Some recent evidence suggests that microglia activation and inflammatory cytokine
production in the hippocampus are associated with the development of pain behavior
following peripheral nerve injury. We observed sciatic nerve chronic constriction
injury (CCI)-induced inflammation-related gene expression changes that are modulated
by minocycline in rat hippocampus. Intra-CA1 administration of minocycline was applied
after nerve injury. Genome-wide mRNA expression in the hippocampus was evaluated to
monitor the fundamental gene expression levels. We found that minocycline treatment
produces a pronounced inhibition of CCI-induced mechanical allodynia. We identified
790 genes differentially expressed in CCI vs. sham rats. Among these changed genes,
the 425 differentially expressed genes showed a significantly different effect in
CCI vs. minocycline-treated rats. Moreover, 390 transcripts were characterized by
an increase in mRNA abundance after nerve injury, and minocycline treatment reduced
the level of these changes. Only 35 transcripts were characterized by a decrease in
mRNA after nerve injury, and minocycline treatment reversed the decrease in the hippocampus.
Noteworthily, cytokine-cytokine receptor interaction and the toll-like receptor signaling
pathway are the top two most significantly enriched KEGG (kyoto encyclopedia of genes
and genomes) terms in comparing the sham vs. CCI group and CCI vs. minocycline-treated
group. Nine kinds of transcription factor gene transcripts (Runx3, Tfec, Pax-1, Batf3,
Sp5, Hlx, Nfkbiz, Spil, Fli1) increased in abundance after nerve injury, and minocycline
treatment reversed these changes. Afterwards, we selected some genes for further validation
by using quantitative PCR: interleukins (Il1β), chemokines (Cxcl13, Cxcl1, Ccl2, Cxcl11,
Ccl7, Ccl20), toll-like receptors (Tlr8 and Tlr1), and transcription factors (Runx3,
Nfkbiz and Spil). We suggested that the transcriptional changes of these inflammation-related
genes are strongly related to the processes of microglia activation underlying neuropathic
pain development.
Introduction
Neuropathic pain is a chronic pain condition that is usually induced by peripheral
nerve injury. Recent reports suggest that the inflammation-related cytokines accumulation
in dorsal root ganglion, dorsal spinal cord, hippocampus, thalamus, and somatosensoric
cortex are paralleled by pain responses in different animal models of neuropathic
pain (Al-Amin et al., 2011; Sun et al., 2016; Chang et al., 2018; Liu et al., 2018).
In the chronic constriction injury (CCI) and the spared nerve injury models of neuropathic
pain in rats, an increase in interleukin 1 beta (IL-1β), interleukin 6 (IL-6), nerve
growth factor (NGF), and glial cell-derived neurotrophic factor (GDNF) was observed
in most brain regions (Al-Amin et al., 2011). The overproduction of tumor necrosis
factor-α (TNF-α) may regulate synaptic plasticity in the rat hippocampus through microglia-dependent
mechanism after spared nerve injury of the sciatic nerve (Liu et al., 2017). However,
it is not clear whether other inflammation-related neuroactive substances will be
affected after microglia activation in the rat hippocampus after peripheral nerve
injury.
It is clear that many kinds of toll-like receptors (TLRs) are expressed in the hippocampus
and act as a type of pattern-recognition receptor that participate in inflammatory
responses. TLR1 expression in the hippocampus was increased in the neurons, microglia,
and astrocytes in seizure mice (Wang et al., 2015). TLR (2, 3, 4, 7, and 9) expression
was upregulated in the hippocampus of restraint stressed rats (Timberlake et al.,
2018). TLR2 and TLR4 in the rat hippocampus are related to the lipopolysaccharide
(LPS)-induced neuron cell death (He et al., 2013; Henry et al., 2014). That TLR3-induces
the increased expression of IL-1β in the rat hippocampus was suggested by Henry et
al. (2014). TLR8, expressed in most regions of the brain, is associated with injury
and neurite outgrowth (Ma et al., 2006). It is well known that TLR-dependent signaling
is often associated with the overproduction and release of inflammatory cytokines
in many different types of cells. However, the relationship between the changes of
TLRs expression and microglia activation in hippocampus of CCI rats is not known.
Previous studies reveal that chemokine production is enhanced in some neuroimmunological
diseases accompanied by pathological pain (Cartier et al., 2005). CXCL13 is obviously
upregulated in the spinal cord after spinal nerve ligation and induces astrocyte activation
via its receptor CXCR5 (Zhang et al., 2017). Chemokine CCL2 (C-C motif ligand 2) in
the rostral ventromedial medulla is related to the descending pain facilitation in
nerve-injured rats (Guo et al., 2012). Expression of chemokines CCL2 and CCL3 was
increased in the thalamus and hippocampus after severe spinal cord injuries (Knerlich-Lukoschus
et al., 2011). The overproduction of IL-1β and CCL2 was found in the hippocampus of
CCI rats (Fiore and Austin, 2018). Moreover, Lanfranco et al. reported that CCL5 gene
expression was found in neurons and glial cells in the rat hippocampus (Lanfranco
et al., 2018). However, no evidence directly addresses the relationship between microglia
activation and chemokine accumulation in neuropathic hypersensitivity.
It is clear that minocycline is an important modulator of the immune response and
easily permeates the blood-brain barrier (Stolp et al., 2007; Vonder Haar et al.,
2014). Clinically, minocycline can be administered by the intravenous route in patients
with traumatic brain injury (Rojewska et al., 2014). More recent evidence suggest
that minocycline is effective at reducing the spontaneous pain behavior in animal
models of neuropathic pain, and that means it appears to be a promising analgesic
drug (LeBlanc et al., 2011; Rojewska et al., 2014). In the present study, minocycline
is applied to identify what inflammation-related genes at the hippocampus are closely
related to the increased microglia activity in CCI-induced neuropathic pain rats.
Materials and Methods
Experimental Animals
In the experiments, adult male Sprague-Dawley (SD) rats (200–220 g) were housed under
a 12: 12 h revised light/dark cycle. The protocol was prepared from SD rats in accordance
with the National Institutes of Health guidelines in a manner that minimized animal
suffering and animal numbers. All experiments involving animals were approved by the
Zunyi Medical University Committee on Ethics in the Care and Use of Laboratory Animals.
Intra-hippocampal Injection
Rats were anesthetized by pentobarbital sodium (40 mg/kg, i.p.) and mounted in a David
Kopf stereotaxic frame (Model 1900, Tujunga, CA, USA) with a flat skull position.
An incision was made along the midline and the scalp was retracted. The area surrounding
the bregma was cleaned. Stainless steel guide cannulae were unilaterally implanted
1 mm above the CA1 according to rat brain atlases. Two holes were drilled through
the skull and two stainless steel needles (28 gauge) were inserted through the holes
(A/P-3.3 mm caudal to the bregma, L/R ± 2.0 mm lateral to the midline, D/V2.8 mm ventral
to the skull surface) (Paxinos and Watson, 1998). These rats were allowed to recover
for 6 days before CCI operation. A total of 0.5 μl of either PBS or minocycline was
infused (0.167 μl/min, 3 min) (Zhang et al., 2016). After infusion, needles remained
in place for an additional 3 min to avoid reflux. After nerve injury, the rats received
bilateral intra-hippocampal treatment of 0.5 μl of either vehicle or minocycline (1,
2, 5, 10, and 15 μg/μl, twice a day) for 7 days consecutively.
The Chronic Constriction Injury (CCI) Model
Rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.), and the sciatic
nerve (left) was exposed. The left sciatic nerve was exposed and a 15-mm length of
sciatic nerve proximal to the sciatic trifurcation was dissected. Four loose ligatures
(4.0 braided silk) were made around the sciatic nerve at 1-mm intervals. Sham rats
underwent the same procedure but without nerve ligation. After surgery, rats were
housed in separate cages (at room temperature for 24 h) to avoid scratching each other
(Safakhah et al., 2017; Liu et al., 2018). Rats that exhibited motor deficits such
as hind-limb paralysis, impaired righting reflexes, and hind-limb dragging were excluded.
That is to say, after implantation of a cannula into the hippocampus, the hind limb
function of rats used for CCI and behavioral testing was not to be impaired (Huang
et al., 2018). Hernández-López et al. also reported that stereotactic surgery for
cannula placement in the dorsal hippocampus does not impair the motor coordination
of rats (Hernández-López et al., 2017). In addition, rats not exhibiting pain hypersensitivity
after nerve injury were excluded.
Behavioral Assessment
Mechanical withdrawal threshold (MWT) was recorded to assess the response of the paw
to mechanical stimulus. An electronic von Frey plantar aesthesiometer (IITC, Wood
Dale, IL, USA) was used (Huang et al., 2018). After habituation to the test environment,
the measurements were made. Baseline values were obtained before surgery. Mechanical
stimulation was applied against the mid-plantar area of the left hind paw, and brisk
withdrawal or paw flinching was considered to be positive behavior. The MWT was recorded
and the cut-off force was set at 60 g. Three successive stimuli were applied, and
MWT was represented by the mean values.
Transcriptional Profile Analysis
Male SD rats were divided into Sham, CCI+0.01M PBS and CCI+ Minocycline groups (n
= 3 per group). Three subjects from each group who met all inclusion criteria (see
below) were subjected to microarray analyses. At 7 days following CCI or sham surgery,
the rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). The hippocampus
of rats was dissected, flash-frozen (in liquid nitrogen) and stored at −80°C for analysis.
According to the procedures described in the manual, total RNA was isolated from hippocampal
tissue using TRI Reagent (Sigma Aldrich, USA). RNA degradation and contamination were
checked by gel electrophoresis. The quantity of each RNA sample obtained was checked
using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) with pass criteria of
absorbance ratios of A260/A280 ≥ 1.8 and A260/A230 ≥ 1.6. RNA concentrations were
assessed using Qubit® RNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies,
CA, USA). A total amount of 2 μg RNA per sample was used to construct the cDNA library.
First-strand cDNA was synthesized using a HiFiScript gDNA Removal cDNA Synthesis Kit
(CWBIO, Beijing, China) according to the standard protocols. Quantitative real-time
PCR was carried out using a QuantStudio™ 6 Flex Real-Time System (Applied Biosystems,
USA) with UltraSYBR Mixture (CWBIO, Beijing, China). The following PCR amplification
program was used: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s, 50–54°C
(changed according to the primer sequences) for 20 s and 72°C for 20 s. A dissociation
curve was performed (55–95°C) after the last PCR cycle to ascertain the specificity
of the amplification reactions. The abundance of each mRNA was normalized with respect
to the endogenous housekeeping gene β-actin, and the relative gene expression levels
were determined by the 2−ΔΔCt method.
Microarray experiments were performed to determine gene-expression profiles in rat
hippocampus. Based on the differentially expressed gene (DEG) results, the heat maps
were constructed using Multiexperiment Viewer (MeV; http://mev.tm4.org/). Gene ontology
(GO) and pathway enrichment analyses were carried out with the aid of the NCBI COG
(http://www.ncbi.nlm.nih.gov/COG/), Gene Ontology Database (http://www.geneontology.org/)
and KEGG pathway database (http://www.genome.jp/kegg/).
The DEGs were ascertained using the DESeq R package (1.10.1) as detailed in a previous
study (Wang et al., 2010). False discovery rate (FDR) was used to correct the results
for P-value. FDR ≤ 0.05 and an absolute value of log2 (fold-change) ≥1 were used as
the threshold for screening DEGs. Pathway functional enrichment analysis was performed
using the “phyper” function in R. The P-value calculating formula is:
P
=
1
-
∑
i
=
0
m
-
1
(
M
i
)
(
N-M
n-i
)
(
N
n
)
Here, M is the number of genes in the pathway, N is the total number of genes in the
genome, m is the number of target gene candidates in M and n is the number of differentially
expressed genes. In addition, i = 1, 2, 3, … (M-1) where M represents the number of
genes in the pathway. The Fisher's score indicates the ratio of genes (number m) belonging
to the functional pathway out of the total differentially expressed genes (number
n) (Zhang et al., 2018). Subsequently we calculate the value of FDR. FDR ≤ 0.01 is
considered as significantly enriched.
RT-PCR
Male SD rats were divided into Sham, Sham+Minocycline, CCI+0.01M PBS and CCI+ Minocycline
groups (n = 6 per group). The changes in the abundance of some gene transcripts in
the rat hippocampus after nerve injury and the modulatory effects of minocycline should
be further investigated by PCR analysis of samples independent from those used for
the microarray studies. According to the methods mentioned above, four groups of animals
were treated and killed by cutting their necks. Brain tissue was quickly dissected
on the ice platform and was immersed and washed with phosphate buffered solution (PBS).
The hippocampus was isolated and rapidly transferred into separate RNase-free 1.5
ml Eppendorf tubes. Total RNA was immediately isolated using the TRIzol Reagent (MRC
Co., Cincinnati, USA). The concentration and purity of RNA samples were measured using
Spectrophotometer (Thermo Fisher Scientific). The ratios of OD260/OD280 were between
1.9 and 2.1. cDNA was synthesized from RNA by reverse transcription reaction using
the SuperScript II reverse transcriptase kit (Invitrogen). All primers are shown in
Table 1. qPCR was performed in a final volume of 20 μl (8 μl H2O, 10 μl mastermix,
1 μl assay-mix, and 1 μl cDNA) on a Linegene Real-time PCR detection system (Bioer
Technology, China). PCR reaction conditions were as follows: (1) 95°C 8 min 1 Cycle;
(2) 95°C 15 s and 60°C 1 min, 40 Cycles. The experimental data analysis was carried
out using the 2−ΔΔCt method (Livak and Schmittgen, 2001).
Table 1
Primers used for RT-PCR.
Gene
Forward
Reverse
Cxcl13 (NM-001017496.1)
5′-TTTGGTAACCATCTGGCAGTA-3′
5′-GCTCGACCTTTATCAATCTAAT-3′
Cxcl1 (NM-030845.1)
5′-TGGCTATGACTTCGGTTTGGGT-3′
5′-GGCAGGGATTCACTTCAAGAACA-3′
Ccl2 (NM-031530.1)
5′-GTGCTGAAGTCCTTAGGGTTG-3′
5′-GTCGGCTGGAGAACTACAAGA-3′
Cxcl11 (NM-182952.2)
5′-CCAGGCACCTTTGTCCTTTAT-3′
5′-GGTTCCAGGCTTCGTTATGTT-3′
Ccl7 (NM-001007612.1)
5′-CACCGACTACTGGTGATCTTTC-3′
5′-TTCATCCACTTGCTGCTATGT-3′
Ccl20 (NM-019233.1)
5′-GACAAGACCACTGGGACA-3′
5′-AGCCTAAGAACCAAGAAG-3′
Iba-1 (NM-017196.3)
5′-CAAGGATTTGCAGGGAGGA-3′
5′-CAGCATTCGCTTCAAGGACATA-3′
Cd68 (NM-001031638.1)
5′-TCAAACAGGACCGACATCAGA-3′
5′- ATTGCTGGAGAAAGAACTATGCT-3′
iNOS (NM-012611.3)
5′-GATGTGCTGCCTCTGGTCCT-3′
5′-GAGCTCCTGGAACCACTCGT-3′
IL-1β (NM-031512.2)
5′-CAGCCTTACTGGCCTGCTAC-3′
5′-CTGCTACCACGACAGCCATA-3′
Tlr8 (NM-001101009.1)
5′-TGCTTCATTTGGGATTTG-3′
5′-TGGCATTTACACGCTCAC-3′
Tlr1 (NM-001172120.2)
5′-CAGTTTCTGGGATTGAGCGGT-3′
5′-TAATGTGCTGAAGACACTTGGGATC-3′
Runx3 (NM-130425.1)
5′-GGCTTTGGTCTGGTCCTCTATC-3′
5′-GCAACGCTTCCGCTGTCA-3′
Nfkbiz (NM-001107095.1)
5′-CCGTAGAAGTAAGCGAGGTT-3′
5′-GAGCATGATCGTGGACAAG-3′
Spil (NM-001005892.2)
5′-CAATCTTTGCTCCTCTTT-3′
5′-CTACCAATCCTGGCTTCA-3′
β-actin (NM-031144.3)
5′-AGCCATGTACGTAGCCATCC-3′
5′-ACCCTCATAGATGGGCACAG-3′
Statistical Analysis
All data were presented as mean ± standard deviation (SD.). The behavioral and PCR
data were analyzed by one- (compared within the group) or two-way (compared between
groups) ANOVA. If significance was established, post-hoc Dunnett or Bonferroni's multiple
comparisons were performed. All statistical tests were carried out using SPSS 18.0
software (IBM, Armonk, NY). The level of significance was set as p < 0.05.
Results
Intra-CA1 Administration of Minocycline Attenuates CCI-Induced Mechanical Allodynia
To investigate the antinociceptive effect of minocycline on the mechanical nociceptive
threshold in neuropathic pain rats, the MWT was recorded on the day before and after
surgery (at POD 1, 3, 5, and 7). A total of five doses (1, 2, 5, 10, and 15 μg/μl,
twice a day) were administered. We compared the changes of MWT between the different
time points (Figure 1). Application of minocycline at 1, 2, and 5 μg/μl for 30 min
showed increased MWT in comparison to CCI rats (P < 0.05). Application of minocycline
at 1, 2, and 5 μg/μl for 1 h also showed more significant increase in MWT (vs. CCI
rats: P < 0.01; vs. 30 min: P < 0.05). Application of minocycline at 10 and 15 μg/μl
for 30 min showed a slight increase but was not significantly different from that
of the vehicle-treated CCI group. Application of minocycline at 10 μg/μl for 1 h showed
obvious increased MWT (vs. CCI rats: P < 0.05; vs. 30 min: P < 0.05). Application
of minocycline at 15 μg/μl for 1 h also showed increased MWT (vs. CCI rats: P < 0.05).
Application of minocycline at 5 μg/μl for 2 h showed slightly increased MWT (vs. CCI
rats: P < 0.05; vs. 1 h: P < 0.01). These results suggest that minocycline produced
a reversal of MWT, with maximal effect at 1 h after minocycline administration.
Figure 1
MWT was determined in different groups. All values represent mean ± SD (n = 8). (A)
Decreased MWT was exhibited in the CCI rats on day 1 after surgery compared to sham
rats (++
P < 0.01). Compared with CCI rats, minocycline (1, 2, 5, 10, and 15 μg/μl) treatment
exerted anti-hyperalgesic effects (*
P < 0.05). Minocycline treatment showed obvious increased MWT (vs. 1 μg/μl: #
P < 0.05; vs. 2 μg/μl: @
P < 0.05; vs. 5 μg/μl: %
P < 0.05; vs. 10 μg/μl: &
P < 0.05). Greater analgesic effect of minocycline occurs 1 h after its administration
(vs. Pre: O
P < 0.05; vs. 30 min: $
P < 0.05; vs. 1 h: ▴
P < 0.05); (B) The MWT was measured at 1, 3, 5, and 7 days after surgery. Decreased
MWT was exhibited in the CCI rats on days 1, 3, 5, and 7 after nerve injury compared
to sham rats (++
P < 0.01). Compared with CCI rats, minocycline (1, 2, 5, 10, and 15 μg/μl) treatment
exerted anti-hyperalgesic effects (*
P < 0.05). Minocycline treatment showed the more obvious increase in MWT (vs. 1 μg/μl:
#
P < 0.05; vs. 2 μg/μl: @
P < 0.05; vs. 5 μg/μl: %
P < 0.05; vs. 10 μg/μl: &
P < 0.05).
As shown in Figure 1B, decreased MWT was observed in rats on day 1 after surgery compared
to sham rats (P < 0.05), and the allodynia was sustained throughout the experimental
period. Compared to the vehicle-treated CCI rats, minocycline at 1 μg/μl induced significant
analgesic effect (P < 0.05). Minocycline at doses of 2 and 5 μg/μl showed better analgesic
effects in comparison with minocycline at dose 1 μg/μl (P < 0.05). We also noticed
that minocycline at a dose of 5 μg/μl showed apparent elevations of the mechanical
pain threshold in comparison with minocycline at a dose of 2 μg/μl (P < 0.05). On
the other hand, minocycline at 10 μg/μl produced moderate antinociceptive effect in
CCI rats. Minocycline at 15 μg/μl induced a slight but significant antinociceptive
effect in CCI rats. Minocycline at a dose of 5 μg/μl showed better analgesic effects
in comparison with minocycline at doses of 10 and 15 μg/μl (P < 0.05). In a short,
three main conclusions can be drawn: (1) The decreased MWT in CCI rats and the analgesic
effect of minocycline in minocycline-treated CCI rats are maintained over 7 days;
(2) a greater analgesic effect of minocycline occurs 1 h after its administration;
(3) the highest analgesic effect of minocycline occurs at a dose of 5 μg/μl. Then,
the minimum dose of minocycline (5 μg/μl) showing maximum effect was selected in the
following experiments.
Identification of Differentially Expressed Genes Between Different Groups
To explore the possible role of microglia activation and inflammation within the hippocampus
in the development of peripheral neuropathic pain, the DEGs between different groups
were identified. According to the results, in the rat hippocampus, there were 790
DEGs between the sham group and the CCI group. Among them, 613 genes were increased
and 177 were decreased (as shown in Figure 2A and Table S1). There were 840 DEGs between
the CCI group and minocycline-treated group, among them 143 genes were increased and
697 were decreased (as shown in Figure 2B and Table S2). Between the sham group vs.
CCI group and minocycline-treated group vs. CCI group, 448 DEGs were shared (as shown
in Figure 2D and Table S3). Among these 448 DEGs, 398 transcripts were characterized
by an increase in mRNA abundance after nerve injury, and minocycline application decreased
the level of these changes. Only 34 transcripts were characterized by a decrease in
mRNA after nerve injury, and minocycline treatment reversed the decrease in hippocampus
of CCI rats (as shown in Table S3). It seems that these 432 genes may be associated
with the effect of minocycline in CCI rats. In addition, only two transcripts were
upregulated in CCI and upregulated by minocycline. Fourteen transcripts were downregulated
in CCI and downregulated by minocycline. We also found that there were 766 DEGs between
the sham group and the minocycline-treated group. Among them 342 genes were increased
and 424 were decreased (as shown in Figure 2C and Table S4). Between the sham group
vs. CCI group and sham group vs. minocycline-treated group, 252 DEGs were shared,
among them 86 genes were increased and 166 were decreased (as shown in Figure 2D and
Table S5).
Figure 2
The DEGs were identified. In (A–C), red dots represent increased DEGs and blue dots
represent decreased DEGs. In addition, gray dots represent non-DEGs. (A) MA plot for
DEG analysis between sham and CCI groups. (B) MA plot of DEGs among the hippocampus
between the CCI and minocycline-treated group. (C) MA plot of DEGs among the hippocampus
between sham and minocycline-treated group. (D) Comparisons of the number and overlapping
DEGs between different experimental groups (the Venn diagram of DEGs). (D1) Blue circle
represents number of DEGs between sham and CCI group; red circle represents number
of DEGs between CCI and minocycline-treated group; the overlapping area represents
shared DEGs of two comparable groups. (D2) Blue circle represents number of DEGs between
sham group and minocycline-treated group; red circle represents number of DEGs between
CCI group and minocycline-treated group; the overlapping area represents shared DEGs
of two comparable groups.
Differential Expression Analysis at the Gene Ontology Annotation Level
The DEGs were annotated covering molecular biological function, cellular component
and biological process. As shown in Figure 3, the DEGs in the sham, CCI and minocycline-treated
groups can be mostly classified into biological processes. The five most enriched
GO terms of the DEGs for biological process were the cellular process, biological
regulation, regulation of biological process, response to stimulus, and metabolic
process. The five most enriched GO terms of the DEGs for the cellular component were
cell, cell part, organelle, membrane, and membrane part. The five most enriched GO
terms of the DEGs for molecular function were binding, catalytic activity, signal
transducer activity and molecular function regulator. Compared with the sham group,
the differentially expressed annotated genes in biological process, molecular function,
and cellular component were mainly increased in CCI rats (Figure 3A). As far as the
minocycline-treated and CCI groups were concerned, the differentially expressed annotated
genes in biological process, molecular function, and cellular component were mainly
decreased in the minocycline-treated group (Figure 3B). As a result, as shown in Figure
3C, between sham and minocycline-treated group, the numbers of DEGs in biological
process, molecular function, and cellular component are decreased.
Figure 3
GO term classification of increased and decreased genes on DEGs for each pairwise.
X axis represents GO term. Y axis represents the number of increased/decreased genes.
(A) The most enriched GO terms between the sham and CCI groups. (B) The most enriched
GO terms between the CCI and minocycline-treated groups. (C) The most enriched GO
terms between the sham and minocycline-treated groups.
KEGG Pathway Analysis of Differentially Expressed Genes
Compared with the sham-operated group, the CCI group had 20 differential gene-involved
significant pathways. DEGs contained in these pathways (top 14) are shown in Table
2. Some pathogenic microorganism infection-related pathways (herpes simplex infection,
tuberculosis, influenza A, malaria, Pertussis, and Leishmaniasis infection) were also
involved in the process. As far as the sham and CCI groups were concerned, the most
enriched KEGG pathways were the cytokine-cytokine receptor interaction pathway and
the TLR signaling pathway. The cytokine-cytokine receptor interaction pathway was
significantly affected, with 31 increased genes and 1 decreased gene involved in the
hippocampus of CCI rats. The TLR signaling pathway was significantly affected, with
17 increased genes and 2 decreased genes involved in the hippocampus of CCI rats.
Table 2
The top 14 most significant KEGG pathways identified with increased and decreased
genes among different groups.
Pathway (FDR ≤ 0.01)
CCI group vs. sham group
Minocycline-treated group vs. CCI group
Cytokine-cytokine receptor interaction
Increased: Cxcl11, Cxcl13, Cxcl16, Cxcl4, Ccl2, Ccl3, Ccl7, Ccl5, Ccr2, Ccr5, Osmr,
Csf3, Csf3r, Il4r, Il13ra1, Csf2rb, Il2ra, Il2rb, Il2rg, Csf1r, Il10rb, IL20rb, Sf1b,
Sf1a, Sf14, Fas, Cd40, Tgfβ1, Il1β, Il1r1, Il18r, Ap
Decreased: Tnfsf15
Decreased: Il8ra, Cxcr3, Cxcl11, Cxcl13, Cxcl16, Cxcl4, Ccr7, Ccl2, Ccl3, Ccl5, Ccl7,
Osmr, Bsf3, Csf3, Csf2rb, Il2rb, Il2ra, Il2rg, Il4r, Il21r, Il10ra, Il20rb, Ltb, Sf1b,
Sf1a, Sf14, Fas, Sf9, Tnfsf4, Tnfsf13b, Il-1b, Il1r1, Il18rap
Toll-like receptor signaling pathway
Increased: Tlr1, Tlr2, Tlr6, Md2, P13k, Tlr7. Tlr9, Trif, Opn, Tp12, Il1β, Rantes,
Mip1α, Cd40, Cd86, Itac
Decreased: Mkk3,Irf7
Increased: Mkk3, Mkk6
Decreased: Lbp, Tlr1, Tlr2, Tlr6, Cd14, Md-2, Tlr7, Tlr8, Tlr9, Tab1, Iκbα, Il-1β,
Ccl5, Mip-1α, Cd86, I-Tac
Phagosome
Increased: MhcI, MhcII, Fcyr, Ic3b, collectins, Tlr2, Cd14, Tlr6, Mr, Dectin1, Sra1,
Tuba, Tubb, Cyba, Nox1, Ncf1, Ncf2, Ncf4,
Decreased: Tap, Stx7
Increased: Stx7
Decreased: MhcI, MhcII, Tuba, Tubb, M6pr, Fcyr, C3, Tsp, Tlr2, Tlr6, Cd14, Mr, Dectin1,
Sra1, P22phox, Gp91, P67phox, P40phox
Fc gamma R-mediated Phagocytosis
Increased: Fcgr2b, Cd45, Src, Lat, Pi3k, Fcyri, Fcgr2a, Plcy, Sphk, Cpkc, Ncf1, Wasp,
Arpc5, Vav, Rac, Dock2, Pag3
Decreased: Crk
Increased: CrkII
Decreased: Fcgr2b, Cd45, Fcyri, FcyrIIa, Src,Pld, Vav, CrkII, Sphk,Rac, Pag3, Wasp,
Arp2, Arp3, Gsn
TNF signaling pathway
Increased: Tnfr1, Ciap1/2, Tp12, Rip3, Mlkl, Ccl2, Ccl5, Cxcl1, Cxcl2, Cxcl3, Fas,
Il1β, Bcl3, Socs3, Ifi47, Tnfr2
Decreased: Mkk3
Increased: Mkk3, Mkk6
Decreased: Tnfr1, Ciap1, Ciap2, Tab1, Tab2, Tab3, Iκba, C/ebpb, Rip3, Mlkl, Ccl2,
Ccl5, Cxc11, Cxc12, Cxc13, Fas, Il-1β, Bcl3, Nfkbia, Socs3, IfI47, Icam1, Tnfr2,
Complement and coagulation cascades
Increased: Tfpi, F10, Vwf, Par3, A2m, Pai, C3, Fd, Fb, C1qrs, C1inh, Mbl, C2, C4,
C6, Cr4, C5ar1
Decreased: Klkb1, Tfpi, Fh
Increased: F5, Fh, A2m, Fga
Decreased: F10, Par3, Par4, A2m, Pai, Upar, Bdkrb1, Bdkrb2, Cfi, Cfb, Cfd, C3, C6,
C7, C8,C9, C1qa, C1inh C2, C4, C3ar1, Cr4, C5ar1
Cell adhesion molecules
Increased: Cd86, MhcII, MhcI, Pvrl2, Cd40, Itgal, Cd2, Cd4, Cd8, Cd6, Ptprc, Selp,
Sell, Sdc, Pvrl1
Decreased: Cntnap2, Mpz, Mhc1
Increased: Cldn, Cdh2, Cdh41
Decreased: Cd2, Cd86, Icos, MhcII, MhcI, Cd8, Cd6. Itgal, Icam3, PtprC, Selp, Icam1,
Icam2, Ngl1, Sdc, Mag
Natural killer cell mediated cytotoxicity
Increased: Bid, Fas, Trailr, Itgal, Shp1, Dap12, Fcer1y, FcyrIII, Nkp30, Lat, Vav,
Rac, Pi3k, Plcy, Pkc
Increased: Rae-1
Decreased: Icam1, Icam2, Trailr, Fas, Itgal, FcyrIII, Shp-1, Dap-12, Fcer1g, Sap,
Vav, Rac
NF-κB signaling pathway
Increased: Il1β, Il1r, Lyn, Lat, Btk, Plcy2, Cd14, Md2, Cd40, Trif, Ciap1/2, Carma,
Bcl2a1, Baff
Decreased: Il1β, Il-1r, Tnf-r1, Lbp, Cd14, Md-2, Ltb, Baff, Btk, Ciap1, Ciap2, Tab,
Carma, Iκbα, Il1β, Icam
Chemokine signaling pathway
Increased: Ac, Chemokine, Cxcr2, Gnb1, Src, Pi3k, Dock2, Rac, Vav, Wasp, Ncf1
Decreased: Ac, Gnb1, Crk
Increased: Gnb1, Crk
Decreased: Gro, Cxcr2, Gnb1, Src, Vav, Crk,Rac, Wasp, Iκb
Osteoclast differentiation
Increased: Ii-1, Tgfb, Il-1r, Tnfr1, Oscar, Fcry, Dap12, Btk, Socs1, Plcy, Pi3k, Nadph,
Spi1
Decreased: Ii-1, Il-1r, Tnfr1, Oscar, Fcry, Dap12, Btk, Socs1, Socs3, Tab1, Tab2,
Nadph, Iκb, SpI1
B cell receptor signaling pathway
Increased: Cd72, Shp1, Lyn, Btk, FcgrIIb, Leu13, Vav, Plc-y2, Calma, Rac, Pi3k
Decreased: Bam32
Decreased: Iga, Cd72, Shp1, Fcgr2b, Leu13, Btk, Vav, Rac, Card11, Iκb
Primary immunodeficiency
Increased: Yc, Btk, Cd45, Cd4, Cd8, Cd8α, CIIta, Cd40
Decreased: Tap2,Rfxap
Decreased: Cd3e, Il2rg, Iga, Btk, Cd45, Cd4, Cd8, Rfxap, CIIta, Cd8a, Rfxap, CIIta,
Icos
Platelet activation
Increased: Collagen, Vwf, Par1, Fcry, Lyn, Pi3k, Btk, Plcy2, Ac, Kind3, Fcgr2a, Tbxas
Decreased: Ac,Collagen
Increased: Col1a,Fg
Decreased: Cll1a, Par1, Par4, P2x1, Fcry, Pi3k, Btk, Tbxas1, Fermt3, Fcgr2a
We also noticed that the minocycline-treated group had 20 differential gene-involved
significant pathways in comparison with the CCI group. DEGs contained in these pathways
(top 14) are also shown in Table 2. These results reveal that minocycline administration
can regulate the expression of genes in these pathways, and reversing the gene expression
changes in these pathways may be considered as one of the important mechanisms of
minocycline against CCI-induced neuropathic pain. Some pathogenic microorganism infection-related
pathways were also significantly downregulated. As far as the CCI and minocycline-treated
groups were concerned, the most enriched KEGG pathways were the cytokine-cytokine
receptor interaction pathway and the TLR signaling pathway. Among them, the cytokine-cytokine
receptor interaction pathway was obviously affected, with 33 decreased genes involved.
The TLR signaling pathway was significantly affected, with 16 decreased genes and
2 increased genes involved in the hippocampus of minocycline-treated CCI rats.
mRNA Expression Profile of Inflammation-Related Genes in Rat Hippocampus
Cytokines and chemokines were originally identified as essential mediators for inflammatory
and immune responses in the formation of neuropathic pain (White and Wilson, 2008;
Totsch and Sorge, 2017). As shown in Table 3, in the rat hippocampus, higher transcript
levels of Cxcl13, Cxcl1, Ccl2, Cxcl11, Ccl7, Ccl20, Ccl3, Ccl6, Ccl5, and Cxcl16 (Top
10 upregulated chemokine genes) were found in the CCI group as compared with sham
rats. Except for Cxcl1, this upregulation of chemokine genes was almost diminished
after repeated treatment with minocycline. We noticed that pro-inflammatory biomarker
Il1β and iNOS are robustly upregulated at the transcriptional level after nerve injury.
After minocycline treatment, iNOS and Il1β were obviously downregulated as compared
with CCI rats. Il-18rap (rCG22315) transcript was also upregulated in the hippocampus,
and minocycline suppressed upregulation in CCI rats.
Table 3
mRNA expression profile of inflammation-related genes among different groups.
Gene symbol
Sham vs. CCI
CCI vs. minocycline
Sham vs. minocycline
Fold
change
FDR
Fold
change
FDR
Fold
change
FDR
Cxcl13
+7.03
7.02E-236
−8.42
2.75 E-207
−1.39
0.13
Cxcl1
+5.44
2.07E-31
−1.87
1.75 E-13
+3.57
2.18E-08
Ccl2
+5.35
1.35E-48
−6.65
1.77 E-47
−1.30
0.26
Cxcl11
+4.80
2.95E-26
−4.45
2.75 E-25
+0.35
0.72
Ccl7
+4.27
1.02E-17
−7.25
4.73E-18
0
0
Ccl20
+3.46
0.003003
−3.44
0.0029
0.02
0.99
Ccl3
+2.40
0.000603
−3.38
4.47 E-05
−0.98
0.42
Ccl6
+2.22
3.47E-12
−3.20
2.39 E-17
−0.98
0.07
Ccl5
+1.61
3.03E-07
−3.48
6.45 E-16
−1.87
0
Cxcl16
+1.27
6.02E-31
−1.88
1.70 E-53
−0.61
5.23E-05
INos
+5.46
2.72E-06
−5.44
2.67 E-06
0
0
Il1β
+4.34
9.98E-19
−3.15
4.47E-15
+1.19
0.15
Il18rap
+4.13
6.00 E-16
−6.11
2.35 E-17
−1.98
0.17
Socs3
+3.67
2.94 E-243
−3.08
5.23E-209
+0.58
0
C3
+3.61
0
−3.83
0
−0.22
0
Tlr8
+3.49
1.05E-11
−4.20
4.72 E-13
−0.71
0.49
Ptges
+3.47
4.28E-49
−2.48
2.02 E-35
+0.99
0
Mt1
+2.15
4.94E-228
−1.81
5.21E-181
+0.34
0
Il20rb
+2.08
1.32 E-18
−1.11
3.61 E-08
+0.97
0
Tlr1
+1.93
1.91E-09
−2.12
1.57 E-10
−0.18
0.66
Il21r
+1.92
1.10E-18
−2.01
1.21 E-19
−0.09
0.77
Il2rb
+1.69
2.99 E-06
−1.78
1.14 E-16
−0.10
0.84
Tnfrsf1b
+1.66
5.44E-32
−1.20
6.16 E-20
+0.46
0.01
Hpgds
+1.64
1.35E-07
−1.56
3.59 E-07
+0.08
0.84
Tlr13
+1.61
8.94E-20
−1.37
1.03 E-15
+0.24
0.27
I11r1
+1.48
8.55E-62
−1.15
6.06 E-42
+0.32
0
Irf8
+1.57
7.14E-85
−1.35
5.28 E-27
+0.23
0.03
Card11
+1.49
1.38E-28
−1.65
9.27 E-33
−0.16
0.36
P2ry6
+1.49
4.35E-44
−1.33
8.20 E-37
+0.17
0.21
Tlr7
+1.48
7.32E-34
−1.15
4.02 E-23
+0.33
0.02
Casp4
+1.38
3.52E-14
−1.34
1.52 E-13
+0.04
0.86
Fas
+1.22
1.79E-05
−1.37
2.68 E-06
−0.15
0.68
Tlr2
+1.06
1.61E-19
−1.05
3.82 E-19
+0.10
0.94
Tlr9
+1.06
0.0001
−1.65
8.08 E-08
−0.60
0.10
Tifab
+1.01
8.91 E-29
−1.05
4.44 E-30
−0.03
0.76
Cd68
+3.45
9.91E-95
−2.90
1.19E-80
+0.54
0.05
Msr-1
+2.01
7.93E-21
−1.94
7.27 E-20
0.07
0.82
Iba-1
+1.16
3.94E-46
−1.16
1.28E-45
0
0.98
Ox-42 (Cd11b)
+0.72
9.34E-30
−0.55
7.70E-19
+0.17
0.01
Ptges
+3.47
4.28E-49
−2.48
2.02 E-35
+0.99
0.01
Mrc1
+2.85
4.43 E-129
−2.71
6.94 E-122
+0.13
0.48
Cd86
+1.46
1.41E-07
−1.86
3.20 E-10
−0.41
0.27
Tgfβ1
+1.16
2.58 E-49
−0.96
4.06E-36
+0.20
0.03
Arg1
−1.06
8.19 E-17
+0.43
0
−0.62
5.18E-08
IL4r
+1.03
5.18 E-70
−0.14
0
+0.90
3.18E-51
Runx3
+6.39
7.42 E-11
−3.79
1.51 E-09
0
0
Tfec
+3.95
5.47 E-17
−5.25
7.13 E-19
−1.30
0.26
Pax-1
+3.70
0.0009
−4.68
0.0004
0
0
Batf3
+2.67
0.0005
−1.03
4.69 E-08
−0.11
0.62
Sp5
+2.17
6.15 E-06
−1.42
0.00076
+0.76
0.20
Hlx
+1.58
9.94 E-28
−1.26
1.85 E-11
+0.32
0.18
Nfkbiz
+1.46
9.25E-28
−2.02
1.12 E-42
−0.56
0
Spi1 (Pu.1)
+1.33
2.57E-34
−1.19
1.12 E-28
+0.15
0.27
Fli1
+1.21
3.04 E-35
−1.11
1.33 E-30
+0.10
0.39
Lst1
+2.39
0.0001
−1.29
0.01
+1.10
0.14
Maff
+1.67
1.72 E-09
−0.45
0.04
+1.23
2.50E-05
Elf4
+1.39
6.46 E-11
−0.95
1.93E-06
+0.45
0.07
Vgl13
+1.38
3.40 E-51
+0.66
2.52E-24
+2.04
2.25E-140
Nr2f2
+1.12
2.89 E-144
+0.59
1.82E-71
+1.72
0
Cartpt
−2.30
2.14 E-70
+0.17
0.23
−2.13
0
Six3
−1.68
6.84 E-06
−0.57
0.20
−2.25
1.48E-08
Meox1
−1.64
7.13 E-05
+1.11
0.01
−0.53
0.10
Tfap-2c
−1.55
1.84 E-05
+0.28
0.31
−1.27
0
Ebf3
−1.40
2.05 E-05
−0.44
0.22
−1.84
7.32E-08
Mkx
−1.12
2.27 E-08
−0.003
0.49
−1.12
7.17E-09
Mei4
−1.00
2.91 E-05
+0.09
0.4
−0.91
6.09E-05
Cxcl13, C-X-C motif chemokine ligand 13; Cxcl1, C-X-C motif chemokine ligand 1; Ccl2,
C-C motif chemokine ligand 2; Cxcl11, C-X-C motif chemokine ligand 11; Ccl7, C-C motif
chemokine ligand 7; Ccl20, C-C motif chemokine ligand 20; Ccl3, C-C motif chemokine
ligand 3; Ccl6, C-C motif chemokine ligand 6; Ccl5, C-C motif chemokine ligand 5;
Cxcl16, C-X-C motif chemokine ligand 16; Nos, nitric oxide synthase 2; Il1β, interleukin
1 beta; Il18rap, interleukin 18 receptor accessory protein; Socs3, suppressor of cytokine
signaling 3; C3, complement C3; Tlr8, toll-like receptor 8; Ptges, prostaglandin E
synthase; Mt1, metallothionein 1; Il20rb, interleukin 20 receptor subunit beta; Tlr1,
toll-like receptor 1; Il21r, interleukin 21 receptor; Il2rb, interleukin 2 receptor
subunit beta; Tnfrsf1b, TNF receptor superfamily member 1B; Hpgds, hematopoietic prostaglandin
D synthase; Tlr13, toll-like receptor 13; Il1r1, interleukin 1 receptor type 1; Irf8,
interferon regulatory factor 8; Card11, caspase recruitment domain family, member
11; P2ry6, pyrimidinergic receptor P2Y6; Tlr7, toll-like receptor 7; Casp4, caspase
4; Fas, Fas cell surface death receptor; Tlr2, toll-like receptor 2; Tlr9, toll-like
receptor 9; Tifab, TIFA inhibitor; Cd68, Cd68 molecule; Msr-1, macrophage scavenger
receptor 1; Iba-1, ionized calcium binding adaptor molecule 1; Cd11b, Complement receptor
3; Mrc1, mannose receptor, C type 1; Cd86, CD86 molecule; Tgfβ1, transforming growth
factor, beta 1; Arg1, arginase 1; IL4rα, interleukin 4 receptor; Runx3, runt-related
transcription factor 3; Pax-1, paired box 1; Batf3, basic leucine zipper ATF-like
transcription factor 3; Sp5, Sp5 transcription factor; Hlx, H2.0-like homeobox; Nfkbiz,
NFKB inhibitor zeta; Spi1, Spi-1 proto-oncogene; Fli1, Fli-1 proto-oncogene; Lst1,
leukocyte specific transcript 1; Maff, MAF bZIP transcription factor F; Elf4, E74
like ETS transcription factor 4; Vgl13, vestigial-like family member 3; Nr2f2, nuclear
receptor subfamily 2; Cartpt, CART prepropeptide; Six3, SIX homeobox 3; Meox1, mesenchyme
homeobox 1; Tfap-2c, transcription factor AP-2 gamma; Ebf3, EBF transcription factor
3; Mkx, mohawk homeobox; Mei4, meiotic double-stranded break formation protein 4).
In addition, cytokine signaling-3 (SOCS3) and TLR gene transcripts were upregulated
in the hippocampus, and minocycline suppressed the upregulation of SOCSs and Tlr8,
Tlr1, Tlr13, Tlr7, Tlr2, and Tlr9 gene transcripts in CCI rats. We found that, after
nerve injury, Tlr4 transcripts were only upregulated by <1 fold (Tlr4: fold = 0.56,
FDR = 0.0167). The Tnf-α and Nlrp3 transcripts were upregulated by >1 fold (Tnf-α:
fold = 1.492, FDR = 0.037; Nlrp3: fold = 1.21, FDR = 4.18E-23) in the hippocampus
of CCI rats. Moreover, the upregulated Tnf-α and Nlrp3 gene transcripts were only
moderately suppressed by minocycline (Tnf-α: fold = 0.469, FDR = 0.275; Nlrp3: fold
= 0.79, FDR = 5.55E-12). Besides, Nlrp1a gene transcripts were only slightly upregulated
(Nlrp1a: fold = 0.29, FDR = 0.007). For these reasons, the changes of Tnf-α, Tlr4,
and Nlrp gene transcripts are not listed in Table 3. At last, we noted that C3, Ptges,
Mt1, Il20rb, Il21r, Il2rb, Hpgds, Il1r1, Tlr8, Card11, P2ry6, Casp4, Fas, and Tifab
were upregulated in the CCI group as compared with sham rats. Afterward, the upregulation
of these gene transcripts was almost diminished after repeated treatment with minocycline.
More studies have suggested that Cd68, Iba-1 (ionized calcium-binding adaptor molecule-1,
involved in microglial motility), Ox-42 (Cd11b, involved in microglial plasticity
and motility), Msr-1(macrophage scavenger receptor 1, involved in phagocytosis), and
Mhc-II (major histocompatibility complex II) are common markers of microglia activation
(Booth and Thomas, 1991; Minett et al., 2016). As shown in Table 3, we found that,
compared with sham rats, the Cd68, Msr-1, and Iba-1 transcripts were upregulated by
>1 fold in hippocampus of CCI rats. After repeated treatment with minocycline, the
Cd68, Msr-1, and Iba-1 transcripts were all obviously downregulated by >1 fold as
compared with CCI rats. To our surprise, the Cd11b transcripts were upregulated by
<1 fold (fold = 0.72, FDR = 9.34E-30) as compared with sham rats. Moreover, after
minocycline treatment, the Cd11b transcripts were only downregulated by <1 fold (fold
= 0.55, FDR = 7.70E-19) as compared with CCI rats.
It is clear that microglia/macrophages respond to acute brain injury by becoming activated
and developing a pro-inflammatory profile of M1-like or anti-inflammatory profile
of M2-like phenotypes (Perego et al., 2013; Luo et al., 2018). According to previous
studies, M1 polarization could be determined by the expression levels of Cd86, as
well as Il1β, Ccl2, Ccl3, and iNOS. M2 polarization could be ascertained by the increased
expression of Arg1, Tgfβ1 (transforming growth factor beta 1), and Il4rα (Pusic et
al., 2014; Wu et al., 2014; Ji et al., 2018; Luo et al., 2018). Cd206 (mannose receptor
1, Mrc1) is present in M1 and M2a microglia (Pusic et al., 2014; Wu et al., 2014;
Ji et al., 2018; Luo et al., 2018). We noticed that peripheral nerve injury increased
the expression of Cd86, Il1β, iNos, Ptges, Ccl2, Ccl3, and Mrc-1. At the same time,
we also observed the increased expression of Tgfβ1, Il4rα, and Socs3. However, the
expression of Arg1, another M2 marker, is decreased. It appears that minocycline obviously
inhibits M1 activation (decreased expression of Cd86, Il1β, iNos, Ptges, and Mrc-1),
thus reducing production of cytokines including Il1β, NO, PGs, Ccl2, and Ccl3 in CCI
rats. On the other hand, as shown in Table 3, we found that, compared with sham rats,
the Tgfβ1 and Il4rα transcripts were upregulated >1 fold (Tgfβ1: fold = 1.16, FDR
= 2.58 E-49; Il4rα: fold = 1.03, FDR = 5.18 E-70) in the hippocampus of CCI rats.
After repeated treatment with minocycline, the Tgfβ1 and IL4rα transcripts were downregulated
by <1 fold as compared with CCI rats. Compared with sham rats, the Arg1 transcripts
were downregulated by >1 fold in the hippocampus of CCI rats. Treatment with minocycline
only slightly upregulated the transcriptional level of Arg1.
It is well known that some transcription factors have been shown to be directly or
indirectly associated with the expression of inflammation-related cytokine genes.
Compared with the sham group, the upregulated transcription factor genes were maff,
Elf4, Nr2f2, Vgl13, Lst1, Runx3, Tfec, Sp5, Nfkbiz, Hlx, Spi1 (Pu.1), Fli1, Batf3,
and Pax-1. Among them, we would like to mention that the levels of gene transcripts
of Runx3, Tfec, Sp5, Nfkbiz, Hlx, Spi1, Fli1, Batf3, and Pax-1 were largely suppressed
by minocycline. It is noteworthy that gene transcripts of Tfec, Nfkbiz, Hlx, Spil,
Fli1, and Batf3 and Pax-1 were obviously increased in the CCI group compared with
those of sham rats, and the expression of these genes returned to normal level after
minocycline administration. On the other hand, as shown in Table 3, we found that,
compared with sham rats, the Maff, Elf4, Vgl13, and Nr2f2 transcripts were upregulated
by >1 fold in the hippocampus of CCI rats. After repeated treatment with minocycline,
the Maff, Elf4, Vgl13, and Nr2f2 transcripts were slightly downregulated by <1 fold
as compared with CCI rats. In addition, compared with sham rats, the Cartpt, Six3,
Meox1, Tfap-2c, Ebf3, Mkx, and Mei4 transcripts were downregulated by >1 fold in the
hippocampus of CCI rats. Among these transcripts, the Meox1 were obviously reversed
by minocycline by >1 fold in the hippocampus of minocycline-treated CCI rats. However,
the Cartpt, Six3, Tfap-2c, Ebf3, mkx, and Mei4 transcripts were not obviously reversed
by <1 fold in minocycline-treated CCI rats. Individual genes in each category are
listed below:
only upregulated in CCI rats: Maff, Elf4, Nr2f2, Vgl13, Lst1;
only downregulated in CCI rats: Cartpt, Six3, Ap-2c, Ebf3, Mei4;
upregulated in CCI and downregulated by minocycline: Runx3, Tfec, Sp5, Nfkbiz, Hlx,
Spi1 (Pu.1), Fli1, Batf3, Pax-1; and
downregulated in CCI and upregulated by minocycline: Meox1.
Validation of Microarray Results
Many of the genes that were identified by microarray analysis should be subject to
validation by RT-PCR. As shown in Figure 4, we observed that there has been a tacit
agreement between the microarray and the PCR gene expression data in terms of changes
in both magnitude and direction. The PCR data show that CCI induced the increased
expression of cytokines (CXCL13, CXCL1, CCL2, CXCL11, CCL7, and CCL20), TLRs (TLR8
and TLR1), Iba-1 and M1 polarization markers (Cd68, iNOS, IL-1β), and transcription
factors (Runx3, Nfkbiz, and Spil). The administration of minocycline did not change
the expression of these inflammation-related genes in sham-operated rats (Figure 4).
In agreement with the microarray data, minocycline treatment obviously suppressed
the elevation in mRNA levels of these genes. Minocycline significantly diminished
the upregulated Cd68, iNOS, and Il1β. It appears that transcription factors Runx3,
Nfkbiz, and Spil may be involved in the minocycline-mediated analgesic effect and
the increased production of inflammation-related cytokines in the hippocampus of neuropathic
pain rats. Finally, we found that, between sham and minocycline-treated CCI rats,
the expression of the inflammatory-related cytokines (Cxcl13, Ccl2, Cxcl11, Ccl7,
and Ccl20), TLRs (Tlr8 and Tlr1), Iba-1 and M1 polarization markers (Cd68, iNOS, and
Il1β), and transcription factor (Nfkbiz and Spil) have no statistical significance
(Figure 4), which imply that, after minocycline treatment, the upregulated gene expression
in CCI rats has returned to normal. On the other hand, between sham and minocycline-treated
CCI rats, the expression of Cxcl1 and Runx3 was only partly suppressed by minocycline,
which implies that these two genes' expression may be only partly modulated by microglia
activity.
Figure 4
RT-PCR showing the expression of Cxcl13, Cxcl1, Ccl2, Cxcl11, Ccl7, Ccl20, Iba-1,
CD68, iNOS, IL-1β, TLR8, TLR1, Runx3, Nfkbiz, and Spil mRNA in the rat hippocampus
(n = 6). +
P < 0.05 and ++
P < 0.01, compared with both the sham and sham+Minociclyne groups; **
P < 0.01, compared with the CCI 7d group; this applies for all the genes. (A) The
expression of CXCL13, CXCL1, CCL2, CXCL11, CCL7, CCL20 in the hippocampus. (B) The
expression of Iba-1, CD68, iNOS, and IL-1β in the hippocampus. (C) The expression
of TLR8 and TLR1 in the hippocampus. (D) The expression of Runx3, Nfkbiz, and Spil
in the hippocampus.
Discussion
We reported here the hippocampal genome-wide transcriptome profiling of rats in neuropathic
pain status to elucidate minocycline-mediated analgesic effect at the molecular level.
It is well known that the CCI model of neuropathic pain displays some symptoms that
are very common in neuropathic pain patients including mechanical and thermal allodynia.
Then, we screened the hippocampus of the CCI rats for DEGs.
It has also been proved that minocycline exerts an anti-nociceptive effect in different
pain models. Recent studies revealed that the hippocampal CA1 region is more sensitive
to ischemic injury and peripheral inflammatory stimulation (Sun et al., 2016; Song
et al., 2018). In the present study, CCI operation reduced the threshold of paw withdrawal
to a mechanical stimulation. After minocycline treatment, this mechanical allodynia
was progressively reduced from 1 to 7 days, which suggests that minocycline reduced
pain hypersensitivity by modulating the microglia function within the hippocampus
at the early stage of neuropathic pain. On the other hand, it was reported that the
onset of depressive-like behavior in CCI animals was 2 weeks following peripheral
nerve injury (Xie et al., 2017; Gong et al., 2018). Moreover, minocycline treatment
suppressed hippocampal cytokine accumulation and depression-like behaviors in different
animal models of chronic pain, such as posttraumatic stress disorder-pain comorbidity
(Sun et al., 2016), visceral pain (Zhang et al., 2016), bone Cancer Pain (Dai et al.,
2019), and infant nerve injury (Gong et al., 2018). For this reason, we may propose
here that minocycline treatment might reduce the risk of nerve injury-induced depression.
More studies should be performed to detect the relationship between depression and
chronic pain and the effects of minocycline.
We observed that minocycline at 1 μg/μl induced significant analgesic effect in comparison
to CCI rats. Minocycline at doses of 2 and 5 μg/μl showed better analgesic effects
in comparison to minocycline at a dose of 1 μg/μl. Minocycline at a dose of 5 μg/μl
showed apparent elevations of the MWT in comparison with minocycline at a dose of
2 μg/μl. On the other hand, minocycline at 10 μg/μl produced a moderate antinociceptive
effect in CCI rats. Minocycline at 15 μg/μl produced a slight but significant nociceptive
effect. It seems the minimum dose of minocycline at 5 μg/μl shows the maximum analgesic
effect. Recently, several studies also showed the negative action of minocycline in
animal or cellular models for nervous system disorders. Similarly to what we observed
in CCI rats, Matsukawa et al. also support the idea that neuroprotection is dose-dependent,
in that only low doses of minocycline inhibit neuronal cell death cascades at the
acute stroke phase, whereas high doses exacerbate ischemic injury (Matsukawa et al.,
2009). A low dose of minocycline (25 mg/kg) showed protective effects, with reduced
retinal ganglion cell loss and microglial activation, while a high dose of minocycline
(100 mg/kg) showed damage effects, with more retinal ganglion cell loss and microglial
activation in mice with retinal ischemia-reperfusion injury (Huang et al., 2018).
An in vivo experiment from Li et al. also showed that intraperitoneal minocycline
treatment (45 mg/kg) may induce delayed activation of microglia in aged rats and thus
cannot prevent postoperative cognitive dysfunction (Li et al., 2018). For this reason,
although our results do not directly investigate the influence and relevant mechanism
of high doses of minocycline (10 and 15 μg/μl) on the neuronal excitability or synaptic
strength, the present study suggests the possibility that a high dose of minocycline
might regulate cell function in neuronal or non-neuronal cells within the hippocampus
of CCI rats. In our experiments, the molecular weight of minocycline hydrochloride
was 493.94. Then, 494 μg/μl corresponds to 1 M and 5 μg/μl to 104 μM, 10 μg/μl corresponds
to 2 × 104 μM and 15 μg/μl corresponds to 3 × 104 μM. Pinkernelle et al. reported
that application of 10 μM minocycline (24 h) was deleterious for spinal motor neuron
survival (Pinkernelle et al., 2013). Incubation with 50 μM minocycline (24 h) resulted
in increased cell metabolic activity in primary glial cultures. Application of 100
μM minocycline inhibited astroglia migration (24 h) and upregulated the elevated Cx43
protein expression (72 h) in rat spinal cord slices (Pinkernelle et al., 2013). A
high dose of minocycline attenuated reductions in O1- and O4-positive oligodendrocyte
progenitor cells and myelin content in hypoxia-ischemia-induced neuroinflammation
and white matter injury in rats (Carty et al., 2008). However, we also noticed that
10 and 15 μg/μl showed poor analgesic effects within 2 h after minocycline treatment.
A study on the protective effect of minocycline on ischemic stroke from Matsukawa
et al. indicated that 75 min incubation with 10 μM minocycline induced increased Bcl-2
protein expression in striatum neurons. Moreover, application of 10 or 100 μM minocycline
for 4 h displayed toxicity to both neurons and astrocytes in the striatum (Matsukawa
et al., 2009). We also noticed that minocycline (30 min, 1 h, and 2 h after its injection)
at doses of 10 and 15 μg/μl showed poor analgesic effects in comparison with minocycline
at a dose of 5 μg/μl, and the poor effect was sustained for 7 days. It seems that
the adverse effects of minocycline on neurons or non-neuronal cells may have occurred
in a short period of time. Of course, the influence of different doses of minocycline
on neurons or non-neuronal cells in the hippocampus remains to be further studied.
We found that, in the sham group vs. CCI group and minocycline-treated group vs. CCI
group, the top 2 items of KEGG pathway are cytokine-cytokine receptor interaction
and TLR pathway, which indicates that minocycline administration can regulate the
expression of genes in these two pathways, and reversing these gene expression changes
may be considered as one of the important reasons for minocycline-mediated analgesic
effect. Nerve damage leads to glial activation and thus facilitates the production
and release of pronociceptive factors such as interleukins and chemokines from glial
cells. We noticed that, after sciatic nerve injury, IL-1β was the most striking interleukin
that increased most seriously in hippocampus of CCI rats. Moreover, the increased
gene expression of CXCL13, CXCL1, CCL2, CXCL11, and CCL7 in the rat hippocampus was
observed after nerve damage. The increased chemokine expression was obviously suppressed
by intra-hippocampal injection of minocycline. It appears that minocycline was able
to reduce microglia activity efficiently, which led to the decreased expression of
these genes. In addition, the increased expression of interleukins and chemokines
should be regulated by some transcription factors. For example, the elevated expression
of IL-1β was associated with binding of transcription factor Spil/Pu.1 to IL-1β promoter
in activated inflammatory macrophage (Vanoni et al., 2017). Spil/Pu.1 can also bind
to the CCL2 promoter and stimulate its expression (Sarma et al., 2014). Runx3 knockdown
can induce the downregulation of CXCL11 in lung cancer cells (Kim et al., 2015). IκBz
can function as a transcriptional activator of CXCL1 and CCL2, which are involved
in inflammatory responses (Hildebrand et al., 2013; Brennenstuhl et al., 2015). Similarly,
we also found that, compared to sham rats, IL-1β, CXCL1, CXCL11, CCL2, and transcription
factor (Spil/Pu.1, Runx3, and IκBz) are obviously elevated at 7 days following nerve
injury. After treatment with minocycline, these interleukins and chemokines and transcription
factor were obviously decreased. It seems that the increased expression of interleukins
and chemokines may be regulated by these transcription factors in the rat hippocampus
after nerve injury.
In addition, nerve injury evoked the elevated expression of many different kinds of
TLRs (TLR8, TLR1, TLR13, TLR7, TLR2, and TLR9) in the rat hippocampus. After treatment
with minocycline, the elevated expression of these TLRs in the hippocampus was significantly
lower compared to the CCI group. More recent studies suggest that TLRs play an important
role in immune response by producing inflammatory cytokines and chemokines under pathological
conditions. For example, TLR1, TLR2, TLR7, and TLR9 activation stimulated the production
of IL-1β and MCP-1 in B cells (Agrawal and Gupta, 2011). TLR2 activation led to the
accumulation of IL-1β and chemokines (CCL7, CCL8, CCL9, CXCL1, CXCL2, CXCL4, and CXCL5)
in primary mouse microglial cells (Aravalli et al., 2005). TLR7 and TLR9 stimulation
led to the accumulation of IL-1β, CCL2, CCL3, CXCL1, CXCL9, and CXCL10 in mouse brain
(Butchi et al., 2011). It is reasonable to speculate that, in the hippocampus of CCI
rats, activation of TLR signaling in the hippocampus by peripheral nerve injury may
partially participate in the increased expression of these inflammatory cytokines
or chemokines.
Some previous studies demonstrate that IκBz can serve as a nuclear inhibitor of NF-κB
and is thought to have a key role in inflammatory responses. On the other hand, IκBz
is induced quickly in monocytes and macrophages after LPS stimulation (Yamazaki et
al., 2001). In the present experiments, the CCI-induced increased expression of IκBz
was completely impaired in minocycline-treated CCI rats, suggesting a role for microglia
activation in upregulated IκBz expression. It was reported that IκBz is obviously
induced in macrophages after TLR or IL-1R stimulation (Hanihara et al., 2013). In
chronic lymphocytic leukemia cells, TLR9 activation can lead to the increased IκBz
expression and IgM release (Fonte et al., 2017). Inhibition of TLR1/TLR2 signaling
suppressed D39-evoked IκBz expression in human monocyte (Sundaram et al., 2016). On
the other hand, promoting IκBz degradation inhibits TLR-mediated inflammation and
disorders (Hanihara-Tatsuzawa et al., 2014). Similarly, the absence of IκBz obviously
suppressed B-cell activation and proliferation after TLR activation (Kimura et al.,
2018). We also noted that TLR8, TLR1, TLR13, TLR7, TLR2, TLR9, and IκBz gene expression
robustly increased in the hippocampus, and the expression was obviously impaired in
minocycline-treated CCI rats. One possible explanation is that the upregulated IκBz
gene expression may be associated with the increased TLR expression. In addition,
microglia-mediated inflammatory reaction plays a double role in some nervous diseases
due to two distinct phenotypes, including the neurotoxic reactive phenotype (M1) and
neuroprotective M2 (Kobayashi et al., 2013; Tang and Le, 2016). In the present study,
minocycline inhibits M1 activation, thus leading to decreased expression of inflammatory
factors including IL-1β, CCL2, CCL3, and iNOS. Thus, it can be seen that dampening
of M1 polarization is another possible mechanism of minocycline-medicated analgesia.
In summary, the DEGs were identified, and many inflammation-related genes including
TLRs and chemokines were considered as important genes in the formation of neuropathic
pain through pathway analysis of microarray data, which may help us to further understand
the underlying molecular mechanisms of chronic pain. After the bioinformatics analysis
of gene expression profiles, the expression of inflammation-related genes was further
identified via the RT-PCR method. Although the results obtained from our experiments
indicate that intra-hippocampal injection of minocycline exerts an analgesic effect
and many inflammation-related genes may be involved in the formation of neuropathic
pain, the study we conducted also has certain limitations that should be considered
in future studies. In other words, further studies are required to further explore
the roles of these inflammation-related genes in the hippocampus, where it is implicated
in the formation of the neuropathic pain.
Data Availability Statement
Publicly available datasets were analyzed in this study. This data can be found here:
https://pan.baidu.com/s/1dZ4ImpqLIEqWkF3gN2llDw.
Ethics Statement
The protocol was prepared from SD rats in accordance with the National Institutes
of Health guidelines in a manner that minimized animal suffering and animal numbers.
All experiments were carried out in accordance with China animal welfare legislation
and were approved by the Zunyi Medical University Committee on Ethics in the Care
and Use of Laboratory Animals.
Author Contributions
JZ, YC, and XL: conceived and designed the experiments. LH, RX, and HT: animal experiment.
LH and TX: behavioral assessment of pain. LH, RX, YP, and SC: analyzed the data. JZ,
LH, and XL: wrote the paper.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.