This study aimed to investigate the effects of Shugan Xiaozhi decoction (SX) on nonalcoholic steatohepatitis (NASH) induced by high-fat diet in rats. The rats were randomly divided into 6 groups, namely, control, model, fenofibrate, and three different dosage of SX (10, 20, and 40 g/kg/day, p.o.). After establishing the NASH model, at 8 weeks of the experiment, treatments were administrated intragastrically to the fenofibrate and SX groups. All rats were killed after 4 weeks of treatment. Compared with the model group, alanine aminotransferase (ALT), aspartate aminotransferase (AST), free fatty acid (FFA), total cholesterol (TC), triacylglycerol (TG), and low-density lipoprotein cholesterol (LDL) serum in the serum were significantly reduced in all SX treatment groups in a dose-dependent manner. Evidence showed that SX could protect the liver by upregulating the gene and protein expressions of peroxisome proliferator-activated receptor alpha (PPAR
The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing worldwide and the disease is estimated to affect approximately 20% of the population in the developed countries [
The world’s overweight population has increased significantly over the past few decades [
In TCM theory, NASH is described in a perspective very different from Western medical terms. The symptoms of NASH could be categorized as hypochondriac “xietong” or liver stuffiness “ganpi.” It is a result of overnutrition in diet (including high-fat diet) or dysfunction of the spleen (our digestive function) in transporting (hence utilizing) the nutrients to other parts of the body, leading to accumulating “dampness” and “heat” in the liver and blocking
In this study, we aimed to investigate the underlying mechanism of SX in rats with NASH induced by high-fat diet, in order to gather pharmacological data to support its modern clinical uses. Biological parameters and endpoints including alanine aminotransferase (ALT), aspartate aminotransferase (AST), free fatty acid (FFA), total cholesterol (TC), triacylglycerol (TG), and low-density lipoprotein cholesterol (LDL) levels were measured, and some gene and protein expressions from the liver that might be related to the actions of SX were further evaluated. Our results demonstrated for the first time that SX upregulated peroxisome proliferator-activated receptor alpha (PPAR
Male Sprague–Dawley rats weighing about 200 ± 20 g were obtained from Experimental Animal Science Center of Guangzhou University of Chinese Medicine (license number SYXK (Yue) 2013-0001). The use of animals in this study was approved by the Ethics Committee of Guangzhou University of Chinese Medicine. The rats were housed in specific-pathogen-free grade animal facility under standard conditions (temperature of 22 ± 2°C, 12/12 h light/dark cycle, and humidity of 50 ± 10%) with free access to water.
90 rats were randomly divided into 6 groups of 15: these groups were control group, high-fat diet group (HFD), treatment groups with low dosage of SX (10 g/kg/day, p.o.) (HFD + LSX), medium dosage of SX (20 g/kg/day, p.o.) (HFD + MSX), and high dosage of SX (40 g/kg/day, p.o.) (HFD + HSX), and treatment group with fenofibrate (0.1 g/kg/day, p.o.). At the beginning of the first 8 weeks of the experiment, the control group was fed with a normal diet [composition: protein (14%), fat (10%), and carbohydrate (76%)], while the other groups were all fed with a high-fat diet (HFD) which is a standard rat chow supplement with cholesterol (1%), protein (10%), and lard (10%). All chow was obtained from Guangdong Provincial Medical Laboratory Animal Center (Guangdong, China). Model evaluation was done by point counting, in which 5 rats of each group were selected randomly and their liver tissues were observed under a microscope for confirmation. After the model building stage, all the rats in the treatment groups were ig given fenofibrate or SX extracts while the rats in the normal and high-fat diet groups were fed with the same dose of distilled water. The course of treatment was 4 weeks. At the end of the treatment period, rats were fasted overnight and sacrificed by cervical dislocation. Tissue samples (blood and livers) were collected and stored in freezer at −80°C.
SX is composed of twelve Chinese Materia Medica (CMMs): Bupleuri Radix, Paeoniae radix alba (stir-baked), Aurantii Fructus Immaturus, Glycyrrhizae Radix et Rhizoma, Artemisiae Scopariae Herba, Gardeniae Fructus, Poria, Alismatis Rhizoma, Crataegi Fructus, Cassiae Semen, Nelumbinis Folium, and Pumex in a ratio of 2 : 1 : 3 : 1 : 6 : 2 : 4 : 6 : 6 : 6 : 6 : 6. All the CMMs were in the form of formula granules and purchased from Guangdong Yifang Pharmaceutical Co., Ltd. Fenofibrate capsules were purchased from Laboratoires Fournier S.A. (batch number H20140369).
ALT, AST, TG, TC, and LDL-C kits were purchased from Shanghai Kehua Bio-Engineering Co., Ltd; FFA kits and Trizol were purchased from AmyJet Scientific Inc and TAKARA, respectively. qRT-PCR kits and solvent were purchased from DBI Bioscience. Cell lysis buffer and antibodies against PPAR
HPLC grade acetonitrile, formic acid, and analytical grade ethanol were purchased from Tedia (USA). Double deionized water was prepared by a Milli-Q-water-purification system (Millipore, MA, USA). All other chemicals used were of analytical grade.
4% paraformaldehyde-fixed livers were processed and embedded in paraffin according to routine histologic techniques. Liver sections, 5
ALT, AST, FFA, TC, TG, and LDL-C were determined with an automatic biochemical analyzer (ADVIA 2400 Clinical Chemistry System, Siemens, Germany).
To determine the gene expression levels, total RNA was isolated from the liver using Trizol reagent according to the standard protocol. 2.0
Total reaction mixture (20
The primer sets used for qRT-PCR.
Gene name | Forward primer | Reverse primer |
---|---|---|
PPAR | 5′-ATTTGCCAAGGCTATCCCA-3′ | 5′-AAGGAGGACAGCATCGTGAAG-3′ |
L-FABP | 5′-TTCTCCGGCAAGTACCAAGT-3′ | 5′-ATGCACGATTTCTGACACCC-3′ |
LCAD | 5′-AAGAAGTGATCCCTACCACGA-3′ | 5′-CTCCACCAAAAAGAGGCTAAT-3′ |
CPT-1 | 5′-AGCCATGGAGGTTGTCTACG-3′ | 5′-GGCTTGTCTCAAGTGCTTCC-3′ |
ACO | 5′-CCTTTGTTGTCCCTATCC-3′ | 5′-GACTCGGCAGGTCATTCA-3′ |
GAPDH | 5′-CCTCGTCTCATAGACAAGATGGT-3′ | 5′-GGGTAGAGTCATACTGGAACATG-3′ |
The qRT-PCR reaction procedures are as follows: 94°C, 2 min; 94°C, 20 s; 58°C, 20 s; 72°C, 20 s; 40 cycles. Each qRT-PCR reaction was performed in triplicate.
Briefly, rat livers were homogenized in lysis buffer and centrifuged at 14,000 rpm at 4°C for 10 min. Protein was quantified by the BCA assay (Pierce, Rockford, IL, USA). The protein (20
Around 0.3 g powder sample was placed into a 50 mL centrifuge tube and then sonicated with 10 mL 70% ethanol for 15 minutes at room temperature. Then, the mixture was centrifuged at 14000 rpm in 4°C for 15 minutes. 100
UPLC-ESI-MS analysis was performed on Orbitrap Fusion Lumos Tribrid Mass Spectrometer (ThermoFisher Scientific, USA) with UPLC (ACQUITY UPLC® System, Waters, USA). A UPLC C18 analytical column (2.1 mm × 100 mm, ID 1.8
Heated Electrospray ionization (HESI) was used in both positive and negative ion modes. The operation parameters were as follows: spray voltage static (positive ion 3600 V, negative ion 3000 V) sheath gas flow rate 20 units, aux gas flow rate 10 units, sweep gas 10 units, ion transfer tube temperature 300°C, and vaporizer temperature 200°C. The mass spectra were acquired in full scan mode from 100 to 1200 in mass to charge ratio (
All data were shown as the mean ± standard deviation (SD), and one-way ANOVA was used to calculate significant differences between the groups. Statistical differences were considered to be significant if
According to the results of H&E staining (Figure
Histological changes of liver section in different groups (H&E staining): (a) control, (b) HFD, (c) treatment of LSX, (d) treatment of MSX, (e) treatment of HSX, and (f) treatment of fenofibrate; bar = 100
Intracellular lipid deposition in hepatocytes was clearly shown via Sudan III staining and the results were illustrated in Figure
Histological changes of liver section in different groups (Sudan III staining): (a) control, (b) HFD, (c) treatment of LSX, (d) treatment of MSX, (e) treatment of HSX, and (f) treatment of fenofibrate; bar = 20
The results of the treatment of SX on the activities of ALT and AST were summarized in Table
ALT and AST activities in the different groups of rats.
Group | Dosage [g/kg/day] | ALT (U/L) | AST (U/L) |
---|---|---|---|
Control | — | 61.25 ± 0.58 | 101.77 ± 0.69 |
Model | — | 78.99 ± 1.93 | 130.64 ± 1.40 |
HFD + LSX | 10 | 69.97 ± 3.91 | 118.30 ± 0.79 |
HFD + MSX | 20 | 67.14 ± 1.37 | 114.93 ± 2.11 |
HFD + HSX | 40 | 64.61 ± 0.73 | 113.30 ± 2.23 |
Fenofibrate | 0.1 | 63.43 ± 0.84 | 112.31 ± 1.46 |
Compared with the normal group,
The results of the treatment of SX on the level of FFA, TC, TG, and LDL-C were summarized in Table
Levels of FFA, TC, TG, and LDL-C in the different groups of rats.
Group | Dosage [g/kg/day] | FFA (nmol/ | TC (nmol/L) | TG (nmol/L) | LDL-C (nmol/L) |
---|---|---|---|---|---|
Control | — | 4.16 ± 0.32 | 3.07 ± 0.20 | 1.45 ± 0.20 | 0.93 ± 0.15 |
| — | 7.77 ± 0.70 | 5.30 ± 0.62 | 3.17 ± 0.41 | 2.80 ± 0.37 |
HFD + LSX | 10 | 5.49 ± 0.39 | 4.11 ± 0.14 | 2.48 ± 0.40 | 2.08 ± 0.09 |
HFD + MSX | 20 | 4.88 ± 0.18 | 4.01 ± 0.27 | 2.27 ± 0.43 | 1.83 ± 0.23 |
HFD + HSX | 40 | 4.70 ± 0.40 | 3.97 ± 0.13 | 1.95 ± 0.16 | 1.69 ± 0.20 |
Fenofibrate | 0.1 | 4.55 ± 0.56 | 3.68 ± 0.41 | 1.87 ± 0.27 | 1.48 ± 0.18 |
Compared with the normal group,
The results of qRT-PCR for determining the expression level of various genes in rat livers under different treatments were shown in Figure
Effects of SX on the gene expressions of (a) PPAR
After investigating how SX affected the gene expressions of PPAR
Effects of SX on the protein expressions of PPAR
In order to provide a better characterization of SX, its chemical profile was determined using UPLC-ESI-MS. Figure
Compounds identified in SX by UPLC-ESI-MS.
Number | Retention time (min) | Molecular formula | [M − H]− ( | [M + HCOO]− ( | [M + H]+ ( | Identification | Origins |
---|---|---|---|---|---|---|---|
1 | 2.48 | C7H6O5 | 169.0142 (3.55) | Gallic acid | Paeoniae radix alba [ | ||
2 | 4.46 | C27H32O14 | 579.1719 (4.83) | Naringin | Aurantii Fructus Immaturus [ | ||
3 | 5.86 | C28H34O15 | 609.1825 (4.76) | Neohesperidin | Aurantii Fructus Immaturus [ | ||
4 | 6.27 | C28H34O14 | 593.1876 (4.38) | Poncirin | Aurantii Fructus Immaturus [ | ||
5 | 6.40 | C17H24O11 | 449.1327 (5.79) | Gardenoside | Gardeniae Fructus [ | ||
6 | 8.26 | C23H34O15 | 595.1915 (5.88) | Genipin-1- | Gardeniae Fructus [ | ||
7 | 8.40 | C16H18O9 | 353.0899 (5.95) | Chlorogenic acid | Gardeniae Fructus [ | ||
8 | 9.14 | C17H24O10 | 433.1379 (5.77) | Geniposide | Gardeniae Fructus [ | ||
9 | 9.82 | C23H28O11 | 481.1676 (−5.82) | Albiflorin | Paeoniae radix alba [ | ||
10 | 11.63 | C26H30O13 | 549.1646 (5.18) | Liquiritin-apioside or isomer | Glycyrrhizae Radix et Rhizoma [ | ||
11 | 11.81 | C21H22O9 | 417.1216 (5.99) | Liquiritin | Glycyrrhizae Radix et Rhizoma [ | ||
12 | 12.43 | C41H32O26 | 939.1170 (6.50) | Pentagalloylglucose | Paeoniae radix alba [ | ||
13 | 13.09 | C25H24O12 | 515.1225 (5.82) | Isochlorogenic acid A | Gardeniae Fructus [ | ||
14 | 13.47 | C23H28O11 | 525.1644 (5.71) | Paeoniflorin | Paeoniae radix alba [ | ||
15 | 15.99 | C32H40O16 | 725.2346 (6.62) | 6′′-O-trans-p-cinnamoyl genipingentiobioside | Gardeniae Fructus [ | ||
16 | 17.87 | C44H64O18 | 881.412 (−6.24) | 22 | Glycyrrhizae Radix et Rhizoma [ | ||
17 | 18.11 | C32H44O14 | 651.2700 (6.45) | cis-Crocin2/trans-crocin2/cis-crocin2′/trans-crocin2′ | Gardeniae Fructus [ | ||
18 | 19.68 | C30H48O6 | 505.3493 (−6.13) | 16-Oxo-alisol A | Alismatis Rhizoma [ | ||
19 | 20.25 | C42H62O16 | 823.4068 (−5.22) | Glycyrrhizic acid | Glycyrrhizae Radix et Rhizoma [ | ||
20 | 20.26 | C44H70O14 | 867.4799 (5.88) | O-Acetyl-saikosaponin or isomer | Bupleuri Radix [ | ||
21 | 20.41 | C32H50O7 | 547.3599 (−5.48) | 16-Oxo-alisol A acetate | Alismatis Rhizoma [ | ||
22 | 20.76 | C17H14O7 | 329.0685 (5.47) | Aurantio-obtusin | Cassiae Semen [ | ||
23 | 20.86 | C42H68O13 | 825.4694 (6.30) | Saikosaponin a | Bupleuri Radix [ | ||
24 | 21.08 | C42H68O13 | 825.4693 (6.18) | Saikosaponin d | Bupleuri Radix [ | ||
25 | 21.26 | C44H70O14 | 867.4799 (5.88) | O-Acetyl-saikosaponin or isomer | Bupleuri Radix [ | ||
26 | 23.47 | C21H20O11 | 367.1187 (4.90) | Kaempferol 3-O-galactoside | Nelumbinis Folium [ | ||
27 | 23.52 | C31H50O4 | 487.3391 (−5.54) | Tumulosic acid | Poria [ | ||
28 | 23.55 | C30H48O5 | 489.3576 (0.20) | Alisol F | Alismatis Rhizoma [ | ||
29 | 24.65 | C16H12O5 | 283.0627 (5.30) | Obtusifolin | Cassiae Semen [ | ||
30 | 25.05 | C22H22O6 | 383.1468 (−6.00) | Licoricone | Glycyrrhizae Radix et Rhizoma [ |
The base peak chromatogram (BPC) of the 70% ethanolic SX extract by UPLC-ESI-MS in the positive (a) and negative (b) ion modes.
NASH is a chronic inflammation of the liver, which is related to environmental factors genetics and metabolisms of the body. The accumulation of lipids in the liver is the major cause of oxidative stress and inflammation leading to hepatic steatosis [
From the “two hits” theory, the increased flux of free fatty acid to the liver in obese and insulin resistance conditions may be important in the development of the disease. The free fatty acids may either go through betaoxidation or esterification to form triglycerides leading to hepatic lipid accumulation. Thus, regulating lipid metabolism could be an important means to manage the disease. Clinically, TC, TG, and LDL-C all increase in NASH patients [
The present animal study clearly showed that SX (low, medium, and high dosages) could reduce the serum ALT, AST, FFA, TC, TG, and LDL-C of rats fed with high-fat diet. In addition, results of histopathological examination of the liver using Sudan III staining indicated that accumulation of fat droplets in the liver was significantly suppressed in the SX treatment groups of all dosages with a dose-dependent trend. This provided strong evidence that SX could regulate lipid metabolism and hence it could protect the liver and prevent the progression of NASH in the rat model. The overall results are consistent with our earlier clinical observation.
The energy expenditure of the body has a close relationship with the oxidation of fatty acids and mitochondrial
L-FABP and PPAR
There is currently no approved pharmaceutical agent targeting the treatment of NASH. Therefore, developing therapy for the prevention and/or treatment of NASH is an important research area. NASH was not known in Traditional Chinese Medicine theory. The upregulation of L-FABP and PPAR
In the present study, the chemical constituents of SX were further examined by mass spectrometry and multiclass compounds (a total of 30 compounds) including saponins, iridoids and iridoid glycosides, triterpenes and triterpene acids, anthraquinones, flavanone glycosides, flavonoids, and phenolic acids were tentatively identified. Many of these compounds have been reported to possess different biological activities that are related to liver protection. For example, tumulosic acid showed a moderate inhibition effect on nitric oxide (NO) release from lipopolysaccharide- (LPS-) induced RAW 264.7 cells [
Treatment of SX could lower ALT, AST, FFA, TC, TG, and LDL-C in serum and protect HFD-induced liver injury and NASH by upregulation of PPAR
The authors declare that there are no competing interests regarding the publication of this paper.
This work was supported by Department of Hepatology, Shenzhen Traditional Chinese Medicine Hospital (Shenzhen, China), Guangzhou University of Chinese Medicine (Guangzhou, China), State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Shenzhen, China), Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University (Hong Kong, China), Grants of the National Natural Science Foundation of China (no. 81303203), and basic research of Shenzhen Government (JCYJ20160229173844278, JCYJ20151030164008764). The authors are also very grateful for the Large Equipment Funds and the University Research Facility in Chemical and Environmental Analysis of the Hong Kong Polytechnic University that supported the instruments used in this study.