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\begin{document}

\title{The effects of astaxanthin supplementation on the expression of miR-146a
and miR-126, oxidative stress and inflammation in patients with type 2
diabetes mellitus: A randomized double blind placebo controlled clinical
trial}



\author[1]{Amirhossein farokhzad}%
\author[2]{Nafiseh  Sokri Mashhadi}%
\author[3]{Maryam  Tahmasebi}%
\author[3]{Javad  Mohammadi-asl}%
\author[3]{Mehrnoosh  Zakerkish}%
\author[3]{Majid  Mohammadshahi}%
\affil[1]{Affiliation not available}%
\affil[2]{Isfahan University of Medical Sciences}%
\affil[3]{Ahvaz Jondishapour University of Medical Sciences}%


\vspace{-1em}



  
  \date{June 18, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: The pathogenesis of type 2 diabetes (T2D) is associated with
chronic oxidative stress and inflammation. It is well known that the
expression of some miRNAs such as miRNA-146a up- regulate in diabetic
and hyperglycemic patients whereas, circulating miRNA-126 reduces.
Therefore, the purpose of this study was to determinate the effects of
Astaxanthin supplementation on the expression of miR-146a and miR-126,
MDA and inflammation in patients with type 2 diabetes. Methods: This
randomized double-blind placebo-controlled clinical trial was conducted
on 44 type 2 diabetes patients who were randomly assigned to received
8mg of oral Astaxanthin/d (n=22) or placebo(n=22) for 8 weeks. Results:
We observed that Astaxanthin supplementation could decrease plasma
levels of MDA and IL-6 (P \textless{} 0.05) and declined the expression
level of miR-146a over time (Fold change: -1/388) (P \textless{} 0.05).
Conclusion: Astaxanthin supplementation might be beneficial for improving
circulating MDA and IL-6 and down regulation of miR-146a. However,
future investigations are suggested to confirm these results.%
\end{abstract}%



\sloppy


\textbf{What's known?}

Some previous studies showed the antioxidant and antinflamatory effects
of astaxanthin.

There have been no human studies to investigate the effect of
astaxanthin or its constituents on the expression of miR-146a and
miR-126, oxidative stress and inflammation in patients with type 2
diabetes mellitus.

\textbf{What new}

This study is the first human investigation that was designed to
evaluate the effect of astaxanthin or its constituents on the expression
of miR-146a and miR-126, oxidative stress and inflammation in patients
with type 2 diabetes mellitus.

The present study suggests that astaxanthin supplementation might be
beneficial for improving circulating MDA and IL-6 and down regulation of
miR-146a.

\textbf{Introduction}

Type 2 diabetes mellitus (T2DM) is a metabolic diseases recognized by
hyperglycemia due to insulin resistance and insufficient insulin
secretion \textsuperscript{1}. Growing evidence indicated that chronic
oxidative stress and inflammation closely involved in the pathogenesis
of T2DM and the progression of related
complications\textsuperscript{2,3}. Systemic inflammation can affect
\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}cell functionality and promote mitochondrial dysfunction that results
in precedes T2D manifestations and predicts its
progression\textsuperscript{4}.

MicroRNAs (miRNAs) are small noncoding RNA which are usually transcribed
by RNA polymerase II \textsuperscript{5}. They modulate the gene
expression level through their interaction by 3'UTR of the genes which
is eventually lead to RNA degradation \textsuperscript{6}. The miRNAs
expression is tissue-specific, but they may be observed in the
circulation \textsuperscript{7}. Previous studies detected the up
regulation of some miRNAs such as miRNA-146a in diabetic and
hyperglycemic patients whereas, circulating miRNA-126 are
reduced\textsuperscript{8,9}. In addition, participating of MicroRNAs in
T2D pathogenesis is indicated through the decline of insulin release and
an increase of pancreatic \selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}cell apoptosis \textsuperscript{10}. So,
from a clinical perspective, the levels of miR-26a and miR-146a can be
evaluated in the circulation at different disease stages related to
T2D\textsuperscript{11}.

Recently, there has been significant interest in the potential role of
food antioxidants in controlling lipid peroxidation, diabetes-induced
inflammation and modulating microRNAs \textsuperscript{12,13}.
Astaxanthin (AST) is a carotenoid belongs to the xanthophyll family and
common pigment in algae and fish \textsuperscript{14}. Recent studies
demonstrated the antioxidant effect of Astaxanthin on in the treatment
and prevention of diabetes \textsuperscript{15}. However, few studies
assessed the efficacy~of AST on circulating miRNAs, and reducing
inflammation and oxidative stress in diabetes patients. Therefore, this
study was aimed to determinate the effects of AST supplementation on the
expression of miR\emph{-146a} and miR\emph{-126} , MDA and inflammation
in patients with type 2 diabetes.

\textbf{Materials and Methods}

\emph{Population and Study design}

In the double-blind, randomized placebo-controlled trial manner,
Forty-four volunteers with type 2 diabetes were joined to this study.
These individuals referred to the Diabetes Research Center at the Ahvaz
University of Medical Sciences and met our inclusion criteria including
30-60 years old subjects, definitive diagnosis of type II diabetes with
no insulin therapy, no pregnancy and lactation, no self-reported
specific diseases and malignancies, no kidney failure, no heart disease,
no thyroid and other inflammatory disease, no vitamin and antioxidant
supplement during the last 6 months, and no smoking and drinking. The
written consent was obtained from each individual before joining to the
project.

All processes were performed under the supervision of a nutritionist at
each step, during which the participants were counseled to eat an exact
diet, perform physical activity, and take blood glucose lowering drugs.
Qualified researchers performed all the data collection and
measurements. Basal characteristics were collected by the interviewers
which is presented before for subjects (\emph{n} = 22) or placebo
(\emph{n} = 22) for 8 weeks. In a double-blinded fashion, subjects
received 8mg of oral Astaxanthin/d (nature vision; USA) which was
derived from Haematococcus pluvialis (microalgae) and inactive
ingredients which were used for both supplement and placebo were
Dicalcium Phosphate, Microcrytalline Cellulose, Stearic Acid, Silicon
Dioxide, Magnesium Stearate. Food consumption was recorded before,
during and after the study period (3-day food records), according to
volume or weight and type of food. To assess energy and macronutrient
intake, the dietary data were analyzed using Nutritionist IV software
(Version 4.1, First Databank Division, The Hearst Corporation, and San
Bruno, CA, USA). During each 2-week appointment, the participants'
compliance to the study protocol was assessed by numeration the residual
AST tablets, and asking them to preserve their habitual diet, lifestyle,
and medication ingestion.

\emph{Blood Sampling and Primary Measures}

Venous blood samples were collected in the fasting state for biochemical
measurements. Samples were centrifuged and serums were frozen and stored
at -80 \selectlanguage{ngerman}°C. The serum level of IL-6 was determined using the method
described by the \emph{Boster} human IL-6 ELISA kit. Malondialdehyde
(MDA) level was measured as an index of lipid peroxidation using TBRAS
assay protocol. The MDA reacts with thiobarbituric acid (TBA) as a
thiobarbituric acid reactive substance (TBARS) to produce a red colored
complex. The absorbance of the samples was measured at 532nm using ELISA
plate reader. 3 ml phosphoric acid (1\%) and 1 ml TBA (0.6\%) was added
to 0.5 ml of homogenate in a centrifuge tube and the mixture was heated
for 45 min in a boiling water bath. After cooling, 4 ml of n-butanol was
added the mixture and vortex-mixed for 1 min followed by centrifugation
at 20000 rpm for 20 min. The organic layer was transferred to a fresh
tube and its absorbance was measured at 532 nm \textsuperscript{16}.

\emph{RNA extraction and MicroRNA analysis}

Out of 44 participants, 22 participants from the active treatment group,
and 21 subjects from the placebo group were randomized and evaluated
regarding the microRNA. The microRNAs were analyzed at the study start
and at the end of study in both the intervention and the placebo groups.
The vials were retained on ice before chilled centrifugation at 3000g
and then frozen at -70°C, and no sample was melted more than twice.

Total RNA was extracted from 100\selectlanguage{greek}μ\selectlanguage{english}L of serum sample using RNX-Plus
reagent (Cinnagen, Iran) followed by DNase I digestion (Takara Bio Inc,
Shiga, Japan) according to the user manual. The concentration and purity
of extracted RNAs was evaluated by Nano drop ND-1000 spectrophotometer
(Nano drop Technologies, Wilmington, DE, USA) reading at wave length
230, 260 and 280 nm. The RNA samples were liquated in RNase free tubes
and stored at -70\selectlanguage{ngerman}°C before use.

Complementary DNA (cDNA) was synthesized by Prime Script\selectlanguage{english} RT reagent kit
(Takara Bio Inc, Shiga, Japan).~Based on manufacturer protocol.
Real-time PCR was performed using the SYBR(r) Premix Ex Taq\selectlanguage{english} II (Takara)
and relative gene expression was calculated as 2\selectlanguage{english}\textsuperscript{-ΔΔCt}.
The list of primers for specific genes has been illustrated in Table4.
The miRNA Snordu6 was used as an internal control.

\emph{Statistical analysis}

The results are presented as a mean \selectlanguage{ngerman}± standard error for continuous
variables, and as numbers (percentages) for categorical variables. A
one-way multivariate analysis of covariance was used to control the
pretest differences, followed by Dunnett's post hoc comparison for
multiple comparisons between the groups and paired-sample student t
tests for comparisons within each group. Finally, the data were tested
for normality using the Kolmogorov--Smirnov test. Because of the
non-normality of the studied variables (positively skewed distribution),
logarithmic transformation was performed and homogeneity of the
covariance matrix was examined using Box's M statistics. Levels of
relative expression of the miRNAs were evaluated using 2\selectlanguage{english}-\selectlanguage{greek}ΔΔ\selectlanguage{english}CT method.
The comparison between groups was carried out with the one-way ANOVA or
Kruskal-Wallis test was performed with a Turkey or Dunn's post-test
pairwise comparison. All statistical analyses were performed using SPSS,
(Version 20, SPSS Inc., Chicago, IL), and significance was set at P
\textless{} 0.05.

\textbf{Results}

\subsubsection*{\texorpdfstring{\emph{Clinical and anthropometric
characteristics of the study
population}}{Clinical and anthropometric characteristics of the study population}}

{\label{clinical-and-anthropometric-characteristics-of-the-study-population}}

Forty-four subjects with type 2 diabetes were enrolled in this study; 43
of them completed the 8 weeks' intervention. Exclusion from the study
were none-compliance of study. The baseline physical and medical
characterization of subjects are detailed in \textbf{Table1} . As
indicated, no statistically significant difference was observed in terms
of basic characteristics including age, gender, the body mass index
(BMI), diabetes duration, basal metabolic rate (BMR), total body fat,
visceral body fat, oral antidepressant drugs, and oral glucose lowering
drugs between the two 8mg ASX or placebo groups.

\emph{Dietary and Physical activity}

Dietary records were analyzed for total dietary energy, fat, protein,
carbohydrate, and micronutrient intake among the participants with type
2 diabetes mellitus before and after the intervention. The obtained data
showed that mentioned dietary parameters did not significantly change
during the intervention. Moreover, the best sources of natural AST are
algae, yeast, wild sockeye salmon, trout, krill, and shrimp and these
were not included in the daily diet of our participants. Thus, food
sources of AST were limited, and intake of other carotenoids did not
differ between the groups during the study \textsuperscript{17}. In
addition, no significant changes were detected in the participants'
physical activity through the intervention (\emph{P} = 0.062).

\subsubsection*{\texorpdfstring{\emph{Circulating miR-146a, miR-126, MDA
and
IL-6}}{Circulating miR-146a, miR-126, MDA and IL-6}}

{\label{circulating-mir146a-mir-126-mda-and-il-6}}

Mean plasma levels of MDA and IL-6, before and after intervention is
shown in~Table 2. There were significant decreases (paired~\emph{t}
-test) in MDA and IL-6 levels in AST group compared with the placebo
group (\emph{P} ~\textless{} 0.05). Although the comparison within the
groups indicated that AST could decrease MDA level and IL-6 values over
time (\emph{P} ~\textless{} 0.05).

On the other hand, following the 8-week administration of AST, we
observed that the expression level of miR-146-a significantly decreased
in comparison to the placebo group (Fold change: -1/388) (P-value
\textless{}0.05). Meanwhile the increased level of the miR-126 was
detected but was not statistically significant. The expression changes
of miR-126 and miR-146 in the AST group before and after intervention
was shown in \textbf{Table3.}

However, regarding placebo group, the mean expression of miR-126 was
significantly decreased (Target gene up-regulated by factor: -1.233).
But, circulating miR-146-a was increased in comparison to the expression
of the reference gene (Target gene up-regulated by factor: 1.185). The
expression changes of miR-126 and miR-146 in the placebo group before
and after intervention was shown in \textbf{Table3.}

\textbf{Discussion}

This study was conducted in order to determinate the effect of 8 mg/day
AST supplementation for eight weeks on the relative expression of
miR-146a and miR-126a, and also improvement of MDA and IL-6 levels.

We observed that 8 mg of the AST supplementation significantly reduced
the expression level of miR-146-a. Moreover, our results displayed that
inhibition of miR-146a expression associated with a moderated release of
IL-6 and MDA. However, the high expression level of miR-126 in
AST-supplemented patients was not significant. This specific effect of
AST on miR-146a expression behavior might be the consequence of a
pro-inflammatory component of the T2D. In this regard, the results from
the other studies showed that miR-146a relative expression was
associated with an inflammatory marker in the T2D
patients\textsuperscript{18}. Also, the modulatory effect of some
antioxidants on circulating MicroRNAs was examined in previous studies
and suggested to consider as a novel therapeutic target in disease
management in many literature\textsuperscript{19,20}. Morales et al,
stated the potential effect of polyphenols extracted from red wine as
antioxidant increased plasma MicroRNAs in a dose-dependent manner. Also,
the up regulation of MicroRNAs and attenuated the expression of
pro-inflammatory cytokines was confirmed by treating cells with mango
extracting rat\textsuperscript{21}.

{\label{we-observed-that-8-mg-of-the-ast-supplementation-significantly-reduced-the-expression-level-of-mir-146-a.-moreover-our-results-displayed-that-inhibition-of-mir146a-expression-associated-with-a-moderated-release-of-il-6-and-mda.-however-the-high-expression-level-of-mir-126-in-ast-supplemented-patients-was-not-significant.-this-specific-effect-of-ast-on-mir146a-expression-behavior-might-be-the-consequence-of-a-proinflammatory-component-of-the-t2d.-in-this-regard-the-results-from-the-other-studies-showed-that-mir146a-relative-expression-was-associated-with-an-inflammatory-marker-in-the-t2d-patients18.-also-the-modulatory-effect-of-some-antioxidants-on-circulating-micrornas-was-examined-in-previous-studies-and-suggested-to-consider-as-a-novel-therapeutic-target-in-disease-management-in-many-literature1920.-morales-et-al-stated-the-potential-effect-of-polyphenols-extracted-from-red-wine-as-antioxidant-increased-plasma-micrornas-in-a-dose-dependent-manner.-also-the-up-regulation-of-micrornas-and-attenuated-the-expression-of-pro-inflammatory-cytokines-was-confirmed-by-treating-cells-with-mango-extracting-rat21.}}

Moreover, in context to placebo group, we indicated that the expression
of miR-146a was augmented during the intervention and associated with
raised IL-6 and MDA which is consistent with previous
studies\textsuperscript{18}. Also in a study conducted
by\textsuperscript{22}, a positive correlation was observed between
miR-146a levels and concentration of inflammatory mediators, whereas,
elevated miR-146a inhibited the production of IL-6 and IL-21 in T cells.
These results in consistent with our obtained results. In addition,
several studies revealed that miR-146a, is overexpressed in type 2
diabetes patients~compared with healthy controls\textsuperscript{8,22},
which it described serum miRNAs as a explore DM biomarkers. Furthermore,
the results from the present study were in line with previous report and
show that high hyperglycemia significantly reduced miR-126 release, ~the
most abundant miRNAs in endothelial apoptotic bodies, from endothelial
cells\textsuperscript{23}. And loss of miR\emph{-} 126 has been proposed
as a predictor of type 2 diabetes onset\textsuperscript{24}.

However, this is the first study that assesses the effect of AST on
circulating miRNAs and we have a lack of experiments comparing the
potential effect of AST supplementation on diabetes patients with other
studies. In addition, short study duration may affect the process of
genetic outcome and we could not demonstrate antioxidant agents of AST
on modulating plasma miR-126 levels through reducing inflammatory
pathways. So further larger sample size studies are urgent to achieve
this goal and our findings should be considered preliminary and the
requirement for further studies is indicated.

\textbf{Conclusion}

In summary, our results introduced 8 mg/ day of AST supplementation for
8 weeks as a new agent for decreasing the circulating MDA, IL-6 and
relative expression of MiR-146a. However, further larger sample size
studies are needed to confirm these outcomes.

\textbf{Acknowledgment}

This study is part of the registered protocol in the Iranian Registry of
Clinical Trials (IRCT2015081623645N1) which was approved by the Research
Ethics Committee of Ahvaz University of Medical Sciences.

\textbf{Conflict of interest}

None of the authors has any potential financial conflict of interest
related to this manuscript.

\textbf{Funding source}

This research did not receive any specific grant from funding agencies
in the public, commercial, or not-for-profit sectors

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