Protection of hUC-MSCs against Neuronal Complement C3a
Receptor-mediated NLRP3 Activation in CUMS-induced Mice
Jing Li (Writing – original draft), Shujuan Tian, Hualong Wang (Data
curation), Yanyong wang (Visualization) (Investigation), Chongbo
Du (Software), Jiyu Fang (Validation), Xiaoxiao Wang (Supervision),
Yufeng Wang, Zhexuan Gong, Baoyong Yan (Methodology)
(Software), Mingwei Wang (Conceptualization)
PII: S0304-3940(20)30755-2
Reference: NSL 135485
To appear in: Neuroscience Letters
Received Date: 12 May 2020
Revised Date: 21 September 2020
Accepted Date: 1 November 2020
Please cite this article as: Li J, Tian S, Wang H, wang Y, Du C, Fang J, Wang X, Wang
Gong Z, Yan B, Wang M, Protection of hUC-MSCs against Neuronal Complement C3a
Receptor-mediated NLRP3 Activation in CUMS-induced Mice, Neuroscience Letters (2020),
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© 2020 Published by Elsevier.
Protection of hUC-MSCs against Neuronal Complement C3a Receptor-mediated
NLRP3 Activation in CUMS-induced Mice
Jing Li
, Shujuan Tian1,2#
, Hualong Wang1,2, Yanyong wang1,2, Chongbo Du1,2, Jiyu Fang1,2, Xiaoxiao Wang3
Yufeng Wang
, Zhexuan Gong4
, Baoyong Yan
and Mingwei Wang1,2*
1 Department of Neurology, the First Hospital of Hebei Medical University, Shijiazhuang, Hebei, China
2 Brain Aging and Cognitive Neuroscience Key Laboratory of Hebei Province, Shijiazhuang, Hebei, China
3Department of Internal Medicine, Hebei Medical University, Shijiazhuang, Hebei, China
4 Basic Medical College, Hebei Medical University, Shijiazhuang, Hebei, China.
5 Cell Therapy Laboratory, The First Hospital of Hebei Medical University, Shijiazhuang, Hebei, China
Correspondence to: Mingwei Wang, MD, Ph.D., Department of Neurology, The First Hospital of Hebei Medical
University, 89, Dong-gang Road, Shijiazhuang 050031, Hebei province, PR China. E-mail:
[email protected].
BaoyongYan, Ph.D., Cell Therapy Laboratory, The First Hospital of Hebei Medical University, 89,
Dong-gangRoad, Shijiazhuang 050031, Hebei province, PR China. E-mail: [email protected]
Li and Tian contributed equally to this work.
Highlights
 hUC-MSCs facilitated resilience to depressive-like behaviors.
 hUC-MSCs rescued synaptic plasticity after CUMS exposure.
 3.Neuronal complement C3aR participated in NLRP3 Journal Pre-proof inflammasome activation, which was inhibited by hUC-MSCs.
1Abstract
Background: Hyperactivation of complement C3 and inflammation-related activation
of NLR family pyrin domain containing 3 (NLRP3) inflammasome are implicated in
the etiology of stress-related disorders. Studies have shown that human umbilical cord
mesenchymal stromal cells (hUC-MSCs) have immunomodulatory and
anti-inflammatory effects; however, the mechanism remains unclear.
Methods: hUC-MSCs were administered to chronic unpredictable mild stress (CUMS)
model mice once a week for four weeks. After the administration of hUC-MSCs,
several parameters were assessed, including behavioral performance, synapse-related
proteins, complement C3 receptors (C3aR) in neurons, and the NLRP3 inflammasome.
Then, CUMS mice were injected with SB290157, a complement C3aR antagonist,
and the behavioral index and NLRP3 inflammasome activation were tested.
Results: The open-field and forced swimming behavioral tests showed an
improvement in depression-like behaviors in the CUMS-exposed mice after the
administration of hUC-MSCs. Treatment with hUC-MSCs significantly decreased the
neuronal C3aR levels and alleviated synaptic damage. Furthermore, the levels of the
NLRP3 inflammasome and inflammatory factors were reduced after hUC-MSC
administration. In particular, treatment with a C3aR antagonist also decreased NLRP3
inflammasome expression and inflammation, which was consistent with the observed
improvements after hUC-MSC treatment.
Conclusion: hUC-MSCs can attenuate NLRP3 activation in CUMS-induced mice,
which may be correlated with C3aR in neurons.
1Abbreviations
NLRP3: NLR family pyrin domain containing 3 CUMS: chronic unpredictable mild
stress hUC-MSCs: Human umbilical cord mesenchymal stem cells C3aR: C3a
receptor IL-18:Interleukin-18 OFT: Open-field test NSFT: Novelty-suppressed
feeding test FST: Forced swimming test
Keywords: human umbilical cord mesenchymal stromal cells, chronic unpredictable
mild stress, complement C3a receptor, NLRP3 inflammasome
Introduction
Accumulating evidence has indicated that severe or prolonged chronic stress results in
an increased risk for physical and psychiatric disorders, such as mood disorder, which
is called stress-related disease1
. Hyperactivation of innate immunity and
neuroinflammation has been demonstrated to be involved in the pathophysiology of
stress-related diseases 2, 3
.The complement system represents one of the primary
effector mechanisms of the innate immune system 4
. Complement activation mainly
focuses on the cleavage of the central complement component C3a 5
, which mediates
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immunomodulatory signals via the seven-transmembrane domain receptor, C3aR 6
. In
recent years, dysregulation of C3 in stress-related patients or animals has received
attention. For example, the expression of C3 was significantly upregulated in
postmortem depressed subjects and stress-related model mice 7, 8
. Knockdown of the
C3or C3aR gene in mice alleviated inflammatory infiltration and depressive-like
behavior 9
. Studies have confirmed that NLRP3 inflammasome cascades emerge in
depression 10, 11
. Complement C3a is considered an essential regulatory component of
the cellular alarm system that triggers NLRP3 inflammasome activation12
.NLRP3
inflammasome activation leads to the maturation of caspase-1, which triggers the
release of interleukin-1 (IL-1) and IL-18 in immune cells upon cellular stress 13
Human umbilical cord mesenchymal stromal cells (hUC-MSCs), typical adult stem
cells and a promising candidate for cell-based therapy, have excellent advantages in
regulating the innate and adaptive immune systems and provide a tolerogenic
environment for improving inflammation 14
. The immunomodulatory and
anti-inflammatory capabilities of hUC-MSCs have been recognized in neurological
disorders, including CUMS15, 16. Previous studies indicated that the
immunosuppressive and anti-inflammatory properties of MSCs reduced
neuroinflammation in a stroke model by deregulating C3 expression 17; however, little
is known about the role of intravenously delivered hUC-MSCs and complement C3 in
CUMS. In this study, we established a model of CUMS injected with hUC-MSCs or a
C3aR antagonist and found that neural complement C3 may be correlated with
NLRP3 activation, leading to synapse loss and depression phenotypes, which might
be significantly attenuated by hUC-MSCs administration.
2. Materials and methods
2.1 Animals
Male ICR mice (aged 6 weeks) were obtained from Beijing HFK Bioscience
Corporation, China. The animals were maintained in a specific pathogen-free animal
laboratory with free access to water and food under a 12 h light/dark cycle. The
experiments were strictly performed in accordance with the NIH Guidelines for the
Care and Use of Laboratory Animals. All experimental procedures on animals were
approved by the Institutional Animal Care and Use Committee of Laboratory Animals
at the First Hospital of Hebei Medical University. The mice were acclimatized to the
laboratory conditions for two weeks before beginning the experimental procedures.
2.2 CUMS procedure
The experimental chronic unpredictable mild stress protocols were performed as
previously described 18, with minor modifications. Briefly, the mice were
administered the following mild stressors 2 times a day for 6 weeks in a
pseudorandom manner: food deprivation for 24h; water deprivation for 24h; overnight
illumination for 24h; sawdust removal for 24h; soiled cage (200 ml water in 100 g
sawdust bedding) for 24h; forced swimming at 8°C for 6min; tail nipping (1 cm from
the tip of the tail) for 1min;and physical restraint (50 ml centrifuge tube) for 2h.
2.3 Isolation and transplantation of hUC-MSCs
MSC lines from human umbilical cords and the characterization of hUC-MSCs were
established as previously described 19, 20
. hUC-MSCs were provided by the Cell
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Therapy Laboratory of the First Hospital of Hebei Medical University. The procedure
was approved by the ethical committee of the First Hospital of Hebei Medical
University. The cells were expanded in essential media with alpha modification, 10%
fetal bovine serum, and 1% penicillin/streptomycin. hUC-MSCs were passaged at a
ratio of 1:2. Before tail vein administration, 1×106
hUC-MSCs (at passage 5) were
suspended as a single-cell suspension in 0.2 ml of normal saline.
2.4 Experimental groupings
Eight-week-old ICR mice were randomly grouped as follows: control group (CON
group, mice received 0.2 mL of NS as an injection control once a week for the latter 4
weeks); CUMS (CUMS group, mice underwent the CUMS procedure every day for 6
weeks and received 0.2 mL of NS once a week for the latter 4 weeks); hUC-MSCs
(MSC group, a single infusion of 1×106
hUC-MSCs at passage 5 were administered in
0.2 ml of NS once a week by tail vein administration in CUMS-induced mice for the
latter 4 weeks); and C3aR antagonist(SB group, CUMS-induced mice were
administered intraperitoneally with a C3aR antagonist(SB290157, 1 mg/kg, Sigma, St.
Louis, MO, USA) three times a week for the latter 4 weeks)
. The animals were
sacrificed during the 7th week for further analyses.
2.5 Behavioral measurements
2.5.1. Open-field test (OFT)
To assess the response to locomotor activity in a novel stressful environment, the mice
were placed individually in an open arena for 5 min of spontaneous activity. The
experiments were videotaped using a ceiling camera for further parameter analysis
using the ANY-maze Behavior Analysis System22. The distance traveled was scored.
2.5.2. Forced swimming test (FST)
The FST was performed on the day after the OFT. The FST is a behavioral test used to
assess antidepressant-like behavior and was performed as previously described 22
experimental animals were individually placed in a clear glass cylinder filled with
water (24 °C) to a depth of 45 cm. The total immobility time for 6 min and the latency
during the initial 2 min of the 6min task were recorded by the ANY-maze Behavior
Analysis System. Immobility was defined as floating in the water without struggling
and making only movements that were necessary to maintain the head above the
water.
2.5.3. Novelty-suppressed feeding test (NSFT)
The NSFT was performed on the day after the FST. The NSFT is a conflict test in
which food is removed for 24 h before the experiment. During the experiment, the rats
were placed in an apparatus consisting of a plastic box (50 × 50 × 30 cm), and a small
amount of food was placed at the center of the arena. The time of the first bite was
recorded as the latent period of eating within 5 min 23
2.6 Tissue preparation
Forty-eight hours after the behavioral tests, the mice were anesthetized and
transcardially perfused with 50 ml 0.9% NaCl and 50 ml 4% paraformaldehyde. The
brains were postfixed in 4% PFA for 24 h followed by graded dehydration. The brain
sections for immunohistochemical and immunofluorescencestaining were embedded
and cut into serial coronal sections (5μm). Brain tissues for western blotting and
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RT-PCR were collected after sacrifice by cervical dislocation and were frozen at
-80 °C for subsequent analysis.
2.7 Western blot analysis
Hippocampi were immediately collected and homogenized for 10 min in RIPA buffer,
and the supernatant was collected and quantified by a BCA protein assay kit. Equal
amounts of protein were loaded and electrotransferred to PVDF membranes. All blots
were incubated with primary antibodies, including anti-microtubule-associated protein
2 (MAP-2, 17490-1-AP, Proteintech), anti-ionotropic glutamate receptor 2(GLU-2,
ab206293, Abcam), anti-synaptophysin (SYN, ab32127, Abcam),
anti-NLRP3(12446SS, Novus), anti-caspase-1(22915-1-AP, Proteintech),
anti-IL-18(ab71495, Abcam) and anti-β-actin. The protein levels were quantified by
densitometry using ImageJ software.
2.8 Immunostaininganalysis
For immunohistochemical analysis, paraffin-embedded sections were stained
according to standard protocols. Briefly, the slides were stained with primary
antibodies against GLU-2, NLRP3, and caspase-1. Imaging of the CA3 region of the
hippocampus (200×) was performed under a light microscope (Olympus, Japan).The
statistical results of quantifying intensity were expressed as percentage ratios relative
to the control group.
For immunofluorescence double-labeling analysis, frozen slices were incubated with
primary antibodies against C3aR (1:100; HM1123, HycultBiotech) and NeuN (1:50;
Cell Signaling Technology) and examined under a fluorescence microscope. The
intensity percentage of the C3aR+
/NeuN+
colocalization region in the NeuN+
channel
was measured.
2.7 Quantitative reverse transcriptase PCR (qRT-PCR)
Total RNA was extracted from brain tissue using an RNA extraction kit (Promega
reagent, Life Technologies, China). Total RNA (500 ng) was reverse-transcribed into
cDNA using the PrimeScript RT master mix reverse transcription kit (Tokyo, Japan),
and mRNA expression was quantified by RT-qPCR using a Bio-Rad thermocycler and
SYBR green kit (Tokyo, Japan). The samples were tested in triplicate with 18S, and
the mRNA expression levels were evaluated using the 2 (ΔΔCt) method. The sequences
of the specific primers are listed in Supplementary Table 1.
2.8 Statistical analysis
The data were analyzed by IBM SPSS software (version 22, IBM Inc.). All values are
expressed as the mean ± standard error of the mean (SEM). The data were analyzed
using one-way ANOVA and Tukey’s post hoc test for multiple comparisons of means.
P-values< 0.05 were considered statistically significant.
3. Results
3.1 hUC-MSCs facilitated resilience to depressive-like behaviors
In the open field paradigm, CUMS exposure has been shown to reduce total
movement distance, a measure of the locomotive activity (P< 0.05). After 4 weeks of
hUC-MSCs and C3aR antagonist infusion, a significant increase in distance was
observed (P< 0.05). Mice with CUMS exposure displayed a reduced desire for food in
a new environment, as quantified by an increase in feeding latency. Both the MSC and
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SB groups displayed shorter feeding latencies than the CUMS group (P< 0.05). In the
FST, the CUMS-exposed mice displayed shorter latency and longer immobility time
(responses indicative of depression)than the CON group mice (Fig. 1C and D);
however, these CUMS-induced behavioral changes were significantly reversed by
administration of either hUC-MSCs or the C3aR antagonist (P< 0.05). These data
suggest that intravenous delivery of MSCs during stress reduces stress-induced
behavioral perturbations.
3.2 hUC-MSCs rescued synaptic plasticity after CUMS exposure
Decreased locomotive activity and increased depression-like behaviors following
CUMS exposure are thought to be driven by neuronal damage. As shown in Fig. 2,
one-way ANOVA revealed that MAP-2 and SYN protein levels were decreased in the
CUMS group (P< 0.05). Post hoc analysis indicated a significant hUC-MSC
treatment-induced restoration of the levels of MAP-2 (P< 0.05) and SYN (P<
0.05).Further examination of excitatory synapses using GLU-2 revealed a significant
decrease in CUMS-induced mice(P< 0.05), which was partly reversed by hUC-MSCs
treatment or C3aR antagonist administration. Immunohistochemical staining indicated
that mice in the MSC and SB groups had increased GLU-2 immunoreactivity in the
CA3 subfields of the hippocampus (P< 0.05).
3.3hUC-MSCs attenuated the expression of C3aR in neurons
RT-PCR showed that the C3aR mRNA level was significantly increased in the
hippocampus of CUMS-induced mice (Fig. 3C) and partly restored in the MSC mice.
C3aR mRNA expression changes in the MSC group were consistent with those in the
SB group (P> 0.05). To explore whether C3aR was involved in neuronal damage in
CUMS-induced mice, C3aR+
/NeuN+
double staining in the hippocampal CA3 area of
the mice was examined. CUMS mice displayed a high degree of colocalization and
strong intensity in the CA3 subfield. Immunostaining colocalization showed
significantly reduced neuronal C3aR expression in the MSC (P< 0.05) and SB (P<
0.05) groups.
3.4Complement C3aR participated in NLRP3 inflammasome activation, which
was inhibited by hUC-MSCs
To test whether NLRP3 inflammasome activation is required for the observed effects
of neuronal C3aR activation in the presence of neuronal damage, CUMS-induced
mice were systemically treated with a C3aR antagonist and the protein and mRNA
levels of the NLRP3 inflammasome and released cytokines were measured. Compared
with those in the CUMS group, the expression levels of NLRP3, caspase-1, and IL-18
were significantly decreased in the C3aR antagonist treated group (P< 0.05). The
administration of hUC-MSCs markedly deregulated the expression of the NLRP3
inflammasome and cytokines (P< 0.05, Fig. 4A-B, C-E), which was consistent with
the SB group. Furthermore, hUC-MSCs (P< 0.05) or C3aR antagonist (P< 0.05)
administration counteracted these decreases in NLRP3 and caspase-1 with significant
differences in the CA3 subfields of CUMS-induced mice (Fig. 4F-I).
4. Discussion
Basic and clinical research indicates that long-term chronic stress initiates immunity
and neuroinflammation through continuous activation of the
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hypothalamic-pituitary-adrenal (HPA) axis, leading to mood disorders including
depressive-like behaviors18, 24
. In this report, we observed that CUMS exposure
resulted in the upregulation of complement C3a receptor in neurons of the
hippocampus, which mediated the activation of NLRP3 inflammasome and the release
of inflammatory cytokines. Intravenous infusion of hUC-MSCs significantly
attenuated neuroinflammation by inhibiting the complement C3a receptor in neurons,
and eventually alleviated neuronal injury and depressive behaviors. These
observations suggest that targeting hUC-MSCs might provide a novel and valuable
way for future therapeutic strategies for stress related diseases.
Recent studies have revealed that injecting MSCs into the central nervous system
alleviated depressive-like behavior in traumatic brain injury and depression models 25,
26. This study demonstrated that hUC-MSCs improved locomotion in the OFT
and chronic stress-related depressive-like phenotypes in the FST and NSFT, which
was consistent with previous studies27
. Importantly, our previous research found that
aberrant stress-induced behavioral patterns were associated with deficits in
neuroplasticity of the hippocampus28
. This study observed that hUC-MSCs improved
the expression of synaptic related proteins and glutamate receptors in CUMS mice,
providing pathological evidence for the improvement in depression-like behaviors.
Furthermore, Cao et al. also demonstrated that hUC-MSCs improved synaptic
plasticity, enhanced dendritic spine, and postsynaptic density in the hippocampus of
aged mice 29
Both preclinical and clinical evidence demonstrated that the assembly of the NLRP3
complex and the release of the proinflammatory cytokines occurs in chronic stress
models and patients with depression disorders
11, 30. NLRP3 complex activation leads
to synaptic dysfunction and behavioral deficits, which is comparable with our data in
the CUMS-induced model31. Recent studies have demonstrated that hUC-MSCs
blocked NLRP3 inflammasome activation in rheumatoid arthritis model and diabetes
model32, 33. Furthermore, our results indicated that the expression of NLRP3 and
caspase-1 and the release of IL-18 were restored by the chronic administration of
hUC-MSCs in the CUMS-induced mice. Behavioral performance alterations of the
hUC-MSCs-treated mice could be related to the decreased NLRP3 activation
mentioned above.
Complement C3a is considered an essential regulator of NLRP3 inflammasome
activation 34. Asgari also demonstrated that engagement of complement C3a
modulated NLRP3 inflammasome activation and IL-1β secretion in human monocytes
. Additionally, our research found that C3a signaling through C3aR inhibited NLRP3
inflammasome activation and the release of inflammatory cytokines in the
CUMS-induced model upon C3aR antagonist administration. Additionally,
complement C3, a potential immunomodulator, has been implicated in neuroplasticity
deficits leading to depressive-like behaviors 9, 36
. More specifically, studies in C3
knockout mice showed that C3 was involved in inducing behavioral deficits and
synaptic regulation 37. This characteristic is consistent with our finding that C3aR
signaling inhibition using a C3aR antagonist inhibited CUMS-induced depressive-like
behaviors and neuronal impairments. In pathological conditions, complement C3aR
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hyperactivation mobilizes intraneuronal calcium regulation, mediating excitatory
synaptic density and dendritic morphology21
.Jung reported that mesenchymal stem
cells reduced the expression of complement C3 after transient focal cerebral ischemia
in mice 17. This characteristic is consistent with our finding that hUC-MSCs treatment
reversed C3aR overexpression in neurons caused by stress exposure. These results are
consistent and reinforce the hypothesis that hUC-MSCs could attenuate NLRP3
activation in CUMS-induced mice, which may be correlated with C3aR in neurons.
5. Conclusion
In summary, hUC-MSCs could attenuate NLRP3 activation in CUMS-induced mice,
which may be correlated with C3aR in neurons.
Author statement
Li Jing：Writing- Original draft preparation. Tian Shujuan：Perform the
scientific design. Wang Hualong: Data curation. Wang Yanyong:
Visualization, Investigation. Du Chongbo: Software. Fang Jiyu:
Validation. Wang Xiaoxiao: Supervision. Wang Yufeng: Perform the
animal experiment. Gong Zhexuan: Perform the cell experiment. Wang
Mingwei: Conceptualization. Yan Baoyong：Methodology, Software .
Declarations of interest
None.
Funding
This work was supported by the Hebei Medical Applied Technology Tracking Project
(grant number G2019019) and the Hebei Health Commission Project (grant number
20170063).
Acknowledgments
The authors would like to express their gratitude to the Cell Therapy Laboratory, The
First Hospital of Hebei Medical University.
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mice fail to display age-related hippocampal decline. J Neurosci. 2015;35:13029-13042
Fig.1 hUC-MSCs facilitated resilience to depressive-like behaviors. A. Total
movement distance (A) in the open field test during week 7. (B)Feeding latency in the
novelty suppressed feeding test during week 7. (C-D) The latency and immobility
time in the forced swimming test during week 7. The values are expressed as the
means ± SEM. N = 12/group. #P< 0.05 vs. the CON group. *P<0.05 vs. the CUMS
group. [F(3,44)= 5.681, p = 0.002, Fig. 1A] [F(3,44)= 5.547, p = 0.003, Fig. 1B] [F(3,44)= 11.692, p =
0.000, Fi. 1C] [F(3,44)= 9.798, p = 0.008, Fig. 1D]
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Fig.2 hUC-MSCs rescued synaptic plasticity after CUMS exposure. (A-B) Western
blotting and histograms showing the protein levels of SYN, MAP-2, and GLU2 in the
hippocampus normalized to β-actin. (C-D) Representative images and histograms
showing the immunoreactivities of GLU2 in the CA3 subfield of the hippocampus by
IHC staining. N=3/group. Scale bars= 25 μm. The data are shown as the mean ± SEM.
#P< 0.05 vs the CON group. *P< 0.05 vs the CUMS group.[F(3,8)= 33.297, p = 0.000, Fig.
2B SYN] [F(3,8)= 203.731, p = 0.000, Fig. 2B MAP-2] [F(3,8)= 16.257, p = 0.001, Fig. 2B GLU2]
[F(3,8)= 54.649, p = 0.000, Fig. 2D GLU2]
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Fig.3 hUC-MSCs attenuated the expression of C3aR in neurons. (A) C3aR is
expressed inneurons. Representative double immunostaining for C3aR and NeuN in
the CA3 subfield of the hippocampus. (B) Quantification of C3aR+
/NeuN+
densityin
the CA3 subfield of the hippocampus. (C) C3aR mRNA expression in the
hippocampus. N=3/group. Scale bars=50μm. The data are shown as the mean ± SEM.
#P< 0.05 vs. the CON group; *P< 0.05 vs. the CUMS group. [F(3,8)= 17.273, p = 0.001, Fig.
3B] [F(3,8)Journal Pre-proof =68.420, p = 0.000, Fig. 3C]
Fig.4 Complement C3aR participated in NLRP3 inflammasome activation, which was
inhibited by hUC-MSCs. (A-B) The protein levels of NLRP3, caspase-1, and IL-1β
were analyzed by Western blotting andwere normalized to β-actin. (C-F)
Representative images and histograms showing the immunoreactivities of NLRP3 and
caspase-1 in the CA3 subfield of the hippocampus by IHC staining. (G-I) The mRNA
expression of NLRP3, caspase-1, and IL-18. Scale bar = 25 μm. The values are
expressed as the mean ± SEM. N = 3/group. #P< 0.05 vs the CON group. *P< 0.05 vs
the CUMS group. [F(3,8)= 11.540, p = 0.003, Fig. 4B NLRP3] [F(3,8)= 47.414, p = 0.000, Fig. 4B
caspase-1] [F(3,8)= 47.585, p = 0.000, Fig. 4B IL-18][F(3,8)= 240.443, p = 0.000, Fig. 4C] [F(3,8)=
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29.873, p = 0.000, Fig. 4D] [F(3,8)= 48.115, p = 0.000, Fig. 4E] [F(3,8)= 33.897, p = 0.000, Fig. 4H]
[F(3,8)= 72.865, p = 0.000, Fig. 4I]
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