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Akt mediated suppression of inflammation
Journal Pre-proof Crocin ameliorates chronic obstructive pulmonary disease-induced depression via PI3K/Akt mediated suppression of inflammation Yupeng Xie, Qiuxiang He, Hong Chen, Zijiang Lin, Yi Xu, Chuang Yang PII:
S0014-2999(19)30592-8
DOI:
https://doi.org/10.1016/j.ejphar.2019.172640
Reference:
EJP 172640
To appear in:
European Journal of Pharmacology
Received Date: 6 August 2019 Revised Date:
30 August 2019
Accepted Date: 2 September 2019
Please cite this article as: Xie, Y., He, Q., Chen, H., Lin, Z., Xu, Y., Yang, C., Crocin ameliorates chronic obstructive pulmonary disease-induced depression via PI3K/Akt mediated suppression of inflammation, European Journal of Pharmacology (2019), doi: https://doi.org/10.1016/j.ejphar.2019.172640. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
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Crocin ameliorates chronic obstructive pulmonary disease-induced
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depression via PI3K/Akt mediated suppression of inflammation
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Yupeng Xie 1*, Qiuxiang He 2, Hong Chen 3, Zijiang Lin 3, Yi Xu 4, Chuang Yang 3*
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1. Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital
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of Wenzhou Medical University, Wenzhou 325000, China
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2. Department of Pathology, The First Affiliated Hospital of Wenzhou Medical
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University, Wenzhou 325000, China
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3. Department of Psychiatry, The First Affiliated Hospital of Wenzhou Medical
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University, Wenzhou 325000, China
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4. School of Pharmacy, Wenzhou Medical University, Wenzhou 325000, China
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*Corresponding author:
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Yupeng Xie ([email protected])
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Yang Chuang ([email protected])
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1 2
Abstract Clinical studies have indicated the co-occurrence of chronic obstructive
3
pulmonary disease (COPD) and psychiatric disorders, for example, comorbid
4
depression. However, the underlying mechanism is rarely addressed. The present
5
study aimed to investigate the mechanism of COPD-induced depression and the
6
psychological and physiological effects of crocin, an active constituent of Crocus
7
sativus L.. C57BL/6 mice were randomly exposed to cigarette smoke for 7 weeks to
8
establish COPD animal model. Crocin (50 mg/kg), Dexamethasone (2 mg/kg) and
9
IGF-1 (2 mg/kg) were respectively injected to mice once a day. The FEV1/FVC ratio
10
and the mean alveolus area of lung tissue demonstrated the COPD model was
11
successfully established by cigarette smoke. Crocin administration significantly
12
reversed markers of depression [loss of body weight, sucrose preference, and
13
elevation of immobile time in tail-suspension tests (TST) and in forced swimming
14
tests (FST)]. Besides, crocin treatment significantly inhibited the numbers of
15
inflammatory cells (macrophages, neutrophils, and lymphocytes), suppressed the
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infiltration of peribronchial inflammatory cells, and reduced the concentration of
17
proinflammatory cytokines in bronchoalveolar lavage (BAL) fluid and lung tissue.
18
Crocin also reduced proinflammatory cytokines in the hippocampus. In exploring
19
associated mechanisms, we discovered that crocin blunted cigarette smoke-induced
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IκB phosphorylation and degradation, and NF-κBp65 nuclear translocation. IGF-1, an
21
activator of PI3K, abrogated the effect of crocin against cigarette smoke-induced
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activation of the NF-κB pathway. Together, these results showed that an inflammatory 2
1
mechanism might be involved in the pathogenesis of COPD with comorbid
2
depression. Crocin exhibited significant effects through the regulation of
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PI3K/Akt-mediated inflammatory pathways.
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Keywords: Chronic obstructive pulmonary disease; Depression; Inflammation;
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PI3K/Akt/NF-κB; Crocin
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1 2
1. Introduction Chronic obstructive pulmonary disease (COPD) is a common chronic disease of
3
the respiratory system characterized by airflow obstruction (Huertas and Palange,
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2011). With increasing emissions of particulate matter (i.e., PM2.5) and other types of
5
air pollution, more and more people suffer from respiratory disease, and it is predicted
6
that COPD will become the third leading cause of death by 2030 (Vijayan, 2008;
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Zhao et al., 2019). Cigarette smoke is considered as the principle factor driving the
8
pathogenesis and progression of pulmonary diseases (Arunachalam et al., 2010). The
9
components generated by smoking, such as carbon monoxide, nicotine, oxidants, fine,
10
were closely related to the onset of COPD (Alwis et al., 2015). Innate immune
11
response could be activated by cigarette smoke and then multiple chemotactic factors
12
is released to recruit neutrophils and inflammatory monocytes to the lungs (Chen et al.,
13
2019). Interestingly, increased accumulation of neutrophils, macrophages were
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observed in the lungs of COPD patients and COPD mouse models (Barnes, 2016;
15
Profita et al., 2010).
16
Depression is a highly debilitating and life-threatening mental disorder, which is
17
a huge burden of the society (Deng et al., 2015). Meta-analysis studies have reported
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that depression was highly correlated with COPD (Tsiligianni et al., 2011). In fact,
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depression is a common clinical comorbidity of COPD (Di Marco et al., 2006; Du et
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al., 2014; Quint et al., 2008). COPD patients suffer from comorbid depression often
21
continuously smoke and frequently medical complications (Hillas et al., 2015). Those
22
could increase their persistent depressive and pulmonary symptoms and signs, leading 4
1
to greater disability and mortality. However, the pathobiology of depression in COPD
2
is still unclear. More and more evidences suggest that inflammatory cytokines play
3
core roles in the aetiology of both COPD and depression (Leonard, 2000).
4
Inflammatory cytokine in the brain is involved in the regulation of neurotransmitter
5
metabolism, synaptic plasticity, and neuroendocrine function, which are known to be
6
important in the aetiopathogenesis of depression (Wang and Campbell, 2002).
7
Inhibiting cytokines induced by cigarette smoke might thus be a therapeutic strategy
8
for COPD with depression.
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Crocin is a natural carotenoid that has exhibited beneficial effects in the
10
treatment of central nervous system disorders, including depression, convulsion, and
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hypomnesia (Farkhondeh et al., 2018; Finley and Gao, 2017). In an animal model,
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crocin showed significant and dose-dependent anti-depressant activities, and may
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have acted via the inhibition of dopamine and norepinephrine uptake (Wang et al.,
14
2010). Crocin also attenuated malathion-induced depressive behaviours by increasing
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the level of brain-derived neurotrophic factor (BDNF) in mice hippocampus and
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cerebral cortex (Dorri et al., 2015). Beside its neuroprotective properties, crocin has
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also exhibited protective effects during lung injury (Wang et al., 2015) as well as
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anti-asthma potential in allergic airway disease (Xiong et al., 2015).
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Therefore, mice model of COPD was established by exposed to cigarette smoke,
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and the underlying pathogenic mechanism of COPD with comorbid depression was
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explored. Meantime, the psychopathological responses to crocin, a clinical
22
antidepressant with beneficial effects against lung injury were also investigated. 5
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2. Materials and Methods
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2.1. Reagents
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Crocin and Dexamethasone (Dex) were obtained from Sigma-Aldrich (Saint
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Louis, MO, USA). Insulin-like growth factor 1 (IGF-1), the activator of PI3K, was
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purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). IL-1β、IL-6
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and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were provided by
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Nanjing KeyGEN Biotech. CO., LTD. (Nanjing, China). Primary antibodies against
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p-PI3K、PI3K、p-Akt、Akt、p-NF-κBp65, NF-κBp65, p-IκBα and IκBα were produced
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by Cell Signaling Technology (Danvers, USA).
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2.2. Animals Male C57BL/6 mice, 7-8 weeks, were obtained from the Animal Center of
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Wenzhou Medical University (Wenzhou, China). The mice were maintained under
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controlled temperature (22-24oC) and 12/12 h light/dark cycle conditions with ad lib
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access to sterile standard laboratory chow. All animal experiments were performed
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according to protocols approved by Medicine Animal Care and Use Committee of
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Wenzhou Medical University.
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2.3. The establishment of CS-induced COPD model and experimental design C57BL/6 mice were exposed to cigarette smoke of 5 3R4F Kentucky reference
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cigarettes (without filter, University of Kentucky, Lexington, KY, USA), one after
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another, four times a day (total of 20 cigarettes per day) with a smoking machine as 6
1
previously described (Xu et al., 2018). The smoking machine consists of an animal
2
containment system and a cigarette smoking control system. Animals were alternately
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exposed to the smoke for 30 min and smoke-free interval for 30 min. The procedure
4
lasts for 7 consecutive weeks.
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Animals in this study were divided into five groups (n=8): the control group; the
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CS exposed group (the CS group); the CS + Dex (2 mg/kg) group; the CS + CRO (50
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mg/kg) group; the CS + CRO (50 mg/kg) + IGF-1 (2 mg/kg) group. Animals in the
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control group were exposed to the fresh air. The mice in different group were treated
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respectively with vehicle, Dex (2 mg/kg) or IGF-1 (2 mg/kg) via a single i.p. injection
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or 50 mg/kg of crocin orally 1h before exposure to cigarette smoke once a day. Dex, a
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widely used anti-inflammatory medicine, was used here as a positive control. The
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dosage of Dex was determined by reference to clinical dosage. The dosages of crocin
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and IGF-1 were determined by preliminary experiments. The behavior tests were
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determined 24 h after the last cigarette smoke exposure. After that, the lung function
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of mice in control group and CS groups were detected to verify the establishment of
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COPD model. Then, the blood samples were collected from the retroorbital plexus.
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Finally, we collected the bronchoalveolar lavage fluid (BAL fluid), lung and brain
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tissue from the mice.
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2.4. Determination of lung function in cigarette smoke -induced COPD mice
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To test lung function, all mice were anesthetized with 1% pentobarbital (60
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mg/kg) and fixed in a forced pulmonary maneuvre system (Buxco, NY, USA). The 7
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forced expiratory volume in the first second (FEV1)/forced vital capacity (FVC) ratio
2
was then determined as previously described (Wang et al., 2018).
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2.5. Sucrose preference test
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In the experiment, the mice were kept in cages, and the water was forbidden and
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fasted for 12 h, then 1% sucrose solution were given for 1 h. Calculate the percentage
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of sugar water consumption: sugar water consumption/total liquid
8
consumption×100%.
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2.6. Open field test (OFT)
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The behavior tests were determined at 24 h after the last cigarette smoke
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exposure. The test was used to measure anxiety levels in mice. Mice were placed in
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an open field, which was divided into symmetrical sectors. In the beginning, the mice
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were placed in the center of the device. And each mouse is free to explore for 6 min,
14
counting the number of crossings.
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2.7. Tail suspension test (TST) The tape was stuck to the end of the mouse tail 1 cm, and the mouse was hung
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upside down on a hook 40 cm from the floor. The camera was recorded for 6 min. The
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cumulative resting time of the last 4 min of the mouse was counted.
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2.8. Forced swimming test (FST) 8
FST was performed as previously described (Detke and Lucki, 1996; Porsolt et
1 2
al., 1978). During the test, the mice were placed in a glass cylinder of 60 cm in height,
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20 cm in diameter and 30 cm in water depth to maintain the water temperature (24 ±
4
1) °C. The test time was 7 min, and the cumulative time of the mice was recorded 5
5
min after the recording.
6 7
2.9. Evaluation of blood cellularity After behavioural tests, the blood samples were collected from the retroorbital
8 9
plexus and prepared for the assessment of whole blood cellularity. The count of total
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cells, lymphocytes, neutrophils and macrophages were measured using a haematology
11
analyser.
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2.10.
Lungs were excised and fixed with 4% paraformaldehyde. After embedded, the
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Histological examination
slices were cut into 4 µm thickness, and subjected to hematoxylin-eosin (HE) staining.
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2.11.
Measurement of bronchoalveolar lavage (BAL) fluid At the end of the experiment, mice were euthanized after the behavioral
19
measurement is completed. The lungs were lavaged with a total of 1 ml ice-cold PBS
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for three times. After centrifuged, the supernatants of BAL fluid were stored at −80 °C
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till cytokine analyses.
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1
2.12.
Analysis of proinflammatory cytokines in lung and brain Lung and brain tissues were homogenized in physiological saline, ant then
2 3
centrifuged at 4°C at 12000 g for 10 min. The supernatant was frozen at -80°C for
4
later use. The contents of TNF-α, IL-6 and IL-1β in lung and brain were detected
5
according to ELISA kit instructions. The results of the samples were expressed as
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picograms per milligram protein.
7 8
2.13.
Immunohistochemistry Immunohistochemistry staining was used to detect the expression of p-PI3K and
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p-NF-κBp65 in the hippocampus. Briefly, the paraffin sections of hippocampus were
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deparaffinized, rehydrated and incubated in 3% hydrogen peroxide (H2O2). The
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sample was incubated with the corresponding primary antibody at 4 °C overnight
13
after blocked with 3% BSA. Secondary antibody and three antibodies for were
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incubated for 20 min at 37 °C, respectively. Then, samples were stained with DAB
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and restained with hematoxylin. After dehydrated and dried, the sections were
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observed under a light microscopy (200 ×) (Nikon, Tokyo, Japan), and analyzed with
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Image J software (National Institutes of Health, Bethesda, MD, USA),
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2.14.
Western blot analysis The total hippocampus protein was extracted using 1% NP40 and 1 mM
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phenylmethylsulfonyl fluoride (PMSF) and quantified via a bicinchoninic acid (BCA)
22
assay (Sigma, USA). The lysates were then separated using SDS–PAGE and 10
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electroblotted onto polyvinylidene difluoride (PVDF) membranes. Bolts were blocked
2
with 5% BSA for 1.5 h at 37 °C followed by incubated overnight with the primary
3
antibodies, anti-p-PI3K, anti-PI3K, anti-p-Akt, anti-Akt, anti-p-NF-κBp65,
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anti-NF-κBp65, anti-p-IκBα, anti-IκBα (CST, Danvers, USA), at 4°C. After that,
5
bands were incubated with peroxidase-conjugated secondary antibody for 1 h at room
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temperature the bands were visualized via enhanced chemiluminescence using an
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ImmobilonTM Western Kit (Millipore, USA).
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2.15.
Statistical analysis All experiments in this study were performed at least in triplicate independently.
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Data were expressed as mean ± S.E.M. All statistical tests were performed using
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SPSS software version 22.0 (SPSS, USA). Means were compared by one-way
13
analysis of variance (ANOVA) with Tukey’s multiple comparisons test. A P value <
14
0.05 was considered to be statistically significant.
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3. Results
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3.1. COPD was induced by cigarette smoke exposure in mice.
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The FEV1/FVC ratio and the mean alveolus area of lung tissue pathology of
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control group and CS exposed group were tested. As was shown in Fig.1A, the
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FEV1/FVC ratio was significantly down-regulated with the treatment of cigarette
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smoke in comparison with the control group. The mean alveolus area of CS group
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also displayed a declining trend (Fig.1B). These results indicated that cigarette smoke 11
1
exposure gave rise to the onset of pulmonary emphysema and leaded to the air space
2
stenosis of lung tissues, which were important characteristics of COPD model. Thus,
3
it was demonstrated that the COPD animal model was induced by cigarette smoke.
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3.2. Crocin improves cigarette smoke-induced depression-related behaviors. The body weights (Fig.2A) and sucrose preference (Fig.2B) in different groups
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were first detected. In the CS group, the weights of mice and the intake of sucrose
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were markedly down-regulated. However, the administration of crocin effectively
9
increased the body weights and sucrose preference. When crocin was given together
10
with IGF-1, the upregulation effects of crocin were blocked. Furthermore, effects of
11
crocin on locomotor activity were shown in the open field test. The crossing number
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(Fig.2C), rearing number (Fig.2D) and grooming number (Fig.2E) decreased in CS
13
group, while crocin administration dramatically increased the open field behaviors.
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For the FST (Fig.2F) and TST (Fig.2G) tests, cigarette smoke-induced mice exhibited
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a decrease in immobile duration versus the control group. Dex (2 mg/kg) and crocin
16
(50 mg/kg) treatment significant restore the cigarette smoke-induced elevation of
17
immobile time in both TST and FST tests. Crocin (50 mg/kg) + IGF-1 (2 mg/kg)
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group also showed a shorter immobility duration in the TST and FST tests.
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3.3. Crocin inhibits the inflammation in lung tissue of cigarette smoke-induced
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COPD model mice.
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As seen in Fig.3A, B and C, the number of inflammatory cells, including 12
1
neutrophils, macrophages and lymphocytes significantly increased in cigarette
2
smoke-induced COPD model compared to the control group. In contrast, Dex (2
3
mg/kg) or crocin (50 mg/kg) group showed remarkable reduced total cell numbers,
4
neutrophils, and macrophages versus cigarette smoke challenged group. IGF-1 (2
5
mg/kg), an activator of PI3K, abrogated the anti-inflammatory effects of crocin
6
against cigarette smoke-induced increase of inflammatory cells in cigarette
7
smoke-induced model of acute lung inflammation.
8 9
H&E staining of lung tissue (Fig.3D) was used to analyze the histomorphological changes induced by cigarette smoke. It was found an increased inflammatory cell
10
infiltration in the CS group versus to control group. Meanwhile, airspace enlargement
11
was observed in the model group. In contrast, Dex (2 mg/kg) or crocin (50 mg/kg)
12
administration showed a weakened degree of inflammation and lessened airspace
13
compared to the CS group. IGF-1 (2 mg/kg) abrogated the anti-inflammatory effects
14
of crocin against infiltration of inflammatory cells and airspace enlargement in lung
15
tissue of cigarette smoke-exposed mice.
16 17
3.4. Crocin inhibits the cigarette smoke-induced production of proinflammatory
18
cytokines in the BAL fluid and lung tissue.
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To evaluate the anti-inflammatory effects of crocin, ELISA kits were used to
20
detect the contents of proinflammatory cytokines in the BAL fluid and lung tissue. As
21
shown Fig.4, smoke was a stimulator of IL-1β, IL-6 and TNF-α both in BAL fluid and
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lung tissue. Treatment with Dex (2 mg/kg) or crocin (50 mg/kg) 1 h prior to smoke 13
1
exposure strongly decreased the concentration of IL-1β, IL-6 and TNF-α compared
2
with those observed in the CS group, while the reductions in the concentration of
3
these cytokines were abrogated after crocin (50 mg/kg) plus IGF-1 (2 mg/kg)
4
treatment. These remarkable changes in IL-1β, IL-6 and TNF-α powerfully
5
demonstrated that crocin reduced the level of proinflammatory mediators in BAL
6
fluid and lung tissue.
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3.5. Crocin inhibits the cigarette smoke-induced secretion of proinflammatory cytokines in hippocampus.
10
Next, we investigated whether crocin also altered the proinflammatory cytokine
11
responses in the brain after cigarette smoke exposure. As shown in Fig.5A, B and C,
12
the level of hippocampal proinflammatory cytokines in CS group mice increased
13
significantly compared to the air-exposed mice. In contrast, Dex (2 mg/kg) or crocin
14
(50 mg/kg) administration showed a reduced secretion of IL-1β, IL-6 and TNF-α
15
compared with the cigarette smoke exposed group. IGF-1 (2 mg/kg) abrogated the
16
anti-inflammatory effects of crocin against cigarette smoke-induced secretion of
17
proinflammatory cytokines in hippocampus.
18 19
3.6. Crocin prevents the activation of PI3K/Akt mediated NF-κB inflammatory
20
pathways in hippocampus.
21
To characterize the intracellular signaling pathway responsible for the
22
antidepressant effect of crocin in cigarette smoke-induced acute lung inflammation, 14
1
the PI3K/Akt mediated NF-κB signaling pathway was tested. The decreased p-PI3K,
2
p-Akt and increased p-NF-κBp65, p-IκBα levels were observed in cigarette
3
smoke-exposed group, while Dex (2 mg/kg) or crocin (50 mg/kg) administration
4
dramatically restored their alterations with PI3K, Akt, NF-κBp65 and IκBα as internal
5
controls respectively (Fig.6). Immunohistochemistry analysis (Fig.7) suggested the
6
up-regulation of p-PI3K and p-NF-κBp65 in the hippocampus of CS group mice. Dex
7
(2 mg/kg) or crocin (50 mg/kg) administration decreased the expression of p-PI3K
8
and p-NF-κBp65. Of note, IGF-1 (2 mg/kg) administration abrogated all the
9
neuroprotective effects of crocin. These results suggested that crocin-medicated
10
protection against smoke-induced depression involves with the PI3K/Akt mediated
11
NF-κB inflammatory pathways.
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4. Discussion COPD is a complex pulmonary chronic disease which is characterized by
15
chronic inflammation, irreversible airflow limitation and emphysema. Recently, it has
16
become increasingly recognized that patients with COPD with comorbidities may die
17
prematurely compared with the COPD alone patients (Yohannes and Alexopoulos,
18
2014). In these comorbidities, depression places a heavy burden on patients and their
19
families, especially by impairing quality of life and reducing compliance with
20
treatment. Researches have also suggested that current smoker status was one of the
21
most central risk factors of depression symptoms in COPD patients (Lou et al., 2012;
22
Matte et al., 2016). The patients of COPD associated with smoke are more susceptible 15
1
to depression (Zhang et al., 2014). Other studies have reported that the amount of
2
exposure to cigarette smoke was also closely correlated with declines in
3
psycho-emotional state and in cognitive function (Iyer et al., 2016; Negewo et al.,
4
2015) However, the interrelationship among smoking, COPD, and depression has not
5
been fully characterized until now.
6
The COPD model established by cigarette smoke was used in our study. Our
7
results showed that the FEV1/FVC ratio and the mean alveolus area of lung tissue,
8
which were important characteristics of COPD model and significantly decreased by
9
cigarette smoke. It was demonstrated that the COPD animal model was induced by
10
cigarette smoke. Besides, the mice exposed to cigarette smoke showed increased
11
inflammation, increased infiltration of inflammatory cells in the bronchial and
12
peribronchial layers, enlarged airspaces in lung tissues, and elevated concentrations of
13
proinflammatory cytokines in BAL fluid and lung. Cigarette smoke exposure also
14
caused activation of brain inflammatory responses, and led to depressive behaviours.
15
Increased immobile time in tail suspension and forced swimming tests, and elevated
16
levels of the proinflammatory cytokines (IL-1β, IL-6, and TNF-α) were observed in
17
cigarette smoke-exposed mice. These results proved that depression related
18
behaviours were observed in cigarette smoke induced COPD mice.
19
Crocin is a major active ingredient of Chinese traditional herb saffron (Crocus
20
sativus). As a nature product, crocin exhibits multiple beneficial effects in the
21
treatment of various kinds of diseases, which making it attractive for drug discovery
22
(Bukhari et al., 2018). Recent studies have demonstrated the antidepressant effects of 16
1
crocin in animal model as well as clinical trial (Talaei et al., 2015; Wang et al., 2010).
2
Besides, crocin also exhibited anti-inflammation effects in lung injury and
3
anti-asthma potential in allergic airway disease (Wang et al., 2015; Xiong et al., 2015).
4
Therefore, the anti-depression effects and underlying mechanism of crocin was further
5
studied in cigarette smoke induced COPD model mice in our study. The
6
administration of crocin (50 mg/kg) significantly suppressed the production of
7
proinflammatory cytokines and alleviated the depressive associated behaviours, with
8
the similar effects of Dex (2 mg/kg).
9
Nuclear factor-κB (NF-κB), an important transcription factor in a wide range of
10
inflammatory networks, plays a key role in regulating immune response and cytokine
11
activity in airway pathology (Di Stefano et al., 2002; Schuliga, 2015). Researches
12
have demonstrated that NF-κB signaling pathway was activated in bronchial biopsies
13
and inflammatory cells of COPD individuals (Uysal et al., 2019). NF-κB and the
14
phosphorylation of inhibitor of NF-κB were also proved to be increased in cigarette
15
smoke-exposed mice (Shin et al., 2018). Therefore, we further tested whether NF-κB
16
levels were also increased and played a regulation role in the protective effects of
17
crocin in cigarette smoke induced mice model. In quiescent cells, NF-κB activity is
18
severely restricted by interacting with IκB in the cell cytoplasm. In response to
19
external stimuli, phosphorylation by the IκB kinase complex induces IκBα
20
ubiquitination and proteasomal degradation, which allows NF-κB to translocate to the
21
nucleus and bind to its DNA target elements (Jiang et al., 2015). In this study, we
22
found that crocin blunted cigarette smoke induced IκB phosphorylation and 17
1
degradation, as well as NF-κBp65 nuclear translocation. Increases in proinflammatory
2
cytokines in the hippocampus were reversed by crocin treatment. This was probably
3
through crocin inhibition of NF-κB activation, which consequently decreased the gene
4
expression of proinflammatory cytokines, chemokines, and other enzymes, ultimately
5
reducing the severity of the inflammatory response.
6
NF-κB signalling can be activated through multiple pathways, including PI3K
7
and its downstream Akt kinase. In fact, the PI3K/Akt signalling pathway has been
8
shown to be an upstream activator of the NF-κB signalling cascade, both in COPD
9
and in depression (Kim et al., 2012; Ma et al., 2015). Our results showed that the
10
expressions of p-PI3K and p-Akt were significantly upregulated by cigarette smoke,
11
but notably reversed by crocin, indicating that crocin most likely modulates NF-κB by
12
inhibiting PI3K/Akt-mediated signalling. Interestingly, the PI3K/Akt activator IGF-1
13
abrogated the effects of crocin against pulmonary inflammation and activation of
14
inflammatory signalling in the brain, which suggested that the protective effect of
15
crocin may be PI3K-dependent.
16
In conclusion, in this study, animals with cigarette smoke-induced COPD
17
exhibited higher levels of depressive symptoms than did a control group. We found
18
that cigarette smoke-induced pulmonary obstruction was associated with measurable
19
depression-related indicators; the deterioration of pulmonary function resulted in
20
hippocampal dysfunction and depressive symptoms. Our results also demonstrated the
21
protective effects of crocin against cigarette smoke-induced COPD with comorbid
22
depression, the underlying mechanism was partly due to its inhibition of the 18
1
inflammatory response via PI3K/Akt-mediated NF-κB signalling. Our study provides
2
an evidence that crocin exhibits therapeutic potential in inflammatory lung disease
3
with comorbid depression.
4 5 6 7
Acknowledgements This study was supported by the Wenzhou Science and Technology Project (Y20190095).
8 9 10
Data Availability The data used to support the findings of this study are included within the article.
11 12 13
Conflicts of interest The authors declare that they have no competing interests
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pathway. International immunopharmacology 29, 370-376. Kim, D.I., Kim, S.R., Kim, H.J., Lee, S.J., Lee, H.B., Park, S.J., Im, M.-J., Lee, Y.C., 2012. PI3K-γ inhibition ameliorates acute lung injury through regulation of IκBα/NF-κB pathway and innate immune responses. Journal of clinical immunology 32, 340-351. Leonard, B., 2000. Stress, depression and the activation of the immune system. The world journal of biological psychiatry : the official journal of the World Federation of Societies of Biological Psychiatry 1, 17-25. Lou, P., Zhu, Y., Chen, P., Zhang, P., Yu, J., Zhang, N., Chen, N., Zhang, L., Wu, H., Zhao, J., 2012. Prevalence and correlations with depression, anxiety, and other features in outpatients with chronic obstructive pulmonary disease in China: a cross-sectional case control study. BMC Pulm Med 12, 53. Ma, C., Zhu, L., Wang, J., He, H., Chang, X., Gao, J., Shumin, W., Yan, T., 2015. Anti-inflammatory effects of water extract of Taraxacum mongolicum hand.-Mazz on lipopolysaccharide-induced inflammation in acute lung injury by suppressing PI3K/Akt/mTOR signaling pathway. Journal of ethnopharmacology 168, 349-355. Matte, D.L., Pizzichini, M.M., Hoepers, A.T., Diaz, A.P., Karloh, M., Dias, M., Pizzichini, E., 2016. Prevalence of depression in COPD: A systematic review and meta-analysis of controlled studies. Respir Med 117, 154-161. Negewo, N.A., McDonald, V.M., Gibson, P.G., 2015. Comorbidity in chronic obstructive pulmonary disease. Respiratory investigation 53, 249-258. Porsolt, R.D., Anton, G., Blavet, N., Jalfre, M., 1978. Behavioural despair in rats: a new model sensitive to antidepressant treatments. Eur J Pharmacol 47, 379-391. Profita, M., Sala, A., Bonanno, A., Riccobono, L., Ferraro, M., La Grutta, S., Albano, G.D., Montalbano, A.M., Gjomarkaj, M., 2010. Chronic obstructive pulmonary disease and neutrophil infiltration: role of cigarette smoke and cyclooxygenase products. Am J Physiol Lung Cell Mol Physiol 298, L261-269. Quint, J.K., Baghai-Ravary, R., Donaldson, G.C., Wedzicha, J., 2008. Relationship between depression and exacerbations in COPD. European respiratory journal 32, 53-60. Schuliga, M., 2015. NF-kappaB Signaling in Chronic Inflammatory Airway Disease. Biomolecules 5, 1266-1283. Shin, N.R., Kim, C., Seo, C.S., Ko, J.W., Cho, Y.K., Shin, I.S., Kim, J.S., 2018. Galgeun-tang Attenuates Cigarette Smoke and Lipopolysaccharide Induced Pulmonary Inflammation via IkappaBalpha/NF-kappaB Signaling. Molecules 23. Talaei, A., Hassanpour Moghadam, M., Sajadi Tabassi, S.A., Mohajeri, S.A., 2015. Crocin, the main active saffron constituent, as an adjunctive treatment in major depressive disorder: a randomized, double-blind, placebo-controlled, pilot clinical trial. J Affect Disord 174, 51-56. Tsiligianni, I., Kocks, J., Tzanakis, N., Siafakas, N., van der Molen, T., 2011. Factors that influence disease-specific quality of life or health status in patients with COPD: a systematic review and meta-analysis of Pearson correlations. Prim Care Respir J 20, 257-268. Uysal, P., Simsek, G., Durmus, S., Sozer, V., Aksan, H., Yurt, S., Cuhadaroglu, C., Kosar, F., Gelisgen, R., Uzun, H., 2019. Evaluation of plasma antimicrobial peptide LL-37 and nuclear factor-kappaB levels in stable chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis 14, 321-330. Vijayan, V., 2008. Global surveillance, prevention and control of chronic respiratory diseases: a comprehensive approach. Medknow Publications. Wang, G., Mohammadtursun, N., Sun, J., Lv, Y., Jin, H., Lin, J., Kong, L., Zhao, Z., Zhang, H., Dong, J., 2018. Establishment and Evaluation of a Rat Model of Sidestream Cigarette Smoke-Induced Chronic 21
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Obstructive Pulmonary Disease. Front Physiol 9, 58. Wang, J., Campbell, I.L., 2002. Cytokine signaling in the brain: putting a SOCS in it? Journal of neuroscience research 67, 423-427. Wang, J., Kuai, J., Luo, Z., Wang, W., Wang, L., Ke, C., Li, X., Ni, Y., 2015. Crocin attenuates lipopolysacchride-induced acute lung injury in mice. Int J Clin Exp Pathol 8, 4844-4850. Wang, Y., Han, T., Zhu, Y., Zheng, C.J., Ming, Q.L., Rahman, K., Qin, L.P., 2010. Antidepressant properties of bioactive fractions from the extract of Crocus sativus L. J Nat Med 64, 24-30. Xiong, Y., Wang, J., Yu, H., Zhang, X., Miao, C., 2015. Anti-asthma potential of crocin and its effect on MAPK signaling pathway in a murine model of allergic airway disease. Immunopharmacol Immunotoxicol 37, 236-243. Xu, F., Lin, J., Cui, W., Kong, Q., Li, Q., Li, L., Wei, Y., Dong, J., 2018. Scutellaria baicalensis Attenuates Airway Remodeling via PI3K/Akt/NF-kappaB Pathway in Cigarette Smoke Mediated-COPD Rats Model. Evid Based Complement Alternat Med 2018, 1281420. Yohannes, A.M., Alexopoulos, G.S., 2014. Depression and anxiety in patients with COPD. European respiratory review : an official journal of the European Respiratory Society 23, 345-349. Zhang, Q., Liao, J., Liao, X., Wu, X., Wan, M., Wang, C., Ma, Q., 2014. Disease knowledge level is a noteworthy risk factor of anxiety and depression in patients with chronic obstructive pulmonary disease: a cross-sectional study. BMC pulmonary medicine 14, 1. Zhao, J., Li, M., Wang, Z., Chen, J., Zhao, J., Xu, Y., Wei, X., Wang, J., Xie, J., 2019. Role of PM2.5 in the development and progression of COPD and its mechanisms. Respir Res 20, 120.
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22
1
Figure legends
2
Figure 1. Comparison of lung function indicators between the control group and
3
cigarette smoke exposure group. (A) The FEV1/FVC ratio. (B) The mean alveolus
4
area. (n=8) Values are expressed as means ± S.E.M. Compared with control group:
5
#
P<0.05, ##P<0.01. CS: cigarette smoke.
6 7
Figure 2. Effects of crocin on cigarette smoke-induced depression-related
8
behaviors. (A) Body weight. (B) sucrose preference. The crossing number (C),
9
rearing number (D) and grooming number (E) in Open field test. (F) Immobility time
10
in the FST(s). (F) Immobility time in the TST(s). (n=8) Values are expressed as means
11
± S.E.M. Compared with control group: ##P<0.01; Compared with CS group: *P<0.05,
12
**
13
Dex: dexamethasone, CRO: Crocin.
P<0.01; Compared with CS+CRO group: @P<0.05, @@P<0.01. CS: cigarette smoke,
14 15
Figure 3. Effects of crocin on the inflammation in lung tissue of cigarette
16
smoke-induced COPD model mice. The amounts of neutrophils (A), macrophages
17
(B), lymphocytes (C). (n=8) Values are expressed as means ± S.E.M. Compared with
18
control group: ##P<0.01; Compared with CS group: *P<0.05, **P<0.01; Compared
19
with CS+CRO group: @P<0.05, @@P<0.01. (D) Effects of crocin on pulmonary
20
histopathological examination (×200). CS: cigarette smoke, Dex: dexamethasone,
21
CRO: Crocin.
22 23
1
Figure 4. Effects of crocin on the levels of proinflammatory cytokines. IL-1β (A)，
2
IL-6 (B) and TNF-α (C) in BAL fluid and IL-1β (D)，IL-6 (E) and TNF-α (F) in lung.
3
(n=8) Values are expressed as means ± S.E.M. Compared with control group:
4
##
5
group: @P<0.05, @@P<0.01. CS: cigarette smoke, Dex: dexamethasone, CRO: Crocin.
P<0.01; Compared with CS group: *P<0.05, **P<0.01; Compared with CS+CRO
6 7
Figure 5. Effects of crocin on the level of proinflammatory cytokines in
8
hippocampus. (A) IL-6, (B) IL-1β and (C) TNF-α. (n=8) Values are expressed as
9
means ± S.E.M. Compared with control group: ##P<0.01; Compared with CS group:
10
**
P<0.01; Compared with CS+CRO group: @P<0.05, @@P<0.01. CS: cigarette smoke,
11
Dex: dexamethasone, CRO: Crocin.
12 13
Figure 6. Effects of crocin on PI3K/Akt mediated NF-κB inflammatory pathways.
14
The expressions of p-PI3K and PI3K (A), p-Akt and Akt (B), p-NF-κBp65 and
15
NF-κBp65 (C) and p-IκBα and IκBα (D) were tested by western blot analysis. (n=8)
16
Values are expressed as means ± S.E.M. Compared with control group: ##P<0.01;
17
Compared with CS group: *P<0.05, **P<0.01; Compared with CS+CRO group:
18
@
P<0.05. CS: cigarette smoke, Dex: dexamethasone, CRO: Crocin.
19 20
Figure 7. Immunohistochemistry analysis of p-PI3K (A) and p-NF-κBp65 (B) in
21
hippocampus. Quantitative summary showing the density of p-PI3K and
22
p-NF-κBp65 in hippocampus (C). Values are expressed as means ± S.E.M. Compared 24
1
with control group: ##P<0.01; Compared with CS group: **P<0.01; Compared with
2
CS+CRO group: @P<0.05, @@P<0.01. CS: cigarette smoke, Dex: dexamethasone,
3
CRO: Crocin.
4 5 6
25
S0014-2999(19)30592-8
DOI:
https://doi.org/10.1016/j.ejphar.2019.172640
Reference:
EJP 172640
To appear in:
European Journal of Pharmacology
Received Date: 6 August 2019 Revised Date:
30 August 2019
Accepted Date: 2 September 2019
Please cite this article as: Xie, Y., He, Q., Chen, H., Lin, Z., Xu, Y., Yang, C., Crocin ameliorates chronic obstructive pulmonary disease-induced depression via PI3K/Akt mediated suppression of inflammation, European Journal of Pharmacology (2019), doi: https://doi.org/10.1016/j.ejphar.2019.172640. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Crocin ameliorates chronic obstructive pulmonary disease-induced
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depression via PI3K/Akt mediated suppression of inflammation
3 4 5
Yupeng Xie 1*, Qiuxiang He 2, Hong Chen 3, Zijiang Lin 3, Yi Xu 4, Chuang Yang 3*
6 7
1. Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital
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of Wenzhou Medical University, Wenzhou 325000, China
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2. Department of Pathology, The First Affiliated Hospital of Wenzhou Medical
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University, Wenzhou 325000, China
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3. Department of Psychiatry, The First Affiliated Hospital of Wenzhou Medical
12
University, Wenzhou 325000, China
13
4. School of Pharmacy, Wenzhou Medical University, Wenzhou 325000, China
14 15
*Corresponding author:
16
Yupeng Xie ([email protected])
17
Yang Chuang ([email protected])
18 19
1
1 2
Abstract Clinical studies have indicated the co-occurrence of chronic obstructive
3
pulmonary disease (COPD) and psychiatric disorders, for example, comorbid
4
depression. However, the underlying mechanism is rarely addressed. The present
5
study aimed to investigate the mechanism of COPD-induced depression and the
6
psychological and physiological effects of crocin, an active constituent of Crocus
7
sativus L.. C57BL/6 mice were randomly exposed to cigarette smoke for 7 weeks to
8
establish COPD animal model. Crocin (50 mg/kg), Dexamethasone (2 mg/kg) and
9
IGF-1 (2 mg/kg) were respectively injected to mice once a day. The FEV1/FVC ratio
10
and the mean alveolus area of lung tissue demonstrated the COPD model was
11
successfully established by cigarette smoke. Crocin administration significantly
12
reversed markers of depression [loss of body weight, sucrose preference, and
13
elevation of immobile time in tail-suspension tests (TST) and in forced swimming
14
tests (FST)]. Besides, crocin treatment significantly inhibited the numbers of
15
inflammatory cells (macrophages, neutrophils, and lymphocytes), suppressed the
16
infiltration of peribronchial inflammatory cells, and reduced the concentration of
17
proinflammatory cytokines in bronchoalveolar lavage (BAL) fluid and lung tissue.
18
Crocin also reduced proinflammatory cytokines in the hippocampus. In exploring
19
associated mechanisms, we discovered that crocin blunted cigarette smoke-induced
20
IκB phosphorylation and degradation, and NF-κBp65 nuclear translocation. IGF-1, an
21
activator of PI3K, abrogated the effect of crocin against cigarette smoke-induced
22
activation of the NF-κB pathway. Together, these results showed that an inflammatory 2
1
mechanism might be involved in the pathogenesis of COPD with comorbid
2
depression. Crocin exhibited significant effects through the regulation of
3
PI3K/Akt-mediated inflammatory pathways.
4 5
Keywords: Chronic obstructive pulmonary disease; Depression; Inflammation;
6
PI3K/Akt/NF-κB; Crocin
7
3
1 2
1. Introduction Chronic obstructive pulmonary disease (COPD) is a common chronic disease of
3
the respiratory system characterized by airflow obstruction (Huertas and Palange,
4
2011). With increasing emissions of particulate matter (i.e., PM2.5) and other types of
5
air pollution, more and more people suffer from respiratory disease, and it is predicted
6
that COPD will become the third leading cause of death by 2030 (Vijayan, 2008;
7
Zhao et al., 2019). Cigarette smoke is considered as the principle factor driving the
8
pathogenesis and progression of pulmonary diseases (Arunachalam et al., 2010). The
9
components generated by smoking, such as carbon monoxide, nicotine, oxidants, fine,
10
were closely related to the onset of COPD (Alwis et al., 2015). Innate immune
11
response could be activated by cigarette smoke and then multiple chemotactic factors
12
is released to recruit neutrophils and inflammatory monocytes to the lungs (Chen et al.,
13
2019). Interestingly, increased accumulation of neutrophils, macrophages were
14
observed in the lungs of COPD patients and COPD mouse models (Barnes, 2016;
15
Profita et al., 2010).
16
Depression is a highly debilitating and life-threatening mental disorder, which is
17
a huge burden of the society (Deng et al., 2015). Meta-analysis studies have reported
18
that depression was highly correlated with COPD (Tsiligianni et al., 2011). In fact,
19
depression is a common clinical comorbidity of COPD (Di Marco et al., 2006; Du et
20
al., 2014; Quint et al., 2008). COPD patients suffer from comorbid depression often
21
continuously smoke and frequently medical complications (Hillas et al., 2015). Those
22
could increase their persistent depressive and pulmonary symptoms and signs, leading 4
1
to greater disability and mortality. However, the pathobiology of depression in COPD
2
is still unclear. More and more evidences suggest that inflammatory cytokines play
3
core roles in the aetiology of both COPD and depression (Leonard, 2000).
4
Inflammatory cytokine in the brain is involved in the regulation of neurotransmitter
5
metabolism, synaptic plasticity, and neuroendocrine function, which are known to be
6
important in the aetiopathogenesis of depression (Wang and Campbell, 2002).
7
Inhibiting cytokines induced by cigarette smoke might thus be a therapeutic strategy
8
for COPD with depression.
9
Crocin is a natural carotenoid that has exhibited beneficial effects in the
10
treatment of central nervous system disorders, including depression, convulsion, and
11
hypomnesia (Farkhondeh et al., 2018; Finley and Gao, 2017). In an animal model,
12
crocin showed significant and dose-dependent anti-depressant activities, and may
13
have acted via the inhibition of dopamine and norepinephrine uptake (Wang et al.,
14
2010). Crocin also attenuated malathion-induced depressive behaviours by increasing
15
the level of brain-derived neurotrophic factor (BDNF) in mice hippocampus and
16
cerebral cortex (Dorri et al., 2015). Beside its neuroprotective properties, crocin has
17
also exhibited protective effects during lung injury (Wang et al., 2015) as well as
18
anti-asthma potential in allergic airway disease (Xiong et al., 2015).
19
Therefore, mice model of COPD was established by exposed to cigarette smoke,
20
and the underlying pathogenic mechanism of COPD with comorbid depression was
21
explored. Meantime, the psychopathological responses to crocin, a clinical
22
antidepressant with beneficial effects against lung injury were also investigated. 5
1
2. Materials and Methods
2
2.1. Reagents
3
Crocin and Dexamethasone (Dex) were obtained from Sigma-Aldrich (Saint
4
Louis, MO, USA). Insulin-like growth factor 1 (IGF-1), the activator of PI3K, was
5
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). IL-1β、IL-6
6
and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were provided by
7
Nanjing KeyGEN Biotech. CO., LTD. (Nanjing, China). Primary antibodies against
8
p-PI3K、PI3K、p-Akt、Akt、p-NF-κBp65, NF-κBp65, p-IκBα and IκBα were produced
9
by Cell Signaling Technology (Danvers, USA).
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2.2. Animals Male C57BL/6 mice, 7-8 weeks, were obtained from the Animal Center of
13
Wenzhou Medical University (Wenzhou, China). The mice were maintained under
14
controlled temperature (22-24oC) and 12/12 h light/dark cycle conditions with ad lib
15
access to sterile standard laboratory chow. All animal experiments were performed
16
according to protocols approved by Medicine Animal Care and Use Committee of
17
Wenzhou Medical University.
18 19 20
2.3. The establishment of CS-induced COPD model and experimental design C57BL/6 mice were exposed to cigarette smoke of 5 3R4F Kentucky reference
21
cigarettes (without filter, University of Kentucky, Lexington, KY, USA), one after
22
another, four times a day (total of 20 cigarettes per day) with a smoking machine as 6
1
previously described (Xu et al., 2018). The smoking machine consists of an animal
2
containment system and a cigarette smoking control system. Animals were alternately
3
exposed to the smoke for 30 min and smoke-free interval for 30 min. The procedure
4
lasts for 7 consecutive weeks.
5
Animals in this study were divided into five groups (n=8): the control group; the
6
CS exposed group (the CS group); the CS + Dex (2 mg/kg) group; the CS + CRO (50
7
mg/kg) group; the CS + CRO (50 mg/kg) + IGF-1 (2 mg/kg) group. Animals in the
8
control group were exposed to the fresh air. The mice in different group were treated
9
respectively with vehicle, Dex (2 mg/kg) or IGF-1 (2 mg/kg) via a single i.p. injection
10
or 50 mg/kg of crocin orally 1h before exposure to cigarette smoke once a day. Dex, a
11
widely used anti-inflammatory medicine, was used here as a positive control. The
12
dosage of Dex was determined by reference to clinical dosage. The dosages of crocin
13
and IGF-1 were determined by preliminary experiments. The behavior tests were
14
determined 24 h after the last cigarette smoke exposure. After that, the lung function
15
of mice in control group and CS groups were detected to verify the establishment of
16
COPD model. Then, the blood samples were collected from the retroorbital plexus.
17
Finally, we collected the bronchoalveolar lavage fluid (BAL fluid), lung and brain
18
tissue from the mice.
19 20
2.4. Determination of lung function in cigarette smoke -induced COPD mice
21
To test lung function, all mice were anesthetized with 1% pentobarbital (60
22
mg/kg) and fixed in a forced pulmonary maneuvre system (Buxco, NY, USA). The 7
1
forced expiratory volume in the first second (FEV1)/forced vital capacity (FVC) ratio
2
was then determined as previously described (Wang et al., 2018).
3 4
2.5. Sucrose preference test
5
In the experiment, the mice were kept in cages, and the water was forbidden and
6
fasted for 12 h, then 1% sucrose solution were given for 1 h. Calculate the percentage
7
of sugar water consumption: sugar water consumption/total liquid
8
consumption×100%.
9
2.6. Open field test (OFT)
10
The behavior tests were determined at 24 h after the last cigarette smoke
11
exposure. The test was used to measure anxiety levels in mice. Mice were placed in
12
an open field, which was divided into symmetrical sectors. In the beginning, the mice
13
were placed in the center of the device. And each mouse is free to explore for 6 min,
14
counting the number of crossings.
15 16 17
2.7. Tail suspension test (TST) The tape was stuck to the end of the mouse tail 1 cm, and the mouse was hung
18
upside down on a hook 40 cm from the floor. The camera was recorded for 6 min. The
19
cumulative resting time of the last 4 min of the mouse was counted.
20 21
2.8. Forced swimming test (FST) 8
FST was performed as previously described (Detke and Lucki, 1996; Porsolt et
1 2
al., 1978). During the test, the mice were placed in a glass cylinder of 60 cm in height,
3
20 cm in diameter and 30 cm in water depth to maintain the water temperature (24 ±
4
1) °C. The test time was 7 min, and the cumulative time of the mice was recorded 5
5
min after the recording.
6 7
2.9. Evaluation of blood cellularity After behavioural tests, the blood samples were collected from the retroorbital
8 9
plexus and prepared for the assessment of whole blood cellularity. The count of total
10
cells, lymphocytes, neutrophils and macrophages were measured using a haematology
11
analyser.
12 13
2.10.
Lungs were excised and fixed with 4% paraformaldehyde. After embedded, the
14 15
Histological examination
slices were cut into 4 µm thickness, and subjected to hematoxylin-eosin (HE) staining.
16 17 18
2.11.
Measurement of bronchoalveolar lavage (BAL) fluid At the end of the experiment, mice were euthanized after the behavioral
19
measurement is completed. The lungs were lavaged with a total of 1 ml ice-cold PBS
20
for three times. After centrifuged, the supernatants of BAL fluid were stored at −80 °C
21
till cytokine analyses.
22 9
1
2.12.
Analysis of proinflammatory cytokines in lung and brain Lung and brain tissues were homogenized in physiological saline, ant then
2 3
centrifuged at 4°C at 12000 g for 10 min. The supernatant was frozen at -80°C for
4
later use. The contents of TNF-α, IL-6 and IL-1β in lung and brain were detected
5
according to ELISA kit instructions. The results of the samples were expressed as
6
picograms per milligram protein.
7 8
2.13.
Immunohistochemistry Immunohistochemistry staining was used to detect the expression of p-PI3K and
9 10
p-NF-κBp65 in the hippocampus. Briefly, the paraffin sections of hippocampus were
11
deparaffinized, rehydrated and incubated in 3% hydrogen peroxide (H2O2). The
12
sample was incubated with the corresponding primary antibody at 4 °C overnight
13
after blocked with 3% BSA. Secondary antibody and three antibodies for were
14
incubated for 20 min at 37 °C, respectively. Then, samples were stained with DAB
15
and restained with hematoxylin. After dehydrated and dried, the sections were
16
observed under a light microscopy (200 ×) (Nikon, Tokyo, Japan), and analyzed with
17
Image J software (National Institutes of Health, Bethesda, MD, USA),
18 19 20
2.14.
Western blot analysis The total hippocampus protein was extracted using 1% NP40 and 1 mM
21
phenylmethylsulfonyl fluoride (PMSF) and quantified via a bicinchoninic acid (BCA)
22
assay (Sigma, USA). The lysates were then separated using SDS–PAGE and 10
1
electroblotted onto polyvinylidene difluoride (PVDF) membranes. Bolts were blocked
2
with 5% BSA for 1.5 h at 37 °C followed by incubated overnight with the primary
3
antibodies, anti-p-PI3K, anti-PI3K, anti-p-Akt, anti-Akt, anti-p-NF-κBp65,
4
anti-NF-κBp65, anti-p-IκBα, anti-IκBα (CST, Danvers, USA), at 4°C. After that,
5
bands were incubated with peroxidase-conjugated secondary antibody for 1 h at room
6
temperature the bands were visualized via enhanced chemiluminescence using an
7
ImmobilonTM Western Kit (Millipore, USA).
8 9 10
2.15.
Statistical analysis All experiments in this study were performed at least in triplicate independently.
11
Data were expressed as mean ± S.E.M. All statistical tests were performed using
12
SPSS software version 22.0 (SPSS, USA). Means were compared by one-way
13
analysis of variance (ANOVA) with Tukey’s multiple comparisons test. A P value <
14
0.05 was considered to be statistically significant.
15 16
3. Results
17
3.1. COPD was induced by cigarette smoke exposure in mice.
18
The FEV1/FVC ratio and the mean alveolus area of lung tissue pathology of
19
control group and CS exposed group were tested. As was shown in Fig.1A, the
20
FEV1/FVC ratio was significantly down-regulated with the treatment of cigarette
21
smoke in comparison with the control group. The mean alveolus area of CS group
22
also displayed a declining trend (Fig.1B). These results indicated that cigarette smoke 11
1
exposure gave rise to the onset of pulmonary emphysema and leaded to the air space
2
stenosis of lung tissues, which were important characteristics of COPD model. Thus,
3
it was demonstrated that the COPD animal model was induced by cigarette smoke.
4 5 6
3.2. Crocin improves cigarette smoke-induced depression-related behaviors. The body weights (Fig.2A) and sucrose preference (Fig.2B) in different groups
7
were first detected. In the CS group, the weights of mice and the intake of sucrose
8
were markedly down-regulated. However, the administration of crocin effectively
9
increased the body weights and sucrose preference. When crocin was given together
10
with IGF-1, the upregulation effects of crocin were blocked. Furthermore, effects of
11
crocin on locomotor activity were shown in the open field test. The crossing number
12
(Fig.2C), rearing number (Fig.2D) and grooming number (Fig.2E) decreased in CS
13
group, while crocin administration dramatically increased the open field behaviors.
14
For the FST (Fig.2F) and TST (Fig.2G) tests, cigarette smoke-induced mice exhibited
15
a decrease in immobile duration versus the control group. Dex (2 mg/kg) and crocin
16
(50 mg/kg) treatment significant restore the cigarette smoke-induced elevation of
17
immobile time in both TST and FST tests. Crocin (50 mg/kg) + IGF-1 (2 mg/kg)
18
group also showed a shorter immobility duration in the TST and FST tests.
19 20
3.3. Crocin inhibits the inflammation in lung tissue of cigarette smoke-induced
21
COPD model mice.
22
As seen in Fig.3A, B and C, the number of inflammatory cells, including 12
1
neutrophils, macrophages and lymphocytes significantly increased in cigarette
2
smoke-induced COPD model compared to the control group. In contrast, Dex (2
3
mg/kg) or crocin (50 mg/kg) group showed remarkable reduced total cell numbers,
4
neutrophils, and macrophages versus cigarette smoke challenged group. IGF-1 (2
5
mg/kg), an activator of PI3K, abrogated the anti-inflammatory effects of crocin
6
against cigarette smoke-induced increase of inflammatory cells in cigarette
7
smoke-induced model of acute lung inflammation.
8 9
H&E staining of lung tissue (Fig.3D) was used to analyze the histomorphological changes induced by cigarette smoke. It was found an increased inflammatory cell
10
infiltration in the CS group versus to control group. Meanwhile, airspace enlargement
11
was observed in the model group. In contrast, Dex (2 mg/kg) or crocin (50 mg/kg)
12
administration showed a weakened degree of inflammation and lessened airspace
13
compared to the CS group. IGF-1 (2 mg/kg) abrogated the anti-inflammatory effects
14
of crocin against infiltration of inflammatory cells and airspace enlargement in lung
15
tissue of cigarette smoke-exposed mice.
16 17
3.4. Crocin inhibits the cigarette smoke-induced production of proinflammatory
18
cytokines in the BAL fluid and lung tissue.
19
To evaluate the anti-inflammatory effects of crocin, ELISA kits were used to
20
detect the contents of proinflammatory cytokines in the BAL fluid and lung tissue. As
21
shown Fig.4, smoke was a stimulator of IL-1β, IL-6 and TNF-α both in BAL fluid and
22
lung tissue. Treatment with Dex (2 mg/kg) or crocin (50 mg/kg) 1 h prior to smoke 13
1
exposure strongly decreased the concentration of IL-1β, IL-6 and TNF-α compared
2
with those observed in the CS group, while the reductions in the concentration of
3
these cytokines were abrogated after crocin (50 mg/kg) plus IGF-1 (2 mg/kg)
4
treatment. These remarkable changes in IL-1β, IL-6 and TNF-α powerfully
5
demonstrated that crocin reduced the level of proinflammatory mediators in BAL
6
fluid and lung tissue.
7 8 9
3.5. Crocin inhibits the cigarette smoke-induced secretion of proinflammatory cytokines in hippocampus.
10
Next, we investigated whether crocin also altered the proinflammatory cytokine
11
responses in the brain after cigarette smoke exposure. As shown in Fig.5A, B and C,
12
the level of hippocampal proinflammatory cytokines in CS group mice increased
13
significantly compared to the air-exposed mice. In contrast, Dex (2 mg/kg) or crocin
14
(50 mg/kg) administration showed a reduced secretion of IL-1β, IL-6 and TNF-α
15
compared with the cigarette smoke exposed group. IGF-1 (2 mg/kg) abrogated the
16
anti-inflammatory effects of crocin against cigarette smoke-induced secretion of
17
proinflammatory cytokines in hippocampus.
18 19
3.6. Crocin prevents the activation of PI3K/Akt mediated NF-κB inflammatory
20
pathways in hippocampus.
21
To characterize the intracellular signaling pathway responsible for the
22
antidepressant effect of crocin in cigarette smoke-induced acute lung inflammation, 14
1
the PI3K/Akt mediated NF-κB signaling pathway was tested. The decreased p-PI3K,
2
p-Akt and increased p-NF-κBp65, p-IκBα levels were observed in cigarette
3
smoke-exposed group, while Dex (2 mg/kg) or crocin (50 mg/kg) administration
4
dramatically restored their alterations with PI3K, Akt, NF-κBp65 and IκBα as internal
5
controls respectively (Fig.6). Immunohistochemistry analysis (Fig.7) suggested the
6
up-regulation of p-PI3K and p-NF-κBp65 in the hippocampus of CS group mice. Dex
7
(2 mg/kg) or crocin (50 mg/kg) administration decreased the expression of p-PI3K
8
and p-NF-κBp65. Of note, IGF-1 (2 mg/kg) administration abrogated all the
9
neuroprotective effects of crocin. These results suggested that crocin-medicated
10
protection against smoke-induced depression involves with the PI3K/Akt mediated
11
NF-κB inflammatory pathways.
12 13 14
4. Discussion COPD is a complex pulmonary chronic disease which is characterized by
15
chronic inflammation, irreversible airflow limitation and emphysema. Recently, it has
16
become increasingly recognized that patients with COPD with comorbidities may die
17
prematurely compared with the COPD alone patients (Yohannes and Alexopoulos,
18
2014). In these comorbidities, depression places a heavy burden on patients and their
19
families, especially by impairing quality of life and reducing compliance with
20
treatment. Researches have also suggested that current smoker status was one of the
21
most central risk factors of depression symptoms in COPD patients (Lou et al., 2012;
22
Matte et al., 2016). The patients of COPD associated with smoke are more susceptible 15
1
to depression (Zhang et al., 2014). Other studies have reported that the amount of
2
exposure to cigarette smoke was also closely correlated with declines in
3
psycho-emotional state and in cognitive function (Iyer et al., 2016; Negewo et al.,
4
2015) However, the interrelationship among smoking, COPD, and depression has not
5
been fully characterized until now.
6
The COPD model established by cigarette smoke was used in our study. Our
7
results showed that the FEV1/FVC ratio and the mean alveolus area of lung tissue,
8
which were important characteristics of COPD model and significantly decreased by
9
cigarette smoke. It was demonstrated that the COPD animal model was induced by
10
cigarette smoke. Besides, the mice exposed to cigarette smoke showed increased
11
inflammation, increased infiltration of inflammatory cells in the bronchial and
12
peribronchial layers, enlarged airspaces in lung tissues, and elevated concentrations of
13
proinflammatory cytokines in BAL fluid and lung. Cigarette smoke exposure also
14
caused activation of brain inflammatory responses, and led to depressive behaviours.
15
Increased immobile time in tail suspension and forced swimming tests, and elevated
16
levels of the proinflammatory cytokines (IL-1β, IL-6, and TNF-α) were observed in
17
cigarette smoke-exposed mice. These results proved that depression related
18
behaviours were observed in cigarette smoke induced COPD mice.
19
Crocin is a major active ingredient of Chinese traditional herb saffron (Crocus
20
sativus). As a nature product, crocin exhibits multiple beneficial effects in the
21
treatment of various kinds of diseases, which making it attractive for drug discovery
22
(Bukhari et al., 2018). Recent studies have demonstrated the antidepressant effects of 16
1
crocin in animal model as well as clinical trial (Talaei et al., 2015; Wang et al., 2010).
2
Besides, crocin also exhibited anti-inflammation effects in lung injury and
3
anti-asthma potential in allergic airway disease (Wang et al., 2015; Xiong et al., 2015).
4
Therefore, the anti-depression effects and underlying mechanism of crocin was further
5
studied in cigarette smoke induced COPD model mice in our study. The
6
administration of crocin (50 mg/kg) significantly suppressed the production of
7
proinflammatory cytokines and alleviated the depressive associated behaviours, with
8
the similar effects of Dex (2 mg/kg).
9
Nuclear factor-κB (NF-κB), an important transcription factor in a wide range of
10
inflammatory networks, plays a key role in regulating immune response and cytokine
11
activity in airway pathology (Di Stefano et al., 2002; Schuliga, 2015). Researches
12
have demonstrated that NF-κB signaling pathway was activated in bronchial biopsies
13
and inflammatory cells of COPD individuals (Uysal et al., 2019). NF-κB and the
14
phosphorylation of inhibitor of NF-κB were also proved to be increased in cigarette
15
smoke-exposed mice (Shin et al., 2018). Therefore, we further tested whether NF-κB
16
levels were also increased and played a regulation role in the protective effects of
17
crocin in cigarette smoke induced mice model. In quiescent cells, NF-κB activity is
18
severely restricted by interacting with IκB in the cell cytoplasm. In response to
19
external stimuli, phosphorylation by the IκB kinase complex induces IκBα
20
ubiquitination and proteasomal degradation, which allows NF-κB to translocate to the
21
nucleus and bind to its DNA target elements (Jiang et al., 2015). In this study, we
22
found that crocin blunted cigarette smoke induced IκB phosphorylation and 17
1
degradation, as well as NF-κBp65 nuclear translocation. Increases in proinflammatory
2
cytokines in the hippocampus were reversed by crocin treatment. This was probably
3
through crocin inhibition of NF-κB activation, which consequently decreased the gene
4
expression of proinflammatory cytokines, chemokines, and other enzymes, ultimately
5
reducing the severity of the inflammatory response.
6
NF-κB signalling can be activated through multiple pathways, including PI3K
7
and its downstream Akt kinase. In fact, the PI3K/Akt signalling pathway has been
8
shown to be an upstream activator of the NF-κB signalling cascade, both in COPD
9
and in depression (Kim et al., 2012; Ma et al., 2015). Our results showed that the
10
expressions of p-PI3K and p-Akt were significantly upregulated by cigarette smoke,
11
but notably reversed by crocin, indicating that crocin most likely modulates NF-κB by
12
inhibiting PI3K/Akt-mediated signalling. Interestingly, the PI3K/Akt activator IGF-1
13
abrogated the effects of crocin against pulmonary inflammation and activation of
14
inflammatory signalling in the brain, which suggested that the protective effect of
15
crocin may be PI3K-dependent.
16
In conclusion, in this study, animals with cigarette smoke-induced COPD
17
exhibited higher levels of depressive symptoms than did a control group. We found
18
that cigarette smoke-induced pulmonary obstruction was associated with measurable
19
depression-related indicators; the deterioration of pulmonary function resulted in
20
hippocampal dysfunction and depressive symptoms. Our results also demonstrated the
21
protective effects of crocin against cigarette smoke-induced COPD with comorbid
22
depression, the underlying mechanism was partly due to its inhibition of the 18
1
inflammatory response via PI3K/Akt-mediated NF-κB signalling. Our study provides
2
an evidence that crocin exhibits therapeutic potential in inflammatory lung disease
3
with comorbid depression.
4 5 6 7
Acknowledgements This study was supported by the Wenzhou Science and Technology Project (Y20190095).
8 9 10
Data Availability The data used to support the findings of this study are included within the article.
11 12 13
Conflicts of interest The authors declare that they have no competing interests
14 15
19
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22
1
Figure legends
2
Figure 1. Comparison of lung function indicators between the control group and
3
cigarette smoke exposure group. (A) The FEV1/FVC ratio. (B) The mean alveolus
4
area. (n=8) Values are expressed as means ± S.E.M. Compared with control group:
5
#
P<0.05, ##P<0.01. CS: cigarette smoke.
6 7
Figure 2. Effects of crocin on cigarette smoke-induced depression-related
8
behaviors. (A) Body weight. (B) sucrose preference. The crossing number (C),
9
rearing number (D) and grooming number (E) in Open field test. (F) Immobility time
10
in the FST(s). (F) Immobility time in the TST(s). (n=8) Values are expressed as means
11
± S.E.M. Compared with control group: ##P<0.01; Compared with CS group: *P<0.05,
12
**
13
Dex: dexamethasone, CRO: Crocin.
P<0.01; Compared with CS+CRO group: @P<0.05, @@P<0.01. CS: cigarette smoke,
14 15
Figure 3. Effects of crocin on the inflammation in lung tissue of cigarette
16
smoke-induced COPD model mice. The amounts of neutrophils (A), macrophages
17
(B), lymphocytes (C). (n=8) Values are expressed as means ± S.E.M. Compared with
18
control group: ##P<0.01; Compared with CS group: *P<0.05, **P<0.01; Compared
19
with CS+CRO group: @P<0.05, @@P<0.01. (D) Effects of crocin on pulmonary
20
histopathological examination (×200). CS: cigarette smoke, Dex: dexamethasone,
21
CRO: Crocin.
22 23
1
Figure 4. Effects of crocin on the levels of proinflammatory cytokines. IL-1β (A)，
2
IL-6 (B) and TNF-α (C) in BAL fluid and IL-1β (D)，IL-6 (E) and TNF-α (F) in lung.
3
(n=8) Values are expressed as means ± S.E.M. Compared with control group:
4
##
5
group: @P<0.05, @@P<0.01. CS: cigarette smoke, Dex: dexamethasone, CRO: Crocin.
P<0.01; Compared with CS group: *P<0.05, **P<0.01; Compared with CS+CRO
6 7
Figure 5. Effects of crocin on the level of proinflammatory cytokines in
8
hippocampus. (A) IL-6, (B) IL-1β and (C) TNF-α. (n=8) Values are expressed as
9
means ± S.E.M. Compared with control group: ##P<0.01; Compared with CS group:
10
**
P<0.01; Compared with CS+CRO group: @P<0.05, @@P<0.01. CS: cigarette smoke,
11
Dex: dexamethasone, CRO: Crocin.
12 13
Figure 6. Effects of crocin on PI3K/Akt mediated NF-κB inflammatory pathways.
14
The expressions of p-PI3K and PI3K (A), p-Akt and Akt (B), p-NF-κBp65 and
15
NF-κBp65 (C) and p-IκBα and IκBα (D) were tested by western blot analysis. (n=8)
16
Values are expressed as means ± S.E.M. Compared with control group: ##P<0.01;
17
Compared with CS group: *P<0.05, **P<0.01; Compared with CS+CRO group:
18
@
P<0.05. CS: cigarette smoke, Dex: dexamethasone, CRO: Crocin.
19 20
Figure 7. Immunohistochemistry analysis of p-PI3K (A) and p-NF-κBp65 (B) in
21
hippocampus. Quantitative summary showing the density of p-PI3K and
22
p-NF-κBp65 in hippocampus (C). Values are expressed as means ± S.E.M. Compared 24
1
with control group: ##P<0.01; Compared with CS group: **P<0.01; Compared with
2
CS+CRO group: @P<0.05, @@P<0.01. CS: cigarette smoke, Dex: dexamethasone,
3
CRO: Crocin.
4 5 6
25