nutrients

Article
Dietary Chrysin Suppresses Formation of Actin
Cytoskeleton and Focal Adhesion in AGE-Exposed
Mesangial Cells and Diabetic Kidney: Role
of Autophagy
Eun-Jung Lee, Min-Kyung Kang, Yun-Ho Kim, Dong Yeon Kim, Hyeongjoo Oh, Soo-Il Kim,
Su Yeon Oh and Young-Hee Kang *
Department of Food and Nutrition, Hallym University, Chuncheon, Kangwon-do 24252, Korea;
reydmswjd@naver.com (E.-J.L.); mitholy@hallym.ac.kr (M.-K.K.); royalskim@hallym.ac.kr (Y.-H.K.);
ehddus3290@naver.com (D.Y.K.); ohhyeongju@gmail.com (H.O.); ky4850@naver.com (S.-I.K.);
suy0411@naver.com (S.Y.O.)
* Correspondence: yhkang@hallym.ac.kr; Tel.: +82-33-248-2132; Fax: 82-33-254-1475




Received: 9 November 2018; Accepted: 4 January 2019; Published: 9 January 2019


Abstract: Advanced glycation end products (AGE) play a causative role in the development of aberrant
phenotypes of intraglomerular mesangial cells, contributing to acute/chronic glomerulonephritis.
The aim of this study was to explore mechanistic effects of the flavonoid chrysin present in bee
propolis and herbs on actin dynamics, focal adhesion, and the migration of AGE-exposed mesangial
cells. The in vitro study cultured human mesangial cells exposed to 33 mM glucose and 100 µg/mL
AGE-bovine serum albumin (AGE-BSA) for up to 5 days in the absence and presence of 1–20 µM
chrysin. The in vivo study employed db/db mice orally administrated for 10 weeks with 10 mg/kg
chrysin. The presence of ≥10 µM chrysin attenuated mesangial F-actin induction and bundle
formation enhanced by AGE. Chrysin reduced the mesangial induction of α-smooth muscle actin
(α-SMA) by glucose, and diminished the tissue α-SMA level in diabetic kidneys, indicating its
blockade of mesangial proliferation. The treatment of chrysin inhibited the activation of vinculin and
paxillin and the induction of cortactin, ARP2/3, fascin-1, and Ena/VASP-like protein in AGE-exposed
mesangial cells. Oral administration of chrysin diminished tissue levels of cortactin and fascin-1
elevated in diabetic mouse kidneys. Mesangial cell motility was enhanced by AGE, which was
markedly attenuated by adding chrysin to cells. On the other hand, chrysin dampened the induction
of autophagy-related genes of beclin-1, LC3 I/II, Atg3, and Atg7 in mesangial cells exposed to
AGE and in diabetic kidneys. Furthermore, chrysin reduced the mTOR activation in AGE-exposed
mesangial cells and diabetic kidneys. The induction of mesangial F-actin, cortactin, and fascin-1
by AGE was deterred by the inhibition of autophagy and mTOR. Thus, chrysin may encumber
diabetes-associated formation of actin bundling and focal adhesion and mesangial cell motility
through disturbing autophagy and mTOR pathway.

Keywords: actin cytoskeleton; advanced glycation end products; autophagy; chrysin; focal adhesion;
mesangial migration

1. Introduction
Mesangial cells possess irregular structures comprised of flattened cylinder-like cell bodies and
contain actin, myosin, and α-actinin at both ends, granting them contractile features [1]. The anchoring
filaments from mesangial cells to the glomerular basement membrane can influence glomerular
capillary blood flow [2,3]. The structural support of mesangial cells in kidney glomeruli counteracts the

Nutrients 2019, 11, 127; doi:10.3390/nu11010127 www.mdpi.com/journal/nutrients

Nutrients 2019, 11, 127 2 of 15

expansible forces created by pressure gradients in capillary vessels and manipulates the capillary blood
flow [4]. Glomerular mesangial cells migrate in response to platelet-derived growth factor (PDGF)
and angiotensin II, which is crucial for glomerulopathy and glomerular development [5,6]. There is
emerging evidence for the role of the abnormal migratory polarity of mesangial cells due to glomerular
injury in the pathogenesis of proliferative glomerulonephritis [4,7,8]. Glomerular injury-triggered
molecular changes in mesangial cells in glomerulonephritis still remain elusive. In general, directional
cell migration entails the establishment of cytoskeletal alterations that are essential for actin polarization
and focal adhesion turnover [9,10]. In the same context, the migratory process is controlled by
coordinated actin dynamics and focal adhesion turnover at the peripheral ruffles in migrating
mesangial cells [6]. However, identifying the key molecular players in motility and clear-cut molecular
functions remains challenging.
Glucose toxicity is a primary cause of glomerular injury in diabetic kidney diseases, and involves
the production of advanced glycation end products (AGE) [11]. Increased glucose levels result in
glomerular oxidative stress via activation of metabolic pathways, which consequently produces
and accumulates AGE, leading to glomerular injury [12,13]. The activation of the AGE-receptor for
advanced glycation end products (RAGE) influences endothelial actin cytoskeletal rearrangement and
dysfunction [14,15]. A recent report shows that AGE-induced oxidative stress stimulates proliferation
and migration of vascular smooth muscle cells [16]. Dynamics of focal adhesions containing vinculin
and paxillin is entailed for cell polarization and motility, and extracellular matrix remodeling [17,18].
Cell migration entails coordination between focal adhesion and actin cytoskeleton via F-actin-binding
vinculin abundant in integrin-based focal adhesions [17]. Exposure of retinal pericytes to AGE induces
cell migration via phosphorylation of focal adhesion kinase and paxillin [19]. Connective tissue growth
factor prompts mesangial cell migration via disassembly of focal adhesion complexes and activation of
cell polarization [20]. On the other hand, autophagy is responsible for the degradation of AGE by the
upregulation of lysosomal biogenesis and function in diabetic nephropathy (DN) [21]. Accordingly,
strategies aimed at enhancing the lysosomal function of autophagy-related proteins can hold promise
for treating DN. One study shows that autophagy is likely to be a mechanism triggered to repair the
reactive oxygen species-induced loss of the AGE-treated cells and thereby prompts cell survival [22].
Numerous studies have demonstrated that natural compounds display renoprotective effects by
diverse mechanisms [23–25]. Polyphenolic flavonoids are known to attenuate hyperglycemia-induced
renal endothelial barrier dysfunction, urinary albumin excretion, and glomerular hyperfiltration [23].
Our previous studies showed that several natural compounds counteracted renal mesangial fibrosis
and inflammation, renal tubulointerstitial fibrosis, glomerulosclerosis, and podocyte injury [26–28].
Chrysin (5,7-dihydroxyflavone, Figure 1A), a flavonoid abundant in edible plants such as passion
flowers, mushrooms, honey, and bee propolis, is known to exhibit multiple biological effects, including
anti-inflammatory, anti-atherogenic, and neuroprotective properties [29–31]. In addition, growing
evidence suggests that chrysin may display nephroprotective activities in rodents [32,33]. Thus,
the present study attempted to explore the renoprotective effects of chrysin on the formation of
F-actin cytoskeleton and focal adhesion complex in AGE-exposed mesangial cells and diabetic mouse
kidneys. Our recent study demonstrates that chrysin may inhibit glucose-mediated AGE-associated
glomerulosclerosis and fibrosis [34]. However, little is known about the renoprotective role of
chrysin in actin cytoskeleton rearrangement, focal adhesion formation, and cell migration due to
AGE. One investigation shows that the flavonoid myricetin suppresses retinal pericytes migration
through suppressing activation of ERK1/2-focal adhesion kinase-paxillin by AGE [19]. Whether the
presence of chrysin modulated mesangial cell proliferation and motility by AGE was also examined.
This study further elucidated how chrysin manipulated autophagy as a molecular player involved in
the actin cytoskeleton, focal adhesion assembly, and motility.
Nutrients 2019, 11, 127 3 of 15

2. Materials and Methods

2.1. Chemicals
Fetal bovine serum (FBS), penicillin–streptomycin and trypsin–EDTA, were provided by
BioWhittaker (San Diego, CA, USA). 3-(4, 5-Dimetylthiazol-yl)-diphenyl tetrazolium bromide (MTT)
was obtained from DUCHEFA Biochemie (Haarlem, The Netherlands). Dulbecco’s modified Eagle
media (DMEM), nutrient mixture F-12 Ham medium, mannitol, and D-glucose, were supplied
by Sigma-Aldrich Chemical (St. Louis, MO, USA), as were all other reagents unless specifically
stated otherwise. Antibodies of F-actin, α-smooth muscle actin (α-SMA), Arp2/3 antibody, mTOR,
and phospho-mTOR were supplied by Abcam (Cambridge, UK). Antibodies of beclin-1, cortactin,
Fascin1, and Ena/VASP (EVL) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).
Phospho-vinculin antibody was obtained from Biorbyt (Cambridge, UK). Atg7 antibody was purchased
from Aviva system Biology (San Diego, CA, USA). Antibodies of Atg3 and phospho-paxillin were
obtained from Cell Signaling Technology (Beverly, CA, USA). LC3 antibody was supplied by MBL
International Corporation (Woburn, MA, USA). AGE-bovine serum albumin (AGE-BSA) was provided
by Merck Millipore (Billerica, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit
IgG, goat anti-mouse, and donkey anti-goat IgG were obtained from Jackson ImmunoResearch
Laboratories (West Grove, PA, USA). SB03580 (MAP kinase inhibitor) was provided from Calbiochem
(Billerica, MA, USA)
Chrysin (Sigma-Aldrich Chemical, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide
(DMSO) for live culture with cells; a final culture concentration of DMSO was <0.5%.

2.2. Culture of Human Renal Mesangial Cells (HRMC)

HRMC (Sciencell Research Laboratories, Carlsbad, CA, USA) were cultured at 37 ◦ C humidified
atmosphere of 5% CO2 in air. Routine culture of HRMC was performed in DMEM/F12 (7:1) media
containing 15% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. HRMC
in 6-10th passage were sub-cultured at 80% confluence and used for further experiments. To mimic
diabetic glomerular injury caused by chronic hyperglycemia, HRMC was incubated in 33 mM glucose-
or 100 µg/mL AGE-BSA-supplemented DMEM containing 2% FBS and 2–8 µg/mL insulin for 3 days
in the absence and presence of 1–20 µM chrysin. For osmotic control incubation, another set of HRMC
was cultured in DMEM (5.5 mM) containing 2% FBS (+2 µg/mL insulin) and supplemented with
27.5 mM mannitol. Culture media was collected and stored at −20 ◦ C.
After the 3-day incubation in 33 mM glucose and 100 µg/mL AGE-BSA, the MTT assay was
routinely carried out for measuring cell proliferation. After the unconverted MTT was removed, cells
were dissolved in isopropanol with gentle shaking. The absorbance of formazan dye was measured
at λ = 570 nm with background subtraction using λ = 690 nm. There was no cytotoxicity of 1–20 µM
chrysin per se observed [34].

2.3. In Vivo Animal Experiments

Adult male db/db mice (C57BLKS/+Leprdb Iar; Jackson Laboratory, Sacramento, CA, USA) and
their age-matched non-diabetic db/m littermates (C57BLKS/J; Jackson Laboratory, Sacramento, CA,
USA) were used in the present study. Mice were kept on a 12 h light/12 h dark cycle at 23 ± 1 ◦ C
with 50 ± 10% relative humidity under specific pathogen-free conditions, fed a standard laboratory
chow diet (CJ Feed, Seoul, Korea), and were provided with water ad libitum at the animal facility of
Hallym University. This study included db/db mice at 7 weeks of age because they begin to develop
diabetes (hyperglycemia) at the age of 7–8 weeks. The animals were allowed to acclimatize for a
week before beginning the experiments. Mice were divided into three subgroups (n = 7–9 for each
subgroup). The first group of mice was non-diabetic db/m control mice and db/db mice were divided
into two groups. One group of db/db mice was daily supplemented 10 mg/kg BW chrysin via gavage
for 10 weeks. Food intake, body weight, and drinking water intake of db/db mice increased, relative to
Nutrients 2019, 11, 127 4 of 15

those of db/m controls. However, the supplementation of chrysin led to a decline in water drinking after
the 6–7th week. The measurement of fasting blood levels of glucose and glycated hemoglobin HbA1C
were conducted every other week from mouse tail veins. Chrysin treatment diminished plasma levels
of glucose and HbA1C elevated in db/db mice. The 24 h urine samples were collected in metabolic
cages during the 10-week chrysin supplementation. The urine volume was reduced in chrysin-treated
mice by ~50–60%, and diabetic proteinuria was alleviated.
All experiments were approved by the Committee on Animal Experimentation of Hallym
University and performed in compliance with the University’s Guidelines for the Care and Use
of Laboratory Animals (hallymR1 2016-10). No mice died and no apparent signs of exhaustion were
observed during the experimental period.

2.4. Western Blot Analysis

Western blot analysis was conducted using whole-cell lysates and tissue extracts prepared from
HRMC at a density of 3.0 × 105 cells. Whole-cell lysates and mouse renal tissue extracts were
prepared in a lysis buffer containing 1 M β-glycerophosphate, 1% β-mercaptoethanol, 0.5 M NaF, 0.1 M
Na3 VO4 , and protease inhibitor cocktail. Cell lysates and tissue extracts containing equal amounts
of total proteins were electrophoresed on 6–15% SDS-PAGE and transferred onto a nitrocellulose
membrane. Non-specific binding was blocked by soaking the membrane in a TBS-T buffer (50 mM
Tris–HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20) supplemented 3% BSA for 3 h. The membrane
was incubated with an antibody to F-actin, α-SMA, phospho-paxillin, phospho-vinculin, cortactin,
Arp2/3, fascin-1, EVL, beclin-1, LC3, Atg3, Atg7, mTOR, or phospho-mTOR. The membrane was
then incubated with a secondary antibody of goat anti-rabbit IgG, goat anti-mouse IgG, or donkey
anti-goat IgG conjugated to HRP. Each protein level was determined by using Supersignal West Pico
Chemiluminescence detection reagents (Pierce Biotechnology, Rockford, IL, USA) and Konica X-ray
film (Konica Co., Tokyo, Japan). Incubation with mouse anti-human β-actin was conducted for the
comparative control.

2.5. Rhodamine-Phalloidin Staining of F-Actin

HRMC (7 × 104 cells) grown on 24 well glass chamber slides were exposed to 33 mM glucose
or 100 µg/mL AGE-BSA in the absence and presence of 1–20 µM chrysin. Cells were fixed in 4%
formaldehyde for 10 min and washed with pre-warmed phosphate-buffered saline (PBS). Subsequently,
10 units of the fluorescent dye rhodamine phalloidin were added to cells and incubated for 20 min.
Nuclear staining was also conducted by using 4 mg/mL 4’,6-diamidino-2-phenylindole (DAPI).
Each slide was mounted in VectaMount mounting medium (Vector Laboratories, Burlingame, CA,
USA). Fluorescent images were taken with an Axiomager Optical fluorescence microscope (Zeiss,
Oberkochen, Germany).

2.6. Mesangial Cell Motility

To assess the effects of glucose and AGE on mesangial cell motility in the absence and presence
of chrysin, an in vitro scratch wound assay was employed. Briefly, mesangial cells were seeded onto
a 12-well plate and incubated for 24 h in 10% FBS-containing media. Subsequently, confluent cells
were scratched away horizontally on each well using a pipette tip. After scratching, injured cells were
incubated for another 24 h in serum-free culture media containing 100 µg/mL AGE-BSA with and
without 1–20 µM chrysin. Images of the scratches were photographed in the 2–3 microscopic fields per
well using a microscope with CCD camera (Motic® , Wetzlar, Germany). A reduction of the scratched
area indicates a sign of mesangial cell migration.

2.7. Immunocytochemical Staining

Immunofluorescent cytochemical staining was conducted to reveal the co-localization of Atg7 to
F-actin fibers in mesangial cells grown on 24-well chamber slides. Cells were fixed with 4% formaldehyde
Nutrients 2019, 11, 127 5 of 15

for 20 min and permeated with 0.1% Triton X-100 for 10 min on ice. Cells were specifically blocked with
20% FBS for 1 h, and a primary antibody of Atg7 and a secondary antibody of FITC-conjugated IgG
were applied to cells. Subsequently, the fluorescent dye rhodamine phalloidin was added to cells,
and incubated for 20 min for F-actin staining. Nuclear counterstaining was performed with DAPI.
EachNutrients
slide was
2019,mounted
11, 127 in a mounting medium and images of each slide were taken using5 an optical
of 15

Axiomager microscope (Zeiss, Oberkochen, Germany).

was performed with DAPI. Each slide was mounted in a mounting medium and images of each slide
wereAnalysis
2.8. Data taken using an optical Axiomager microscope (Zeiss, Oberkochen, Germany).

TheData
2.8. results are presented as mean ± SEM for each treatment group. Statistical analyses were
Analysis
performed using Statistical
The results Analysis
are presented Systems
as mean statistical
± SEM for eachsoftware
treatmentpackage version 6.12
group. Statistical (SASwere
analyses Institute
Inc., performed
Cary, NC, using
USA).Statistical
Significance
Analysis Systems statistical software package version 6.12 (SAS Instituterange
was determined by one-way ANOVA, followed by Duncan
test for multiple
Inc., comparisons.
Cary, NC, Differences
USA). Significance were considered
was determined by one-waysignificant
ANOVA,at p < 0.05.
followed by Duncan range
test for multiple comparisons. Differences were considered significant at p < 0.05.
3. Results
3. Results
3.1. Effect of Chrysin on the Induction of Cytoskeletal Actin Proteins
3.1. Effect of Chrysin on the Induction of Cytoskeletal Actin Proteins
This study attempted to investigate if chrysin retarded diabetes-induced mesangial actin
This study
polymerization attemptedWhen
in kidneys. to investigate if chrysin
33 mM glucose or retarded
100 µg/mL diabetes-induced
AGE-BSA was mesangial actin
supplemented to
mesangial cells in culture, the F-actin induction was elevated (Figure 1B). However, the elevated to
polymerization in kidneys. When 33 mM glucose or 100 μg/mL AGE-BSA was supplemented F-actin
mesangial cells in culture, the F-actin induction was elevated (Figure 1B). However, the elevated
induction was attenuated in the presence of ≥10 µM chrysin. In addition, high glucose and AGE
F-actin induction was attenuated in the presence of ≥10 μM chrysin. In addition, high glucose and
enhanced the F-actin formation, as evidenced by visualization with rhodamine phalloidin (Figure 1C).
AGE enhanced the F-actin formation, as evidenced by visualization with rhodamine phalloidin
In contrast, the In
(Figure 1C). reddish staining
contrast, of AGE-exposed
the reddish mesangialmesangial
staining of AGE-exposed cells wascells
diminished by the
was diminished bypresence
the
of chrysin.
presence of chrysin.

FigureFigure 1. Chemical
1. Chemical structure
structure of chrysin
of chrysin (A), inhibition
(A), and and inhibition of F-actin
of F-actin induction
induction by chrysin
by chrysin in
in advanced
advanced glycation end products (AGE)-bovine serum albumin (BSA)-exposed human renal
glycation end products (AGE)-bovine serum albumin (BSA)-exposed human renal mesangial cells
mesangial cells (HRMC, B and C). HRMC were challenged with 5.5 mM glucose, 5.5 mM glucose
(HRMC, B and C). HRMC were challenged with 5.5 mM glucose, 5.5 mM glucose plus 27.5 mM
plus 27.5 mM mannitol as osmotic controls or with 33 mM glucose or 100 μg/mL AGE-BSA in the
mannitol as osmotic controls or with 33 mM glucose or 100 µg/mL AGE-BSA in the absence and
absence and presence of 1–20 μM chrysin. The F-actin induction was measured by Western blot
presence of 1–20
analysis usingµM cell chrysin.
lysates withThea F-actin
primary induction
antibody ofwas measured
F-actin by Western
(B). β-Actin blotused
protein was analysis
as anusing
cell lysates
internalwith a primary
control. antibody
Representative blotsofshown
F-actinare(B). β-Actin
typical protein
of three was used
independent as an internal
experiments. control.
The bar
Representative
graphs (mean blots shown
± SEM) in theare typical
right panel of three independent
represent experiments.
quantitative results obtained fromTheabar graphs (mean
densitometer.
± SEM) in the
Values right panel
not sharing represent
a letter quantitative
are different results
at p < 0.05. obtained from
Red-rhodamine a densitometer.
phalloidin Values not
staining for F-actin
formation
sharing was
a letter areconducted
differentinatAGE-BSA-exposed HRMC (C).phalloidin
p < 0.05. Red-rhodamine Nuclear counter-staining was done
staining for F-actin by
formation
using blue 4’,6-diamidino-2-phenylindole. Scale bar = 50 μm. Each photograph
was conducted in AGE-BSA-exposed HRMC (C). Nuclear counter-staining was done by using is representative of at blue
least four animals. Magnification: 200-fold.
4’,6-diamidino-2-phenylindole. Scale bar = 50 µm. Each photograph is representative of at least four
animals. Magnification: 200-fold.
The temporal induction of the filamentous F-actin and the contractile α-SMA was examined in
mesangial
The temporal cells induction
treated with 33 mM
of the glucose for
filamentous 5 days.
F-actin andAsthe
expected, the mesangial
contractile α-SMA was proteins of
examined in
F-actin and α-SMA were highly induced by the 3-day culture of 33 mM glucose
mesangial cells treated with 33 mM glucose for 5 days. As expected, the mesangial proteins of F-actin (Figure 2A). In
addition, high glucose promoted the formation of F-actin fibers in mesangial cells, which was
reduced by 1–20 μM chrysin (Figure 2B). Similarly, the α-SMA level was enhanced in diabetic
kidneys, indicating mesangial cell proliferation and expansion (Figure 2C). Unexpectedly, the renal
Nutrients 2019, 11, 127 6 of 15

and α-SMA were highly induced by the 3-day culture of 33 mM glucose (Figure 2A). In addition, high
glucose promoted the formation of F-actin fibers in mesangial cells, which was reduced by 1–20 µM
chrysin (Figure 2B). Similarly, the α-SMA level was enhanced in diabetic kidneys, indicating mesangial
Nutrients 2019, 11, 127 6 of 15
cell proliferation and expansion (Figure 2C). Unexpectedly, the renal level of F-actin was reduced
in diabetic kidneys (Figure 2C). Accordingly, one can assume that glucose-mediated AGE
level of F-actin was reduced in diabetic kidneys (Figure 2C). Accordingly, one can assume that
produced
mesangial actin polymerization
glucose-mediated and mesangial
AGE produced mesangialproliferation. Increasedand
actin polymerization formation of F-actin
mesangial fibers may
proliferation.
be a cause of renal dysfunction implicated in DN.
Increased formation of F-actin fibers may be a cause of renal dysfunction implicated in DN.

Figure 2. Temporal
Figure responses
2. Temporal responsesofofprotein
proteininduction of F-actin
induction of F-actinand
andα-SMA
α-SMA to glucose
to glucose (A), (A), inhibition
inhibition
of F-actin
of F-actin formation
formation by by chrysinininglucose-exposed
chrysin glucose-exposed human
humanrenal
renalmesangial
mesangial cellscells
(HRMC,
(HRMC, B) andB) and
tissue levels of F-actin and α-SMA in chrysin-treated diabetic kidneys (C). HRMC
tissue levels of F-actin and α-SMA in chrysin-treated diabetic kidneys (C). HRMC were challenged were challenged
with with 5.5 mM
5.5 mM glucose,
glucose, 5.55.5
mM mM glucoseplus
glucose plus 27.5
27.5 mM
mM mannitol
mannitolasasosmotic
osmotic controls
controlsor with 33 mM
or with 33 mM
glucose in the absence and presence of 1–20 μM chrysin. Diabetic mice were orally administrated
glucose in the absence and presence of 1–20 µM chrysin. Diabetic mice were orally administrated with
with 10 mg/kg chrysin for 10 weeks. The induction of F-actin and α-SMA was measured by Western
10 mg/kg chrysin for 10 weeks. The induction of F-actin and α-SMA was measured by Western blot
blot analysis using cell lysates and renal tissue extracts with a primary antibody of F-actin or α-SMA
analysis using cell lysates and renal tissue extracts with a primary antibody of F-actin or α-SMA
(A and C). β-Actin protein was used as an internal control. Representative blots shown are typical of
(A,C).three
β-Actin protein was used as an internal control. Representative blots shown are typical
independent experiments. The bar graphs (mean ± SEM) in the right panel represent
of three independent
quantitative experiments.
results obtained The bar graphs
from a densitometer. (mean
Values ± SEM)
not sharing in the
a letter right panel
are different at p <represent
0.05.
quantitative results obtained
Red-rhodamine phalloidinfrom a densitometer.
staining Values not
for F-actin formation wassharing
conducted a letter different at p < 0.05.
areglucose-exposed
in high
Red-rhodamine phalloidin
HRMC (B). Nuclear staining forwas
counter-staining F-actin
done formation
by using bluewas conducted in high glucose-exposed
4’,6-diamidino-2-phenylindole. Scale
HRMC bar(B).
= 50 Nuclear
μm. Eachcounter-staining was done of
photograph is representative byatusing blueanimals.
least four 4’,6-diamidino-2-phenylindole.
Magnification: 400-fold. Scale
bar = 50 µm. Each photograph is representative of at least four animals. Magnification: 400-fold.
3.2. Blockade of Focal Adhesion Formation by Chrysin
3.2. Blockade of Focal Adhesion Formation by Chrysin
Dynamics of integrin-based focal adhesions containing F-actin-binding vinculin and paxillin is
Dynamics of process
an unceasing involvingfocal
integrin-based coordination
adhesionsbetween focal adhesion
containing and the actin
F-actin-binding cytoskeleton,
vinculin and paxillin
which is vital for cell migration, polarization, and extracellular matrix remodeling
is an unceasing process involving coordination between focal adhesion and the actin cytoskeleton, [17,18]. High
glucose and AGE-BSA markedly promoted the phosphorylation of vinculin and
which is vital for cell migration, polarization, and extracellular matrix remodeling [17,18]. High glucosepaxillin in
mesangial cells (Figure 3A). The activation of vinculin and paxillin was dose-dependently inhibited
and AGE-BSA markedly promoted the phosphorylation of vinculin and paxillin in mesangial cells
by treating 1–20 μM chrysin. Accordingly, chrysin may attenuate the focal adhesion clustering in an
(Figure 3A). The activation of vinculin and paxillin was dose-dependently inhibited by treating 1–20 µM
active conformation and focal adhesion-actin interaction in the diabetic milieu.
chrysin. Accordingly,
The F-actin-linkedchrysin maycortactin
protein attenuate the focal
is known adhesion
to activate the clustering in an active
actin polymerization conformation
mediated by
and focal adhesion-actin interaction in the diabetic milieu.
Arp2/3 complex, a branched actin filament nucleator [35]. The induction of cortactin and ARP2/3
The
was F-actin-linked
highly prompted protein cortactin
in glucose- is known tomesangial
or AGE-exposed activate the actin
cells polymerization
(Figure mediated
3B). In contrast, such by
Arp2/3 complex,
induction a branched actin
dose-dependently filament
declined nucleator
in 1–20 [35]. The induction
μM chrysin-exposed of cortactin
mesangial and ARP2/3
cells. Thus, chrysin was
may
highly block branched
prompted actin network
in glucose- assembly and
or AGE-exposed actin polymerization
mesangial cells (Figure promoted by glucose.
3B). In contrast, such induction
Nutrients 2019, 11, 127 7 of 15

dose-dependently declined in 1–20 µM chrysin-exposed mesangial cells. Thus, chrysin may block
branched actin network assembly and actin polymerization promoted by glucose.

3.3. Inhibition of F-Actin

Nutrients 2019, 11, 127 Bundling and Mesangial Migration by Chrysin 7 of 15

Fascin, a filamentous
3.3. Inhibition of F-Actin actin-crosslinking/bundling
Bundling and Mesangial Migrationprotein,
by Chrysin is associated with cell motility and
recruited to filopodia [36]. This study examined whether chrysin inhibited the F-actin crosslinking
Fascin, a filamentous actin-crosslinking/bundling protein, is associated with cell motility and
responsible
recruited fortoactin bundling.
filopodia Both
[36]. This glucose
study examinedandwhether
AGE increased mesangial
chrysin inhibited induction
the F-actin of fascin-1,
crosslinking
whichresponsible
was reduced by the presence of ≥ 10 µM chrysin (Figure 3C). Additionally, chrysin
for actin bundling. Both glucose and AGE increased mesangial induction of fascin-1, encumbered
the induction
which was of EVL involved
reduced by theelongation
presenceofofactin
≥10filaments
μM chrysinin glucose-
(Figure or AGE-treated
3C). Additionally,mesangial
chrysincells
(Figure 3C). On the
encumbered theother hand,ofchrysin
induction counteracted
EVL involved theoftissue
elongation levels of in
actin filaments cortactin
glucose-and fascin-1 highly
or AGE-treated
mesangial
diminished cells (Figure
in diabetic mouse3C).kidneys,
On the other
alonghand,
with chrysin
F-actin counteracted
(Figure 3D). the tissue levels of cortactin
and fascin-1 highly diminished in diabetic mouse
This study attempted to investigate that chrysin interruptedkidneys, along withmesangial
F-actin (Figure
cell 3D).
migration in the
diabetic glomerular microenvironment. Mesangial cell motility was enhancedmigration
This study attempted to investigate that chrysin interrupted mesangial cell in the to
by an exposure
diabetic glomerular microenvironment. Mesangial cell motility was enhanced by an exposure to
glucose or AGE, evidenced by the in vitro scratch wound healing assay (Figure 4). When the mesangial
glucose or AGE, evidenced by the in vitro scratch wound healing assay (Figure 4). When the
cells were supplemented with 1–20 µM chrysin, the cell migration was notably attenuated. Accordingly,
mesangial cells were supplemented with 1–20 μM chrysin, the cell migration was notably
chrysin may hamper
attenuated. mesangial
Accordingly, cell migration
chrysin may hamper through disturbing
mesangial focal adhesion
cell migration through formation
disturbing and
focalactin
bundling due to
adhesion glucose-mediated
formation AGE. due to glucose-mediated AGE.
and actin bundling

3. Western
FigureFigure blot blot
3. Western datadata
showing
showing inhibitory
inhibitoryeffects
effects of
of chrysin
chrysin ononAGE-induced
AGE-induced cellular
cellular induction
induction
of phospho-vinculin
of phospho-vinculin andandphospho-paxillin
phospho-paxillin(A),
(A),cortactin
cortactin and Arp2/3(B)
and Arp2/3 (B) and
and fascin-1
fascin-1 andand
EVLEVL (C) in
(C) in
human human
renal renal mesangial
mesangial cellscells (HRMC).
(HRMC). HRMCHRMCwerewere challenged
challenged forfor 3 days
3 days with5.5
with 5.5mM
mMglucose,
glucose, 5.5
5.5 mM
glucosemM glucose
plus 27.5 plus
mM27.5 mM mannitol
mannitol as osmotic
as osmotic controls,
controls, 33 mM33 mM glucose
glucose or 100
or 100 μg/mLAGE-BSA
µg/mL AGE-BSA in in the
the absence and presence of 1–20 μM chrysin. db/db mice were orally administrated
absence and presence of 1–20 µM chrysin. db/db mice were orally administrated with 10 mg/kg chrysin with 10 mg/kg
for 10 chrysin
weeks (D).for 10 weeks (D). Cellular induction of phospho-vinculin, phospho-paxillin, cortactin,
Cellular induction of phospho-vinculin, phospho-paxillin, cortactin, Arp2/3, fascin-1
Arp2/3, fascin-1 and Ena/VASP (EVL) were measured by Western blot analysis with each of their
and Ena/VASP (EVL) were measured by Western blot analysis with each of their primary antibodies.
primary antibodies. The renal tissue levels of cortactin and fascin-1 were also measured (D). The bar
The renal tissue levels of cortactin and fascin-1 were also measured (D). The bar graphs (mean ± SEM,
graphs (mean ± SEM, n = 3) in the bottom or right panels represent quantitative results obtained from
n = 3) aindensitometer.
the bottom orβ-Actin
right panels
proteinrepresent
was usedquantitative results
as an internal obtained
control. from
Values notasharing
densitometer.
a letter β-Actin
are
protein was used
different at p as an internal control. Values not sharing a letter are different at p < 0.05.
< 0.05.
Nutrients 2019, 11, 127 8 of 15
Nutrients 2019, 11, 127 8 of 15

Figure4.4. In
Figure Invitro
vitroscratch
scratch wound
wound healing
healing assay
assay showing
showing inhibition
inhibition ofof migration
migration of of glucose-
glucose- and
and
AGE-exposed human renal mesangial cells (HRMC) by chrysin. HRMC were challengedfor
AGE-exposed human renal mesangial cells (HRMC) by chrysin. HRMC were challenged for33days
days
with5.5
with 5.5mM
mMglucose,
glucose, 5.5
5.5 mM
mM glucose
glucoseplus
plus27.5
27.5mM
mMmannitol
mannitolasasosmotic
osmoticcontrols, 33 mM
controls, 33 mMglucose, or
glucose,
or100
100μg/mL
µg/mL AGE-BSA
AGE-BSA in the
in absence and presence
the absence of 1–20
and presence ofμM chrysin.
1–20 In vitro In
µM chrysin. scratch
vitro wound
scratchhealing
wound
healing assay was performed (see the Materials and Methods). Scale bar = 200 µm. Each photographisis
assay was performed (see the Materials and Methods). Scale bar = 200 μm. Each photograph
representativeofofatatleast
representative leastfour
fouranimals.
animals.Magnification:
Magnification: 200-fold.
200-fold.

3.4.
3.4.Inhibition
InhibitionofofAutophagic
AutophagicResponses
Responses to
to AGE
AGE by
by Chrysin
Chrysin
Thisstudy
This studyinvestigated
investigated that chrysin
chrysinmodulated
modulatedautophagic
autophagicactivity
activityin in
mesangial
mesangial cells placed
cells in
placed
inthe
thediabetic
diabetic milieu
milieuby by
evaluating induction
evaluating of the
induction of autophagy
the autophagy markers of beclin-1
markers and LC3.
of beclin-1 andHigh
LC3.
glucose
High enhanced
glucose enhancedthe the
induction of beclin-1
induction and
of beclin-1 andLC3
LC3 I/III/II
in in
a temporal
a temporal manner
mannerfor for5-day
5-daytreatment
treatment
(Figure5A).
(Figure 5A). Moreover,
Moreover, thethe induction
induction of beclin-1
of beclin-1 and I/II
and LC3 LC3was I/IIobserved
was observed in mesangial
in mesangial cells
cells exposed
toexposed to 100AGE-BSA
100 µg/mL μg/mL AGE-BSA for (Figure
for 4 days 4 days (Figure
5B). The5B).increased
The increased induction
induction of beclin-1
of beclin-1 and and
LC3LC3I/II
was dose-dependently diminished by chrysin. The induction of the autophagy-related genes of
I/II was dose-dependently diminished by chrysin. The induction of the autophagy-related genes of
Atg3and
Atg3 andAtg7
Atg7 was
was promoted
promotedin inAGE-exposed
AGE-exposedmesangial
mesangial cells, andand
cells, such induction
such was was
induction reduced in a
reduced
indose-dependent
a dose-dependent manner
manner(Figure 5C). 5C).
(Figure Furthermore,
Furthermore,renalrenal
tissuetissue
levelslevels
of beclin-1 and LC3
of beclin-1 andI/II
LC3 were
I/II
elevated in diabetic kidneys (Figure 5D). Oral administration of 10 mg/kg
were elevated in diabetic kidneys (Figure 5D). Oral administration of 10 mg/kg chrysin significantly chrysin significantly
loweredthese
lowered theselevels
levelsinindiabetic
diabeticmice.
mice. Therefore,
Therefore, autophagy
autophagy activation
activation maymay contribute
contribute toto mesangial
mesangial
proliferation and an aberrant actin cytoskeleton, and targeting autophagy genes may be a potential
proliferation and an aberrant actin cytoskeleton, and targeting autophagy genes may be a potential
therapeuticstrategy.
therapeutic strategy.

3.5.Suppression
3.5. SuppressionofofAGE-Induced
AGE-InducedmTOR
mTORActivation
Activation by
by Chrysin
Chrysin

AnAnemerging
emerging body
body of evidence
of evidence has suggested
has suggested that protein
that mTOR mTOR plays
protein plays
a key role ain key role in the
the development
ofdevelopment of diabetes
diabetes [37,38]. This study[37,38]. This whether
examined study examined whetherrenal
chrysin deterred chrysin deterred
mesangial renal mesangial
activation of mTOR
activation of mTOR under diabetic conditions. In 33 mM glucose-exposed mesangial cells
under diabetic conditions. In 33 mM glucose-exposed mesangial cells for 5 days the mTOR was temporally for 5 days
phosphorylated (Figure 6A). The mTOR phosphorylation increased in mesangial cells subjectedin
the mTOR was temporally phosphorylated (Figure 6A). The mTOR phosphorylation increased to
mesangial
AGE (Figurecells
6B). subjected
In contrast,to the
AGE (Figure mTOR
increased 6B). Inactivation
contrast, was
the increased
attenuatedmTOR activation was
by adding/chrysin to
attenuated
diabetic by adding/
mesangial cells. chrysin
When 10tomg/kg
diabetic mesangial
chrysin cells. administrated
was orally When 10 mg/kg chrysin
to db/db was
mice, theorally
renal
administrated to db/db mice, the renal level of phosphorylated
level of phosphorylated mTOR was markedly suppressed (Figure 6C). mTOR was markedly suppressed
(Figure 6C).
Nutrients 2019, 11, 127 9 of 15
Nutrients2019,
Nutrients 2019,11,
11,127
127 99 of
of 15
15

Figure
Figure
Figure Temporal
5. 5.
5.Temporal
Temporal responses
responsesofof
responses ofcellular
cellularinduction
cellular inductionof
induction of autophagy-related
ofautophagy-related
autophagy-related proteins proteinsto
proteins toto high
high
high glucose
glucose
glucose (A)(A)
(A)
and and
and inhibitory
inhibitory
inhibitory effects
effects
effects of
ofof chrysinon
chrysin
chrysin onmesangial
on mesangialinduction
mesangial induction of
induction autophagy-related proteins
of autophagy-related
autophagy-related proteins
proteins byby
by AGE-BSA
AGE-BSA
AGE-BSA
(B and
(B,C).
(B and
Human C). Human
C). Human renal mesangial
renal mesangial
renal mesangial
cells werecellschallenged
cells were challenged
were challenged
for 3 days for 33with
for days5.5
days with
mM
with 5.5 mM glucose,
glucose,
5.5 mM glucose,
5.5 mM 5.5glucose
5.5 mM
mM
plusglucose
27.5 mMplus 27.5
mannitol mM asmannitol
osmotic as osmotic
controls, 33 controls,
mM 33
glucose mM or glucose
with
glucose plus 27.5 mM mannitol as osmotic controls, 33 mM glucose or with 100 μg/mL AGE-BSA in100 or with
µg/mL 100 μg/mL
AGE-BSA AGE-BSA
in the absencein
and the absence
presence ofand
1–20presence
µM of
chrysin.1–20 μM
Cellular chrysin.
induction Cellular
of induction
beclin-1, LC3,
the absence and presence of 1–20 μM chrysin. Cellular induction of beclin-1, LC3, Atg3, and Atg7of beclin-1,
Atg3, and LC3,
Atg7 Atg3,
were and Atg7
measured
were
bywere
Western measured by Western
blot analysis
measured by Western blot analysis
with each
blot analysis
of with each
their primary
with each of their
their primary
antibodies.
of primary
db/db mice antibodies.
were orally
antibodies. db/db
db/db mice were
were
administrated
mice
withorally
orally administrated
10 mg/kg with
chrysinwith
administrated for 10 10 mg/kg
10 weeks. chrysin
The renal
mg/kg chrysin for 10 weeks.
for tissue levels
10 weeks. The renal
Theofrenal tissue
beclin-1
tissueandlevels of beclin-1
LC3ofwere
levels and
alsoand
beclin-1 LC3
measured
LC3
were
bywere
Western also measured
alsoblotting
measured (D).by
byThe Western blotting
bar graphs
Western (mean
blotting (D).
(D). ± The
The bar graphs
bar ngraphs
SEM, (mean
= 3) in(mean
the right± SEM, n =
or bottom
± SEM, 3) in
n = 3) panels the right or
represent
in the right or
bottom
quantitative panels
bottom panels represent
represent
results obtained quantitative
quantitative results obtained
results obtained
from a densitometer. from a densitometer.
from a protein
β-Actin densitometer. β-Actin
β-Actin
was used protein
protein
as an was used
was control.
internal used
as an
as
Values annotinternal
internal
sharing control.
control. Values
Values
a letter not sharing
not
are differentsharing
at paa<letter
letter are different
0.05.are different at at pp <<0.05.
0.05.

Figure
Figure
Figure 6. Temporal
Temporal
Temporal
6. 6. activationofof
activation
activation ofmTOR
mTORto
mTOR tohigh
to highglucose
high glucose (A),
glucose (A), and
(A), and inhibitory
and inhibitory effects
inhibitoryeffects ofof
effectsof chrysin
chrysin
chrysin onon
on itsits
its
activation
activation
activation in
in in AGE-exposed
AGE-exposedhuman
AGE-exposed human
humanrenalrenal mesangial
renalmesangial cells (HRMC,
mesangial cells (HRMC,
(HRMC,B) B) and
B)and diabetic
anddiabetic kidneys
diabetickidneys
kidneys (C).
(C). HRMC
HRMC
(C). HRMC
were
were
were challenged
challenged
challenged for
forfor 3 days
days
3 3days with5.5
with
with 5.5mM
5.5 mMglucose,
mM glucose,5.5
glucose, 5.5mMmM glucose
glucose plus
glucose plus 27.5
plus27.5
27.5mMmM mannitol
mMmannitol
mannitol asas
as osmotic
osmotic
osmotic
controls, 33
controls,
controls, 33 33
mM mM glucose
mM glucose or
glucose oror100
100 μg/mL
100 μg/mL AGE-BSA
µg/mL AGE-BSA in
AGE-BSAinin the absence
the absence and
the and presence
absence presence of
and presenceof 1–20
1–20 μMμM chrysin
of 1–20 chrysin (A
µM chrysin (A
and B). Diabetic
and Diabetic
B). Diabetic mice
mice were
were orally
orally administrated
administratedwithwith
with10 10 mg/kg
10mg/kg chrysin
mg/kg chrysin for 10 weeks (C). The mTOR
(A,B). mice were orally administrated chrysinforfor1010weeks
weeks(C). The
(C). The mTOR
mTOR
phosphorylation in
phosphorylation in cells
cells and
and kidney
kidney tissues
tissues was
was measured
measured by by Western
Western blot
blot analysis
analysis with
with aa primary
primary
phosphorylation in cells and kidney tissues was measured by Western blot analysis with a primary
antibodyagainst
antibody againstmTOR
mTORor orphospho-mTOR.
phospho-mTOR.The Thebar
bargraphs
graphs(mean
(mean±±SEM,
SEM, n = 3) inthethebottom
bottompanels
panels
antibody against mTOR or phospho-mTOR. The bar graphs (mean ± SEM,nn= =3)3)inin the bottom panels
represent
represent quantitative results
quantitativeresults obtained
resultsobtained
obtainedfromfrom a densitometer.
from aa densitometer. β-Actin
densitometer. β-Actin protein was used as an internal
represent quantitative β-Actinprotein
proteinwaswasused
used asasananinternal
internal
control. Values
control. Values not
not sharing
sharing aletter
letter are
are different
different at
at p <0.05.
0.05.
control. Values not sharing a aletter are different at pp << 0.05.
Nutrients 2019, 11, 127 10 of 15
Nutrients 2019, 11, 127 10 of 15

3.6.3.6.
Association
AssociationofofAutophagy
Autophagywith
withMesangial
Mesangial Motility
AArecent paper
recent has has
paper reviewed the cytoskeleton-autophagy
reviewed the cytoskeleton-autophagyconnection [39]. The
connection current
[39]. study elucidated
The current study
that Atg7 contributed
elucidated that Atg7 to actin assembly
contributed to under pathological
actin assembly diabetic
under conditions.
pathological The coexisting
diabetic induction
conditions. The
coexisting induction of both F-actin and Atg7 was highly augmented in
of both F-actin and Atg7 was highly augmented in 33 mM glucose- or 100 µg/mL AGE-BSA-treated 33 mM glucose- or 100
μg/mL AGE-BSA-treated
mesangial cells, indicating thatmesangial cells, indicating
green FITC-Atg7 that green
co-localized FITC-Atg7
with red co-localizedinwith
phalloidin-F-actin red
mesangial
phalloidin-F-actin
cells (Figure 7A). However,in mesangial cells (Figure
the presence 7A). However,
of chrysin attenuatedthethe
presence
inductionof chrysin
of Atg7attenuated thethe
localized to
induction of Atg7 localized to the actin
actin cytoskeleton in mesangial cells (Figure 7A). cytoskeleton in mesangial cells (Figure 7A).
Since
Since p38MAPK
p38 MAPKregulates
regulatesdistinct
distinct phases
phases ofof autophagy
autophagy[40,41],
[40,41],this
thisstudy
studyemployed
employed thethe
p38p38
MAPK inhibitor for the inhibition of mesangial autophagy. As expected, the beclin-1
MAPK inhibitor for the inhibition of mesangial autophagy. As expected, the beclin-1 induction and induction and
LC3 I/II activation by AGE was near-completely abated in mesangial cells by 10 μM SB203580, a p38
LC3 I/II activation by AGE was near-completely abated in mesangial cells by 10 µM SB203580, a p38
MAPK inhibitor (Figure 7B). In addition, the mesangial mTOR activation by AGE was abrogated by
MAPK inhibitor (Figure 7B). In addition, the mesangial mTOR activation by AGE was abrogated by
the inhibition of p38 MAPK (Figure 7B). Accordingly, p38 MAPK may elicit autophagy via beclin-1
the inhibition of p38 MAPK (Figure 7B). Accordingly, p38 MAPK may elicit autophagy via beclin-1
induction and LC3 I/II activation. This study further investigated that autophagy and mTOR
induction and LC3 I/II activation. This study further investigated that autophagy and mTOR activation
activation were involved in mesangial protrusion and migration under diabetic conditions. The
were involved in mesangial protrusion and migration under diabetic conditions. The induction of
induction of mesangial F-actin, cortactin, and fascin-1 was dampened by the inhibition of p38
mesangial
MAPK, as F-actin,
with the cortactin,
presenceand fascin-1
of 20 was dampened
μM chrysin (Figure 7C).by the inhibition
Therefore, chrysinofmay
p38 inhibit
MAPK,mesangial
as with the
presence of 20 µM chrysin (Figure 7C). Therefore, chrysin may inhibit mesangial
actin assembly and cell migration by interfering with autophagy via the blockade of beclin-1 and actin assembly and
cellLC3
migration
I/II. by interfering with autophagy via the blockade of beclin-1 and LC3 I/II.

Figure
Figure7. 7.Involvement
Involvement of of autophagy
autophagy andand mTOR
mTOR in in F-actin
F-actinformation
formationand andactin
actinpolymerization.
polymerization.
Human renal mesangial cells (HRMC) were challenged for 3 days with
Human renal mesangial cells (HRMC) were challenged for 3 days with 5.5 mM glucose, 5.5 mM glucose, 5.55.5
mM mM
glucose
glucose plus27.5
plus 27.5mM
mMmannitol
mannitolas asosmotic
osmotic controls,
controls, 33
33 mMmM glucose
glucoseoror100
100μg/mL
µg/mL AGE-BSA
AGE-BSA in in
thethe
absenceand
absence andpresence
presenceofof1–20
1–20µMμM chrysin
chrysin or
or 10
10 µM
μM SB203580.
SB203580. The TheF-actin
F-actinformation
formation and
and Atg7
Atg7
inductionininglucose-
induction glucose-ororAGE-exposed
AGE-exposed HRMCHRMC werewere visualized
visualized with
withrhodamine
rhodaminephalloidine-red
phalloidine-red
staining
staining andand FITC-greenimmunostaining,
FITC-green immunostaining, andand nuclear
nuclear counter-staining
counter-stainingwas wasdone
done with
with thethe
blue
bluestain
stain
4',6-diamidino-2-phenylindole (A). Each photograph is representative of four different
4’,6-diamidino-2-phenylindole (A). Each photograph is representative of four different experiments and experiments
and images
images were takenwerewith
taken with a fluorescence
a fluorescence microscope.
microscope. 200-fold.200-fold.
Scale barScale
= 100barµm.=Cellular
100 μm.induction
Cellular of
induction of beclin-1, LC3, phospho-mTOR, F-actin, cortactin, and fascin-1 were
beclin-1, LC3, phospho-mTOR, F-actin, cortactin, and fascin-1 were measured by Western blot analysis measured by
Western blot analysis with each of their primary antibodies (B and C). The bar graphs
with each of their primary antibodies (B,C). The bar graphs (mean ± SEM, n = 3) in the bottom panels (mean ± SEM,
n = 3) in the bottom panels represent quantitative results obtained from a densitometer. β-Actin
represent quantitative results obtained from a densitometer. β-Actin protein was used as an internal
protein was used as an internal control. Values not sharing a letter are different at p < 0.05.
control. Values not sharing a letter are different at p < 0.05.
Nutrients 2019, 11, 127 11 of 15

4. Discussion
Ten major findings were observed in this study. (1) The enhanced mesangial F-actin induction
and bundle formation by 33 mM glucose or 100 µg/mL AGE-BSA were attenuated by the presence of
≥10 µM chrysin in mesangial cells. (2) The mesangial induction of α-SMA by glucose and the tissue
level of α-SMA in diabetic kidneys were reduced by chrysin, indicating its blockade of mesangial
proliferation. (3) Treating chrysin inhibited the activation of vinculin and paxillin and the induction of
cortactin, ARP2/3, fascin-1, and EVL in glucose- or AGE-exposed mesangial cells. (4) Oral treatment
of 10 mg/kg chrysin diminished renal tissue levels of cortactin and fascin-1 elevated in diabetic
mouse kidneys. (5) Mesangial cell motility was enhanced in glucose- or AGE-exposed mesangial
cells, which was notably attenuated by adding chrysin. (6) Chrysin dampened the induction of
autophagy-related genes of beclin-1, LC3 I/II, Atg3, and Atg7 in mesangial cells exposed to AGE.
(7) Oral administration of chrysin lessened the tissue levels of beclin-1 and LC3 I/II enhanced in
diabetic kidneys. (8) Chrysin reduced the mTOR activation in AGE-exposed mesangial cells and
diabetic kidneys. (9) The presence of chrysin attenuated the induction of Atg7 localized to the F-actin
cytoskeleton in mesangial cells. (10) The induction of mesangial F-actin, cortactin, and fascin-1 by
AGE was near-completely abolished by the inhibition of autophagy. Therefore, chrysin may hamper
diabetes-associated mesangial cell protrusion and migration through disturbing actin dynamics by
interfering with autophagy via the blockade of beclin-1 and LC3 I/II.
The irregular mesangial structures in kidney glomeruli influence the blood capillary flow through
manipulating expansible forces created by pressure gradients in glomerular vessels [4]. A growing
body of evidence shows that the aberrant proliferation and migratory polarity of mesangial cells plays a
role in the pathogenesis of glomerulonephritis due to glomerular injury [4,7,8]. However, the underlying
molecular changes for mesangial aberrant proliferation and motility during glomerular injury remain
elusive. The cell migratory process is controlled by well-coordinated actin dynamics and focal adhesion
turnover at the peripheral ruffles in migrating cells [6]. Numerous studies have shown that glomerular
mesangial cells migrate in response to pathophysiological stimuli such as PDGF, angiotensin II,
oxidants and inflammatory cytokines [5,6]. Accordingly, alterations in induction and localization of
cytoskeletal regulators trigger mesangial phenotypic alterations in glomerulonephritis [6]. However,
identifying the key molecular components steering motility and actin functions in mesangial cells
remains challenging. This study investigated the mesangial dynamics of F-actin cytoskeleton and
integrin-based focal adhesions in glomerular injury due to glucose and AGE implicated in the
pathogenesis of diabetic complications. It was found that glucose and AGE prompted aberrant
F-actin polymerization, bundling, and motility in mesangial cells. AGE induces alterations in cell
morphology and function by affecting the cytoskeletal structure through activation of the receptor for
AGE (RAGE) [14,15].
Several studies have demonstrated that actin-targeting natural plant compounds influence actin
dynamics, prompting either polymerization or depolymerization in signaling pathways through
diverse mechanisms [42,43]. A recent study shows that salvianolic acid A inhibits renal cytoskeletal
dysfunction through inhibiting the AGE–RAGE signaling, thus effectively ameliorating early-stage
DN [44]. The polyphenol honokiol counteracts with cisplatin-induced cytoskeletal structure disruption
in renal epithelial cells and preserves epithelial cell polarity and morphology [45]. Our previous study
found that chrysin, a naturally-occurring flavonoid, inhibited AGE-induced kidney fibrosis in renal
mesangial cells and diabetic kidneys through inhibition of AGE–RAGE activation, contributing to
blockade of glucose-mediated AGE-associated glomerulosclerosis and fibrosis [34]. The current study
examined whether chrysin inhibited glucose- and AGE-stimulated actin cytoskeleton rearrangement
and focal adhesion dysfunction in renal mesangial cells. Herein, chrysin diminished the F-actin fiber
formation highly enhanced by glucose and AGE. Chrysin attenuated the activation of F-actin-binding
focal adhesion proteins of vinculin and paxillin in diabetic mesangial cells. This study also found
that this compound encumbered the mesangial cell induction of cortactin, Arp2/3 complex, EVL,
and fascin-1, all involved in actin polymerization and actin-crosslinking/bundling. The EVL proteins
Nutrients 2019, 11, 127 12 of 15

enhance in the cellular processes including axon guidance and cell migration as modulators of the
actin cytoskeleton and cell migration [46]. Unexpectedly, the renal tissue levels of F-actin, cortactin,
and fascin-1 were reduced in diabetic mouse kidney, which was counteracted by chrysin. The kidney
is composed of several different types of cells such as podocytes and glomerular vascular cells, and the
mesangial cell population in the whole kidney is very small. Accordingly, the overall renal tissue levels
of F-actin, cortactin, and fascin-1 might be lowered in diabetic kidneys comprised of diverse types of
glomerular cells. Unlike mesangial cells, the induction of F-actin and cortactin was reduced in high
glucose-exposed podocytes. Nevertheless, these findings shed light on a therapeutic role for chrysin in
modulating aberrant actin remodeling and abnormal coordination between F-actin and focal adhesion
in mesangial cells during glucose or AGE-induced glomerular injury.
Autophagy serves as a vital mechanism to maintain kidney homeostasis, and its impairment is
implicated in the pathogenesis of DN [47]. One investigation shows that autophagy is involved
in tumor cell motility and focal adhesion disassembly through targeted degradation of paxillin
interacting with processed LC3 [48]. The autophagy inhibition reduces tumor cell migration and
invasion and attenuates metastasis [48]. The present study demonstrated that glucose and AGE
enhanced mesangial cell autophagy through inducing the beclin-1-LC3 I/II-Atg pathway, and that
chrysin encumbered such diabetic induction of autophagy. This study further attempted to reveal
that autophagy contributed to F-actin polymerization/bundling and focal adhesion dysfunction in the
diabetic milieu. The presence of chrysin in mesangial cells reduced the induction of Atg7 localized
to the F-actin cytoskeleton. Consistently, one study shows that a mouse Atg7 knockout inhibiting
autophagosome formation displays severe defects in actin assembly owing to reduced expression of
proteins involved in controlling actin dynamics [49]. Additionally, the inhibition of autophagy deterred
the induction of mesangial F-actin, cortactin, and fascin-1 by AGE, indicating that actin polymerization
and bundling entailed autophagic activity. On the other hand, chrysin suppressed the mTOR activation
in AGE-exposed mesangial cells and diabetic kidneys. Similarly, one study shows that paeoniflorin
attenuates AGE-induced mesangial cell damage through inhibiting the RAGE/mTOR/autophagy
pathway [50]. Moreover, the blockade of mTOR activation highly attenuated the mesangial induction
of F-actin, cortactin, and fascin-1. Growing evidence has suggested the interconnections of mTOR to
autophagy in cellular signaling pathways in kidneys [51]. Unfortunately, this study did not examine
reciprocal interconnection of mTOR to autophagy in mesangial proliferation and motility due to AGE.
Inhibition of mTOR attenuates the actin crosslinking protein filamin A-dependent focal adhesion
formation and cell migration [52].

5. Conclusions
This study elucidated that chrysin influenced glucose- or AGE-stimulated actin polymerization,
focal adhesion disassembly and cell migration contributing to phenotypic alterations in mesangial
cells. These diabetic stimuli induced mesangial cell proliferation, aberrant actin cytoskeleton, and focal
adhesion formation. However, chrysin ameliorated dysregulation of mesangial actin assembly
and bundling through blocking the induction of F-actin, cortactin, EVL, and fascin-1. In addition,
this flavonoid hampered diabetic AGE-enhanced formation of focal adhesions involving paxillin and
vinculin. Furthermore, the autophagy and mTOR pathways were involved in actin polymerization,
focal adhesion disassembly and cell migration in AGE-stimulated mesangial cells. Therefore, chrysin
may counteract diabetes-associated mesangial cell protrusion and migration through disturbing
actin polymerization/bundling and focal adhesion formation by interfering with autophagy/mTOR
activation. Since the mesangial cell population is very small in whole kidneys, the dissociation between
the in vitro and in vivo results may take place. Accordingly, the immunohistochemical sequential
double staining is needed to examine the mesangial level of F-actin, cortactin or fascin-1 in kidneys,
along with mesangial α-SMA level. Western blotting with primary culture mesangial cells or glomeruli
can be considered by using sieving methods at least cortex part of kidneys.
Nutrients 2019, 11, 127 13 of 15

Author Contributions: E.-J.L., M.-K.K. and Y.-H.K. (Young-Hee Kang) designed research; E.-J.L., M.-K.K., D.Y.K.,
Y.-H.K. (Yun-Ho Kim), H.O., S.-I.K. and S.Y.O. conducted research; E.-J.L. and M.-K.K. analyzed data and E.-J.L.
and Y.-H.K. (Young-Hee Kang) wrote the paper. Y.-H.K. (Young-Hee Kang) had primary responsibility for final
content. All authors read and approved the final manuscript.
Funding: This work was supported by Hallym University Research Fund, 2018 (HRF-201810-008).
Conflicts of Interest: The authors declare no conflict of interest.

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