Development of rhodamine-based fluorescent probes for sensitive detection of Fe3+ in water: spectroscopic and computational investigations

Article abstract of DOI:10.1039/c8nj05366a

Four novel rhodamine-based fluorescent probes (RE1-RE4) were designed and synthesized for sensitive detection of Fe³⁺ in water. RE1-RE4 exhibited low detection limits in the range of 18.6 nM to 29.4 nM. In addition, the binding modes of the pro

Full text of DOI:10.1039/c8nj05366a

8letter2018 18952EJI0010.1177/2058739218818952European Journal of InflammationWu
Letter to the Editor
European Journal of Inflammation
Volume 16: 1–5
©
The Author(s) 2018
Expression of HIF-1α in keloids and its
correlation with inflammatory responses
and apoptosis
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Xiaolong Wu
Abstract
The aim of this study was to investigate the expression of hypoxia-inducible factor-1α (HIF-1α) in keloids and its
correlation with inflammatory responses and apoptosis. The keloid specimens resected in our hospital from November
2015 to February 2017 were selected as the pathological group, and the normal skin tissues from our hospital during
the same period were selected as the control group. The expression of HIF-1α, inflammatory response cytokines, and
apoptotic molecules in the tissues of two groups were detected. The messenger RNA (mRNA) expression of HIF-1α in
the keloids in the pathological group was significantly higher than that in the control group, and the mRNA expression of
interleukin (IL)-1β, IL-2, IL-6, and tumor necrosis factor (TNF)-α in the pathological group was significantly higher than
those in the control group. The mRNA expression of Bax in the pathological group was significantly higher than that in
the control group. The mRNA expression of Bcl-2, livin, and hPEBP4 in the pathological group was significantly lower
than that in the control group. Pearson test showed that there was a positive correlation between the mRNA expression
of HIF-1α and inflammatory cytokines including IL-1β, IL-2, IL-6, and TNF-α. There were also a positive correlation
between the mRNA expression of HIF-1α and Bax and a negative correlation between the mRNA expression of HIF-1α
and Bcl-2, livin, and hPEBP4. In conclusion, HIF-1α was highly expressed in keloids and closely related to inflammatory
response cytokines and apoptosis molecules. Increased expression of HIF-1α in keloids may be an important factor in
inflammatory responses and increased apoptosis in skin tissues.
Keywords
apoptosis, hypoxia-inducible factor-1α, inflammatory responses, keloids
Date received: 26 June 2018; accepted: 15 October 2018
Introduction
research focus in the field of plastic surgery. At
present, the clinical treatment of keloids is not
ideal and is easy to relapse after resection.2
Microenvironmental hypoxia is a common feature
of many diseases and can cause the production of a
series of adaptive responses to maintain oxygen
Keloids are formed when hyperplasia occurs in
connective tissues and exceeds the original dam-
age area in the healing process of skin injury such
as trauma, surgery, inflammation, or burns.1
Keloids have persistent proliferation characteris-
tics and are fibrous tissue tumors. In the field of
plastic surgery, keloid is the most prevalent dis-
ease. Its existence is extremely unfavorable for
psychological condition of patients and affects the
patients’ social activities. It needs timely treatment
to restore skin smoothness and improve symptoms.
And the treatment difficulty has become the
Department of Plastic Surgery, Linzi District People’s Hospital, Zibo,
China
Corresponding author:
Xiaolong Wu, Department of Plastic Surgery, Linzi District People’s
Hospital, No.139 Huan Road, Linzi District, Zibo 255400, Shandong,
China.
Email: weeshrupengwa@163.com
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use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and
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European Journal of Inflammation
3
homeostasis. A large number of transcription fac-
tors are involved in hypoxia response, while
hypoxia-inducible factor (HIF)-1 is considered to
be an important transcription factor mediating
converted to complementary DNA (cDNA) by
reverse transcription using cDNA synthesis kits.
Detection of messenger RNA expression
4
hypoxia response. HIF-1α plays an important role
in immune and inflammatory responses. In fact,
HIF-1α can regulate the differentiation of immune
cells, promote adhesion, affect cell metabolism,
induce angiogenesis and regulate cell proliferation
5
The resultant cDNA samples were used to perform
reverse transcription polymerase chain reaction
(RT-PCR) under the following conditions: dena-
turation at 95°C for 10min, then 40 cycles at 95°C
for 15s, and 60°C for 1min. The mouse GAPDH
gene expression value was normalized as an endog-
enous control for HIF-1α, interleukin (IL)-1β,
IL-2, IL-6, tumor necrosis factor (TNF)-α, Bax,
Bcl-2, livin and hPEBP4 gene expression.
6
and apoptosis. At present, there are few researches
on the relationship between HIF-1α and keloids. In
this study, we compared the difference of HIF-1α
in keloid tissues and normal skin tissues and fur-
ther explored the relationship between HIF-1α and
skin tissue inflammation and skin cell apoptosis.
Statistical analysis
Materials and methods
SPSS 20 software was used to analyze the data.
Comparisons on measurement data between the
two groups were conducted using t test, and corre-
lation analysis between the two groups was per-
formed using Pearson test. P<0.05 indicates a
significant difference.
Subjects
A total of 50 keloid specimens resected in our hos-
pital from November 2015 to February 2017 were
selected as the pathological group, and the general
information of the patients was as follows: 31
males and 19 females, age from 37 to 65years with
a mean age of 54.6±1.7years. A total of 50 cases
of the normal skin tissues from our hospital during
the same period were selected as the control group,
and the general information of the patients was as
follows: 30 males and 20 females, age from 36 to
Results
Comparisons on messenger RNA expression of
HIF-1α between the two groups
The messenger RNA (mRNA) expression of HIF-
1
2
α in keloid tissues in the pathological group was
.73±.35, while mRNA expression of HIF-1α in
6
4years with a mean age of 54.3±1.5years. All
patients voluntarily participated in the study and
signed informed consent. There were no signifi-
cant differences between the two groups on sex,
age, and other characteristics at baseline (P>0.05).
keloid tissues in the control group was 1.01±0.26.
The mRNA expression of HIF-1α in keloid tissues
in the pathological group was significantly higher
than that in the control group, as shown in Figure 1.
RNA extraction methods
Comparisons on mRNA expression of
TRIzol lysates were added to the pathological
keloid specimens and normal skin tissues and the
tissue was fully ground. A volume of 200μL of
chloroform was added to the suspension and the
suspension was kept unchanged after full shock.
The suspension was centrifuged after liquid strati-
fication at 12,000r/min speed for 20min. The
supernatant was absorbed adding equal volume of
isopropanol; a slight shock was applied and kept
static for 20min and then centrifuged at 12,000r/
min speed for another 20min to get RNA precipita-
tion. Add diethyl pyrocarbonate (DEPC) water
after drying RNA for the following study. Some
sample of RNA was dissolved in DEPC water and
inflammatory cytokines between the two groups
The mRNA expression of IL-1β, IL-2, IL-6, and
TNF-α in keloids in the pathological group was
significantly higher than that in the control group,
as shown in Figure 2.
Comparisons of mRNA expression of apoptosis
molecules between the two groups
The mRNA expression of apoptosis molecule Bax
in the pathological group was significantly higher
than that in the control group. The mRNA expres-
sion of anti-apoptotic molecules, including Bcl-2,

Wu
3
livin, and hPEBP4, in the pathological group was
significantly lower than those in the control group,
as shown in Figure 3.
Correlation analysis
Pearson test showed that there was a positive cor-
relation between the mRNA expression of HIF-1α
and inflammatory cytokines, including IL-1β,
IL-2, IL-6, and TNF-α. There was also a positive
correlation between the mRNA expression of HIF-
1
α and Bax and a negative correlation between the
mRNA expression of HIF-1α, Bcl-2, livin, and
hPEBP4 (P<0.05), as shown in Table 1.
Discussion
Keloid is a special type of scar, which has the char-
acteristics of continuous growth. Although it is
benign hyperplasia of fibrous tissue, it has aggres-
sive behavior of malignant tumor, and it is easy to
Figure 1. Comparisons of mRNA expression of HIF-1α
between the two groups (**P<0.01).
7
relapse after resection. At present, the pathogenesis
Figure 2. qT-PCR was performed to examine the mRNA expression of inflammatory cytokines (a) IL-β, (b) IL-2, (c) IL-6, and (d)
TNF-1α between control groups and pathological groups (**P<0.01, ***P<0.001).

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European Journal of Inflammation
Figure 3. qT-PCR was performed to examine the mRNA expression of (a) Bax, (b) Bcl-2, (c) livin, and (d) Hpebp4 between
control groups and pathological groups (**P<0.01, ***P<0.001).
Table 1. Correlation between mRNA expression of HIF-1α and inflammatory cytokines and apoptosis molecules.
HIF-1α mRNA IL-1β mRNA IL-2 mRNA IL-6 mRNA TNF-α mRNA Bax mRNA Bcl-2 mRNA Livin mRNA hPEBP4 mRNA
r
0.627
0.653
0.584
0.609
0.581
–0.125
–0.172
–0.166
P
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
HIF: hypoxia-inducible factor; IL: interleukin; mRNA: messenger RNA.
of keloids has not yet been fully elucidated. In fact,
keloid formation can cause inflammatory responses,
cell proliferation, and apoptosis. HIF-1α is the
effector molecule of cells under hypoxia, and con-
onaffectingthedownstreaminflammatoryresponses
and cell apoptosis maybe the possible pathway in
keloids.
8
The activation of inflammatory responses and
the secretion of inflammatory cytokines involved
in keloid formation can significantly promote the
tinuous hypoxia can increase the expression of HIF-
9
1
α. By analyzing the difference of HIF-1α
10
expression in keloid specimens and normal skin tis-
sues, we found that the mRNA expression of HIF-
proliferation of fibroblasts. After the formation of
keloids, the function of mononuclear macrophage
is activated, resulting in secretion of IL-1β, IL-2,
and IL-6 and inducing neutrophil aggregating in
local lesions. It can further promote the synthesis
1
α in keloid tissues was significantly higher. This
indicated that the increased expression of HIF-1α
was related to the formation of keloids, and its effect

Wu
5
of inflammatory mediators such as TNF-α and
increase inflammatory responses. By analyzing the
differences of inflammatory cytokines in keloids
and normal skin tissues, we found that the mRNA
expression of IL-1β, IL-2, IL-6, and TNF-α in
keloids was significantly higher, which indicated
that the increased expression of pro-inflammatory
factors is related to the formation of keloids.
Correlation analysis showed that there was a posi-
tive correlation between the mRNA expression of
HIF-1α and the expression of IL-1β, IL-2, IL-6,
and TNF-α, which meant that the increased expres-
sion of HIF-1α in keloids could affect the expres-
sion of inflammatory cytokines and promote
inflammation responses.
Funding
The author(s) received no financial support for the
research, authorship, and/or publication of this article.
References
1
. Butler PD, Longaker MT and Yang GP (2008) Current
progress in keloid research and treatment. Journal of
the American College of Surgeons 206(4): 731–741.
2. Carantino I, Florescu IP and Carantino A (2010)
Overview about the keloid scars and the elaboration
of a non–invasive, unconventional treatment. Journal
of Medicine and Life 3(2): 122–127.
3
. Zheng L, Kelly CJ and Colgan SP (2015) Physiologic
hypoxia and oxygen homeostasis in the healthy
intestine. A review in the theme: Cellular responses
to hypoxia. American Journal of Physiology Cell
Physiology 309(6): C350–C360.
The proliferation of fibroblasts in keloids is also
closely related to the abnormality of apoptosis pro-
4
. Zeitouni NE, Chotikatum S, Köckritz-Blickwede
MV et al. (2016) The impact of hypoxia on intesti-
nal epithelial cell functions: Consequences for inva-
sion by bacterial pathogens. Molecular and Cellular
Pediatrics 3(1): 14.
11
gram. Bax is a typical apoptosis gene that can
promote tumor cell apoptosis, while Bcl-2, livin,
and hPEBP4 are genes that can inhibit apoptosis.12
By comparing the difference in gene expression of
apoptosis and anti-apoptotic genes, we found that
the mRNA expression of the apoptosis molecule
Bax in pathological group was significantly higher.
However, the mRNA expression of anti-apoptotic
molecules including Bcl-2, livin, and hPEBP4 in
the pathological group was significantly lower.
The results indicated that the upregulated expres-
sion of apoptosis molecule and downregulated
expression of anti-apoptotic molecules were related
to the formation of keloids. Pearson test showed
that there was a positive correlation between the
mRNA expression of HIF-1α and inflammatory
cytokines including IL-1β, IL-2, IL-6, and TNF-α.
There was also a positive correlation between the
mRNA expression of HIF-1α and Bax and a nega-
tive correlation between the mRNA expression of
HIF-1α, Bcl-2, livin, and hPEBP4, which meant
that the increased expression of HIF-1α could
affect the expression of cell apoptosis molecules
and inhibit cell apoptosis.
5. Chen JY, Gu ZJ, Wu MX et al. (2016) C-reactive
protein can upregulate VEGF expression to promote
ADSC-induced angiogenesis by activating HIF-1α
via CD64/PI3k/Akt and MAPK/ERK signaling path-
ways. Stem Cell Research & Therapy 7(1): 114.
6
. Lampert FM, Kütscher C, Stark GB et al. (2016)
Overexpression of Hif-1α in mesenchymal stem cells
affects cell-autonomous angiogenic and osteogenic
parameters. Journal of Cellular Biochemistry 117(3):
7
60–768.
7
. NieZ,BayatA,BehzadFetal.(2010)Positiveresponse
of a recurrent keloid scar to topical methyl aminole-
vulinate-photodynamic therapy. Photodermatology,
Photoimmunology & Photomedicine 26(6): 330–332.
8. Seifert O and Mrowietz U (2009) Keloid scarring:
Bench and bedside. Archives of Dermatological
Research 301(4): 259–272.
9
. Chou CC, Chuang HC, Salunke SB et al. (2015) A
novel HIF-1α-integrin-linked kinase regulatory loop
that facilitates hypoxia-induced HIF-1α expression
and epithelial-mesenchymal transition in cancer cells.
Oncotarget 6(10): 8271–8285.
In summary, HIF-1α was highly expressed in
keloids and closely related to inflammatory
response cytokines and apoptosis molecules.
Increased expression of HIF-1α in keloids may be
an important factor in inflammatory responses and
increased apoptosis in skin tissues.
1
0. Dong X, Mao S and Wen H (2013) Upregulation of pro-
inflammatory genes in skin lesions may be the cause of
keloid formation. Biomedical Reports 1(6): 833–836.
1. Li H, Nahas Z, Feng F et al. (2013) Tissue engineer-
ing for in vitro analysis of matrix metalloproteinases
in the pathogenesis of keloid lesions. JAMA Facial
Plastic Surgery 15(6): 448–456.
1
Declaration of conflicting interests
1
2. Guo T, Li B and Gu C (2013) Expression of hPEBP4
negatively correlates with estrogen and progesterone
receptors in endometrial carcinoma. Journal of the
Balkan Union of Oncology 18(2): 465–470.
The author(s) declared no potential conflicts of interest
with respect to the research, authorship, and/or publication
of this article.