·Basic
Research·
Inhibitive
effect of TAK-242 on Tenon’s capsule fibroblasts proliferation in rat eyes
Liang Liang1,2, Meng-Nan Zhu 1,2,3,
Bao-Ji Chen1,2, Zheng Wang1,2, Li-Ye He1,2,
Rang Zhang1,2
1Department of
Ophthalmology, The First College of Clinical Medical Science, China Three
Gorges University, Yichang 443003, Hubei Province, China
2Department
of Ophthalmology, Yichang Central People’s Hospital, Yichang 443003, Hubei
Province, China
3Department
of Ophthalmology, Xianning Central Hospital, Xianning 437100, Hubei Province,
China
Correspondence
to: Liang
Liang. Department of Ophthalmology, The First College of Clinical Medical
Science, China Three Gorges University and Yichang Central People’s Hospital,
Yiling Road 183, Yichang 443003, Hubei Province, China. liangliang
419519@163.com
Received:
Abstract
AIM: To study the inhibition effect of TAK-242 on the proliferation of rat
eye Tenon’s capsule fibroblasts via the toll-like receptor 4 (TLR4)
signaling pathway.
METHODS: SD rat Tenon’s capsule fibroblasts were extracted
and cultured, then the cells were divided into normal control group,
lipopolysaccharide (LPS) group (
RESULTS: Double immunofluorescent labeling in the extracted
cells showed negative keratin staining and positive vimentin staining. Western
blot showed that the LPS group had the highest expression of TLR4 and TGF-β1 (P<0.01). Enzyme
linked immunosorbent assay (ELISA) also showed that the secretion of IL-6 was
the highest in LPS group (P<0.01). But there was no significant
difference in TLR4 and TGF-1, as well as IL-6 expressions between the TAK-242
group and the normal control group (P>0.05). RT-PCR showed that the
IL-6 mRNA expression in LPS group was the highest in the three groups (P<0.01).
CONCLUSION: TAK-242 inhibits the proliferation of LPS-induced
Tenon’s capsule fibroblasts and the release of inflammatory factors by
regulating the TLR4 signaling pathway, providing a new idea for reducing the
scarring of the filter passage after glaucoma filtration surgery.
KEYWORDS: Tenon’s capsule fibroblasts;
fibrosis; TAK-242; rat
DOI:10.18240/ijo.2019.11.06
Citation: Liang
L, Zhu MN, Chen BJ, Wang Z, He LY, Zhang R. Inhibitive effect of TAK-242 on
Tenon’s capsule fibroblasts proliferation in rat eyes. Int J Ophthalmol
2019;12(11):1699-1707
INTRODUCTION
Glaucoma
filtration surgery (GFS) is the golden standard for lowering intraocular
pressure (IOP) in glaucoma[1]. The success rate is
often limited by postoperative scarring of the filter passage[2].
Tenon’s capsule fibroblasts are the main cellular components of filtration
tract scar, which have been studied to reduce scar formation by inhibiting the
proliferation of human Tenon’s cystic fibroblasts[3-4]. TAK-242 is a cyclohexene derivative that blocks
toll-like receptor 4 (TLR4) signal path specifically and inhibits the
production of cytokines mediated by TLR4 selectively[5].
There are studies proved that TLR4 signal path plays an important role in
various organ fibrosis diseases[6-8].
Moreover, TLR4 may be associated with the pathological development of glaucoma[9], but there was no report about its effect on the
scarring of the filter passage after glaucoma surgery.
In the
study, we examined the effect of TAK-242, a specific antagonist of TLR4, on the
secretion of inflammatory cytokines and cell proliferation by Tenon’s capsule
fibroblasts, in order to verify the role of TAK
MATERIALS AND METHODS
Ethical
Approval All animals were conducted in line
with the Chinese Ministry of Science and Technology Guidelines on the Humane
Treatment of Laboratory Animals and the Association for Research in Vision and
Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision
Research. This study was approved by the Animal Care Committee of China Three
Gorges University.
Primary
Culture of SD Rat Tenon’s Capsule Fibroblasts Two SD rats aged 5-6wk were selected
and weighed. Intraperitoneal injection of chloral hydrate 10% anesthesia
(chloral hydrate 10% / rat body weight =0.35 mL/
SD Rat
Tenon’s Capsule Fibroblast Identification
After the
rat Tenon’s cystic fibroblast slide was taken, the fixed slides were rinsed
with PBS for 3 times. After the glass slide was dried, the cells were evenly
distributed in the cover glass slide with the histochemical pen to circle the
appropriate range, and the membrane breaking liquid was added to 50-100 μL, and
incubated at room temperature for 20min. Cell slides were removed and rinsed
with PBS for 3 times. The slides were placed in 5% milk and sealed at room
temperature for 1h. Then slides into a diluted with PBS containing 1% bovine
serum albumin (BSA) resistance of incubation,
Western Blot
Analysis of TLR4, Transforming Growth Factor-β1 Protein After the primary cells of Tenon’s
sac were cultured in groups, the total protein was extracted, and then the TLR4
protein expression was detected by Western blot. Western blot was used to
detect the expression of transforming growth fautor (TGF)-β1 protein in three
groups of cells by the same method.
Enzyme
Linked Immunosorbent Assay After administration of Tenon’s
cystic fibroblasts in groups, the content of IL
Reverse
Transcriptase-Polymerase Chain Reaction The relative transcriptional levels
of IL
Cell
Counting Kit-8 Detect the Proliferation Activity of the Three Groups Tenon’s capsule fibroblasts were
inoculated into 96-well plate. After cell adherence, the inoculated cells were
divided into three groups: normal control group, lipopolysaccharide (LPS) group
and TAK-242 group. The normal control group did not do any treatment. The LPS
group was treated with 10 μg/mL LPS. The TAK-242 group was treated with 1
μmol/L TAK-242. Next, cell counting kit-8 (CCK-8) was administered and cultured
in the absence of light. After a series of operations, the OD values of the
three groups were detected by enzyme-labelled instrument.
RESULTS
Morphologic
Observation Microscopically, small pieces of
tissue digested by trypsin as chylous are seen scattered in the medium, the
cells dissociate from the tissue in a clear, round shape. On the second day, a
small number of cells start to adhere to the wall and continue to move out of
the tissue mass. After adherent to the wall, the cells become elongated
fusiform, divide and proliferate. About 10d after inoculation, when the cells
covered 75%-80% of the bottle bottom, the cells were digested, centrifuged and
subcultured. After passage, most of the cells were elongated fusiform and
distributed in vortices, and some cells were irregular in shape. The cell
morphology is shown in Figure 1. The results showed that the cells accord with
the morphology of Tenon’s capsule fibroblasts.
Figure 1
Primary culture of SD rat Tenon’s capsule fibroblasts (100×) A: In the center, tissue fragments
digested by trypsin. The cells migrate from the tissue fragments and divide and
proliferate. The cells are spindle shaped or spindle-shaped, and a few are
triangular. B: The cells are arranged in bundles or spirals after confluence.
Cell
Identification Cy3-labeled keratin should be red
fluorescence, FITC-labeled vimentin should be green fluorescence, and DAPI
labeled nucleus should be blue fluorescence (Figure
Figure 2
Identification of keratin vimentin: double immunofluorescent labeling
(400×) A-C: Vimentin staining was positive,
green fluorescence was seen in the cytoplasm (A), blue fluorescence was seen in
the nucleus (B), and C was co-development of cytoplasm and nucleus. D-F:
Keratin staining was negative, cytoplasm red fluorescence was not visible (D),
the nucleus was blue fluorescence (E), and only the nucleus fluorescence could
be seen in the cytoplasm co-development (F).
Western
Blot It was found that the TLR4 protein
expression level of LPS group was significantly higher than that of normal
control group and TAK-242 group. The relative gray value of each band was
determined and statistically analyzed (Figure 3). The results showed that LPS
could cause the increase of TLR4 protein expression, while TAK-242 could
significantly inhibit the increase of TLR4 protein expression induced by LPS.
The expression levels of TGF-β1 protein in the LPS group were significantly
higher than those in the normal control group and the TAK-242 group. We
measured the relative gray value of each band and analyzed statistically
(Figure 4). The results showed that LPS can increase the expression of TGF-β1
protein, while TAK-242 can significantly inhibit the expression of TGF-β1
protein induced by LPS, suggesting that TAK-242 can inhibit the release of
inflammatory factors downstream of TLR4 signal path.
Figure 3
Expression of TLR4 protein in each group was detected by Western blot After LPS intervention, TLR4 protein
expression level in the LPS group increased significantly compared with that in
the normal control group (bP<0.01). In the TAK-242 group,
TLR4 protein expression was significantly lower than that in the LPS group due
to the intervention of TAK-242 (bP<0.01). There was no
significant difference between TAK-242 group and normal control group (P>0.05).
Figure
4 Western blot analysis of protein
expression of TGF-β
Enzyme
Linked Immunosorbent Assay The content of IL
Figure 5
ELISA detect the secretion level of IL
Reverse
Transcriptase-Polymerase Chain Reaction
The relative
transcription levels of IL-6 mRNA in three groups of cells were further
detected. After grouping Tenon’s capsule fibroblasts and total RNA was
extracted, IL-6 mRNA expression was detected by RT-PCR in each group. The
results showed that the relative expression of IL-6 mRNA in LPS group was
significantly higher than that in normal group and TAK-242 group, and the
relative expression level of lL-6 mRNA in TAK-242 group was significantly
higher than that in normal control group (Figure 6).
Figure 6
RT-PCR detect the relative transcription levels of IL-6 mRNA in each group A: Solubility curve. B: The
amplification curve: a stands for LPS group; b stands for group TAK-242; c is
the normal control group. All curves are unimodal distribution and the peak is
concentrated. C: After the intervention of LPS, the relative transcription
level of IL-6 mRNA in the LPS group was significantly raised compared with that
in normal control group (bP<0.01). In the TAK-242 group,
the relative transcription level of Il-6 mRNA was significantly lower than that
in the LPS group due to the intervention of TAK-242 (bP<0.01).
Compared with the normal control group, the relative transcription level of
IL-6 mRNA increased in the TAK-242 group (bP<0.01).
Cell
Counting Kit-8 Tenon’s cystic fibroblasts were
inoculated into 96-well plates, and the inoculated cells were divided into
three groups: normal control group, LPS group, and TAK-242 group. After
pre-culture, groups were treated differently. 1) The normal control group did
not do any treatment; 2) The LPS group add LPS of 10 μg/mL; 3) The TAK-242
group add TAK-242 of 10 μg/mL. After a series of operations, such as CCK-8
administration and light-avoiding culture, OD values of the three groups were
detected by microplate reader. CCK-8 is based on the principle that, under the
action of the carrier, methyl thiazolyl tetrazolium-Water-Soluble Tetrazolium-8
(WST-8) is reduced to yellow formazan (Formazen dye) by cellular dehydrogenase in
the mitochondria, and the number of formazan molecules generated, namely the
gradation of color, is used to reflect the number of living cells. The results
showed that OD value of LPS group was significantly higher than that of normal
control group and TAK-242 group (P<0.05), while OD value of TAK-242
group was slightly higher than that of LPS group, but the difference was
unobvious (P<0.05; Figure 7). This indicated that TAK-242 played a
role in inhibiting cell proliferation induced by LPS.
Figure 7
CCK-8 detect cell proliferation in each group OD value in the LPS group increased
significantly compared with that in the normal control group after LPS
intervention (aP<0.05). In the TAK-242 group, OD value
decreased significantly compared with LPS group due to the intervention of
TAK-242 (aP<0.05). However, there was no significant
difference in OD value between the TAK-242 group and the normal control group (P>0.05).
DISCUSSION
Glaucoma is
one of the leading causes of irreversible blindness worldwide. Glaucoma
filtering operation remains the gold standard for glaucoma in patients who
cannot control IOP with medication
or laser therapy. The success rate of glaucoma filtering operation is often
limited by postoperative scarring of the filtration tract[10].
The excessive synthesis of extracellular matrix (ECM) and the scar formed by
the contraction of subconjunctival tissue block the filtration channel and
prevent the outflow of aqueous humor, leading to the increase of IOP and the
failure of surgery ultimately[11]. Mitomycin C,
5-fluorouracil and other antimetabolites can inhibit fibroblast proliferation
and reduce postoperative scar formation to a certain extent. However, these
drugs can cause more postoperative complications, such as long-term low IOP,
corneal epithelial defect, conjunctival wound leakage, follicular infection,
endophthalmitis and so on[12-13].
Therefore, we need a safer and more effective drug to fight the scarring after
GFS.
TAK-242 is a
cyclohexene compound that can specifically inhibit the TLR4 signal path.
Although some synthetic lipid A analogues have been reported to act as LPS or
TLR4 antagonists, TAK-242 is the first small molecule compound to inhibit the
production of TLR4-mediated cytokines selectively[14-15]. The action principle is inhibiting TLR4 signal
transduction by Cys747 binding to the toll-interleukin-1 receptor (TIR) domain
of target protein TLR4 and disrupting the interaction between TLR4 and its
downstream adaptor molecule TIRAP containing TIR domain and inducing the interaction
between TLR4 and its downstream adaptor molecule TRIF-related adaptor protein
(TRAM) containing interferon beta TIR domain[16].
The TLRs is a pattern recognition receptor that recognizes specific
pathogen-associated molecular patterns (PAMPs) that are present in pathogens
but not in mammalian cells. TLR is known to be highly expressed in innate
immune cells in response to pathogens and environmental stress, to participate
in the detection of foreign pathogens (bacteria, viruses or fungi) and to
regulate innate and immunological adaptive response[17-18], thus becoming the first line of defense against
infection. TLR4, as a member of the family, plays a key role in infection and
fibrosis. It is a type I transmembrane protein, which is composed of three
parts: extracellular domain, transmembrane domain and intracellular domain[19]. TLR4 signal pathway transduction mainly includes
MyD88-dependent pathway and MyD88-non-dependent pathway, the former is not
dependent on MyD88 protein activation, the latter is mainly dependent on MyD88
activation to induce the rapid production of inflammatory factors[19]. When the activation pathway of TLR4 is poorly
regulated, it will lead to further development of the disease. For example,
Astafurov et al[9] found in their study
that TLR4 signal transduction and complement system up-regulation in glaucoma
animal models would further lead to optic nerve atrophy, aggravating the
degeneration of optic nerve in model animals. The level of TLR4 and its
endogenous ligands is increased in systemic sclerosis (SSc), and it has a
strong and effective stimulation effect on the expression of fibrosis genes.
Genetic differences of TLR4 or its endogenous ligands can be targeted to
improve the experimental fibrosis in the SSc model of mice[20-22]. In liver injury, blocking the TLR4 signaling pathway
can inhibit the transformation of Hepatic stellate cell (HSC) into
myofibroblasts and the production of type 1 gels, thereby inhibiting the
formation of liver fibrosis. Studies have shown that TLR4 genetically deficient
mice can also fight experimental liver fibrosis[9,23]. There is increasing evidence that TLR4 is an
important player in the development and progression of inflammatory and
fibrotic diseases and an excellent therapeutic target for fibrotic diseases. We
hypothesized that drugs targeting the inhibition of TLR4 may be of great
significance in the prevention and treatment of glaucoma filtration tract
scarring.
Fort et
al[15] found that TAK-242 inhibited the production
of LPS-induced inflammatory mediators such as tumor necrosis factor-ɑ (TNF-α),
IL-1β, IL-6 and nitric oxide (NO) at similar concentrations. In addition, the
inhibitory effect of TAK-242 on cytokines was similar in both mouse and human
macrophages, which may indicate that the difference in species does not have
much impact on the efficacy of TAK-242. Zhang et al[20]
found that TAK-242 was significantly effective against
aldosterone-induced cardiac remodeling and renal fibrosis. In this study, we
investigated the effect of TLR4 specific blocker TAK-242 on inhibiting the
proliferation of rat eye Tenon’s capsule fibroblasts.
Gram-negative
bacteria LPS is a recognized ligand of TLR4, which can activate tlr4-mediated
signaling pathways. Since Molteni et al[24]
discovered the role of TLR4 as LPS sensor of bacteria, other endogenous and
exogenous ligands have been discovered successively. PAMPs are isolated from
bacteria, viruses, fungi, plants and cyanobacteria. Most of them are
TLR4/(myeloid differential protein-2) MD2 complex agonists, and a few have
blocking effect. There are two main types of damage-associated molecular
patterns (DAMPs): 1) molecules originating from the ECM; 2) intracellular
mediators are released or actively secreted by cells[24-27]. There is no doubt that endogenous molecules can
induce pro-inflammatory responses through TLR4. Under normal conditions,
hyaluronic acid exists in tissues in the form of a polymer. After tissue
damage, it is decomposed into small fragments, which can activate macrophages
in vivo and in vitro through TLR4[28]. Endogenous
TLR4 intracellular triggers include: DNA binding protein high mobility group
protein 1 (HMGB1) and cellular heat shock protein (HSP). After cell injury and
necrosis, these molecules are released into the extracellular environment, thus
causing a strong TLR4-mediated pro-inflammatory response[24].
In addition to its role in aseptic inflammation, HMGB1 is also actively
released after exposure of immunoactive cells to pathogen products, so it is a
common mediator between infectious and non-infectious inflammatory responses[24]. Studies have shown that Tenon’s capsule fibroblasts
are the main components of scar formation after glaucoma filtration, and the
excessive proliferation and fibrosis main causes of scarring in the filter
passage[29]. Therefore, the successful extraction
and culture of SD rat Tenon’s capsule fibroblasts are the basis of our
follow-up study on the scar formation of the filter passage. In this study, we
extracted Tenon’s capsule subconjunctival tissue of SD rats and successfully
isolated the primary cells with elongated spindle shape and fascicular
arrangement by trypsin digestion. Combined with the morphology and arrangement
of primary cells, these were identified by immunofluorescence method as Tenon’s
capsule fibroblasts. In the subsequent experiments, we selected cells with
better vitality of 3-5 generations. The LPS was selected as the TLR4 signaling
pathway agonist. Investigating the effect of TAK-242 on inhibition of mouse eye
Tenon’s capsule fibroblast proliferation, we combined action of LPS and
TAK-242.
As mentioned
above, considering the important role of TLR
The wound
healing response at the subconjunctival follicular site is mediated in part by
matrix metalloproteinases (MMPs) and is regulated by a variety of molecules
including growth factors and inflammatory mediators. Cytokine TGF-β is a key
regulator of wound healing and fibrosis and a major driver of conjunctival scar
formation[31]. Both TGF-β1 and TGF-β2 can be
detected on subconjunctival wounds after GFS[32].
Induction of collagen gel contraction by TGF-β1 and conversion of fibroblasts
into myofibroblasts play an important role in scar formation and contraction of
surgical incision[11]. Therefore, these factors
have become an important target of the development of anti-fibrosis programs.
Stifano et al[33] found in their
experiments that TLR4 and its assistant receptors MD2 and CD14 were
overexpressed in the dermal lesions of patients with diffuse skin SSc, and that
inflammatory chemokines were overexpressed and TGF-β genes were raised in their
chronic skin LPS exposure model. Stifano et al[33]
found in their experiments that TLR4 and its assistant receptors MD2 and CD14
were overexpressed in the dermal lesions of patients with diffuse SSc, and that
inflammatory chemokines were overexpressed and TGF-β genes were raise in their
chronic skin LPS stimulation model. Seki et al[23]
also suggested that LPS could enhance signal transduction at TGF-β by
activating the TLR4-MyD88-NF-κB
axis and thereby promote the development of inflammation and fibrosis.
Our results
indicate that LPS induces increased secretion of TGF-β
IL-6 is a
multifunctional monomolecular glycoprotein cytokine with extensive biological
activities and plays an important role in the occurrence and development of
multiple diseases. It has the function of promoting the proliferation of
fibroblast B cells, T cells and other cells. Studies have found that IL-6 is
closely related to the occurrence and development of periodontitis, oral lichen
planus, renal fibrosis, liver fibrosis and other inflammation and fibrosis
diseases[34-35]. For example,
studies have mentioned that LPS directly acts on periodontal fibroblasts,
which, as helper cells of immune response, produce inflammatory cytokines such
as IL-6, TGF-β1, TNF-α and so on, mediate inflammatory reactions and
participate in the destruction process of periodontal tissue, thus
participating in the whole process of the occurrence and development of
periodontitis. IL-6 plays an important role in the histopathological damage of
periodontitis caused by LPS[35-36].
Other studies have reported that IL-6 can promote fibroblast proliferation and
mRNA expression of collagen I and collagen III. IL-6 can also enhance the
expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA, inhibit
collagenase activity and promote the expression of α2-macroglobulin in
monocytes, thereby reducing the decomposition of ECM. In addition, inflammatory
cytokines IL-6 can also stimulate cells to release more cytokines such as TGF
and PDGF, which can play a positive feedback amplification effect, produce more
ECM and aggravate the course of fibrosis[37-38].
In this
experiment, the cells were divided into normal control group, LPS group and
TAK-242 group, and different treatments were taken respectively. Then, the
secretion of IL
CCK-8, the
principle of which is in carrier under the action of tetrazolium salt-WST-8,
all referred to as: 2-(2-methoxy-4-(phenyl)-3-[4-(phenyl)-5-(2, 4-disulfonic
acid benzene)- 2h-tetrazolium monosodium salt)], by reduction of cells in
mitochondria dehydrogenase yellow formazan (Formazen dye), which used to
generate a formazans is the number of gradation of color to reflect the number
of living cells. In recent years, CCK-8 kit, as a highly sensitive, fast, safe
and effective method, is often used to detect the toxic effects of cell
proliferation[39]. The absorbance value (OD
value) detected by the enzyme marker in the experiment represents the lactate
dehydrogenase (LDH) activity of lactate dehydrogenase in mitochondria, which
can reflect the degree of cell proliferation, the rate of death and the
metabolic rate. Compared with the traditional
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, CCK-8
kit method makes the experimental operation easier, the experimental process
faster and the experimental results more accurate[40-41]. Therefore, CCK-8 kit was used in this experiment to
measure the degree of cell proliferation in the three groups.
In this
experiment, CCK-8 kit was used to detect the degree of proliferation of Tenon’s
capsule fibroblasts in three groups: normal control group, LPS group and
TAK-242 group. The results showed that TAK-242 significantly inhibited cell
proliferation induced by LPS. This indicated that in vitro exposure to LPS can
cause significant proliferation of Tenon’s capsule fibroblasts, and such a
degree of cell proliferation can be inhibited by TLR4 specific blocker TAK-242.
In
conclusion, TAK-242 can inhibit the proliferation and release of inflammatory
cytokines in Tenon’s capsule fibroblasts induced by proinflammatory factor LPS.
TAK-242 acts as an effective specific TLR4 inhibitor, and this inhibition is
accomplished by antagonistic to the up-regulated TLR4 signaling pathway.
TAK-242 provides a new method to solve the scarring of glaucoma filter passage
in the future.
ACKNOWLEDGEMENTS
Foundations: Supported by National Natural
Science Foundation Program of China (No.81770920); Hubei Health and Family
Planning Commission Youth Talent Project (No.WJ2017Q037).
Conflicts of
Interest: Liang L, None; Zhu MN, None; Chen BJ, None; Wang Z, None; He
LY, None; Zhang R, None.
REFERENCES