Scutellarin

Scutellarin ameliorates nonalcoholic fatty liver disease through the PPAR/PGC-1-Nrf2 pathway

Xiaoxue Zhang, Renpeng Ji, Huijun Sun, Jinyong Peng, Xiaodong Ma, ChangYuan Wang, Yufeng Fu, Liuchi Bao & Yue Jin

KEYWORDS
Scutellarin; NAFLD; antioxidation; nuclear factor erythroid-2-related factor; peroxisome proliferator- activated receptor-gamma

Introduction

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in western countries and affects 20–30% of the general population; this percentage is still increasing. This disease is closely associated with central obesity, dyslipidaemia, hypertension, hypergly- caemia, and other metabolic disorders [1]. NAFLD includes a spectrum of liver disorders ranging from lipid accumulation to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma [2]. The “two hits” hypothesis proposed by Day and James indi- cates that oxidative stress (OS) and dysregulation of redox-sensitive signaling pathways are vital to the path- obiology of fatty liver diseases [3,4]. To date, no evi- dence-based pharmacotherapy is available for NAFLD. NAFLD, which primarily manifests by excessive fat accu- mulation in hepatocytes, has become the most com- mon chronic liver disease worldwide [5]. The peroxisome proliferator-activated receptor gamma (PPARc) is a member of the nuclear hormone receptor superfamily, which plays an important role in adipocyte differentiation, cell growth, and inflammation. PPARc regulates transcription by binding to specific peroxisome proliferator-response elements (PPREs) on target genes and thus plays a role in the transcriptional regulation of target genes, inducing antioxidant enzymes and genes involved in removal of molecular oxygen and hydrogen peroxide, thereby improving the antioxidant capacity [6,7]. Peroxisome proliferator- activated receptor gamma coactivator-1 alpha (PGC-1a) is a transcriptional coactivator of PPARc, which is involved in the transcriptional regulation of PPARr. PGC- 1a participates in the activation of nuclear receptors and other transcription factors and plays an important role in mitochondria generation [8]. The nuclear factor erythroid-2-related factor (Nrf2) represents an important.

Materials and methods

Materials and methods
Scutellarin (> 98% purity) was purchased from Chengdu Must Biotechnology Co Ltd. (Sichuan, China). Lovastatin and free fatty acid-free BSA were purchased from Sigma (St. Louis, MO). GW9662 was purchased from MCE (St. Marostica, Milano, Italy). Oleic acid was obtained from Sigma (St. Louis, MO).

Animal

Eight-week old male C57BL/6J mice (20.0 ± 2.00 g), which were purchased from Changsheng Biotechnology Co Ltd, (Liaoning, China) (SCXK 2010-0001), were used in the experiment. All research procedures of this study followed the Institutional guidelines for the care and use Committee of Dalian Medical University and were approved by the Institutional Ethics Committee of Dalian Medical University in accordance with the Guide to the Care and Use of Experimental Animals from the Canadian Council on Animal Care (see Guide to the Care and Use of Experimental Animals, Vol. 1 (2nd ed, 1993) and Vol. 2 (1984). All efforts were made to minimise the pain and stress of the mice. After adaptation for 1 week, the animals were randomly assigned to (A) normal group, (B) high-fat diet (HFD) group, (C) HFD þ scutellarin (12.5 mg/kg/d) group, (D) HFD þ scutellarin (25 mg/kg/d) group, (E) HFD þ scutellarin (50 mg/kg/d) group, and (F) HFD þ lovastatin (10 mg/kg/d) group. The control diet (standard labora- tory rodent chow diet) for normal group was composed of 58% carbohydrate, 20.8% protein, 5.6% fat, 3.656 kcal/kg, (Xietong Organism, Nanjing, Jiangsu, China). The HFD used in this study was 45% fat diet, which consisted of 41% carbohydrate, 24% protein, 24% fat, and the caloric density of this diet was 4.73 kcal/kg (MD12032, Medicience Ltd., Jiangsu, China).
After acclimatisation with the facility for 2 weeks, the animals were weighed weekly. Successful model estab- lishment was confirmed by measurement of the lipid levels. After 10 weeks, blood samples were collected, and the serum was separated for biochemical assays.
The aorta tissues from all mice were stored at —80 ◦C for further analysis.

Biochemical analysis

Blood samples of each animal were centrifuged at 3000g for 10 min at 4 ◦C to produce the serum. Total cholesterol (TC), triglycerides (TG), high-density lipopro- tein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in serum were measured using Nanjing Jiancheng Bioengineering Institute assay kits. The liver homogenate was centrifuged, and the super- natant was collected for biochemical analysis. Protein concentrations of tissues were assayed by the BCA method. Malondialdehyde (MDA), catalase (CAT), glu- tamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), and total antioxidative capacity (T-AOC) in livers were measured with assay kits from the Nanjing Jiancheng Bioengineering Institute.

Histopathological examination

Liver samples were fixed in 10% neutral formalin, alco- hol-dehydrated, and paraffin-embedded. Then, 5-lm sections were prepared, stained with haematoxylin and eosin (H&E), and examined microscopically. Other liver specimens were frozen at —80 ◦C and sec-
tioned using a cryostat for lipids staining using Oil Red O staining. All sections were photographed under a light microscope to assess the degree of hepatic lipid accumulation.

Ultrastructural changes

Liver tissues were collected and cut into sections of approximately 1 mm ×1 mm ×1 mm, fixed with gluta- raldehyde and osmic acid, dehydrated with ethanol and
embedded with ethoxyline resin for electron micro- scopic examination.

ELISA
The level of Lp(a), apoA1, and apoB in the serum were measured by ELISA according to the manufacturer’s protocol (Shanghai Meilian Institute of Biotechnology, Shanghai, China). A standard instrument was used to measure the absorbance (OD) through standard curve calculation according to the manufacturer’s instructions.

Cell culture and treatment

HepG2 cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37 ◦C in DMEM medium con- taining 10% (v/v) foetal bovine serum (FBS, Thermo Fisher HyClone). The medium was renewed every 2–3 days and subcultured every 4 days. Oleic acid (OA) was dissolved in sterile NaOH plus 10% BSA and stored at —20 ◦C. The indicated concen- tration of OA was prepared with 10% BSA solvent. After
serum starvation, cells were treated with 0.3 mmol/L of OA for 24 h to induce lipid accumulation. We added scutellarin to cells at the same time as OA administra- tion and treated the cells for 24 h. Cells were separated to six groups: normal group, OA (0.3 mmol/L) control group, scutellarin (50 mmol/L)þOA (0.3 mmol/L) group, scutellarin (100 mmol/L)þOA (0.3 mmol/L) group, scutel- larin (200 mmol/L)þOA (0.3 mmol/L) group, and lovastatin (10 mmol/L)þOA (0.3 mmol/L) group.

In vitro induction of lipid accumulation HepG2 cells were seeded in six-well tissue culture plates at 5 × 105 cells/well and cultured overnight. After serum starvation, cells were treated with 0.3 mmol/L of OA for 24 h to induce lipid accumulation, and we added scutel- larin for 24 h in some cases. Fat accumulation was con- firmed by measurement of intracellular TC, TG content and the Oil Red O staining in the cells. We investigated the level of MDA, CAT, GOT, GPT, and T-AOC with the kits manufactured by the Nanjing Jiancheng Bioengineering Institute.Quantitative real-time PCR assay Total RNA from liver tissue and cells was extracted with Trizol reagent (Gibco BRL Life Technologies, Inc, Gaithersburg, MD) according to the manufacturer’s instructions. RT was performed to obtain cDNA with the TransStart First-Strand cDNA Synthesis SuperMix kit (TransGen Biotech, Inc, Beijing, China), according to the manufacturer’s instructions. The 25-ml PCR system con- tained 2-ll template, 1 ll each primer, 12.5 ll TransStart Top Green qPCR SuperMix (TransGen Biotech, Inc, Beijing, China), and 8.5 ll double-distilled H2O. The fluorescence signals emitted by fluorophores of TaqMan probes were detected by a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). Cycling reactions were carried out as follows: 30 s at 95 ◦C, followed by 40 cycles of 5 s at 95 ◦C, 34 s at 60 ◦C, and a melt curve stage after the cycling stage. The primers used for PCR were as follows: human PPARc (F 50- GGCGAGG GCGATCTTGACAGG-30, R 50- TGCGGATGGCCACCTCT TTGC-30); human PGC-1a (F 50- GGAACTGCAGGCCTAAC TCC-30, R 50- CACTGTCCCTCAGTTCACCG-30); human Nrf2 (F 50- TCAGCGACGGAAAGAGTATGA-30, R 50- CCACTGGT TTCTGACTGGATGT-30); human bactin (F 50- TGGCACC CAGCACAATGAA-30, R 50- CTAAGTCATAGTCCGCCTA GAAGCA-30); mouse PPARc (F 50- CCAGAGCATGGTGC CTTCGCTG-30, R 50- GAGCTGACCCAATGGTTGCTG-30); mouse PGC-1a (F 50- GCAGGTCGAACGAAACTGAC-30, R 50- CTCAGCCTGGGAACACGTTA-30); mouse Nrf2 (F 50- CGAGATATACGCAGGAGAGGTAAGA-30, R 50- GCTCGACA ATGTTCTCCAGCTT-30); mouse bactin (F 50- ACTGCCGCA TCCTCTTCCT-30,R 50- TCAACGTCACACTTCATGATGGA-30).

Western blot analysis

Liver tissues and HepG2 cells were collected and lysed in lysis buffer for 10 min at 4 ◦C. Protein concentration was determined with a BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Total pro- teins were extracted for western blot analysis of HO-1, NQO1, GST, and Keap1 levels. Nucleoprotein was extracted by a nucleoprotein and cytoplasmic protein extraction kit (KEYGEN, Jiangsu, China). Protein samples (20 mg) were subjected to 10% sodium dodecyl sul- phate-polyacrylamide gel electrophoresis and then elec- tronically transferred onto a nitrocellulose membrane. The protein was resolved by electrophoresis and elec- troblotted onto nitrocellulose membranes. The mem- branes were individually incubated with anti-PPARc, anti-PGC-1a, anti-Nrf2, anti-Keap1, anti-NQO1, anti-HO- 1, anti-GST, or anti-NF-jB. Then, the membranes were incubated with goat anti-rabbit or goat antimouse IgG secondary antibody. Subsequently, the blot was devel- oped using an enhanced chemiluminescence system (cat. No. P0018A; Beyotime Institute of Biotechnology), and the protein bands were analysed with Image Lab software version 3.0 (Bio-Rad Laboratories, Inc, Hercules, CA). The proteins b-actin and histone-H3 were detected as internal controls.

PPARc antagonist GW9662

GW9662 (irreversible PPARc antagonist) was dissolved by dimethyl sulfoxide (DMSO). The cells were assigned to (A) normal group, (B) OA (0.3 mmol/L), (C) OA (0.3 mmol/L) þ scutellarin (100 mmol/L), (D) GW9662 (10 mmol/L), (E) GW9662 (10 mmol/L)þOA (0.3 mmol/L), and (F) GW9662 (10 mmol/L)þOA (0.3 mmol/L)þ scutellarin (100 mmol/L). Then, the protein expression of PPARc, PGC-1a, Nrf2, Keap1, NQO1, HO-1, GST, and NF-jB was detected by western blot analysis.

Statistical analysis

All data are expressed as the mean ± SD. One-way ANOVA was used to estimate overall significance fol- lowed by post hoc Tukey’s tests corrected for multiple comparisons. p Values < .05 and < .01 were considered as statistically significant. All statistical analysis was per- formed using SPSS 19.0 statistical software. Results The effect of scutellarin on hepatic lipid accumulation of NAFLD mice fed an HFD diet The body weights from each group were monitored once a week. As shown in Figure 2(a), the mean body weights of normal group mice increased steadily, while the body weights of the HFD group were significantly greater at 8 weeks after feeding. However, administra- tion of scutellarin significantly reduced the body weight. Compared to the normal mice, mice in the HFD group had magnificent high lipid profiles. As shown in Figure 2(b–e), the HFD group showed significantly increased serum TC, TG, and LDL-C levels, while HDL-C levels were significantly decreased. However, adminis- tration of scutellarin as well as the lovastatin lowered the serum TC, TG, and LDL-C levels and increased HDL- C levels in a dose-dependent manner. Meanwhile, liver sections from the HFD group showed lipid vacuoles, swelling, and degeneration, which scutellarin inhibited in a dose-dependent manner (Figure 2(f)). Oil Red O staining (Figure 2(g)) also showed a significant increase of fat deposition in HFD mice, whereas the administration of scutellarin signifi- cantly decreased the fat accumulation in the liver. These results showed that scutellarin could reduce the fatty liver and showed substantial protective effects. The protective effect of scutellarin on MDA level, GOT, GPT, CAT, and T-AOC activity Compared to the normal mice, significant increase in MDA, GOT, and GPT activities in the liver were observed in the HFD group. However, the activities of MDA, GOT, and GPT (Figure 3(a,c,d)) were dose-dependently decreased by scutellarin treatments. Meanwhile, CAT and T-AOC (Figure 3(b,e)) activities were significantly decreased in the HFD group, but administration of scu- tellarin increased CAT and T-AOC activities in a dose- dependent manner. Effects of scutellarin on the levels of apoA1, apoB, and Lp(a) in the serum As shown in Figure 4(a–c), compared to the normal group, the HFD group showed significantly increased serum apoB and Lp(a) levels, while apoA1 levels decreased significantly. However, administration of scu- tellarin as well as the lovastatin lowered the serum apoB and Lp(a) levels and increased apoA1 levels in a dose-dependent manner. Ultrastructural changes in the hepatocytes of HFD mice As shown in Figure 5, the normal group had fewer lipid vacuoles, and the mitochondria and endoplasmic reticu- lum morphology was normal. In the HFD group, many lipid vacuoles appeared in the liver, and mitochondria were generally swollen. However, administration of scu- tellarin significantly decreased the lipid vacuoles in the liver, and the structure in the liver appeared to be normal. The effects of scutellarin on OA-induced lipid accumulation in HepG2 cells As shown in Figure 6(a,b), the content of TC and TG increased significantly in the OA-induced lipid accumula- tion group. Scutellarin as well as the lovastatin lowered the content of TC and TG in a dose-dependent manner. Compared to the normal group, significant increases in MDA, GOT, and GPT activities in the cells were observed in the OA-induced lipid accumulation group. Compared with the OA group, the MDA, GOT, GPT activities (Figure 6(c,e,f)) were dose-dependently decreased by scutellarin treatments. Meanwhile, CAT and T-AOC (Figure 6(d,g) activities were significantly decreased in the OA group, and scutellarin increased CAT and T-AOC activities in a dose-dependent manner. As shown in Figure 6(h), no obvious Oil Red O staining was observed in the normal cells. The results revealed abundant accumulation of larger lipid droplets (red staining) in the OA group compared with the normal group. Scutellarin reduced the OA-induced lipid accumulation. Scutellarin activates the PPARr-PGC-1a-Nrf2 pathways As shown in Figure 7(a–c), the HFD group showed sig- nificantly downregulated PPARc and PGC-1a mRNA and protein expression and upregulated Nrf2 mRNA and protein expression. Scutellarin enhanced the mRNA and protein expression levels of PPARc, PGC-1a, and Nrf2 in a dose-dependent manner. This resulted in the activa- tion of HO-1, NQO1, and GST. Therefore, we found that the expression of HO-1, NQO1, and GST (Figure 7(e,f,g)) was upregulated after the administration of scutellarin, whereas the expression of Keap1 and NF-jB (Figure 7(d,h)) was downregulated, which was consistent with our results in vitro (Figure 8(a–h)). Thus, we confirmed the stimulatory effect of scutellarin on the PPARc-PGC- 1a-Nrf2 pathway. Effect of GW9662 on the expression of PPARc, PGC-1a, Nrf2, Keap1, HO-1, NQO1, GST, and NF-jB To confirm the stimulatory effect of scutellarin on the PPARc-PGC-1a-Nrf2 pathway, we introduced GW9662, an irreversible antagonist of PPARc. In further studies, we detected the protein expression of PPARc, PGC-1a, Nrf2, Keap1, HO-1, NQO1, GST, and NF-jB by western blot analyses. As shown in Figure 9(a–h), compared with the OA group, scutellarin significantly upregulated PPARc, PGC-1a, Nrf2, HO-1, NQO1, and GST protein expression and downregulated the expression of Keap1 and NF-jB. However, after addition of GW9662, the upregulation of PPARc, PGC-1a, Nrf2, HO-1, NQO1, and GST was significantly attenuated, and the downregula- tion on Keap1 and NF-jB was also attenuated. Therefore, we confirmed the regulatory effect of scutel- larin on the PPARc-PGC-1a-Nrf2 signaling pathway. Discussion The liver is the major organ responsible for lipid synthe- sis and metabolism. NAFLD is a clinical metabolic syndrome characterised by excessive fat deposition in liver cells not due to alcoholic factors [17,18]. Obesity- related conditions are believed to be important factors related to the increased incidence of NAFLD [19]. The increase of free fatty acid (FFA) in liver cells is the basis of NAFLD formation. FFA is an important lipid toxicity factor, which can cause insulin resistance, glycolipid metabolism disorder, and lipid peroxidation in a variety of ways, and play an important role in the pathogenesis of fatty liver. FFA can also induce hepatic synthesis of HMG-CoA and accelerate the synthesis of cholesterol. Oleic acid is the highest content of FFA in the body, and oleic acid is the main long-chain fatty acid in people’s diet. It is also the main component of triglyceride in the liver. In order to better simulate the fatty acid metabolism of hepatocytes in vivo, oleic acid is used in this study. HepG2 is a highly differentiated human embryonic liver cancer cell line. It can not only be subcultured indefinitely, but also maintain many specific functions of normal hepatocytes, including the synthesis of apoli- poprotein, the secretion, and catabolism of lipoproteins. HepG2 cell line lipoprotein synthesis and secretion changes due to different culture conditions, so HepG2 cell line has become a widely used model for studying human liver lipid and lipoprotein metabolism. Peroxisome proliferator activated-receptors (PPARs) are a member of the nuclear receptor superfamily. The PPAR subfamily consists of three isotypes, PPARa, PPARc, and PPARb/d. PPARs are important players in fatty acid metabolism, lipogenesis, and adipogenesis [20,21]. PPARc is found in many tissues, such as adipose tissue, liver, skeletal muscle, kidney, breast, intestine, and endothelium and vascular wall, where it regulates adipocyte differentiation, modulates insulin sensitivity, and mediates anti-inflammatory effects, tissue remodel- ling, and atherosclerosis [22,23]. PPARc participates in the transcriptional activation of numerous adipogenic and lipogenic genes. Peroxisome proliferator-activated receptor c-coactivator-1a is a specific coactivator of PPARc. When PGC-1a is activated, transcription factors and receptors in the nucleus play an important role in lipid metabolism [12,24,25]. Nrf2 is an important tran- scription factor that plays a significant role in mainten- ance of cellular redox homeostasis and antidamage and antitumour processes [26]. Recent research showed that the promoter region of the Nrf2 gene had PPARc-bind- ing site PPREs; thus, PPARc could bind to PPRE on the Nrf2 promoter, which in turn activates Nrf2 gene tran- scription and upregulates the expression of various anti- oxidant genes [11,27]. Scutellarin is a flavonoid extracted from the herbal medication Erigeron breviscapus Hand-Mazz (Figure 1). Previous research showed that scutellarin had a strong anticancer effect in vivo and in vitro [28–30]. In addition, various reports have demonstrated that scutellarin could protect against oxidative damage and inflamma- tory injury [31–35]. However, little is known about the effect of scutellarin on the expression of PPARc. In this study, serum TG, TC, and LDL-C levels were obviously increased, and HDL-C levels were decreased in response to HFD compared with the normal group. These results can trigger a series of biological changes in hepatic lipid metabolism and ultimately lead to hep- atic lipid accumulation. As predicted, the significant elevation of GOT, GPT activities, and MDA content, as well as the significant decrease of CAT and T-AOC activities, reflected the liver damage and decreased antioxidation in HFD-treated mice. Meanwhile, H&E and Oil Red O staining showed that HFD mice had obvious hepatic lipid accumulation. These data showed that we successfully established a NAFLD model in C57BL/6J mice. After administration of 12.5, 25, and 50 mg/kg/d of scutellarin in HFD-fed mice, we found that scutellarin could reverse the parameters of lipid and energy metabolism in a dose-dependent manner. Our results showed that scutellarin significantly increased the mRNA and protein levels of PPARc, PGC-1a, and Nrf2 both in vitro and in vivo. Nrf2 associates with the Keap1 in the cytosol, but various stimuli, such as oxidative or electrophilic stress, promote the release of Nrf2 from Keap1 and its translocation to the nucleus to activate transcription [8,9]. In the nucleus, Nrf2 binds to antioxi- dant response elements and then induces gene expres- sion of phase II detoxification enzymes, such as GST, SOD, CAT, HO-1, and NQO1 [10]. We found that scutel- larin stimulated the nuclear translocation of Nrf2 and increased hepatic expression of HO-1, NQO1, and GST. These data indicate that scutellarin initially activates PPARc, resulting in the upregulation of PGC-1a and Nrf2 protein expression, and finally stimulates the pro- tein expression of HO-1, NQO1, and GST through Nrf2 activation. To confirm the stimulatory effect of scutel- larin on the PPARc-PGC-1a-Nrf2 pathway, we intro- duced GW9662, an irreversible antagonist of PPARc [36]. In further studies, we found that GW9662 attenu- ated the upregulation of PPARc, PGC-1a, Nrf2, HO-1, NQO1, and GST expression by scutellarin and attenu- ated the downregulation of Keap1 and NF-jB. The results suggest that scutellarin improves lipid metabol- ism in NAFLD through the PPARc/PGC-1a-Nrf2 signal- ling pathways. Conclusions Scutellarin has a lipid-lowering effect, and it shows strong antioxidant and liver protective activity. It can increase the level of antioxidants and reduce lipid per- oxidation. Thus, scutellarin can ameliorate NAFLD and oxidative stress via the PPARc/PGC-1a-Nrf2 signalling pathway. Disclosure statement The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. ORCID Yue Jin http://orcid.org/0000-0002-6926-566X References [1] Tarantino G, Saldalamacchia G, Conca P, et al. Non- alcoholic fatty liver disease: further expression of the metabolic syndrome. J Gastroenterol Hepatol. 2007; 22:293–303. [2] Berlanga A, Guiu-Jurado E, Porras JA, et al. Molecular pathways in non-alcoholic fatty liver disease. Clin Exp Gastroenterol. 2014;7:221–239. [3] Feldstein AE, Bailey SM. 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