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Chlorogenic acid-enriched extract from Eucommia ulmoides leaves inhibits hepatic lipid accumulation through regulation of cholesterol metabolism in HepG2 cells a
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Shun Hao , Yuan Xiao , Yan Lin , Zhentao Mo , Yang Chen , Xiaofeng Peng , Canhui Xiang , a
Yiqi Li & Wenna Li a
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Zunyi Medical University, Zhuhai Campus, Zhuhai, Guangdong, China and
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Pharmaceutical Preparation Section, Guizhou Province People's Hospital, Guiyang, Guizhou, China Published online: 05 May 2015.
To cite this article: Shun Hao, Yuan Xiao, Yan Lin, Zhentao Mo, Yang Chen, Xiaofeng Peng, Canhui Xiang, Yiqi Li & Wenna Li (2015): Chlorogenic acid-enriched extract from Eucommia ulmoides leaves inhibits hepatic lipid accumulation through regulation of cholesterol metabolism in HepG2 cells, Pharmaceutical Biology To link to this article: http://dx.doi.org/10.3109/13880209.2015.1029054
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http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–9 ! 2015 Informa Healthcare USA, Inc.. DOI: 10.3109/13880209.2015.1029054
ORIGINAL ARTICLE
Chlorogenic acid-enriched extract from Eucommia ulmoides leaves inhibits hepatic lipid accumulation through regulation of cholesterol metabolism in HepG2 cells Shun Hao1, Yuan Xiao2, Yan Lin1, Zhentao Mo1, Yang Chen1, Xiaofeng Peng1, Canhui Xiang1, Yiqi Li1, and Wenna Li1 Zunyi Medical University, Zhuhai Campus, Zhuhai, Guangdong, China and 2Pharmaceutical Preparation Section, Guizhou Province People’s Hospital, Guiyang, Guizhou, China
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Abstract
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Context: Eucommia ulmoides Oliver (Eucommiaceae) leaf exhibits beneficial lipid-lowering and anti-obesity effects. However, the mechanisms remain unknown. Objective: The objective of this study is to investigate the lipid-lowering effects of chlorogenic acid (CGA)-enriched extract from this plant (CAEF) in human hepatoma HepG2 cells, focusing on cholesterol metabolism. Materials and methods: HepG2 cells were treated with CAEF (10, 20, 25, 40, 60, and 80 mg/L), CGA (0.3, 3, 30, 300, and 600 lmol/L), and simvastatin (0.1, 1, 10, 50, and 100 lmol/L) for 24 or 48 h. The cytotoxicity, Oil red O staining, total cholesterol, and triacylglycerol in supernatants were determined. The mRNA expression of genes involved in cholesterol metabolism was determined with RT-PCR. The protein expression of HMG-CoA reductase (HMGCR) was examined by immunocytochemistry and western-blot. Results: The IC50 values were 59.2 mg/L for CAEF, 335.9 lmol/L for CGA, and 10.5 lmol/L for simvastatin. By treating cells with CAEF (25 mg/L), CGA (30 lmol/L), or simvastatin (10 lmol/L) for 48 h, the efflux of total cholesterol and triacylglycerol was increased (CAEF, 4.06- and 31.00-folds; CGA, 2.94- and 2.17-folds; and simvastatin, 3.94- and 24.67-folds), and the cellular lipid droplets were reduced in Oil red O staining. CAEF and CGA increased mRNA expression of ABCA1, CYP7A1, and AMPKa2, while CAEF and simvastatin decreased SREBP2. However, their effects on LXRa mRNA expression were variable. Importantly, all drugs significantly inhibited protein expression of HMGCR at mRNA and protein levels. Discussion and conclusion: CAEF is a promising dietary supplement to prevent obesity and dyslipidemia and the effects appear to be due, at least in part, to regulating cholesterol metabolism through inhibition of HMGCR in HepG2 cells.
ABCA1, AMPKa, chlorogenic acid, CYP7A1, HMGCR, lipid accumulation, SREBP2
Introduction Obesity is one of the world-wide epidemic diseases affecting human health. A survey showed that from year 2002 to 2010, waist circumference and abdominal obesity have continued to rise in a southern Chinese population of 85 million residents in Guangdong, while in northeast China, obesity in adults reached 37.71% in 2011 (Lao et al., 2015; Shi, 2011). Obesity not only reduces the quality of life but also results in the serious complications. Dysregulation of lipid metabolism plays a key role in the pathology of obesity, which leads to fatty liver and hyperlipidemia as the most common complications. It is reported that improving dyslipidemia is one of the most important strategies for obesity prevention and treatment (Yun, 2010).
Correspondence: Prof. Wenna Li, Zhuhai Campus, Zunyi Medical University, Jinwan District, Zhuhai, Guangdong 519041, PR China. Tel: +86 756 762 3384. Fax: +86 756 763 7938. E-mail: [email protected]
History Received 16 July 2014 Accepted 4 March 2015 Published online 7 April 2015
Eucommia ulmoides Oliver (Eucommiaceae), also called Du-Zhong, is a native traditional medicinal plant in China, which is also widely used in Japan and Korea. Previously, only the bark of E. ulmoides was used as medicinal materials. However, it is reported that the leaves containing many phytochemicals are also found in the bark, and other bioactive components are found only in the leaves (Yuan et al., 2013). The leaf of Eucommia has been recorded officially in Chinese Pharmacopoeia since 2005 (Chinese Pharmacopoeia Committee, 2005). It has been found that the Eucommia leaf extracts exhibit beneficial lipid-lowering effects, significantly decrease the cholesterol and triglycerides, and increase the HDL-C in the serum of high-fat diet mice and hamsters (Choi et al., 2008; Park et al., 2006). Several recent studies have shown that the extracts of Eucommia leaf can also have the anti-obesity and antimetabolic syndrome activity, which were mainly attributed to several mechanisms as follows: (1) stimulating lipolysis and thermogenesis through elevations in adipose tissue sympathetic nerve activity, and suppressing the appetite by
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inhibiting the parasympathetic nerves of the gastrointestinal tract (Horii et al., 2010), (2) diminishing ATP production of white adipose tissue, accelerating fatty acid b-oxidation of liver, and increasing the use of ketone bodies and glucose in the skeletal muscle (Fujikawa et al., 2010), and (3) DNA microarray analysis found that genes related to the peroxisome proliferator-activated receptor a and d signaling pathways were up-regulated (Kobayashi et al., 2012). Those anti-obesity effects were observed in humans. After 8 weeks of given oral Eucommia capsule containing the Eucommia leaf extracts to 15 patients or placebo to control group of 12 patients with abdominal obesity, the visceral fat (21% versus 4.1%) and subcutaneous fat (11.4% versus + 7.2%) were decreased significantly, as evidenced by CT scanner, the body weight, abdominal circumference, and BMI of patients (Zhou et al., 2011). The main active components of Eucommia leaf extracts that produce anti-obesity and hypolipidemia effect are chlorogenic acid (CGA), geniposidic acid, and asperuloside (Hirata et al., 2011; Kobayashi et al., 2012). CGA is one of the active compounds enriched in leaves than in barks, and has been shown to have anti-obesity property and improve lipid metabolism in high-fat induced obese mice (Cho et al., 2010), and stimulate glucose transport in the skeletal muscle via AMPK activation (Ong et al., 2012). We previously isolated and purified of the CGA-enriched extract from Eucommia leaf extracts (CAEF) (Li et al., 2012b) and also found that CAEF, like orlistat, can suppress pancreatic lipase activity both in vitro and in vivo. CAEF is more effective in inhibition of cholesterol micelles formation than CGA and has higher inhibitory potency on HMG-CoA reductase (HMGCR) than simvastatin in vitro. In a rat model of acute fat-loading, CAEF could also significantly increase the excretion of cholesterol and bile acid into feces (Li et al., 2012a,c). To further examine the beneficial effects of CAEF in lipid metabolism, in this study, we used an in vitro model, in which the cellular and molecular mechanisms could be better dissected to evaluate hypolipidemic and beneficial effects of CAEF in human hepatoma HepG2 cells.
Materials and methods Plant materials and extraction The leaves of E. ulmoides were purchased from Zunyi, Guizhou, China, in December 2012, and identified by Dr. Yang Chen. The voucher specimens are deposited at the Department of Pharmacology, Zunyi Medical College, Zhuhai Campus, China. CAEF was prepared as reported (Li et al., 2012b). In brief, the leaves were cleaned, grounded, and extracted in 60% ethanol (1:8 w/v), in which pH was adjusted to 5.0 and ultrasonicated for 40 min at room temperature. After filtration, the supernatant was concentrated, and the concentrate was purified by chitosan flocculation and decolorized by polysaccharide. The crude extract solution was absorbed by NKA-9 macroporous resin, eluted with 50% ethanol. The resultant aqueous extract was evaporated in a rotary evaporator under reduced pressure. After freeze drying at 50 C, the final extracts were obtained and kept in 20 C.
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During the extraction of leaves of E. ulmoides with ethanol, the paper chromatography-UV method was used to qualitatively identify the main ingredients. On the filter paper (Xinhua, Hangzhou, China) and with the water:acetic acid:nbutanol in volume ratio 5:1:4 as a mobile phase, the extracts were chromatophyed, air dried, and observed at 254 nm, the spots were cut and dissolved in 50% methanol and ultrasonicated for 20 min. The quantification of supernatant was performed by UV Spectrophotometry (Shimadzu, UV-2550, Shimadzu Corporation, Kyoto, Japan) at 327 nm. After the final extracts were collected, the amounts of CGA in the extract were analyzed with high-performance liquid chromatography (HPLC) in comparison with standard preparation of CGA by the method of Chinese Pharmacopoeia 2005 edition (Chinese Pharmacopoeia Committee, 2005). Cell culture Human hepatoma HepG2 cells were grown in RPMI-1640 (Invitrogen, Waltham, MA) supplemented with 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 lg/mL streptomycin) in an incubator at 37 C with 5% CO2 with medium changes three times a week. HepG2 cell viability was assayed by trypan blue exclusion method. Cytotoxicity assay The HepG2 cell growth was determined by MTT assay. HepG2 cells were seeded in a 96-well plate at a density of 1 105 cells/well and cultured to reach confluence. Cultures were changed to serum-free medium 12 h before assay. Different concentrations of CAEF (10, 20, 25, 40, 60, and 80 mg/L), CGA (0.3, 3, 30, 300, and 600 lmol/L), and simvastatin (0.1, 1, 10, 50, and 100 lmol/L) were added for another 24 or 48 h in 5% CO2 at 37 C, and then treated with MTT for 4 h. Cells were treated with 150 lL of dimethyl sulfoxide (DMSO) and shaken for 10 min. The cytotoxicity was measured by the absorbance at 490 nm. Oil red O staining To observe intracellular total lipid, HepG2 cells were treated with or without different drugs for 48 h. Then the medium was discarded, and the cells were washed with PBS twice, fixed with 4% polyformaldehyde in PBS at room temperature for 10 min. After washed with distilled water three times, Oil Red O working solution (three parts 0.5% Oil Red O in isopropanol plus two parts distilled water) was added into all wells including the background wells without cells for 30 min. Stained cells were washed with PBS twice. Microscopic images were taken to visualize red oil droplets staining in different cells with the Olympus microscope (Olympus Corporation, Tokyo, Japan). Determination of extracellular cholesterol and triglyceride levels HepG2 cells were seeded in 24-well plates and cultured. All studies were conducted using 80–90% confluent cells. The cells were divided into different groups and treated in the absence or presence of various drugs. After 24 or 48 h, the supernatant of cells was collected for analysis of total
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cholesterol (TC) and triacylglycerol (TG) content using TC and TG kits (Beihuakangtai, Beijing, China).
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Immunocytochemistry HepG2 cells were seeded on poly-L-lysine-coated slides in a six-well plate and divided into five groups. After treatment with different drugs for 48 h, the slides were washed extensively with PBS, fixed in 4% paraformaldehyde for 30 min at room temperature, and then were stained with a kit (Boaoseng, Beijing, China, SP-0023). The slides were treated with 0.3% hydrogen peroxide in PBS for 10 min to inactivate endogenous peroxidase. After washed three times with PBS, the normal goat serum was added into slides and incubated at 37 C for 10 min. Cells were then washed and incubated at 4 C with 1:50 rabbit polyclonal HMGCR antibody (Boaoseng, Beijing, China, LOT. YE0905W, bs-5068R) overnight, washed, and incubated with biotinylated secondary antibody for 30 min, and peroxidase-labeled streptavidin for 30 min. Slides staining was developed with 3,30 -diaminobenzidine (Zhongshanjinqiao, Beijing, China) for 25 min. Then, the nuclei were counterstained with H&E and dehydrated by graded alcohol. For negative controls, cells were treated as above but with PBS instead of the primary antibody. Staining of slides with no drug treatment was used as normal control. RNA extraction and RT-PCR HepG2 cells (5 105 cells/mL) were treated with drugs for 48 h separately. The total RNA was extracted based on the guidelines of the manufacture using an RNA isolation kit (Total RNA Extractor, Sangon, Shanghai, China). Total RNA was reversely transcribed into complementary DNA (cDNA) using a cDNA synthesis kit (TaKaRa PrimeScriptÔ RT reagent Kit, Baosheng, Dalian, China) and were amplified with PCR (TaKaRa Ex Taq PCR Kit, Baosheng, Dalian, China). The primers used in this experiment were as follows: ABCA1 sense 50 -AGGCCCAGACCTGTAAATGC-30 , antisense 50 -CTCACCAACCTTGCCAACTTC-30 ; SREBP2 sense 50 -CAGACGCCAAGATGCACAAG-30 , antisense 50 -T GGCTCATCTTTGACCTTTGC-30 ; CYP7A1 sense 50 -CATT TGGGCACAGAAGCATTG-30 , antisense 50 -AGGCAGCGG TCTTTGAGTTAG-30 ; LXRa sense 50 -AAGAAACTGAAG CGGCAAGA-30 , antisense 50 -AGCAATGAGCAAGGCAAA CT-30 ; AMPKa sense 50 -TCAATCGTTCTGTCGCCAC-30 , antisense 50 -ATACGGTTTGCTCTGACTTCG-30 ; HMGCR sense 50 -CTCCGCAGGCTATTTGTTCAG-30 , antisense 50 -G CTAAGAGCGTTCGTGGGTC-30 . The sense 50 -ACTCCTC CACCTTTGACGCTG-30 , and antisense primers 50 -CTCTCT TCCTCTTGTGCTCTTGC-30 for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as the control for measuring the total RNA content of each sample. PCR was performed at 95 C for 30 s, followed by 56 C (SREBP2, GAPDH, and LXRa), 57 C (ABCA1, AMPKa, and HMGCR), or 58 C (SREBP2) for 30 s, and 72 C for 45 s. The last cycle was followed by a final extension step at 72 C for 5 min. The amplified products were analyzed on 1% agarose gel electrophoresis under 120 V and visualized by staining with SYBR. Scanning densitometry was performed by Quality One Software (SPSS Inc., Chicago, IL).
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Western-blot analysis The cells were harvested by trypsinization and rinsed twice with ice-cold PBS buffer. Then the whole cell proteins were lysed in a protein extraction kit (Protease inhibitor cocktail set I, Calbiochem, San Diego, CA). Insoluble protein was removed by centrifugation at 14 000 rpm for 20 min and soluble protein concentrations were quantified using a BioRad protein kit (Pierce, Rockford, IL). Equal amounts of protein (40 lg/lane) were resolved by 10% SDS-PAGE and transferred to PVDF (polyvinylidene fluoride) (Millipore, IPVH00010, Millipore Corporation, New Orleans, LA) membrane, which was blocked 1 h with 5% non-fat milk and then incubated with the primary antibody for GAPDHHRP (Kangcheng, Shanghai, KC-5G5, China) and rabbit antihuman HMGCR (Cell Signaling Technology, Danvers, MA) at 1:1000 dilution and secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, TX) at 1:10 000 dilution. Enhanced chemiluminescence (ECL) (Millipore, WBKLS0500, Millipore Corporation, New Orleans, LA) was used to develop the blots. The protein bands were detected by ChemiDoc XRS systems and Quality One 4.6.1 (Bio-Rad, Hercules, CA) software and the density of bands were analyzed by the same program. Statistical analysis All data were presented as means ± SD and were obtained from three separate experiments. Statistical analyses were tested by one-way analysis of variance followed by Dunnett’s test. p50.05 was set statistically significant.
Results The content of CGA in CAEF Using the paper chromatography, the Rf value of CGA in the crude extract was 0.70 in accord with standard preparation of CGA observed at 270 nm. The CGA chromatogram from CAEF is shown in Figure 1. Figure 1(A) shows the chromatogram of authentic CGA (10 mg/L). Identification of CGA was based on the comparison of the retention time with the authentic CGA analyzed under the same chromatographic conditions, while the quantitative data were calculated from the calibration curves. At concentrations ranging between 5.0 and 30.0 mg/L, the peak area of CGA showed good linear relationship (g ¼ 0.9998), the average recovery of 98.65% and RSD of 1.88% was measured by HPLC. The content of CGA in the final extract was 42.43%. Effect of CAEF on lipid accumulation on HepG2 cells HepG2 cell viability was tested using the MTT assay. At concentrations up to 25 mg/L, no significant cytotoxicity was observed for CAEF, while the cell survival rate was decreased as the drug concentration increased to 80 mg/L. In HepG2 cells, the IC50 values were 59.2 mg/L for CAEF, 335.9 lmol/L for CGA, and 10.5 lmol/L for simvastatin. Figure 2 shows that the non-cytotoxic concentration of CAEF, either 24 or 48 h, was 25 mg/L of CAEF (42.43%). Based on these results, the 25 mg/L CAEF, the 30 lmol/L CGA (the same concentration contained in CAEF), and 10 lmol/L simvastatin were used for all the subsequent experiments.
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Figure 1. HPLC chromatograms of authentic CGA and CAEF: (A) HPLC elution profiles of authentic CGA (10 mg/L); (B) HPLC elution profiles of CAEF. Detection was carried out at 270 nm.
To observe the effect of CAEF on lipid deposition, Oil red O staining was used to view lipid droplets in HepG2 cells with different treatments. Figure 3 shows that the lipid droplet accumulation of cells treated with oleate was markedly increased compared with normal controls. Treatment with simvastatin slightly attenuated the lipid droplet accumulation; however, lipid accumulation was suppressed significantly when treated with CGA or CAEF. It has been reported that stimulating TG hydrolysis could diminish fat stores, thereby combating obesity (Yun, 2010).
Therefore, we examined the effects of CAEF on the TC and TG accumulation in the culture medium of HepG2 cells. Cells were treated with each drug for 24 or 48 h, the extracellular level of TC and TG was increased, as shown in Figure 4. Addition of CAEF produced a significant increase in the concentrations of the secreted TC and TG, higher than CGA and simvastatin. This up-regulation of TC and TG contents by CAEF is in the line with the finding that the cellular lipid droplet accumulation is reduced in the Oil red O study.
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Effect of CAEF on the HMGCR protein expression Intracellular localization of HMGCR protein was determined by immunostaining. As shown in Figure 5, numerous brown granules are the positive staining of HMGCR, which are predominantly localized in the cytoplasm of HepG2 cells. Simvastatin inhibited the expression of HMGCR. The representative immunocytochemistry data show that the endogenous level of HMGCR protein is decreased markedly compared with untreated cells, after treatment with CAEF or CGA for 48 h. Western-blot analysis showed the expressions of HMGCR in HepG2 cells treated with all drugs were suppressed, corresponding to the decreased mRNA levels (Figure 6).
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Figure 2. The effect of CAEF on the growth of HepG2 cell. Data are expressed as mean ± SD.
Effect of CAEF on the gene expression related to cholesterol metabolism As we know, the most important aspects of cholesterol metabolism are regulated at the transcriptional level. To study the mechanism of cholesterol regulation of CAEF, we
Figure 3. The effects of CAEF on lipid content in HepG2 cells. HepG2 cells were stained with Oil red O solution. Images of cells were captured by microscope at 250 original magnification showing lipid accumulation in cells stained by Oil red O. (A) Control, (B) Oleate (0.1 mmol/L), (C) simvastatin (10 lmol/L), (D) CGA (30 lmol/L), and (E) CAEF (25 mg/L). Each value represents the mean ± SD. n ¼ 4, *p50.05 versus control, # p50.05 versus Oleate.
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Figure 4. Effect of CAEF on the secretion of TC and TG by HepG2 cells in minimum essential medium. (A) TC, (B) TG. simvastatin (10 lmol/L), CGA (30 lmol/L), and CAEF (25 mg/L). Each value represents the mean ± SD. n ¼ 6. After 24 h, *p50.05 versus control; after 48 h, #p50.05 versus control.
Figure 5. Representative immunocytochemical images of HMGCR expression in HepG2 cells treated with CAEF (original magnification 250). (A) Negative, (B) normal, (C) simvastatin (10 lmol/L), (D) CGA (30 lmol/L), and (E) CAEF (25 mg/L). HMGCR positive staining result was the numerous brown granules in the cytoplasm in HepG2 cells. Cell nuclei were counterstained with hematoxylin (blue). The positive staining result was the numerous oil drops in the cytoplasm in HepG2 cells (red). Each value represents the mean ± SD. n ¼ 4, *p50.05 versus control.
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Figure 6. The effect of drugs on the expression of HMGCR. Each value represents the mean ± SD. *p50.05 versus control, #p50.05 versus simvastatin.
examined the gene expression related to cholesterol metabolism in HepG2 cells. Figure 7 shows the effects of CAEF, CGA, and simvastatin on the expression levels of the ABCA1, CYP7A1, and HMGCR in HepG2 cells. Following 48 h of treatment, the mRNA levels of the HMGCR gene in the CAEF, CGA, and simvastatin groups were significantly lower than that of the control group. CAEF and CGA up-regulated the CYP7A1 gene significantly with respect to the control level but not as much as simvastatin. Treatment with CAEF and CGA resulted in higher expression levels of the ABCA1 gene than did simvastatin. Consequently, it was of interest to determine whether CAEF might play a pivotal role in the regulation of cholesterol activities associated with the signal transduction. Thus, we conducted an additional experiment by measuring the mRNA expression for SREBP2, LXRa, and AMPKa2 (Figure 7). All drugs decreased the SREBP2 expression significantly compared with the control level but not as much as simvastatin. The LXRa expression level in the CAEF was not significantly different from those in the control group, but in the CGA, it was lower than the levels of all other groups. Treatment with simvastatin resulted in higher expression of LXRa. In the case of the AMPKa2 gene, all drugs except simvastatin increased the gene expression level compared with the control group.
Discussion This study clearly demonstrated that CAEF had beneficial effects in the molecular and enzymatic regulation on cholesterol metabolism in HepG2 cells, as evidenced by reduced Oil red O staining, increased TC and TG secretion, induction of genes related to cholesterol metabolism (AMPKa2 and CYP7A1), transport (ABCA1), and suppression of cholesterol
Figure 7. The effect of CAEF on expressions of regulation cholesterol activities associated genes in HepG2 cells. (1) Simvastatin (10 lmol/L), (2) CAEF (25 mg/L), (3) CGA (30 lmol/L), and (4) control. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control for modulated gene expression. Each value represents the mean ± SD. *p50.05 versus control; #p50.05 versus simvastatin.
synthesis genes (SREBP2 and HMGCR). The suppressed HMGCR was also confirmed by immunohistochemistry and Western-blot analysis. To our knowledge, this is among the first to demonstrate the beneficial effects of CAEF on cholesterol metabolism at cellular and molecular levels, and these results would add to our understanding of pharmacological basis of using CAEF in the treatment of dyslipidemia. More and more studies found that hundreds of extracts from plants exhibited good effects in anti-obesity and regulated lipid disorders in a multilevel and multi-target way with relatively few side effects (Yun, 2010). Therefore, searching the natural materials from medicinal plants in the management of the obesity epidemic has been a hot area. The profile of CAEF obtained by HPLC analysis showed that CAEF mainly consists of CGA, which accounts for 42.43% of the CAEF by weight. It has been reported that CGA markedly decreased the concentration of TC and TG in plasma and liver, which is most probably due to the increase of the fatty acid utilization in the liver and the alteration of the activities of lipid metabolism enzymes (Cho et al., 2010; Meng et al., 2013; Ong et al., 2012; Wan et al., 2013; Zhang et al., 2013). Hence, CGA may be the active components of CAEF used for the treatment of hyperlipidemia. To verify this hypothesis, we tested CGA and CAEF (containing the same concentration of CGA) for
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lipid-altering activities in HepG2 cells, using simvastatin as a positive control. In Oil red O staining observations, the decreasing effect of CAEF on the neutral lipid accumulation was not only greater than simvastatin but also better than CGA, which was further supported by our findings showing that the efflux of TC and TG were induced by CAEF. Compared with the basal level, in cells treated with three drugs for 48 h, TC levels were increased 3.94-fold by simvastatin, 2.94-fold by CGA, and 4.06-fold by CAEF, while TG levels were increased 24.67-fold by simvastatin, 2.17-fold by CGA, and 31.00-fold by CAEF, respectively, in the culture medium. Although CGA has a potent lipid-lowering effect and CAEF has a high content of CGA, the changes in potency are not in line with the content of CGA, it is reasonable to deduce that other components may also participate in the hypolipidermic effect of CAEF. As we previously reported, as determined with copper soap colorimetry, CAEF had stronger inhibitory activity of pancreatic lipase (IC50 value ¼ 2.6 lg/mL) than CGA (IC50 value ¼ 4.0 lg/mL), but less than Orlistat (IC50 value ¼ 1.7 lg/mL) (Li et al., 2012a). In a rat model of acute fat-loading, the effect of CAEF on reducing the fat absorption through the intestinal tract was obviously stronger than orlistat (Li et al., 2012c); CAEF could also significantly increase the excretion of cholesterol and bile acid into feces (Li et al., 2012c). Pancreatic lipase not only is a key enzyme in exogenous fat digestion but also is one of the most widely studied targets for lipid-lowering and anti-obesity actions (de la Garza et al., 2011). It is apparent from the data of Oil red O staining and the efflux of TC and TG that these mechanisms may be involved in the hypolipidemic effects of CAEF. These effects are also consistent with the literature that multiple mechanisms facilitate endogenous cholesterol conversion into bile acids, control cholesterol absorption and synthesis, and promote TC and TG excretion. The metabolic pathways of liver cholesterol include three parts: secretion of HDL into blood, conversion of cholesterol into bile acid, and direct elimination of cholesterol into feces. ABCA1 is involved in the cholesterol efflux from cells and absorption from the intestinal tract. The redundant cholesterol in peripheral tissues can be eliminated by carrying back to liver, biotransformed, and excreted into the bile, called as reverse cholesterol transport (RCT). ABCA1 plays a crucial role in the transmembrane transport of cholesterol and takes part in the synthesis of HDL and the initial phase of RCT (Khera et al., 2011). RT-PCR analysis showed that all three drugs can increase the ABCA1 gene mRNA level, but CAEF and CGA were more effective than simvastatin. The data are well documented that increased expression of ABCA1 is accompanied with the promoting RCT and the increasing the cholesterol secretion from intestinal mucosa cells, resulting in the decreased of cholesterol assimilation (Favari et al., 2004). Bile acids play a vital role in regulating cholesterol and lipid homeostasis. CYP7A1 catalyzes the first and rate-controlling step in the classic pathway for cholesterol converting to bile acids (Shin et al., 2008). In HepG2 cells, simvastatin, CAEF, and CGA also significantly increased the CYP7A1 mRNA level. Based on the previous results, we deduced that CAEF increased bile acids in feces in vivo and reduced concentration of TC in liver cells may attribute to the activation of CYP7A1
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resulting in the promotion of cholesterol conversion to bile acids. AMP-activated protein kinase (AMPK) is served as a metabolic master switch in response to alterations in cellular energy charge. Over-expression of active AMPKa subunit has been shown to decrease intracellular TG and cholesterol in HepG2 cells (Pan et al., 2012; Spencer-Jones et al., 2006). Thus, identifying medicinal plant extracts that stimulate AMPK activity may give effective options for dyslipidemia and obesity. Liver X receptor-a (LXRa) belongs to a superfamily of the nuclear hormone receptor. Its target genes are involved in regulating lipid homeostasis (Lian et al., 2011; Murase et al., 2010; Zhong et al., 2013). It has been reported that AMPK directly inhibited ligand-induced LXR activity in addition to blocking production of endogenous LXR ligands (Yap et al., 2011). In this study, the expression levels of AMPKa2 mRNA in HepG2 cells were increased by CGA, which was consistent with the previous report (Ong et al., 2012). We found that CGA could also significantly inhibit the expression level of LXRa mRNA. These results suggested that CGA may exert suppressions of TG and TC levels in hepatocytes via modulation of AMPKLXR signaling pathways. Although equally containing CGA, the combined action of other active ingredients of CAEF could activate AMPKa2 mRNA, while CAEF had no effects on LXRa mRNA. Moreover, we observed that simvastatin increased the expression levels of LXRa mRNA, consistent with recent reports (Wang et al., 2014). Maintaining hepatocyte lipid homeostasis relays on the regulation of SREBPs located in endoplasmic reticulum, which is the most important transcription factor regulating de novo cholesterol, free fatty acid, and triglyceride in the liver. SREBP2 is the main regulator of the genes involved in the cholesterol metabolism, including HMGCR. The effects of CAEF, CGA, and simvastatin on the expression of SREPB2 mRNA level were investigated with RT-PCR. The results showed that the expressions of SREBP2 were significantly suppressed by CAEF and simvastatin. Both Oil red O staining and TC showed a decrease in the cellular cholesterol content by CAEF, which was associated with the inhibition of HMGCR, which is in charge of cholesterol synthesis. HMGCR in liver and small intestine is a rate-limiting enzyme in the synthesis of cholesterol (Bommer et al., 2011; Seo et al., 2011; Torres et al., 2006). Statins are classic HMGCR inhibitors available in the market as improving lipid metabolism drugs. It has been reported recently that simvastatin treatment upregulated intestinal lipid secretion pathways in a rodent model of the metabolic syndrome (Borthwick et al., 2014). We previously found that CAEF dose dependently and strongly inhibited the activity of HMGCR from normal pig liver at doses ranging from 0.1 to 100 lg/mL than simvastatin at the same concentrations (Li et al., 2012a). Hence, we investigated the effects of CAEF, CGA, and simvastatin on the expression of HMGCR at mRNA and protein levels. Importantly, all three drug treatments of HepG2 cells significantly decreased the expressions of HMGCR mRNA and protein, which was corroborated with the data of RT-PCR, western-blot, and immunocytochemistry. According to the in vitro results, the effects of inhibiting accumulation of TC and TG by CAEF
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were the strongest among three drugs, although the suppression of the HMGCR expression by CAEF is lower than both simvastatin and CGA. These data suggested that CAEF decreasing lipid accumulation was likely related to the inhibition of the expression of genes involved in cholesterol biosynthesis in the liver by HMGCR signal pathway. Taken together, these findings of the study demonstrated that CAEF improved the lipid metabolism by the transcriptional activating AMPK and inhibiting downstream targets, such as SREBP2 and HMGCR, to suppress the TC synthesis and TG levels in liver cells. CAEF also increased ABCA1 and CYP7A1 to enhance TC efflux and bile acid transport, synthesis, and excretion, and these effects were superior to simvastatin. These findings will add our understanding of how E. ulmoides exerts anti-obesity effects and regulates lipid metabolism. Eucommia ulmoides and CGA-containing extracts could represent a promising dietary supplement to prevent obesity and dyslipidemia.
Declaration of interest This work was supported by project from Guizhou Provincial Modernization of Traditional Chinese Medicine Foundation of China (Grant No. QKHZY [2013] 3018) (W. L.), The Project Natural Science Foundation of China (No. 21162046) (Y. C.); and The Research Funds of Department of Education of Guizhou Province of China (Grant No. QKJ 2010042) (C. X.).
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Pharmaceutical Biology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/iphb20
Chlorogenic acid-enriched extract from Eucommia ulmoides leaves inhibits hepatic lipid accumulation through regulation of cholesterol metabolism in HepG2 cells a
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Shun Hao , Yuan Xiao , Yan Lin , Zhentao Mo , Yang Chen , Xiaofeng Peng , Canhui Xiang , a
Yiqi Li & Wenna Li a
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Zunyi Medical University, Zhuhai Campus, Zhuhai, Guangdong, China and
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Pharmaceutical Preparation Section, Guizhou Province People's Hospital, Guiyang, Guizhou, China Published online: 05 May 2015.
To cite this article: Shun Hao, Yuan Xiao, Yan Lin, Zhentao Mo, Yang Chen, Xiaofeng Peng, Canhui Xiang, Yiqi Li & Wenna Li (2015): Chlorogenic acid-enriched extract from Eucommia ulmoides leaves inhibits hepatic lipid accumulation through regulation of cholesterol metabolism in HepG2 cells, Pharmaceutical Biology To link to this article: http://dx.doi.org/10.3109/13880209.2015.1029054
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http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–9 ! 2015 Informa Healthcare USA, Inc.. DOI: 10.3109/13880209.2015.1029054
ORIGINAL ARTICLE
Chlorogenic acid-enriched extract from Eucommia ulmoides leaves inhibits hepatic lipid accumulation through regulation of cholesterol metabolism in HepG2 cells Shun Hao1, Yuan Xiao2, Yan Lin1, Zhentao Mo1, Yang Chen1, Xiaofeng Peng1, Canhui Xiang1, Yiqi Li1, and Wenna Li1 Zunyi Medical University, Zhuhai Campus, Zhuhai, Guangdong, China and 2Pharmaceutical Preparation Section, Guizhou Province People’s Hospital, Guiyang, Guizhou, China
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Abstract
Keywords
Context: Eucommia ulmoides Oliver (Eucommiaceae) leaf exhibits beneficial lipid-lowering and anti-obesity effects. However, the mechanisms remain unknown. Objective: The objective of this study is to investigate the lipid-lowering effects of chlorogenic acid (CGA)-enriched extract from this plant (CAEF) in human hepatoma HepG2 cells, focusing on cholesterol metabolism. Materials and methods: HepG2 cells were treated with CAEF (10, 20, 25, 40, 60, and 80 mg/L), CGA (0.3, 3, 30, 300, and 600 lmol/L), and simvastatin (0.1, 1, 10, 50, and 100 lmol/L) for 24 or 48 h. The cytotoxicity, Oil red O staining, total cholesterol, and triacylglycerol in supernatants were determined. The mRNA expression of genes involved in cholesterol metabolism was determined with RT-PCR. The protein expression of HMG-CoA reductase (HMGCR) was examined by immunocytochemistry and western-blot. Results: The IC50 values were 59.2 mg/L for CAEF, 335.9 lmol/L for CGA, and 10.5 lmol/L for simvastatin. By treating cells with CAEF (25 mg/L), CGA (30 lmol/L), or simvastatin (10 lmol/L) for 48 h, the efflux of total cholesterol and triacylglycerol was increased (CAEF, 4.06- and 31.00-folds; CGA, 2.94- and 2.17-folds; and simvastatin, 3.94- and 24.67-folds), and the cellular lipid droplets were reduced in Oil red O staining. CAEF and CGA increased mRNA expression of ABCA1, CYP7A1, and AMPKa2, while CAEF and simvastatin decreased SREBP2. However, their effects on LXRa mRNA expression were variable. Importantly, all drugs significantly inhibited protein expression of HMGCR at mRNA and protein levels. Discussion and conclusion: CAEF is a promising dietary supplement to prevent obesity and dyslipidemia and the effects appear to be due, at least in part, to regulating cholesterol metabolism through inhibition of HMGCR in HepG2 cells.
ABCA1, AMPKa, chlorogenic acid, CYP7A1, HMGCR, lipid accumulation, SREBP2
Introduction Obesity is one of the world-wide epidemic diseases affecting human health. A survey showed that from year 2002 to 2010, waist circumference and abdominal obesity have continued to rise in a southern Chinese population of 85 million residents in Guangdong, while in northeast China, obesity in adults reached 37.71% in 2011 (Lao et al., 2015; Shi, 2011). Obesity not only reduces the quality of life but also results in the serious complications. Dysregulation of lipid metabolism plays a key role in the pathology of obesity, which leads to fatty liver and hyperlipidemia as the most common complications. It is reported that improving dyslipidemia is one of the most important strategies for obesity prevention and treatment (Yun, 2010).
Correspondence: Prof. Wenna Li, Zhuhai Campus, Zunyi Medical University, Jinwan District, Zhuhai, Guangdong 519041, PR China. Tel: +86 756 762 3384. Fax: +86 756 763 7938. E-mail: [email protected]
History Received 16 July 2014 Accepted 4 March 2015 Published online 7 April 2015
Eucommia ulmoides Oliver (Eucommiaceae), also called Du-Zhong, is a native traditional medicinal plant in China, which is also widely used in Japan and Korea. Previously, only the bark of E. ulmoides was used as medicinal materials. However, it is reported that the leaves containing many phytochemicals are also found in the bark, and other bioactive components are found only in the leaves (Yuan et al., 2013). The leaf of Eucommia has been recorded officially in Chinese Pharmacopoeia since 2005 (Chinese Pharmacopoeia Committee, 2005). It has been found that the Eucommia leaf extracts exhibit beneficial lipid-lowering effects, significantly decrease the cholesterol and triglycerides, and increase the HDL-C in the serum of high-fat diet mice and hamsters (Choi et al., 2008; Park et al., 2006). Several recent studies have shown that the extracts of Eucommia leaf can also have the anti-obesity and antimetabolic syndrome activity, which were mainly attributed to several mechanisms as follows: (1) stimulating lipolysis and thermogenesis through elevations in adipose tissue sympathetic nerve activity, and suppressing the appetite by
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inhibiting the parasympathetic nerves of the gastrointestinal tract (Horii et al., 2010), (2) diminishing ATP production of white adipose tissue, accelerating fatty acid b-oxidation of liver, and increasing the use of ketone bodies and glucose in the skeletal muscle (Fujikawa et al., 2010), and (3) DNA microarray analysis found that genes related to the peroxisome proliferator-activated receptor a and d signaling pathways were up-regulated (Kobayashi et al., 2012). Those anti-obesity effects were observed in humans. After 8 weeks of given oral Eucommia capsule containing the Eucommia leaf extracts to 15 patients or placebo to control group of 12 patients with abdominal obesity, the visceral fat (21% versus 4.1%) and subcutaneous fat (11.4% versus + 7.2%) were decreased significantly, as evidenced by CT scanner, the body weight, abdominal circumference, and BMI of patients (Zhou et al., 2011). The main active components of Eucommia leaf extracts that produce anti-obesity and hypolipidemia effect are chlorogenic acid (CGA), geniposidic acid, and asperuloside (Hirata et al., 2011; Kobayashi et al., 2012). CGA is one of the active compounds enriched in leaves than in barks, and has been shown to have anti-obesity property and improve lipid metabolism in high-fat induced obese mice (Cho et al., 2010), and stimulate glucose transport in the skeletal muscle via AMPK activation (Ong et al., 2012). We previously isolated and purified of the CGA-enriched extract from Eucommia leaf extracts (CAEF) (Li et al., 2012b) and also found that CAEF, like orlistat, can suppress pancreatic lipase activity both in vitro and in vivo. CAEF is more effective in inhibition of cholesterol micelles formation than CGA and has higher inhibitory potency on HMG-CoA reductase (HMGCR) than simvastatin in vitro. In a rat model of acute fat-loading, CAEF could also significantly increase the excretion of cholesterol and bile acid into feces (Li et al., 2012a,c). To further examine the beneficial effects of CAEF in lipid metabolism, in this study, we used an in vitro model, in which the cellular and molecular mechanisms could be better dissected to evaluate hypolipidemic and beneficial effects of CAEF in human hepatoma HepG2 cells.
Materials and methods Plant materials and extraction The leaves of E. ulmoides were purchased from Zunyi, Guizhou, China, in December 2012, and identified by Dr. Yang Chen. The voucher specimens are deposited at the Department of Pharmacology, Zunyi Medical College, Zhuhai Campus, China. CAEF was prepared as reported (Li et al., 2012b). In brief, the leaves were cleaned, grounded, and extracted in 60% ethanol (1:8 w/v), in which pH was adjusted to 5.0 and ultrasonicated for 40 min at room temperature. After filtration, the supernatant was concentrated, and the concentrate was purified by chitosan flocculation and decolorized by polysaccharide. The crude extract solution was absorbed by NKA-9 macroporous resin, eluted with 50% ethanol. The resultant aqueous extract was evaporated in a rotary evaporator under reduced pressure. After freeze drying at 50 C, the final extracts were obtained and kept in 20 C.
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During the extraction of leaves of E. ulmoides with ethanol, the paper chromatography-UV method was used to qualitatively identify the main ingredients. On the filter paper (Xinhua, Hangzhou, China) and with the water:acetic acid:nbutanol in volume ratio 5:1:4 as a mobile phase, the extracts were chromatophyed, air dried, and observed at 254 nm, the spots were cut and dissolved in 50% methanol and ultrasonicated for 20 min. The quantification of supernatant was performed by UV Spectrophotometry (Shimadzu, UV-2550, Shimadzu Corporation, Kyoto, Japan) at 327 nm. After the final extracts were collected, the amounts of CGA in the extract were analyzed with high-performance liquid chromatography (HPLC) in comparison with standard preparation of CGA by the method of Chinese Pharmacopoeia 2005 edition (Chinese Pharmacopoeia Committee, 2005). Cell culture Human hepatoma HepG2 cells were grown in RPMI-1640 (Invitrogen, Waltham, MA) supplemented with 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 lg/mL streptomycin) in an incubator at 37 C with 5% CO2 with medium changes three times a week. HepG2 cell viability was assayed by trypan blue exclusion method. Cytotoxicity assay The HepG2 cell growth was determined by MTT assay. HepG2 cells were seeded in a 96-well plate at a density of 1 105 cells/well and cultured to reach confluence. Cultures were changed to serum-free medium 12 h before assay. Different concentrations of CAEF (10, 20, 25, 40, 60, and 80 mg/L), CGA (0.3, 3, 30, 300, and 600 lmol/L), and simvastatin (0.1, 1, 10, 50, and 100 lmol/L) were added for another 24 or 48 h in 5% CO2 at 37 C, and then treated with MTT for 4 h. Cells were treated with 150 lL of dimethyl sulfoxide (DMSO) and shaken for 10 min. The cytotoxicity was measured by the absorbance at 490 nm. Oil red O staining To observe intracellular total lipid, HepG2 cells were treated with or without different drugs for 48 h. Then the medium was discarded, and the cells were washed with PBS twice, fixed with 4% polyformaldehyde in PBS at room temperature for 10 min. After washed with distilled water three times, Oil Red O working solution (three parts 0.5% Oil Red O in isopropanol plus two parts distilled water) was added into all wells including the background wells without cells for 30 min. Stained cells were washed with PBS twice. Microscopic images were taken to visualize red oil droplets staining in different cells with the Olympus microscope (Olympus Corporation, Tokyo, Japan). Determination of extracellular cholesterol and triglyceride levels HepG2 cells were seeded in 24-well plates and cultured. All studies were conducted using 80–90% confluent cells. The cells were divided into different groups and treated in the absence or presence of various drugs. After 24 or 48 h, the supernatant of cells was collected for analysis of total
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cholesterol (TC) and triacylglycerol (TG) content using TC and TG kits (Beihuakangtai, Beijing, China).
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Immunocytochemistry HepG2 cells were seeded on poly-L-lysine-coated slides in a six-well plate and divided into five groups. After treatment with different drugs for 48 h, the slides were washed extensively with PBS, fixed in 4% paraformaldehyde for 30 min at room temperature, and then were stained with a kit (Boaoseng, Beijing, China, SP-0023). The slides were treated with 0.3% hydrogen peroxide in PBS for 10 min to inactivate endogenous peroxidase. After washed three times with PBS, the normal goat serum was added into slides and incubated at 37 C for 10 min. Cells were then washed and incubated at 4 C with 1:50 rabbit polyclonal HMGCR antibody (Boaoseng, Beijing, China, LOT. YE0905W, bs-5068R) overnight, washed, and incubated with biotinylated secondary antibody for 30 min, and peroxidase-labeled streptavidin for 30 min. Slides staining was developed with 3,30 -diaminobenzidine (Zhongshanjinqiao, Beijing, China) for 25 min. Then, the nuclei were counterstained with H&E and dehydrated by graded alcohol. For negative controls, cells were treated as above but with PBS instead of the primary antibody. Staining of slides with no drug treatment was used as normal control. RNA extraction and RT-PCR HepG2 cells (5 105 cells/mL) were treated with drugs for 48 h separately. The total RNA was extracted based on the guidelines of the manufacture using an RNA isolation kit (Total RNA Extractor, Sangon, Shanghai, China). Total RNA was reversely transcribed into complementary DNA (cDNA) using a cDNA synthesis kit (TaKaRa PrimeScriptÔ RT reagent Kit, Baosheng, Dalian, China) and were amplified with PCR (TaKaRa Ex Taq PCR Kit, Baosheng, Dalian, China). The primers used in this experiment were as follows: ABCA1 sense 50 -AGGCCCAGACCTGTAAATGC-30 , antisense 50 -CTCACCAACCTTGCCAACTTC-30 ; SREBP2 sense 50 -CAGACGCCAAGATGCACAAG-30 , antisense 50 -T GGCTCATCTTTGACCTTTGC-30 ; CYP7A1 sense 50 -CATT TGGGCACAGAAGCATTG-30 , antisense 50 -AGGCAGCGG TCTTTGAGTTAG-30 ; LXRa sense 50 -AAGAAACTGAAG CGGCAAGA-30 , antisense 50 -AGCAATGAGCAAGGCAAA CT-30 ; AMPKa sense 50 -TCAATCGTTCTGTCGCCAC-30 , antisense 50 -ATACGGTTTGCTCTGACTTCG-30 ; HMGCR sense 50 -CTCCGCAGGCTATTTGTTCAG-30 , antisense 50 -G CTAAGAGCGTTCGTGGGTC-30 . The sense 50 -ACTCCTC CACCTTTGACGCTG-30 , and antisense primers 50 -CTCTCT TCCTCTTGTGCTCTTGC-30 for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as the control for measuring the total RNA content of each sample. PCR was performed at 95 C for 30 s, followed by 56 C (SREBP2, GAPDH, and LXRa), 57 C (ABCA1, AMPKa, and HMGCR), or 58 C (SREBP2) for 30 s, and 72 C for 45 s. The last cycle was followed by a final extension step at 72 C for 5 min. The amplified products were analyzed on 1% agarose gel electrophoresis under 120 V and visualized by staining with SYBR. Scanning densitometry was performed by Quality One Software (SPSS Inc., Chicago, IL).
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Western-blot analysis The cells were harvested by trypsinization and rinsed twice with ice-cold PBS buffer. Then the whole cell proteins were lysed in a protein extraction kit (Protease inhibitor cocktail set I, Calbiochem, San Diego, CA). Insoluble protein was removed by centrifugation at 14 000 rpm for 20 min and soluble protein concentrations were quantified using a BioRad protein kit (Pierce, Rockford, IL). Equal amounts of protein (40 lg/lane) were resolved by 10% SDS-PAGE and transferred to PVDF (polyvinylidene fluoride) (Millipore, IPVH00010, Millipore Corporation, New Orleans, LA) membrane, which was blocked 1 h with 5% non-fat milk and then incubated with the primary antibody for GAPDHHRP (Kangcheng, Shanghai, KC-5G5, China) and rabbit antihuman HMGCR (Cell Signaling Technology, Danvers, MA) at 1:1000 dilution and secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, TX) at 1:10 000 dilution. Enhanced chemiluminescence (ECL) (Millipore, WBKLS0500, Millipore Corporation, New Orleans, LA) was used to develop the blots. The protein bands were detected by ChemiDoc XRS systems and Quality One 4.6.1 (Bio-Rad, Hercules, CA) software and the density of bands were analyzed by the same program. Statistical analysis All data were presented as means ± SD and were obtained from three separate experiments. Statistical analyses were tested by one-way analysis of variance followed by Dunnett’s test. p50.05 was set statistically significant.
Results The content of CGA in CAEF Using the paper chromatography, the Rf value of CGA in the crude extract was 0.70 in accord with standard preparation of CGA observed at 270 nm. The CGA chromatogram from CAEF is shown in Figure 1. Figure 1(A) shows the chromatogram of authentic CGA (10 mg/L). Identification of CGA was based on the comparison of the retention time with the authentic CGA analyzed under the same chromatographic conditions, while the quantitative data were calculated from the calibration curves. At concentrations ranging between 5.0 and 30.0 mg/L, the peak area of CGA showed good linear relationship (g ¼ 0.9998), the average recovery of 98.65% and RSD of 1.88% was measured by HPLC. The content of CGA in the final extract was 42.43%. Effect of CAEF on lipid accumulation on HepG2 cells HepG2 cell viability was tested using the MTT assay. At concentrations up to 25 mg/L, no significant cytotoxicity was observed for CAEF, while the cell survival rate was decreased as the drug concentration increased to 80 mg/L. In HepG2 cells, the IC50 values were 59.2 mg/L for CAEF, 335.9 lmol/L for CGA, and 10.5 lmol/L for simvastatin. Figure 2 shows that the non-cytotoxic concentration of CAEF, either 24 or 48 h, was 25 mg/L of CAEF (42.43%). Based on these results, the 25 mg/L CAEF, the 30 lmol/L CGA (the same concentration contained in CAEF), and 10 lmol/L simvastatin were used for all the subsequent experiments.
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Figure 1. HPLC chromatograms of authentic CGA and CAEF: (A) HPLC elution profiles of authentic CGA (10 mg/L); (B) HPLC elution profiles of CAEF. Detection was carried out at 270 nm.
To observe the effect of CAEF on lipid deposition, Oil red O staining was used to view lipid droplets in HepG2 cells with different treatments. Figure 3 shows that the lipid droplet accumulation of cells treated with oleate was markedly increased compared with normal controls. Treatment with simvastatin slightly attenuated the lipid droplet accumulation; however, lipid accumulation was suppressed significantly when treated with CGA or CAEF. It has been reported that stimulating TG hydrolysis could diminish fat stores, thereby combating obesity (Yun, 2010).
Therefore, we examined the effects of CAEF on the TC and TG accumulation in the culture medium of HepG2 cells. Cells were treated with each drug for 24 or 48 h, the extracellular level of TC and TG was increased, as shown in Figure 4. Addition of CAEF produced a significant increase in the concentrations of the secreted TC and TG, higher than CGA and simvastatin. This up-regulation of TC and TG contents by CAEF is in the line with the finding that the cellular lipid droplet accumulation is reduced in the Oil red O study.
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Effect of CAEF on the HMGCR protein expression Intracellular localization of HMGCR protein was determined by immunostaining. As shown in Figure 5, numerous brown granules are the positive staining of HMGCR, which are predominantly localized in the cytoplasm of HepG2 cells. Simvastatin inhibited the expression of HMGCR. The representative immunocytochemistry data show that the endogenous level of HMGCR protein is decreased markedly compared with untreated cells, after treatment with CAEF or CGA for 48 h. Western-blot analysis showed the expressions of HMGCR in HepG2 cells treated with all drugs were suppressed, corresponding to the decreased mRNA levels (Figure 6).
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Figure 2. The effect of CAEF on the growth of HepG2 cell. Data are expressed as mean ± SD.
Effect of CAEF on the gene expression related to cholesterol metabolism As we know, the most important aspects of cholesterol metabolism are regulated at the transcriptional level. To study the mechanism of cholesterol regulation of CAEF, we
Figure 3. The effects of CAEF on lipid content in HepG2 cells. HepG2 cells were stained with Oil red O solution. Images of cells were captured by microscope at 250 original magnification showing lipid accumulation in cells stained by Oil red O. (A) Control, (B) Oleate (0.1 mmol/L), (C) simvastatin (10 lmol/L), (D) CGA (30 lmol/L), and (E) CAEF (25 mg/L). Each value represents the mean ± SD. n ¼ 4, *p50.05 versus control, # p50.05 versus Oleate.
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Figure 4. Effect of CAEF on the secretion of TC and TG by HepG2 cells in minimum essential medium. (A) TC, (B) TG. simvastatin (10 lmol/L), CGA (30 lmol/L), and CAEF (25 mg/L). Each value represents the mean ± SD. n ¼ 6. After 24 h, *p50.05 versus control; after 48 h, #p50.05 versus control.
Figure 5. Representative immunocytochemical images of HMGCR expression in HepG2 cells treated with CAEF (original magnification 250). (A) Negative, (B) normal, (C) simvastatin (10 lmol/L), (D) CGA (30 lmol/L), and (E) CAEF (25 mg/L). HMGCR positive staining result was the numerous brown granules in the cytoplasm in HepG2 cells. Cell nuclei were counterstained with hematoxylin (blue). The positive staining result was the numerous oil drops in the cytoplasm in HepG2 cells (red). Each value represents the mean ± SD. n ¼ 4, *p50.05 versus control.
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Figure 6. The effect of drugs on the expression of HMGCR. Each value represents the mean ± SD. *p50.05 versus control, #p50.05 versus simvastatin.
examined the gene expression related to cholesterol metabolism in HepG2 cells. Figure 7 shows the effects of CAEF, CGA, and simvastatin on the expression levels of the ABCA1, CYP7A1, and HMGCR in HepG2 cells. Following 48 h of treatment, the mRNA levels of the HMGCR gene in the CAEF, CGA, and simvastatin groups were significantly lower than that of the control group. CAEF and CGA up-regulated the CYP7A1 gene significantly with respect to the control level but not as much as simvastatin. Treatment with CAEF and CGA resulted in higher expression levels of the ABCA1 gene than did simvastatin. Consequently, it was of interest to determine whether CAEF might play a pivotal role in the regulation of cholesterol activities associated with the signal transduction. Thus, we conducted an additional experiment by measuring the mRNA expression for SREBP2, LXRa, and AMPKa2 (Figure 7). All drugs decreased the SREBP2 expression significantly compared with the control level but not as much as simvastatin. The LXRa expression level in the CAEF was not significantly different from those in the control group, but in the CGA, it was lower than the levels of all other groups. Treatment with simvastatin resulted in higher expression of LXRa. In the case of the AMPKa2 gene, all drugs except simvastatin increased the gene expression level compared with the control group.
Discussion This study clearly demonstrated that CAEF had beneficial effects in the molecular and enzymatic regulation on cholesterol metabolism in HepG2 cells, as evidenced by reduced Oil red O staining, increased TC and TG secretion, induction of genes related to cholesterol metabolism (AMPKa2 and CYP7A1), transport (ABCA1), and suppression of cholesterol
Figure 7. The effect of CAEF on expressions of regulation cholesterol activities associated genes in HepG2 cells. (1) Simvastatin (10 lmol/L), (2) CAEF (25 mg/L), (3) CGA (30 lmol/L), and (4) control. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control for modulated gene expression. Each value represents the mean ± SD. *p50.05 versus control; #p50.05 versus simvastatin.
synthesis genes (SREBP2 and HMGCR). The suppressed HMGCR was also confirmed by immunohistochemistry and Western-blot analysis. To our knowledge, this is among the first to demonstrate the beneficial effects of CAEF on cholesterol metabolism at cellular and molecular levels, and these results would add to our understanding of pharmacological basis of using CAEF in the treatment of dyslipidemia. More and more studies found that hundreds of extracts from plants exhibited good effects in anti-obesity and regulated lipid disorders in a multilevel and multi-target way with relatively few side effects (Yun, 2010). Therefore, searching the natural materials from medicinal plants in the management of the obesity epidemic has been a hot area. The profile of CAEF obtained by HPLC analysis showed that CAEF mainly consists of CGA, which accounts for 42.43% of the CAEF by weight. It has been reported that CGA markedly decreased the concentration of TC and TG in plasma and liver, which is most probably due to the increase of the fatty acid utilization in the liver and the alteration of the activities of lipid metabolism enzymes (Cho et al., 2010; Meng et al., 2013; Ong et al., 2012; Wan et al., 2013; Zhang et al., 2013). Hence, CGA may be the active components of CAEF used for the treatment of hyperlipidemia. To verify this hypothesis, we tested CGA and CAEF (containing the same concentration of CGA) for
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lipid-altering activities in HepG2 cells, using simvastatin as a positive control. In Oil red O staining observations, the decreasing effect of CAEF on the neutral lipid accumulation was not only greater than simvastatin but also better than CGA, which was further supported by our findings showing that the efflux of TC and TG were induced by CAEF. Compared with the basal level, in cells treated with three drugs for 48 h, TC levels were increased 3.94-fold by simvastatin, 2.94-fold by CGA, and 4.06-fold by CAEF, while TG levels were increased 24.67-fold by simvastatin, 2.17-fold by CGA, and 31.00-fold by CAEF, respectively, in the culture medium. Although CGA has a potent lipid-lowering effect and CAEF has a high content of CGA, the changes in potency are not in line with the content of CGA, it is reasonable to deduce that other components may also participate in the hypolipidermic effect of CAEF. As we previously reported, as determined with copper soap colorimetry, CAEF had stronger inhibitory activity of pancreatic lipase (IC50 value ¼ 2.6 lg/mL) than CGA (IC50 value ¼ 4.0 lg/mL), but less than Orlistat (IC50 value ¼ 1.7 lg/mL) (Li et al., 2012a). In a rat model of acute fat-loading, the effect of CAEF on reducing the fat absorption through the intestinal tract was obviously stronger than orlistat (Li et al., 2012c); CAEF could also significantly increase the excretion of cholesterol and bile acid into feces (Li et al., 2012c). Pancreatic lipase not only is a key enzyme in exogenous fat digestion but also is one of the most widely studied targets for lipid-lowering and anti-obesity actions (de la Garza et al., 2011). It is apparent from the data of Oil red O staining and the efflux of TC and TG that these mechanisms may be involved in the hypolipidemic effects of CAEF. These effects are also consistent with the literature that multiple mechanisms facilitate endogenous cholesterol conversion into bile acids, control cholesterol absorption and synthesis, and promote TC and TG excretion. The metabolic pathways of liver cholesterol include three parts: secretion of HDL into blood, conversion of cholesterol into bile acid, and direct elimination of cholesterol into feces. ABCA1 is involved in the cholesterol efflux from cells and absorption from the intestinal tract. The redundant cholesterol in peripheral tissues can be eliminated by carrying back to liver, biotransformed, and excreted into the bile, called as reverse cholesterol transport (RCT). ABCA1 plays a crucial role in the transmembrane transport of cholesterol and takes part in the synthesis of HDL and the initial phase of RCT (Khera et al., 2011). RT-PCR analysis showed that all three drugs can increase the ABCA1 gene mRNA level, but CAEF and CGA were more effective than simvastatin. The data are well documented that increased expression of ABCA1 is accompanied with the promoting RCT and the increasing the cholesterol secretion from intestinal mucosa cells, resulting in the decreased of cholesterol assimilation (Favari et al., 2004). Bile acids play a vital role in regulating cholesterol and lipid homeostasis. CYP7A1 catalyzes the first and rate-controlling step in the classic pathway for cholesterol converting to bile acids (Shin et al., 2008). In HepG2 cells, simvastatin, CAEF, and CGA also significantly increased the CYP7A1 mRNA level. Based on the previous results, we deduced that CAEF increased bile acids in feces in vivo and reduced concentration of TC in liver cells may attribute to the activation of CYP7A1
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resulting in the promotion of cholesterol conversion to bile acids. AMP-activated protein kinase (AMPK) is served as a metabolic master switch in response to alterations in cellular energy charge. Over-expression of active AMPKa subunit has been shown to decrease intracellular TG and cholesterol in HepG2 cells (Pan et al., 2012; Spencer-Jones et al., 2006). Thus, identifying medicinal plant extracts that stimulate AMPK activity may give effective options for dyslipidemia and obesity. Liver X receptor-a (LXRa) belongs to a superfamily of the nuclear hormone receptor. Its target genes are involved in regulating lipid homeostasis (Lian et al., 2011; Murase et al., 2010; Zhong et al., 2013). It has been reported that AMPK directly inhibited ligand-induced LXR activity in addition to blocking production of endogenous LXR ligands (Yap et al., 2011). In this study, the expression levels of AMPKa2 mRNA in HepG2 cells were increased by CGA, which was consistent with the previous report (Ong et al., 2012). We found that CGA could also significantly inhibit the expression level of LXRa mRNA. These results suggested that CGA may exert suppressions of TG and TC levels in hepatocytes via modulation of AMPKLXR signaling pathways. Although equally containing CGA, the combined action of other active ingredients of CAEF could activate AMPKa2 mRNA, while CAEF had no effects on LXRa mRNA. Moreover, we observed that simvastatin increased the expression levels of LXRa mRNA, consistent with recent reports (Wang et al., 2014). Maintaining hepatocyte lipid homeostasis relays on the regulation of SREBPs located in endoplasmic reticulum, which is the most important transcription factor regulating de novo cholesterol, free fatty acid, and triglyceride in the liver. SREBP2 is the main regulator of the genes involved in the cholesterol metabolism, including HMGCR. The effects of CAEF, CGA, and simvastatin on the expression of SREPB2 mRNA level were investigated with RT-PCR. The results showed that the expressions of SREBP2 were significantly suppressed by CAEF and simvastatin. Both Oil red O staining and TC showed a decrease in the cellular cholesterol content by CAEF, which was associated with the inhibition of HMGCR, which is in charge of cholesterol synthesis. HMGCR in liver and small intestine is a rate-limiting enzyme in the synthesis of cholesterol (Bommer et al., 2011; Seo et al., 2011; Torres et al., 2006). Statins are classic HMGCR inhibitors available in the market as improving lipid metabolism drugs. It has been reported recently that simvastatin treatment upregulated intestinal lipid secretion pathways in a rodent model of the metabolic syndrome (Borthwick et al., 2014). We previously found that CAEF dose dependently and strongly inhibited the activity of HMGCR from normal pig liver at doses ranging from 0.1 to 100 lg/mL than simvastatin at the same concentrations (Li et al., 2012a). Hence, we investigated the effects of CAEF, CGA, and simvastatin on the expression of HMGCR at mRNA and protein levels. Importantly, all three drug treatments of HepG2 cells significantly decreased the expressions of HMGCR mRNA and protein, which was corroborated with the data of RT-PCR, western-blot, and immunocytochemistry. According to the in vitro results, the effects of inhibiting accumulation of TC and TG by CAEF
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were the strongest among three drugs, although the suppression of the HMGCR expression by CAEF is lower than both simvastatin and CGA. These data suggested that CAEF decreasing lipid accumulation was likely related to the inhibition of the expression of genes involved in cholesterol biosynthesis in the liver by HMGCR signal pathway. Taken together, these findings of the study demonstrated that CAEF improved the lipid metabolism by the transcriptional activating AMPK and inhibiting downstream targets, such as SREBP2 and HMGCR, to suppress the TC synthesis and TG levels in liver cells. CAEF also increased ABCA1 and CYP7A1 to enhance TC efflux and bile acid transport, synthesis, and excretion, and these effects were superior to simvastatin. These findings will add our understanding of how E. ulmoides exerts anti-obesity effects and regulates lipid metabolism. Eucommia ulmoides and CGA-containing extracts could represent a promising dietary supplement to prevent obesity and dyslipidemia.
Declaration of interest This work was supported by project from Guizhou Provincial Modernization of Traditional Chinese Medicine Foundation of China (Grant No. QKHZY [2013] 3018) (W. L.), The Project Natural Science Foundation of China (No. 21162046) (Y. C.); and The Research Funds of Department of Education of Guizhou Province of China (Grant No. QKJ 2010042) (C. X.).
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