Abstract
The aim of this study is to investigate Emodin on alleviating intrahepatic cholestasis by regulation of liver farnesoid X receptor (FXR) pathway. Cell and animal models of intrahepatic cholestatis were established. Biochemical tests and histomorphology were performed. The messenger RNA (mRNA) and protein expression of FXR, small heterodimer partner (SHP), uridine diphosphate glucuronosyltransferase 2 family polypeptide B4 (UGT2B4), and bile salt export pump (BSEP) was detected. As a result, compared with the model group, the serum levels of biochemical test were significantly lower in the Emodin group (P <0.01). The histopathological changes were remitted significantly by Emodin treatment. In the model group, the mRNA and protein expression of FXR, SHP, UGT2B4, and BSEP was significantly lower than in the normal group in cell models (P <0.05). With Emodin intervention, the expression of FXR, SHP, UGT2B4, and BSEP was notably increased (P <0.05). In conclusion, Emodin plays a protective role in intrahepatic cholestasis by promoting FXR signal pathways.
Introduction
Infant cholestasis is a clinical syndrome. So far, ursodeoxycholic acid (UDCA) is recognized as effective medication, but about one-third of patients with cholestasis are invalid for UDCA. 1 Those patients will face cholestasis-caused liver fibrosis, cirrhosis, and liver failure as serious consequences that ultimately require liver transplantation. 2
Recent studies found that a nuclear receptor, farnesoid X receptor (FXR), plays a vital role in the metabolism of bile acid (BA). 3 Presently, FXR has been a recognized therapeutic target in cholestasis, and confirmed as a pivot to maintain the homeostasis of cholesterol and bile acids via regulating target genes related to the metabolism of BA, particularly in its synthesis, detoxification, and transportation. 4
Our previous work revealed that Emodin as the main active ingredient of rhubarb can relieve liver damage by inhibiting inflammatory mediators and antioxidants, improving liver circulation, reducing damage signal, controlling neutrophils infiltration, etc. 5 Compared with the ursodesoxycholic acid group, emodin can obviously alleviate the biochemical indicator and pathological damage in ANIT-induced intrahepatic cholestasis among rats model. 5 But in addition to the inflammatory mechanisms, whether FXR signaling pathways work as well remain unclear. Our preliminary experiments showed that Emodin can improve cholestatic hepatitis and raise the level of FXR. On the basis of previous research, we hope to further investigate Emodin on alleviating intrahepatic cholestasis in infant rat and realizing the molecular mechanisms of liver protection.
Materials and methods
Reagents
Guggulsterones were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Emodin, alpha-naphthylisothiocyanate (ANIT), and peanut oil were purchased from Sigma-Aldrich (Sigma-Aldrich Chemical Co., MO, USA). The serum total bilirubin kits, direct bilirubin kits, bile acid kits, serum serum alanine aminotransferase (ALT) kits, and serum aspertate aminotransferase (AST) kits were bought in Nanjing Jiancheng biological engineering research institution (Nanjing, PR China). Fetal bovine serum (FBS) and RPMI-1640 medium were obtained from HyClone (Logan, UT, USA). Nucleoprotein and plasma protein extraction kits, bicinchoninic acid (BCA) kits, and SDS-PAGE gel preparation kits were purchased form Wuhan Aspen Biological Technology Co., Ltd. (Wuhan, PR China). Anti-FXR antibody, anti-SHP antibody, and anti-BSEP antibody were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-UGT2B4 antibody was obtained from Fitzgerald (Sudbury, MA, USA). SYBR Premix Ex Taq kit was purchased from Takara (Dalian, PR China). TRIzol reagent was provided from Invitrogen (Grand Island, NY, USA)
Animals
Specific pathogen-free neonatal Sprague-Dawley rats (60–80 g) were obtained from the Hubei Provincial Centers for Disease Control, including 12 female rats and 12 male rats. All rats were kept under constant housing conditions (22°C, 60% relative humidity, and a 12-hour light/dark cycle) and had free access to water and food throughout the experiment. 6 All the experimental procedures received approval from the Bioethics Committee.
Animal models and samples
After feeding for 3 days for adaptation, 24 rats were randomly allocated to the control group, model group, and Emodin group (40 mg/ kg•d). 7 Each group included eight rats (4 male, 4 female). The intervention cycle for the model and Emodin groups was 7 days, and 0.5% ANIT was given to both of the two groups on the fifth day through administration by gavage. Animals were terminated by cervical dislocation after 48 h of ANIT treatment; during this time the rats must fast for the last 12 h and be anesthetized for picking 2 mL eyeball blood before their death. The serum was stored at −20°C. The right liver was immediately snap frozen in liquid nitrogen and stored at −80°C, while the left lobe tissue was fixated in 4% formaldehyde and embedded in paraffin.
Cell culture
The human embryohepatocyte L02 cell line was purchased from the Chinese Academy of Sciences. The cells were cultured, passaged, and proliferated in a routine method as in our previous studies.8,9
Establishment of FXR gene suppression cellular model
L02 cells were maintained in medium RPMI-1640 supplemented with 10% FBS for 12 h. Then, in the normal group, the same medium was replaced and the cells continued to be cultured for 48 h. In the model group, the medium was first substituted with 50 μmol/L guggulsterones and 10% FBS modified RPMI-1640 medium for 24 h, and then was replaced with 50 μmol/L guggulsterones and 1% DMSO medium and 10% FBS-modified RPMI-1640 medium for 24 h. In the Emodin group, the medium was first substituted with 50 μmol/L guggulsterones and 10% FBS-modified RPMI-1640 medium for 24 h, and then was replaced with 25 μmol/L Emodin and 10% FBS-modified RPMI-1640 medium for 24 h. Cells were harvested at day 2 after seeding, respectively.
Biochemical tests
Total bilirubin (TBIL), direct bilirubin (DBIL), ALT, and AST were detected through colorimetric method.
Histomorphology
After fixation in 4% formaldehyde, tissues were embedded in paraffin and cut in serial sections of 4 mm for hematoxylin and eosin (H&E) staining as in our previous studies.10,11
Western blot assay
The experiment abided by our previous studies. 12 Nucleus protein used to detect FXR and total protein used to detect SHP, UGT2B4, and BSEP were extracted from the liver tissues and hepatocytes, respectively. The protein concentration was determined using the BCA method. Then the proteins were separated by 10.0% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes then went through a series of procedures including blocking with skim milk, incubating with primary antibodies and second antibodies, chemiluminescence detection, and gel image analysis. Finally, the optical density of the target protein and internal reference were analyzed and compared by AlphaEaseFC software processing system. The dilution rate of primary antibodies was shown as follows: anti-GAPDH, 1:10,000; anti-FXR, 1:500; anti-SHP, 1:500; anti-BSEP, 1:500; and anti-UGT2B4, 1:1000.
RNA extraction and quantitative real-time PCR assay
The experiment abided by our previous studies. 13 Total RNA from tissues and cells was isolated by TRIzol reagent, following the manufacturer’s instructions. To detect mRNA expression levels for the genes investigated in this study, primers were designed using Primer Express software spanning intron/exon boundaries. Quantitative real-time PCR (qRT-PCR) assays were performed in 96-well optical reaction plates, using the Applied BiosystemsStepOne Real-Time PCR System. Relative quantification was done by using the ΔΔCT method, and expression values have been represented as fold change between the groups. The reaction was 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, 58°C for 20 s, and 72°C for 20 s, and then 72°C for 5 min, with 72°C to 95°C for each 20 s elevating 1°C.
Primers for L02 cells β-actin: forward 5’-GTCCACCGCAAATGCTTCTA-3’ reverse 5’-TGCTGTCACCTTCACCGTTC-3’ FXR: forward 5’-AAGAGATGGGAATGTTGGCTG-3’ reverse 5’-CTCCCTGCATGACTTTGTTGTC-3’ SHP: forward 5’-GAAAGGGACCATCCTCTTC-AAC-3’ reverse 5’-TCTCCAATGATAGGGCGAAAG-3’ BSEP: forward 5’-CAACTGCTGGACCGACAACC-3’ reverse 5’-CATCCACTGCTCCCAACAAAC-3’ UGT2B4: forward 5’-GGTCGATGGTCAGTAACA-CGTC-3’ reverse 5’-TTGTACAGCCGAGTATTGAGTCCT-3’
Statistical analysis
All experiment data were processed by SPSS 12.0. All results were expressed as mean ± SD. Multiple groups were tested by one-way analysis of variance (ANOVA), followed by LSD test or Dunnett’s test. P <0.05 indicated a significant difference. 14
Results
Effect of Emodin on serum biochemical indicator
Compared with the normal group, the serum levels of TBIL, DBIL, ALT, and AST in the model group were significantly higher (P <0.01). Compared with the model group, the serum levels of TBIL, DBIL, ALT, and AST were significantly lower in the Emodin group (P <0.01) (Table 1).
The effect of Emodin on serum biochemical indicator among cholestasis rats.
The data were shown as mean ± SD, n = 8.
P <0.01, compared with the normal group.
P <0.01, compared with the model group.
ALT, alanine aminotransferase; AST, aspertate aminotransferase; DBIL, direct bilirubin; TBIL, total bilirubin.
Effects of Emodin on liver morphology
In the normal group, the structure of hepatic lobules was complete. The hepatocytes enclosed the central veins and the sizes were uniform. In the model group, the structure of hepatic lobules was abnormal. The hepatocytes were diffusely altered, with ballooning of cytoplasm, agmination of nuclear chromatin, visible necrotic zones, and hyperplasia of Kupffer cells. Compared with the model group, the histopathological changes were remitted in the Emodin group significantly (Figure 1).
Effects of Emodin on liver morphology in an infant rat suffering from intrahepatic cholestasis. The liver tissue was prepared by H&E staining, 200×.
Effects of Emodin on protein levels of FXR, SHP, UGT2B4, and BSEP in hepatocytes
Interfered L02 cell line with guggulsterones, compared with the normal group, the protein expressions of FXR, SHP, UGT2B4, and BSEP were significantly lower in the model group (P <0.01), while compared with the model group, the protein expressions of FXR, SHP, UGT2B4, and BSEP were significantly elevated in the Emodin group (P <0.01) (Figure 2).
The protein and mRNA expressions of FXR, SHP, UGT2B4, and BSEP in hepatocytes. (a) The protein and mRNA expressions of FXR; (b) the protein and mRNA expressions of SHP; (c) the protein and mRNA expressions of UGT2B4; (d) the protein and mRNA expressions of BSEP. N, normal group; M, model group; E, Emodin group. ΔΔP <0.01, compared with the normal group; ▲▲P <0.01, compared with the model group.
Effects of Emodin on mRNA levels of FXR, SHP, UGT2B4, and BSEP in hepatocytes
Interfered L02 cell line with guggulsterones, compared with the normal group, the mRNA expressions of FXR, SHP, UGT2B4, and BSEP were significantly decreased in the model group (P <0.01), while compared with the model group, the mRNA expressions of FXR, SHP, UGT2B4, and BSEP were significantly elevated in the Emodin group (P <0.01) (Figure 2).
Discussion
ANIT-induced intrahepatic cholestasis among rats has been widely used to simulate human intrahepatic cholestasis. 15 Our study selected 3-week-old ablactated rats, which was closer to the pathophysiology characteristics of infants. According to the results, the biochemical and pathological changes showed that Emodin might play an important role in improving intrahepatic cholestasis and protecting the liver from damage.
Our previous study found that Emodin could alleviate ANIT-induced intrahepatic cholestasis in infant rats. BA as an FXR natural ligand can activate transcription of FXR target genes by binding to a FXR ligand-binding domain. 16 FXR is the key regulator of BA synthesis, detoxification, and transportation. 17 Activation of FXR stimulates multiple downstream ligands, resulting in the inhibition of pathways related to cholestasis. 17 Studies in vitro also investigated Emodin on regulating FXR signaling-related pathway. Human embryo hepatocytes L02 were stimulated by guggulsterones to antagonize FXR. 18 The protein and mRNA expressions of FXR, SHP, UGT2B4, and BSEP in the Emodin group were significantly elevated when compared with the model group, indicating that the expression of FXR signaling-related genes SHP, UGT2B4, and BSEP may share common regulating factors on the promoter, FXR.
This study demonstrates that Emodin can promote the expression of FXR and FXR signaling-related genes, resulting in regulating the metabolism of BA, which preliminary demonstrates that Emodin can regulate the synthesis, detoxification, and transportation process of BA. Emodin can control cholestasis through many pathways and play a protective role in intrahepatic cholestasis. However, it is not clear whether Emodin can also interfere with the expression of RXR. Further studies will be carried out to confirm whether Emodin has an effect on RXR and other molecules in signaling pathways in cholestasis.
Footnotes
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
This project supported by the National Science Foundation of China (Nos. 81403434 and 81371840).