1
Determining the Effect of Geraniol on Liver Regeneration via the NF-κB Pathway after
Partial Hepatectomy
Emre Ceyhan1,*Ph.D. , Mediha Canbek1 Ph.D.
1
Department of Biology, Faculty of Science, Eskisehir Osmangazi University, 26480
Eskisehir, Turkey
*Correesponding author: Emre Ceyhan, Department of Biology, Faculty of Science,
Eskisehir Osmangazi University, 26480 Eskisehir, Turkey. e-mail: [email protected]
Authors:
Dr. Emre Ceyhan
Tel: +90 222 2290433/2429
Fax: +90 222 2393578
e-mail: [email protected]
Dr. Mediha Canbek
Tel: +90 222 2290433/2849
Fax: +90 222 2393578
e-mail: [email protected]
Conflict of interest: I certify that there is no financial contributions or conflicts of interest to
declare in this study.
Financial support: There is no financial support for this work
Authors' contributions: Emre CEYHAN conception and design, analysis and interpretation,
RT-PCR and Western Blott, writing the article; Mediha CANBEK surgery, histological
analysis;
Acknowledgments: The authors thanks Dr. Azmi Yerlikaya for comments on the manuscript.
2
ABSTRACT
Background and study aims: NF-κB is a dimeric transcription factor that is involved in the
regulation of regeneration and apoptosis genes and plays a key role in liver regeneration after
partial hepatectomy. Complementary medicine is used to treat various diseases obtained from
a large number of plants that are found in nature. One such plant is geraniol, and there are as
yet no studies about its in vivo effects on liver regeneration.
Methods: The effects of geraniol on liver regeneration were assessed in Wistar albino male
rats after 70% partial hepatectomy (PH) in vivo, and 8 groups with n = 6 were formed.
Groups I and II were the sham control groups; a single dose of saline, silymarin or geraniol
was injected intraperitoneally to the other 6 groups immediately after PH. Using RT-PCR and
Western blot analysis, the NF-κB, TNF-α and IL-6 gene expression and protein levels were
measured. Moreover, heat shock proteins HSP27 and HSP60 protein levels were examined by
Western blot.
Results: According to the data obtained, geraniol had a significant role (P < .05) in increasing
the process of liver regeneration when given intraperitoneally, and it protected the liver as
assessed by histology and the HSP protein levels.
Conclusions: In rats receiving 100 mg/kg geraniol intraperitoneally, this agent induced
hepatic regeneration 24 and 48 hours after PH (70%).
Keywords: TNF-α IL-6, HSP27, HSP60, Silymarin
3
INTRODUCTION
Liver surpasses most other organs in its ability to regenerate itself, which is manifested
by the proliferative and replicative activity targeting functional mass recovery when the liver
is subjected to injury. Similarly, the surgical removal of some parts of the hepatic lobes, i.e.,
partial hepatectomy (PH), results in increased hepatocyte replication. This unique potential of
hepatocytes is of utmost importance because under normal conditions, these cells rarely
divide 1.
The regeneration process proceeds under the effect of a number of signalling pathways
in which certain cytokines and growth factors play a role. Following liver injury due to partial
hepatectomy or a chemical insult to the liver, TNF-α and IL-6 production are initiated within
Kupffer and endothelial cells. The combined effect of IL-6, and particularly the effect of the
Hepatocyte Growth Factor (HGF) synthesised along with TNF-α, leads to SOR production,
which is also influenced by growth factors originating from surrounding organs 2. The
signalling pathways that are activated following hepatectomy include TNF-TNFR1-NF-κBIL-6-STAT3.
A number of studies have clearly established the role of NF-κB in liver regeneration
after partial hepatectomy 3,4,5,6. NF-κB is a dimeric transcription factor that is involved in a
number of processes, such as regeneration, apoptosis, inflammation, cellular adhesion,
proliferation, and gene regulation, and in the activation of more than 70 genes 7,8,9. The
nuclear factor kappa B (NF-κB) is a transcription factor that is activated in the cytoplasm in
the presence of IL-6 and other growth factors and consists of p65-p50 subunits. It is expressed
in many other cell types and is generally inactive under normal conditions. The I/B inhibitor
bound to the p65 molecular subunit is responsible for this inactive state. The IKK enzyme
complex is triggered by SOR, resulting in I/B molecule catalysis and its cleavage from NFκB. Thus, NF-κB activation following its phosphorylation in the hepatocyte cytoplasm causes
its migration to the cell nucleus. Inhibiting the interaction between NF-κB and the molecule
results in apoptosis. After partial hepatectomy, an augmentation in NF-κB, AP-1, and C/EBP
DNA binding is observed and is followed by augmented STAT3 binding to DNA shortly
thereafter. NF-κB can be detected 10 to 15 minutes after surgery and returns to normal values
within 1-2 hours 1,2,10,11.
The synthesis of heat shock proteins (Hsp), which were originally described in cells
that had been exposed to heat shock, is increased under several stress factors, such as high or
low temperatures and oxygen deficiency. These proteins confer some protection to cells
within the biological system 12. Whereas the inhibition of Hsp27 protein aggregation plays a
role in cellular growth and differentiation, Hsp60 is involved in the blockade of protein
aggregation and protein folding 13,14.
From a medical perspective, several agents are utilised to accelerate the healing
process in the injured liver, although side effects, as observed in many other pharmaceutical
treatments, are not always avoidable. This has led to a search for possible natural treatments
in liver injury.
Geraniol, the subject of this study, is a monoterpenoid alcohol (Fig-1) that possesses
strong antioxidant effects in vivo to protect the mitochondrial membrane against ROS injury
and has additional free-radical binding properties 15.
4
Fig-1 Geraniol formulae.
Geraniol has been previously demonstrated to exert anti-bacterial, anti-tumoural, and
hepato-protective effects 16,17,18. This study was based on the absence of in vivo studies
examining the effect of geraniol on liver regeneration.
Materials and methods
Animals
In the study, male Wistar albino rats with 200-250 g body weights were obtained from
the T.R. Ministry of Health, Refik Saydam National Public Health Agency, Experimental
Animal Production Laboratory. The experiment was performed following a stabilisation
period in the laboratory. The rats were housed in polycarbonate cages in an air-conditioned
room (12 h light/12 h dark, 22±2°C, 50±5% humidity). Throughout the experimental period,
the animals were provided with pellet chow, and water was given ad libitum 19,20,21.
Chemicals
In our experimental studies, 99% pure geraniol was used (Acros Organics –
410900250), which was obtained commercially. Silymarin (Sigma – S0292 – 10G) was used
as a positive regeneration control in our study due to its protective and reparative effect on the
liver. Silymarin was dissolved in saline solution 16. A single dose of a 100 mg.kg-1 of
silymarin and geraniol was injected intraperitoneally.
Experimental Design
Animals were divided into 8 groups, each consisting of 6 animals. These groups are as
follows:
Group I sham - operated 24 h control group.
Group II sham - operated 48 h control group.
Group III partial hepatectomy – operated 24 h saline group.
Group IV partial hepatectomy – operated 48 h saline group.
Group V partial hepatectomy – operated 24 h silymarin group.
Group VI partial hepatectomy – operated 48 h silymarin group.
Group VII partial hepatectomy – operated 24 h geraniol group.
Group VIII partial hepatectomy – operated 48 h geraniol group.
Surgical Procedure
All of the surgical procedures were performed under anaesthesia (xylazine 10 mg per
kg and ketamine 70 mg per kg, intramuscular). A partial hepatectomy (PH) was performed
using the Higgins and Anderson (1931) technique 22.
RT-PCR Assay
RNA isolation from the liver was performed using the kit protocol using a PureLink
RNA Mini Kit and Trizol. cDNA was obtained from the RNA using a High Capacity cDNA
5
kit (Applied Biosystems). TaqMan Gene Expression Master Mix (Applied Biosystems) was
used for RT-PCR. Gene expression was analysed using the Applied Biosystems brand
StepOne Plus RT-PCR tool.
Western Blotting
Amersham Bioscience's ECF detection system was utilised for Western blotting
according to the manufacturer's instructions. Proteins were separated by electrophoresis on
12.5% polyacrylamide gels under denaturing conditions in 0.1% SDS. The proteins were then
transferred to PVDF membranes at 30 V overnight at 4 °C in transfer buffer (25 mM Tris, 192
mM glycine, and 20% methanol). The next morning, voltage was increased to 90 V for an
additional hour to complete the transfer. The blots were then incubated with rabbit polyclonal
anti-AdoMetDC antiserum (1:500 dilution) overnight in TBS-T (Tris-buffered saline-Tween
20). The blots were then extensively washed with TBS-T and incubated with anti-rabbit
alkaline phosphatase-conjugated secondary antibodies (1:10,000 dilution) for 1 h. To detect
HA-ubiquitin-AdoMetDC conjugates or HA-ubiquitin expression, blots were probed with
monoclonal anti-HA monoclonal antibodies (1:500 dilution) for 1 h. The blots were
extensively washed with TBS-T and then incubated with anti-mouse alkaline phosphataseconjugated secondary antibodies (1:5,000 dilution) for 1 h. Finally, the blots were developed
with ECF substrate for 3-5 min. Fluorescence was scanned with an Amersham Bioscience
FluorImager using a 570 nm filter, and specific bands were quantified using ImageQuant
software.
Protein Determination
The protein concentration was determined using the Bio-Rad dye binding microassay.
Bovine serum albumin at 1–20 pmol/l was used as standard.
Histological Evaluation
Liver tissue samples were fixed in 10% neutral formalin for histological investigation.
The tissues were routinely processed and embedded in paraffin. Sections (5-6 μm thick) were
cut and stained with Harris Haematoxylin and Eosin (H&E). H&E-stained liver preparations
were histopathologically examined through light microscopy.
RESULTS
Gene Expression Results
NF-κB Gene Expression
In our study, NF-κB gene expression in groups I and II demonstrated a significant
increase after the first 24 hours (Fig-2a), with a significant decline at the 48th hour compared
with the 24-hour levels (p < 0.05). Additionally, gene expression after 24 and 48 hours of
treatment in the geraniol-treated groups was very close to that observed in the silymarin
groups (silymarin is an agent that has regenerative capabilities).
6
IL-6 Gene Expression
IL-6 gene expression was also examined after partial hepatectomy. Compared with
groups I and II, there was a significant increase in IL-6 gene expression in Groups III, IV, V,
VI, VII, and VIII, with a decrease at the 48th hour. These differences were statistically
significant (p < 0.05). The gene expression at the 24th and 48th hours in the geraniol-treated
groups was similar to that observed in the silymarin groups (Fig-2b).
TNF-α Gene Expression
As demonstrated in Figure 4, except for groups III and IV, there was a significant increase in
TNF-α gene expression following partial hepatectomy in all groups compared with Groups I
and II. Additionally, the gene expression at the 24th and 48th hours in geraniol-treated groups
was very close to that observed in the silymarin groups (Fig-2c).
Fig-2 a: Significant differences in NF-κB1 gene expression according to groups (a and b) and
time (n=6) (P < 0.05). b: Significant differences in IL-6 gene expression according to
groups (a and b) and time (n=6) (P <0.05). c: Significant differences in TNF-α gene
expression according to groups (a and b) and time (n=6) (P <0.05).
Western Blot Analysis Results for NF-κB1
A comparison of Group I and II was performed to assess changes in the amount of NFκB protein, which has a molecular weight of 105 kDa and exists in a free cytosolic form.
Compared with Group I, a significant increase in NF-κB protein was observed in Groups IV,
V, VI, VII, and VIII but not in Group III (Fig-3). In geraniol groups, the NF-κB levels were
higher in geraniol groups compared with the positive control, i.e., silymarin groups (Figure
2.1).
7
1
2
3
4
5
6
7
8
Fig-3 Western blot analysis of NF-κB1 protein levels.
Western Blot Analysis Results for IL-6
Changes in IL-6 protein (molecular weight of 22 kDa) following partial hepatectomy
were examined with respect to Groups I and II using Western blot analysis. Accordingly, a
29% increase was noted only in Group V compared with Group I (Fig-4). There was a timedependent decrease in IL-6 protein in geraniol groups.
1
2
3
4
5
6
7
8
Fig-4 Western blot analysis of IL-6 protein levels
Western Blot Analysis Results for TNF-α
Changes in the TNF-α levels (which has a molecular weight of 22 kDa) following
partial hepatectomy were examined with respect to Groups I and II using Western blot
analysis. There was a decrease at the 24th hour in Group III, i.e., the partial hepatectomy
group. In Group V, there was an increase in protein content compared with Group I, which
was the sham group. Additionally, TNF-α protein levels were higher in Group V than in
Group VII, and there was an increase in Groups V and VII compared with Group III. An
increase in the amount of TNF-α protein in the geraniol group was noted, and the values at the
48th hour were similar to those observed in the silymarin group (Fig-5).
At the 48th hour after partial hepatectomy, the highest TNF-α protein levels were
observed in Group IV, followed by Groups II, VIII and VI, in decreasing order.
1
2
3
4
5
6
7
8
Fig-5 Western blot analysis of TNF-α protein levels
Western Blot Analysis Results for HSP27
A comparison of stress protein HSP27 content following partial hepatectomy with
regard to Groups I and II revealed no change in Group III, whereas a continuous decrease
8
occurred in Groups IV, V, VI, VII and VIII (Fig-6), and the most marked decrease was
observed in Groups V and VII 24 hours after post-partial hepatectomy.
At the 48th hour, the most significant decrease occurred in Group VIII, followed by
Groups IV and VI.
1
2
3
4
5
6
7
8
Fig-6 Western blot analysis of HSP27 protein levels
Western Blot Analysis Results for HSP60
Comparative Western blot analysis of the stress protein HSP60 content following
partial hepatectomy with regard to Groups I and II revealed a continuous decrease in protein
content in Groups III, IV, V, VI, VII and VIII (Fig-7).
The most significant decrease in HSP60 protein levels at the 24th hour after partial
hepatectomy occurred in Group III, followed by Groups VII and V.
At the 48th hour following partial hepatectomy, the most significant decrease was
observed in Group VIII, followed by Groups VI and IV.
1
2
3
4
5
6
7
8
Fig-7 Western blot analysis of HSP60 protein levels
Histologic Analysis
Histological tissue structures were preserved in all of the groups (Fig-8), despite the
universal presence of vacuolisation and mitotic activity. Vacuolisation decreased in Group
VIII (Geraniol 48 hours).
9
Fig-8 Mitotic activity (←) and diffuse vacuolisation (↑) in hepatocytes from microscopic liver
cross-sections. (a) Group V, (b) Group VI, (c) Group VII, (d) Group VIII.
DISCUSSION AND CONCLUSIONS
In the present study, an increase in NF-κB, TNF-α and IL-6 gene expression was noted
in all groups 24 hours after partial hepatectomy, whereas a decrease was noted at the 48th hour
compared with the 24th hour. In the geraniol groups, gene expression was similar to that
observed in the positive controls (i.e., silymarin).
In a study by Arai et al., effective NF-κB expression still existed at the 48th hour after
partial hepatectomy in rats 23, whereas Yang et al. 24 observed that NF-κB activation lasted for
more than 24 hours in Kupffer and non-parenchymal cells after partial hepatectomy. In an
adenovirus liver injury model, Malato et al. observed effective NF-κB activation until the 48th
hour via the IKK-alpha pathway, which stimulated cyclin D and therefore triggered AP-1 and
NF-κB activation 25. Our results are consistent with these previous reports in terms of NF-κB,
IL-6 and TNF-α gene expression.
In this study utilising Western blot analysis, the NF-κB, IL-6 and TNF-α protein
contents were estimated. In the first 24-hour period following partial hepatectomy, increased
NF-κB protein content was observed at the 24th hour compared with Groups I and VII,
whereas high protein levels were detected in Groups VIII, VI and IV. The protein content at
10
the 24th and 48th hours was similar. The groups with most marked increase in the protein
levels were Groups VII and VIII, which were the test groups. In these two groups, the protein
content was similar to that of the positive control groups V and VI. The NF-κB gene
expression data were consistent with the Western blot results. For a period of 48 hours, NFκB gene expression and protein formation were present.
In the first 24-hour period after partial hepatectomy, the TNF-α levels were increased
in Group V compared with Group I, whereas more protein was detected in Group IV
compared with Group II at the 48th hour. In Group VIII, the protein content was similar to that
of Group II. Additionally, the protein content was comparable in these groups at the 24th and
48th hours. Groups IV and VIII had the most significant increases in protein. Test groups VII
and VIII had similar protein levels to Groups V and VI, which were the positive control
groups. The cytokine TNF-α was expressed in the test Groups VII and VIII for 48 hours. The
Western blot results for TNF-α paralleled those obtained using RT-PCR in Groups I, II, III,
IV, V, VI and VIII. The lower level of TNF-α protein in Group VII may be explained by the
requirement for a certain concentration ratio between IL-6 and TNF-α to continue
regeneration.
Western blot analysis indicated a lower protein content in Groups III and VII at the
24 hour compared with Group I, whereas it was higher in Group V than in Group I. At the
48th hour, the protein content was increased in Groups IV and VI compared with Group II,
whereas the protein levels were very similar in Groups VIII and II. The protein levels
decreased in the treatment groups VII and VIII. RT-PCR demonstrated higher IL-6 gene
expression in Groups VII and VIII at the 24th and 48th hours compared with Groups I and II.
In light of these observations, one might consider that the IL-6 protein content is reduced by
post-transcriptional regulation at the transcription or translation levels because IL-6
concentrations play a critical role in the regeneration process.
th
IL-6 is activated via stimulation by TNF-α and exhibits a multitude of biological
activities on different cell types, consistent with the profile of pleiotropic cytokine 26.
Increases in the IL-1 and IL-6 concentrations inhibit TNF-α expression. Thus, there is a
reciprocal balance between IL-6 and TNF-α concentrations because excessive TNF-α or IL-6
inhibits regeneration 3,11,27.
Heat-shock proteins (HSPs) are induced by stress, and HSP27, HSP60, HSP70 and
HSP90 have important hepato-protective effects during hepatic regeneration after partial
hepatectomy 28.
In our study, the role of HSP27 and HSP60 in hepatic regeneration following partial
hepatectomy was explored using Western blotting. The highest HSP concentrations were
detected in the Sham and PS groups, whereas the HSP27 and HSP60 protein levels were low
in the silymarin- and geraniol-treated groups. In the groups in which the therapeutic effect of
geraniol was explored, the HSP27 and HSP60 protein levels were lower compared with those
of the positive control silymarin groups. These results suggest a hepato-protective effect of
geraniol on hepatic stress induced by partial hepatectomy.
A comparison of our results with previous reports indicates similarities in terms of the
Western blot analysis results for HSP27 and HSP60. In the study by Shi et al., the HSP27 and
HSP60 protein levels were evaluated in mice that were given intraperitoneal quercetin after
partial hepatectomy. A comparison of the experimental group with the sham group revealed
an increase in HSP27 and HSP60 up to the 24th hour in all groups, whereas the HSP27 and
HSP60 levels were significantly reduced at the 48th hour in the quercetin group 28.
11
The regenerative capacity of an organ or a tissue is defined as the ability to recover its
previous tissue or organ mass following injury or loss implicated upon the organ or tissue in
question 1. In this regard, the liver represents a unique organ that has the ability to exhibit
hepatocyte regeneration, even after a serious loss of mass, until it returns to its original
volume. A loss of hepatic tissue can induce regeneration, which is delicately controlled by the
relationship between the body mass and hepatic mass.
In our study, an increase in mitotic activity in PS, Silymarin and Geraniol groups was
noted after partial hepatectomy. Particularly in the geraniol groups, the detection of mitotic
cells in histologic cross-sections at the 48th hour suggests an induction of hepatic regeneration
by geraniol.
The establishment of NF-κB, TNF-α and IL-6 gene expression and protein content in
our study may facilitate further the testing of geraniol at different time points. Determining
the TNF-α and IL-6 contents in Kupffer cells after partial hepatectomy using gene expression
and protein quantification analyses represents an additional appropriate methodology for
assessing the geraniol concentration, which may have critical importance for regeneration. In
the groups that received geraniol, the observation of lower HSP27 and HSP60 protein content
compared with another hepato-protective agent, i.e., silymarin, suggests that geraniol plays
some hepato-protective and regulatory roles in hepatic regeneration.
In conclusion, rats receiving 100 mg/kg intraperitoneal geraniol displayed apparent
hepatic regeneration at the 24th and 48th hours after partial (70%) hepatectomy. The NF-κB,
TNF-α and IL-6 gene expressions, as assessed by RT-PCR and protein quantification, lend
support to this possibility.
12
Conflict of Interest
I certify that there is no financial contributions or conflicts of interest to declare in this study.
Dr. Emre CEYHAN
Dr. Mediha CANBEK
13
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