Polysaccharides from Dicliptera chinensis ameliorate liver disturbance by regulating TLR‐4/NF‐κB and AMPK/Nrf2 signalling pathways

Abstract The purpose of this study was to alleviate liver disturbance by applying polysaccharides from Dicliptera chinensis (DCP) to act on the adenosine monophosphate–activated protein kinase/ nuclear factor erythroid 2‐related factor 2 (AMPK/ Nrf2) oxidative stress pathway and the Toll‐like receptor 4 (TLR‐4)/ nuclear factor kappa‐B (NF‐κB) inflammatory pathway and to establish an in vivo liver disturbance model using male C57BL/6J and TLR‐4 knockout (−/−) mice. For this, we evaluated the expression levels of SREBP‐1 and Nrf2 after silencing the expression of AMPK using siRNA technology. Our results show that with regard to the TLR‐4/ NF‐κB inflammatory pathway, DCP inhibits TLR‐4, up‐regulates the expression of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), reduces the expression of phospho(p)‐NF‐κB and leads to the reduction of downstream inflammatory factors, such as tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐6 and IL‐1β, thereby inhibiting the inflammatory response. Regarding the AMPK/ Nrf2 oxidative stress pathway, DCP up‐regulates the expression of p‐AMPK and Nrf2, in addition to regulating glucose and lipid metabolism, oxidative stress and ameliorating liver disturbance symptoms. In summary, our study shows that DCP alleviates liver disturbances by inhibiting mechanisms used during liver inflammation and oxidative stress depression, which provides a new strategy for the clinical treatment of liver disturbance.


| INTRODUC TI ON
Non-alcoholic fatty liver disease (NAFLD) with an incidence of about 24% of the global adult population is one of the most widespread chronic liver diseases worldwide. 1 Notably, NAFLD is associated with the development of obesity and type 2 diabetes. 2 Many studies have found that NAFLD does not only promote end-stage liver disease, but can also accelerate the occurrence of atherosclerosis, insulin resistance (IR) and tumour development. 2,3 Importantly, there are no approved drugs specifically registered for the treatment of NAFLD. 4,5 Based on this, it is clear that identifying novel treatments to improve NAFLD development and treatment is imminent.
The AMPK/ Nrf2 pathway has been shown to play a crucial part in the process of resisting oxidative stress and inflammation in vivo. 6 Notably, AMPK, a crucial cellular regulator, can maintain the energy supply and demand balance of cells in the body.
Activated AMPK promotes activation of Nrf2, which further enhances antioxidant capacity, inhibiting oxidative stress signals. 7 Studies have also shown that activation of Nrf2 promotes the protein expression of phospho (p)-Akt, which enhances antioxidant responses and inhibits hepatocyte apoptosis, thus normalizing the liver function of acetaminophen (APAP)-induced acute liver toxicity in mice. 8 Hence, the regulation of AMPK/ Nrf2 signals is also a potential strategy for the prevention and treatment of liver disturbance.
The TLR-4/ NF-κB signalling pathway acts as an important player in liver inflammation and is closely related to chronic inflammation reactions, such as liver disturbance. 9,10 Inhibiting TLR-4/ NF-κB signals is key in alleviating inflammatory responses in liver tissue, and it is also an indication for down-regulating inflammatory expression related to oxidative stress. 11 In addition, the Gardenia fruit glycoside, geniposide, reduces acetaminophen-induced liver injury in mice, significantly inhibiting inflammatory cell infiltration and reducing the release of inflammatory factors, as well as the protein expression of TLR4 and NF-κB, which suggests that geniposide-mediated inhibition via the TLR4/ NF-κB pathway may help protect hepatocytes. 12 Thus, inhibiting the TLR-4/ NF-κB pathway is a likely candidate target for relieving liver disturbance.
The Dicliptera chinensis (L.) Juss (Acanthaceae) polysaccharide (DCP) is a type of natural macromolecular polymer that consists of more than 10 monosaccharides linked by glucoside bonds, used to treat liver diseases due to its plethora of biological activities, including autoxidation, anti-inflammation and insulin sensitization. 13 Based on the above studies, we hypothesize that DCP could potentially help alleviate the effects brought on by liver disturbances.
The aim of this study was to investigate the effects and underlying mechanisms mediated by DCP via the AMPK/ Nrf2 pathway by blocking the TLR-4/ NF-κB signalling pathway both in vitro and in vivo. This is a crucial step towards exploring novel effective and safe therapies for the treatment of liver disturbances.

| Ethical approval
All experiments in this study were performed in accordance with the guidelines of the Animal Experimentation Ethics Committee of the Guilin Medical University (approval No. 2019-0004).

| Preparation and characterization of DCP
The flowering plant of Dicliptera chinensis (L.) Juss used in the experiment was purchased from the Chinese herbal medicine market.  All mice were fed with standard pellet feed and water under relative humidity (40%-50%) and temperature (22 ± 2℃). The control day and night period were 12:12 hours, and food and water were available to the mice ad libitum.

| Animal modelling and administration
For the experimental procedures, 66 male C57BL/6J mice were randomly divided into three groups at the beginning of the experiment, including a Control group (n = 13), a DCP200 group (n = 10) and a Model group (n = 43). The Control and DCP200 groups were fed standard Chow diet, which provided 10% calories from fat, 70% calories from carbohydrate and 20% calories from protein (1 010 001; Jiangsu Xietong Pharmaceutical Bio-engineering Co.). In contrast, animals in the Model group were fed a high-fat diet (HFD), which provided 60% calories from fat, 20% calories from carbohydrate and 20% calories from protein (D12492; Jiangsu Xietong Pharmaceutical Bio-engineering Co.). After 8 weeks of feeding, 3 mice in the Control group and 3 mice in the Model group were sacrificed to evaluate through pathological examination, whether a liver disturbance lesion was formed. 14 After the liver disturbance model was successfully defined, all mice left in the Model group were further randomly assigned to the HFD group (n = 10) and to the HFD + DCP groups (HFD + DCP50, HFD + DCP100, and HFD + DCP200). The HFD group continued to be fed a HFD for a period of 6 weeks, and the HFD + DCP groups received a combination of a HFD diet and oral DCP for 6 weeks. The HFD + DCP50, HFD + DCP100 and HFD + DCP200 groups were given DCP for intervention at 50, 100 and 200 mg/kg, respectively. At the end of the 14th week, all mice were euthanized to harvest their liver tissues and blood, collected from the abdominal cava vein, for further analysis. In addition, the brain, heart, spleen, lungs and kidneys of the euthanized mice were also fixed with 4% paraformaldehyde.

| Histopathological examination
After 48 hours fixation, the liver tissues were dehydrated and embedded in paraffin. Then, the tissues were cut into sections (4 μm) using the microtome. Sections were then stained according to the haematoxylin-eosin (H&E) staining methods. Subsequently, sections were treated with xylene and gradient alcohol. After soaking the sections in 95% alcohol, they were then sealed with neutral resin.
Moreover, according to the Congo red staining methods (TR-1302; Beijing Zhongshan Jinqiao Biotechnology Co.), the sections were then stained with Congo red stain, differentiated with alkaline ethanol, counterstained with haematoxylin for 2 minutes and then sealed with neutral resin. Following the above staining procedure, tissue structure, oxidative deposits and lipid deposition were sequentially observed and photographed under a light microscope (Olympus).

| Immunohistochemistry
For immunohistochemistry analysis, liver sections were dewaxed and placed in 1x citrate buffer (ThermoFisher Scientific) for highpressure antigen repair over 5 minutes. The sections were then allowed to cool naturally, before moving on to blocking procedures.
Subsequently, the sections were incubated with a p-NF-κBp65 (1:100; Abcam) or an p-IKKα/β (1:100, Abcam) antibody. The next day, after incubation with respective secondary antibodies, the sections were coloured with the 3, 3'diaminobenzidine (DAB) reagent, red dyed with haematoxylin, dehydrated and dried with ethanol, made transparent with xylene and sealed with neutral gum. These sections were then observed and imaged under the BX51 optical light microscope (Olympus).

| in vivo toxicity test
The effect of DCP on liver disturbance remission was observed in our experiments; thus, we further investigated whether a high dose of DCP (eg DCP200) could have an effect on the brain, heart, liver, spleen, lung and kidney tissue structures. Therefore, we collected these organs from the DCP200 group for H&E staining. After sectioning, and fixation procedures, as described above, the sections were stained according to the H&E staining methods. Stained tissue sections were observed and imaged under a light microscope.

| Analysis of liver tissue samples
Liver tissues were ground into a 10% homogenate (w/v) using phosphate-buffered saline (PBS) or 0.9% saline for evaluation of oxidative stress and inflammatory response signals. Hepatic contents of TG, superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) were detected according to the manufacturer's procedure (Nanjing Jiancheng Bioengineering Institute).
The hepatic TNF-α, IL-1β and IL-6 were measured using the ELISA kit following the manufacturer's instructions provided (Wuhan Elabscience Biotechnology Co.). A microplate reader (Bio Tek, Winooski, VT, USA) was used to detect the absorbance at 450 nm.
The TNF-α, IL-1β and IL-6 detected in liver tissue were calculated according to a standard curve.
In addition, a control siRNA was used to test for possible non-specific effects of siRNA. Finally, HepG2 cells were transfected with siRNA using the lipid agent, Lipofectamine 2000 (Invitrogen) reagent and incubated for 36 hours.

| Real-time reverse transcription polymerase chain reaction
A TRIzol reagent was used to extract the total RNA of liver tissues (Beyotime Biotechnology Co.). Next, according to the real- and 60℃ for 1 minutes, and the cycle was performed 40 times.
Expressions of target genes were carried out by a comparative method (2 −ΔΔCt ), with β-actin used as an internal reference. The primer sequences utilized are shown in Table 1 (Huada Gene Research Institute, Shenzhen, China).

| Western blot analysis
For protein analysis, 50 mg of liver tissue samples was lysed using the radioimmunoprecipitation assay (RIPA) buffer containing 1% (v/v) phenylmethylsulphonyl fluoride (PMSF, P0013B; Beyotime Biotechnology Co.). After the protein supernatant was collected, the bicinchoninic acid (BCA) protein assay kit (P0010S; Beyotime Biotechnology Co.) was utilized to detect the protein content of the supernatant. Next, a protein loading buffer was added and protein denaturation was carried out in a 95℃ water bath for 5 minutes.

| Statistical analysis
The GraphPad Prism 5.0 software (GraphPad) was used to analyse the data, which was expressed as the mean ± standard deviation (SD). To assess the differences between groups, one-way analysis of variance was used. The independent sample t test was used for comparison between the two groups. A P value of less than (<) .05 indicated statistical significance.

| Establishment of the liver disturbance model
Compared to the Control group, the body weights and liver indexes of HFD group were markedly increased ( Figure 1A). Moreover, the HFD group exhibited significant enhancement in serum liver enzyme activity, including ALT, AST, AKP and GGT( Figure 1B). In the Control group, the liver surface was bright red and smooth and exhibited integrated cords of hepatocytes which radiated from the central vein and presented a normal hepatic lobular structure ( Figure 1C,D). In the HFD group, liver tissue samples presented a yellowish colour and had increased in volume. In addition, liver sections displayed macroand microvascular steatosis, slight inflammatory cell infiltration and ballooning degeneration ( Figure 1C,D). These data show that we successfully established a liver disturbance model.

F I G U R E 1 Effect of DCP on liver function. A, Bodyweights and liver
indexes of treated mice. B, ALT, AST, AKP and GGT activity levels in collected serum samples. C, Liver morphology and (D) H&E staining. E, H&E staining of the brain, heart, liver, spleen, lungs and kidneys from the DCP200 group. All data are presented as the means ± standard deviation [SD] (n = 10; *P < .05, **P < .01). Bar = 100 μm

| The effect of DCP on liver function
Compared to the HFD group, there are significant decreases in the body weights and liver indexes of the HFD + DCP200 group ( Figure 1A). Moreover, the enzyme activity of ALT, AST, AKP and GGT were markedly reduced ( Figure 1B). Liver tissues of the HFD + DCP group presented a liver colour that was bright red and shiny ( Figure 1C), with the pathological lesions ameliorating distinctly and a lowered accumulation of fat vacuoles and necrotic cells ( Figure 1D). H&E analysis demonstrated that no significant pathological differences ( Figure 1E), indicating that DCP had no obvious toxic side effects to the other main organs in HFD + DCP group.

| DCP effects on lipid accumulation
Serum lipid profiles, including TC, TG, LDL-C, CETP and FFA, were dramatically elevated in the HFD group, whereas the serum content of HDL-C and LCAT decreased markedly, when compared to the Control group (Figure 2A-C). In addition, the FBG, FINS and HOMA-IR levels were markedly increased in the HFD group relative to the Control group ( Figure 2D). Notably, in the HFD group, Oil Red O staining showed a large area of red lipid drops with obvious inflammatory cell infiltration ( Figure 2E). These data demonstrate that lipid accumulation is significant in our liver disturbance model and that DCP treatment has beneficial effects on liver and serum lipid levels.

| DCP effect on liver oxidative stress signals
When compared to those of the HFD group, the enzyme activity of SOD and GSH-Px were markedly increased and the MDA content in the liver was reduced dramatically in the HFD + DCP200 group ( Figure 3A,B). Notably, Congo red staining analysis in the liver of the HFD group showed a large amount of red sediment accumulation.
This accumulation was significantly reduced in the HFD + DCP200

| The effect of DCP on the AMPK/ Nrf2 liver pathways
Notably, the HFD group showed no significant changes in AMPK mRNA levels; however, SREBP-1, acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), stearoyl-coenzyme A desaturase 1 (SCD1) mRNA were significantly increased, when compared to the Control group, (Figure 4A,B). In contrast, p-AMPK protein expression levels decreased and SREBP-1 protein expression levels increased in the HFD group ( Figure 4C). In addition, the expression levels of p-AKT and Nrf2 proteins in the HFD group were lower than those of the Control group ( Figure 4D).

| Effects of DCP on HepG2 AMPK/ Nrf2 signalling pathways
At a concentration of 80 μmol/L of DCP, we observed stable cell viability and high levels of p-AMPK. Therefore, we chose this concentration as the effective concentration for our studies ( Figure 5A,C).
Compared with the Control group, the expression levels of SREBP-1 and Nrf2 in the siAMPK group were not statistically significant ( Figure 5D). After silencing the expression of AMPK using siRNA, SREBP-1expression increased, whereas the expression of Nrf2 decreased in the siAMPK + FFA group, when compared to the FFA group ( Figure 5B).
After DCP treatment, we found no statistically significant differences in the expression levels of SREBP-1 and Nrf2 between the DCP + siAMPK + FFA group and the siAMPK + FFA group ( Figure 5D).
These results demonstrate that DCP can improve liver disturbance by activating AMPK/ Nrf2 signalling pathways.

| The effect of DCP on TLR-4/ NF-κB liver pathways
When compared to the Control group, the hepatic levels of TNF-α, IL-1β and IL-6 in the HFD group were significantly elevated when reflecting the inflammatory response typical of liver disturbance ( Figure 6A). Furthermore, RT-PCR analysis showed that PPAR-γ mRNA levels were remarkedly decreased in the HFD group, whereas TLR-4 and NF-κBp65 mRNA levels were significantly elevated ( Figure 6B). The expression of PPAR-γ proteins in the HFD group was down-regulated whereas TLR-4 and p-NF-κBp65 levels were significantly increased ( Figure 6C). Immunohistochemical analysis also showed that the levels of p-NF-κBp65 and p-IKKα/β in the liver tissue collected from HFD were particularly increased ( Figure 6D).
These data indicate that the inflammatory response mediated by liver disturbance is closely connected with the TLR-4/ NF-κB signalling pathway.
When compared to the TLR-4 −/− + HFD group, the mRNA levels of TNF-α, IL-6, IL-1β, TLR-4 and NF-κBp65 were lower, and PPAR-γ levels were higher, whereas protein expression levels of PPAR-γ were higher, and TLR-4 and NF-κBp65 were lower in the TLR-4 −/− + HFD + DCP group (Figure 7). These results show that DCP can F I G U R E 3 The effect of DCP on oxidative stress pathways in the liver. A, MDA liver content. B, SOD and GSH-Px levels. C, Congo red stain analysis. All data are presented as the means ± SD (n = 10; *P < .05, **P < .01). Bar = 100 μm inhibit the TLR-4/ NF-κB pathway and ameliorate liver disturbance symptoms.

| D ISCUSS I ON
The AMPK/ Nrf2 signalling pathway can regulate lipid metabolism in liver disturbance. 17 Meanwhile, chronic inflammatory responses such as the ones associated with liver disturbance have been shown to be closely connected with the TLR-4/ NF-κB signalling pathway. 12 In this study, C57BL/6J mice were fed a HFD to establish a liver dis- In summary, DCP can block liver inflammation, and also regulate lipid metabolism, thereby reducing liver disturbance symptoms.
In the current study, we successfully established a liver disturbance model using an enriched diet. Notably, this diet is known to increase FFA in blood serum. The continuous accumulation of FFA in the body causes liver damage and can lead to the overflow of ALT and AST enzymes into the blood. 18 In addition, an HFD diet has been shown to elevate ALT, AST, AKP and/ or GGT enzyme activities in slowing cell damage. 28,29 In addition, DCP also inhibited the TLR-4/ NF-κB pathway, reduced inflammatory responses. It inhibited the release of TNF-α, IL-6 and IL-1β, reducing the inflammatory response and protecting liver function ( Figure 6A). Moreover, it significantly up-regulated PPAR-γ expression, down-regulated TLR-4 expression and inhibited p-NF-κBp65 levels ( Figure 6B-C). Therefore, DCP plays a regulatory role in the TLR-4/ NF-κB signalling pathway and inhibits TNF-α, IL-6 and IL-1β levels to alleviate liver disturbance.
Nrf2, NF-κB and PPAR-γ are key nuclear transcription factors that play important roles in the regulation of liver inflammation. 30 F I G U R E 5 The effect of DCP on AMPK/ Nrf2 signalling pathways in HepG2 cells. A, Evaluating a safe DCP concentration for dose administration. B, mRNA expression levels of AMPK. C, Western blot analysis showing the protein expression of p-AMPK and AMPK. D, Nrf2 and SREBP-1 protein levels. All data are presented as the means ± SD (n = 3; *P < .05, **P < .01)

F I G U R E 6
The effect of DCP on TLR-4/ NF-κB signals. A, TNF-α, IL-6 and IL-1β liver expression levels. B, TLR-4, PPAR-γ and NF-κBp65 mRNA expression profiles. C, Hepatic protein expression of PPAR-γ, TLR-4, p-NF-κBp65 and NF-κBp65. D, Immunohistochemical analysis of p-NF-κBp65 and p-IKKα/β expression levels. All data are presented as the mean ± SD (n = 3; *P < .05, **P < .01). Bar = 100 μm F I G U R E 7 Effect of DCP on TLR-4/ NF-κB signals of TLR-4 −/− mice. A, Levels of TNF-α, IL-6 and IL-1β in the liver. B, Expression profiles of liver TLR-4, PPAR-γ and NF-κBp65 mRNA levels. C, Hepatic protein expression of PPAR-γ, TLR-4, p-NF-κBp65 and NF-κBp65. All data are presented as the mean ± SD (n = 3; *P < .05, **P < .01) F I G U R E 8 Proposed model depicting the underlying mechanisms of DCP in regulating liver inflammation responses and oxidative stress in liver disturbance Interestingly, the TLR-4 receptor can bind to its unique ligand to activate NF-κB, thereby releasing TNF-α, IL-6 and IL-1β in the circulation. On the other hand, a HFD diet can cause an increase in fatty acids, which in turn can lead to the increase of inflammation-related factors such as NF-kB a and TNF-α. 31 Decreased gene expression and PPAR transcription factor family activities are closely related to an increase of the nuclear transcription factor activity of NF-kB. [31][32][33] In order to further investigate the target of DCP inhibition on

DATA AVA I L A B I L I T Y S TAT E M E N T
All data included in this study are available upon request by contact with the corresponding author.