Gunaydin-Akyildiz et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-023-00486-1(2023) 24:23

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

Emodin and aloe-emodin, two potential
molecules in regulating cell migration of skincells through the MAP kinase pathway
and affecting Caenorhabditis elegans
thermotolerance
Aysenur Gunaydin-Akyildiz1*, Rabia Sare Yanikoglu2, Meltem Gulec3, Gulbahar Ozge Alim-Toraman4,
Ebru Didem Kuran5, Sezen Atasoy2, Abdullah Olgun6 and Gulacti Topcu4
Abstract
Background Emodin and aloe-emodin are two anthraquinones having positive effects in wound healing. However,their mechanism of action of wound healing is not fully understood. The MAP kinase family, which plays an activerole in wound healing, is a well-characterized large family of serine/threonine kinases and regulates processes suchas proliferation, oncogenesis, differentiation, and inflammation in the cell. The aim of this study is to comparatively
elucidate the mechanisms of action of emodin and aloe-emodin, which are potential agents in wound healing.
Methods The mechanism of the effects of emodin and aloe-emodin on cell viability and cell migration was
examined using the human skin fibroblast (CCD-1079Sk) cell line. The gene expression levels of the MAP kinases (JNK,
P38, ERK) in the skin fibroblast cells along with a molecular docking study analyzing their interaction potential were
evaluated. Furthermore, the molecules’ effects on the lifespan of Caenorhabditis elegans were studied.
Results Emodin and aloe-emodin inhibited the ATP content of the cells in a concentration dependent manner andaccelerated cell migration at the lower concentrations while inhibiting cell migration in the higher concentration
treatment groups. The expressions of JNK and P38 were upregulated at the low concentrations and downregulated at
the higher concentrations. The molecular docking studies of the molecules gave high docking scores indicating theirinteraction potential with JNK and P38. C. elegans lifespan under heat stress was observed longer after 75 µM emodin
and was significantly reduced after 150 µM aloe-emodin treatment.
Conclusion Aloe-emodin was found to be more potent on cell viability, cell migration, gene expression levels of the
MAP kinases in healthy fibroblastic skin cells, and on the lifespan of C. elegans. This study reveals the functional effects
and the biological factors that interact in the wound healing process of emodin and aloe-emodin, and give a possible
treatment alternative to shorten the duration of wound care.

*Correspondence:
Aysenur Gunaydin-Akyildiz
gunaydinaysenur@gmail.com
Full list of author information is available at the end of the article© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
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in this article, unless otherwise stated in a credit line to the data.

Gunaydin-Akyildiz et al. BMC Molecular and Cell Biology(2023) 24:23

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Keywords Emodin, Aloe-emodin, Molecular docking, MAP kinase, JNK, P38, ERK, C. elegans, Thermotolerance

Introduction
The skin represents the largest organ of the human bodyand its main function is protection from external stresses.
This protective feature can be compromised when
the skin deteriorates as a result of mechanical attacks.
Wound healing is an evolutionarily conserved physiological process that aims to preserve the integrity of the skinand restore the skin barrier after trauma. The normal
wound healing process includes three consecutive andoverlapping phases: the hemostasis/inflammatory phase,the proliferative phase, and the remodeling phase. Eachstage of wound healing is essential for a normal healing
progress because the interruption of any stage delays
healing and can cause various skin pathologies, including
failure of recovery or chronic ulceration [1, 2]. Various
specialized cells such as platelets, macrophages, fibroblasts, epithelial and endothelial cells are involved in thewound healing process. These cells interact with eachother and with the extracellular matrix. Fibroblasts, inparticular, play an active role in the proliferation phase of
the healing process [3].

Fig. 1 MAP kinase signaling pathways related with cell migration.
Created with BioRender.com

The anthraquinone derivatives emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) and aloe-emodin(1,8-dihydroxy-3-(hydroxymethyl)-anthraquinone) arefound in many plants traditionally used in wound healing such as Aloe vera, Polygonum cuspidatum, Rheum
officinale [4] which are among the secondary metabolitesof the species. The aim of this study is to comparatively
elucidate the mechanisms of action of emodin and aloeemodin, which are potential agents in wound healing.
The MAP kinase family, which plays an active role inwound healing, is a well-characterized large family ofserine/threonine kinases and regulates processes such as
proliferation, oncogenesis, differentiation, and inflammation in the cell. The MAP kinase family, which is examined in three major groups; Jun N-terminus kinase (JNK)
pathway, extracellular signal regulatory kinases (Erk1/2)
pathway and P38 group protein kinase pathway, controls
important cellular functions which are also the basis ofwound healing [5–7]. Figure 1. shows the diagram ofthese MAP kinase signaling pathways.
JNK activity plays an important role in the migrationof fibroblasts in wound healing experiments [8]. Erk1/2
is one of the main molecules of the MAPK signaling
pathway activated through the three-component protein
kinase cascade Raf → MEK → Erk which is critical inpromoting angiogenesis during wound healing [6, 9]. P38
is known to play a role in apoptosis, cell differentiationand cell migration [5].
In traditional folk medicine, herbs are widely used as
natural molecules and derivatives from plants are foundto be effective and safe. The extracts prepared frommedicinal plants and the molecules/derivatives isolated
from these extracts create an important potential for scientific research considering the developing phytochemical research and technology Although the use of natural
products in wound healing is common, studies investigating the mechanisms of action are insufficient [4]. The
elucidation of the mechanisms of action of various effective wound healing natural compounds may offer alternatives for the treatment of very persistent wounds such as
diabetic neuropathic lower extremity ulcers [6].
In this study, the gene expression levels of the MAP
kinase (JNK, P38, ERK) signaling pathway were evaluatedafter emodin and aloe-emodin treatment in CCD-1079Sk
human skin fibroblast cells. Additionally, a moleculardocking study was conducted analyzing the interaction
potential of emodin and aloe-emodin with the MAP
kinases. Molecular docking is a method that allows us
to determine the interactions of a protein -whose crystal structure has been reported in the literature- andour original molecules under optimal conditions. The inGunaydin-Akyildiz et al. BMC Molecular and Cell Biology(2023) 24:23

vitro wound healing properties of the natural molecules
emodin and aloe-emodin in the CCD-1079Sk skin fibroblast cell line was investigated. The effects of emodin andaloe-emodin on intracellular ATP content were examined. Understanding the mechanisms that regulate cell
migration and proliferation of dermal fibroblast cells by
the action of a natural compound will be useful in designing new treatments to regulate fibrosis and wound contraction by accelerating the wound healing process [10].
Furthermore, the molecules’ effects on the lifespan of
Caenorhabditis elegans (C.elegans) under heat stress(thermotolerance) were studied to obtain in vivo preclinical data.

Materials and methods
Cell Culture

Human skin fibroblasts CCD-1079Sk (CRL-2097™) werepurchased from American Type Culture Collection(ATCC, VA, USA), and cultured in DMEM-F12 medium
supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Gibco, USA). Sub-culturing wasdone every 2–3 days.
Treatment

Emodin and aloe-emodin were purchased from SigmaAldrich (MO, USA) and dissolved in DMSO. The studied
concentrations were chosen according to the cytotoxicity
assay which was done in the range of 0-1000 µM with the
ATP assay. According to the IC50 values, for the evaluation of the gene expression levels 25 µM, 12.5 µM, and6.25 µM were selected for emodin; 10 µM, 5 µM, and 2.5µM for aloe-emodin. The control group was the solvent
containing the same percentage of DMSO (0.1%) with thetreatment groups.
ATP Bioluminescence Assay

ATP content of the cells was determined with recording the bioluminescence using CellTiter-Glo® 2.0 (Promega, USA). The luminescent signal is proportional to
the ATP content of the cells which is an indicator of cell
viability [11]. Opaque white 96-well plates were used.
CCD-1079Sk cells were seeded 104/well and treatment
was conducted after a 24 h incubation. Following 24 htreatment with emodin and aloe-emodin, the cells wereshaken for 2 min with CellTiter-Glo® 2.0 Reagent which
was added the same amount with the culture medium
present in each well. After 10 min incubation in room

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temperature, the luminescence was recorded using a
BioTek Synergy H1 (Epoch, Germany) microplate reader.
The results are given as a percentage relative to the solvent control group.
In vitro wound healing (scratch) assay

For evaluating the migration of the CCD-1079Sk cells.
The CytoSelect™ 24-well Wound Healing Assay Kit (Cell
Biolabs Inc, USA) was used. The inserts were placed
inside every well of the 24 well plate using a sterile forceps with their “wound field” aligned in the same direction. 500 µL of medium containing 2 × 105 cells wasadded to the gaps of the inserts. After 24 h incubation ina cell culture incubator overnight for the cells to provide
a monolayer, the inserts were removed, and the images ofeach well were taken before treatment. After taking theimages, emodin (6.25 µM, 12.5 µM, and 25 µM) and aloeemodin (2.5 µM, 5 µM, and 10 µM) treatments were donefor 24 h and the images of the same area of the wounds
were taken. For calculating the wound healing percentage, the program ImageJ (National Institutes of Health,
USA) was used, and comparison was done between thetreatment groups with the vehicle control group.
Evaluation of gene expression levels

Gene expressions of JNK, P38, and ERK were evaluatedby reverse transcription polymerase chain reaction (RTPCR) and Beta actin (βactin) was used as house-keeping
gene. Total RNA extraction was done according to the
manufacturer’s instructions of Quick RNA Mini Prep
Kit (Zymo Research). cDNA synthesis was done using
SensiFAST™ cDNA Synthesis Kit (Bioline). RT-PCR was
performed with the SensiFAST™ SYBR No-ROX Kit(Bioline). Gene expression levels were calculated by the
2-ΔΔCT method [12]. The studied primer sequences can befound in Table 1.
Molecular docking study

The docking study was carried out using the Schrödinger
Software Suite (Maestro Schrödinger Release 2022-2:
Meastro, Schrödinger, LLC, New York, NY, 2021). The
crystal structure of N-terminus kinase-1 (JNK1) complexed with its inhibitor quercetagetin at 2.70 A ̊ resolution and the crystal structure of p38alpha complexedwith pyrazolobenzothiazine inhibitor COXH11 (9Y5)
at 2.50 A ̊ (PDB ID: 3V3V and 5OMH, respectively) was
retrieved from the RCSB Protein Data Bank [13, 14]. The

Table 1 Primer sequences for determination of gene expression levels
Gene
JNK
P38
ERK
βactinForward Primer
GACGCCTTATGTAGTGACTCGCGAGCGTTACCAGAACCTGTCTCTGGCAAGCACTACCTGGATCAG
CATGTACGTTGCTATCCAGGC

Reverse Primer
TCCTGGAAAGAGGATTTTGTGGCAGTAACCGCAGTTCTCTGTAGGTGCAGAGACTGTAGGTAGTTTCGG
CTCCTTAATGTCACGCACGAT

Melting Temperature (ºC)
62.1
62.2
62
62

Gunaydin-Akyildiz et al. BMC Molecular and Cell Biology(2023) 24:23

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crystal structures of the enzymes were prepared using
the multi-step Protein Preparation Wizard Module ofSchrödinger Software Suite (Schrödinger Release 20222: Protein Preparation Wizard; Epik, Schrödinger, LLC,
New York, NY, 2021; Impact, Schrödinger, LLC, New
York, NY; Prime, Schrödinger, LLC, New York, NY, 2021)[15]. The retrieved crystal structure was optimized byremoving water molecules, heteroatoms, and co-factors.
The hydrogens, missing atoms, bonds, and charges werecomputed and corrected through Maestro. The studied compounds (emodin and aloe-emodin) and the cocrystallized ligands (quercetagetin and 9Y5) were also
prepared and optimized (generating various tautomers
and ring conformations, assigning bond orders, and stereochemistries) using the LigPrep module of Schrödinger
Software Suite (Schrödinger Release 2022-2: LigPrep,
Schrödinger, LLC, NY, 2021). All the conformations generated were minimized using the OPLS4 force field. The
receptor grid was generated around the co-crystalized
ligand of the enzyme to specify the binding site, where
the grid box size was determined with the receptor Grid
Generation implemented in Glide (Schrödinger Release2022-2: Glide, Schrödinger, LLC, NY, 2021) [16–18]. The
Glide docking was performed in the Standard Precision(SP) mode.

control group. The seeded plates were left overnight atroom temperature to allow the E. coli OP50-1 to grow.
Our experiment used the thermotolerance test that ispart of survival assays to evaluate and screen the stressevolution of the experimental worms exposed to emodin and aloe-emodin compared to the control group on
worm’s lifespan within hours. The experiment started at
the end of the L4 larval stage; the plates were incubated at
35°±0.8 °C for 12 h in an incubator, and images were captured with a high-resolution scanner (Epson Perfection,
V800 Photo) starting from T0 the plates were scanned inevery 20 min, till every worm dies. The worms that were
not moving in two consecutive scans were accepted asdead. The results were scored and analyzed using a statistical method, The Kaplan-Meier, to estimate the survival
function.

C. elegans Survival (Thermotolerance) assay under heatstressIntracellular ATP content

C. elegans used in this experiment were obtained fromthe Caenorhabditis Genetics Center CGC. The typeused was the wild N2 strain and was maintained in the
Nematode Growth Medium (NGM). One-liter volume
of NGM was prepared as follows: First agar 17 g, NaCl
3 g, and peptone 2.5 g were mixed and autoclaved at
121 °C for 15 min. After the flask cooled down to 55 °C;
1 M MgSO4.7H2O 1 mL, 1 M CaCl2.2H2O 1 mL, 5 mg/
mL cholesterol in ethanol 1 mL, 1 M KPO4 buffer 25
mL, and 100 µg/mL penicillin-streptomycin 1 mL wereadded under the laminar flow cabinet using sterile techniques with a constant mixing to make sure the mixture
was homogeneous, and later it was poured into 100 mm
diameter Petri plates and left for 24 h under 22 ± 2 °C temperature conditions. After synchronizing, a total of 210
synchronized worms were prepared, and 35 were divided
into each plate separately. For this experiment, 15 plates
were prepared and divided into five groups, three as thecontrol group and three as the experimental groups. The
NGM plates were seeded with 500 µL of Escherichia coli
OP50-1 strain (OD:0.4; treated at 65 °C for 10 min tokill) under sterile conditions as a food source; the control
groups only contained the E. coli and a concentration of
75 µM and 150 µM emodin and aloe-emodin was added
to the experimental groups. DMSO was included in theStatistical analysis

Statistical analysis was performed using Graphpad Prism
6. Statistical differences of the treatment groups vs. theuntreated control group were evaluated with one-way
ANOVA followed by the Tukey test. The results wererepresented as mean ± standard deviation (SD)., *p < 0.05
and **p < 0.01 versus the control group.

Results
Emodin and aloe-emodin inhibited the ATP content of
the CCD-1079Sk skin fibroblast cells after 24 h treatment
in a concentration dependent manner. The IC50 values
are 29.42 µM and 10 µM, respectively (Fig. 2). The ATPcontent of the cells indicates cell viability.
In vitro wound healing (scratch) assay

Emodin and aloe-emodin accelerated cell migrationof skin cells after 24 h treatment compared to the control group (Fig. 3). Emodin and aloe-emodin similarly
induced cell migration of skin cells in the lower concentration groups (6.25 µM and 12.5 µM concentrationgroups of emodin, 2.5 µM and 5 µM of aloe-emodin).
Evaluation of gene expression levels of the MAP kinase
pathway

JNK, P38, and ERK gene expression levels of the skin cells
were evaluated after 24 h emodin and aloe-emodin treatment (Fig. 4). Emodin induced JNK and P38 at 6.25 µM
concentration and showed significant downregulation at
25 µM. The change in the ERK level was not significant(p > 0.05). Similarly, aloe-emodin induced JNK and P38
gene expressions in the lowest treatment group (2.5 µM)
and showed significant P38 downregulation at 10 µM.
Although the change in the ERK level was not significant(p > 0.05), it was observed to be downregulated in thehigher concentrations of emodin and aloe-emodin.

Gunaydin-Akyildiz et al. BMC Molecular and Cell Biology(2023) 24:23

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Fig. 2 : Effects of emodin and aloe-emodin on cellular ATP content of CCD-1079Sk skin fibroblasts after 24 h treatment. The cells were treatedwith 0-1000 µM concentrations and the IC50 values are presented. Data are expressed as mean ± SD

Fig. 3 : Effects of emodin and aloe-emodin on cell migration of CCD-1079Sk skin fibroblasts after 24 h treatment. The images of the wound areas
were taken before and after treatment. The cells were treated with 6.25 µM, 12.5 µM, and 25 µM emodin; 2.5 µM, 5 µM, 10 µM aloe-emodin concentrations.
Data are expressed as mean ± SD, *p < 0.05 and **p < 0.01 versus the control group

Molecular docking study

Molecular docking studies were carried out to examine
the states of emodin and aloe-emodin in the active siteof the enzymes Jun N-terminus kinase-1 (JNK1) and

p38alpha. Emodin and aloe-emodin interactions withthe binding sites of JNK1 and docking scores are given
in Fig. 5; Table 2. In the active site of the JNK1 receptor,
there are hydrogen bonds between the hydroxyl groups ofGunaydin-Akyildiz et al. BMC Molecular and Cell Biology(2023) 24:23

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Fig. 4 : Effects of emodin and aloe-emodin on MAP kinase genes (JNK, P38, ERK) after 24 h treatment to CCD-1079Sk skin fibroblasts. The cells
were treated with 6.25 µM, 12.5 µM, and 25 µM emodin; 2.5 µM, 5 µM, 10 µM aloe-emodin concentrations. Data are expressed as mean ± SD, *p < 0.05
and **p < 0.01 versus the control group

Table 2 The docking scores of the emodin, aloe-emodin andquercetagetin and their interactions with the active site of Jun
N-terminus kinase-1 (JNK1) enzyme crystal structure (PDB ID:
3V3V)
LigandQuercetagetin

Docking Score(kcal/mol)-9.37

Emodin-8.46

Aloe-emodin-8.76

H-bond
GLU73, ASN114,
ASP169, MET111
GLU73, ASN114,
ASP169SER34, GLU73,
ASN114, ASP169

emodin with the GLU73, ASN114, ASP169 amino acids.
Similarly, there are hydrogen bonds between the hydroxyl
groups of aloe-emodin with GLU73, ASN114, ASP169,
and a hydrogen bond between one of the carbonyl groupsof aloe-emodin with the SER34.
Emodin and aloe-emodin interactions with the binding sites of p38alpha and docking scores are given in
Fig. 5; Table 3. In the active site of the p38alpha receptor,
there are hydrogen bonds between the hydroxyl groupsof emodin with GLY110, ASP112 and there are hydrogenbonds between the hydroxyl groups of aloe-emodin with
LYS53, GLY110, MET109.
To verify the reliability of the docking study, the cocrystalized ligands were redocked to the active sites ofproteins and the root- mean-square deviation (RMSD)
was calculated (Fig. 6). Glide successfully reproduced theexperimental binding conformations of quercetagetin in
the binding site of 3V3V with an acceptable RMSD value
0.0652 Å. Furthermore, the RMSD value was determined
as 0.77 Å for 5OMH which was also an acceptable value
which indicates the validity of the studied docking model.
C. elegans thermotolerance study

The effects of emodin and aloe-emodin on the lifespanof C.elegans worms under heat stress are given in Fig. 7.,
where emodin shows an insignificant (p > 0.05) positiveeffect in the lower concentration and aloe-emodin showsa significant (p < 0.01) inhibitory effect in the higher concentration, compared to the control group.

Gunaydin-Akyildiz et al. BMC Molecular and Cell Biology(2023) 24:23

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Fig. 5 : 2D interactions with the active site of the crystal structure of JNK1 and P38alpha (PDB ID:3V3V, 5OMH). (a) Quercetagetin and its 2D interactions with the active site of Jun N-terminus kinase-1 (PDB ID: 3V3V), (b) Emodin and its 2D interactions with the active site of Jun N-terminus kinase-1(PDB ID: 3V3V), (c) Aloe-emodin and its 2D interactions with the active site of Jun N-terminus kinase-1 (PDB ID: 3V3V), (d) 9Y5 and its 2D interactions withthe active site of p38alpha (PDB ID: 5OMH), (e) Emodin and its 2D interactions with the active site of p38alpha (PDB ID: 5OMH), (f) Aloe-emodin and its
2D interactions with the active site of p38alpha (PDB ID: 5OMH). NL: Native ligand

Table 3 The docking scores of the emodin, aloe-emodin and
9Y5 and their interactions with the active site of p38alphaenzyme crystal structure (PDB ID: 5OMH)
LigandDocking Score(kcal/mol)H-bond9Y5-5.9

ASP168

Emodin-6.5

Aloe-emodin-6.16

GLY110,
ASP112
LYS53, GLY110,
MET109

Halogenbond
HIE107,
MET109-Discussion
In traditional folk medicine, herbs are widely used andnatural molecules/derivatives from plants are foundto be effective in wound healing. Although the use ofnatural products in wound healing is common, studies
investigating the mechanisms of action are insufficient[4]. Emodin and aloe-emodin which are two anthraquinone derivatives found in the content of plants havepositive effects in the wound healing process. The aim of
this study is to elucidate the mechanisms of emodin andaloe-emodin comparatively, which are potential agents inwound healing.
This is the first study to evaluate emodin and aloeemodin’s effects on the human CCD-1079Sk fibroblastic
skin cell line. Fibroblasts play an important role in thewound healing process; the fibroblasts of damaged tissue
are recognized as the key cells in the proliferative phaseof healing by enabling cell proliferation, differentiationand migration. Among these processes, cell migration isa critical biological response during wound healing andallows measurement of wound healing ability by analyzing the migration of cells in vitro [19]. The IC50 values of
emodin and aloe-emodin against the human fibroblasts
were found to be 29.42 µM and 10 µM, respectively.

Gunaydin-Akyildiz et al. BMC Molecular and Cell Biology(2023) 24:23

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Fig. 6 : Validation of the docking models. A: Superposition of docked pose (green) and experimental binding conformation (pink) of quercetagetinin the binding pocket of 3V3V. B: Superposition of docked pose (green) and experimental binding conformation (pink) of 9Y5 in the binding pocket of
5OMH

Fig. 7 : Effects of emodin and aloe-emodin onC.eleganslifespan (A). Survival / Log Cumulative Hazard Plot (B). The cells were treated with 75 µM
and 150 µM emodin and aloe-emodin concentrations. Data are expressed as mean ± SD, **p < 0.01 versus the control group

Therefore, aloe-emodin is more potent in terms of cytotoxicity. In a recent study, the molecules’ effects on different skin cell lines were similar showing that aloe-emodin
is more potent in growth inhibition compared to emodin[20].
The presence of the hydroxy methyl group in aloeemodin instead of the hydroxyl group in the C-3 positionof emodin, and the absence of the methyl group in aloeemodin in the C-6 position potentially affected the activities of the compounds. These differences in the functionalgroups at the C-3 and C-6 positions of the two moleculeswith the same skeleton structure had significant effect on

the potence of the compounds as seen in the in silico andin vitro results.
There are current studies using fibroblast cells in in
vitro wound healing testing [9, 10]. According to theresults obtained from the in vitro scratch assay in ourstudy, low concentrations of both emodin and aloe-emodin accelerate cell migration whilst higher concentrations
tend to attenuate the process. Emodin and aloe-emodin
was observed to inhibit cell migration in 20 µM concentration in which the HaCaT cell line was used as healthy
human skin cells [20]. In an in vitro wound healing study
on human fibroblasts, it was shown that emodin is apotent profibrinolytic and wound healing agent [21]. In a

Gunaydin-Akyildiz et al. BMC Molecular and Cell Biology(2023) 24:23

study on rats, it was determined that the wounds shrunk
significantly after treatments of emodin was applied topically in excisional injuries [22]. Another in vivo study
on mice with excision wounds, it was found that woundhealing rates were increased and the time to epithelization was decreased in aloe-emodin-treated mice [23].
The MAP kinase family, which is actively involved inwound healing, is a well-characterized large family of serine/threonine kinases and regulates important cellular
processes such as proliferation, migration, oncogenesis,
differentiation, and inflammation. The MAP kinase family is examined in three major groups: Jun N-terminus
kinase (JNK) pathway, extracellular signal regulatory
kinases (ERK1/2) pathway and P38 group protein kinase
pathway. [5–7]. JNK activity is shown to play an important role in the migration of fibroblasts in wound healing
experiments [8]. ERK1/2 is one of the main molecules ofthe MAPK signaling pathway which has been implicatedin cell proliferation and plays a critical role in promoting
angiogenesis during wound healing [6]. Likewise, P38 is
known to play a role in apoptosis, cell differentiation andcell migration, and furthermore, a strong link has been
established between the P38 pathway and inflammation and has been associated with cytokine-induced cell
migration [5, 7, 9].
We conducted a molecular docking study to roughly
evaluate the binding potential of emodin and aloe-emodin against the proteins JNK and P38 which were significantly affected at the gene level after in vitro treatment
with the molecules. The docking study shows that emodin and aloe-emodin may have a great potential to inhibit
the proteins giving high docking scores and aloe-emodin
was again observed to be more potent compared to emodin against JNK1, and in fact, was even more potent thanthe positive control against P38. According to the docking studies, JNK1 enzyme inhibition docking score wasvery close to the native ligand and this may be due to thesimilarity with the structure with quercetagetin which isan anthraquinone derivative. However, even though thestructure-activity relationship with p38 is not in correlation with its native ligand (9Y5) structure, the two molecules’ docking scores were higher than 9Y5. Althoughpreliminary, the results of the in vitro studies are in linewith the in silico results, the observed JNK and P38 genelevels indicate that aloe-emodin is more potent comparedto emodin in enzyme interaction activities along with cell
viability and cell migration.
The gene expression level after emodin and aloe-emodin treatment of the fibroblasts supports the dual effectseen in the cell migration study, i.e., JNK and P38 was
observed to be induced in the studied lowest concentrations whilst higher concentrations inhibited the gene
expression levels. Many phytochemicals induce hormetic
responses on physiological functions, i.e., positive effects

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are seen at low concentrations and negative effects areobserved at high concentrations. Emodin is listed among
the compounds as a molecule that produces hormetic
effects on health and longevity in vivo [24].
Some previous studies also show significant changes
after emodin and aloe-emodin [25, 26]. In a study using
hepatic stellate cells, P38 activity was observed to be
inhibited after emodin treatment in a concentrationdependent matter while JNK was not [27] suggestingemodin as a potential candidate for the treatment of
hepatic fibrosis. Similarly, the P38 expression was inhibited after emodin and aloe-emodin treatments in the highconcentration groups in our study. Although the study
concentration range is some-what similar to our study,
the inducement of P38 and JNK in the lowest concentration group in our study may be due to cell line difference and/or expression analyses in the gene level. In the
study of Cui et al., after emodin treatment to HepG2 cellline, P38 phosphorylation was observed to be induced in
the protein level while ERK was inhibited, furthermore,
JNK level was not observed to change [26]. P38 and JNK
expressions were induced in the low concentrations in
our study, although ERK, on the other hand, seemed not
to be affected significantly. This may be because ERK is
one of the main molecules of the MAPK signaling pathway activated through the three-component protein
kinase cascade Ras → Raf → MEK1/2 and although thecascade may actually be affected ERK may not seem tobe affected in the gene level of the cells in our study [28].
Collectively, these results suggest the concentrationdependent effects of the molecules, which should be verified with further in-depth molecular methods and future
in vivo studies.
Next, to see the comparative toxic effects of emodin
and aloe-emodin in vivo, we used the C. elegans nematode model and observed that emodin had little unsignificant effects in the studied concentrations (75 µM and 150µM), although the lower concentration slightly increased
the lifespan. A study conducted with the same model
reports extension of the lifespan after 150 µM emodin
treatment while 300 µM emodin had little effect, which isin some way in line with our results (lower concentrationslightly extended lifespan while higher concentration had
no effect) [29]. When we look at aloe-emodin, the highconcentration of aloe-emodin shortened the lifespan significantly in our study. This suggests that aloe-emodin
should be used in lower concentrations due to its potent
activity compared to emodin. In C. elegans, thermotolerance assays are preferred for in aging studies because of
its short experimental since there is a proven significant
correlation between thermotolerance and life span phenotypes in C. elegans [30–32].

Gunaydin-Akyildiz et al. BMC Molecular and Cell Biology(2023) 24:23

Conclusions
In this study we aimed to analyze the mechanistic effectsof emodin and aloe-emodin, and we focused on evaluating the related pathways in the CCD-1079Sk humanfibroblastic skin cell line. Indeed, the findings should beverified with other cell lines along with further molecular methods, and further in silico, in vitro, in vivo studies
to conclude the molecules’ wound healing effects. This
study provides a great opportunity to better understandthe mechanism of the wound healing process, and tocompare the two molecules’ biological effects where theonly molecular differences between them are the presence of a hydroxy methyl molecule at position C-3 andan absence of a methyl group at C-6 yet showing a significant influence. Overall, aloe-emodin was found to be
more potent on cell viability, cell migration, gene expression levels of the MAP kinases in healthy fibroblastic skincells, and on the lifespan of C. elegans. This study contributes to the accumulating studies showing the potential of emodin and aloe-emodin to be used as therapeutic
wound healing agents. In conclusion, these results reveal
the functional effects and the biological factors thatinteract in the wound healing process of emodin andaloe-emodin, and give a possible treatment alternative toshorten the duration of wound care.
Acknowledgements
None.
Author contributions
A.G.A., R.S.Y., G.O.A.T, and S.A. designed the experiments. A.G.A., R.S.Y., G.O.A.T,
M.G., and E.D.K. performed the experiments. A.G.A., S.A., G.T., and A.G. analyzed
the data. A.G.A. wrote the main manuscript. All of the authors reviewed andedited the manuscript.
Funding
This work was supported by the Scientific Research Projects Coordination
Unit and Drug Application and Research Center (İLMER) of Bezmialem Vakif
University (Project Number: 20200905). The funding bodies played no role inthe design of the study and collection, analysis, interpretation of data, and inwriting the manuscript.
Data availability
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Faculty of Pharmacy, Department of Pharmaceutical Toxicology,
Bezmialem Vakif University, Istanbul 34093, Turkey
2
Faculty of Pharmacy, Department of Biochemistry, Bezmialem Vakif
University, Istanbul 34093, Turkey
3
Faculty of Pharmacy, Istinye University, Istanbul 34010, Turkey

Page 10 of 11

4

Faculty of Pharmacy, Department of Pharmacognosy, Bezmialem Vakif
University, Istanbul 34093, Turkey
5
Faculty of Pharmacy, Department of Pharmaceutical Chemistry,
Bezmialem Vakif University, Istanbul 34093, Turkey
6
Faculty of Medicine, İstinye University, Istanbul 34010, Turkey
Received: 20 February 2023 / Accepted: 19 July 2023

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