Chicoric acid ameliorate inflammation and oxidative stress in Lipopolysaccharide and d‐galactosamine induced acute liver injury

Abstract Chicoric acid is polyphenol of natural plant and has a variety of bioactivity. Caused by various kinds of stimulating factors, acute liver injury has high fatality rate. The effect of chicoric acid in acute liver injury induced by Lipopolysaccharide (LPS) and d‐galactosamine (d‐GalN) was investigated in this study. The results showed that CA decreased the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum and reduced the mortality induced by LPS/d‐GalN. CA can restrain mitogen‐activated protein kinases (MAPKs) and nuclear factor‐kappa B (NF‐κB) to alleviate inflammation. Meanwhile, the results indicated CA can active nuclear factor‐erythroid 2‐related factor 2 (Nrf2) pathway with increasing the level of AMP‐activated protein kinase (AMPK). And with the treatment of CA, protein levels of autophagy genes were obvious improved. The results of experiments indicate that CA has protective effect in liver injury, and the activation of AMPK and autophagy may make sense.

synthase kinase 3β (GSK-3β). 3 MAPKs, which include extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun NH2-terminal kinases 1, 2 and 3 (JNK1/2/3) and p38α/β/δ/γ, participate in numerous metabolic processes in liver. The activated JNK mediates liver injury though TNF and oxidative stress, which lead to hepatocytosis. 4 Phosphorylated ERK is significant increased in models of ALF , 5 and the disordered P-ERK can aggravate liver injury. It has been proven that p38 is associated with inflammation 6,7 and oxidative stress. Besides, inflammasome complexes NLRP3 can activate IL-1β, which is essential in congenital and adaptive immunity. Previous study has proven that the activated NLRP3 can aggravate the liver injury by intense inflammation. 8 AMPK makes sense of maintaining energy balance in cell.
Present study reports that AMPK is activated to respond the oxidative stress in cancer cells and neurons. 9,10 Meanwhile, it is reported that AMPK can increase the nuclear accumulation of Nrf2 though activating phosphorylation of GSK3β. 11 GSK3β is a key enzyme in glucose metabolism, and the participation of it in cell regulation has been proven. Nrf2 is a transcription factor which can response oxidative stress. After transferring into nucleus, Nrf2 can coordinate a large amount of gene expression which responds to oxidative stress. Nrf2-dependent genes and proteins, such as Heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase 1 (NQO1), are increased whereas Nrf2 transfers to nucleus and binds with Cis-acting androgen responsive elements (AREs) to activate the expression of phase II detoxifying enzymes. 12

| Method
The mice were given LPS (30 μg/kg) and

| Serum ALT, AST and ROS level
The blood was pleased in 37°C for 2 hours, and then, it was centrifuged for 10min with 1000g (4°C). Under the instruction, we detected the ALT, AST and ROS in serum with kit.

| Biochemical analyses
The removed liver tissue was operated with instruction. Then, we detected absorbance with spectrophotometer at special wavelength. And the activity of MPO and GSH was measured though the formula.

| Histopathological evaluation
Part of liver tissue in each group was isolated and fixed for 48 hours with normal 10% neutral buffered formalin. After treated by ethanol and xylene, the liver was encapsulated in wax. We used slice to evaluate the injury after staining sections by haematoxylin and eosin.

| Western blot
The collected cell and liver tissue was treated by cell lysis solution.
And then, we used BCA protein assay kit (Thermo, America) to detect the concentration. With the help of 10% SDS-polyacrylamide gel, target substance was obtained and it was transferred to polyvinylidene fluoride membrane. After blocked, incubated by particular primary antibody and secondary antibody, we analysed the bolt with Image-Pro Plus, and we detected the signal with the assistance of ECL (Dalian Meilun Biotechnology Co., Ltd).

| Statistical analysis
Three independent data of experiment were collected and applied for one-way ANOVA by Statistical Product and Service Solutions. P < .05 or P < .01 makes sense in statistics. The data were presented as mean ± SD.

| CA decreases serum AST, ALT and ROS
Serum ALT, AST and ROS increased with LPS/d-GalN treatment.
3 hours later, ALT, AST and ROS were slightly increased, while after 6 hours, the level of AST, ALT and ROS was significant increased shown in Figure 1C,D,E. And with the CA treatment, the AST, ALT and ROS were decreased.

| CA restrains the level of MPO and increase GSH in mice
MPO, one of the peroxisomes, plays an important role in inflammation. Combined with three amino acids, GSH is an important antidote and antioxidant in organism. As it is shown in Figure 1F and G, CA can restrain the MPO which was increased after treated by LPS/D-GalN. Meanwhile, treated with CA, level of GSH in liver increased.

| CA decreases the mortality of LPS/D-GalNinduced ALF
Acute liver failure is a kind of infrequent and lethal disease. After injected CA and LPS/d-GalN, mice were monitored for 24 hours. The results indicated that mice only treated by CA (50 mg/kg) and the control group were all alive after 24 hours. However, in LPS/d-GalN group, mice died at 6 hours, and all mice in this group died in 8 hours. Although the mice treated by CA (12.5, 25, 50 mg/kg), the mortality was decreased to 30%, 50% and 80% with dosage dependent ( Figure 1B).

| CA protects mice form LPS/d-GalN-induced ALF in hepatotoxicity
H&E stain sections (5-μm-thick) of liver were detected to obtain visual pathological picture. In Figure 1H, the liver of mice had severer intrahepatic haemorrhage and necrosis over time in LPS/d-GalN group, whereas CA can protect mice form ALF. However, only treated by CA had not found hepatotoxicity compared to control group.

| CA activated Nrf2-HO-1 pathway in mice
In this study, we detected the Nrf2 in nucleus and the expression of antioxidase such as GCLC and GCLM. As the results showed in

| CA suppress phosphorylation of MAPKs in mice
MAPKs play important roles in growth, differentiation and death of cell. As it is pictured in Figure 4, LPS/d-GalN induced the excessive Meanwhile, the results of MPO and GSH in liver are pictured in F and G. Besides, we collected the blood of mice to detect the serum AST, ALT and ROS is shown in C, D and E. We took three independent experiments, and the data were shown as means ± SD. # P < .05 vs the control group; ## P < .01 vs the control group; *P < .05 vs the LPS/d-GalN group; **P < .01 vs the LPS/d-GalN group P-JNK, P-ERK and P-P38. After treatment of CA, P-ERK and P-P38 were obvious decreased at 3 hours. Furthermore, at 6 hours, CA decreased the level of P-MAPKs.

| CA suppress phosphorylation of NF-κB in mice
Responding activated TLR4 by stimulation of LPS though the Myeloid differentiation primary response gene (MYD88), NF-κB is a pivotal regulator in inflammation. Figure 4 shows that CA can restrain MYD88, P-NF-κB and P-IκB.

| CA regulated NLRP3 pathway in mice
NLRP3 can be activated by series of stimulate, such as virus and ATP, and is significant in inflammation. As Figure 5 shows that the CA restrained the ASC and NLRP3, so the caspase1 in treatment groups was lower than LPS/d-GalN groups.

| CA activated autophagy in mice
Autophagy participates in various activities in cells, such as clearance of damaged organelles and causative agent. The results presented in Figure 5 indicate that CA can activate autophagy. The content of Atg16, 12, 7, 5 and 3 Beclin-1 had varying degrees of increase.

| Toxicity of CA on HepG2 cell
To evaluate the toxicity of CA, we plated HepG2 cell into 96 plates with 5% CO 2 , 37°C. Twenty-four hours later, we treated the HepG2 F I G U R E 2 CA activated Nrf2 -HO-1 pathway in mice. After the treatment of CA, LPS/d-GalN was given to mice. After 3 and 5 h, we used the liver tissue for Western blot. The results were compared with the internal control (lamin B and β-actin). The data were collected for data shown as means ± SD of three independent experiments. # P < .05 vs the control group; ## P < .01 vs the control group; **P < .01 vs the LPS/d-GalN group by CA (0-800 μm). The result shows in Figure 6H that CA (0-50 μm) has no toxicity.
Then, protein was collected. As it is shown in Figure 6, CA (50 and 100 μm) accelerated the transfer of Nrf2. Meanwhile, we used 50 μm CA treated HepG2 cells, the results indicated that CA can activated the Nrf2 in 6-18 hours. Moreover, with the increased of Nrf2, HO-1, NQO1, GCLC and GCLM were significant increased (Figure 7). Besides, we treated Nrf2 −/− HepG2 cells with CA to verify the mechanism. As it has been judged, whether using the CA or not, the expressions of HO-1, NQO1, GCLC and GCLM were lower compared with those in WT cells.

| CA regulated AMPK pathway in HepG2 cells
We have detected the expression of AMPK pathway. After the treatment of CA, the P-AMPKα was increased (shown in Figure 7). And, phosphoryltion of ACC and GSK3β was improved. Besides, treated by inhibitors of AMPK (compound c), the phosphorylation of AMPKα and the expression of Nrf2 were down-regulation as it is shown in Figure 7I, M and N.  In a word, we mainly proved that CA in activating the AMPK/ Nrf2 pathway in ALF in mice while the effects of CA on AMPK/Nrf2 pathway in HepG2 cell were proved. The result indicated that CA can relieve oxidative stress and inflammation, whereas CA can activate the Nrf2 pathway and autophagy. Therefore, our results proved the positive effects of CA in ALF induced by LPS/GalN, which may provide new insights into liver injury treatment.

ACK N OWLED G EM ENT
This work was supported by grants from the National Natural Science Foundation of China (No. 31772798, No.31572347, and No.31672621).

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

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.