European Journal of Pharmacology 744 (2014) 52–58
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Celastrol suppresses obesity process via increasing antioxidant capacity and improving lipid metabolism Chaoyun Wang, Chunfeng Shi, Xiaoping Yang, Ming Yang, Hongliu Sun, Chunhua Wang n School of Pharmaceutical Sciences, Binzhou Medical University, Yantai, Shandong 264003, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 July 2014 Received in revised form 24 September 2014 Accepted 25 September 2014 Available online 7 October 2014
High fat diet, as an important risk factor, plays a pivotal role in atherosclerotic process. Celastrol is one of the active triterpenoid compounds with antioxidative and anti-inflammatory characters. The aims of this study were to evaluate the effect of celastrol on weight, blood lipid and oxidative injury induced by high fat emulsion, and investigate its potential pharmacological mechanisms. Male Sprague–Dawley rats were fed with high fat emulsion for 6 wk to mimic high fat mediated oxidative injury. The effects of celastrol on weight and blood lipid were evaluated, and its mechanisms were disclosed by applying western blot, ELISA and assay kits. Long-term consumption of high fat emulsion could significantly increase weight by enhancing total cholesterol (TC), triacylglycerol (TG), apolipoprotein B (Apo B), low-density lipoprotein cholesterol (LDL-c) levels, attenuating ATP-binding cassette transporter A1 (ABCA1) expression, and decreasing the levels of high-density lipoprotein cholesterol (HDL-c) and apolipoprotein A-I (Apo A-I), and inhibit antioxidant enzymes activities, improve nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Comparing with model group, celastrol was able to effectively suppress weight and attenuate high fat mediated oxidative injury by improving ABCA1 expression, reducing the levels of TC, TG, LDL-c and Apo B in plasma, and increasing antioxidant enzymes activities and inhibiting NADPH oxidase activity, and decreasing the serum levels of Malondialdehyde (MDA) and reactive oxygen species in dose-dependent way. These data demonstrated that celastrol was able to effectively suppress weight and alleviate high-fat mediated cardiovascular injury via mitigating oxidative stress and improving lipid metabolism. & 2014 Elsevier B.V. All rights reserved.
Chemical compounds studied in this article: Celastrol (Pubchem CID: 122724) Keywords: Celastrol High fat emulsion ATP-binding cassette transporter A1 Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase Apolipoprotein A-I Apolipoprotein B
1. Introduction Daily dietary types had been proven to closely associate to the production of reactive oxygen (Conforti et al., 2009). Some literatures had been found that long-term high-fat diet treatment could increase the production of reactive oxygen species, promote lipid peroxidation, and then lead to oxidative injury (Nolan et al., 2005; Xu et al., 2009). Dyslipidemia was involved in the development of atherosclerosis, cardiovascular disease and renal ailments. Low-density lipoprotein (LDL), cholesterol, and high fat diet, as important risk factors, can change health status and accelerate obesity process through disturbing lipid metabolism and promoting the formation of lipid-rich plaques in the wall of arterial blood vessels. Oxidative stress plays a key role in the process, low-density lipoprotein (LDL) is oxidized to ox-LDL, and the latter bound with Lectin-like oxidized low-density lipoprotein receptor-1
n Correspondence to: School of Pharmaceutical Sciences, Binzhou Medical University No. 346, Guanhai Road, Laishan District Yantai, Shandong 264003, P.R. China. Tel.: þ86 535 6913216; fax: þ86 535 6913718. E-mail address: [email protected] (C. Wang).
http://dx.doi.org/10.1016/j.ejphar.2014.09.043 0014-2999/& 2014 Elsevier B.V. All rights reserved.
(LOX-1) and promote the obesity process (Yan et al., 2011). In addition, accumulating evidences indicated that obesity was also accompanied with a state of chronic inflammation, so it may be a very effective method to suppress obesity and cure obese-related disease by using antioxidants or applying anti-inflammatory agents (Kang, 2013). Thunder God Vine (Tripterygium wilfordii Hook F.) is a perennial vine of the Celastraceae family. The plant contains several therapeutically active compounds, including terpenoids, alkaloids, and steroids, which has been used as a traditional Chinese medicine to promote blood circulation, suppress rheumatism and relieve pain for hundreds of years. Celastrol [3-hydroxy-24nor-2-oxo-1(10),3,5,7-friedelatetraen-29-oic acid] is one of the active triterpenoid compound isolated from Tripterygium wilfordii Hook F. Previous studies have shown that celastrol was widely used to treat rheumatoid arthritis, allergic asthma, and systemic lupus erythematosus due to its potential anti-inflammatory and antioxidant effects (Tao et al., 2002; Qiu and Kao, 2003; Kim et al., 2009). In this study, we investigate the effect of celastrol on oxidative stress and blood lipid profiles after long term consumption of high fat emulsion, reveal its primary mechanisms, and then extend our opinion.
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2. Materials and methods
2.4. Determination of blood lipids
2.1. Drug and chemicals
Immediately after euthanasia, whole blood samples from abdominal aorta were drawn into tubes containing 3.8% trisodium citrate solution at a volume ratio of 9:1, and centrifuged at 1500g for 15 min. The levels of TC, TG, HDL-c and LDL-c in the supernatant were quantified using assay kits (Jiancheng Bioengineering Institute, Nanjing, P.R. China)
Celastrol (Fig. 1), extracted from root pulp of Tripterygium wilfordii Hook F., is the red amorphous powder and unsoluble compound with purity of more than 98%, whose molecular formula is C29H38O4 with a molecular weight of 450.61 Da, which was purchased from KMST Pharmaceutical Technology Development Co., Ltd.( Tianjing,P.R. China). Simvastatin was purchased from Shandong Lukang Pharmaceutical Group Co., Ltd. (Jining, P.R. China). All regents were AR degree grand, Assay kits of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c), lowdensity lipoprotein cholesterol (LDL-c), apolipoprotein A-I (Apo A-I), apolipoprotein B (Apo B), Malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) were provided from the Jiancheng Institute of Biotechnology (Nanjing, P.R. China). The specific antibodies analyzed target proteins were supplied by Santa Cruz Biotechnologies, USA 2.2. Animals and treatment Sixty male Sprague–Dawley rats with weight 200–250 g were purchased from Experimental Animal Department of Shandong Luye-Pharmcautical Co. Ltd., Shandong province, P.R. China. All animals were bred in temperature controlled animal facility with 12 h light–dark cycle. Rats were randomly divided into the following six groups: control group; high fat group (high fat diet); Celastrol treated groups (1 mg/kg, 3 mg/kg and 9 mg/kg); and simvastatin treated group (10 mg/kg). Except for control group, all rats were given oral administration of high fat emulsion (HFE ) containing 15% lard oil (W/V), 5% cholesterol (W/V), 2.5% yolk powder (W/V), 0.5% Propylthiouracil (W/V), 1% Tween-80 (V/V) and 76% distilled water (V/V) at a single dosage of 1.0 mL/100 g daily for 6 wk. Rats in celastrol treated group or simvastatin treated group were orally administered with celastrol or simvastatin in the doses indicated above every day, respectively. All rats were weighed at different times. At the end of 6 wk, all rats were briefly anesthetized and killed by bleeding from abdominal aorta. All animals were treated in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. Animal care and experimental procedures were approved by the Ethics Committee in Animal and Human Experimentation of Binzhou Medical University. 2.3. Observation of weight The weights of all rats from different groups were observed at preliminary stage, at the end of 3 wk and 6 wk.
Fig. 1. Molecular structure of celastrol.
2.5. Measurement of apolipoprotein A-I, apolipoprotein B The blood samples were collected from abdominal aorta and centrifuged at 1500g for 15 min. The levels of Apo A-I and Apo B in the supernatant were analyzed using assay kits (Jiancheng Bioengineering Institute, Nanjing, P.R. China) 2.6. Assessment of the activities of antioxidant enzyme and the levels of reactive oxygen species and Malondialdehyde Whole blood samples with 3.8% trisodium citrate solution were centrifuged at 1500g for 15 min. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) and the concentration of Malondialdehyde (MDA) in the supernatant were detected by using assay kits according to the manufacturer's instructions (Jiancheng Bioengineering Institute, Nanjing, P.R., China). The levels of reactive oxygen species in the supernatant were measured using the OxiSelect™ In Vitro Reactive Oxygen Species/Rns Assay Kit (Cell Biolabs Inc., USA). 2.7. Measurement of nicotinamide adenine dinucleotide phosphate oxidase activity The live tissues were washed, lysed and homogenized, and then were centrifuged at 16,000g for 25 min at 4 1C (Wang et al., 2013). The pellets (membrane fractions) were stored at 80 1C until use. An aliquot (50 mg) of the pellet was diluted in 500 ml of phosphate buffer. Dark-adapted lucigenin (20 mmol/L) was added to the sample, and the chemiluminescence measurements were immediately initiated. Chemiluminescence (in arbitrary units) was measured at 30-s intervals for 5 min by using the GloMax-20/20 Luminometer (Turner Biosystems, Inc., USA). NADPH was used as an electron donor substrate. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activities are expressed as the percentage of the control value. 2.8. Analysis of β-actin, ATP-binding cassette transporter A1 and p22phox expression by using western blotting The rats were killed, and the livers were collected on ice and were kept at 80 1C before use. The liver tissues were homogenized and treated with trizol protein extraction reagent (Invitrogen), and the protein contents of supernatant fluids were determined by the BCA method (BCA protein assay kit, Thermo Scientific). The protein samples were denatured by mixing with sample buffer at 95 1C for 15 min, and 15 mg of equal amounts of protein samples were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). The membranes were incubated with blocking buffer containing 1% bovine serum albumin for 2 h at 37 1C, and then were incubated with primary antibodies against β-actin, ABCA1 and p22phox (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in blocking buffer for 2 h at 37 1C or overnight at 4 1C. Membranes were then washed with washing buffer 3 times for 5 min. The membranes were subsequently incubated with a secondary antibody for 2 h at 37 1C,and followed by washing with
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Table 1 Effect of celastrol on the weight of rats treated with high fat emulsion. Group
Dosage (mg/kg)
– – 1 3 9 Simavastatin 10
Control High fat Celastrol
Preliminary stage weight (g)
3 weekend weight (g)
6 weekend weight (g)
230.5 7 14.2 226.8 7 15.2 228.7 7 14.2 228.3 7 15.2 225.17 14.2 227.6 7 13.6
313.9 7 17.8 402.3 7 20.7a 394.6 7 35.9 355.57 36.1b 336.27 35.0b 325.5 7 27.1b
388.9 7 15.9 560.8 7 28.5a 533.7 7 37.6 474.7 7 53.7b 455.7 7 49.1b 456.8 7 37.4b
Sixty male rats were treated according to the procedure described in Section 2. The weights of all rats were detected. After administration of high fat emulsion for 3 wk and 6 wk, animal weights of all rats treated with high fat emulsion were measured. Data were presented as mean7 standard deviation (S.D.) (n ¼10). a b
Significant difference relative to the control group is indicated as P o 0.01. Significant differences relative to the high fat group are indicated as Po 0.01.
washing buffer for 5 min. Protein bands on the membrane were detected by using a chemiluminescence reagent (ECL kit; Amersham Corporation, Arlington Heights, CA) in accordance with the manufacturer's instructions, and exposed to X-ray film. The intensity of each protein band on the film was analyzed with the ImageJ software. The actual levels of target protein were expressed as the percentage of target protein value in control group.
2.9. Statistical analysis Data are presented as means 7 SD. One-way analysis of variance (ANOVA) followed by Dunnett's test was used for multiple comparisons (SPSS10.0 software). Differences were considered significant at P o0.05.
Fig. 2. Effect of celastrol on the levels of total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c) in plasma of rat treated with high fat emulsion. Sixty male rats were treated according to the procedure described in Section 2. After administration of high fat emulsion for 6 wk, all rats were briefly anesthetized and sacrificed by exsanguination of the abdominal aorta. Total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c) in plasma were detected according to the procedure described in the assay kits. A, B, C, and D represent TC, TG, LDL-c and HDL-c respectively. Data were presented as mean7standard deviation (S.D.) (n¼ 10). Significant difference relative to the control group is indicated as þ þ Po0.01; significant differences relative to the high fat group are indicated as nPo0.05 and nnPo0.01.
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Fig. 3. Effect of celastrol on the levels of apolipoprotein A-I (Apo A-I) and apolipoprotein B (Apo B) in plasma of rats treated with high fat emulsion. After long-term consumption of high fat emulsionall rats were briefly anesthetized and sacrificed by bleeding from abdominal aorta. Apolipoprotein A-I (Apo A-I) and apolipoprotein B (Apo B) in plasma were quantified by using assay kits. A and B represent Apo A-I and Apo B. Data were presented as mean 7 standard deviation (S.D.) (n¼10). Significant difference relative to the control group is indicated as þþ Po 0.01; significant differences relative to the high fat group are indicated as n P o0.05 and nnPo 0.01.
3. Results 3.1. Effect of celastrol of weight As shown in Table 1, high fat emulsion remarkably enhanced animal body weight at the end of 3 wk and 6 wk (Po 0.01). After oral treatment with celastrol, the increasing trend of body weight was effectively inhibited comparing with model group (P o0.01), which depended on the dosage of celastrol.
Fig. 4. Effect of celastrol on antioxidant enzymes activities and Malondialdehyde (MDA) content in plasma of rat treated with high fat emulsion. The activities of SOD and GSH-Px, and MDA content in plasma were detected according the procedure descried in the assay kits. A, B, and C represent the activities of SOD and GSH-Px, and MDA content in plasma respectively. Data were presented as mean7 standard deviation (S.D.) (n¼10). Significant difference relative to the control group is indicated as þ þ Po 0.01; significant differences relative to the high fat group are indicated as nPo 0.05 and nnPo 0.01.
3.2. Effect of celastrol on TC, TG, HDL-c and LDL-c levels
3.3. Effect of celastrol on Apo A-I and Apo B levels
Comparing with the control group, high fat emulsion significantly elevated the levels of TC, TG and LDL-c and reduced HDL-c level in plasma (P o0.01). As shown in Fig. 2, celastrol significantly decreased the levels of TC, TG and LDL-c in plasma (P o0.01), and raised HDL-c level (P o0.01) in dose-dependent way. Likewise, simvastatin was able to significantly decrease the levels of TC, TG and LDL-c in plasma (P o0.01), and increase plasma HDL-c level compared to the model group (P o0.01).
As shown in Fig. 3, the concentrations of Apo A-I and Apo B in plasma were remarkably reduced or increased after orally administering high fat emulsion (P o0.01). Compared to model group, celastrol and simvastatin effectively decreased Apo B level (P o0.01), which showed a dose dependent way. Although there was the upward trend for Apo A-I levels in the plasma after treating with celastrol, the increase was insignificant as compared to high fat group (P 40.05) (Fig. 3).
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Fig. 5. Effect of celastrol on nicotinamide adenine dinucleotide phosphate oxidase activity and p22phox expression in the liver tissue of rats treated with high fat emulsion. A representative western blot is shown in panel (A). The expression level of p22phox (B) the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (C) in the liver tissue and reactive oxygen species level (D) in plasma were detected using an assay kit and western blotting method, respectively. Data were presented as mean 7standard deviation (S.D.) (n ¼10). Significant difference relative to the control group is indicated as þ þ Po 0.01; significant differences relative to the high fat group are indicated as nPo 0.05 and nnP o0.01.
3.4. Effect of celastrol on antioxidant capacity Compared to the control group, long-term high fat emulsion significantly inhibited the activities of SOD and GPx and increased the levels of reactive oxygen species and MDA in plasma (Po0.05 or Po0.01). As shown in Figs. 4 and 5D, celastrol and simvastatin markedly increased the activities of SOD and GPx and obviously decreased the levels of reactive oxygen species and MDA in plasma comparing with the model group (Po0.05 or Po0.01, Figs. 4 and 5D). 3.5. Effect of celastrol on NADPH oxidase activity and p22phox expression in live tissue As shown in Fig. 5A–C, long-term high fat emulsion significantly increased the expression of p22phox and NADPH oxidase activity in liver tissue (Po 0.01). Compared to the model group,
celastrol was able to significantly suppress p22phox expression, and then inhibit NADPH oxidase activity at the dosage of 3 mg/kg and 9 mg/kg (Po 0.01). Likewise, the data showed that simavastatin remarkably decrease NADPH oxidase activity and p22phox expression in live tissue (P o0.05) (Fig. 5A–C).
3.6. Effect of celastrol on ATP-binding cassette transporter A1 expression in liver tissue Compare to the control group, the ABCA1 expression was significantly inhibited in live tissue after oral treatment with high fat emulsion (Po 0.01). As shown in Fig. 6, celastrol and simavatatin were able to effectively up regulate the ABCA1 expression, and ameliorate the inhibition of high fat diet comparing with the model group (P o0.05 or P o0.01).
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Fig. 6. Effect of celastrol on the expression level of ATP-binding cassette transporter A1 in the liver tissues of rats treated with high fat emulsion. A representative western blot is shown in panel (A). The ABCA1 expression level in liver tissue was measured via applying the western blotting method. Data were presented as mean 7 standard deviation (S.D.) (n¼ 10). Significant difference relative to the control group is indicated as þ þ P o0.01; significant differences relative to the high fat group are indicated as nPo 0.05 and nnPo 0.01.
4. Discussion Dietary habits play a key role in maintaining health. Excessive intake of high fat diet should speed up the process of liver fibrogenesis (De Minicis et al., 2014), cause the significant increases of TC, LDL, LDL-c and TG and a sharp decrease of HDL-c in plasma and stimulate oxidative stress and inflammatory responds, and then result in cardiovascular diseases (CVDs), including coronary artery disease and most cases of heart failure and stroke (Barone et al., 1998; Meetoo, 2008; Christopher et al., 2010). In the study, the data have shown that long-term consumption of HFE was able to lead to the prominent increases in TC, LDL, LDL-c and TG, and cause a notable decrease in HDL-c concentration. High-density lipoprotein (HDL) level is significantly and inversely correlated with risk for development of CVD (LIPID Study Group, 2002; Colhoun et al., 2004), whereas low concentration of HDL cholesterol was proved to be a more valuable and accurate predictor of CVDs risk (Williams and Tabas, 1995; Steinberg, 2005). Previous studies had shown that HDL reduced cholesterol concentrations of peripheral tissues and alleviated cholesterol deposition in atherosclerotic lesions by transporting cholesterol from extrahepatic tissues to the liver for its catabolism to bile acids (Brewer, 2004; Yokoyama, 2006; Staels and Fonseca, 2009; Fitzgerald et al., 2010). Apolipoprotein A-I (ApoA-I), as a well known apolipoprotein which is synthesized and secreted by the liver and small intestine (Brunham et al., 2006; Lewis, 2006), is a major protein component of high density lipoprotein (HDL), and promotes cholesterol efflux from tissues to the liver for excretion. Hepatic ATP-binding cassette transporter A1 (ABCA1) plays an important role in regulating and maintaining plasma HDL and is the major transporter for cholesterol efflux from peripheral tissues and lipidation of ApoA-I in the liver (Bodzioch et al., 1999; Orsó et al., 2000; McNeish et al., 2000). Recent studies have shown that selective knock-down of hepatic ABCA1 in mice induced lower HDL level, and overexpression of hepatic ABCA1 in mice increased
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HDL level in plasma (Singaraja et al., 2006). Therefore, it was assumed that hepatic ABCA1 maybe an essential first step in cholesterol transport pathway. Apolipoprotein B (Apo B) is the primary apolipoprotein of low density lipoproteins (LDL or “bad cholesterol”) and is absolutely required for LDL formation, which is responsible for carrying cholesterol to tissues. In the present study, long-term consumption of HFE effectively up regulated Apo B expression, inhibited ABCA1 and Apo A-I expression, blocked the cholesterol transport pathway from peripheral tissue to live, destroyed the normal lipid metabolism, and then enhanced the levels of TC, LDL, LDL-c and TG in plasma and decreased plasma HDL-c concentration. After treatment with celastrol, the HDL level in plasma and ABCA1 expression in hepatic tissue were remarkably enhanced, and then the lipid metabolism of rats fed with high-fat diet was markedly improved. Finally, the levels of TC, LDL, TG and LDL-c in plasma were significantly decreased, which all were attributed to the capacity of celastrol to regulate cholesterol transport pathway and promote cholesterol efflux from peripheral tissues to the liver. In addition, there is a lot of evidence indicating that high-fat diet can decrease antioxidant enzymes activities, and up regulate NAD(P)H oxidase expression and improve its activities (CarmielHaggai et al., 2005; Roberts et al., 2006). Tölle et al. (2008) proved that HDL was able to inhibit reactive oxygen species generation by directly influencing the activation of NADPH oxidase. NADPH oxidase is a membrane-localized, multiunit protein that is an especially important source of reactive oxygen species production (Griendling et al., 2000; Lassègue and Clempus, 2003). p22phox as one of NADPH oxidase subunits provides a docking site for cytosolic factors and stimulates the activation of NADPH oxidase (Delbosc et al., 2005). In contrast, SOD and GPx are important antioxidant enzymes present in all cells that maintain cell function by inhibiting reactive oxygen species generation and promoting reactive oxygen species degradation. Thus, inhibition of antioxidant enzymes and activation of NADPH oxidase result in excessive reactive oxygen species production and induction of oxidative stress-mediated cell injury (Didion et al., 2002). The data showed that long-term consumption of HFE broke the oxidant–antioxidant balance via up regulating the expression level of p22phox, enhancing NADPH oxidase activity and inhibiting the activities of SOD and GPx, and then led to excessive reactive oxygen species generation. Celastrol effectively reduced reactive oxygen species level in plasma, and attenuated HFE-mediated oxidative stress injury by increasing the antioxidant enzymes activities, suppressing the expression of p22phox and inhibiting NADPH oxidase activity. Taken together, our data showed that celastrol effectively attenuates long-term consumption of HFE mediated oxidative stress by inhibiting reactive oxygen species generation and regulating cholesterol transport pathway. Further studies on celastrol will elucidate the detailed mechanisms of action of HFE and will provide a theoretical basis for its clinical application.
Acknowledgments We would like to thank all members of the Department of Pharmacology, Binzhou Medical University, for helpful discussions and technical assistance. The present study was supported by Project of Shandong Province Higher Educational Science and Technology Program (No. J10LF89), Project of Shandong Province Medicine & Health Science and Technology Program (2011HZ003), Natural Science Foundation of Shandong Province (No. ZR2012HM076) and Foundation of Taishan Scholar (tshw20110515).
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Celastrol suppresses obesity process via increasing antioxidant capacity and improving lipid metabolism Chaoyun Wang, Chunfeng Shi, Xiaoping Yang, Ming Yang, Hongliu Sun, Chunhua Wang n School of Pharmaceutical Sciences, Binzhou Medical University, Yantai, Shandong 264003, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 July 2014 Received in revised form 24 September 2014 Accepted 25 September 2014 Available online 7 October 2014
High fat diet, as an important risk factor, plays a pivotal role in atherosclerotic process. Celastrol is one of the active triterpenoid compounds with antioxidative and anti-inflammatory characters. The aims of this study were to evaluate the effect of celastrol on weight, blood lipid and oxidative injury induced by high fat emulsion, and investigate its potential pharmacological mechanisms. Male Sprague–Dawley rats were fed with high fat emulsion for 6 wk to mimic high fat mediated oxidative injury. The effects of celastrol on weight and blood lipid were evaluated, and its mechanisms were disclosed by applying western blot, ELISA and assay kits. Long-term consumption of high fat emulsion could significantly increase weight by enhancing total cholesterol (TC), triacylglycerol (TG), apolipoprotein B (Apo B), low-density lipoprotein cholesterol (LDL-c) levels, attenuating ATP-binding cassette transporter A1 (ABCA1) expression, and decreasing the levels of high-density lipoprotein cholesterol (HDL-c) and apolipoprotein A-I (Apo A-I), and inhibit antioxidant enzymes activities, improve nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Comparing with model group, celastrol was able to effectively suppress weight and attenuate high fat mediated oxidative injury by improving ABCA1 expression, reducing the levels of TC, TG, LDL-c and Apo B in plasma, and increasing antioxidant enzymes activities and inhibiting NADPH oxidase activity, and decreasing the serum levels of Malondialdehyde (MDA) and reactive oxygen species in dose-dependent way. These data demonstrated that celastrol was able to effectively suppress weight and alleviate high-fat mediated cardiovascular injury via mitigating oxidative stress and improving lipid metabolism. & 2014 Elsevier B.V. All rights reserved.
Chemical compounds studied in this article: Celastrol (Pubchem CID: 122724) Keywords: Celastrol High fat emulsion ATP-binding cassette transporter A1 Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase Apolipoprotein A-I Apolipoprotein B
1. Introduction Daily dietary types had been proven to closely associate to the production of reactive oxygen (Conforti et al., 2009). Some literatures had been found that long-term high-fat diet treatment could increase the production of reactive oxygen species, promote lipid peroxidation, and then lead to oxidative injury (Nolan et al., 2005; Xu et al., 2009). Dyslipidemia was involved in the development of atherosclerosis, cardiovascular disease and renal ailments. Low-density lipoprotein (LDL), cholesterol, and high fat diet, as important risk factors, can change health status and accelerate obesity process through disturbing lipid metabolism and promoting the formation of lipid-rich plaques in the wall of arterial blood vessels. Oxidative stress plays a key role in the process, low-density lipoprotein (LDL) is oxidized to ox-LDL, and the latter bound with Lectin-like oxidized low-density lipoprotein receptor-1
n Correspondence to: School of Pharmaceutical Sciences, Binzhou Medical University No. 346, Guanhai Road, Laishan District Yantai, Shandong 264003, P.R. China. Tel.: þ86 535 6913216; fax: þ86 535 6913718. E-mail address: [email protected] (C. Wang).
http://dx.doi.org/10.1016/j.ejphar.2014.09.043 0014-2999/& 2014 Elsevier B.V. All rights reserved.
(LOX-1) and promote the obesity process (Yan et al., 2011). In addition, accumulating evidences indicated that obesity was also accompanied with a state of chronic inflammation, so it may be a very effective method to suppress obesity and cure obese-related disease by using antioxidants or applying anti-inflammatory agents (Kang, 2013). Thunder God Vine (Tripterygium wilfordii Hook F.) is a perennial vine of the Celastraceae family. The plant contains several therapeutically active compounds, including terpenoids, alkaloids, and steroids, which has been used as a traditional Chinese medicine to promote blood circulation, suppress rheumatism and relieve pain for hundreds of years. Celastrol [3-hydroxy-24nor-2-oxo-1(10),3,5,7-friedelatetraen-29-oic acid] is one of the active triterpenoid compound isolated from Tripterygium wilfordii Hook F. Previous studies have shown that celastrol was widely used to treat rheumatoid arthritis, allergic asthma, and systemic lupus erythematosus due to its potential anti-inflammatory and antioxidant effects (Tao et al., 2002; Qiu and Kao, 2003; Kim et al., 2009). In this study, we investigate the effect of celastrol on oxidative stress and blood lipid profiles after long term consumption of high fat emulsion, reveal its primary mechanisms, and then extend our opinion.
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2. Materials and methods
2.4. Determination of blood lipids
2.1. Drug and chemicals
Immediately after euthanasia, whole blood samples from abdominal aorta were drawn into tubes containing 3.8% trisodium citrate solution at a volume ratio of 9:1, and centrifuged at 1500g for 15 min. The levels of TC, TG, HDL-c and LDL-c in the supernatant were quantified using assay kits (Jiancheng Bioengineering Institute, Nanjing, P.R. China)
Celastrol (Fig. 1), extracted from root pulp of Tripterygium wilfordii Hook F., is the red amorphous powder and unsoluble compound with purity of more than 98%, whose molecular formula is C29H38O4 with a molecular weight of 450.61 Da, which was purchased from KMST Pharmaceutical Technology Development Co., Ltd.( Tianjing,P.R. China). Simvastatin was purchased from Shandong Lukang Pharmaceutical Group Co., Ltd. (Jining, P.R. China). All regents were AR degree grand, Assay kits of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c), lowdensity lipoprotein cholesterol (LDL-c), apolipoprotein A-I (Apo A-I), apolipoprotein B (Apo B), Malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) were provided from the Jiancheng Institute of Biotechnology (Nanjing, P.R. China). The specific antibodies analyzed target proteins were supplied by Santa Cruz Biotechnologies, USA 2.2. Animals and treatment Sixty male Sprague–Dawley rats with weight 200–250 g were purchased from Experimental Animal Department of Shandong Luye-Pharmcautical Co. Ltd., Shandong province, P.R. China. All animals were bred in temperature controlled animal facility with 12 h light–dark cycle. Rats were randomly divided into the following six groups: control group; high fat group (high fat diet); Celastrol treated groups (1 mg/kg, 3 mg/kg and 9 mg/kg); and simvastatin treated group (10 mg/kg). Except for control group, all rats were given oral administration of high fat emulsion (HFE ) containing 15% lard oil (W/V), 5% cholesterol (W/V), 2.5% yolk powder (W/V), 0.5% Propylthiouracil (W/V), 1% Tween-80 (V/V) and 76% distilled water (V/V) at a single dosage of 1.0 mL/100 g daily for 6 wk. Rats in celastrol treated group or simvastatin treated group were orally administered with celastrol or simvastatin in the doses indicated above every day, respectively. All rats were weighed at different times. At the end of 6 wk, all rats were briefly anesthetized and killed by bleeding from abdominal aorta. All animals were treated in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. Animal care and experimental procedures were approved by the Ethics Committee in Animal and Human Experimentation of Binzhou Medical University. 2.3. Observation of weight The weights of all rats from different groups were observed at preliminary stage, at the end of 3 wk and 6 wk.
Fig. 1. Molecular structure of celastrol.
2.5. Measurement of apolipoprotein A-I, apolipoprotein B The blood samples were collected from abdominal aorta and centrifuged at 1500g for 15 min. The levels of Apo A-I and Apo B in the supernatant were analyzed using assay kits (Jiancheng Bioengineering Institute, Nanjing, P.R. China) 2.6. Assessment of the activities of antioxidant enzyme and the levels of reactive oxygen species and Malondialdehyde Whole blood samples with 3.8% trisodium citrate solution were centrifuged at 1500g for 15 min. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) and the concentration of Malondialdehyde (MDA) in the supernatant were detected by using assay kits according to the manufacturer's instructions (Jiancheng Bioengineering Institute, Nanjing, P.R., China). The levels of reactive oxygen species in the supernatant were measured using the OxiSelect™ In Vitro Reactive Oxygen Species/Rns Assay Kit (Cell Biolabs Inc., USA). 2.7. Measurement of nicotinamide adenine dinucleotide phosphate oxidase activity The live tissues were washed, lysed and homogenized, and then were centrifuged at 16,000g for 25 min at 4 1C (Wang et al., 2013). The pellets (membrane fractions) were stored at 80 1C until use. An aliquot (50 mg) of the pellet was diluted in 500 ml of phosphate buffer. Dark-adapted lucigenin (20 mmol/L) was added to the sample, and the chemiluminescence measurements were immediately initiated. Chemiluminescence (in arbitrary units) was measured at 30-s intervals for 5 min by using the GloMax-20/20 Luminometer (Turner Biosystems, Inc., USA). NADPH was used as an electron donor substrate. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activities are expressed as the percentage of the control value. 2.8. Analysis of β-actin, ATP-binding cassette transporter A1 and p22phox expression by using western blotting The rats were killed, and the livers were collected on ice and were kept at 80 1C before use. The liver tissues were homogenized and treated with trizol protein extraction reagent (Invitrogen), and the protein contents of supernatant fluids were determined by the BCA method (BCA protein assay kit, Thermo Scientific). The protein samples were denatured by mixing with sample buffer at 95 1C for 15 min, and 15 mg of equal amounts of protein samples were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). The membranes were incubated with blocking buffer containing 1% bovine serum albumin for 2 h at 37 1C, and then were incubated with primary antibodies against β-actin, ABCA1 and p22phox (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in blocking buffer for 2 h at 37 1C or overnight at 4 1C. Membranes were then washed with washing buffer 3 times for 5 min. The membranes were subsequently incubated with a secondary antibody for 2 h at 37 1C,and followed by washing with
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Table 1 Effect of celastrol on the weight of rats treated with high fat emulsion. Group
Dosage (mg/kg)
– – 1 3 9 Simavastatin 10
Control High fat Celastrol
Preliminary stage weight (g)
3 weekend weight (g)
6 weekend weight (g)
230.5 7 14.2 226.8 7 15.2 228.7 7 14.2 228.3 7 15.2 225.17 14.2 227.6 7 13.6
313.9 7 17.8 402.3 7 20.7a 394.6 7 35.9 355.57 36.1b 336.27 35.0b 325.5 7 27.1b
388.9 7 15.9 560.8 7 28.5a 533.7 7 37.6 474.7 7 53.7b 455.7 7 49.1b 456.8 7 37.4b
Sixty male rats were treated according to the procedure described in Section 2. The weights of all rats were detected. After administration of high fat emulsion for 3 wk and 6 wk, animal weights of all rats treated with high fat emulsion were measured. Data were presented as mean7 standard deviation (S.D.) (n ¼10). a b
Significant difference relative to the control group is indicated as P o 0.01. Significant differences relative to the high fat group are indicated as Po 0.01.
washing buffer for 5 min. Protein bands on the membrane were detected by using a chemiluminescence reagent (ECL kit; Amersham Corporation, Arlington Heights, CA) in accordance with the manufacturer's instructions, and exposed to X-ray film. The intensity of each protein band on the film was analyzed with the ImageJ software. The actual levels of target protein were expressed as the percentage of target protein value in control group.
2.9. Statistical analysis Data are presented as means 7 SD. One-way analysis of variance (ANOVA) followed by Dunnett's test was used for multiple comparisons (SPSS10.0 software). Differences were considered significant at P o0.05.
Fig. 2. Effect of celastrol on the levels of total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c) in plasma of rat treated with high fat emulsion. Sixty male rats were treated according to the procedure described in Section 2. After administration of high fat emulsion for 6 wk, all rats were briefly anesthetized and sacrificed by exsanguination of the abdominal aorta. Total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c) in plasma were detected according to the procedure described in the assay kits. A, B, C, and D represent TC, TG, LDL-c and HDL-c respectively. Data were presented as mean7standard deviation (S.D.) (n¼ 10). Significant difference relative to the control group is indicated as þ þ Po0.01; significant differences relative to the high fat group are indicated as nPo0.05 and nnPo0.01.
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Fig. 3. Effect of celastrol on the levels of apolipoprotein A-I (Apo A-I) and apolipoprotein B (Apo B) in plasma of rats treated with high fat emulsion. After long-term consumption of high fat emulsionall rats were briefly anesthetized and sacrificed by bleeding from abdominal aorta. Apolipoprotein A-I (Apo A-I) and apolipoprotein B (Apo B) in plasma were quantified by using assay kits. A and B represent Apo A-I and Apo B. Data were presented as mean 7 standard deviation (S.D.) (n¼10). Significant difference relative to the control group is indicated as þþ Po 0.01; significant differences relative to the high fat group are indicated as n P o0.05 and nnPo 0.01.
3. Results 3.1. Effect of celastrol of weight As shown in Table 1, high fat emulsion remarkably enhanced animal body weight at the end of 3 wk and 6 wk (Po 0.01). After oral treatment with celastrol, the increasing trend of body weight was effectively inhibited comparing with model group (P o0.01), which depended on the dosage of celastrol.
Fig. 4. Effect of celastrol on antioxidant enzymes activities and Malondialdehyde (MDA) content in plasma of rat treated with high fat emulsion. The activities of SOD and GSH-Px, and MDA content in plasma were detected according the procedure descried in the assay kits. A, B, and C represent the activities of SOD and GSH-Px, and MDA content in plasma respectively. Data were presented as mean7 standard deviation (S.D.) (n¼10). Significant difference relative to the control group is indicated as þ þ Po 0.01; significant differences relative to the high fat group are indicated as nPo 0.05 and nnPo 0.01.
3.2. Effect of celastrol on TC, TG, HDL-c and LDL-c levels
3.3. Effect of celastrol on Apo A-I and Apo B levels
Comparing with the control group, high fat emulsion significantly elevated the levels of TC, TG and LDL-c and reduced HDL-c level in plasma (P o0.01). As shown in Fig. 2, celastrol significantly decreased the levels of TC, TG and LDL-c in plasma (P o0.01), and raised HDL-c level (P o0.01) in dose-dependent way. Likewise, simvastatin was able to significantly decrease the levels of TC, TG and LDL-c in plasma (P o0.01), and increase plasma HDL-c level compared to the model group (P o0.01).
As shown in Fig. 3, the concentrations of Apo A-I and Apo B in plasma were remarkably reduced or increased after orally administering high fat emulsion (P o0.01). Compared to model group, celastrol and simvastatin effectively decreased Apo B level (P o0.01), which showed a dose dependent way. Although there was the upward trend for Apo A-I levels in the plasma after treating with celastrol, the increase was insignificant as compared to high fat group (P 40.05) (Fig. 3).
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Fig. 5. Effect of celastrol on nicotinamide adenine dinucleotide phosphate oxidase activity and p22phox expression in the liver tissue of rats treated with high fat emulsion. A representative western blot is shown in panel (A). The expression level of p22phox (B) the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (C) in the liver tissue and reactive oxygen species level (D) in plasma were detected using an assay kit and western blotting method, respectively. Data were presented as mean 7standard deviation (S.D.) (n ¼10). Significant difference relative to the control group is indicated as þ þ Po 0.01; significant differences relative to the high fat group are indicated as nPo 0.05 and nnP o0.01.
3.4. Effect of celastrol on antioxidant capacity Compared to the control group, long-term high fat emulsion significantly inhibited the activities of SOD and GPx and increased the levels of reactive oxygen species and MDA in plasma (Po0.05 or Po0.01). As shown in Figs. 4 and 5D, celastrol and simvastatin markedly increased the activities of SOD and GPx and obviously decreased the levels of reactive oxygen species and MDA in plasma comparing with the model group (Po0.05 or Po0.01, Figs. 4 and 5D). 3.5. Effect of celastrol on NADPH oxidase activity and p22phox expression in live tissue As shown in Fig. 5A–C, long-term high fat emulsion significantly increased the expression of p22phox and NADPH oxidase activity in liver tissue (Po 0.01). Compared to the model group,
celastrol was able to significantly suppress p22phox expression, and then inhibit NADPH oxidase activity at the dosage of 3 mg/kg and 9 mg/kg (Po 0.01). Likewise, the data showed that simavastatin remarkably decrease NADPH oxidase activity and p22phox expression in live tissue (P o0.05) (Fig. 5A–C).
3.6. Effect of celastrol on ATP-binding cassette transporter A1 expression in liver tissue Compare to the control group, the ABCA1 expression was significantly inhibited in live tissue after oral treatment with high fat emulsion (Po 0.01). As shown in Fig. 6, celastrol and simavatatin were able to effectively up regulate the ABCA1 expression, and ameliorate the inhibition of high fat diet comparing with the model group (P o0.05 or P o0.01).
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Fig. 6. Effect of celastrol on the expression level of ATP-binding cassette transporter A1 in the liver tissues of rats treated with high fat emulsion. A representative western blot is shown in panel (A). The ABCA1 expression level in liver tissue was measured via applying the western blotting method. Data were presented as mean 7 standard deviation (S.D.) (n¼ 10). Significant difference relative to the control group is indicated as þ þ P o0.01; significant differences relative to the high fat group are indicated as nPo 0.05 and nnPo 0.01.
4. Discussion Dietary habits play a key role in maintaining health. Excessive intake of high fat diet should speed up the process of liver fibrogenesis (De Minicis et al., 2014), cause the significant increases of TC, LDL, LDL-c and TG and a sharp decrease of HDL-c in plasma and stimulate oxidative stress and inflammatory responds, and then result in cardiovascular diseases (CVDs), including coronary artery disease and most cases of heart failure and stroke (Barone et al., 1998; Meetoo, 2008; Christopher et al., 2010). In the study, the data have shown that long-term consumption of HFE was able to lead to the prominent increases in TC, LDL, LDL-c and TG, and cause a notable decrease in HDL-c concentration. High-density lipoprotein (HDL) level is significantly and inversely correlated with risk for development of CVD (LIPID Study Group, 2002; Colhoun et al., 2004), whereas low concentration of HDL cholesterol was proved to be a more valuable and accurate predictor of CVDs risk (Williams and Tabas, 1995; Steinberg, 2005). Previous studies had shown that HDL reduced cholesterol concentrations of peripheral tissues and alleviated cholesterol deposition in atherosclerotic lesions by transporting cholesterol from extrahepatic tissues to the liver for its catabolism to bile acids (Brewer, 2004; Yokoyama, 2006; Staels and Fonseca, 2009; Fitzgerald et al., 2010). Apolipoprotein A-I (ApoA-I), as a well known apolipoprotein which is synthesized and secreted by the liver and small intestine (Brunham et al., 2006; Lewis, 2006), is a major protein component of high density lipoprotein (HDL), and promotes cholesterol efflux from tissues to the liver for excretion. Hepatic ATP-binding cassette transporter A1 (ABCA1) plays an important role in regulating and maintaining plasma HDL and is the major transporter for cholesterol efflux from peripheral tissues and lipidation of ApoA-I in the liver (Bodzioch et al., 1999; Orsó et al., 2000; McNeish et al., 2000). Recent studies have shown that selective knock-down of hepatic ABCA1 in mice induced lower HDL level, and overexpression of hepatic ABCA1 in mice increased
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HDL level in plasma (Singaraja et al., 2006). Therefore, it was assumed that hepatic ABCA1 maybe an essential first step in cholesterol transport pathway. Apolipoprotein B (Apo B) is the primary apolipoprotein of low density lipoproteins (LDL or “bad cholesterol”) and is absolutely required for LDL formation, which is responsible for carrying cholesterol to tissues. In the present study, long-term consumption of HFE effectively up regulated Apo B expression, inhibited ABCA1 and Apo A-I expression, blocked the cholesterol transport pathway from peripheral tissue to live, destroyed the normal lipid metabolism, and then enhanced the levels of TC, LDL, LDL-c and TG in plasma and decreased plasma HDL-c concentration. After treatment with celastrol, the HDL level in plasma and ABCA1 expression in hepatic tissue were remarkably enhanced, and then the lipid metabolism of rats fed with high-fat diet was markedly improved. Finally, the levels of TC, LDL, TG and LDL-c in plasma were significantly decreased, which all were attributed to the capacity of celastrol to regulate cholesterol transport pathway and promote cholesterol efflux from peripheral tissues to the liver. In addition, there is a lot of evidence indicating that high-fat diet can decrease antioxidant enzymes activities, and up regulate NAD(P)H oxidase expression and improve its activities (CarmielHaggai et al., 2005; Roberts et al., 2006). Tölle et al. (2008) proved that HDL was able to inhibit reactive oxygen species generation by directly influencing the activation of NADPH oxidase. NADPH oxidase is a membrane-localized, multiunit protein that is an especially important source of reactive oxygen species production (Griendling et al., 2000; Lassègue and Clempus, 2003). p22phox as one of NADPH oxidase subunits provides a docking site for cytosolic factors and stimulates the activation of NADPH oxidase (Delbosc et al., 2005). In contrast, SOD and GPx are important antioxidant enzymes present in all cells that maintain cell function by inhibiting reactive oxygen species generation and promoting reactive oxygen species degradation. Thus, inhibition of antioxidant enzymes and activation of NADPH oxidase result in excessive reactive oxygen species production and induction of oxidative stress-mediated cell injury (Didion et al., 2002). The data showed that long-term consumption of HFE broke the oxidant–antioxidant balance via up regulating the expression level of p22phox, enhancing NADPH oxidase activity and inhibiting the activities of SOD and GPx, and then led to excessive reactive oxygen species generation. Celastrol effectively reduced reactive oxygen species level in plasma, and attenuated HFE-mediated oxidative stress injury by increasing the antioxidant enzymes activities, suppressing the expression of p22phox and inhibiting NADPH oxidase activity. Taken together, our data showed that celastrol effectively attenuates long-term consumption of HFE mediated oxidative stress by inhibiting reactive oxygen species generation and regulating cholesterol transport pathway. Further studies on celastrol will elucidate the detailed mechanisms of action of HFE and will provide a theoretical basis for its clinical application.
Acknowledgments We would like to thank all members of the Department of Pharmacology, Binzhou Medical University, for helpful discussions and technical assistance. The present study was supported by Project of Shandong Province Higher Educational Science and Technology Program (No. J10LF89), Project of Shandong Province Medicine & Health Science and Technology Program (2011HZ003), Natural Science Foundation of Shandong Province (No. ZR2012HM076) and Foundation of Taishan Scholar (tshw20110515).
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