Key words
icariin -
Epimedium brevicornum
- Berberidaceae - asthma - depression - glucocorticoid resistance
Abbreviations
AHR:
airway hyperresponsiveness
BALF:
bronchoalveolar lavage fluid
CCK-8:
cell count kit-8
CUMS:
chronic unpredictable mild stress
DEP:
depression
Dex:
dexamethasone
GR:
glucocorticoid receptor
H&E:
hematoxylin and eosin
IgE:
immunoglobulin E
IL:
interleukin
INF:
interferon
MAPK:
mitogen-activated protein kinase
OD:
optical density
OFT:
open field test
OVA:
ovalbumin
Penh:
enhanced pause
Introduction
Asthma affects more than 300 million individuals around the world, and its global burden has increased by almost 30% in the past 20 years [1]. Individuals with
asthma were reported to have a higher prevalence of depression than the general population [2]. Depression is closely related to poorer control and quality of life
in adults with asthma, as well as a high rate of asthma exacerbation [3], [4], which is attributed to increased airway inflammation
aggravated by depression [5]. A diminished inhibitory action of endogenous glucocorticoid was involved in the pathogenesis of chronic stress-induced exacerbation of
airway inflammation in patients with asthma [6]. A significant proportion of depressed patients exhibited steroid resistance with elevated levels of inflammatory
biomarkers, as well as increased secretion of the stress hormone cortisol [7]. However, the mechanisms through which depression-induced steroid resistance in asthma
have yet to be discovered.
p38 MAPK is a class of evolutionarily conserved serine/threonine mitogen-activated protein kinases, and it links extracellular signals, such as stress, to the intracellular machinery to
influence a variety of cell responses [8]. The p38 MAPK-induced phosphorylation of GR S226 in cytoplasm can result in defective GR nuclear translocation, which
leads to weakened GR function and thus steroid insensitivity [9]. Furthermore, activation of p38 MAPK has been shown to influence behaviorally relevant
pathophysiologic activities, and p38 MAPK was found to be significantly activated in patients with depression [10]. Thus, p38 MAPK might be a good target for
improvement of depression-induced steroid resistance in asthma. Indeed, a p38 MAPK inhibitor (SB203580) has been demonstrated to preferentially restore steroid sensitivity [11]. However, the adverse effects of the p38 MAPK inhibitor remain uncertain, and new drugs are still needed for asthma with steroid resistance that target p38 MAPK
signaling with little side effects.
Epimedium brevicornum (Berberidaceae) Maxim has been widely used as a tonic herb to nourish the kidney Qi according to the famous Chinese medicine document Ben Cao Gang Mu [12]. Icariin ([Fig. 1]), a main active constituent of E. brevicornum Maxim, exerts a wide range of biological activities and
pharmacological properties, including anti-inflammatory, immunomodulatory, estrogenic, and anti-osteoporotic effects [13], [14], [15]. Recently, icariin was demonstrated to effectively inhibit the activity of p38 MAPK in vivo and in vitro
[16], [17]. However, its effects on depression-induced steroid resistance in asthma and the mechanisms involved have not been fully illustrated. Therefore, the objective of
this study was to investigate the possible mechanisms of icariin action on steroid resistance in a murine model of asthma with depression, with particular emphasis on the p38 MAPK signaling
pathway.
Fig. 1 Chemical structure of icariin.
Results
As shown in [Fig. 2], the asthma-DEP group displayed depressive behavior, as evidenced by increased duration of immobility in both the forced swim test
(p < 0.01) ([Fig. 2 a]) and the tail suspension test (p < 0.05) ([Fig. 2 b]) when compared to the single asthma group and saline
control group. These results suggested that 4 weeks of CUMS induced depression-like behaviors. Furthermore, the time in the center area in the OFT test was significantly reduced in the
asthma-DEP group compared to the saline control group. However, the results of the above behavioral tests were not significantly changed after administration with icariin, thereby illustrating
that icariin had limited effects on the depression-like behaviors in this study.
Fig. 2 Behavioral assessment of depression-like symptoms. a The immobility time in the forced swimming test (FST). b The immobility time in the tail suspension test
(TST). c The travel distance in the center zone in the open field test. d The time in the center in the open field test. Asthma: single asthma group, AD: asthma with
depression group, Dex: dexamethasone group. IC25, IC50, IC100: the low, median, and high dosage of icariin groups treated with 25, 50 and 100 mg/kg,
respectively. The asthma with depression group is a control for the treatment groups. All data are expressed as the mean ± SEM (n = 6 – 8 each group); *p < 0.05 and **p < 0.01 versus
asthma in depression mice.
To evaluate the effects of icariin on OVA-induced AHR, the Penh was used as an indicator of airway responsiveness at baseline and following delivery of increasing concentrations of inhaled
methacholine (6.25 to 25 mg/mL). As shown in [Fig. 3], mice in the asthma-DEP group displayed increased Penh when compared to mice in both the single asthma group
and saline control group (p < 0.01), thereby demonstrating that AHR was aggravated by depression induced by CUMS. The oral administration of icariin at 25, 50, and 100 mg/kg and Dex caused
an obvious reduction in Penh to methacholine at 6.25, 12.5, and 25 mg/mL compared to mice in the asthma-DEP group (p < 0.01).
Fig. 3 Icariin suppressed methacholine-induced airway hyperresponsiveness (AHR). Airway responsiveness to aerosolized methacholine was evaluated by Buxcoʼs whole-body barometric
plethysmography in awake unrestrained mice. Mice were nebulized with PBS followed by increasing doses (6.25 to 25 mg/mL) of methacholine. The enhanced pause (Penh) index of airway
hyperreactivity was used as an indicator of changes in airway resistance. Treatment with icariin at 25, 50, and 100 mg/kg and dexamethasone (Dex) (0.5 mg/kg) caused a marked decrease in
Penh compared to mice in the asthma with depression group (p < 0.01). Asthma: single asthma group, AD: asthma with depression group, Dex: dexamethasone group. IC25,
IC50, IC100: the low, median, and high dosage of icariin groups treated with 25, 50, and 100 mg/kg, respectively. The asthma with depression group is a control for
the treatment groups. Data are expressed as means ± SEM (n = 8 – 10 in each group); **p < 0.01 versus asthma with depression group.
As shown in [Fig. 4], lung tissues were further examined after H&E staining to confirm the inhibitory effects of icariin on airway inflammation. When compared
to the single asthma group and saline control group, mice in the asthma-DEP group displayed aggravated airway inflammation, as evidenced by the increased inflammatory score of H&E staining
(p < 0.01). However, oral administration of icariin at 50 and 100 mg/kg and Dex dramatically attenuated the inflammation infiltrated around the airways and blood vessels (p < 0.05 and
p < 0.01, respectively).
Fig. 4 Icariin attenuated airway inflammation in lung tissues. Lung tissue slices were stained with H&E and then observed under a microscope (100 ×). The inflammation score
represents the severity of inflammatory cell infiltration in the airway. Representative photomicrographs of each group (n = 8 per group) are shown as follows: (a) saline,
(b) asthma, (c) asthma with depression, (d) dexamethasone (Dex; 0.5 mg/kg/day), (e) icariin 25, (f) icariin 50, and (g) icariin 100. Asthma:
single asthma group, AD: asthma with depression group, Dex: dexamethasone group. IC25, IC50, IC100: the low, median, and high dosage of icariin groups
treated with 25, 50, and 100 mg/kg, respectively. The asthma with depression group is a control for the treatment groups. The size of the scale bars is 100 µm. Data are expressed as means
± SEM (n = 10 in each group); *p < 0.05 and **p < 0.01 versus the asthma with depression group.
As shown in [Fig. 5], mice in the asthma-DEP group showed remarkable airway inflammation with increased levels of IL-4, IL-5, IL-13, IL-6, and TNF-α
(p < 0.05), and a decreased level of INF-γ in BALF (p < 0.05), as well as a higher level of antigen-specific IgE in serum (p < 0.01) when compared to mice in the saline control
group. Furthermore, mice in the asthma-DEP group displayed higher levels of IL-4 and IL-5 in BALF compared to the single asthma group (p < 0.05 and p < 0.01, respectively). However,
after the oral administration of icariin or Dex, levels of IL-4, IL-5, and IL-6 in BALF and IgE in serum were dramatically reduced (p < 0.05 and p < 0.01, respectively).
Fig. 5 Icariin inhibited the secretion of inflammatory cytokines in BALF and IgE and corticosterone in serum. Secreted levels of IL-4, IL-5, IL-13, INF-γ, IL-6, and
TNF-α cytokines in BALF and serum antigen-specific IgE and corticosterone levels were measured by ELISA. Asthma: single asthma group, AD: asthma with depression group, Dex:
dexamethasone group. IC25, IC50, IC100: the low, median, and high dosage of icariin groups treated with 25, 50, and 100 mg/kg, respectively. The asthma
with depression group is a control for the treatment groups. Data are expressed as means ± SEM (n = 10 in each group); *p < 0.05 and **p < 0.01 versus the asthma with depression
group.
The results presented in [Figs. 5 h] and [6] demonstrate the beneficial effects of icariin on steroid resistance. [Fig. 5 h] shows that the corticosterone level in the group of asthma-DEP was significantly higher than that in the single asthma group and saline control group
(p < 0.01), whereas Dex and icariin at doses of 50 and 100 mg/kg remarkably inhibited corticosterone production (p < 0.01 and p < 0.05, respectively). [Fig. 6] clearly shows the dose-dependent inhibition of the steroid on splenocyte function in the saline group. The inhibitory effects of corticosterone were markedly abolished on
splenocyte proliferation and cytokine secretion in the asthma-DEP group when compared to both the saline and single asthma groups (p < 0.01 and p < 0.05, respectively). Treatment with
icariin, but not Dex, facilitated the responsiveness of splenocytes to corticosteroid, as evidenced by icariin at doses of 50 and 100 mg/kg, which markedly ameliorated steroid sensitivity in
the inhibition of splenocyte proliferation and cytokine secretion of IL-6 and TNF-α (p < 0.01 and p < 0.05, respectively).
Fig. 6 Icariin facilitated the inhibitory effects of corticosterone on LPS-stimulated splenocytes. Data are presented as the percentage of cell viability or cytokine concentration
of control values treated without corticosterone. Treatment of icariin facilitated corticosteroid responsiveness in splenocytes. The doses of 50 and 100 mg/kg of icariin markedly improved
glucocorticoid sensitivity in the inhibition of splenocyte proliferation and inflammatory cytokine secretion. Asthma: single asthma group, AD: asthma with depression group, Dex:
dexamethasone group. IC25, IC50, IC100: the low, median, and high dosage of icariin groups treated with 25, 50, and 100 mg/kg, respectively. The asthma
with depression group is a control for the treatment groups. Data are expressed as means ± SEM (n = 6 in each group); *p < 0.05 and **p < 0.01 versus the asthma with depression
group.
To determine whether the beneficial effects of icariin on glucocorticoid sensitivity were attributed to the improved GR function or GR expression, GR DNA-binding activity and GR expression in
splenocytes were evaluated. As shown in [Fig. 7], splenocytes in the asthma-DEP group displayed significantly lower GR DNA-binding activity and decreased GR
expression when compared to the single asthma group and the saline control group (p < 0.01 and p < 0.05, respectively). However, administration with icariin at doses of 50 and 100 mg/kg
remarkably enhanced GR DNA-binding activity and total GR expression in splenocytes.
Fig. 7 Icariin enhanced the DNA-binding activity of the glucocorticoid receptor (GR) and GR expression and inhibited the phosphorylation of GR S226. a Evaluation of GR
DNA-binding activity. Splenocytes from each group were isolated and incubated in the presence of dexamethasone (10−7 M) for 1 h in vitro. After preparation of nuclear
extracts from splenocytes, GR nuclear translocation was then quantitated by the TransAM GR DNA-binding ELISA kit. b, c Western blotting analysis was performed to investigate GR S226
phosphorylation and GR expression in splenocytes. c Western blotting analysis was performed to investigate p38 MAPK phosphorylation in splenocytes. The expression of these proteins
was quantified and represented as the band intensity of phosphorylated proteins normalized to the relevant total proteins. Asthma: single asthma group, AD: asthma with depression group,
Dex: dexamethasone group. IC25, IC50, IC100: the low, median, and high dosage of icariin groups treated with 25, 50, and 100 mg/kg, respectively. The
asthma with depression group is a control for the treatment groups. Data are expressed as means ± SEM (n = 6 in each group); *p < 0.05, **p < 0.01 versus the asthma with depression
group.
Increased GR S226 phosphorylation can result in the inhibition of GR nuclear translocation, thereby inducing reduced GR DNA-binding activity. p38 MAPK signaling has been previously shown to
play an important role in regulating GR S226 phosphorylation. Therefore, the phosphorylation of GR S226 and p38 MAPK in splenocytes was further determined. As shown in [Fig. 7], GR S226 phosphorylation was significantly increased in splenocytes in the asthma-DEP group when compared to the single asthma and saline control groups (p < 0.01 and
p < 0.05, respectively). The p38 MAPK phosphorylation was significantly increased in the asthma-DEP group compared to the saline group (p < 0.01), but when compared to the single asthma
group, no obvious change of p38 MAPK phosphorylation was observed. However, icariin at doses of 50 and 100 mg/kg remarkably inhibited phosphorylation of GR S226 and p38 MAPK in splenocytes
(p < 0.05).
Discussion
The present study revealed that depression aggravated airway inflammation and steroid resistance in a murine model of asthma. Notably, administration with icariin dramatically improved airway
inflammation and steroid sensitivity, which was associated with its enhancing GR function and GR expression. Its beneficial effects on the GR function could possibly be attributed to the
inhibition of phosphorylation of GR S226 and p38 MAPK.
Patients with asthma often experience depression and vice versa [2]. Depression can decrease the control of asthma symptoms and increase the asthma exacerbations
rate [4], [18]. In this study, OVA exposure combined with CUMS remarkably increased immobility duration in both the forced swim test
and tail suspension test compared to single OVA exposure, indicating that CUMS successfully induced depression-like behaviors. As compared to the single asthma group, mice in the asthma-DEP
group displayed more severe AHR, increased inflammatory cell infiltrates in the airways in histopathological examination, and higher IL-4 and IL-5 levels in the BALF, indicating that
depression could aggravate airway inflammation. Our results showed that icariin had little effects on the depression-like behaviors. However, icariin significantly alleviated AHR, infiltration
of inflammatory cells in airways, and levels of inflammatory cytokines in BALF, consistent with reduced levels of OVA-specific IgE in serum, which contributed to the anti-inflammatory effects
of icariin in mice with asthma and depression.
It is now recognized that chronic inflammation in asthma is not just attributed to an exposure to provocative stimuli, leading to excess airway inflammation, but also to insufficient
engagement of endogenous pro-resolving mediators [19]. Endogenous glucocorticoid exerts as a key anti-inflammatory mediator in asthma to restrain and resolve
allergic inflammation [20]. However, a diminished inhibitory action of endogenous glucocorticoid, namely steroid resistance, was suggested to be involved in the
pathogenesis of chronic stress-induced exacerbation of airway inflammation [6]. In this study, when compared to the saline control group, the corticosterone level
in serum in the asthma-DEP group was significantly increased, however, the inhibitory effects of corticosterone in the asthma-DEP group were significantly abolished on both splenocyte
proliferation and cytokine secretion. These results suggest that OVA exposure combined with CUMS contributes to the glucocorticoid resistance. Furthermore, there was still statistical
significance between the asthma-DEP group and the single asthma group in terms of the inhibitory effects of corticosterone, which indicates that CUMS exposure might aggravate glucocorticoid
resistance. However, this study showed that administration with icariin at 50 and 100 mg/kg significantly ameliorated hypercortisolemia and enhanced inhibitory effects of corticosterone on
splenocyte proliferation and cytokine production, which suggests that icariin could improve steroid sensitivity in an asthma model with depression. However, the mechanisms through which
icariin ameliorates steroid insensitivity have not been fully elucidated.
Glucocorticoid exerts its effects by binding to a single cytoplasmic GR, followed by efficient GR translocation to the nucleus and GR DNA-binding under normal signal transduction [21], [22]. Therefore, GR function and GR expression were evaluated to investigate the mechanism of icariin on glucocorticoid resistance in
asthma, and GR function was reflected by evaluation of GR DNA-binding activity in nuclear fractions of splenocytes. When compared to the single asthma group, mice in the asthma-DEP group
displayed further impaired GR DNA-binding activity and decreased total GR expression in this study, which indicates that depression could further impair GR function and expression. Icariin at
doses of 50 and 100 mg/kg remarkably improved GR DNA-binding activity and increased GR expression in splenocytes. A previous study showed that hypercortisolemia could lead to decreased GR mRNA
levels in patients with mood disorders [23]. In the present study, icariin alleviated hypercortisolemia, which therefore might be associated with its enhancement
of GR expression. The phosphorylation of GR S226 contributes to the inhibition of GR nuclear translocation, thereby resulting in reduced GR DNA-binding activity. When compared to the single
asthma group, mice in the asthma-DEP group displayed significantly increased GR S226 phosphorylation. Thus, the impaired GR function aggravated by depression might be associated with the
increased GR S226 phosphorylation. However, icariin at doses of 50 and 100 mg/kg remarkably inhibited GR S226 phosphorylation, which possibly contributed to its effects on the GR DNA-binding
activity in splenocytes.
The activation of p38 MAPK can lead to the GR S226 phosphorylation in the cytoplasm, thus resulting in reduced GR DNA binding [24]. In this study, phosphorylation
of p38 MAPK was significantly increased in the asthma-DEP groups compared to the saline group, indicating the possible role of p38 MAPK pathway activation in the increased phosphorylation of
GR S226. However, phosphorylation of p38 MAPK was significantly inhibited by icariin treatment in splenocytes. Therefore, our findings indicate that the effects of icariin on glucocorticoid
resistance were associated with suppression of the phosphorylation of GR S226 and p38 MAPK.
Taken together, this study suggests that icariin alleviated airway inflammation by improving endogenous glucocorticoid sensitivity, which, in addition to the increase of GR expression, was
partially attributed to the enhancement of GR function associated with the decreased phosphorylation of GR S226 and p38 MAPK.
Materials and Methods
Animals
Seventy specific pathogen-free male BALB/c mice (aged 6 weeks, weighed 20 – 22 g) were purchased from Shanghai SLAC Laboratory Animal Company. Animals were maintained in a
temperature-controlled environment (22 ± 2 °C) on a 12-h light-dark schedule with free access to food and water under pathogen-free conditions. Mice were randomly assigned into the following
seven groups (n = 10 per group): (1) normal saline control group (saline), (2) single asthma group (asthma), (3) asthma with depression group (AD), (4) dexamethasone (Dex) group (0.5 mg/kg),
(5) low-dose icariin group (25 mg/kg, IC25), (6) median-dose icariin group (50 mg/kg, IC50), and (7) high-dose icariin group (100 mg/kg, IC100). All
experimental protocols were approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (permit number: SYXK-2018-0012; December 30, 2018).
Reagents and materials
Icariin (purity 98%), corticosterone, and LPS were purchased from TargetMol. OVA (grade V) and methacholine were provided by Sigma-Aldrich. Injectable Dex sodium phosphate was obtained from
Tianjin Pharmaceutical Group Xinzheng Company. Imject alum adjuvant, a formulation of aluminum hydroxide and magnesium hydroxide, was provided by Thermo Fisher Scientific Company. ELISA kits
for IL-4, IL-5, IL-6, IL-13, TNF-α, interferon (INF)-γ, and OVA-specific IgE were purchased from eBioscience Company. The CCK-8 assay was obtained from Dojindo Laboratories.
TransAM GR kits were purchased from Active Motif. Antibodies for phosphorylated p38 MAPK (Thr180/Tyr182, Cat. no. 4511), p38 MAPK (Cat. no.8690), and phosphorylated GR (S226, Cat. no.97 285)
were obtained from Cell Signaling Technology, and GR alpha (ab3580) was purchased from Abcam.
Model establishment and drug administration
Animals were sensitized and challenged by OVA according to a modified protocol for the asthma model as described previously [25]. In brief, mice were immunized
on days 0 and 7 with a peritoneal injection of 0.2 mL of sterile saline containing 40 µg of OVA and 0.05 mL of alum adjuvant. One week after sensitization, mice were challenged with
aerosolized 1% OVA for 30 min/day for 5 consecutive weeks from day 14. For mice in the asthma-DEP group and treatment groups, mice were further subjected to CUMS from day 21 for 4 weeks. The
stress conditions applied in this experiment included a wet pad for 8 h, ice-water bath for 5 min at 6 °C, constraint for 1 h, tilted cage (30°) for 3 h, exposed to light overnight, noise
for 3 h, deprivation of water and food for 12 h, and tail clamping for 1 min. Two types of stress inducers were randomly selected and applied each day. From days 21 to 48, mice in the
treatment groups were administrated icariin at doses of 25, 50, and 100 mg/kg or Dex at 0.5 mg/kg/day once a day by oral gavage. Mice in the saline control group were challenged and treated
with normal saline instead. The protocols for sensitization, challenge, stress exposure, and drug administration are summarized in [Fig. 8].
Fig. 8 Experimental protocols. Mice were subjected to OVA exposure combined with continuous chronic unpredictable mild stress (CUMS). Briefly, mice were sensitized twice with OVA
followed with 1% OVA inhalation each day for 5 consecutive weeks. From days 21 to 48, mice were subjected to CUMS, and, simultaneously, mice in the treatment groups received oral icariin
at 25, 50, and 100 mg/kg or 0.5 mg/kg of dexamethasone (Dex) for 4 weeks.
Open field test
The OFT was applied to assess the spontaneous movement of mice. As previously described [26], the test system consisted of a square arena (50 cm length × 50 cm
width × 50 cm depth) with a digital camera positioned directly above the center of the field. Each animal was gently placed in the center of the cages and tracked for 5 min. Locomotor
activity, including travel distance and time in the center zone, was quantified using the automatic video tracking system (SMART 3.0; Panlab S. I.). The center field was defined as the
central 25 × 25 cm2 area of the open field, which was one-fourth of the total area.
Forced swim test
A modified version of the forced swim test was performed to assess the effects of icariin treatment on depression-like behavior. Briefly, subjects were gently placed in a glass cylinder
filled with 15 cm of water at room temperature (25 ± 1 °C). The mice were placed in the cylinders for 6 min and the immobility time of the last 4 min was determined by the automatic video
tracking system (SMART V3.0; Panlab S. I.). Immobility, in which the mice remained immobile or made only small limb movements necessary to float, was regarded as depression-like
behavior.
Tail suspension test
The tail suspension test was conducted to assess the behavioral despair of mice, as previously described [27]. The mice were suspended by their tails using an
elastic band attached to the tail (approximately 1 cm from the tip of the tail). The distance between the tip of the nose of the mouse and the laboratory bench was approximately 20 cm. Mice
were suspended for a period of 6 min and the time spent immobile during the last 4 min of 6 min was scored for each mouse by an observer blinded to the test.
Measurement of airway hyperresponsiveness
Buxcoʼs unrestrained whole-body plethysmography systems were performed to evaluate AHR to methacholine [28]. Briefly, mice were kept in a closed chamber and the
pressure fluctuations were recorded during the breathing cycle. After mice reached a stable baseline, aerosolized PBS or various concentrations of methacholine (6.25, 12.5, or 25 mg/mL) were
administered to the mice via a jet nebulizer in the chamber. The “enhanced pause” (Penh), as a dimensionless parameter, was used to indicate changes in airway resistance [29].
Serum collection and analysis
Blood samples were collected from all experimental animals following full anesthesia by an intraperitoneal injection of pentobarbital sodium. Serum was collected after centrifugation at
1500 × g for 10 min and then stored at − 80 °C. OVA-specific IgE and corticosterone levels were then measured by ELISA according to the manufacturerʼs instructions.
Cytokine analysis in bronchoalveolar lavage fluid
To obtain BALF, a tracheal tube was inserted in the mice and lung lavage was performed twice with 0.8 mL of sterilized normal saline. The collected BALF was centrifuged at 500 × g at
4 °C for 10 min. The supernatants were then collected and stored at − 80 °C for subsequent cytokine measurements. Subsequently, interleukin levels (IL-4, IL-5, IL-13, INF-γ, IL-6,
TNF-α) in the BALF supernatant were detected by ELISA according to the instructions of the manufacturer.
Histopathological evaluation of the lung
After BALF collection, lung tissue slices were fixed with 4% neutral-buffered formalin. The fixed tissue blocks were then cut into thin sections (3 – 4 µm) and stained with H&E. The
histopathological evaluation was performed blindly using randomized sections with Image-Pro Plus software at a magnification of 100 ×. A five-point scoring system was adopted to analyze the
severity of inflammatory cell infiltration in the airway: 0, no cells; 1, a few cells; 2, a ring of cells, 1 cell layer deep; 3, a ring of cells, 2 to 4 cells deep; and 4, a ring of cells
which was > 4 cells deep [30].
Splenocyte isolation and corticosterone sensitivity assay
The corticosterone sensitivity assay was evaluated as previously described [31]. A single-cell splenocyte suspension was prepared and then cultured in
triplicate in 96-well plates (2.5 × 105 cells/well) to perform cell proliferation assays and cytokine evaluation at a volume of 100 and 300 µL/well, respectively. Cells were
treated with corticosterone (dose range 0, 0.05, 0.5, 5 µM) and 1 µg/mL LPS at 37 °C with 5% CO2. Cell supernatants were collected after 18 h of incubation and frozen at − 20 °C
for cytokines analysis of IL-6 and TNF-α by ELISA. At the end of the 48-h culture, a CCK-8 assay was performed to evaluate cell viability, and absorbance was read at 490 nm by an
ELISA plate reader. Cell viability is expressed as percentage of OD of the saline-treated control.
Glucocorticoid receptor DNA-binding activity
To determine GR DNA-binding activity in nuclear extracts, splenocytes were treated with 1 × 10−7 M Dex for 2 h. Then nuclear extraction was performed as previously described
[32]. GR DNA-binding activity was assessed by adding 20 µg of nuclear extract to TransAM GR kits, according to the manufacturerʼs instructions. Briefly, nuclear
extracts were incubated in wells of the 96-well plates coated with a GR-binding consensus oligonucleotide sequence for 1 h, then incubated with the supplied primary anti-GR antibody
(1 : 1000) for 1 h, and finally with a peroxidase-conjugated secondary antibody (1 : 1000) for 1 h. The color development was read at 450 nm following addition of the substrate, and then the
OD of the GR was recorded.
Western blotting analysis
Splenocytes and lung tissues were homogenized to extract total protein using RIPA protein extraction reagent. An amount of 50 µg proteins was subjected to SDS-PAGE and electroblotted to a
PVDF membrane (Bio-Rad). Immunoblotting was then performed by incubating the membranes with the following antibodies: phosphor-GR ser226 (1 : 1000 dilution), GRα (1 : 1000 dilution),
phosphophorylated-p38 MAPK Thr180/Tyr182 (1 : 1000 dilution), p38 MAPK (1 : 1000 dilution), and GAPDH (1 : 2000 dilution), followed with secondary HRP-conjugated antibodies (1 : 1000
dilution). After the incubation, the membrane was then developed by enhanced chemiluminescence according to the manufacturerʼs instructions. The protein bands were then quantified and
expressed as the band intensity of the phosphorylated proteins normalized to the relevant total proteins.
Statistical analysis
Results were analyzed by GraphPad Prism v.9.0 (GraphPad software). Data are expressed as the mean ± standard error (SEM). The significance of differences between groups was determined by
one-way ANOVA followed by post hoc Dunnett tests. A value of p < 0.05 was accepted as statistically significant.
Contributorsʼ Statement
Data collection: H. L. Jin, Y. Zhou, J. Ye, C. H. Qiu, W. Z. Jin; design of the study: L. M. Wang, H. L. Jin, Y. Zhou; statistical analysis: H. L. Jin, J. Ye, C. H. Qiu, W. Z. Jin; analysis
and interpretation of the data: H. L. Jin, Y. Zhou, L. M. Wang, J. Ye, C. H. Qiu, W. Z. Jin; drafting the manuscript: H. L. Jin, Y. Zhou, L. M. Wang, C. H. Qiu; critical revision of the
manuscript: L. M. Wang, J. Ye, W. Z. Jin.
