ORIGINAL PAPER

International Journal of Occupational Medicine and Environmental Health 2018;31(4):1 – 13

https://doi.org/10.13075/ijomeh.1896.01037

SUBCUTANEOUS ADMINISTRATION
OF INFLIXIMAB-ATTENUATED
SILICA-INDUCED LUNG FIBROSIS
HUA ZHANG1,2, JUN-NA SUI3, LEI GAO4, and JIAN GUO3
1
Qilu Hospital of Shandong University, Jinan, China
Department of Respiratory Medicine
2
Qingdao Central Hospital, Qingdao, China
Department of Occupational Disease
3
Qingdao Central Hospital, Qingdao, China
Department of Clinical Laboratory
4
Qingdao Blood Center, Qingdao, China
Department of Clinical Laboratory

Abstract
Objectives: To investigate the influence of the anti-tumor necrosis factor-α infliximab (IFX) in the case of rats with silicosis.
Material and Methods: Forty-eight Wistar rats were randomly divided into 3 groups. The study group (N = 16) – silicosis
was induced by intratracheal instillation of  50  mg  silica on day  1, and  IFX was subcutaneously administered at a  dose
of 15 mg/kg of body weight from day 2 to day 6, the vehicle group (N = 16) – silica used as the study group but without IFX,
the sham group (N = 16) – 1 ml of saline was intratracheal-used. Eight rats in each group were euthanized on day 7 and on
day 14, respectively. Lung tissue sections were stained with hematoxylin and eosin or Masson’s trichromedye. The nuclear
factor-κB p65 (NF-κB p65) positioning in the lung tissues were determined by immunohistochemical staining. Levels of tu-
mor necrosis factor α (TNF-α) in rat serum and bronchoalveolar lavage fluid were measured with enzyme linked immuno-
sorbent assay. The inducible nitric oxide synthase (iNOS) mRNA in the lung tissues was measured by quantitative real-time
polymerase chain reaction, as well as inhibitor protein-κB (I-κB) and NF-κB p65 expression were measured quantitatively
by western blotting. Results: Silica installation increased the lung tissues inflammation reaction, oxidative stress and pul-
monary fibrosis. Infliximab treatment significantly improved silica-induced lung pathological changes (inflammatory cells,
collagen deposition), decreased the TNF-α inhibited NF-κB signaling (I-κB, NF-κB p65) as well as oxidant status (iNOS).
Conclusions: Infliximab may improve silica-induced pulmonary inflammation by decreasing the TNF-α, inhibiting NF-κB
signaling (I-κB, NF-κB p65) as well as oxidant status (iNOS), which suggest that IFX has potential role in the treatment of
silica-induced lung damage. Int J Occup Med Environ Health 2018;31(4)

Key words:
Infliximab, Silicosis, TNF-α, Rat model, NF-κB, iNOS

Funding: this work was supported by Qingdao Science and Technology Development (project No. KJZD-12-23-nsh entitled “Effects of Tumor Necrosis Factor-alpha
in Pathogenesis and Therapy of silica Exposed animal Model,” project manager: Hua Zhang, Ph.D.).
Received: June 12, 2016. Accepted: September 25, 2017.
Corresponding author: J. Guo, Qingdao Central Hospital, Department of Clinical Laboratory, 127  Siliu Nan Road, Qingdao  260042, China (e-mail:  Wudao-
[email protected]).

Nofer Institute of Occupational Medicine, Łódź, Poland 1

 ORIGINAL PAPER     H. ZHANG ET AL.

INTRODUCTION emia/reperfusion, airway smooth muscle hyper-reactivity

Silicosis caused by inhalation of crystalline silica is an ir­ of asthma and cigarette smoking-induced emphysema by
reversible potentially fatal lung pneumoconiosis, charac- reducing lung inflammation and improving pathological
terized by inflammation and fibrosis of the lung [1,2]. De- changes [23–26]. It is also effective in attenuating bleomy-
spite significant progress in its research, the exact mecha- cin-induced  (BLC-induced) pulmonary fibrosis and  IFX
nism of silicosis has not yet been elicited. Silicosis is be- per se does not result in pulmonary fibrosis [27].
lieved to be initiated by an increased number of activated Since no more biochemical and histopathological changes
alveolar macrophages, releasing numerous inflammatory on pulmonary fibrosis induced by silicosis in the case of rats
mediators, which play crucial roles in the occurrence and by  IFX pretreatment had been reported, the aim of this
development of the disease [3]. Despite extensive effort, study was to examine the protective effects and mechanism
no significant effective treatment method is available to of action of subcutaneously-administered IFX on the silica-
reverse or slow the disease process [4–6]. Novel approach- induced lung inflammation and fibrosis in the rat model.
es for key cytokines targeted at treatment of silica-induced
inflammation and fibrosis are urgently needed. MATERIAL AND METHODS
Tumor necrosis factor  α  (TNF-α) as an activator, plays Dust sampling and suspension preparation
a  key role in the inflammatory and fibrogenic responses Silica content of the quartz dust (Sigma-Aldrich, St  Lou-
of silicosis [7]. Exogenous recombinant TNF-α augments is, MO, USA) was > 99% and the particle size of the dust
silicotic fibrosis and anti-TNF antibody attenuates silico- was 0.5–10 μm with 80% of the dust particles were 1–5 μm.
sis, and silicosis are absent in TNF-α receptor knockout Crystalline silica particles were suspended in saline at
mice [8–10]. Tumor necrosis factor α in a canonical man- 50 mg/ml after sterilization (121.3°C, 103.4 kPa for 90 min),
ner triggers nuclear factor-κB  (NF-κB) activation which and then added with penicillium to make the final con­
may occur in several pathways [11–13]. Nuclear factor-κB centration of 8000 U/ml in silicon suspension [28].
as a transcription factor plays a fundamental role in the
pathogenesis of silicosis. Binding of  NF-κB to  DNA Animals and experimental design
promotes up-regulation of inducible genes involved in Male Wistar rats (6–8-week old, 180–220 g in weight) were
mediating the inflammatory and fibrotic responses to obtained from the Food and Drug Administration, Qing­
silica  [14–16]. Tumor necrosis factor  α also activates in- dao, China and kept on an alternating 12 h light / 12 h dark
ducible nitric oxide synthase  (iNOS) to produce nitric cycle under standard temperature (22±2°C) and humid-
oxide (NO) in respiratory epithelium [17]. As a reactive ity (60±5%). The rats were acclimatized for 7 days before
nitrogen species,  iNOS-derived  NO contributes to the the start of the experiment with standard chew food and
pathogenesis of silica-induced lung disease, up-regulating water freely available. The animal experimental protocol
inflammatory cytokines and amplifies the inflammatory complied with the Animal Management Rules of the Chi-
response [18,19]. nese Ministry of Health (document No. 55, 2001) and was
Infliximab (IFX) is a chimeric monoclonal immunoglobu- approved by the Animal Care Committee, Qingdao Food
lin G1, neutralizing TNF-α biologic activity with a high af- and Drug Administration.
finity and specificity to human membrane-bound and solu- Forty-eight rats were randomly divided into 3 experimental
ble TNF-α [20–22]. Infliximab has demonstrated a protec- groups according to their experimental treatment as follows:
tive effect on acute lung injury induced by intestinal isch- the treatment group (N = 16), the vehicle group (N = 16)

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and the sham group (N = 16). The vehicle group was anes- average score was calculated for each group  [15]. The
thetized with  40  mg/kg pentobarbital sodium via intra- Masson’s trichrome staining method was used for deter-
peritoneal injection and received a  single trans-oral  (TO) mining the severity of fibrosis and the quantitative grade
intratracheal instillation of silica suspension of 1 ml as of pulmonary fibrosis was blindly scored by randomly se-
described previously  [29]. The sham group received the lecting regions according to the method introduced by Or-
same procedure but received an equal volume of saline in- tiz et al. [16]. A score ranging from 0 to 8 was assigned and
stead of silica. The treatment group received a single dose the scores were averaged per group.
of 15 mg/kg IFX (Remicade, Cilag AG Pharmaceutical Com-
pany, Switzerland, registration certificate No. for the import- Immunohistochemical assessment
ed drug: s20120012) dissolved in 0.2 ml of a diluent consisting of nuclear factor-κB p65 (NF‑κB p65) in lung tissue
of normal saline subcutaneously on day 2 to 6 for 5 days over- Lung sections, 4 μm thick, were deparaffinized and then
all after silica installation as described previously [30]. Ani- soaked in hydrogen peroxide  (3%) for  15  min to inacti-
mals were sacrificed using sodium pentobarbital (120 mg/rat) vate endogenous peroxidase antigens. Antigen retrieval
on days 7 or 14 after silica instillation. was performed by means of a pressure cooker (ethylene
diaminetetraacetic acid, pH  =  9; for  15  min at  90°C).
Peripheral blood Following serum sealing for  30  min, primary anti-
and bronchoalveolar lavage fluid (BALF) NF‑κB  p65  (Proteintech group,  Inc.,  USA) was added
Blood samples were harvested from abdominal aorta. and incubated for  1  h  at  37°C. The slides were washed
Serum was separated by centrifugation and then stored with phosphate buffered saline (PBS) and then incubated
at –80°C for analyses. with peroxidase-conjugated anti-rabbit  IgG (Proteintech
After the blood had been sampled, the right lung was group, Inc., USA) for 0.5 h at 37°C. Staining was then per-
ligated at the bronchi with surgical wire and  BALF was formed using a 3,3’-diaminobenzidine staining kit (ZSGB-
collected by washing the left lung with 3 separate aliquots Bio Co. Ltd., China). Phosphate buffered saline was used
of 2 ml saline at 37°C through a tracheal cannula. Broncho­ in the negative control group instead of primary antibody.
alveolar lavage fluid was centrifuged and stored at –80°C. A positive reaction was indicated by brown staining in the
nuclei and cytoplasm. The immunohistochemical staining
Pathologic examination was evaluated under an Olympus light microscope, Image-
of lung inflammation and fibrosis Pro Plus software v. 6.0 (Media Cybernetics, Inc., USA).
The intact right lung lobes were removed, blotted dry,
the middle lobe was fixed in 10% formalin and processed Determine TNF-α level in serum and BALF
for routine histology in paraffin wax. Sections were pre- Tumor necrosis factor α was measured in BALF and se-
pared of 4 mm in size, stained with hematoxylin and eo- rum using commercial enzyme-linked immunosorbent
sin (H&E) for histopathology and Masson’s trichrome for assay (ELISA) kits (R&D Systems, USA) used according
collagen deposition. The lung tissues were examined in to the manufacturer’s instructions.
random and blind conditions with standard light micros­
copy. The degree of lung tissue inflammation was evalu- Determine iNOS mRNA
ated quantitatively by  H&E staining according to its se- The quantitative analysis of mRNA for iNOS and β-actin
verity. A score ranging from 0 to 4 was designed and the was performed by real-time polymerase chain reaction

IJOMEH 2018;31(4) 3
 ORIGINAL PAPER     H. ZHANG ET AL.

(PCR) using CFX96 real-time PCR detective system (Bio- gel)  (40  μg/well) and electrophoretically transferred to
Rad Laboratories, Inc., USA) and SYBR Green technol- polyvinylidenedifluoride  (PVDF) membranes. After
ogy. Total RNA was extracted from lungs with the RNA­ blocking for 3 h with 5% skim milk in phosphate buffered
isoPlus Kit (Takara Bio, Inc., Japan) according to the man- saline with tween (PBST) (PBS with 1% polyoxyethylene-
ufacturer’s instructions. Ribonucleic acid purity and con- sorbitan fatty acid esters) buffer at room temperature,
centration were determined using a  spectrophotometer membranes were incubated overnight at  4°C  with poly-
NanoDrop2000c (Thermo Fisher Scientific  Inc.,  USA). clonal rabbit anti-rat  NF-κB  p65 (1:5000) (Proteintech
Complementary DNA was synthesized from deoxyribonu- Group,  USA), polyclonal rabbit anti-rat I-κB (1:1000)
clease-treated total RNA (1 μg) with the PrimeScript RT (Immunoway Inc., USA) in skim-milk PBST. The PVDF
reagent kit with gDNA Eraser (Takara Bio, Inc., Japan) membranes were washed for 3 times with PBST and in-
and reverse transcription-real-time  PCR was performed cubated with peroxidase-conjugated polyclonal goat
with the SYBR Premix EX Taq II (Takara Bio, Inc., Japan) anti-rabbit secondary antibody (1:5000) (Beijing Biosyn-
according to the manufacturer’s protocol. thesis Biotechnology  Co.,  Ltd., China) for  1  h  at room
The primers (SangonBioteck,  Co.,  Ltd, China) had the temperature.
following sequences: Following final washes, protein signals were visualized
1. iNOS – forward 5’-ACGGAGAACAGCAGAGTTGG-3’ using the enhanced chemiluminescence reagents (Bei-
and reverse 5’-TGTTGGGCTGGGAATAGCAC-3’; jing ComWin Biotech Co., Ltd, China) and detected by
2. β-actin – forward  5’-CCCATCTATGAGGGTTAC-3’ the fusion imaging system (Vilber Lourmat Company,
and reverse 5’-TTTAATGTCACGCACGATTTC-3’. France). Beta-actin protein was visualized and detected
The following thermocycling conditions were used: in the same way. The intensity of each signal spot was
initial polymerase activation step for  30  s at  95°C, fol- quantified using the ImageJ  2x  software (Wayne Ras-
lowed by  40  cycles of  5  s  at  95°C  for template denatur- band, National Institutes of Health, USA) that was used
ation, 30 s at 60°C for annealing and extension. All sam- for semiquantifing mean optical density of  NF-κB p65
ples were amplified by fluorescence measurement in trip- and I-κB expression.
licates and the mean was used for quantitative real-time
polymerase chain reaction  (RT-qPCR) analysis. Relative Statistical analysis
levels of target  mRNA expression were calculated using All data is presented as mean (M) ± standard error (SE).
the 2–ΔΔCT method and normalized by endogenous control The statistical analyses were performed using  SPSS  22.0
(β-actin). software (SPSS Inc., USA) and GraphPad Prism 6.0 (Graph-
Pad, USA). Statistical significance was determined by one-
Analysis of NF-κB p65 and inhibitor protein-κB (I-κB) way analysis of variance (ANOVA) followed by the post hoc
Total protein was extracted from lung tissue using pro- least significant difference test (LSD-t) and p < 0.05 was
tein extraction reagents (Shanghai Solarbio Bioscience considered statistically significant.
& Technology Company, China), and protein concentra-
tions measured using a BCA protein assay kit (Solarbio, RESULTS
China) according to the manufacturer’s instructions. Histopathological findings
Protein extracts were loaded onto a  12%  sodium do- Representative sections of rat lung tissue are shown in the
decyl sulphate- polyacrylamide gel  (SDS-polyacrylamide Figure 1. Normal alveolar structure, lack of inflammatory

4 IJOMEH 2018;31(4)
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a) b) c)

d) e) f) Group:
5 control

Pulmonary inflammation [pts]

vehicle
4 treatment
* *
3
2
1
0
7 14
Time after silica instillation [days]
g) h) i)

j) k) l) Group:
5 control
Pulmonary fibrosis [pts]

vehicle
4 treatment *
*
3
2
1
0
7 14
Time after silica instillation [days]

Hematoxylin and eosin (H&E) staining: a) sham group, day 7; b) vehicle group, day 7; c) treatment group, day 7; d) vehicle group, day 14;
e) treatment group, day 14.
Pulmonary inflammation and fibrosis score (0 – normal lung, 8 – total fibrous obliteration of the microscope field): f) on day 7 after instillation
of silica or saline control, l) on day 14 after instillation of silica or saline control. Data presented as mean ± standard error.
* p < 0.05 vehicle group vs. sham group (one-way analysis of variance and least significant difference test).
Masson’s trichrome staining: g) sham group, day 7; h) vehicle group, day 7; i) treatment group, day 7; j) vehicle group, day 14; k) treatment group,
day 14.
Treatment group (N = 16) – silicosis was induced by intratracheal instillation of 50 mg silica on day 1, and IFX was subcutaneously administered
at a dose of 15 mg/kg of body weight from day 2 to day 6.
Vehicle group (N = 16) – received silica as treatment group but without IFX.
Sham group (N = 16) – received the same procedure as vehicle group but with equal volume of saline instead of silica.
Eight rats in each group were euthanized on day 7 and the next 8 rats on day 14 after silica instillation.

Fig. 1. Representative sections (4-μm thickness) of rat lung tissue stained with H&E for histopathology and Masson’s trichrome
for collagen deposition (original magnification ×200)

IJOMEH 2018;31(4) 5
 ORIGINAL PAPER     H. ZHANG ET AL.

cellular infiltration and fibrous thickening, was observed (Figure 1g). In contrast, the untreated vehicle group showed
in the case of the sham rats (Figure 1a). weak collagen deposition on day 7 (Figure 1h and 1i). In-
On day 7 after instillation, acute inflammation and alveo- creases in collagen deposition began remarkable following
lar wall thickening with infiltration of macrophages, lym- instillation on day  14  (Figure  1j). The degree of fibrosis
phocytes and neutrophils into the alveoli were observed in was suppressed in the lungs of the silica-administered rats
lung tissue of the vehicle group (Figure 1b). On the con- with IFX treatment (Figure 1k). Inflammation (day 7) and
trary,  IFX  treatment provided protection against silica- fibrosis (day 14) score were decreased significantly in the
induced lung tissue damage with the reduction of alveoli- IFX-treatment group as compared to the vehicle group
tis severity on day 7 (Figure 1c). (p < 0.05) (Figure 1f and 1l).
On day 14, animals in the untreated vehicle group showed
extensive pulmonary lesions including multifocal areas of Immunohistochemical findings
intense fibrosis with the number of neutrophils decreasing Sections as shown in the Figure  2a, the sham rats lung
and the number of lymphocytes increasing. What’s more, tissue section showed a faint degree of immune staining
granulomas containing macrophages, neutrophils and fibro- for  NF-κB  p65. Silica exposure significantly increased
blasts apparently increased and enlarged, as compared with the NF-κB p65 levels of nuclear protein in the lung tissues,
day 7 (Figure 1d and 1e). In Masson’s trichrome stained sec- which was evident from intense brown staining distributed
tions, the sham group had normal collagen fibers distribution in nucleus and cytoplasm of alveolar macrophages and

a) b) c)

d) e) f) Group:
0.4 control
NF-κB p65 optical density

vehicle
treatment *
0.3 * *
0.2
0.1
0
7 14
Time after silica instillation [days]

a) section of lung in sham group showing normal architecture, day 7; b) and c) section of lung in vehicle group showing extensive NF-κB p65
expression (brown color) in tissue, day 7 and day 14, respectively; d) and e) in contrast, section of lung in infliximab (IFX) treated group showing
limited NF-κB p65 expression, day 7 and day 14, respectively; f) mean optical density of NF-κB p65 staining in immunohistochemical sections
represents quantification of NF-κB p65 expression (data presented as mean ± standard error).
* p < 0.05, compared with sham/vehicle group (one-way analysis of variance and least significant difference test).
Groups as in Figure 1.

Fig. 2. Immunohistochemical analysis of nuclear factor-κB p65 (NF-κB p65) in rats’ lung sections (original magnification ×400)

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Table 1. Serum and bronchoalveolar lavage fluid (BALF) levels of tumor necrosis factor α (TNF-α) in rats measured
using enzyme-linked immunosorbent assay (ELISA)

TNF-α concentration
[ng/l]
(M±SE)
Sample
on day 7 on day 14
sham group vehicle group treatment group sham group vehicle group treatment group
(N = 8) (N = 8) (N = 8) (N = 8) (N = 8) (N = 8)
Serum 42.97±4.38 86.41±7.25 66.57±7.02* 45.58±5.16 77.06±4.18 58.73±5.78*
BALF 36.46±4.40 69.46±8.34 49.13±6.09* 38.55±4.57 50.76±5.86 47.35±4.59

M – mean; SE – standard error.

* p < 0.05, compared with sham/vehicle group (one-way analysis of variance and least significant difference test).
Groups as in Figure 1.

epithelial cells (Figure  2b  and  2c). However, treatment Infliximab treatment significantly decreased TNF-α level
with  IFX markedly inhibited the expression of  NF-κB as compared with the vehicle group (Figure 3a) (p < 0.05).
p65 (Figure 2d and 2e), as compared to the vehicle group However, upon  IFX treatment,  BALF  TNF-α level was
(Figure 2f) (p < 0.05). down-regulated and found only on day 7 (p < 0.05) but
not on day 14 (Figure 3b).
ELISA to determine cytokine levels
in serum and BALF Quantitative real-time PCR analysis
Serum and BALF levels of TNF-α were shown in the Ta- of iNOS mRNA expression
ble 1. Serum TNF-α level was found considerably higher Lung tissues-based iNOS gene expression was shown in
in the vehicle group than in the sham group at both times. the Table  2. Inducible nitric oxide synthase  mRNA ex-

a) b)
100 100
TNF-α concentration [ng/l]

TNF-α concentration [ng/l]

Group: Group:
sham sham
80 vehicle 80 vehicle
treatment treatment
60 60

40 40

20 20

0 0
7 14 7 14
Time after silica instillation [days] Time after silica instillation [days]

Data presented as mean ± standard error.

* p < 0.05, compared with sham/vehicle group (one-way analysis of variance and least significant difference test).
Groups as in Figure 1.

Fig 3. Tumor necrosis factor α (TNF-α) concentration measured using enzyme linked immunosorbent assay (ELISA) in rats’:
a) serum, b) bronchoalveolar lavage fluid (BALF)

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 ORIGINAL PAPER     H. ZHANG ET AL.

Table 2. Inducible nitric oxide synthase (iNOS) mRNA expression level in lung tissues of rats measured
using real-time quantitative polymerase chain reaction (RT-qPCR)

iNOS mRNA expression level

(M±SE)
Sample on day 7 on day 14
sham group vehicle group treatment group sham group vehicle group treatment group
(N = 8) (N = 8) (N = 8) (N = 8) (N = 8) (N = 8)
Lung tissue 10.29±0.19 12.91±0.21 11.66±0.14* 10.31±0.16 12.60±0.18 12.03±0.22*

M – mean; SE – standard error.

* p < 0.05, compared with sham/vehicle group (one-way analysis of variance and least significant difference test).
Groups as in Figure 1.

15 duced rats, as compared with the sham group. Treatment

iNOS mRNA expression

Group:
sham
vehicle
treatment
with IFX significantly increased the I-κB protein level on
10 day  7  and  14  (Figure  5b) (p  <  0.05). In contrast to the
downtrend of I-κB levels, relative NF-κB p65 levels (ra-
5 tios of NF-κB p65 to β-actin) were increased in the case
of the silica rats to a larger extent than in the case of the
0 sham animals  (p  <  0.05). However, the increasing was
7 14
Time after silica instillation [days] significantly attenuated in the case of IFX-treatment rats
on day 7 (p < 0.05) but not on day 14 (Figure 5d).
Data presented as mean ± standard error.
* p < 0.05, compared with sham/vehicle group (one-way analysis
of variance and least significant difference test). DISCUSSION
Groups as in Figure 1.
Silicosis is associated with persistent inflammation which
Fig. 4. Inducible nitric oxide synthase (iNOS) mRNA leads to fibroblast formation and excessive collagen de-
expression level in lung tissues of rats measured using real-time
position resulting in interstitial fibrosis [31]. In this study,
quantitative polymerase chain reaction (RT-qPCR)
histopathological analyses have revealed that a  single-
pression level was significantly higher in the vehicle group dose intratracheal silica instillation could produce early
rats than in the sham group, IFX treatment significantly acute alveolitis, characterized by inflammatory cells in-
reduced iNOS mRNA expression on day 7 and 14 as com- filtration into the alveoli, accompanied by early alveolar
pared to the vehicle group (Figure 4) (p < 0.05). walls thickening. Furthermore, granulomas apparently
formed and extensive collagen deposition increased over
Western blot analysis time indicated progressive pulmonary inflammation and
of NF-κB p65 and I-κB protein expression pulmonary fibrosis as compared with the sham group,
The Western blot analysis was employed to semi-quan- closely mimicking acute silicosis injury  [30]. Infliximab-
titatively determine protein expression levels of  NF-κB attenuated silica induced the early inflammation and the
p65 (65 kDa), I-κB (39 kDa), and β-actin (43 kDa) (Fig- subsequent fibrosis, supported by a  marked decrease in
ure 5). Relative I-κB level (ratios of I-κB to β-actin) ana- the distribution and severity of infiltrates, organized gran-
lyzed by densitometry was lower in the case of silica-in- ulomas and collagen deposition following  IFX adminis­

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 INTERVENTION OF PNEUMOCONIOSIS     ORIGINAL PAPER

a) tration. This result is consistent with previous studies

39 kDa I-κB that indicated the ability of IFX to decrease collagen lev-
els in lung diseases [23–27].
43 kDa β-actin
To explore the possible mechanism(s) of the protective
1 2 3 4 5 6 action of IFX against silica-induced fibrosis, factors of in-
b)
1.5 flammatory, nuclear transcriptional and oxidative stress
I-κB expression [%]

Group:
sham
vehicle were evaluated in the lung tissue.
treatment
1.0 The cytokine TNF-α was believed to be an upstream fac-
tor, which could initiate a cascade complex of inflammato-
0.5 ry mediators in the early inflammatory event and develop
silica-induced fibrosis. Exogenous recombinant  TNF-α
0 augments silicotic fibrosis and anti-TNF antibody attenu-
7 14
Time after silica instillation [days]
ates silicosis, silicosis is absent in the case of TNF-α re-
c) ceptor knockout mice, suggesting that  TNF-α  receptor
signaling is needed for the development of pulmonary
65 kDa NF-κB p65
inflammation and fibrosis [8–10]. In confirmation of the
43 kDa β-actin previous study, we observed that TNF-α level in serum
1 2 3 4 5 6 over the time and in BALF on day 7 increased in the ve-
1.5
d) hicle group to an extent greater than in the sham group,
NF-κB p65 expression [%]

Group:
sham while IFX treatment significantly decreased TNF-α lev-
vehicle
treatment els, which is similar to the trend of histopathological re-
1.0
sults. However, BALF TNF-α level on day 14 didn’t show
the remarkable decreasing upon IFX treatment. We ana-
0.5
lyzed the IFX administration of intraperitoneal injection
which might result in the more unstable drug concentra-
0
7 14 tion distribution in local lung tissues, as compared with
Time after silica instillation [days]
the in-the-blood system.
Nuclear factor-κB has been considered a primary target to
a) a representative western blot.
c) the analysis of the ratio between I-κB and β-actin
accelerate silica-induced inflammation and fibrosis in the
and between NF-κB p65 and β-actin by densitometry. lung [32]. Tumor necrosis factor α induces NF-κB transac-
1 – vehicle group, day 7; 2 – treatment group, day 7; 3 – sham group, tivation by a mechanism involving I-κB degradation [33].
day 7; 4 – vehicle group, day 14; 5 – treatment group, day 14;
6 – sham group, day 14. Tumor necrosis factor-persistent stimulation may lead
Data presented as mean ± standard error. new I-κB–NF-κB complexes to enter the feedback loop,
* p < 0.05, compared with sham/vehicle group (one-way analysis beginning with phosphorylation of I-κB [34]. A pharma-
of variance and least significant difference test).
Groups as in Figure 1. cologic inhibitor  (BAY  11-7085) of  I-κBα  (NF-κBIA)
phosphorylation, inhibiting systemically NF-κB activation,
Fig. 5. Effect of different treatments in the rats’ lung tissues
on the expression of: a) and b) inhibitor protein-κB (I-κB), significantly decreases silica-induced inflammation and fi-
c) and d) nuclear factor-κB p65 (NF-κB p65) brosis by reducing apoptosis and collagen deposition [35].

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 ORIGINAL PAPER     H. ZHANG ET AL.

In the current study, silica installation induced the ac- mation  [22]. In our study, silica administration in-
tivation of  NF-κB signaling pathway, which was con- creased iNOS expression, which was shown by the quan-
firmed as followed. Firstly, by immunohistochemical titative real-time PCR analysis of iNOS mRNA expres-
staining of the lung tissue, silica exposure significantly sion, basically identifying with the previous conclusions.
increased the  NF-κB  p65 level of nuclear protein dis- However,  IFX  treatment decreased  INOS expression
tributed in nucleus and cytoplasm of alveolar macro- on both day  7  and  14. We have supposed that it may
phages and epithelial cells. Secondly, by protein band be one of the mechanisms, due to which IFX improves
analysis of western blotting, silica exposure apparently inflammation and fibrosis by decreasing transcriptional
increased I-κB degradation and NF-κB p65 expression. activation of oxidant-stress index iNOS.
Infliximab administration considerably inhibited  NF- In clinical studies, it has been shown that IFX diminishes
κB signaling activation by decreasing protein level and pulmonary fibrosis via blocking  TNF-α  [41,42], and pul-
nuclear translocation of NF-κB p65, I-κB phosphoryla- monary fibrosis is not a  contraindication for  TNF-α in-
tion as well. However, the result of NF-κB p65 expres- hibitor therapy of rheumatology [43]. Altintas et al. [27]
sion by  IHC staining kept inconsistency with that by have evaluated the preventive effect of IFX in bleomycin-
the  WB analysis on day  14. Considering the method- induced pulmonary fibrosis, eliminating the harmful effect
ological difference between the 2 technologies, compa- of IFX per se on pulmonary fibrosis. However, there is no
nied with possibly weakened NF-κB signaling feedback study to explain the treatment effect of IFX on silica-in-
loop over time, IHC staining result, mainly measuring duced pulmonary inflammation and fibrosis.
combining and dissociative  NF-κB  p65 with intracyto- This study has shown that IFX treatment prevents the
plasmic  I-κB and nucleous  DNA, appeared more re- inflammatory and fibrotic response as assessed from
markable than that by the western blot (WB), which is quantitative histopathological evaluation of silica-
measuring merely dissociative ones. induced lung fibrosis model in the case of rats. In ad-
Silica-induced inflammation and oxidative stress envi- dition, it has also been found that  IFX attenuates the
ronment contributes to lung injury. It has been demon- silica-induced inflammatory by decreasing  TNF-α lev-
strated that iNOS (iNOS)-dependent formation of NO el, I-κB degradation, NF-κB p65 expression and nuclear
has a  key role in the initiation of pulmonary inflam- translocation, oxidative stress relative enzyme iNOS ac-
mation and fibrosis  [36]. Quartz instillation into rat tivities in the damaged lung tissue of rats. To the best
lungs resulted in increasing of mRNA for  iNOS in al- of our knowledge, this is the first study that evaluates
veolar macrophages [37]. Tumor necrosis factor α may the treatment effect of  IFX on silica-induced lung fi-
activate  iNOS to produce  NO in respiratory epithe- brosis in terms of the animal model, but it needs more
lium [17], which has been reported in alveolar macro- indicators and data of pulmonary fibrosis to confirm.
phages and neutrophils [36,38]. This study hopes to provide drug treatment for pneu-
The over-produced iNOS-dependent formation of NO moconiosis patients to alleviate the pain and improve
may cause serious lung damage though the formation of the quality of life, so as to provide some help for clinical
peroxynitrite (reactive nitrogen species – RNS) combin- diagnosis and treatment. With the Chinese government
ing with superoxide  [36,39,40]. Furthermore,  NO  pro- to further strengthen the occupational precautionary
duces and amplifies the inflammatory response by measures, the occurrence of occupational diseases will
up-regulating the key cytokine  TNF-α during inflam- be significantly reduced.

10 IJOMEH 2018;31(4)
 INTERVENTION OF PNEUMOCONIOSIS     ORIGINAL PAPER

CONCLUSIONS 7. Miyazaki Y, Araki K, Vesin C, Garcia I, Kapanci Y, Whit-

In summary, IFX may improve silica-induced pulmonary sett  JA, et al. Expression of a tumor necrosis factor-alpha
inflammation by decreasing the TNF-α, inhibiting NF-κB transgene in murine lung causes lymphocytic and fibrosing
signaling (I-κB, NF-κB p65) as well as oxidant status (iNOS), alveolitis. A mouse model of progressivepulmonary fibro-
which suggest that IFX has potential role in the treatment sis. J Clin Invest. 1995;96(1):250–9, https://doi.org/10.1172/
of silica-induced lung damage. JCI118029.
8. Piguet PF, Collart MA, Grau GE, Sappino AP, Vassalli P.
ACKNOWLEDGMENTS Requirement of tumour necrosis factor for development of
The authors would like to acknowledge with deep apprecia- silica-induced pulmonary fibrosis. Nature. 1990;344(6263):
tion and gratitude to the invaluable help of the following per- 245–7, https://doi.org/10.1038/344245a0.
sons: You-xin  Ji, M.D.,  Ph.D (North Shore University Hospi- 9. Gossart S, Cambon C, Orfila C, Séguélas MH, Lepert JC,
tal, Manhasset, NY) for this article text corrections. Rami J, et al. Reactive oxygen intermediates as regulators of
TNF-alpha production in ratlung inflammation induced by
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