Background
ConA-induced liver injury is a well-established mouse model used to detect immune cell-mediated acute hepatitis, and closely resembles the pathology of human autoimmune hepatitis [
1]. ConA-induced liver injury is characterised by a marked increase in plasma alanine transaminase (ALT) levels from 8 to 24 h after injection, as well as simultaneous hepatic infiltration by CD4
+ T immune cells, natural killer T cells (NKT), Kupffer cells, and neutrophils [
2]. The activation of lymphocytes and production of inflammatory cytokines and chemokines are well studied in conA-induced liver injury [
3,
4]; however, it remains unclear whether the kidney is damaged in conA-treated mice.
Paeoniflorin (PF), a monoterpene glycoside, is the principal bioactive component of
Radix Paeoniae Rubra, a traditional Chinese herbal medicine [
5]. PF inhibits hepatocyte apoptosis and the potential mechanism underlying this is associated with the regulating mediators in endoplasmic reticulum (ER) stress and mitochondria-dependent pathways [
6]. PF reduces dextran sodium sulphate (DSS)-induced colitis by suppressing the expression of toll-like receptor 4 (TLR4) and reducing the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways [
7]. PF has also been used for the treatment of liver fibrosis induced by the
Schistosomiasis japonica egg [
8]. We have recently reported that PF inhibits liver fibrosis induced by dimethylnitrosamine (DMN) in rats [
9]. Renal macrophages, similar to hepatic Kupffer cells, increased significantly after two weeks of DMN treatment, then decreased after four weeks of DMN administration. Therefore, PF could inhibit renal macrophage activation in DMN-induced liver fibrosis. As a result, it has been hypothesised that the kidney is damaged in conA-induced hepatitis, and PF could reduce conA-induced renal damage by inhibiting macrophage infiltration. It was investigated 1) whether the kidney was damaged, and if so, the macrophage involvement was assessed; 2) whether PF reduced renal damage and macrophage infiltration in conA-induced injury; and 3) whether the CXCR3/CXCL11 signalling pathway was involved in macrophage infiltration in conA-induced injury. This study describes a newly discovered effect of PF and a previously unknown functional mechanism in renal diseases.
Methods
Major materials
Paeoniflorin (PF, >95 % purity), DAPI fluorescent stain, and conA type IV were obtained from Sigma (St Louis, MO, USA). The SABC kit for immunohistochemical analysis was obtained from Boster (Wuhan, China). The IL1β ELISA kit was from R&D system (Minneapolis, MN, USA). The antibodies used for the immunohistochemical and western blot analyses were rabbit polyclonal IL1β (sc-7884), goat polyclonal monocyte chemotactic protein 1 (MCP1) (sc-1785), rabbit polyclonal F4/80 (sc-25830), mouse monoclonal CXCR3 (sc-137140) and rabbit polyclonal CXCL11 (sc-28874) purchased from Santa Cruz Biotechnology (La Jolla, CA, USA). Mouse monoclonal CD68 (MCA31R) was obtained from Serotec (Oxfordshire, OX51GE, UK). Secondary fluorescence-labelling goat anti-mouse Cy3 and goat anti-rabbit FITC second antibodies were obtained from Jackson (West Grove, PA, USA).
Ethics statement
All of the study protocols complied with the current ethical considerations of Shanghai University of Traditional Chinese Medicine’s Animal Ethic Committee and the procedural and ethical guidelines of the Chinese Animal Protection Act, which is in accordance with the National Research Council criteria. All animal experiments and procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai University of Traditional Chinese Medicine and were performed in accordance with the relevant guidelines and regulations.
Animals
60 Female BALB/C mice at (18 ± 2 g) were supplied by the Central Animal Care Facility of Shanghai University of Traditional Chinese Medicine and housed in an air-conditioned room at 25 °C with a 12 h darkness/light cycle. The mice received humane care with unlimited access to food and water during the study.
ConA-induced tissue damage in mice
Mice received conA injection via the tail vein at 15 mg/kg body weight. PF (6 mg/kg, 30 mg/kg, or 150 mg/g) was orally administered 2 h before conA injection and control mice received vehicle (distilled water) or PF (30 mg/kg). There was 10 mice in each group. At 8 h after conA injection, all the mice were euthanized under 2 % pentobarbital sodium, and all efforts were made to minimise suffering; kidney and liver samples were taken for the following investigations.
Histology analysis
The kidney and liver specimens were preserved in 4 % paraformaldehyde and dehydrated in a graded alcohol series. The specimens were embedded in paraffin blocks, cut into 3 μm-thick sections, and placed on glass slides. The sections were then stained with haematoxylin-eosin (HE).
Liver and kidney function tests
Serum levels of ALT, BUN, and Cr were measured in samples obtained at the end of the experiment. Activity and content were evaluated using a commercial clinical test kit (Jiancheng Institute of Biotechnology, Nanjing, China) according to manufacturer’s instructions.
Measurement of cytokine levels in the kidneys
The kidneys were homogenised in 5 ml ice-cold physiological saline and the supernatant was obtained by centrifugation at 3000 g for 10 min. Samples were analysed and absolute values were obtained by comparison with standards.
Immunohistochemistry
Embedded tissue was deparaffinised in xylene and rehydrated. Microwave antigen retrieval was carried out for 5 min before quenching the peroxidase with 3 % H2O2 in phosphate-buffered saline (PBS) for 10 min at room temperature. The sections were blocked using 5 % BSA for 30 min at 37 °C, and then incubated with the respective primary antibodies (anti-CD68, anti-IL1β, anti-MCP1, and anti-CXCR3) at room temperature for 1 h. After washing with PBS, sections were subsequently incubated with species-specific biotinylated secondary antibodies at room temperature for 30 min. After washing, the sections were stained with 3,3′-diaminobenzidine (DAB). Counterstaining was performed with haematoxylin before dehydration and mounting.
Immunofluorescent staining
Double staining for F4/80 and CXCR3, and, CXCR3 and CXCL11 were performed on paraffin sections. Sections were deparaffinised in xylene, rehydrated and incubated with protease K (20 μg/ml) for 10 min at 37 °C. Thereafter, the slides were incubated with 5 % BSA for 30 min followed by incubation with anti-F4/80 primary antibody at 37 °C for 1 h. Slides were then washed three times with PBS and incubated with the secondary FITC-conjugated Affinipure goat anti-rabbit antibody for 30 min. After washing, the sections were incubated with the CXCR3 antibody followed by Cy3-conjugated Affinipure goat anti-mouse antibody. Nuclei were labelled with DAPI. Imaging analyses were performed using an Olympus (Osaka, Japan) BX43 system.
Real-time PCR analysis
Total RNA was extracted from kidney tissue using Trizol reagent (Invitrogen, Carlsbad, CA). A high-capacity cDNA reverse transcription kit (Applied Biosystems, Forster City, CA) was used to synthesise the cDNA. PCR amplification was conducted in 10 μl of solution containing 3 μl cDNA, 5 μl SYBR mixture, 1.6 μl H2O, and 0.4 μl primer (10 μM). The primers used were as follows: CXCR3, 5′-TCTCGTTTTCCCCATAATCG-3′ (forward) and 5′-AGCCAAGCCATGTACCTTGA-3′ (reverse); CXCL11, 5′-CATTTTGACGGCTTTCATCC-3′ (forward) and 5′-AAGGTCACAGCCATAGCCCT-3′ (reverse). CD68, 5′-ACCGCCATGTAGTCCAGGTA-3′ (forward) and 5′-ATCCCCACCTGTCTCTCTCA-3′ (reverse). MCP1 5′-ATTGGGATCATCTTGCTGGT-3′ (forward) and 5′-CCTGCTGTTCACAGTTGCC-3′(reverse). IL1β 5′ -GGCTCATCTGGGATCCTCTC-3′ (forward) and 5′-TCATCTTTTGGGGTCCGTCA-3′ (reverse).18 S rRNA, 5′-AGTCCCTGCCCTTTGTACAC-3′ (forward) and 5′ -CGATCCGAGGGCCTCACTA-3′ (reverse). Amplification steps consisted of 40 cycles of denaturation at 94 °C for 40 s, annealing at 55 °C for 40 s, and extension at 72 °C for 40 s using a DNA cycler CFX96 real-time system (Bio Rad, Hercules, CA, USA).
Western blot analysis
Kidney samples were prepared in ice-cold radio-immune precipitation assay (RIPA) buffer with protease inhibitors. Samples were centrifuged for 10 min at 12,000 rpm. The supernatant was collected, and the protein concentration was measured using a BCA commercial kit. Protein lysates were separated by SDS-PAGE and subsequently transferred onto nitrocellulose membranes. Membranes were blocked with 5 % non-fat dry milk buffer and incubated with antibodies against CXCR3 and CXCL11. A secondary antibody was used for chemiluminescent detection. The loading accuracy was evaluated by monoclonal antibody against GAPDH.
Statistical analysis
Each experiment was performed at least three independent times. All the results are expressed as mean ± s.d. The statistical test was performed with SPSS software version 18.0. Groups were compared using one-way analysis of variance with Dunnett’s multiple comparison test or the Student-Newman-Keuls test. P < 0.05 was considered statistically significant.
Discussion
In this study, it was demonstrated that PF protected mice from renal damage in conA-induced injury, and the mechanism of PF was shown to be closely associated with its suppressive effect on the CXCR3/CXCL11 chemokine axis in macrophage recruitment.
Intravenous injection of conA (at a dose of more than 10 mg/kg body weight) in mice can cause severe liver injury with high mortality rate [
12], which is similar to acute hepatic failure. Experimental and clinical studies have demonstrated that liver diseases with liver dysfunction, especially in hepatic failure, are frequently accompanied by acute kidney injury (AKI) [
13]. For example, renal damage consists of proximal tubular nephrosis in acute hepatic coma [
14]. Furthermore, patients with acute hepatic failure usually suffer renal damage with higher levels of creatinine [
14]. The kidney was damaged with higher tryptophan levels in the lipopolysaccharide (LPS)-induced acute hepatic failure model [
15]. All these reports confirm that the kidneys are damaged in acute hepatitis. The data presented here also shows that BUN and Cr increases significantly after 8 h of conA administration. Renal tubular necrosis and inflammatory cell infiltration were also observed by HE staining. From these results, it could be concluded that the kidneys were also damaged in conA-treated mice.
ConA-induced liver injury in mice is characterised by inflammatory infiltration of macrophages, neutrophils, and T cells into the liver [
16]. It has been shown that conA-induced liver injury depends on the production of inflammatory cytokines and chemokines, such as tumour necrosis factor-α (TNF-α) and IL1β [
17]. Thus, macrophage infiltration has been considered to be a hallmark of all forms of injury [
4,
18]. Macrophage deletion causes a decrease in pro-inflammatory cytokines and liver injury is suppressed in the conA mouse model [
19]. These results demonstrate that macrophages are involved in the production of pro-inflammatory cytokines and played an important role in conA-induced liver injury [
20]. It was reported that one of the initial events of renal disease was macrophage infiltration into the kidney [
21,
22]. The results showed that CD68-positive macrophages increased significantly in kidneys 8 h after conA administration, meanwhile, the inflammatory cytokines such as IL1β and MCP1 increased notably in the damaged kidneys. However, surprisingly, the results showed that IL1β and MCP1 originated mostly from tubular epithelial cells, and not macrophages. These results are consistent with other reports [
23,
24].
CXCR3 was not expressed in tubular epithelial cells, unlike IL1β and MCP1, and the immunofluorescent staining demonstrated that almost all of the macrophages could express CXCR3. This result indicates that CXCR3 may play an important role in macrophage infiltration. It has been suggested that CXCR3 and its ligands is one of the most important chemokine axes that promotes the arrival of cells into inflamed tissues [
11]. This receptor interacts with three ligands: CXCL9, CXCL10, and CXCL11 [
25]. However, in our preliminary experiments using real-time PCR analysis, CXCR9 and CXCR10 did not significantly increase 8 h after conA stimulation compared with non-conA mice (data not shown here), indicating that CXCL9 and CXCL10 may not be important in conA-induced renal damage. CXCL11 increased more than seven-fold in conA-vehicle mice compared with non-conA mice. Importantly, CXCL11 binds to CXCR3 with a much higher affinity than it does CXCL9 and CXCL10 [
25]. Our results showed that CXCL11 was strong stained in tubular epithelial cells and was bound to CXCR3 after conA stimulation. Combined with these results it could be demonstrated that the CXCR3/CXCL11 signalling axis was over-activated and played a key role in macrophage infiltration in the kidneys in conA-treated mice.
Paeoniflorin is one of the principal bioactive components derived from the root of
P. lactiflora Palls/Paeoniae Radix, a traditional Chinese herbal medicine which has been widely used in the treatment of liver and renal diseases. It has been reported that PF is widely used in the treatment of central nervous system diseases and serves as an antioxidant to protect neurons against oxidative stress [
5]. PF is also able to alleviate acute lung injury, and the underlying mechanisms are probably attributed to a decrease in the production of pro-inflammatory cytokines through down-regulating the activation of p38, JNK, and NF-κB pathways in lung tissues [
26]. We previously reported that PF administration attenuated DMN-induced liver fibrosis by regulating macrophage activation in the main organs [
9]. In this study, PF had positive effects on liver function and renal function, with histopathological improvement at doses of 30 mg/kg and 150 mg/kg. Pre-treatment with PF significantly reduced macrophage infiltration and expression of pro-inflammatory cytokines IL1β and MCP1. PF inhibited the elevated expression of CXCR3 and CXCL11 and also suppressed the interactions between CXCR3 and CXCL11.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CL and DX conceived of the study. ZC and YW carried out data collection. CL and DX participated in the design of the study. XD and JZ carried out data analysis. All authors read and approved the final manuscript.