Biochemical and Biophysical Research Communications xxx (2018) 1e5

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Effects of calcium gluconate on lipopolysaccharide-induced acute lung

injury in mice
Lian Liu a, 1, Dan Xu a, 1, Pan Liu b, 1, Fangyu Liu b, 1, Luqi Dai b, Hou Yan c, Fuqiang Wen a, *
a
Laboratory of Pulmonary Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China
b
West China School of Medicine, Sichuan University, Chengdu, 610041, China
c
Department of Respiratory Medicine, Lanzhou General Hospital of Lanzhou Command, PLA, Lanzhou Gansu, 730050, China

a r t i c l e i n f o a b s t r a c t

Article history: Lipopolysaccharide (LPS), which can cause acute airway inflammatory reactions, constitutes one of the
Received 3 August 2018 most common substances to establish acute lung injury (ALI) models in mice. Studies suggest that cal-
Accepted 7 August 2018 cium gluconate offers the possibility of suppressing the immune response, and this study was intended
Available online xxx
to explore the effects of calcium gluconate on LPS-induced ALI in mice. Mice inhaled with LPS were
intraperitoneally injected with calcium gluconate (12.5, 25, 50 mg/kg). IL-1b, IL-6 and TNF-a levels in
Keywords:
bronchoalveolar lavage fluid (BALF) were determined by ELISA. The expression of signaling proteins,
Lipopolysaccharide
phosphorylation extracellular regulated protein kinases (p-ERK), was detected using Western Blot in lung
Acute lung injury
Calcium gluconate
tissues. In our study, the release of inflammatory cytokines IL-1b, IL-6 and TNF-a in BALF increased after
inhalation of LPS. Post-treatment with calcium gluconate inhibited LPS-induced airway inflammatory
injury and the release of inflammatory cytokines. In addition, LPS promoted the expression of signaling
protein p-ERK while calcium gluconate was capable of reversing this change. Overall, calcium gluconate
inhibits LPS-induced ALI in mice, which may take effects through the inhibition of ERK phosphorylation.
© 2018 Published by Elsevier Inc.

1. Introduction Currently, commonly used medications for the treatment of ALI

Acute lung injury (ALI), usually caused by bacterial infections, is
one of the most perilous lung diseases, which accompanies with a
series of pulmonary pathological changes reflecting general
excessive inflammatory reactions [1,2]. Endotoxin is a key factor
leading to ALI. Lipopolysaccharide(LPS), the main component of
endotoxin, plays a major role in ALI [3]. It is currently recognized
that overreacted LPS-TLR (Toll-like receptor) response resulting in
the uncontrolled inflammatory reactions is one of the principal
causes of diffuse alveolar damage in LPS-induced ALI [4]. A study
showed that anti-inflammatory effects in ALI mice could happen
through suppressing TLR4-mediated MAPK (mitogen-activated
protein kinase) pathway in activated macrophages [5]. Therefore,
the inhibition of the inflammatory response is critical in the
treatment of ALI caused by LPS.
mainly include adrenal glucocorticoids, vasodilators and so on [6].
Studies suggested that calcium ion possesses the capacity of regu-
lating MAPK signaling pathway, which plays an important role in
the inflammatory response [7,8]. Calcium gluconate provides us an
opportunity to inhibit the exudation of inflammatory cells, alveolar
histopathological changes and the secretion of inflammatory cy-
tokines such as IL-1b, IL-6 and TNF-a [9,10]. However, there has
been no study exploring the effects of calcium gluconate towards
inflammatory on airways. Therefore, as a new attempt to relieve the
inflammatory injury in airway, we speculate that the calcium glu-
conate may possess the potential to relieve the LPS-induced ALI in
mice. The purpose of this experiment was to investigate the effects
of calcium gluconate on LPS-induced ALI in an experimental mouse
model.

2. Materials and methods

* Corresponding author. Department of Respiratory and Critical Care Medicine, 2.1. Animals
West China Hospital of Sichuan University and Division of Pulmonary Diseases,
State Key Laboratory of Biotherapy of China, Chengdu, 610041, China.
E-mail address: wenfuqiang.scu@gmail.com (F. Wen). Animal preparation work was carried out under the adminis-
1
Authors contributed equally to this study. tration of Institutional Animal Care and Use Committee of Sichuan

https://doi.org/10.1016/j.bbrc.2018.08.072
0006-291X/© 2018 Published by Elsevier Inc.

Please cite this article in press as: L. Liu, et al., Effects of calcium gluconate on lipopolysaccharide-induced acute lung injury in mice, Biochemical
and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.072
2 L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e5
University. Male C57BL/6J mice (4e6 weeks) were purchased from
Dashuo Biological Technology Co, Ltd (Chengdu, China). Mice were
housed in a pathogen-free, humidity controlled facility at 22e24
C
with a 12-h light/dark cycle. Water and laboratory feed were free to
access.
Mice were randomly divided into the following six groups
(n ¼ 10 per group): Control group with no inhalation of LPS; Cal-
cium gluconate group (CG group) without inhalation of LPS but
with 50 mg/kg calcium gluconate (purchased from West China
Hospital, Chengdu, China); LPS-induced group (LPS group), with
LPS-induced ALI; LPS-induced low-dose calcium gluconate group
(LPS þ CG(L)), which was exposed to LPS and injected intraperi-
toneally with 12.5 mg/kg calcium gluconate; LPS-induced medium-
dose calcium gluconate group (LPS þ CG(M)) was induced ALI by
LPS and injected with 25 mg/kg calcium gluconate; and the LPS-
induced high-dose calcium gluconate group (LPS þ CG(H))group
was used LPS and injected with 50 mg/kg calcium gluconate.
The mice in LPS, LPS þ CG(L), LPS þ CG(M) and LPS þ CG(H)
group were anesthetized and then the glottis was exposed with a
laryngoscope. LPS 5 mg/kg (Sigma-L2880, purchased from Univ
Bio-Technology Co., Ltd., Shanghai, China) at a concentration of
5 mg/ml was injected into the airway from the nebulizer inlet to
establish the LPS-induced ALI model. The same amount of normal
saline (NS, purchased from West China Hospital, Chengdu, China)
was sprayed into Con and CG group in the formerly mentioned way.
Thirty minutes after modeling, the mice in Con and LPS group were
intraperitoneally injected with 0.25 ml NS, while the mice in the
other groups were dosed by 0.25 ml calcium gluconate solution
prepared at various concentrations.
Twenty-four hours after successful modeling, mice in all groups
were anesthetized respectively. Bronchoalveolar lavage fluid (BALF)
was collected by lavaging the right lung and cytokines were
detected by ELISA. Then the complete lung tissue was removed, left
lung was used for pathological examination, while right lung tissue
was used for Western Blot analysis.
2.2. Bronchoalveolar lavage fluid analysis
Inflammatory cell count: Mice were exsanguinated via the
abdominal aorta and cannulated in the trachea. Then the chest
cavity was opened with a midline incision. The left main bronchus
was ligated and the right lung was lavaged three times with 1.5 ml
of NS in total, with a recovery rate of 90%. A 0.5-ml aliquot of BALF
sample was used for cell counting and the remaining BALF was
centrifuged at 1000 g for 5 min at 4
C, and the cell-free superna-
tants were stored at 80
C for cytokines detection. The 0.5-ml
BALF samples were processed in RBC lysis buffer (C3702, pur-
chased from Beyotime Co., Ltd., Shanghai, China) in room temper-
ature for 5 min after being centrifuged at 2000 g for 5 min at 4
C.
Then the samples were resuspended in 800 ml phosphate-buffered
saline and performed by centrifugation at 500g for 5 min. Then the
cells were adjusted to a concentration of 5  105/ml in phosphate-
buffered saline. After cytocentrifugation (Cytopro7620, Wescor,
Utah, USA) at 700 rpm for 10 min, the cells were stained with
Wright's stain. An experienced investigator blinded to the experi-
mental conditions performed all counts on standard morphological
criteria. (200 cells were counted for each mouse).
Inflammatory cytokines detection: Levels of IL-1b, IL-6 and TNF-
a in BALF were measured using commercially available enzyme-
linked immunosorbent assay (ELISA) kit for mouse cytokines
(Xitang Bio-Technology Co., Ltd., Shanghai, China). ELISA experi-
ments strictly followed the instructions.
Please cite this article in press as: L. Liu, et al., Effects of calcium gluconate on lipopolysaccharide-induced acute lung injury in mice, Biochemical
and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.072
2.3. Lung tissue analysis
Evaluation of ALI severity: The left lung tissue which is not
lavaged was drawn and fixed in 4% paraformaldehyde for 24 h. After
paraffin embedding, the tissues were cut into 4 mm sections. The
sections were deparaffinized in xylene, and then washed with
ethanol. The samples were stained with hematoxylin and eosin
(H&E) to evaluate morphological changes in the lungs. Evaluation
of ALI severity: The left lung tissue which is not lavaged was drawn
and fixed in 4% paraformaldehyde for 24 h. After paraffin embed-
ding, the tissues were cut into 4 mm sections. The sections were
deparaffinized in xylene, and then washed with ethanol. The
samples were stained with hematoxylin and eosin (H&E). The ALI
severity scored for each lung tissue by a board-certified pathologist
using previously published criteria [11]. Briefly, the scoring items
included: (1) alveolar capillary congestion; (2) hemorrhage; (3)
infiltration or aggregation of neutrophils in the air space or the
vessel wall; and (4) thickness of the alveolar wall/hyaline mem-
brane formation. Each item was graded according to the following
five-level scale: 0 ¼ minimal (little) damage; 1 ¼ mild damage;
2 ¼ moderate damage; 3 ¼ severe damage; and 4 ¼ maximal dam-
age. Based on five randomly selected high-power fields (HPF, 400X
magnification) in each section, the mean score of each field was
compared between the groups.
Western blot analysis: Lung tissues were lysed in RIPA buffer
containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5%
sodium deoxycholate, 2 mM sodium fluoride, 2 mM EDTA, 0.1% SDS,
and PMSF. Total protein concentrations of lung extracts were
determined by a BCA protein assay kit (Thermo Fisher Scientific
Inc., MA, USA). To analyze p-ERK, total protein (20 mg) was frac-
tionated by 10% SDS-polyacrylamide gel electrophoresis and
transferred to PVDF membranes. Membranes were blocked for 1 h
at room temperature with 5% BSA in TBS-Tween and incubated
overnight at 4
C with the appropriate primary antibodies. Primary
antibody was anti-phospho-ERK mAb. After incubation with
horseradish peroxidase-conjugated second antibodies (Cell
Signaling Technology, Beverly, MA, USA), the immune complexes
were identified with enhanced chemiluminescence reagents (Mil-
lipore, Billerica, USA). Band intensities were quantified using
computerized image analysis (QuantityOne-software, BioRad,
Hercules, CA).
2.4. Statistical analysis
Statistical analysis was performed using one-way ANOVA fol-
lowed by the LSD significant difference test (SPSS for Windows
version 24.0, Chicago, IL, USA) and all values are expressed as
Mean ± SD. A significant difference was determined at P < 0.05.
3. Results
3.1. Calcium gluconate prevented LPS-induced mouse airway
histopathological changes
In this study, ALI score was used to evaluate the histopatho-
logical transformation in mouse airways. LPS inhalation induced
intensive airway inflammatory response manifested as thickening
of the airway epithelium, lumen obstruction by mucus and cell
debris, and inflammatory cell infiltration (Fig. 1AeF). Such changes
were attenuated by calcium gluconate treatment, especially with
high-dose calcium gluconate.
 L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e5
Fig. 1. The effect of calcium gluconate on lipopolysaccharide-induced lung histological
changes.
Lung tissues were analyzed by hematoxylin and eosin staining. A. Control group, no
exposure to LPS and no calcium gluconate treatment; B. Calcium gluconate group (CG
group), without inhalation of LPS but with 50 mg/kg calcium gluconate post-
treatment; C. LPS-induced group (LPS group), only with LPS-induced acute lung
injury; D. LPS-induced low-dose calcium gluconate group (LPS þ CG(L)), with inhala-
tion of LPS and injected with 12.5 mg/kg calcium gluconate; E. LPS-induced medium-
dose calcium gluconate group (LPS þ CG(M)), with inhalation of LPS and injected with
Please cite this article in press as: L. Liu, et al., Effects of calcium gluconate on lipopolysaccharide-induced acute lung injury in mice, Biochemical
and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.072
3
Fig. 2. The effect of calcium gluconate on BALF cellularity in LPS-inhaled mice.
Cell count in BALF was determined by cytocentrifugation and Wright's staining. A.
Control group, no exposure to LPS and no calcium gluconate treatment; B. Calcium
gluconate group (CG group), without inhalation of LPS but with 50 mg/kg calcium
gluconate post-treatment; C. LPS-induced group (LPS group), only with LPS-induced
acute lung injury; D. LPS-induced low-dose calcium gluconate group (LPS þ CG(L)),
with inhalation of LPS and injected with 12.5 mg/kg calcium gluconate; E. LPS-induced
medium-dose calcium gluconate group (LPS þ CG(M)), with inhalation of LPS and
injected with 25 mg/kg calcium gluconate; F. LPS-induced high-dose calcium gluconate
group (LPS þ CG(H)), with inhalation of LPS and injected with 50 mg/kg calcium
gluconate. eP>0.05 with respect to the control group, þP < 0.001 with respect to the
control group, *P < 0.05 with respect to the LPS group, **P < 0.001 with respect to the
LPS group.
3.2. Calcium gluconate attenuated LPS-induced inflammatory cell
influx and inflammatory cytokines release in BALF
Cell count was performed in BALF to investigate the effects of
calcium gluconate on LPS-induced inflammation in mouse airways.
The neutrophils were meaningfully higher in LPS-inhaled mice
than mice in the control group. High-dose calcium gluconate
treatment significantly reduced the LPS-induced exudation of
neutrophils to BALF (Fig. 2). BALF levels of IL-1b, IL-6, and TNF-a
were comparatively higher in samples from LPS-inhaled mice than
samples from the control group. Injection with high-dose calcium
gluconate greatly mitigated the LPS-induced elevated levels of IL-
1b, IL-6, and TNF-a in BALF (Fig. 3).
3.3. Calcium gluconate inhibited LPS-induced ERK phosphorylation
To explore the possible mechanism involved in LPS-induced
airway inflammation, the expression level of p-ERK was exam-
ined. Lung tissues of LPS-inhaled mice showed higher levels of p-
ERK than the lung tissues of the control group, and calcium glu-
conate treatment attenuated this effect of LPS (Fig. 4).
4. Discussion
In this study, LPS was used to establish ALI model in mouse,
which led to histopathological changes, conspicuous release of in-
flammatory cytokines in BALF and ERK phosphorylation in mouse
lung. In comparison, post-treatment with calcium gluconate
dramatically mitigated these effects in vivo.
In our study, calcium gluconate protected mouse airway from
25 mg/kg calcium gluconate; F. LPS-induced high-dose calcium gluconate group
(LPS þ CG(H)), with inhalation of LPS and injected with 50 mg/kg calcium gluconate.
The degree of lung injury in tissue sections was evaluated based on an ALI score.
e
P>0.05 with respect to the control group, þP < 0.001 with respect to the control
group, *P < 0.001 with respect to the LPS group.
4 L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e5
Fig. 3. The effects of calcium gluconate on the production of BALF cytokines.
BALF was collected from different groups of mouse lungs. The concentrations of IL-1b,
IL-6, and TNF-a in BALF were analyzed by ELISA. Values are expressed as mean ± SD. A.
Control group, no exposure to LPS and no calcium gluconate treatment; B. Calcium
gluconate group (CG group), without inhalation of LPS but with 50 mg/kg calcium
gluconate post-treatment; C. LPS-induced group (LPS group), only with LPS-induced
acute lung injury; D. LPS-induced low-dose calcium gluconate group (LPS þ CG(L)),
with inhalation of LPS and injected with 12.5 mg/kg calcium gluconate; E. LPS-induced
medium-dose calcium gluconate group (LPS þ CG(M)), with inhalation of LPS and
injected with 25 mg/kg calcium gluconate; F. LPS-induced high-dose calcium gluconate
Please cite this article in press as: L. Liu, et al., Effects of calcium gluconate on lipopolysaccharide-induced acute lung injury in mice, Biochemical
and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.072
Fig. 4. Expression of p-ERK in mice lungs.
P-ERK levels in mouse lungs were measured by Western blotting and quantified by
densitometry. The histogram shows the relative intensity of p-ERK protein bands
normalized to the b-actin band, A. Control group, no exposure to LPS and no calcium
gluconate treatment; B. Calcium gluconate group (CG group), without inhalation of LPS
but with 50 mg/kg calcium gluconate post-treatment; C. LPS-induced group (LPS
group), only with LPS-induced acute lung injury; D. LPS-induced low-dose calcium
gluconate group (LPS þ CG(L)), with inhalation of LPS and injected with 12.5 mg/kg
calcium gluconate; E. LPS-induced medium-dose calcium gluconate group
(LPS þ CG(M)), with inhalation of LPS and injected with 25 mg/kg calcium gluconate; F.
LPS-induced high-dose calcium gluconate group (LPS þ CG(H)), with inhalation of LPS
and injected with 50 mg/kg calcium gluconate. eP>0.05 with respect to the control
group, þP < 0.001 with respect to the control group, *P < 0.001 with respect to the LPS
group.
LPS-induced inflammatory response through inhibiting the release
of inflammatory cytokines, including IL-1b, IL-6, and TNF-a, which
is of great importance in the pathogenesis of ALI [12e14]. Recent
studies suggested that IL-1b was strongly increased in total lung
tissue and alveolar exudate [15]. The level of pulmonary edema was
obviously higher in high IL-1b expression rats. IL-1b should be
regarded as an important mediator of LPS-induced ALI. Our previ-
ous study found that LPS increases IL-6 expression in the serum and
bronchoalveolar fluid of ALI patients with poor outcomes. IL-6 is
associated with the pathogenesis of ALI, which may serve as an
immediate cause of injury [16]. TNF-a is a cytokine that regulating
inflammatory activities, it is an important chemotactic protein for
neutrophils. Studies suggested that TNF-a levels were increased in
the blood and alveolar exudate of ALI patients, which may account
for the pathogenesis and progression of ALI [17,18]. Many clinical
trials have investigated the therapeutic potential of specific cyto-
kines in inhibition of ALI [12], but with unsatisfactory results.
group (LPS þ CG(H)), with inhalation of LPS and injected with 50 mg/kg calcium
gluconate. eP>0.05 with respect to the control group, þP < 0.001 with respect to the
control group, *P < 0.05 with respect to the LPS group, **P < 0.01 with respect to the
LPS group, ***P < 0.001 with respect to the LPS group.
 L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e5
Anyway, further studies need to be performed to determine the
specific role of each inflammatory cytokine in ALI. A specific group
of ALI patients should be targeted with specific anti-cytokine
therapy if there are evidence of high expression of that cytokine
and clinical indications of ALI patients who will respond to therapy
well.
MAPK signaling pathways containing ERK phosphorylation have
crucial effects on the pathogenesis of ALI. Studies showed a highly
increased expression of p-ERK in the lung samples of the ALI pa-
tients, and the MAPK activity was found remarkably correlated
with neutrophils infiltration in the airway [19,20]. Zhang et al. re-
ported that through inhibition of the Toll-like receptors (TLRs), ALI
could be alleviated with the suppression of NF-kB pathway [21].
While MAPK/NF-kB pathway is exactly the approach that how the
inflammation is triggered from the excitation of TLRs to various
injuries towards alveolar capillaries and epithelial cells, phos-
phorylated ERK is involved in pro-inflammatory cytokines pro-
duction and airway mucus hypersecretion [22,23]. Our studies
confirmed that ERK phosphorylation should be involved in LPS-
induced airway inflammation, therapy or chemicals targeting on
this signaling pathway may help to attenuate LPS-induced airway
inflammation. What should be noticed is that the present study
included a limited number of mice and further studies are imper-
ative to confirm the protective role of calcium gluconate on LPS-
induced airway inflammation and investigate its accurate mecha-
nism and how to transform the preclinical study findings into
clinical applications.
Acknowledgement
This work was supported by grants from the National Natural
Science Foundation of China (81230001, 81470236, 81001010 and
81670038), and National Key Research and Development Program
in China (2016YFC0903600, 2016YFC0901100, 2016YFC1303900
and 2016YFC1304500). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of
the manuscript.
Transparency document
Transparency document related to this article can be found
online at https://doi.org/10.1016/j.bbrc.2018.08.072.
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