# Effects of Ubiquinol-10 on MicroRNA-146a Expression In Vitro and In Vivo

Authors: Constance Schmelzer; Mitsuaki Kitano; Gerald Rimbach; Petra Niklowitz; Thomas Menke; Kazunori Hosoe; Frank Döring
Journal: Mediators of Inflammation
 (2009)

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## Abstract

MicroRNAs (miRs) are involved in key biological processes via suppression of gene expression at posttranscriptional levels. According to their superior functions, subtle modulation of miR expression by certain compounds or nutrients is desirable under particular conditions. Bacterial lipopolysaccharide (LPS) induces a reactive oxygen species-/NF-κB-dependent pathway which increases the expression of the anti-inflammatory miR-146a. We hypothesized that this induction could be modulated by the antioxidant ubiquinol-10. Preincubation of human monocytic THP-1 cells with ubiquinol-10 reduced the LPS-induced expression level of miR-146a to 78.9± 13.22%. In liver samples of mice injected with LPS, supplementation with ubiquinol-10 leads to a reduction of LPS-induced miR-146a expression to 78.12 ± 21.25%. From these consistent in vitro and in vivo data, we conclude that ubiquinol-10 may fine-tune the inflammatory response via moderate reduction of miR-146a expression.


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## Body

## 1. Introduction

MicroRNAs (miRs) are endogenous~22
nucleotide noncoding RNAs which suppress gene expression at
posttranscriptional levels by binding to the 3′-untranslated region of specific
mRNA targets through base pairing [1]. A number of recent studies reveal that miRs have critical functions in
key biological processes such as cell proliferation and cell death during
development [2], fat metabolism [3, 4], or insulin secretion [5]. Expression profiling approaches implicated a
differential expression of several miRs in cancer cells [1]. Regulated expression of miRs is scarcely understood
but is of special interest in the context of innate immunity and inflammation [6]. Expression profiling of more than 200 miRs in human
monocytic THP-1 cells revealed that miR-146a is substantially inducible by LPS
or other Toll-like receptor ligands [7]. Moreover, promoter analysis of the miR-146a gene
suggests a role of NF-κB in LPS-dependent upregulation of miR-146a [7].Modulation of the LPS→reactive
oxygen species→NF-κB signalling pathway and dependent miR-146a expression could
be an approach in order to fine-tune the inflammatory response. The importance
of such a modulation is of special interest because the balance between pro- and anti-inflammatory signals is critical in certain inflammatory diseases such
as atherosclerosis. Here we hypothesized that the reduced form of Coenzyme Q10 (QH2, ubiquinol-10) affects LPS-inducible expression of the
anti-inflammatory miR-146a. The hypothesis was based on functions of CoQ10 as a potent antioxidant and putative modulator of inflammation via gene
expression [8, 9]. In the present work, we tested our hypothesis using an in vitro and an in vivo model. Initially,
we utilized the human monocytic cell line THP-1 as a well-established model of
LPS-induced miR-146a expression and applied ubiquinol-10. Additionally, liver
samples of LPS-injected mice supplemented with ubiquinol-10 were used to
strengthen the hypothesis that ubiquinol-10 has a relevance in fine-tuning
LPS-induced miR-146a expression levels.


## 2. Materials and Methods

### 2.1. Reagents

Ubiquinol-10 aqueous
solution (PEG-60 hydrogenated castor oil, ubiquinol-10, glycerol, water) and
corresponding vehicle (no ubiquinol-10 supplement) were purchased from KANEKA
Corporation, Japan.
Lipopolysaccharide (LPS,E. coli O55:B5)
was obtained from Sigma-Aldrich (Germany).


#### 2.2. Cell Culture

THP-1 cells were routinely cultivated in RPMI medium
1640 supplemented with 10% FCS and 1% antibiotics (penicillin/streptomycin) in
a humidified incubator containing 5% CO2 at 37°C. Twenty four hours
before preincubation, cells were plated at a density of 1×106 cells per well in a 6-well plate. Subsequently, cells were preincubated with
10 μM ubiquinol-10 or the respective vehicle control. After 24 hours, cell
culture medium was removed and fresh LPS-containing medium (1 μg/mL) was added
for 4 hours. Finally, cells were collected in Qiazol-lysis buffer (Qiagen,  Germany)
for isolation of total RNA or alternatively scraped in Phosphate Buffered
Saline (PBS) for HPLC-analysis.


#### 2.3. Animals

Male C57/BL6J mice (10–12 weeks old, 25 g weight)
were purchased from Charles River Lab., Inc., Japan. Animals were separated into
two groups: (1) intervention group(n=6), which was given a diet
enriched with
ubiquinol-10 (QH2, 250 mg/kg/d)
for one week and (2) the control group (n=6), which received a diet
prepared by using corn oil in equal proportions to 1% (v/w) of the diet as a vehicle. In other respects all animals were maintained on a standard laboratory
diet (powdered
CE-2, CREA Japan) and housed under conditions at 22 ± 2°C  with a 12-hour light/dark cycle. Food intake and body
weight were monitored daily but indicated no relevant differences between
animals. After the 7-day supplementation or control diet period, an
intraperitoneal
injection (1mg/kg BW) of lipopolysaccharide (LPS, E. coli, O55:B5, Sigma-Aldrich,
Japan) was
administered for further 4 hours. However, for HPLC-experiments, only saline-injected
mice were used. In all other aspects, animal treatment was identical. Subsequently,
all mice were sacrificed, and liver samples were collected and stored at −80°C in RNAlater
Storage Solution (Qiagen, Japan) until use.


#### 2.4. Cytotoxicity of THP-1 Cells

Determination
of cell viability was performed using the Cell-Titer Glo Luminescent Assay. Thus, the total ATP levels were measured as an index of the
viable cell number. The luminescence was detected on a GloMax (Promega). Data (Figure1) are means ± SD of three biological experiments
performed in triplicate.Figure 1
Effect of ubiquinol-10 on viability of THP-1 monocytes. THP-1 cells were treated with medium, 0.1–100μM
ubiquinol-10, or 10% DMSO (PC, positive control) for 24 hours. Afterwards, cell
viability was determined based on ATP measurements. The viability of medium
control was set to 100%, and the other values were referenced to it. Data are
means ± SD of three independent experiments performed in triplicate.


#### 2.5. Determination of CoQ10 In Vitro and In Vivo

The different treated cells of each well were washed
with Phosphate Buffered Saline (PBS) and counted with Trypan blue at −80°C
before measurement of cellular CoQ10. Liver homogenates of 10 QH2-supplemented
or nonsupplemented mice (5 from each group, resp.) were stored at −80°C
(in 0.9% sodium chloride, 10 mg/mL) until further analysis. The method is
based on high-pressure liquid chromatography (HPLC) with electrochemical
detection and internal standardisation using ubihydroquinone-9 and ubiquinone-9
as standards and is described elsewhere [10]. In brief, as internal standard 15 pmol of ubihydroquinone-9 in 50 μL
ethanol were added to a 100 μL monocyte or liver homogenate suspension. The
cells and homogenates were disintegrated by adding of 300 μL of cold methanol.
Subsequently, the sample was mixed for 1 minute, and the suspension was immediately
extracted with 500 μL hexane after mixing for further 2 minutes. After
centrifugation (1000 xg, 5 minutes, 4°C), 300 μL of the supernatant were
transferred to a separate tube and dried under a stream of argon. Finally, the
dried residue was redissolved in 40 μL ethanol and injected into the HPLC
system. For each liver homogenate sample, the analyzed CoQ10 concentration was related to its respective protein level.


#### 2.6. Protein Quantification of Liver Homogenate Solutions

For calculation of differences in sample
preparation, protein concentration was determined in liver homogenate samples
(inμg/ml). Thus, homogenate samples were collected into NET-buffer (50 mM TRIS [pH 7.5], 150 mM NaCl, 1 mM EDTA [pH 8.0], 0.5%  NP-40). In each case,
homogenate solutions were treated with ultrasonics (“n” vs. “m”) and then centrifuged by
14.000 rpm at 4°C for 20 minutes. The protein concentration was determined by the Bradford method according to the manufacturer's
instructions.


#### 2.7. RNA Isolation and Quantitative RT-PCR of THP-1 Cells.

Total RNA was
isolated by using the miRNeasy Isolation Kit (Qiagen, Germany),
and cDNA was converted by the TaqManMicroRNA Reverse Transcription
Kit (Applied Biosystems). miRNA-146a expression was measured and quantified by
using the TaqManMicroRNA Assays (Applied Biosystems) according to
the manufacturer's protocol and normalised by snoRNA202 (Applied Biosystems).
Quantitative RT-PCR reaction was performed on an Applied Biosystems 7300
Real-Time PCR System.


#### 2.8. RNA Isolation of Liver Samples and Quantitative RT-PCR

Total RNA was isolated
with Qiazol lysis reagent obtained with the miRNeasy Isolation Kit (Qiagen, Germany).
cDNA was converted by the TaqMan MicroRNA Reverse Transcription (RT)
Kit (Applied Biosystems). The RT reaction product was diluted 10 times in water
and subsequently used for RT-PCR amplification of miRNA-146a by using the
TaqMan MicroRNA Assays (Applied Biosystems) according to the manufacturer's
protocol and normalized by snoRNA202 (Applied Biosystems). A 6-fold total
RNA-dilution series from a control-treated (+LPS) mouse liver served as
standard to ensure a linear range of the amplification. Quantitative RT-PCR
reaction was performed on an Applied Biosystems 7300 Real-Time PCR System.


#### 2.9. Statistics

Results were
analyzed by an unpaired, two- or one-sided
Student'st-test using SPSS 11.5 for Windows and GraphPad Prism 4.0 software.
P-values less than or equal to .05 were considered statistically significant.


### 3.Results and Discussion

#### 3.1. No Cytotoxic Effects but Cellular Accumulation of Ubiquinol-10 in the Human Monocytic Cell Line THP-1

To exclude cytotoxic side effects in our experimental
set-up, vitality of THP-1 cells was determined after incubation with
ubiquinol-10. As shown in Figure1, incubation of THP-1 cells with increasing
ubiquinol-10 concentrations (0.1–100 μM) for 24 hours led to no significant effects
on cell vitality. Thus, in THP-1 cells no cytotoxicity was found for
ubiquinol-10 at physiological (1.0 μM), supraphysiological (10 μM), and
pharmacological (>10 μM) concentrations. The putative effects of extracellular ubiquinol-10 on miR expression
depend on its capability to reach cellular concentrations above background
level. Therefore, we determined the cellular concentration of CoQ10 as a function of medium ubiquinol-10. As shown in Table 1, cellular CoQ10 levels arose with increasing extracellular ubiquinol-10 concentrations (0.1–100 μM). As mainly the reduced form of CoQ10 can function as an
antioxidant [11], we determined the proportion between the oxidized and reduced form of
CoQ10. Depending on the extracellular ubiquinol-10 concentration,
about 75–90% of cellular CoQ10 was present in its reduced form (Table 1). Moreover, our previous results have shown that monocytic cells are
able to convert oxidized CoQ10 effectively into its reduced form [12]. Thus, at physiological and supraphysiological CoQ10 levels
in the medium (0.1–10 μM), the intracellular CoQ10 distribution is
clearly in favor of the reduced form (75–90%). Other in vitro and in vivo studies also revealed an intracellular incorporation of CoQ10 after
supplementation in blood cells, thereby leading, for example, to a reduction of DNA
strand breakdowns [11, 13]. Taken together, we were able to increase the cellular ubiquinol-10
concentration without any cytotoxic side-effects in human THP-1 cells.Table 1
Concentration and redox state of CoQ10 in
the human monocytic cell line THP-1 after incubation with various
concentrations of ubiquinol-10 for 24 hours. Data are given as means ± SD of two independent measurements performed in duplicate.
ExtracellularCellularμM ubiquinol-10pmol CoQ10/106 cells% ubiquinol-10μM ubiquinol-10((a)-(b))0, medium control25.62±4.4274.40±0.144.560, vehicle control23.07±0.4674.70±1.564.120.124.68±2.0175.25±0.354.441.026.48±3.3679.35±0.075.0310.072.35±1.1486.10±0.7114.90100627.46±138.0890.35±1.48140.35(a)Suggested diameter of monocytes: 20 μm(b)Related to mean
values.


#### 3.2. Ubiquinol-10 Attenuates the LPS-Induced Expression of miR-146a in the Human Monocytic Cell Line THP-1

In order to
study the effect of ubiquinol-10 on LPS-induced miR-146a expression,
appropriate conditions were established. For this purpose, the LPS-induced
response of THP-1 cells was examined. Unstimulated THP-1 cells did not secrete
relevant amounts of TNF-α into the medium (4.22 pg/mg protein ± SD). However,
stimulation with 1 μg/mL LPS for 4 hours resulted in an increase of medium TNF-α
levels (351.94 pg/mg protein ± SD). Thus, we obtained a
substantial and anticipated LPS-response of THP-1 cells [14]. As already described in the literature [7], the expression level of miR-146a is upregulated by LPS.
As shown in Figure 2, in comparison to unstimulated THP-1 cells, LPS challenge
induced a 19-fold induction of miR-146a expression (P=.0007). With respect
to our results regarding cytotoxicity and cellular accumulation of
ubiquinol-10, THP-1 cells were preincubated with 10 μM ubiquinol-10 for 24 hours
as an effective dose. This is nearly in accordance to other in vitro studies performed with CoQ10 [15, 16] and is only 2-fold
higher than CoQ10 serum levels after supplementation in humans [17]. Thereafter, cells
were stimulated with 1 μg/mL LPS for 4 hours and the resulting steady-state
expression level of miR-146a was determined. As shown in Figure 2, preincubation of
THP-1 cells with ubiquinol-10 reduced the LPS-induced expression level of miR-146a to 78.9±13.22%. Although these effects were not statistically
significant (P≤.05), the reduced expression levels of miR-146a in
the ubiquinol-10-pretreated cells (QH2 + LPS) might be a first hint
for a fine-tuning mechanism of QH2 on miR-146a expression when compared to the high significant induction levels in control cells after
LPS-stimulation (Veco LPS).Figure 2
Effect of ubiquinol-10 on LPS-induced miR-146a expression in THP-1
cells. THP-1-cells were preincubated
without (−) or with (+) 10μM ubiquinol-10 for 24 hours. Afterwards, cells were
treated without (−) or with (+) 1 μg LPS/mL for 4 hours. After this treatment,
total RNA was extracted, converted to cDNA and miRs were assayed by
TaqMan-based qRT-PCR. Observed expression levels of miRs in the respective
treated cells were normalized to the corresponding levels of the endogenous
control (snoRNA202). Data (two-sided t-test)
are means ± SEM of three biological experiments performed in quadruplicate. P*<.05 versus  unstimulated cells.
P***<.001 versus unstimulated cells.


#### 3.3. Accumulation of CoQ10 Levels in the Liver of Ubiquinol-10 Supplemented Mice

To test putative
effects of CoQ10 on miRNA 146a regulation in a more physiological
manner, liver tissues of QH2-supplemented C57BL6/J mice were used.
However, to mediate these effects, a tissue-specific accumulation of CoQ10 is essential. Therefore, total CoQ10 levels were determined in liver
homogenate samples of QH2-supplemented and control mice. As shown in
Figure 3(a), total CoQ10 levels increased about 12-fold (P=.0193) in
liver tissues of QH2-supplemented mice when related to control
samples. Because CoQ9 is the predominant CoQ form in rodents [18], CoQ9 was used as an internal standard for HPLC-analysis. Thus, the CoQ9 level was not significantly changed between treatment and control group (P=.51, data not shown). Accordingly, the CoQ9/CoQ10 ratio
was significantly different between groups, corresponding to a 6.5-fold
increase in control tissue samples (P=.0019, Figure 3(b)). In general, all HPLC measurements
were related to protein levels in the respective tissue homogenate samples. In
summary, we could significantly increase the CoQ10 concentration in liver
tissues of ubiquinol-10-supplemented mice. An effective uptake of exogenously
applied CoQ10 in liver tissues of rodents has been already described
earlier [19].Effect of ubiquinol-10 concentration (a) on CoQ10 levels and (b) the CoQ9/CoQ10 ratio in liver tissues
of mice.  Mice were fed with ubiquinol-10 (250 mg/kg/d) or a respective control diet (no ubiquinol-10 supplement) for 7 days
followed by a sublethal intraperitoneal injection with saline (0.9% NaCl, LPS
negative control) for further 4 hours. Thereafter,
liver samples were collected, homogenized, and used for HPLC analysis with
electrochemical detection.  CoQ9 was used as an internal standard. Observed CoQ levels were related to the corresponding
protein levels of the respective liver homogenate samples (single data not
shown). All data (one-sided t-test) are
means ± SEM of 5 animals in the intervention group (+QH2) and
control group (−QH2), respectively.  Protein-related CoQ10 levels and
CoQ9/CoQ10 ratio of ubiquinol-10-supplemented and control animals are depicted in Figures 3(a) and 3(b), respectively.
(a)(b)


#### 3.4. Reduced Expression Levels of miR-146a in Liver Tissues of Ubiquinol-10-Supplemented Mice

To test the
relevance of a putative ubiquinol-10-dependent downregulation of LPS-inducible
miR-146a expression in vivo, we used
mice receiving either a diet enriched with ubiquinol-10 (QH2, 250 mg/kg/d) or a respective control diet for seven days. This dosage is in
accordance with previous CoQ10 studies in mice [20–22]. Thereafter, an
intraperitoneal injection of lipopolysaccharide (LPS, 1 mg/kg BW) was administered
for further 4 hours. In contrast to the control non-LPS-injected mice (no detectable TNF-α levels), TNF-α levels
increased significantly in the serum of LPS-treated animals (612.46 ± SD).
Finally, mice were sacrificed and livers were collected for miR isolation and
determination of miRNA-146a expression levels. As shown in Figure 4(a), the
LPS-induced expression level of miR-146a is generally lower in animals
supplemented with ubiquinol-10. In average, ubiquinol-10 reduces the
LPS-induced miR-146a expression to 78.12±21.25% (+QH2/+LPS) when compared to control animals (−QH2/+LPS) (Figure 4(b)).
This effect was statistically not significant but was consistent to those
obtained in cell culture experiments.Effect of ubiquinol-10 supplementation on miR-146a
expression in liver samples of LPS-treated mice. Mice were fed with ubiquinol-10 (250 mg/kg/d) or a respective control diet (no ubiquinol-10 supplement) for 7 days. Afterwards, a sublethal intraperitoneal injection of LPS (1 mg/kg BW)
was administered for further 4 hours Thereafter, liver samples were collected,
homogenized, and total RNAs including miRs were isolated as described by the manufacturer's
instructions. Subsequently, miRs were converted to cDNA and assayed by the
TaqMan-based qRT-PCR. Observed miR-146a expression levels of each animal were
related to the corresponding levels of the endogenous control (snoRNA202). Data
(two-sidedt-test) are means ± SEM of 6 animals in the intervention group (+QH2/+LPS) and control group (+LPS), respectively. miR-146a expression levels are
depicted as single bars of each animal (Figure 4(a)) or as total mean ± SEM of
each group (Figure 4(b)).
(a)(b)Regulation of miRs by certain
compounds or nutrients is of general interest, because this class of noncoding
RNAs is involved in central biological processes such as development,
inflammation and innate immunity, and signalling networks [1, 23]. So far, only a small number of environmental modulators of miR
expression have been identified. Expression levels of several miRs (i.e.,
miR-15a/b, miR-16, miR-107) are regulated by retinoic acid-induced
differentiation in human acute promyelocytic leukemia patients and cell lines [24]. Compounds such as sulphate, phosphate, and amino acids regulate the
expression of special miRs in plants [25] and human liver cells [26]. Toll-like receptor (TLR) ligands such as LPS induce the expression of
miR-146a significantly in human monocytes/macrophages [7].


### 4. Conclusion

Here we identified ubiquinol-10 as a
putative modulator of miR-146a expression. Preincubation of THP-1 cells with
ubiquinol-10 reduced the LPS-induced expression level of miR-146a. These
results are consistent to our in vivo data, where the expression of miR-146a was reduced in liver samples of mice
supplemented with ubiquinol-10 before LPS-injection (+QH2/+LPS)
when compared to control animals (+LPS). Although the observed effects are
statistically not significant, we postulated a fine-tuning mechanism of
ubiquinol-10 on the inflammatory response via a moderate reduction of miR-146a
expression. According to the superior function of miR-146a in the inflammatory
response, the observed moderate reduction of its expression by ubiquinol-10
seems to be desirable based on the following mechanisms. First, the LPS induced
upregulation of miR-146a in human monocytes/macrophages functions as a
negative regulator of the innate immune response because miR-146a targets
TRAF6, a regulator protein within the TLR-signalling pathways involved in the
formation and accumulation of reactive oxygen species [7]. Second, we have recently shown that ubiquinol-10
lowers the LPS-stimulated release of some proinflammatory cytokines and
chemokines relevant in inflammatory processes [27]. The observed effects were comparable to those of the potent and
characterized antioxidants N-acetyl-cysteine (NAC) or pyrrolidine-dithiocarbamate
(PDTC) [12, 14, 27].  Moreover,
mir-146a has been shown to be induced by proinflammatory cytokines such as
TNF-α, interleukin 1-beta (IL-1β), and TLRs [7, 28, 29]. miR-146a was also detected in tissues related to
inflammatory diseases including, for example, synovial fibroblasts and rheumatoid
synovial tissue [28]. Thus, we conclude that ubiquinol-10 reduces both the secretion of
proinflammatory agents and the expression of the anti-inflammatory miR-146a.
As a consequence, ubiquinol-10 acts as an anti-inflammatory compound but
perpetuates the essential inflammatory response via moderate reduction of
miR-146a expression. This dual effect could be due to the radical scavenging
activity of ubiquinol-10 since reactive oxygen species are involved in the
TLR-signalling pathways.In conclusion, the consistent in vitro and in vivo data suggest that ubiquinol-10 may fine-tune the
inflammatory response via moderate reduction of miR-146a expression.


## 2.1. Reagents

Ubiquinol-10 aqueous
solution (PEG-60 hydrogenated castor oil, ubiquinol-10, glycerol, water) and
corresponding vehicle (no ubiquinol-10 supplement) were purchased from KANEKA
Corporation, Japan.
Lipopolysaccharide (LPS,E. coli O55:B5)
was obtained from Sigma-Aldrich (Germany).


### 2.2. Cell Culture

THP-1 cells were routinely cultivated in RPMI medium
1640 supplemented with 10% FCS and 1% antibiotics (penicillin/streptomycin) in
a humidified incubator containing 5% CO2 at 37°C. Twenty four hours
before preincubation, cells were plated at a density of 1×106 cells per well in a 6-well plate. Subsequently, cells were preincubated with
10 μM ubiquinol-10 or the respective vehicle control. After 24 hours, cell
culture medium was removed and fresh LPS-containing medium (1 μg/mL) was added
for 4 hours. Finally, cells were collected in Qiazol-lysis buffer (Qiagen,  Germany)
for isolation of total RNA or alternatively scraped in Phosphate Buffered
Saline (PBS) for HPLC-analysis.


### 2.3. Animals

Male C57/BL6J mice (10–12 weeks old, 25 g weight)
were purchased from Charles River Lab., Inc., Japan. Animals were separated into
two groups: (1) intervention group(n=6), which was given a diet
enriched with
ubiquinol-10 (QH2, 250 mg/kg/d)
for one week and (2) the control group (n=6), which received a diet
prepared by using corn oil in equal proportions to 1% (v/w) of the diet as a vehicle. In other respects all animals were maintained on a standard laboratory
diet (powdered
CE-2, CREA Japan) and housed under conditions at 22 ± 2°C  with a 12-hour light/dark cycle. Food intake and body
weight were monitored daily but indicated no relevant differences between
animals. After the 7-day supplementation or control diet period, an
intraperitoneal
injection (1mg/kg BW) of lipopolysaccharide (LPS, E. coli, O55:B5, Sigma-Aldrich,
Japan) was
administered for further 4 hours. However, for HPLC-experiments, only saline-injected
mice were used. In all other aspects, animal treatment was identical. Subsequently,
all mice were sacrificed, and liver samples were collected and stored at −80°C in RNAlater
Storage Solution (Qiagen, Japan) until use.


### 2.4. Cytotoxicity of THP-1 Cells

Determination
of cell viability was performed using the Cell-Titer Glo Luminescent Assay. Thus, the total ATP levels were measured as an index of the
viable cell number. The luminescence was detected on a GloMax (Promega). Data (Figure1) are means ± SD of three biological experiments
performed in triplicate.Figure 1
Effect of ubiquinol-10 on viability of THP-1 monocytes. THP-1 cells were treated with medium, 0.1–100μM
ubiquinol-10, or 10% DMSO (PC, positive control) for 24 hours. Afterwards, cell
viability was determined based on ATP measurements. The viability of medium
control was set to 100%, and the other values were referenced to it. Data are
means ± SD of three independent experiments performed in triplicate.


### 2.5. Determination of CoQ10 In Vitro and In Vivo

The different treated cells of each well were washed
with Phosphate Buffered Saline (PBS) and counted with Trypan blue at −80°C
before measurement of cellular CoQ10. Liver homogenates of 10 QH2-supplemented
or nonsupplemented mice (5 from each group, resp.) were stored at −80°C
(in 0.9% sodium chloride, 10 mg/mL) until further analysis. The method is
based on high-pressure liquid chromatography (HPLC) with electrochemical
detection and internal standardisation using ubihydroquinone-9 and ubiquinone-9
as standards and is described elsewhere [10]. In brief, as internal standard 15 pmol of ubihydroquinone-9 in 50 μL
ethanol were added to a 100 μL monocyte or liver homogenate suspension. The
cells and homogenates were disintegrated by adding of 300 μL of cold methanol.
Subsequently, the sample was mixed for 1 minute, and the suspension was immediately
extracted with 500 μL hexane after mixing for further 2 minutes. After
centrifugation (1000 xg, 5 minutes, 4°C), 300 μL of the supernatant were
transferred to a separate tube and dried under a stream of argon. Finally, the
dried residue was redissolved in 40 μL ethanol and injected into the HPLC
system. For each liver homogenate sample, the analyzed CoQ10 concentration was related to its respective protein level.


### 2.6. Protein Quantification of Liver Homogenate Solutions

For calculation of differences in sample
preparation, protein concentration was determined in liver homogenate samples
(inμg/ml). Thus, homogenate samples were collected into NET-buffer (50 mM TRIS [pH 7.5], 150 mM NaCl, 1 mM EDTA [pH 8.0], 0.5%  NP-40). In each case,
homogenate solutions were treated with ultrasonics (“n” vs. “m”) and then centrifuged by
14.000 rpm at 4°C for 20 minutes. The protein concentration was determined by the Bradford method according to the manufacturer's
instructions.


### 2.7. RNA Isolation and Quantitative RT-PCR of THP-1 Cells.

Total RNA was
isolated by using the miRNeasy Isolation Kit (Qiagen, Germany),
and cDNA was converted by the TaqManMicroRNA Reverse Transcription
Kit (Applied Biosystems). miRNA-146a expression was measured and quantified by
using the TaqManMicroRNA Assays (Applied Biosystems) according to
the manufacturer's protocol and normalised by snoRNA202 (Applied Biosystems).
Quantitative RT-PCR reaction was performed on an Applied Biosystems 7300
Real-Time PCR System.


### 2.8. RNA Isolation of Liver Samples and Quantitative RT-PCR

Total RNA was isolated
with Qiazol lysis reagent obtained with the miRNeasy Isolation Kit (Qiagen, Germany).
cDNA was converted by the TaqMan MicroRNA Reverse Transcription (RT)
Kit (Applied Biosystems). The RT reaction product was diluted 10 times in water
and subsequently used for RT-PCR amplification of miRNA-146a by using the
TaqMan MicroRNA Assays (Applied Biosystems) according to the manufacturer's
protocol and normalized by snoRNA202 (Applied Biosystems). A 6-fold total
RNA-dilution series from a control-treated (+LPS) mouse liver served as
standard to ensure a linear range of the amplification. Quantitative RT-PCR
reaction was performed on an Applied Biosystems 7300 Real-Time PCR System.


### 2.9. Statistics

Results were
analyzed by an unpaired, two- or one-sided
Student'st-test using SPSS 11.5 for Windows and GraphPad Prism 4.0 software.
P-values less than or equal to .05 were considered statistically significant.


## 2.2. Cell Culture

THP-1 cells were routinely cultivated in RPMI medium
1640 supplemented with 10% FCS and 1% antibiotics (penicillin/streptomycin) in
a humidified incubator containing 5% CO2 at 37°C. Twenty four hours
before preincubation, cells were plated at a density of 1×106 cells per well in a 6-well plate. Subsequently, cells were preincubated with
10 μM ubiquinol-10 or the respective vehicle control. After 24 hours, cell
culture medium was removed and fresh LPS-containing medium (1 μg/mL) was added
for 4 hours. Finally, cells were collected in Qiazol-lysis buffer (Qiagen,  Germany)
for isolation of total RNA or alternatively scraped in Phosphate Buffered
Saline (PBS) for HPLC-analysis.


## 2.3. Animals

Male C57/BL6J mice (10–12 weeks old, 25 g weight)
were purchased from Charles River Lab., Inc., Japan. Animals were separated into
two groups: (1) intervention group(n=6), which was given a diet
enriched with
ubiquinol-10 (QH2, 250 mg/kg/d)
for one week and (2) the control group (n=6), which received a diet
prepared by using corn oil in equal proportions to 1% (v/w) of the diet as a vehicle. In other respects all animals were maintained on a standard laboratory
diet (powdered
CE-2, CREA Japan) and housed under conditions at 22 ± 2°C  with a 12-hour light/dark cycle. Food intake and body
weight were monitored daily but indicated no relevant differences between
animals. After the 7-day supplementation or control diet period, an
intraperitoneal
injection (1mg/kg BW) of lipopolysaccharide (LPS, E. coli, O55:B5, Sigma-Aldrich,
Japan) was
administered for further 4 hours. However, for HPLC-experiments, only saline-injected
mice were used. In all other aspects, animal treatment was identical. Subsequently,
all mice were sacrificed, and liver samples were collected and stored at −80°C in RNAlater
Storage Solution (Qiagen, Japan) until use.


## 2.4. Cytotoxicity of THP-1 Cells

Determination
of cell viability was performed using the Cell-Titer Glo Luminescent Assay. Thus, the total ATP levels were measured as an index of the
viable cell number. The luminescence was detected on a GloMax (Promega). Data (Figure1) are means ± SD of three biological experiments
performed in triplicate.Figure 1
Effect of ubiquinol-10 on viability of THP-1 monocytes. THP-1 cells were treated with medium, 0.1–100μM
ubiquinol-10, or 10% DMSO (PC, positive control) for 24 hours. Afterwards, cell
viability was determined based on ATP measurements. The viability of medium
control was set to 100%, and the other values were referenced to it. Data are
means ± SD of three independent experiments performed in triplicate.


## 2.5. Determination of CoQ10 In Vitro and In Vivo

The different treated cells of each well were washed
with Phosphate Buffered Saline (PBS) and counted with Trypan blue at −80°C
before measurement of cellular CoQ10. Liver homogenates of 10 QH2-supplemented
or nonsupplemented mice (5 from each group, resp.) were stored at −80°C
(in 0.9% sodium chloride, 10 mg/mL) until further analysis. The method is
based on high-pressure liquid chromatography (HPLC) with electrochemical
detection and internal standardisation using ubihydroquinone-9 and ubiquinone-9
as standards and is described elsewhere [10]. In brief, as internal standard 15 pmol of ubihydroquinone-9 in 50 μL
ethanol were added to a 100 μL monocyte or liver homogenate suspension. The
cells and homogenates were disintegrated by adding of 300 μL of cold methanol.
Subsequently, the sample was mixed for 1 minute, and the suspension was immediately
extracted with 500 μL hexane after mixing for further 2 minutes. After
centrifugation (1000 xg, 5 minutes, 4°C), 300 μL of the supernatant were
transferred to a separate tube and dried under a stream of argon. Finally, the
dried residue was redissolved in 40 μL ethanol and injected into the HPLC
system. For each liver homogenate sample, the analyzed CoQ10 concentration was related to its respective protein level.


## 2.6. Protein Quantification of Liver Homogenate Solutions

For calculation of differences in sample
preparation, protein concentration was determined in liver homogenate samples
(inμg/ml). Thus, homogenate samples were collected into NET-buffer (50 mM TRIS [pH 7.5], 150 mM NaCl, 1 mM EDTA [pH 8.0], 0.5%  NP-40). In each case,
homogenate solutions were treated with ultrasonics (“n” vs. “m”) and then centrifuged by
14.000 rpm at 4°C for 20 minutes. The protein concentration was determined by the Bradford method according to the manufacturer's
instructions.


## 2.7. RNA Isolation and Quantitative RT-PCR of THP-1 Cells.

Total RNA was
isolated by using the miRNeasy Isolation Kit (Qiagen, Germany),
and cDNA was converted by the TaqManMicroRNA Reverse Transcription
Kit (Applied Biosystems). miRNA-146a expression was measured and quantified by
using the TaqManMicroRNA Assays (Applied Biosystems) according to
the manufacturer's protocol and normalised by snoRNA202 (Applied Biosystems).
Quantitative RT-PCR reaction was performed on an Applied Biosystems 7300
Real-Time PCR System.


## 2.8. RNA Isolation of Liver Samples and Quantitative RT-PCR

Total RNA was isolated
with Qiazol lysis reagent obtained with the miRNeasy Isolation Kit (Qiagen, Germany).
cDNA was converted by the TaqMan MicroRNA Reverse Transcription (RT)
Kit (Applied Biosystems). The RT reaction product was diluted 10 times in water
and subsequently used for RT-PCR amplification of miRNA-146a by using the
TaqMan MicroRNA Assays (Applied Biosystems) according to the manufacturer's
protocol and normalized by snoRNA202 (Applied Biosystems). A 6-fold total
RNA-dilution series from a control-treated (+LPS) mouse liver served as
standard to ensure a linear range of the amplification. Quantitative RT-PCR
reaction was performed on an Applied Biosystems 7300 Real-Time PCR System.


## 2.9. Statistics

Results were
analyzed by an unpaired, two- or one-sided
Student'st-test using SPSS 11.5 for Windows and GraphPad Prism 4.0 software.
P-values less than or equal to .05 were considered statistically significant.


## 3.Results and Discussion

### 3.1. No Cytotoxic Effects but Cellular Accumulation of Ubiquinol-10 in the Human Monocytic Cell Line THP-1

To exclude cytotoxic side effects in our experimental
set-up, vitality of THP-1 cells was determined after incubation with
ubiquinol-10. As shown in Figure1, incubation of THP-1 cells with increasing
ubiquinol-10 concentrations (0.1–100 μM) for 24 hours led to no significant effects
on cell vitality. Thus, in THP-1 cells no cytotoxicity was found for
ubiquinol-10 at physiological (1.0 μM), supraphysiological (10 μM), and
pharmacological (>10 μM) concentrations. The putative effects of extracellular ubiquinol-10 on miR expression
depend on its capability to reach cellular concentrations above background
level. Therefore, we determined the cellular concentration of CoQ10 as a function of medium ubiquinol-10. As shown in Table 1, cellular CoQ10 levels arose with increasing extracellular ubiquinol-10 concentrations (0.1–100 μM). As mainly the reduced form of CoQ10 can function as an
antioxidant [11], we determined the proportion between the oxidized and reduced form of
CoQ10. Depending on the extracellular ubiquinol-10 concentration,
about 75–90% of cellular CoQ10 was present in its reduced form (Table 1). Moreover, our previous results have shown that monocytic cells are
able to convert oxidized CoQ10 effectively into its reduced form [12]. Thus, at physiological and supraphysiological CoQ10 levels
in the medium (0.1–10 μM), the intracellular CoQ10 distribution is
clearly in favor of the reduced form (75–90%). Other in vitro and in vivo studies also revealed an intracellular incorporation of CoQ10 after
supplementation in blood cells, thereby leading, for example, to a reduction of DNA
strand breakdowns [11, 13]. Taken together, we were able to increase the cellular ubiquinol-10
concentration without any cytotoxic side-effects in human THP-1 cells.Table 1
Concentration and redox state of CoQ10 in
the human monocytic cell line THP-1 after incubation with various
concentrations of ubiquinol-10 for 24 hours. Data are given as means ± SD of two independent measurements performed in duplicate.
ExtracellularCellularμM ubiquinol-10pmol CoQ10/106 cells% ubiquinol-10μM ubiquinol-10((a)-(b))0, medium control25.62±4.4274.40±0.144.560, vehicle control23.07±0.4674.70±1.564.120.124.68±2.0175.25±0.354.441.026.48±3.3679.35±0.075.0310.072.35±1.1486.10±0.7114.90100627.46±138.0890.35±1.48140.35(a)Suggested diameter of monocytes: 20 μm(b)Related to mean
values.


### 3.2. Ubiquinol-10 Attenuates the LPS-Induced Expression of miR-146a in the Human Monocytic Cell Line THP-1

In order to
study the effect of ubiquinol-10 on LPS-induced miR-146a expression,
appropriate conditions were established. For this purpose, the LPS-induced
response of THP-1 cells was examined. Unstimulated THP-1 cells did not secrete
relevant amounts of TNF-α into the medium (4.22 pg/mg protein ± SD). However,
stimulation with 1 μg/mL LPS for 4 hours resulted in an increase of medium TNF-α
levels (351.94 pg/mg protein ± SD). Thus, we obtained a
substantial and anticipated LPS-response of THP-1 cells [14]. As already described in the literature [7], the expression level of miR-146a is upregulated by LPS.
As shown in Figure 2, in comparison to unstimulated THP-1 cells, LPS challenge
induced a 19-fold induction of miR-146a expression (P=.0007). With respect
to our results regarding cytotoxicity and cellular accumulation of
ubiquinol-10, THP-1 cells were preincubated with 10 μM ubiquinol-10 for 24 hours
as an effective dose. This is nearly in accordance to other in vitro studies performed with CoQ10 [15, 16] and is only 2-fold
higher than CoQ10 serum levels after supplementation in humans [17]. Thereafter, cells
were stimulated with 1 μg/mL LPS for 4 hours and the resulting steady-state
expression level of miR-146a was determined. As shown in Figure 2, preincubation of
THP-1 cells with ubiquinol-10 reduced the LPS-induced expression level of miR-146a to 78.9±13.22%. Although these effects were not statistically
significant (P≤.05), the reduced expression levels of miR-146a in
the ubiquinol-10-pretreated cells (QH2 + LPS) might be a first hint
for a fine-tuning mechanism of QH2 on miR-146a expression when compared to the high significant induction levels in control cells after
LPS-stimulation (Veco LPS).Figure 2
Effect of ubiquinol-10 on LPS-induced miR-146a expression in THP-1
cells. THP-1-cells were preincubated
without (−) or with (+) 10μM ubiquinol-10 for 24 hours. Afterwards, cells were
treated without (−) or with (+) 1 μg LPS/mL for 4 hours. After this treatment,
total RNA was extracted, converted to cDNA and miRs were assayed by
TaqMan-based qRT-PCR. Observed expression levels of miRs in the respective
treated cells were normalized to the corresponding levels of the endogenous
control (snoRNA202). Data (two-sided t-test)
are means ± SEM of three biological experiments performed in quadruplicate. P*<.05 versus  unstimulated cells.
P***<.001 versus unstimulated cells.


### 3.3. Accumulation of CoQ10 Levels in the Liver of Ubiquinol-10 Supplemented Mice

To test putative
effects of CoQ10 on miRNA 146a regulation in a more physiological
manner, liver tissues of QH2-supplemented C57BL6/J mice were used.
However, to mediate these effects, a tissue-specific accumulation of CoQ10 is essential. Therefore, total CoQ10 levels were determined in liver
homogenate samples of QH2-supplemented and control mice. As shown in
Figure 3(a), total CoQ10 levels increased about 12-fold (P=.0193) in
liver tissues of QH2-supplemented mice when related to control
samples. Because CoQ9 is the predominant CoQ form in rodents [18], CoQ9 was used as an internal standard for HPLC-analysis. Thus, the CoQ9 level was not significantly changed between treatment and control group (P=.51, data not shown). Accordingly, the CoQ9/CoQ10 ratio
was significantly different between groups, corresponding to a 6.5-fold
increase in control tissue samples (P=.0019, Figure 3(b)). In general, all HPLC measurements
were related to protein levels in the respective tissue homogenate samples. In
summary, we could significantly increase the CoQ10 concentration in liver
tissues of ubiquinol-10-supplemented mice. An effective uptake of exogenously
applied CoQ10 in liver tissues of rodents has been already described
earlier [19].Effect of ubiquinol-10 concentration (a) on CoQ10 levels and (b) the CoQ9/CoQ10 ratio in liver tissues
of mice.  Mice were fed with ubiquinol-10 (250 mg/kg/d) or a respective control diet (no ubiquinol-10 supplement) for 7 days
followed by a sublethal intraperitoneal injection with saline (0.9% NaCl, LPS
negative control) for further 4 hours. Thereafter,
liver samples were collected, homogenized, and used for HPLC analysis with
electrochemical detection.  CoQ9 was used as an internal standard. Observed CoQ levels were related to the corresponding
protein levels of the respective liver homogenate samples (single data not
shown). All data (one-sided t-test) are
means ± SEM of 5 animals in the intervention group (+QH2) and
control group (−QH2), respectively.  Protein-related CoQ10 levels and
CoQ9/CoQ10 ratio of ubiquinol-10-supplemented and control animals are depicted in Figures 3(a) and 3(b), respectively.
(a)(b)


### 3.4. Reduced Expression Levels of miR-146a in Liver Tissues of Ubiquinol-10-Supplemented Mice

To test the
relevance of a putative ubiquinol-10-dependent downregulation of LPS-inducible
miR-146a expression in vivo, we used
mice receiving either a diet enriched with ubiquinol-10 (QH2, 250 mg/kg/d) or a respective control diet for seven days. This dosage is in
accordance with previous CoQ10 studies in mice [20–22]. Thereafter, an
intraperitoneal injection of lipopolysaccharide (LPS, 1 mg/kg BW) was administered
for further 4 hours. In contrast to the control non-LPS-injected mice (no detectable TNF-α levels), TNF-α levels
increased significantly in the serum of LPS-treated animals (612.46 ± SD).
Finally, mice were sacrificed and livers were collected for miR isolation and
determination of miRNA-146a expression levels. As shown in Figure 4(a), the
LPS-induced expression level of miR-146a is generally lower in animals
supplemented with ubiquinol-10. In average, ubiquinol-10 reduces the
LPS-induced miR-146a expression to 78.12±21.25% (+QH2/+LPS) when compared to control animals (−QH2/+LPS) (Figure 4(b)).
This effect was statistically not significant but was consistent to those
obtained in cell culture experiments.Effect of ubiquinol-10 supplementation on miR-146a
expression in liver samples of LPS-treated mice. Mice were fed with ubiquinol-10 (250 mg/kg/d) or a respective control diet (no ubiquinol-10 supplement) for 7 days. Afterwards, a sublethal intraperitoneal injection of LPS (1 mg/kg BW)
was administered for further 4 hours Thereafter, liver samples were collected,
homogenized, and total RNAs including miRs were isolated as described by the manufacturer's
instructions. Subsequently, miRs were converted to cDNA and assayed by the
TaqMan-based qRT-PCR. Observed miR-146a expression levels of each animal were
related to the corresponding levels of the endogenous control (snoRNA202). Data
(two-sidedt-test) are means ± SEM of 6 animals in the intervention group (+QH2/+LPS) and control group (+LPS), respectively. miR-146a expression levels are
depicted as single bars of each animal (Figure 4(a)) or as total mean ± SEM of
each group (Figure 4(b)).
(a)(b)Regulation of miRs by certain
compounds or nutrients is of general interest, because this class of noncoding
RNAs is involved in central biological processes such as development,
inflammation and innate immunity, and signalling networks [1, 23]. So far, only a small number of environmental modulators of miR
expression have been identified. Expression levels of several miRs (i.e.,
miR-15a/b, miR-16, miR-107) are regulated by retinoic acid-induced
differentiation in human acute promyelocytic leukemia patients and cell lines [24]. Compounds such as sulphate, phosphate, and amino acids regulate the
expression of special miRs in plants [25] and human liver cells [26]. Toll-like receptor (TLR) ligands such as LPS induce the expression of
miR-146a significantly in human monocytes/macrophages [7].


## 3.1. No Cytotoxic Effects but Cellular Accumulation of Ubiquinol-10 in the Human Monocytic Cell Line THP-1

To exclude cytotoxic side effects in our experimental
set-up, vitality of THP-1 cells was determined after incubation with
ubiquinol-10. As shown in Figure1, incubation of THP-1 cells with increasing
ubiquinol-10 concentrations (0.1–100 μM) for 24 hours led to no significant effects
on cell vitality. Thus, in THP-1 cells no cytotoxicity was found for
ubiquinol-10 at physiological (1.0 μM), supraphysiological (10 μM), and
pharmacological (>10 μM) concentrations. The putative effects of extracellular ubiquinol-10 on miR expression
depend on its capability to reach cellular concentrations above background
level. Therefore, we determined the cellular concentration of CoQ10 as a function of medium ubiquinol-10. As shown in Table 1, cellular CoQ10 levels arose with increasing extracellular ubiquinol-10 concentrations (0.1–100 μM). As mainly the reduced form of CoQ10 can function as an
antioxidant [11], we determined the proportion between the oxidized and reduced form of
CoQ10. Depending on the extracellular ubiquinol-10 concentration,
about 75–90% of cellular CoQ10 was present in its reduced form (Table 1). Moreover, our previous results have shown that monocytic cells are
able to convert oxidized CoQ10 effectively into its reduced form [12]. Thus, at physiological and supraphysiological CoQ10 levels
in the medium (0.1–10 μM), the intracellular CoQ10 distribution is
clearly in favor of the reduced form (75–90%). Other in vitro and in vivo studies also revealed an intracellular incorporation of CoQ10 after
supplementation in blood cells, thereby leading, for example, to a reduction of DNA
strand breakdowns [11, 13]. Taken together, we were able to increase the cellular ubiquinol-10
concentration without any cytotoxic side-effects in human THP-1 cells.Table 1
Concentration and redox state of CoQ10 in
the human monocytic cell line THP-1 after incubation with various
concentrations of ubiquinol-10 for 24 hours. Data are given as means ± SD of two independent measurements performed in duplicate.
ExtracellularCellularμM ubiquinol-10pmol CoQ10/106 cells% ubiquinol-10μM ubiquinol-10((a)-(b))0, medium control25.62±4.4274.40±0.144.560, vehicle control23.07±0.4674.70±1.564.120.124.68±2.0175.25±0.354.441.026.48±3.3679.35±0.075.0310.072.35±1.1486.10±0.7114.90100627.46±138.0890.35±1.48140.35(a)Suggested diameter of monocytes: 20 μm(b)Related to mean
values.


## 3.2. Ubiquinol-10 Attenuates the LPS-Induced Expression of miR-146a in the Human Monocytic Cell Line THP-1

In order to
study the effect of ubiquinol-10 on LPS-induced miR-146a expression,
appropriate conditions were established. For this purpose, the LPS-induced
response of THP-1 cells was examined. Unstimulated THP-1 cells did not secrete
relevant amounts of TNF-α into the medium (4.22 pg/mg protein ± SD). However,
stimulation with 1 μg/mL LPS for 4 hours resulted in an increase of medium TNF-α
levels (351.94 pg/mg protein ± SD). Thus, we obtained a
substantial and anticipated LPS-response of THP-1 cells [14]. As already described in the literature [7], the expression level of miR-146a is upregulated by LPS.
As shown in Figure 2, in comparison to unstimulated THP-1 cells, LPS challenge
induced a 19-fold induction of miR-146a expression (P=.0007). With respect
to our results regarding cytotoxicity and cellular accumulation of
ubiquinol-10, THP-1 cells were preincubated with 10 μM ubiquinol-10 for 24 hours
as an effective dose. This is nearly in accordance to other in vitro studies performed with CoQ10 [15, 16] and is only 2-fold
higher than CoQ10 serum levels after supplementation in humans [17]. Thereafter, cells
were stimulated with 1 μg/mL LPS for 4 hours and the resulting steady-state
expression level of miR-146a was determined. As shown in Figure 2, preincubation of
THP-1 cells with ubiquinol-10 reduced the LPS-induced expression level of miR-146a to 78.9±13.22%. Although these effects were not statistically
significant (P≤.05), the reduced expression levels of miR-146a in
the ubiquinol-10-pretreated cells (QH2 + LPS) might be a first hint
for a fine-tuning mechanism of QH2 on miR-146a expression when compared to the high significant induction levels in control cells after
LPS-stimulation (Veco LPS).Figure 2
Effect of ubiquinol-10 on LPS-induced miR-146a expression in THP-1
cells. THP-1-cells were preincubated
without (−) or with (+) 10μM ubiquinol-10 for 24 hours. Afterwards, cells were
treated without (−) or with (+) 1 μg LPS/mL for 4 hours. After this treatment,
total RNA was extracted, converted to cDNA and miRs were assayed by
TaqMan-based qRT-PCR. Observed expression levels of miRs in the respective
treated cells were normalized to the corresponding levels of the endogenous
control (snoRNA202). Data (two-sided t-test)
are means ± SEM of three biological experiments performed in quadruplicate. P*<.05 versus  unstimulated cells.
P***<.001 versus unstimulated cells.


## 3.3. Accumulation of CoQ10 Levels in the Liver of Ubiquinol-10 Supplemented Mice

To test putative
effects of CoQ10 on miRNA 146a regulation in a more physiological
manner, liver tissues of QH2-supplemented C57BL6/J mice were used.
However, to mediate these effects, a tissue-specific accumulation of CoQ10 is essential. Therefore, total CoQ10 levels were determined in liver
homogenate samples of QH2-supplemented and control mice. As shown in
Figure 3(a), total CoQ10 levels increased about 12-fold (P=.0193) in
liver tissues of QH2-supplemented mice when related to control
samples. Because CoQ9 is the predominant CoQ form in rodents [18], CoQ9 was used as an internal standard for HPLC-analysis. Thus, the CoQ9 level was not significantly changed between treatment and control group (P=.51, data not shown). Accordingly, the CoQ9/CoQ10 ratio
was significantly different between groups, corresponding to a 6.5-fold
increase in control tissue samples (P=.0019, Figure 3(b)). In general, all HPLC measurements
were related to protein levels in the respective tissue homogenate samples. In
summary, we could significantly increase the CoQ10 concentration in liver
tissues of ubiquinol-10-supplemented mice. An effective uptake of exogenously
applied CoQ10 in liver tissues of rodents has been already described
earlier [19].Effect of ubiquinol-10 concentration (a) on CoQ10 levels and (b) the CoQ9/CoQ10 ratio in liver tissues
of mice.  Mice were fed with ubiquinol-10 (250 mg/kg/d) or a respective control diet (no ubiquinol-10 supplement) for 7 days
followed by a sublethal intraperitoneal injection with saline (0.9% NaCl, LPS
negative control) for further 4 hours. Thereafter,
liver samples were collected, homogenized, and used for HPLC analysis with
electrochemical detection.  CoQ9 was used as an internal standard. Observed CoQ levels were related to the corresponding
protein levels of the respective liver homogenate samples (single data not
shown). All data (one-sided t-test) are
means ± SEM of 5 animals in the intervention group (+QH2) and
control group (−QH2), respectively.  Protein-related CoQ10 levels and
CoQ9/CoQ10 ratio of ubiquinol-10-supplemented and control animals are depicted in Figures 3(a) and 3(b), respectively.
(a)(b)


## 3.4. Reduced Expression Levels of miR-146a in Liver Tissues of Ubiquinol-10-Supplemented Mice

To test the
relevance of a putative ubiquinol-10-dependent downregulation of LPS-inducible
miR-146a expression in vivo, we used
mice receiving either a diet enriched with ubiquinol-10 (QH2, 250 mg/kg/d) or a respective control diet for seven days. This dosage is in
accordance with previous CoQ10 studies in mice [20–22]. Thereafter, an
intraperitoneal injection of lipopolysaccharide (LPS, 1 mg/kg BW) was administered
for further 4 hours. In contrast to the control non-LPS-injected mice (no detectable TNF-α levels), TNF-α levels
increased significantly in the serum of LPS-treated animals (612.46 ± SD).
Finally, mice were sacrificed and livers were collected for miR isolation and
determination of miRNA-146a expression levels. As shown in Figure 4(a), the
LPS-induced expression level of miR-146a is generally lower in animals
supplemented with ubiquinol-10. In average, ubiquinol-10 reduces the
LPS-induced miR-146a expression to 78.12±21.25% (+QH2/+LPS) when compared to control animals (−QH2/+LPS) (Figure 4(b)).
This effect was statistically not significant but was consistent to those
obtained in cell culture experiments.Effect of ubiquinol-10 supplementation on miR-146a
expression in liver samples of LPS-treated mice. Mice were fed with ubiquinol-10 (250 mg/kg/d) or a respective control diet (no ubiquinol-10 supplement) for 7 days. Afterwards, a sublethal intraperitoneal injection of LPS (1 mg/kg BW)
was administered for further 4 hours Thereafter, liver samples were collected,
homogenized, and total RNAs including miRs were isolated as described by the manufacturer's
instructions. Subsequently, miRs were converted to cDNA and assayed by the
TaqMan-based qRT-PCR. Observed miR-146a expression levels of each animal were
related to the corresponding levels of the endogenous control (snoRNA202). Data
(two-sidedt-test) are means ± SEM of 6 animals in the intervention group (+QH2/+LPS) and control group (+LPS), respectively. miR-146a expression levels are
depicted as single bars of each animal (Figure 4(a)) or as total mean ± SEM of
each group (Figure 4(b)).
(a)(b)Regulation of miRs by certain
compounds or nutrients is of general interest, because this class of noncoding
RNAs is involved in central biological processes such as development,
inflammation and innate immunity, and signalling networks [1, 23]. So far, only a small number of environmental modulators of miR
expression have been identified. Expression levels of several miRs (i.e.,
miR-15a/b, miR-16, miR-107) are regulated by retinoic acid-induced
differentiation in human acute promyelocytic leukemia patients and cell lines [24]. Compounds such as sulphate, phosphate, and amino acids regulate the
expression of special miRs in plants [25] and human liver cells [26]. Toll-like receptor (TLR) ligands such as LPS induce the expression of
miR-146a significantly in human monocytes/macrophages [7].


## 4. Conclusion

Here we identified ubiquinol-10 as a
putative modulator of miR-146a expression. Preincubation of THP-1 cells with
ubiquinol-10 reduced the LPS-induced expression level of miR-146a. These
results are consistent to our in vivo data, where the expression of miR-146a was reduced in liver samples of mice
supplemented with ubiquinol-10 before LPS-injection (+QH2/+LPS)
when compared to control animals (+LPS). Although the observed effects are
statistically not significant, we postulated a fine-tuning mechanism of
ubiquinol-10 on the inflammatory response via a moderate reduction of miR-146a
expression. According to the superior function of miR-146a in the inflammatory
response, the observed moderate reduction of its expression by ubiquinol-10
seems to be desirable based on the following mechanisms. First, the LPS induced
upregulation of miR-146a in human monocytes/macrophages functions as a
negative regulator of the innate immune response because miR-146a targets
TRAF6, a regulator protein within the TLR-signalling pathways involved in the
formation and accumulation of reactive oxygen species [7]. Second, we have recently shown that ubiquinol-10
lowers the LPS-stimulated release of some proinflammatory cytokines and
chemokines relevant in inflammatory processes [27]. The observed effects were comparable to those of the potent and
characterized antioxidants N-acetyl-cysteine (NAC) or pyrrolidine-dithiocarbamate
(PDTC) [12, 14, 27].  Moreover,
mir-146a has been shown to be induced by proinflammatory cytokines such as
TNF-α, interleukin 1-beta (IL-1β), and TLRs [7, 28, 29]. miR-146a was also detected in tissues related to
inflammatory diseases including, for example, synovial fibroblasts and rheumatoid
synovial tissue [28]. Thus, we conclude that ubiquinol-10 reduces both the secretion of
proinflammatory agents and the expression of the anti-inflammatory miR-146a.
As a consequence, ubiquinol-10 acts as an anti-inflammatory compound but
perpetuates the essential inflammatory response via moderate reduction of
miR-146a expression. This dual effect could be due to the radical scavenging
activity of ubiquinol-10 since reactive oxygen species are involved in the
TLR-signalling pathways.In conclusion, the consistent in vitro and in vivo data suggest that ubiquinol-10 may fine-tune the
inflammatory response via moderate reduction of miR-146a expression.



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*Source: 415437-2009-04-16.xml*