Abstract
The protective effects of the total glycosides from Ligustri Lucidi Fructus against nonalcoholic fatty liver (NAFL) in mice were investigated. Liver injury was induced by the administration of high fat diet for 60 days. During this period, the model group received high fat diet only; the treatment groups received various drugs plus high fat diet. Compared with the model group, the total glycosides significantly decreased the contents of triglyceride (TG) and cholesterol (TC), as well as the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum. Moreover, the contents of TG and TC in liver tissue and the liver index were reduced. Histological findings also confirmed antisteatosis. Compared with the model group, total glycosides significantly reduced the levels of the sterol regulatory element binding protein-1c (SREBP-1c) and liver X receptor-a (LXR-α) protein, and down-regulated the expression of SREBP-1c, LXR-α and interleukin-6 (IL-6) mRNA in the liver. These results suggest that the total glycosides are effective in the treatment of NAFL of mice. Their mode of action is associated with inhibiting SREBP-1c, LXR-α and IL-6 mRNA, reducing lipid synthesis factor SREBP-1c and LXR-α protein and gene expression, suppressing inflammatory responses, then decreasing serum lipid and hepatic lipid.
1 Introduction
With the changes of life style, nonalcoholic fatty liver disease (NAFLD) has been on the increase and turns out to be an important disease in many developed and developing countries. NAFLD influences people’s health and efficiency of labour seriously, so it is a social problem which has aroused general concern to prevent and cure the disease early and effectively. Hence it is important to develop specific pharmacological or nutritional agents to treat NAFL and prevent the development of more severe forms of liver injury [1]. The fruits of Ligustrum lucidum Ait. (Ligustri Lucidi Fructus, LLF) are a common traditional Chinese health food, mainly to nourish livers and kidneys. Water-soluble glycosides are the main pharmacological components of LLF, which have a variety of pharmacological effects such as liver-protecting, anti-inflammatory and antioxidative [2–6]. Our previous studies showed that LLF can prevent fatty liver and fibrosis in rats [7]. The effects of the total glycosides from LLF on mice suffering from NAFLD induced by high fat diet were investigated in this study, which deals with the pathological changes and lipid levels. Ultimately, the putative mechanism underlying the hepatoprotective effects was evaluated. The study can also help to provide experimental information for the development of active constituents of LLF.
2 Experimental
2.1 Experimental animals
Fifty male 4-week-old ICR mice (20±1 g) were obtained from the Zhejiang Laboratory Animal Center (No. SCXK [Zhe] 2011-0001) and allowed to acclimate in quarantine for a week prior to experimentation. This study was carried out in strict accordance with the recommendations in the Guide for Animal Experiments of Nanjing University of Chinese Medicine. The protocol was approved by the Animal Ethics Committee of the Nanjing University of Chinese Medicine. The animals were handled under the standard laboratory conditions of a 12-h light/dark cycle in a room with controllable temperature and humidity. Food and water were available ad libitum.
2.2 Drugs and reagents
High fat diet, containing 15 % lard, 10 % yolk powder, 4 % cholesterol, 1 % sodium cholate, and 70 % common feed, was prepared by the Experimental Animal Center of Nanjing University of Chinese Medicine. The kits for the determination of triglyceride (TG), cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and interleukin-6 (IL-6) were provided by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against sterol regulatory element binding protein-1c (SREBP-1c) and liver X receptor (LXR-a) were the products of SantaCruz Biotechnology (Shanghai, China). β-Actin antibody and RIPA lysis buffer were from Beyotime Institute of Biotechnology (Shanghai, China). The 2×Taq PCR Master Mix was purchased from Real-Times Biotechnology (Beijing, China). Simvastatin was the product of Hangzhou MSD Pharmaceutical Company (Hangzhou, China).
2.3 Extraction and isolation of total glycosides
The air-dried and powdered LLF (3 kg) was collected in November 2010 from Nanjing, and extracted with 80 % (v/v) aqueous alcohol for 2 h under reflux; the extract was reduced to dryness under reduced pressure. The resulting extract (920 g) was suspended in H2O and repeatedly extracted with petroleum ether. The H2O layer was evaporated under vacuum to leave the residue (670 g), which was absorbed on D-101 macroporous resin (1 kg) and then eluted consecutively with H2O, 40 % EtOH, and EtOH, each repeatedly. The combined 40 % EtOH elutropic fraction was concentrated under vacuum to afford the total glycoside extract (TGE, 190 g). The content of total glycosides was determined to be 68 % by ultraviolet–visible spectrophotometry. HPLC analysis revealed that TGE consisted mainly of salidroside, nuezhenoside, Gl3 and p-hydroxyphenethyl 7-β-D-glucoside elenolic acid ester (Figure 1), which were confirmed by comparison with reference compounds based, on their retention times and UV spectra.
HPLC chromatogram of total glycosides extract (TGE) from Ligustri Lucidi Fructus.
a, Salidroside; b, nuezhenoside; c, Gl3; d, p-hydroxyphenethyl 7-β-D-glucoside elenolic acid ester.
2.4 Animal experimental design
The animals were randomly divided into five groups of 10 mice per group. Group I served as a normal control and was given normal diet. To induce fatty liver, the animals in Groups II–V received high fat diet. Group II served as the fatty liver model group. In addition to high fat diet, Group III was given simvastatin (10 mg/kg) orally daily for a period of 60 days, and served as a positive control. In addition to high fat diet, Groups IV–V received TGE orally (250,500 mg/kg, representative of TGE low and high dosage, respectively) daily for 60 days. At the end of the experiment, the animals were weighed and sacrificed by cervical dislocation. Blood samples were drawn from eye sockets. Liver samples were dissected, washed immediately with ice-cold saline to remove as much blood as possible, and weighed to calculate the liver index. One part of each liver sample was immediately stored at –80 °C until analysis, and another part was excised and fixed in 10 % formalin solution for histopathological analysis.
2.5 Biochemical assay and determination of liver index
Blood samples were immediately centrifuged at 10,000 g for 10 min at 4 °C, and the supernatant serum was collected. Each 200 mg liver sample was homogenised with 1.8 mL isopropanol or normal saline, and centrifuged at 10,000 g for 10 min at 4 °C, and the supernatants were collected. The serum and hepatic tissue TG, TC, ALT and AST levels were measured according to the kit instructions. The liver index was calculated as the ratio of hepatic wet weight to body mass.
2.6 Histological analysis of liver
After formalin fixation for 36 h, the liver samples were sectioned and stained with haematoxylin–eosin (H&E) and subsequently examined under a light microscope (IX51, Olympus, Tokyo, Japan) for general histopathology examination. The extent of hepatic damage was evaluated on H&E slides. The histological changes were observed and scored.
2.7 Western blot analysis of LXR-α and SREBP-1c protein levels in liver tissue
The liver tissue was homogenised and lysed in RIPA lysis buffer. Samples were incubated on a rotator at 4 °C for 30 min, and centrifuged at 10,000 g for 10 min at 4 °C. The supernatant protein concentration was measured by the bicinchoninic acid method, and 20 μg of protein were loaded onto a gel, subjected to SDS-polyacrylamide gel electrophoresis, and then electroblotted onto nitrocellulose membranes. Membranes were incubated with LXR-α or SREBP-1c antibodies according to the manufacturer’s protocol. Protein bands were visualized by enhanced chemiluminescence (ECL) with a kit from Santa Cruz. The apparent molecular masses of proteins were determined using electrophoresis colour markers. Image Tools 3.0 provided the means for processing protein band images, and the ratio of the optical density of the target protein level to that of β-actin was calculated to represent its relative level, as shown in Figure 2.
Effect of TGE on expression of the LXR-α (top) and SREBP-1c (bottom) protein, respectively.
I, Normal group; II, model group (high fat diet); III, simvastatin group; IV, TGE low dosage group; V, TGE high dosage group.
2.8 Real-time quantitative polymerase chain reaction (RQ-PCR) analysis of LXR-α, SREBP-1c and IL-6 mRNA
Total RNA was extracted with Trizol, and the A260/A280 value of the preparation was in the range of 1.8-2.0. Each sample (5 μg) was taken to produce cDNA by reverse transcription. The PCR primers were designed as follows: LXR-α, sense: 5′-CCTACAGAACTTCGTCCACAGA-3′, antisense: 5′-GCAGC CACCAACTTCTCAATC-3′, 93bp; SREBP-1c, sense: 5′-GGAGCCATGGATTGCACATT-3′, antisense: 5′-AGGAAGGCTTCCAGAGAGGA-3′, 93bp; IL-6 sense: 5′-AATCATCACTGGTCTTTTG GAG-3′, antisense: 5′-GCATTTGTGGTTGGGTCA-3′, 97bp; β-actin, sense: 5′-TACAACTCCTTGCA GCTCC-3′, antisense: 5′-ATCTTCATGAGGTAGTCAGTC-3′. The PCR reaction was carried out in a 25 μL reaction volume. Final concentrations of reactants were as follows: 1 μL sense primer, 0.4 μM; 1 μL antisense primer, 0.4 μM; 2×Taq PCR Master Mix 12.5 μL; ddH2O 9.5 μL. PCR amplification were carried using β-actin primers as the internal control. The relative level of the transcript of the target gene was determined.
2.9 Statistical analysis
Data were presented as mean values ± SD. The significance of differences between the means among different groups was analyzed by one-way ANOVA with SPSS13.0 and the level of P<0.05 or P<0.01 was taken as statistically significant.
3 Results
3.1 Effects of TGE on serum lipid, serum transaminase levels, liver lipid and liver index
The effects of TGE treatment on high fat diet-induced changes in the serum lipid levels are shown in Tables 1 and 2. Adipose degeneration was induced by high fat diet in mice, as indicated by an increase in the serum levels of TG, TC, ALT and AST. In contrast, the animals treated with simvastatin or TGE exhibited a significant dose-dependent decrease in the activities of these serum lipids and transaminase levels (P<0.05~P<0.01). Simvastatin or TGE administration also individually reversed the alterations of the liver index and lipid levels compared with the model group (P<0.05~P<0.01).
Effect of TGE on serum lipid and transaminase levels of NAFL mice.
| Group | TG (mmol·L-1) | TC (mmol·L-1) | ALT (U·L-1) | AST (U·L-1) |
|---|---|---|---|---|
| I | 0.86±0.13** | 2.35±0.21** | 20.17±5.18** | 45.41±9.12** |
| II | 1.52±0.26 | 3.61±1.50 | 41.20±9.82 | 60.19±11.36 |
| III | 0.95±0.20** | 2.40±0.30** | 27.45±6.83** | 45.67±8.68** |
| IV | 0.99±0.17** | 2.68±0.91** | 38.56±8.10 | 51.10±9.78* |
| V | 0.89±0.12** | 2.42±0.43** | 36.33±7.38* | 49.29±10.05* |
I, Normal group; II, model group (high fat diet); III, simvastatin group; IV, TGE low dosage group; V, TGE high dosage group. Compared with value of the model group. *P<0.05, **P<0.01.
Effect of TGE on liver index and liver lipid levels of NAFL mice.
| Group | Liver index (%) | TG (mg/g LW) | TC (mg/g LW) |
|---|---|---|---|
| I | 4.52±0.85** | 10.45±2.06** | 4.45±0.77** |
| II | 6.60±1.19 | 21.11 ±3.46 | 5.71±1.21 |
| III | 5.33±0.99* | 12.82±2.34** | 4.67±0.87** |
| IV | 5.50±1.01 | 16.90±3.31** | 5.05±0.96** |
| V | 5.02±0.78* | 12.55±2.10** | 4.40±0.80** |
I, Normal group; II, model group (high fat diet); III, simvastatin group; IV, TGE low dosage group; V, TGE high dosage group. Compared with that of the model group. *P<0.05, **P<0.01. LW, Liver fresh weight.
3.2 Histopathological examination
Examination of liver histology (not shown) revealed that high fat diet induced changes including cell swelling, inflammatory infiltration, increased fat vacuoles and obvious degeneration. All mice except those in the normal group exhibited the ballooning degeneration and inflammation reaction. The damage in the model group was more severe than in the groups treated with simvastatin or TGE. High fat diet causes steatosis in liver; TGE appears to ameliorate the liver injuries by reducing fat deposit and enhancing fat metabolization, which is further confirmed by the reduced extent of histopathologically visible injury.
3.3 Effects of TGE on LXR-α and SREBP-1c levels
Figure 2 indicates that compared with the normal group, the levels of LXR-α and SREBP-1c in liver tissue of the model (high fat diet) group were significantly higher (P<0.01), but were reduced by TGE administration with significant difference (P<0.05 and P<0.01, respectively).
3.4 Effects of TGE on transcript levels of LXR-α, SREBP-1c and IL-6
The levels of the LXR-α, SREBP-1c and IL-6 transcripts in the model (high fat diet) group were higher than those of the controls, but their increase was significantly suppressed by TGE in a dose-dependent manner (P<0.05) (Table 3), corresponding to a similar trend of the protein levels observed in the Western blot analysis (Figure 2).
Effect of TGE on transcript levels in livers of NAFL mice.
| Group | LXR-α | SREBP-1c | IL-6 |
|---|---|---|---|
| I | 0.14±0.03** | 0.26±0.03** | 1.00±0.10** |
| II | 0.56±0.10 | 0.92 ±0.04 | 10.33±0.71 |
| III | 0.28±0.04* | 0.50±0.04** | 7.01±0.96** |
| IV | 0.39±0.05* | 0.33±0.05** | 5.06±0.62** |
| V | 0.23±0.03* | 0.46±0.04** | 3.03±0.21** |
I, Normal group; II, model group (high fat diet); III, simvastatin group; IV, TGE low dosage group; V, TGE high dosage group. Compared with that of the model group. *P<0.05, **P<0.01.
4 Discussion
Ligustri lucidi fructus is a common liver protectant in Chinese medicine. LLF and its chemical components have a variety of pharmacological effects, such as protecting the liver, being anti-inflammatory and antioxidative, and lowering lipid levels. Our previous studies showed that LLF mainly contains water-soluble glycosides, such as salidroside, nuezhenoside, Gl3 and p-hydroxyphenethyl 7-β-D-glucoside elenolic acid ester [8]. As an extension of our previous investigation, one of our current research priorities is the detailed characterization and quantitative determination of representative biomarkers for the anti-steatohepatic properties of the total glycosides from LLF.
NAFLD is a clinical syndrome characterized by excessive fat storage in the liver and by hepatic cellular degeneration, not resulting from alcohol ingestion. The results of this study show that the levels of TG and TC in the liver of NAFL mice had increased significantly, along with a diffuse severe degeneration and inflammatory cell infiltration. The pathogenesis of NAFLD is still not very clear, but the “two hits” theory is approved by most scientists in the field [9]. The “first hit” is insulin resistance, which leads to fatty degeneration of liver cells through increased hyperinsulinaemia. The “second hit” is lipid peroxidation caused by excess peroxide and some abnormal cytokines such as TNF-α and IL-6, which cause inflammation, necrosis and fibrosis of liver cells [10–14]. Recent research suggests that PPAR-a, SREBP-lc and LXR-a are the main regulating factors balancing lipid synthesis and decomposition, and their signaling pathways are closely linked to the two hits [15]. In this study, in mice with fatty livers induced by a long time high fat diet, TGE could significantly reduce the degree of degeneration, and mitigated the inflammation response. TGE intervention also lowered the levels of TG, TC, ALT and AST in the serum. Moreover, the levels of TG and TC in liver tissue as well as the liver index were reduced. The results suggest that the mode of action of TGE on NAFL mice might be related to regulating lipid metabolism and relieving lipid peroxidation injury. This study also revealed that TGE can significantly reduce the levels of the lipid synthesis factor SREBP-lc and of the LXR-a protein through decreasing the expression of the respective genes, and it reduced the expression of IL-6 as well. So we can infer that TGE has a beneficial effect in preventing NAFLD by regulating lipid metabolism, reducing lipid peroxidation, inhibiting pro-inflammatory cytokines, and down-regulating the expression of SREBP-lc and LXR-α. It can be assumed that TGE predominantly contributes to the therapeutic effects of LLF on NAFLD.
Acknowledgments
This research was financially supported by the Ph.D. Programs Foundation of the Ministry of Education of China (20113237120008) and the Priority academic program development of Jiangsu Higher Education Institutions.
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