Effects of Gynura divaricate (L.) DC on Improving Non-alcoholic Fatty Liver by Regulating NF-κB and Nrf-2/HO-1 Pathways

Xian YANG

Pharmaceutical Research Institute of Shijiazhuang No.4 Pharmaceutical Co., Ltd., Shijiazhuang 050000, China

Abstract [Objectives] To study the effect and mechanism of Gynura divaricate (L.) DC (GD) on non-alcoholic fatty liver disease (NAFLD). [Methods] Male mice were randomly divided into 2 groups: normal group and model group. The mice were fed with high-fat diet (HFD) for 4 weeks to induce NAFLD in the model group. The successfully modeled mice were divided into model group, positive drug group, GD high dose group, and GD low dose group. After 4 weeks of administration, the liver index, serum AST, ALT and blood lipid levels, liver tissue pathological changes, antioxidant enzymes, non-enzymatic antioxidants and inflammatory factors levels were measured in each group, and the expression of NF-κB, Nrf-2 and HO-1 in liver tissues were compared. [Results] GD significantly reduced the serum AST, ALT and blood lipid levels, increased enzyme antioxidant and non-enzymatic antioxidant content, reduced the steatosis and inflammatory infiltration of liver cells, down-regulated the level of inflammatory factors, and inhibited the expression of NF-κB, Nrf -2 and HO-1 in liver tissue. [Conclusions] GD has a protective effect on NAFLD in mice and its mechanism may be related to the regulation of NF-κB and Nrf-2/HO-1 pathways.

Key words Gynura divaricate (L.) DC, Non-alcoholic fatty liver disease (NAFLD), Oxidative stress

1 Introduction

Non-alcoholic fatty liver disease (NAFLD), as the most common chronic liver disease, influences about 20%-30% of the global population, and its pathological feature is the accumulation of lipid particles in liver cells[1-3]. The increase in accumulation of fat in liver cells leads to increased oxidative stress in animal and human liver cells, consequently leading to liver cell damage associated with NAFLD[4-5]. Studies have indicated that antioxidants in Chinese herbal medicines and foods play an important role in alleviating NAFLD[6-7].

Gynuradivaricata(L.) DC (GD) is the root and rhizome of Divaricate Gynura in the Compositae family, and it is a Chinese medicinal plant widely distributed in southern China[8]. GD is rich in many active components such as flavonoids, polysaccharides, and alkaloids, and has high medicinal and edible value. GD is often taken for the prevention and treatment of high blood pressure, hyperlipidemia and cancer,etc., and its tender stems and leaves are often used to make tea to treat diabetes[9]. Modern pharmacological studies have shown that GD has biological activities such as lowering the "three highs" (hyperlipidemia, hypertension, and hyperglycemia), preventing cancer, anti-oxidation, anti-inflammatory, antibacterial, protecting liver and choleretics, and regulating immune function[10-12]. In this experiment, we used high-fat diet (HFD) to induce NAFLD model in mice, explored the protective effect of GD on non-alcoholic fatty liver, and explored its action mechanism.

2 Materials and methods

2.1 Experimental animalsForty two 6-8 weeks old SPF male Kunming mice weighing 18-20 g were purchased from the Laboratory Animal Center of Huazhong University of Science and Technology (animal certificate number: SCXK (E) 2010-0009). The mice were fed under the conditions of temperature (25±5) ℃, humidity (65±5)%, and 12 h light (12 h light-dark cycle). All experiments were carried out after approval by the Medical Laboratory Animal Management Committee.

2.2 DrugsThe herbs were collected in Guangdong Province (China) in April 2013 and identified by Professor Wang Jianping from School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology as fresh ground parts ofGynuradivaricata(L.) DC (GD).

2.3 Reagents and instrumentsMetformin was purchased from Bristol-Myers Squibb Company; antioxidant enzymes, non-enzymatic antioxidants, malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG) and total cholesterol (TC) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute; tumor necrosis factor (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) ELISA kits were purchased from Neobioscience Co., Ltd.; NF-κB antibody was purchased from Cell Signaling Technology, nuclear factor E2-related factor 2 (Nrf2) and heme oxidase-1 (HO-1) were purchased from Abcam.

Formula of high-fat feed: 47% normal feed, 20% egg yolk, 20% lard, 2% cholesterol, 1% cholate, 10% sucrose[13]. Inverted microscope: Nanjing Jiangnan Optics & Electronics (Group) Joint Stock Co., Ltd.; microplate reader: Sanyuan Technology Co., Ltd.; UV-visible spectrophotometer: Shanghai Lengguang Technology Co., Ltd.; Rotary evaporator: Gongyi City Yingyu Gaoke Instrument Factory; Gel electrophoresis instrument and gel imaging system: Bio-Rad Laboratories

2.4 Drug preparationFresh GD (10 kg) was pulverized and then extracted with 70% ethanol (1∶20,W/V) percolation extraction 3 times. The filtrate was concentrated using a rotary evaporator at 40 ℃ to obtain 224 g of ethanol extract. The ethanol extract was dispersed in distilled water, and then extracted with petroleum ether and ethyl acetate. Then, the ethyl acetate was concentrated and freeze-dried to obtain 20 g of ethyl acetate extract.

2.5 Modeling and groupingAfter one week of adaptive feeding, 42 mice were randomly divided into normal group (n=8) with 8 mice and model group with 34 mice. The normal group was provided with normal diet, and the model group was treated with HFD to induce NAFLD[10]. After high-fat diet feeding for 4 weeks, 2 mice in the model group were randomly selected and killed by spinal dislocation. The liver was taken to observe the pathological changes of the liver. When the liver HE staining shows obvious different sizes of fat vacuoles, it is deemed that the model is successfully replicated.

The successfully modeled mice were randomly divided into model group (M), positive drug group (P), GD high-dose group (GDH) and GD low-dose group (GDL), with 8 mice in each group. The positive drug group was given 200 mg/kg metformin by intragastric administration, the GD high-dose group was given 400 mg/kg GD by intragastric administration, the GD low-dose group was given 200 mg/kg GD by intragastric administration, and the normal group and model group were given an equal volume of 0.5% CMC-Na by intragastric administration, for 4 consecutive weeks. During the administration period, the normal group was given normal diet, and the other groups were given high-fat diet.

2.6 Observation indicators

No sooner had the nyamatsanes tumbled off to sleep than the man stole softly down and fled away as fast as his legs would carry him, and by the time his enemies were awake he was a very long way off

2.6.1Determination of body weight, liver weight and liver index. After the last administration, the mice in each group were fasted for 12 h, weighed, and blood was taken from the eyeballs, and killed by cervical dislocation. The liver was completely taken out, washed with physiological saline, the surface water was absorbed by the filter paper, weighed, and the liver index was calculated. Liver index=Liver weight (g)/Body weight (g) × 100%.

2.6.2Detection of serum AST, ALT and blood lipid levels in mice. The blood was taken through removing eyeballs and centrifuged at 3 500 rpm at 4 ℃ for 15 min to separate the serum. Then, the contents of AST, ALT, TC, and TG were detected in accordance with the instructions of assay kits.

2.6.3Detection of oxidative stress related indicators. The chopped liver tissue was put into 4 ℃ pre-cooled normal saline at the ratio of 1∶9 (M/V), centrifuged at 4 ℃ 3 500 rpm for 20 min, and the supernatant was taken. Then, the contents of MDA, VC, VD, GSH, SOD, CAT, GR, GST and GSH-Px were detected in accordance with the instructions of assay kits.

2.6.4Liver histological observation and immunohistochemical staining. The liver tissue was fixed with 4% paraformaldehyde, dehydrated, transparentized, immersed, and embedded in paraffin, and then sectioned with a thickness of 5 μm. The sections were stained with hematoxylin-eosin (HE), mounted, and the morphological change in liver tissue were observed under a ×200 magnification optical microscope. Besides, deparaffinized sections were used for immunohistochemical analysis. The above paraffin-embedded tissue was cut into small slices, dried in a constant temperature oven at 37 ℃, deparaffinized, soaked in 3% H2O2at room temperature, sectioned in 5% goat serum, washed with phosphate buffered saline (PBS), and NF-κB (1∶200) was added to 4 ℃ refrigerator and stored overnight for incubation, then HRP-labeled secondary antibody was added at 37 ℃ for 30 min. After washing with PBS, added color developing agent, rinsed the colored slices with distilled water, stained the slices with HE, dehydrated with alcohol, and observed under the ×200 microscope.

2.6.5Detection of liver inflammatory factors. The liver supernatant was prepared using the method stated in Section2.6.3, and the ELISA kit was used to detect the contents of TNF-α, IL-1β and IL-6. For specific steps, refer to the kit instructions.

2.6.6Detection of Nrf2 and HO-1 proteins in mouse liver tissue. After the liver tissue was ground, the concentration was measured with the BCA kit, SDS-PAGE gel was prepared, samples were loaded at 50 μg, 100 V electrophoresis at constant voltage, membrane transferred at 1.5 h, after blocking, added the primary antibody [Nrf2 (1∶500), HO-1 (1∶500); β-actin (1∶500)], incubated overnight at 4 ℃. Washed with TBST, added the secondary antibody, and incubated at room temperature for 1 h. The developer solution was prepared in accordance with the instructions of the electrochemiluminescence kit.

3 Results and analysis

3.1 Effects of GD on the body weight, liver weight and liver index of each group of miceCompared with the normal group, the body weight, liver weight and liver index of mice in the model group were significantly increased (P<0.05); compared with the model group, the body weight, liver weight and liver index of mice in the positive drug group and the GD high-dose group were significantly reduced (P<0.05), while the body weight, liver weight and liver index of the GD low-dose group did not change significantly; compared with the positive drug group, the body weight, liver weight and liver index of the GD high-dose group did not change significantly, and the above values of the GD low-dose group increased (P<0.05); compared with the GD high-dose group, the body weight, liver weight and liver number of the mice in the GD low-dose group increased (P<0.05), as indicated in Table 1.

Table 1 Comparison of body weight, liver weight and liver index of mice in each group n=8)

3.2 Effects of GD on the serum AST, ALT and blood lipid levels of mice in each groupCompared with the mice in the normal group, the AST, ALT, TC and TG of the model group were significantly increased (P<0.05), while the AST, ALT, TC and TG of the positive drug group, GD high-dose group and GD low-dose group were significantly reduced (P<0.05); compared with the mice in the positive drug group, the AST of the mice in the GD high-dose group increased significantly (P<0.05), while the contents of ALT, TC and TG did not change significantly; the AST, ALT, TC and TG of mice in the GD low-dose group were higher than those in the GD high-dose group and the positive drug group (P<0.05), as shown in Table 2.

Table 2 Comparison of serum AST, ALT and blood lipid levels of mice in each group n=8)

3.3 Effects of GD on liver pathological changes and NF-κB expression in each group of miceAs shown in the liver tissue section (Fig.1A), the hepatocyte cords of the mice in the normal group were arranged neatly, the liver cell structure was intact, and there was no obvious fat granule and inflammatory cell infiltration in the cells. Compared with the normal group, the hepatocyte cords of the mice in the model group were arranged disorderly, and there were a large number of fatty vacuoles in the liver tissues, but they have not yet merged into large fatty vacuoles, accompanied by inflammatory cell infiltration. Compared with the model group, the number of fatty vacuoles and the degree of inflammatory cell infiltration in the liver tissue of mice in the positive drug group and the GD high-dose group and GD low-dose group were reduced. There was no significant difference between the GD high-dose group and the positive drug group. The number of fatty vacuoles and the degree of inflammatory cell infiltration in the GD low-dose group were higher than those in the positive drug group and the GD high-dose group. As shown in Fig.1B, compared with the normal group, the model group was dark brown and there was higher expression of NF-κB. Compared with the model group, the positive drug group and the GD high-dose group and GD low-dose group became lighter brown, and the expression of NF-κB decreased. Between GD high-dose group and the positive drug group, there was no difference in the expression of NF-κB. Compared with positive drug group and GD high-dose group, the expression of NF-κB in GD low-dose group was reduced.

3.4 Effects of GD on MDA and non-enzymatic antioxidants in liver tissues of NAFLD miceCompared with the normal group, the content of MDA in the liver of the model group mice was significantly increased, and the content of VC, VEand GSH was significantly decreased (P<0.05); compared with the model group, the content of MDA in the positive drug group, GD high-dose group and GD low-dose group was significantly reduced (P<0.05), and the content of VC, VEand GSH was significantly increased (P<0.05). Compared with the positive drug group, the content of MDA in the GD high-dose group and GD low-dose group was decreased (P<0.05), the contents of VCand VEin the high-dose group and GD low-dose group were increased (P<0.05), and the content of GSH in the GD high-dose group did not change significantly, while the content of GSH in the GD low-dose group was decreased (P<0.05); the content of MDA in the liver of mice in the GD low-dose group was higher than that in the GD high-dose group (P<0.05), and the contents of VC, VEand GSH were lower than that in the GD high-dose group (P<0.05), as shown in Table 3.

Note: N denotes normal group, M denotes model group, P denotes positive drug group, GDH denotes GD high-dose group, and GDL denotes GD low-dose group.

Table 3 Comparison of the contents of MDA, VC, VE and GSH in the liver tissues of mice in each group n=8)

3.5 Effects of GD on antioxidant enzymes in liver tissues of NAFLD miceCompared with the normal group, the contents of SOD, CAT, GSH-Px, GST and GR in the liver of the model group were decreased (P<0.05); compared with the model group, the levels of antioxidant enzymes in the positive drug group, GD high-dose group and GD low-dose group were significantly increased (P<0.05). Compared with the positive drug group, the contents of SOD and GSH-Px in the GD high-dose group were decreased (P<0.05), and the contents of SOD, CAT, GSH-Px and GST in the GD low-dose group were decreased (P<0.05), and the other contents did not change significantly; the levels of GSH-Px and GST in the liver of mice in the GD low-dose group were lower than those in the GD high-dose group (P<0.05), and the other contents had no significant changes, as shown in Table 4.

Table 4 Comparison of SOD, CAT, GSH-Px, GST and GR contents in liver tissues of mice in each group n=8)

3.6 Effects of GD on inflammatory factors in liver tissue of NAFLD miceCompared with the normal group, the contents of TNF-α, IL-1β and IL-6 in the liver of the model group were increased (P<0.05). Compared with the model group, the levels of inflammatory factors in the positive drug group, GD high-dose group and GD low-dose group were significantly decreased (P<0.05). Compared with the positive drug group, the levels of inflammatory factors in the GD high-dose group did not change significantly, while the levels of TNF-α and IL-1β in the GD low-dose group were significantly increased (P<0.05), and the IL-6 content has no significant change. The levels of inflammatory factors in the liver of mice in the GD low-dose group were higher than those in the GD high-dose group (P<0.05), as shown in Table 5.

Table 5 Comparison of the contents of TNF-α, IL-1β and IL-6 in the liver tissues of mice in each group n=8)

3.7 Effect of GD on the expression of Nrf2 and HO-1 protein in each group of miceCompared with the normal group, the expression of Nrf2 and HO-1 in the liver of the model group was decreased (P<0.05). Compared with the model group, the expression of Nrf2 and HO-1 in the liver of mice in the positive drug group and the GD high-dose group was increased (P<0.05). Compared with the positive drug group, the expression of Nrf2 in the liver of mice in the GD high-dose group was increased (P<0.05), and the expression of HO-1 did not change significantly, as shown in Fig.2 and Table 6.

Fig.2 Comparison of expression of Nrf2 and HO-1 proteins in mice of each group

Table 6 Comparison of expression of Nrf2 and HO-1 proteins in mice of each group n=8)

4 Discussion

Nowadays, NAFLD has become an increasingly serious global public health concern[14]. There has been report of participation of oxidative stress in the development of NAFLD[15]. This study indicates that GD can alleviate the NAFLD through reducing the oxidative stress. High dietary fat and cholesterol intake are known risk factors for NAFLD in humans and animals[16]. In this study, we established the NAFLD model through feeding healthy Kunming mice with HFD. The levels of TC, TG, ALT, and AST in the model group were increased, and liver pathology observations showed that a large number of fatty vacuoles and inflammatory cell infiltration were seen in the liver tissue of the model group, indicating that the modeling was successful. Compared with the model group, GD can reduce mouse body weight, liver weight and liver index, improve lipid metabolism disorders, and relieve the NAFLD. High fat and cholesterol in the diet are related to the oxidative stress produced in the cell membrane, and there is a strong relationship between the severity of NAFLD and the degree of oxidative stress[16-18]. In both NAFLD experimental models and human patients, it was found that there is increase of the oxidation marker MDA[19-21]. In this experiment, the MDA level of the model group was significantly higher than that of the normal group, and the increase of MDA level was reversed after GD administration. The reduction of antioxidant defense is also a main factor that promotes oxidative stress in NAFLD patients[22]. According to epidemiological studies, compared with normal people, the first line of defense, the antioxidant defense system, in patients with NAFLD had the reduced content of antioxidant enzymes[23]. In this experiment, the contents of SOD, CAT, GSH-Px, GST and GR in the model group were decreased. After 4 weeks of GD administration, the content of antioxidant enzymes was increased significantly. In addition, studies have shown that the level of endogenous non-enzymatic antioxidants in NAFLD was reduced[24]. The serum non-enzymatic antioxidant VC, VEand GSH levels in the model group were significantly reduced, while the serum non-enzymatic antioxidant levels in the GD group were significantly increased. HO-1 acts as a defense system against oxidative stress in the liver. In the HFD diet model, both hepatic steatosis and increased MDA concentration inhibited the expression of HO-1[25]. Nrf2 is a transcription factor mediated by oxidative stress[26]. As an antioxidant pathway, the Nrf2/HO-1 signaling pathway plays an important role in the progression of the NAFLD[7]. In this experiment, the expression of Nrf2 and HO-1 in the model group was decreased. The expression of Nrf2 and HO-1 was increased after GD administration. According to the above results, GD can reduce oxidative stress and improve the NAFLD by activating the Nrf2/HO-1 pathway.

Reactive oxygen species (ROS) and lipid peroxidation products can promote the production of pro-inflammatory cytokines and induce inflammation. Inflammation mediates the progression of NAFLD and causes various pathological changes of NASH. Recent studies have shown that the levels of pro-inflammatory cytokines including IL-6 and TNF-α are significantly up-regulated in the NAFLD[27]. In this study, the liver pathology results of the model group showed high inflammatory cell infiltration, while the levels of TNF-α, IL-1β, and IL-6 in the liver tissue increased. The liver pathology results of the GD groups showed low inflammatory cell infiltration and reduced level of TNF-α, IL-1β and IL-6 in the tissues. The maturation and secretion of pro-inflammatory cytokines are regulated by activating NF-κB[28]. NAFLD animal models and NASH patients have shown sustained NF-κB pathway activation[29]. The results of immunohistochemistry showed that the expression of NF-κB in the model group was significantly increased. Compared with the model group, when the GD dose was 400 mg/kg/d, the expression of NF-κB was significantly reduced. According to the above results, GD improves the inflammatory response induced by oxidative stress in the liver of NAFLD mice by inhibiting the expression of NF-κB.

In summary, GD has a protective effect on mouse NAFLD, and its mechanism may be related to the regulation of NF-κB and Nrf-2/HO-1 pathways.