Abstract

Introduction

   Prefrontal cortex (PFC) and striatal neurotransmitter homeostasis is
   affected by alcohol dependence. In this study, the microarray dataset
   from the Gene Expression Omnibus (GEO) database were downloaded. The
   prefrontal and striatum data were cross-analyzed to reveal the
   co-effects of alcohol dependence on the two brain regions of mice.

Methods

   The [46]GSE123114 microarray profile was downloaded from the GEO
   database, and differentially expressed genes (DEGs) between the two
   groups were acquired by GEO2R. KEGG analyses were performed to identify
   the pivotal pathways of these DEGs. Key differential gene expressions
   and their mechanism associated with alcohol exposure were investigated
   by an intraperitoneal alcohol model.

Results

   A total of 13 overlapping DEGs from the PFC and striatal datasets of
   the [47]GSE123114 microarray profile were identified, and they were
   significantly enriched in the morphine addiction pathway. The
   transcript levels and protein expression of Gabrb3 were consistent with
   the microarray data both in the PFC and striatum. The transcript levels
   of HMGB1, TLR4, TNFα and IL-1β were upregulated in the PFC and striatum
   of mice in the alcohol group. The HMGB1 inhibitor decreased Gabrb3
   transcript and protein levels as well as TNFα and IL-1β transcript
   levels both in the PFC and striatum in the intraperitoneal alcohol
   model mice.

Discussion

   Through the reanalysis of [48]GSE123114 microarray profile, we found
   that Gabrb3 is a key gene associated with alcohol exposure. In further
   experiments, our findings suggest that alcohol exposure modulates
   Gabrb3 expression through the HMGB1/TLR4 pathway. Moreover,
   inflammation-associated factors, such as IL-1β and TNFα, may be related
   to the HMGB1/TLR4-mediated regulation of GABRB3 expression in alcohol
   exposure.

   Keywords: alcohol exposure, prefrontal cortex, striatum, Gabrb3,
   HMGB1/TLR4 pathway

Introduction

   Alcohol dependence is a chronic recurrent brain disease, with negative
   health, economic, and social consequences for millions of adults
   worldwide.[49]^1–3 Emerging evidence suggests that alcohol dependence
   is related to dysregulation of limbic circuits, including pathways that
   connect with the prefrontal cortex (PFC) and striatum. It has been
   reported that repeated alcohol intake increases PFC dopamine release in
   mice, which plays a key role in facilitating learning associated with
   drug dependence.[50]^4 In particular, upregulation of GLT-1 and xCT
   expression in the PFC is related to attenuation of chronic and
   relapse-like alcohol drinking.[51]^1 In mammal, alcohol exposure is
   associated with reduced excitability in the prefrontal cortex.[52]^5

   Several studies suggest that dysregulation of the striatal
   neurotransmitter system is closely associated with alcohol dependence.
   For example, extracellular glutamate levels are increased[53]^6 and
   GLT-1 expression is decreased in the striatum after alcohol
   withdrawal.[54]^7 Ceftriaxone-induced upregulation of GLT-1 in the
   striatum also leads to reduced alcohol consumption in mice.
   Furthermore, upregulation of GLT-1 in the striatum induced by
   ceftriaxone also results in reduced alcohol drinking in mice. Together,
   these studies suggest that PFC and striatal neurotransmitter
   homeostasis are closely associated with alcohol dependence.

   Although several researchers have investigated the impact of alcohol
   dependence on PFC or striatal gene expression individually, the
   co-effects of alcohol dependence on these two brain regions have not
   been reported. In this study, the [55]GSE123114 microarray profile was
   downloaded from the GEO (Gene Expression Omnibus) database. The PFC and
   striatal data were cross-analyzed to identify the co-effects of alcohol
   dependence on the two brain regions of mice to provide insight into the
   molecular pathogenesis of the disorder.

Materials and Methods

Microarray Data

   The [56]GSE123114 microarray profile datasets were obtained from GEO
   database which is based on the [57]GPL1261 (Mouse430_2) platform of the
   Affymetrix Mouse Genome 430 2.0 Array that includes 12 mouse samples,
   including three inflexible drinkers PFC, three light drinkers PFC,
   three inflexible drinkers striatum and three light drinkers striatum.
   In this part of the experiment, the three-bottle free-choice method (5
   and 10% of ethanol (v/v) and water for 16 weeks) was carried out on all
   experimental male mice. On the last two weeks of the experiment, the
   bitter and aversive substance, quinine was adulterated in both ethanol
   solutions. According to their individual ethanol intake and preference
   mice were classified in three groups: “light drinkers” (preference for
   water at all experimental stages); “heavy drinkers” (ethanol preference
   decreased after adulteration of the ethanol solution with quinine), and
   “inflexible drinkers” (preference for ethanol at all experimental
   stages).[58]^8^,[59]^9

DEG Identification and Pathway Enrichment Analysis

   Differentially expressed genes (DEGs) between the two groups were
   acquired by GEO2R. A P-value <0.05 and FC=1.5 were set as the cutoff
   criteria. KEGG pathway enrichment analyses were performed to identify
   the potential pathways of the DEGs, with P<0.01 as the threshold.

Animals

   All experiments were approved by the Animal Ethics Committee of Jiamusi
   University and Chongqing Medical University and were performed in
   accordance with the National Institute of Health’s Guide for the Care
   and Use of Laboratory Animals (NIH Publication No. 80–23, revised in
   1996). Six to eight week old C57BL/6J mice (males) were obtained from
   the Department of Laboratory Animal Science of Chongqing Medical
   University. All mice, with free access to food and water, were
   maintained in a temperature (21–22°C) and humidity-controlled room
   (55±5%) under a 12/12 h light/dark cycle.

Drug Treatment

   All mice were randomly divided into control, alcohol and glycyrrhizin
   groups. Glycyrrhizic is a triterpenoid saponin which is present in
   large quantities in licorice root (Glycyrrhiza radix), and has been
   suggested to act as a direct HMGB1 antagonist. We used 0.9% saline to
   dissolve all drugs. Mice were treated with saline (control group),
   alcohol (20% v/v, 2 g/kg) or alcohol+glycyrrhizin (50
   mg/kg)[60]^10^,[61]^11 by intraperitoneal (ip) injection once per day
   for seven days.[62]^12–14 Glycyrrhizin was administered on days six and
   seven after alcohol injection. Glycyrrhizin and alcohol were
   administered as separate injections. On day eight, all mice were
   sacrificed and PFC and striatum were quickly isolated from mouse brain
   on ice and stored at −80°C.

Quantitative Real-time PCR (qRT-PCR) and Western Blot Analysis

   TRIzol was used to isolate RNA from the PFC and striatal samples
   (Thermo Fisher Scientific, Rockford, IL, USA) and the PrimeScript RT
   master mix kit was used to reverse-transcribe the total RNA from the
   PFC and striatal samples (Takara, Otsu, Japan). The qRT-PCR primer
   sequences are listed in [63]Supplementary materials Table S1. qRT-PCR
   which using SYBR Premix Ex Taq II (Takara) was performed on a
   LightCycler 96 System (Roche, Basel, Switzerland). Fold change was
   calculated using the ΔΔCt method. Transcript levels were calculated
   relative to β-actin.

   As previously described,[64]^15 the PFC and striatum stored at −80°C
   were homogenized in ice-cold RIPA lysis buffer with the presence of
   phosphatase and protease inhibitors, then centrifuged at 4°C to extract
   total protein. Primary antibodies, GABRB3 (gamma-aminobutyric acid type
   A receptor subunit beta3; 1:1000) and GAPDH (1:10,000), were used for
   Western blotting analysis (Abcam, Cambridge, MA, USA). GAPDH was used
   as a loading control and enhanced chemiluminescence was used to detect
   signals.

Statistical Analysis

   For qRT-PCR and Western blot analysis, one-way ANOVA with the LSD post
   hoc test was used to assess the differences among more than two groups;
   Student’s t-test was used to assess the differences between two groups.
   P<0.05 was considered statistically significant.

Results

Alcohol Exposure Increases Expression of Morphine Addiction-related Genes in
the PFC and Striatum

   Using the cutoff criteria described above, we extracted 229 DEGs
   between inflexible drinker mice and light drinker mice from the PFC
   datasets of the [65]GSE123114 microarray profile ([66]Figure 1C). The
   most enriched KEGG pathways related to these 229 DEGs were “morphine
   addiction” (mmu05032, P=0.0000), “amphetamine addiction” (mmu05031,
   P=0.0000) and “dopaminergic synapse” (mmu04728, P=0.0000) ([67]Figure
   1A). Using the same cutoff criteria, we extracted 32 DEGs between
   inflexible drinkers and light drinkers from the striatum datasets of
   the [68]GSE123114 microarray profile ([69]Figure 1C). The most enriched
   KEGG pathways related to the 32 DEGs were “arrhythmogenic right
   ventricular cardiomyopathy (ARVC)” (mmu05412, P=0.0032), “GABAergic
   synapse” (mmu04727, P=0.0043) and “morphine addiction” (mmu05032,
   P=0.0045) ([70]Figure 1B). The 13 overlapping DEGs from the PFC and
   striatum were subjected to KEGG enrichment analysis ([71]Figure 1C).
   Again, the most enriched KEGG pathway was “morphine addiction”
   (mmu05032, P=0.0006) ([72]Figure 1D). More data about KEGG analysis
   results were shown in [73]supplementary materials Table S2-S4.

Figure 1.

   [74]Figure 1
   [75]Open in a new tab

   Alcohol exposure promote morphine addiction-related gene expressions in
   the PFC and striatum. (A) The most enriched KEGG pathways related to
   DEGs in PFC. (B) The most enriched KEGG pathways related to DEGs in
   striatum; (C) Venn diagram of 13 overlapping DEGs from PFC and
   striatum; (D) The most enriched KEGG pathways related to the
   overlapping differentially genes from PFC and striatum.

Alcohol Exposure Increases Gabrb3 Expression in the PFC and Striatum

   According to KEGG enrichment analysis ([76]Figure 1D), two genes,
   Gabrb3 and Drd1, among the 13 overlapping DEGs were enriched in the
   morphine addiction pathway. The effect of alcohol exposure on the
   expression of Gabrb3 and Drd1 was investigated by intraperitoneal
   alcohol model. The mRNA expression of Gabrb3 was consistent with the
   microarray data, both in the PFC and striatum (Gabrb3: F1, 10=0.011,
   P=0.024; [77]Figure 2C) (Gabrb3: F1, 10=1.429, P=0.015; [78]Figure 2D).
   The transcript levels of Drd1 in the striatum were increased in the
   alcohol group mice, but no difference was detected in the PFC (Drd1:
   F1, 10=0.230, P=0.013; [79]Figure 2D) (Drd1: F1, 10=0.168, P=0.745;
   [80]Figure 2C). The protein expression of Gabrb3 were both increased in
   the PFC and striatum of the Alcohol group mice (F1, 4=1.198, P=0.015,
   [81]Figure 2A) (F1, 4=3.467, P=0.037, [82]Figure 2B). These results
   indicate that alcohol exposure regulates Gabrb3 signaling in the PFC
   and striatum.

Figure 2.

   [83]Figure 2
   [84]Open in a new tab

   Alcohol exposure promoted Gabrb3 expression in prefrontal cortex and
   striatum. (A) Effect of alcohol exposure on Gabrb3 protein levels in
   prefrontal cortex (n=3); (B) Effect of alcohol exposure on Gabrb3
   protein expression in striatum (n=3); (C) Gabrb3 and Drd1 transcript
   levels in prefrontal cortex (n=6). (D) Gabrb3 and Drd1 transcript
   levels in striatum (n=6). *P<0.05.

Alcohol Exposure Upregulates HMGB1 and TLR4 in the PFC and Striatum

   It has been reported that the HMGB1/TLR4 pathway is related to the
   alcohol-mediated regulation of GABRB3 expression in the mouse
   brain,[85]^16 and HMGB1 induces TNFα and IL-1β expression through TLR4
   activation in alcohol dependence.[86]^17 To analyze the mechanisms by
   which alcohol exposure regulates Gabrb3 expression in the PFC and
   striatum, we examined HMGB1/TLR4 signaling, which is closely associated
   with alcohol exposure. The transcript levels of HMGB1 and TLR4 were
   both upregulated in the PFC and striatum of the alcohol group mice
   (HMGB1: F1, 10=11.673, P=0.017; TLR4: F1, 10=0.779, P=0.030; [87]Figure
   3A) (HMGB1: F1, 10=6.003, P=0.009; TLR4: F1, 10=1.857, P=0.004;
   [88]Figure 3B). Also, the expression levels of inflammatory
   response-related factors, IL-1β and TNFα, were significantly increased
   in the PFC and striatum of mice in the alcohol group [TNFα: (F2,
   15=7.465, P=0.006); control vs alcohol, P=0.005; IL-1β: (F2, 15=7.399,
   P=0.006); control vs alcohol, P=0.011; [89]Figure 4C] [TNFα: (F2,
   15=7.519, P=0.005); control vs alcohol, P=0.005; IL-1β: (F2, 15=4.492,
   P=0.030); control vs alcohol, P=0.017; [90]Figure 4D]. These results
   indicate that alcohol exposure regulates HMGB1/TLR4 signaling in the
   PFC and striatum.

Figure 3.

   [91]Figure 3
   [92]Open in a new tab

   Alcohol exposure promoted HMGB1 and TLR4 expression in prefrontal
   cortex and striatum. (A) Effect of alcohol exposure on HMGB1 and TLR4
   transcript levels in prefrontal cortex (n=6); (B) Effect of alcohol
   exposure on HMGB1 and TLR4 transcript levels in striatum (n=6).
   *P<0.05; **P<0.01;.

Figure 4.

   [93]Figure 4
   [94]Open in a new tab

   HMGB1 inhibitor decreased inflammatory response and Gabrb3 expression
   in PFC and striatum of alcohol exposure mice. (A) Effect of HMGB1
   inhibitor on Gabrb3 protein levels in PFC (n=3); (B) Effect of HMGB1
   inhibitor on Gabrb3 protein expression in striatum (n=3); (C) Effect of
   HMGB1 inhibitor on Gabrb3, TNFα and IL-1β transcript levels in PFC
   (n=6). (D) Effect of HMGB1 inhibitor on Gabrb3, TNFα and IL-1β
   transcript levels in striatum (n=6). *P<0.05; **P<0.01.

HMGB1 Inhibitor Decreases the Inflammatory Response and Gabrb3 Expression in
the PFC and Striatum of Alcohol Exposure Mice

   In order to further analyze the regulation mechanism of GABRB3
   expression mediated by alcohol exposure, the effect of HMGB1 inhibitor
   on GABRB3 expression was detected by qRT-PCR and Western blotting. The
   HMGB1 inhibitor decreased Gabrb3 protein expression and transcript
   levels both in the PFC and striatum of alcohol exposure mice [Gabrb3:
   (F2, 6=7.747, P=0.022); alcohol vs glycyrrhizin, P=0.011; [95]Figure
   4A] [Gabrb3: (F2, 15=5.982, P=0.012); alcohol vs glycyrrhizin, P=0.005;
   [96]Figure 4C] [Gabrb3: (F2, 6=5.226, P=0.048); alcohol vs
   glycyrrhizin, P=0.026; [97]Figure 4B] [Gabrb3: (F2, 15=3.731, P=0.048);
   alcohol vs glycyrrhizin, P=0.045; [98]Figure 4D]. Also, the expression
   of inflammatory response-related factors, IL-1β and TNFα, was
   significantly inhibited by HMGB1 inhibitor in the PFC and striatum of
   the alcohol group mice [TNFα: (F2, 15=7.465, P=0.006); alcohol vs
   glycyrrhizin, P=0.004; IL-1β: (F2, 15=7.399, P=0.006); alcohol vs
   glycyrrhizin, P=0.002; [99]Figure 4C] [TNFα: (F2, 15=7.518, P=0.005);
   alcohol vs glycyrrhizin, P=0.004; IL-1β: (F2, 15=7.337, P=0.006);
   alcohol vs glycyrrhizin P=0.024; [100]Figure 4D]. These results
   indicate that HMGB1/TLR4 signaling may be related to the regulation of
   Gabrb3 expression by alcohol exposure in the PFC and striatum.

Discussion

   By analyzing [101]GSE123114 microarray profile, we found that the
   morphine addiction pathway was significantly different in both the PFC
   and striatum. The chronic use of morphine is characterized by adaptive
   changes in neurons and neuronal communication. Morphine can cause
   indirect excitation of dopamine neurons by reducing inhibitory synaptic
   transmission mediated by GABAergic neurons. Differences in the morphine
   addiction pathway suggested that there are similar pathological
   processes and corresponding gene expression changes in
   alcohol-dependent mice. Furthermore, we identified Gabrb3 of morphine
   addiction pathway as a key gene. GABRB3 is a gamma-aminobutyric acid
   receptor, a major inhibitory neurotransmitter in human central nervous
   system.[102]^18 As a GABA-gated chloride ion channel,[103]^19 it plays
   an important role in the formation of functional inhibitory GABAergic
   synapses and participates in synaptic inhibition.[104]^19 Also, it
   participates in mediating the cellular response to histamine,[105]^20
   somatosensation, and the production of antinociception.[106]^21

   Alleles of GABRB3 in Caucasian (non-Hispanic) subjects have been
   associated with alcohol dependence.[107]^22 Alcohol dependence patients
   with the GABRB3 G1+ allele reported significantly lower drinking
   refusal self-efficacy in situations of stronger mood-related alcohol
   expectancy (AE) and social pressure relating to negative affective
   change.[108]^23 In Korea, the GABRB3 G1− alleles are more commonly in
   children of alcoholics (COAs) than in non-COAs.[109]^24 In African
   Americans, CpGs in GABRB3 are hypermethylated in alcohol dependence
   patients compared with controls.[110]^25 Together, these studies
   indicate that GABRB3 is closely related to alcohol dependence. In in
   vitro experiments, alcohol increases the expression of the GABRB3 gene
   in human embryonic stem cells.[111]^26 In in vivo experiments, adult
   male offspring born to Sprague Dawley dams that received ethanol had
   elevated expression of the Gabrb3 gene in the hippocampus.[112]^27
   These studies provide further evidence that GABRB3 is related to the
   regulation of brain function by alcohol. In this research, functional
   analysis of DEGs from [113]GSE123114 microarray profile revealed that
   Gabrb3 is the key gene of alcohol dependence in both the PFC and
   striatum. In the PFC and striatum of alcohol exposure mice, GABRB3
   expression was significantly increased at both the transcript and
   protein levels, consistent with the microarray profile. Together, all
   these findings suggest that Gabrb3 is a critical novel gene in alcohol
   exposure.

   It has been reported that the expression of Gabrb3 is elevated in the
   male offspring frontal cortex of ethanol-fed wild-type dams, but is
   unaffected in the offspring of ethanol-fed TLR4(-/-) dams.[114]^16
   These results indicate that the TLR4 pathway is related to the
   alcohol-mediated regulation of GABRB3 expression in the mouse brain.

   Toll-like receptors are the most well characterized pattern recognition
   receptors that recognize PAMPs and DAMPs, including HMGB1 and heat
   shock proteins.[115]^28 TLR4 has been identified as a critical receptor
   related to ethanol-induced neuropathology.[116]^28^,[117]^29 HMGB1, a
   nuclear chromatin binding protein, is a endogenous TLR4 agonist which
   has been identified as a key immune mediator in alcohol
   dependence.[118]^28 Extracellular HMGB1 released as an innate immune
   mediator activates innate immune responses through direct binding to
   TLR4 or RAGE.[119]^30 It has been reported that the expression of TLR4
   and HMGB1 is increased both in chronic ethanol-administered mice and
   the human postmortem alcoholic brain.[120]^31 In this experiment, the
   expression levels of HMGB1 and TLR4 were significantly increased in the
   PFC and striatum of mice in the alcohol group. These results are
   consistent with previous research indicating increased HMGB1/TLR4
   signaling in the alcoholic brain. To examine whether changes in
   HMGB1/TLR4 signaling are associated with increased Gabrb3 gene
   expression in alcoholic brain, we assessed the effects of an HMGB1
   inhibitor on Gabrb3 expression in alcohol exposure mice. The HMGB1
   inhibitor decreased Gabrb3 expression in both the PFC and striatum of
   these mice. Together, these results reveal that alcohol exposure
   modulates Gabrb3 expression through the HMGB1/TLR4 pathway.

   Previous studies have reported that HMGB1 induces TNFα and IL-1β
   expression through TLR4 activation in alcohol dependence,[121]^17 and
   that inflammation inhibits GABA transmission in CNS diseases.[122]^32
   In this study, the HMGB1 inhibitor decreased TNFα and IL-1β expression
   both in the PFC and striatum of alcohol exposure mice, consistent with
   previous studies. These results indicate that HMGB1 may regulate Gabrb3
   expression through the inflammatory response pathway. It has been
   reported that activation of Akt is involved in GABA(A) receptor
   anchoring in neurons,[123]^33 and that Akt1 deficiency modulates
   GABAergic functions and reduces GABAAR subunit expression in the
   hippocampus.[124]^34 In glioma cells, TNFα-induced oxidative
   stress-dependent Akt signaling affects actin cytoskeletal
   organization,[125]^35 and in the CNS, IL-1β enhances the expression of
   GABAA receptor in cell surface by the Akt pathway.[126]^36
   Collectively, these findings suggest that inflammation-related factors
   in the PFC and striatum of alcohol-dependent mice, such as TNFα and
   IL-1β, may regulate Gabrb3 expression through the Akt pathway. However,
   further study is needed to test this hypothesis.

Conclusion

   Our findings suggest that Gabrb3 is the key gene associated with
   alcohol exposure in mice, and reveal that alcohol exposure modulates
   Gabrb3 expression through the HMGB1/TLR4 pathway. Moreover,
   inflammation-related factors, such as IL-1β and TNFα, may be related to
   the regulation of GABRB3 expression by the HMGB1/TLR4 pathway in
   alcohol exposure.

Funding Statement

   This work was supported by Doctoral Initiation Project of Jiamusi
   University (grant numbers: JMSUBZ2019-06); talent training project for
   basic scientific research of Heilongjiang Province Educational
   Commission of China (grant numbers: 2019-KYYWF-1357); The National Key
   R&D Program of China (grant numbers: 2018YFC1314404); National Natural
   Science Foundation of China (grant numbers: 81901398). Natural Science
   Foundation of Chongqing, China (grant numbers: cstc2019jcyj-msxmX0025).

Author Contributions

   All authors made substantial contributions to conception and design,
   acquisition of data, or analysis and interpretation of data; took part
   in drafting the article or revising it critically for important
   intellectual content; agreed to submit to the current journal; gave
   final approval of the version to be published; and agree to be
   accountable for all aspects of the work.

Disclosure

   The authors report no conflicts of interest in this work.

References