 10 ␮M group (P < 0.05).

4. Discussion The present study demonstrated the inhibitory effect of OA on high fat diet induced atherosclerosis in quail. Further in vitro study demonstrated that OA counteracted ox-LDL induced cytotoxicity in HUVECs via modulation of LOX-1 activity and downstream gene expressions. 4.1. The antiatherosclerotic effect of OA in vivo The in vivo work utilized classical high fat diet quail model, which had been used in multiple studies (He et al., 2005; Shanmugasundaram and Selvaraj, 2011). Simvastatin was included as a positive control, which effectively counteracted the atherosclerosis-related pathological changes induced by high fat

diet, including serum and aorta lipid profile changes and morphological changes in aorta. Our results suggest that OA is an effective anti-atherosclerosis agent in high-fat diet fed quails, which is consistent with Buus et al. (2011). Interestingly, Buus et al. reported no significant hypolipidemic effect yet effective anti-atherosclerosis effect. This difference might be contributed by the ApoE knock out mouse model used in that study, and suggested hypolipidemc effect-independent role of oleanolic acid in its anti-atherosclerosis effect. Meanwhile, both simvastatin and OA had been shown to improve the antioxidant status in high fat diet fed quails. Our result is consistent with published results that simvastatin was reported to possess antioxidant effects (Abbas and Sakr, 2013). Meanwhile, OA had been demonstrated to possess similar antioxidant properties as simvastatin, which might contribute to its antiatherosclerosis effects.

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Fig. 6. Western blotting for LOX-1 in LOX-1 silenced HUVECs exposed to ox-LDL lentiviral transfection. HUVECs were grown to 70–80% confluence and infected with lentivirus with LOX-1-shRNA or scramble RNA, incubated for five days, and then exposed to vehicle or ox-LDL for 24 h. Western blotting was then carried out for LOX-1. (A) Representative blot picture for LOX-1 in lentivirus infected HUVECs. (B) Quantification of LOX-1 expression level by western blotting (N = 3 from three independent experiments). (a) Statistically different from control group (P < 0.05). (b) Statistically different from ox-LDL group (P < 0.05).

4.2. Ox-LDL It has been generally accepted that ox-LDL is more important than native LDL in the atherosclerosis pathogenesis (Fraley and Tsimikas, 2006). As a result, multiple studies had used oxLDL in atherosclerosis studies (Li and Mehta, 2000; Harris et al., 2006; Mehta et al., 2004;). Ox-LDL had been shown to stimulate superoxide anion formation in HUVECs cell line: at low dose and short exposure duration, it induced cell proliferation; but higher dose and duration resulted in apoptosis and necrosis (Galle et al., 2001; Seibold et al., 2004). The dose we used (200 ␮g/mL) is based on published evidence of circulating ox-LDL in human (Holvoet et al., 2004) and previous experiments in our lab (data not shown).

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Fig. 8. Western blotting for gp91phox, p47phox and p67phox in LOX-1 silenced HUVECs exposed to ox-LDL. HUVECs were grown to 70–80% confluence and infected with lentivirus with LOX-1-shRNA or scramble RNA, incubated for five days, and then exposed to vehicle or ox-LDL for 24 h. Western blotting was then carried out for gp91phox, p47phox and p67phox. (A) Representative blot picture for gp91phox, p47phox and p67phox in lentivirus infected HUVECs. (B) Quantification of gp91phox, p47phox and p67phox expression levels by western blotting (N = 3 from three independent experiments). (a): Statistically different from control group (P < 0.05). (b) Statistically different from ox-LDL group (P < 0.05).

4.3. The protective effect of OA in vitro Our in vitro work in ox-LDL exposed HUVECs demonstrated cytoprotective effects of OA. Ox-LDL was observed to induce cytotoxicity, decreasing viability and inducing ROS generation, consistent with published results (Ou et al., 2006). In current study, OA was found to reverse the decrease in viability and ROS generation, providing evidence that OA could protect endothelial cells in presence of ox-LDL.

Fig. 7. Assessment of cytotoxicity and ROS generation in LOX-1 silenced HUVECs exposed to ox-LDL. HUVECs were grown to 70–80% confluence and infected with lentivirus with LOX-1-shRNA or scramble RNA, incubated for five days, and then exposed to vehicle or ox-LDL for 24 h. Cells were then subjected to MTT assay for cell viability and DCFDA staining for ROS generation assessment. (A) Cell viability normalized to control group (N = 3 from three independent experiments). (B) ROS generation assessment (N = 3 from three independent experiments). (a) Statistically different from control group (P < 0.05). (b) Statistically different from ox-LDL group (P < 0.05).

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generation is a LOX-1 dependent process. Silence of LOX-1 also abolished the increase of NADPH oxidase subunits, nrf2 and ho-1 expression, confirming that these genes are regulated by LOX-1. 4.5. NADPH oxidase

Fig. 9. Western blotting for nrf2 and ho-1 in LOX-1 silenced HUVECs exposed to oxLDL. HUVECs were grown to 70–80% confluence and infected with lentivirus with LOX-1-shRNA or scramble RNA, incubated for five days, and then exposed to vehicle or ox-LDL for 24 h. Western blotting was then carried out for gp91phox, p47phox and p67phox. (A) Representative blot picture for nrf2 and ho-1 in lentivirus infected HUVECs. (B) Quantification of nrf2 and ho-1 expression levels by western blotting (N = 3 from three independent experiments). (a) Statistically different from control group (P < 0.05). (b) Statistically different from ox-LDL group (P < 0.05).

4.4. LOX-1 LOX-1 is a type II trans-membrane glycoprotein that belongs to the C-type lectin family, which was found expressed in all cell types involved in the atherosclerotic lesion and primarily expressed in endothelial cells (Mitra et al., 2011). Its binding to ox-LDL is involved in atherosclerosis generation (Sawamura et al., 2015) and associated risk factors such as decreased nitric oxide release (Cominacini et al., 2001) and increased secretion of chemokines (Inoue et al., 2005). Various molecular targets, such as membrane type 1 matrix metalloproteinase (MT1-MMP), rhoA, rac1 and protein kinase C were involved in LOX1 signaling (Sugimoto et al., 2009; Besler et al., 2011). The anti-atherosclerotic action of many drugs such as statins was associated with direct or indirect inhibition of LOX-1 expression in vascular lesions; (Mehta et al., 2001; Hofnagel et al., 2006). Therefore, LOX-1 is a promising target for anti-atherosclerotic drug discovery. The present study focused on LOX-1 to determine its role in ox-LDL induced cytotoxicity and ROS generation in HUVECs. Time course study revealed that following ox-LDL exposure, LOX-1 expression increased over time in HUVECs. Interestingly, a double-peak manner was observed for LOX-1, one at 3 h post exposure and one at 24 h post exposure, suggesting a fast and a slow response to LOX-1 that worth further exploration. Considering the ROS peak was observed 6 h post ox-LDL exposure, our hypothesis is that the first peak was induced by ox-LDL itself, while the second peak was mediated via ROS generation. OA treatment suppressed the elevation of LOX-1 gene expression, which corresponded well with the alleviation of ox-LDL mediated cytotoxicity and ROS generation, suggesting the mechanism of OA involves decreased LOX-1 expression. Further information regarding to LOX-1 comes from lentivirus-mediated silencing. Once LOX-1 expression was silenced, ox-LDL exposure no longer caused decrease of cell viability and increase of ROS generation, indicating that ox-LDL mediated cytotoxicity and ROS

NADPH oxidase is a main cause of atherosclerosis by ROS generation and macrophage attraction (Park et al., 2009). As reported by Khaidakov et al. (2010), NADPH oxidase could be induced by LOX-1, which was confirmed by our finding. Moreover, our finding further revealed information about LOX-1 mediated NADPH oxidase induction: as mentioned before, ox-LDL exposure resulted in two expression peaks of LOX-1 at 3 and 24 h, in between which the expression level returned to baseline; in contrast, NADPH oxidase subunits expression were stably induced after 3 h ox-LDL exposure and stayed at induced level; the expression level further increased after 24 h ox-LDL exposure, suggesting that NADPH oxidase subunits expression depends on LOX-1 and is persistent once induced. LOX-1 silencing further confirmed the LOX-1 dependence of NADPH oxidase subunits: NADPH oxidase subunits no longer were induced following ox-LDL exposure once LOX-1 was silenced. The fact that NADPH oxidase subunits expression decrease accompanied the cytoprotection suggests that NADPH oxidase participates in the cytotoxicity. 4.6. Nrf2 and ho-1 Previous studies have shown that nrf2/ho-1 signal pathway plays a critical role in the cellular protection under stress conditions (Alfieri et al., 2011; Takabe et al., 2011; Singh et al., 2010). It has been reported that ox-LDL activates the nrf2/ho-1 signaling pathway in HUVECs (Zhang et al., 2013), suggesting a possible vasoprotective effect of nrf2/ho-1 in ox-LDL-induced cell injury. In current study, ox-LDL exposure upregulated the expression of nrf2/ho-1 is likely the compensative action in response to oxidative stress. Furthermore, such upregulation was absent in LOX-1 knockdown cells, suggesting that nrf2 and ho-1 expression are dependent on LOX-1. Interestingly, OA treatment further enhanced the expression of nrf2/ho-1. In light of OA pretreatment can also prevent ox-LDL induced increase of LOX-1, it is likely that OA modulate, other than simply inhibit, LOX-1 expression under variety of situations. This modulation is definitely beneficial, and could indeed contribute to its antioxidant and cytoprotective effects. 5. Conclusions In this study, OA was found to counteract high fat diet induced atherosclerosis in quails. In HUVECs, LOX-1 was found to be a critical mediator of ox-LDL-induced damage. OA counteracts oxLDL induced cytotoxicity and ROS generation. Mechanism involves inhibition of LOX-1 induction, which then suppressed induction of NADPH oxidase subunits. Moreover, OA significantly increased expression of nrf2/ho-1, which may also contribute to its cytoprotective effects. Further study on OA’s anti-atherosclerosis effect is warranted. Disclosures There are none. Acknowledgments This study was funded by the National Natural Science Foundation of China (Grant Nos. 81173593 and 81274126).

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