Aucubin inhibited lipid accumulation and oxidative stress via Nrf2/HO‐1 and AMPK signalling pathways

Abstract Aucubin (AU) is the main active ingredient of Aucuba japonica which has showed many positive effects such as anti‐inflammation and liver protection. Non‐alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. In this research, we explored the effects of AU on the tyloxapol‐induced NAFLD in mice and apolipoprotein C‐III (apoC‐III) induced‐3T3L1 cells. Tyloxapol (300 mg/kg) was injected to C57BL/6 mice with aucubin. The differentiated 3T3‐L1 cells were treated with or without aucubin after stimulation of apoC‐III (100 μg/mL). In results, aucubin inhibited hyperlipidaemia, oxidative stress and inflammation by influencing the content of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), very low density lipoprotein (VLDL), myeloperoxidase (MPO), superoxide dismutase (SOD), tumour necrosis factor receptor‐α (TNF‐α), interleukin‐1β (IL‐1β), and IL‐6 in blood. AU activated NF‐E2‐related factor 2 (Nrf2), peroxisome proliferator‐activated receptor α (PPARα), PPARγ and hemeoxygenase‐1 (HO‐1) and promoted the phosphorylation of adenosine 5′‐monophosphate‐activated protein kinase (AMPKα), AMPKβ, acetyl‐CoA carboxylase (ACC) and protein kinase B (AKT). In conclusion, AU performed the function of hypolipidaemic by its obvious anti‐inflammation and antioxidant activity, which may become a kind of new drug targeting at NAFLD.

when HCC occurs. 4 It also has been reported that toll-like receptor 4 (TLR-4), which is an activator of NF-κB, may up-regulate expression by gut microbiota-derived ligand (like lipopolysaccharide). 5 In the progress of NAFLD, it is often accompanied by ectopic fat accumulation and this is usually associated with increasing secretion of hepatokines, 6,7 gluconeogenesis, decreased glycogen synthesis and inhibition of insulin signalling. 8 Transcription factor nuclear factor erythrocyte 2-related factor 2 (Nrf2) is a positive regulator of a group of gene expression involved in antioxidant/electrophilic stress protection. Nrf2 regulates downstream antioxidant stress genes such as hemeoxygenase-1 (HO-1) and superoxide dismutase (SOD). It is a key regulator of cellular stress defense mechanisms by binding to antioxidant redox elements (ARE) when challenged by cellular stress. 9,10 In rodents, Nrf2 is also involved in hepatic fatty acid metabolism as a negative regulator of genes that promote hepatic ossification. Several recent works suggest the nuclear receptors peroxisome proliferator-activated receptor (PPAR) α and PPARγ are involved in anti-inflammation reactions in atheroma associated cells. [11][12][13][14][15][16] PPAR regulates gene expression by acting as a transcription factor for specific ligands. PPARα and PPARγ catalysts could reduce inflammatory proteins such as adhesion molecules, cytokines, chemokines, in monocytes/macrophages, ECs and vascular smooth muscle cells. 17 In addition, it has been revealed that PPAR activators can limit experimental atherosclerosis in animal models, although its expression is considered to be restricted to the tissues like liver and fat. 18,19 AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis. It can sense cellular ATP starvation and is an important regulator of autophagy. 20 AMPK is prevalent as a heterotrimeric complex containing α catalytic subunits and β and γ regulatory subunits, which occur in multiple subtypes (α1/α2; β1/β2; γ1/γ2/γ3) encoded by different genes. 21 In skeletal muscle and liver, AMPK activates fatty acid oxidation by inhibiting the acetyl-CoA carboxylase (ACC1)/ACC2 subtype of ACC, thereby reducing malonyl-CoA, a substance that inhibits fatty acid entry into mitochondria. 22,23 AMPK promotes the expression of the tricarboxylic acid cycle enzyme, as well as the mitochondrial biological origin, which achieves transcriptional co-activator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) by increasing expression/activity. 24 PGC-1α could coactive several transcription factors associated with oxidative stress, including Nrf2 and PPARα. 21 Aucubin (1, 4, 5, 7 a-tetra-5-hydroxy-7-(hydroxymethyl) cyclopenta (c) pyran 1-yl-β-D-glucopyranoside, Figure 1), a natural compound extracted from various plants including Aucuba indica and Eucommia leaves, is proven to have a variety of pharmacological effects, especially anti-inflammatory effects. [25][26][27][28][29][30][31] Aucubin could inhibit the RNA and protein synthesis in the liver of mice, and protect liver from the damage caused by carbon tetrachloride or alpha-amanitin in mice and rats, and has antibacterial activity. 25,26 In this experiment, we examined the potential regulatory effects of aucubin on NAFLD via Nrf2 and AMPK family. It was found that aucubin significantly regulated the lipid accumulation and oxidative stress both in vivo and in vitro to inhibit the damage caused by NAFLD.

| Animals
Male C57/BL6 mice (6-8 weeks), weighing approximately 20 g, were purchased from Liaoning Changsheng Biotechnology (Liaoning, China). The mice were fed a standard diet and housed under a specific pathogen free (SPF) condition (temperature: 24 ± 1°C, relative humidity: 40%-80%, n = 3 per cage). The animal studies are reported as recommended by the ARRIVE guidelines. 32 All animal experiments were performed in accordance with the National Institutes of Health (NIH) guide for the Care and Use of Laboratory Animals and approved by the Jilin University animal administration committee.

| Measurement of SOD in serum
The serum was collected after 24 hours of aucubin treatment. The serum of all groups was pre-tested to determine the optimal inhibition rate. The preliminary results showed that the inhibition rate was 46% when the serum was diluted 15 times for the following experiments. The SOD activity was calculated following the instruction of kit (Jiancheng Nanjing, Jiangsu, China).

| Measurement of TNF-α, IL-1β and IL-6 in serum
The levels of TNF-α, IL-1β and IL-6 in serum were detected by ELISA kits according to the manufacturer's instruction (BioLegend, CA, USA).

| Western blotting
The liver (about 20 mg) was ground using Electric Tissue Grander

| Oil-red staining for liver
The middle of the fresh liver lobe was put into OCT embedding and storing at −20°C. The tissue sections were made by freezing microtome using embedded wax. The sections were placed in 75% ethanol for 3 minutes and 0.3% oil red dye for 10 minutes. The sections were placed in the essence of cypress for 15 seconds and covered by the glass slides. The sections were observed under the microscope.

| Haematoxylin and eosin staining for liver
The left lobe of liver was collected after the mice were killed and put into 4% formaldehyde buffer. The tissues were embedded in paraffin, sliced and stained with haematoxylin and eosin. The slices were observed with a light microscope.

| Measurement of liver index
The weight and total liver of each mouse were measured accurately.
Liver index = liver weight/body weight, and the mean value was calculated.

| Observation of blood character
After 24 hours of the treatment, the blood was taken and put at 4°C in 1.5 mL EP overnight. The images were collected.

| 3T3-L1 cell differentiation
3T3-L1 cells were inoculated with complete culture medium in 35 mm petri dishes (1 × 10 6 /well) at 37 °C and 5% CO 2 incubator. When the cells grew to 80%-90%, the medium was replaced with it containing 0.5 mM IBMX (storage density of 0.5 mol/L), 1 μmol/L DEX (storage density of 2.5 mol/L), and 1.7 μmol/L insulin (storage density of 1.7 mol/L). The medium was exchanged with the medium containing 1 μmol/L insulin after 48 hours and cultured for another 48 hours. According to the state of cell growth and differentiation, complete culture medium should be replaced every 48 hours to continue culture. After 8-10 days, the complete differential cells could be used for subsequent experiments.

| Cell count kits -8
The differential 3T3-L1 cells were cultured in the 96-well plate (4 × 10 5 cells/mL) for 1 hour. DMEM medium was added to the control group, and 20, 35, 70, 140 and 280 μg/mL aucubin were added to the aucubin group, respectively. Each group was added with 10 μL CCK-8 and cultured for 3-4 hours. OD value was determined by 450 nm enzyme assays.

| Detection of the expression of mRNA
Software Primer Express 5.0 was used to design the sequences of TNF-α, IL-1β, IL-6 and β-actin (Table 1). Trizol was used to extract the total RNA from the differential 3T3-L1 cells treated with aucubin and apoC-III. Total RNA content and purity were measured by

| Western blot
The differential 3T3-L1 cells were used to extract protein. The method was the same as mentioned in Western blotting.

| Statistics analysis
The obtained data were analysed using IBM SPSS Statistics 19.0 and the results were shown as mean plus or minus standard deviation. ONE way-ANOVA was used for multi-group comparisons and Student's t test was used between the two groups. ## P < 0.01 indicates a significant difference from the control group. *P < 0.05 indicates a significant difference from the model group and **P < 0.01 indicates a significant difference from the model group.

| Aucubin decreased the accumulation of TC, TG, LDL-C and VLDL induced by tyloxapol
The results showed that TC, TG, LDL-C and VLDL content of mice in tyloxapol group increased significantly compared with the control group (P < 0.01), and the content of HDL-C decreased significantly The results showed that there was no significant variation in the four indexes between the only aucubin group with the control group. As shown above, the tyloxapol can significantly increase blood lipids, while aucubin can significantly reduce high blood lipids.

| Aucubin elevated the level of SOD
SOD is a free radical scavenger, which can scavenge superoxide anion free radicals in the body. Lower SOD content in the body implies that the body's ability to remove harmful free radicals is reduced. As shown in Figure 2A, SOD content decreased significantly after tyloxapol stimulation (P < 0.01). However, the content of SOD in aucubin groups (20 and 40 mg/kg) increased significantly (P < 0.01) and the application of aucubin significantly increased the SOD content, which suggested that aucubin could improve the body's antioxidant capacity.

| Aucubin reduced the increasing level of MPO induced by tyloxapol
As shown in Figure 2B, MPO content in tyloxapol group increased significantly compared to the control group (P < 0.01). The content of MPO in the aucubin groups (10, 20 and 40 mg/kg) and fenofibrate group significantly decreased compared with tyloxapol group (P < 0.01). There was no difference between the aucubin only group and the control group.

| Aucubin inhibited the increase in TNF-α, IL-1β and IL-6 caused by tyloxapol
The content of pro-inflammatory cytokines in the blood can directly reflect the immune response ability of the body. TNF-α, IL-1β and IL-6 expression of mice was determined by ELISA. As shown in

| Aucubin regulated the phosphorylation of ACC, AMPKα AMPKβ and AKT in tyloxapol-induced hyperlipaemia of mice
As is shown in Figure 3G, H, I, J and K, aucubin could enhance the phosphorylation of ACC, AMPKα, AMPKβ and AKT significantly on tyloxapol-induced NAFLD of mice.

| Aucubin reduced the accumulation of lipid in the liver of mice
Oil-red O could dye the lipid into red, while haematoxylin dyes the cell nucleus into blue. As shown in Figure 4A, in the tyloxapol group the normal nucleus (blue) had been covered by the lipid droplets (red). The application of fenofibrate and aucubin (10, 20 and 40) can clearly reduce the fat in the liver and the nuclear structure was gradually clear.

| Aucubin reduced the inflammation damages of liver
The liver pathological injury was examined to detect the anti-inflammation effect of aucubin. As is shown in Figure 4B

| Aucubin reduced the lipid gathering in liver and serum
As shown in Figure 4C, the serum of the control group was clear and transparent, while that of the tyloxapol group was milk fat and opaque. However, after the application of aucubin, the content of serum lipid decreased and gradually turned translucent.
As shown in Table 2, the mice weight, the liver weight and liver index of tyloxapol group all increased compared with the control group. Compared with the tyloxapol group, the liver index of three concentrations of aucubin groups (10, 20 and 40 mg/kg) decreased in different degrees which explained that aucubin could break the lipid production both in liver and blood.

| Aucubin affected the cell viability
The  Figure 5A, aucubin had no effect on the cell quantities and viability with or without apoC-III.

| Aucubin inhibited the release of TG, TNF-α, IL-1β and IL-6 induced by apoC-III
We concluded from Figure 5A that apoC-III could promote 3T3-L1 cells release TG, TNF-α, IL-1β and IL-6 while aucubin suppressed this increasing trend in a dose-independent manner in Figure 5C-H.

| D ISCUSS I ON
It is widely accepted and confirmed that atherosclerosis adhered to monocytes/lymphocyte activation of endothelial cells (EC) of chronic inflammatory diseases, whose early symptoms are usually hyperlipidaemia. Inflammation is a complex interaction between soluble factors and cells that can occur in any tissue to cope with trauma, infectivity, ischaemia, poisoning or autoimmune damage.
Inflammation seems to be involved in all stages of atherosclerosis. 33 When endothelial cells are activated and express chemokines, in- Apolipoprotein C-III is a small surface protein found on many TRLs and is considered to be a key factor in hypertriglyceridaemia because of its inhibitory effect on apolipoprotein catabolism. 35 ApoC-III has been shown to be an important factor in regulating plasma triglyceride concentrations in vitro. In rodent and human subjects, increased expression of apoC-III is associated with elevated plasma triglyceride levels, and decreased triglyceride levels. 36,37 In our study, the results

TA B L E 2 Liver index of each group
showed that the level of apoC-III was increasing with the higher TC, TG, LDL and VLDL in tyloxapol group while aucubin inhibited the rising trend ( Figure 1A-C, E and H). In vitro, lipoprotein lipase and liver lipase can be inhibited by apoC-III via blocking the binding of apoB100 or apoE to liver receptors, delaying the clearance of very low density lipoprotein (VLDL), 38,39 so we used apoC-III as the stimulant in differential 3T3-L1 cells.
Some studies report pro-inflammatory cytokines could stimulate atherosclerosis chemokines and adhesion molecules that enable early culture of monocytes and lymphocytes of endometrium. In addition, the cytokine activated macrophages and blood vessel cells of matrix metalloproteinases (MMPS) and promoted cell apoptosis.
Cytokines play a potentially harmful role in advanced atherosclerosis, often causing plaques to rupture or erode more easily. The balance between pro-inflammatory cytokines and anti-inflammatory cytokines is a major determinant of plaque stability. 40 Therefore, it is necessary to explore the role of inflammatory cytokines and inflammatory signalling pathways in early atherosclerosis, which F I G U R E 5 Aucubin affected on the cell viability inhibited the releasing of tumour necrosis factor receptor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 induced by apoC-III. A, The differentiation 3T3-L1 cells were exposed to aucubin (20,35,70,140 and 280 μg/mL) in absence or presence of apoC-III. The effect of aucubin on cell viability was detected using CCK-8 assay. B, The differentiation 3T3-L1 cells were exposed to aucubin (35, (Figures 3 and 6).
Besides, we also detected the effect of aucubin on the AMPK signalling pathway. Many studies have reported that AMPK could be activated by many natural products and inhibited by lipid overload, and can modulate the oxidative stress response via regulating ROS production. In our study, we found that tyloxapol and apoC-III could obviously inhibit the phosphorylation of ACC, AMPKα and AKT while aucubin activated the phosphorylation of ACC, AMPKα, AMPKβ and AKT. As the previous study reported, oxidative stress often occurs in the progress of hyperlipaemia and even NAFLD by regulating ROS and MPO production. 48 The level of MPO and ROS in tyloxapol-induced mice decreased significantly compared with the control group while aucubin pretreatment suppressed the decreasing trend indicating that high blood lipid was accompanied by the process of oxidative stress ( Figure 2B). What is more, the activity of AMPK changes with the occurrence of oxidative stress. The observation indicated that aucubin decreased the lipid accumulation and oxidative damage via regulating the expression of Nrf2, PPARα, PPARγ and AMPK relevant proteins.
Hepatic lipid level of liver can be observed through oil red O staining of frozen section. The lipid in the liver of tyloxapol group was increased significantly compared with the control group, while the lipid content decreased in different degrees after the pre-treatment with aucubin. The liver of the aucubin alone group was basically the same as that of the control group, indicating that the application of aucubin alone could not produce toxic and adverse effects on the liver ( Figure 4A).
In conclusion, as shown in Figure 7, aucubin is capable of relieving the lipid accumulation through activating Nrf2 and PPAR both in mice and 3T3-L1 cell. Aucubin also inhibited the release of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 and enhanced the phosphorylation of ACC, AMPKα and AKT which were associated with oxidative stress and inflammation in hyperlipaemia. From the above, aucubin may be a potential therapeutic drug targeting at NAFLD and hyperlipaemia.

ACK N OWLED G EM ENTS
This study was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.