Effects of Long-Term Administration of Q808 on Hippocampal Transcriptome in Healthy Rats

Abstract

Epilepsy treatment with antiepileptic drugs (AEDs) is usually requires for many years. Q808 is an innovative antiepileptic chemical. It exerts effective antiepileptic effect against various epilepsy models. Exploring the gene transcriptomic profile of long-term treatment of Q808 is necessary. In the present study, hippocampus RNA-sequencing was performed to reveal the transcriptome profile of rats before and after treatment of Q808 for 28 d. Results confirmed 51 differentially expressed genes (DEGs) between Q808 and healthy control groups. Gene cluster analysis showed that most upregulated DEGs linked to response to drug and nucleus, most downregulated DEGs linked to locomotory, neuronal cell body, and drug binding. Most of DEGs were enriched in the signaling transduction, substance dependence, nervous system, and neurodegenerative disease pathways. Furthermore, quantitative real-time PCR analysis confirmed that Q808 significantly increased the expression of neuroprotective genes, such as Mdk, and decreased the mRNA levels of Penk, Drd1, and Adora2a, which are highly expressed in epilepsy models. In addition, Q808 decreased the mRNA expression of Pde10A and Drd2, which are known to be closely associated with schizophrenia. Our study may provide a theoretical basis to explore the effect of Q808 on the susceptibility to epilepsy and other neurological diseases.

Introduction

Epilepsy is one of the most common chronic neurological disorders, and approximately 70 million epileptics have been reported worldwide.¹⁾ Antiepileptic drugs (AEDs) are the main treatment modality for most people with epilepsy. Treatment of epilepsy with AEDs usually need to be maintained for a long time.²⁾ Thus, considerable research has focused on exploring the long-term safety of AEDs.³⁾

Treatment with AEDs is frequently associated with side effects.^4–6) Given that the main acting region of AEDs is the brain, the effects of AEDs on neurological disorders and normal brain functions need to be critically evaluated. A research has reported that 40% of psychoses were thought to be triggered by AEDs.⁷⁾ Many AEDs, such as Phenobarbital, Tiagabine, Zonisamide and Levetiracetam, have been associated with development or worsening of depression.⁸⁾ Valproic acid (VPA) can induce parkinsonism even at moderate doses and years into treatment.⁹⁾ AEDs can exert detrimental effects on cognitive function, such as reductions in psychomotor speed, attention, and memory.¹⁰⁾ For example, treatment with Phenobarbital usually induced memory impairment in adults and cognitive dulling in children¹¹⁾; and Topiramate can induce significant cognitive slowing, verbal fluency, and word-finding difficulties.¹²⁾ Thus, evaluating the long-term effect of AEDs on the brain is necessary because it may provide information for avoiding or minimizing long-term adverse effects of AEDs and evaluating the effects of AEDs on the susceptibility to neurological diseases.

Q808 is an innovative chemical with international patent, and it is under research and development. Q808 has been tested to be effective against varies epilepsy models, such as the seizure induced by pentylenetetrazole (PTZ), isoniazid, thiosemicarbazide, and 3-mercaptopropionic acid.^13–15) Thus, Q808 has become a potential AED candidate and is now under clinical research. Our previous study has shown that a single dose of Q808 evidently increased the content of γ-aminobutyric acid (GABA), a predominant inhibitory neurotransmitter, in the rat hippocampal region and enhanced the frequency of spontaneous inhibitory postsynaptic currents in the hippocampus.¹⁵⁾ In addition, we have employed RNA-sequencing (RNA-seq) analysis and observed that Q808 may affect the GABA level by increasing the gene and protein expressions of angiotensin I-converting enzyme (Ace) in the hippocampus of PTZ-kindled rats.¹⁶⁾ Hippocampus, which belongs to the limbic system, plays important roles in epilepsy and some other neurological diseases.¹⁷⁾ Abnormal physical and chemical changes in the hippocampus can cause epileptic discharges that spread to other brain regions to induce a seizure.¹⁸⁾ Thus, hippocampus should be the key region affected by long-term treatment with Q808.

RNA-seq is increasingly used to investigate the gene expression profiling through RNA fragmentation, capture, sequencing, and computational analysis.^19,20) By employing the RNA-seq method, the specific chemokines which could involve in VPA resistance processes have been identified.²¹⁾ We have utilized RNA-seq to explore the transcriptome file changed of chronic epilepsy model rats before and after treatment of Q808.¹⁶⁾ However, the RNA-seq profile of long-term treatment of Q808 on healthy rat has not been clarified.

In the present study, the mRNA profile in the hippocampus of long-term treatment of Q808 on healthy rats was analyzed. Quantitative real-time PCR (qRT-PCR) was used to validate the differentially expressed genes (DEGs) related to epilepsy. Our study will lay a theoretical basis to further explore the effect of Q808 on the susceptibility to epilepsy and other central nervous system diseases.

Results

Comparative Transcriptome Profiling

Overall, a total of 125 genes were differentially expressed between Q808 and control groups at a nominal p value <0.05 (Supplementary Table S1). Of these DEGs, 51 had a |Log 2 fold change|≥1 and comprised of 15 DEGs being upregulated and 36 DEGs being downregulated. The upregulated and downregulated genes are listed in Tables 1, 2, respectively. The raw sequencing data can be found at GEO: GSE202301 and GSE189785.

Table 1. 15 Upregulated DEGs

GENE	Definition	Log 2 FC	p-Value
Bad	BCL2-Associated agonist of cell death	2.4139	0.0007
Camk2n2	Calcium/calmodulin-dependent protein kinase II inhibitor 2	1.4156	<0.0001
Cox8b	Cytochrome c oxidase, subunit VIIIb	1.8994	0.0415
Gfy	Golgi-associated, olfactory signaling regulator	3.9067	0.0025
Hnrnpab	Heterogeneous nuclear ribonucleoprotein A/B	3.6302	0.0001
LOC100363289	LRRGT00022-like	4.3928	0.0003
LOC100911417	ATP synthase subunit b, mitochondrial-like	3.5021	0.0019
LOC103689986	Protein YIF1B	3.0893	0.0468
LOC310926	Hypothetical protein LOC310926	2.6987	0.0458
Mdk	Midkine	1.3498	0.0447
Msx1	Msh homeobox 1	1.7869	0.0266
RGD1560394	RGD1560394	1.2414	0.0056
Slc25a22	Solute carrier family 25 member 22	3.8050	0.0081
Slc3a1	Solute carrier family 3 member 1	4.1793	0.0293
Ttyh3	Tweety family member 3	1.0436	0.0007

Table 2. 36 Downregulated DEGs

GENE	Definition	Log 2 FC	p-Value
Adcy5	Adenylate cyclase 5	−1.9764	0.0203
Adora2a	Adenosine A2a receptor	−6.7452	0.0159
Ano3	Anoctamin 3	−1.0131	0.0495
Arpp19	cAMP-regulated phosphoprotein 19	−1.2905	0.0183
Asic4	Acid sensing ion channel 4	−2.4998	0.0303
Dgkb	Diacylglycerol kinase, beta	−1.0635	0.0425
Dmkn	Dermokine	−4.8626	0.0407
Drd1	Dopamine receptor D1	−4.0047	0.0017
Drd2	Dopamine receptor D2	−5.0493	0.0009
Ermn	Ermin	−1.0011	0.0233
Gnal	G protein subunit alpha L	−1.7141	0.0177
Gng7	G protein subunit gamma 7	−1.7469	0.0070
Gpr6	G protein-coupled receptor 6	−4.0635	0.0088
Gpr88	G-protein coupled receptor 88	−4.4302	0.0102
LOC100360977	TRIMCyp-like	−1.0038	0.0013
LOC100365810	40S ribosomal protein S17-like	−1.0115	0.0085
LOC100910446	Syntaxin-7-like	−4.5377	0.0146
LOC100911238	Proteasome maturation protein-like	−2.0472	0.0010
LOC100912599	NADH dehydrogenase [ubiquinone] iron-sulfur protein 6, mitochondrial-like	−3.7250	0.0030
Nexn	Nexilin (F actin binding protein)	−3.4624	0.0449
Pcp4l1	Purkinje cell protein 4-like 1	−2.0235	0.0205
Pde10a	Phosphodiesterase 10A	−2.8697	0.0030
Pde1b	Phosphodiesterase 1B	−1.8876	0.0092
Pdp1	Pyruvate dehyrogenase phosphatase catalytic subunit 1	−1.6212	0.0243
Penk	Proenkephalin	−3.8989	0.0081
Ppp1r1b	Protein phosphatase 1, regulatory (inhibitor) subunit 1B	−2.8081	0.0232
Ptpn5	Protein tyrosine phosphatase, non-receptor type 5	−1.2252	0.0467
Rarb	Retinoic acid receptor, beta	−3.1196	0.0354
Rasgrp2	RAS guanyl releasing protein 2	−2.2175	0.0424
RGD1562037	Similar to OTTHUMP00000046255	−1.8332	0.0401
Rgs9	Regulator of G-protein signaling 9	−4.3077	0.0072
Scn4b	Sodium voltage-gated channel beta subunit 4	−3.2826	0.0154
Strn	Striatin	−1.4249	0.0355
Sv2c	Synaptic vesicle glycoprotein 2c	−2.1913	0.0474
Syndig1l	Synapse differentiation inducing 1-like	−3.9934	0.0009
Tac1	Tachykinin, precursor 1	−3.2375	0.0201

Gene Oncology (GO) Analysis

In the GO category BP, the upregulated DEGs were enriched in the terms ‘response to drug’; in the category CC, the upregulated DEGs were significantly enriched in ‘nucleus,’ ‘cytoplasm,’ and ‘integral component of membrane’; none of upregulated DEGs were enriched in the GO category MF (Fig. 1A, Supplementary Table S2). On the other hand, in the category BP, the downregulated DEGs were accumulated in the terms ‘locomotory behavior,’ ‘response to amphetamine,’ and ‘response to morphine’; in the category CC, the downregulated DEGs were highly enriched in ‘neuronal cell body,’ ‘heterotrimeric G-protein complex,’ and ‘axon’; and in the category MF, they enriched in the GO terms ‘drug binding,’ ‘protein-containing complex binding,’ and ‘signaling receptor binding’ (Fig. 1B, Supplementary Table S3).

Fig. 1. GO Enrichment Analysis for 51 DEGs

(A) Terms enriched by the upregulated genes. (B) Terms enriched by the downregulated genes

Pathway Analysis

The 51 DEGs were enriched in 26 pathways (p < 0.05), of which the signaling transduction, substance dependence, nervous system, and neurodegenerative disease were the most represented (Fig. 2, Supplementary Table S4). DEGs including Adcy5, Adora2a, Bad, Dgkb, Drd1, Drd2, Gnal, Gng7, Pde1b, Ppp1r1b, Ptpn5, and Rasgrp2 were involved in the signaling transduction.

Fig. 2. Pathway Enriched by the 51 DEGs

PPI Network Analysis

By using STRING and Cytoscape, 190 interactions among 51 DEGs have been revealed (Fig. 3). A literature review in PubMed confirmed that the function of Adora2a, Bad, Drd1, Drd2, Ermn, Gng7, Mdk, Pde10a, Penk, and Slc25a22 are associated with epilepsy^22–31) (Table 3).

Fig. 3. PPI Networks of DEGs. Nodes with a Degree of Connectivity of 1–5, 5–10, 10–20, and 20–30 Were Indicated in Blue, Yellow, Pink, and Green, Respectively

Table 3. Functions of DEGs in Epilepsy

Gene	Function	References
Adora2a	D-Limonene exhibits anticonvulsant activity through modulation of adenosine A2A receptors.	22)
Bad	Knocking out the Bad gene provides seizure resistance in a genetic model of chronic epilepsy.	23)
Drd1	Dopamine D1 agonist SKF-38393 enhances susceptibility to seizures.	24)
Drd2	Dopamine D2 receptor agonists suppress pilocarpine and kindled seizures.	25)
Ermn	Human Ermn is a novel cytoskeleton-related oligodendroglial protein in human epileptogenesis.	26)
Gng7	A progressive seizure disorder has been displayed in Gng3(−/−)Gng7(−/−) double knock-out mice.	27)
Mdk	Mdk functions as an anticonvulsant and neuroprotective agent in hippocampus during kainic acid (KA)-induced seizures.	28)
Pde10a	The PDE10A Inhibitor PF-2545920 enhances seizure activity.	29)
Penk	The up-regulated Penk gene that is induced by KA has proconvulsant properties	30)
Slc25a22	Mutations in Slc25a22 cause neonatal epileptic encephalopathy and migrating partial seizures in infancy.	31)

qRT-PCR Validation of DEGs

In order to validate the accuracy of RNA-seq results and evaluate the influence of Q808 on healthy rats in the aspect of epilepsy susceptibility, the mRNA expression of DEGs in Table 4, which are known to be closely associated with epilepsy, were validated by qRT-PCR. After Q808 treatment, the mRNA expression levels of Bad, Mdk, and Slc25a22 were significantly upregulated; the gene expression of Adora2a, Drd1, Drd2, Ermn, Gng7, Pde10A and Penk were remarkably downregulated (Fig. 4).

Table 4. Sequences of the Primers

Genes	Forward (5′→3′)	Reverse (5′→3′)
Adora2a	CCC AGG GAC ATT TCC TCC AG	TGC AGG CGA CTT CGA AAC TA
Bad	CTT GAG GAA GTC CGA TCC CG	GCT CAC TCG GCT CAA ACT CT
Drd1	GCA TGG CTT GGA TTG CTA CG	AGG AGA AAT CCC TCT CCG CT
Drd2	TAC GTG CCC TTC ATC GTC AC	CCT CAG GGT GGG TAC AGT TG
Ermn	GGT CCC AGC TTT ATG CTT GC	GGC TCA CCA GTC ATC TAG GC
Gng7	AAA CCC TAG TTT GCC GGG G	GGG CGA CGT TGT TAG TAC CT
Mdk	GCA GCA CCG AAG TTT CTT CC	CAG GTC CAC TCC GAA CAC TC
Pde10a	ACA GGG GCT TCA GTA ACA GC	CCC AAA GTA CAG TGC GAG GT
Penk	ACT CCC CCG TGG AAG ATA GG	CAG GAC CAG CAG GGA CAA TC
Slc25a22	GCC GCA TTG GCT GCG TG	CTC AAG TGA CTG AAG CCG AGT

Fig. 4. Validation of DEGs According to the Fold-Changes of qRT-PCR (2^−ΔΔCt)

* p < 0.05, ** p < 0.01, and *** p < 0.001. Data are expressed as mean ± S.E.M. and analyzed using unpaired t-test (n = 3).

Discussion

This work underscores the DEGs after long-term treatment of Q808 on healthy rats. A total of 51 DEGs between Q808 and control groups were initially identified. Among them, 16 were upregulated and 35 were downregulated. Upon reviewing the published literature, a total of 10 out of 51 DEGs were reported to have been associated with epilepsy, and their expression were further confirmed by qRT-PCR. The 10 genes may suggest the effect of long-term use of Q808 on the susceptibility to epilepsy.

Penk mRNA expression in hippocampus has been found to significantly increase in several seizure models.^32,33) Penk also has proconvulsant properties, and it contributes to the development of spontaneous seizures.³⁰⁾Penk is regulated by c-Jun N-terminal kinase (JNK) signaling pathway via c-Jun and c-Fos, and it is related to the activator protein 1 (AP-1) transcriptional activity given that the promoter of Penk contains AP-1 recognition sequences.^34,35) Thus, in the seizure models, an increase in Penk mRNA is often accompanied by the activation of JNK signaling pathway.³⁰⁾ Ketogenic diet (KD), which is an effective therapy for controlling refractory epilepsy, decreases the level of Penk mRNA in the KA-induced mouse hippocampus, and Noh stated that decreased Penk mRNA may be one of the mechanisms involved in the antiepileptic and neuroprotective actions of KD.³⁶⁾ The present study revealed that Q808 treatment decreased the mRNA level of Penk, which indicated that Q808 may influence the JNK signaling pathway and has a protective effect against drug-resistant epilepsy. However, this deduction needs to be further confirmed by animal research.

Previous reports have revealed that Mdk have neurotrophic and neuroprotective properties in the central nervous system (CNS). In the seizure model induced by KA, Mdk showed an anticonvulsant effect and inhibited the neuronal cell death in the hippocampus.²⁸⁾ Q808 can increase the Mdk mRNA levels, which suggested that Q808 could exert neuroprotective effect mediated by Mdk.

Drd1 and Drd2 are the mRNA of dopamine receptors of D1 and D2, respectively. The present study revealed that the mRNA expression of Drd1 and Drd2 were both lower in the hippocampus after long-term Q808 treatment, which may indicate that Q808 could work as dopamine receptor antagonists to some extent. The two receptors mediated the opposing influences on neuronal excitability. D1 agonists could lower the seizure threshold and has proconvulsant properties.³⁷⁾ D2 agonists have anticonvulsant and D2 antagonists are proconvulsant.^24,38) However, the effect of the Drd2 on the seizure activity was inconsistent among different seizure models. Uridine, an endogenous neuromodulator, produced an antiepileptic effect against the status epilepticus model induced by lithium-pilocarpine (PC), and this action is related to the decreased expression of Drd2.³⁹⁾ Given that Q808 also decreased the expression of Drd2, Q808 may be effective in controlling the seizure induced by PC. In addition, Q808 may affect schizophrenia, a neurological disorders which can be controlled with D2 antagonists.⁴⁰⁾ Therefore, future research could focus on the effect of Q808 on schizophrenia. Another reason that Q808 might influence schizophrenia is that Q808 decreased the mRNA expression of Pde10A. Several PDE10A inhibitors have been used for schizophrenia treatment in animal or preclinical study.^41,42) However, some PDE10A inhibitors enhanced the hyperexcitability of neurons in the hippocampus of rats and facilitated seizure activity.^29,43) Recent clinical research has reported that Pde10A expression significantly increased in the cortex of patient with epilepsy.²⁹⁾ Thus, Zhang et al. stated that upregulation of Pde10A in epilepsy patients might decrease seizure.²⁹⁾ Thus, Q808 might increase the seizure susceptibility from Pde10A genes aspect. However, the effect of Q808 on the two genes, Drd2 and Pde10A, may lay a basis for the new clinical use of Q808 on schizophrenia.

Bad, a proapoptotic member of the family of Bcl-2 death regulators, often increased in epilepsy and contributes to neuro death.⁴⁴⁾ Apart from proapoptotic functions, Ziviani and Scorrano claimed that Bad in epilepsy is not really bad. Bad controls the fuel utilization by neuronal mitochondria, and fuel metabolism can profoundly influence neuronal excitability.⁴⁵⁾ The knockout of Bad in mice changed the brain cell fuel metabolism and reduced epileptiform activity, which is mediated by ATP-sensitive potassium channels.⁴⁶⁾ Therefore, Bad has seizure protection function. Our RNA-seq result showed that Bad mRNA expression was increased five-fold after Q808 treatment. Therefore, further study could use terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay to evaluate the effect of Q808 on neuronal apoptosis. At the same time, exploring the effect of Q808 on brain metabolism will also be an attractive project.

When adenosine binds to adenosine A2a receptor (Adora2a), the release of glutamate increases, which promotes the occurrence of epilepsy.⁴⁷⁾ However, the opposite function occurs when adenosine A1a receptor (Adora1a) is activated.⁴⁸⁾ Furthermore, exogenous adenosine can inhibit the mossy fiber sprouting by reversing the DNA methylation process in the hippocampus, and it can control the occurrence of epilepsy.⁴⁹⁾ Thus, increased Adora1a expression and decreased Adora2a expression not only can control the release of excitatory neurotransmitter glutamate but also can inhibit the mossy fiber sprouting. Q808 treatment significantly decreased the Adora2a mRNA levels 10 times the healthy control, and it had no obvious effect on Adora1a. This feature could be one of the advantages of Q808.

Ermn, expressed exclusively in oligodendrocytes, is a novel cytoskeletal molecule and bind with F-actin. Evidence suggests the expression of Ermn in epileptic patients brain specimens is much lower than that of healthy control, and overexpression of Ermn promotes formation of myelin.²⁶⁾ Our result showed that Q808 treatment decreased the mRNA expression of Ermn, which suggested that Q808 may affect oligodendrocyte maturation and myelination stages. Thus, the effect of Q808 on oligodendrocytes mediated by Ermn should be further confirmed.

G-protein γ3 and γ7 subunits play an important role in different receptor signaling pathways.^50,51)Gng3^−/−Gng7^−/− double knock-out mice showed a progressive seizure disorder, which is due to that γ3 subunit is responsible for GABA-regulated neuronal excitability and γ7 subunit is required for A2A adenosine or D1 dopamine-induced protective response.²⁷⁾ Even though Q808 decreased the Gng7 mRNA expression, it cannot achieve the same effect as Gng7 knockout.

Our recent study reported the alterations in the hippocampal transcriptome of the PTZ-kindled seizure model before and after the treatment with Q808.¹⁶⁾ A total of 23 hub genes were identified among the control, PTZ, and Q808 groups. Compared with the results of this study, the differences in the transcriptome between normal and seizure rats after Q808 treatment were observed. Genes, such as Ace, Alas, Enpp2, Faap100, and Sox11, showed the same trend in the two studies. However, a difference in the effect of Q808 was observed on the expressions of Col18a1, Doc2a, Fbl, Hbb, Lmo4, Man2b2, Mvd, and Slc25a22 between normal and seizure rats. Among these eight genes, the first seven genes exhibit no evident differences from those of this research. Only Slc25a22, which is downregulated by Q808 in seizure rats, was significantly upregulated by Q808 in normal rats. This opposite effect may be closely related to the multiple functions of Slc25a22. It is mainly expressed in astrocytes and involved in the import of glutamate.⁵²⁾ Thus, Slc25a22 is important in maintaining astrocytes glutamate homeostasis.⁵³⁾ The inhibition of Slc25a22 leads to intracellular glutamate accumulation in astrocytes.⁵⁴⁾ Thus, the low expression of Slc25a22 in the hippocampus of Q808-treated PTZ-kindled rats may inhibit the release of glutamate thus controlling the seizure. In addition to the effect on glutamate release, Slc25a22 influences the energy metabolism of neural cells by increasing the reduced nicotinamide adenine dinucleotide phosphate (NADPH) production.⁵⁴⁾ A sufficient energy supply of astrocytes is essential for proper brain activities, such as interaction with neuron cells, modulation of neurotransmitter-receptor activity, and conservation of redox status.⁵⁵⁾ Thus, for normal rats, the effect of Q808 can exert a positive effect on energy homeostasis by increasing the expression of Slc25a22.

The 10 DEGs we have discussed were all associated with epilepsy. However, some DEGs are related to other neurological diseases. For example, Sv2c mRNA levels are increased in Parkinson’s disease (PD) models;⁵⁶⁾Slc3a1 has already been associated with the hypotonia–cystinuria syndrome, which is a neurological syndrome;⁵⁷⁾Acis4 plays an important role in Alzheimer’s disease and PD.⁵⁸⁾ These information could aid in exploring the new function of Q808 or the adverse effect of Q808 on other neurological disease.

Compared with traditional AEDs, Q808 may have certain advantages. We previously reported that Q808 has a potential to increase the GABA level and exerts its antiepileptic activity against various epilepsy models. Traditional AEDs, such as VPA, also show pharmacological actions on GABA-mediated inhibitory neurotransmission.⁵⁹⁾ Zhang’s laboratory explored the DEGs between VPA-responsive and non-responsive patients.²¹⁾ Notably, a quarter of these DEGs, such as Fn1, Pdk4, Ptges, Ifi27, and Rrm2 were upregulated in the VPA-resistant group but downregulated in our RNA-seq results on the Q808 treatment group. Thus, Q808 may enhance the effect of VPA by regulating the resistant-related gene expression. Further studies should be performed to evaluate the effect of the combined treatment of Q808 and VPA against seizure models.

Conclusion

The present study revealed that gene changed after long-term treatment of the novel anti-epileptic chemical Q808. We discussed the effect of long-term use of Q808 on the susceptibility to epilepsy and other neurological diseases. Our results are important for the future clinical application of Q808, and they also provide a basis for exploring the new functions and potential protective and adverse effect of Q808.

Experimental

Chemicals

The Q808 compound was a gift from professor Li’s lab, and its synthesis conformation has been verified.⁶⁰⁾ Reagents used in the study were purchased from Sigma-Aldrich chemical company (St. Louis, United States). Prime ScriptTM RT Master Mix for first-strand cDNA synthesis and SYBR Green Master for qRT-PCR were purchased from TaKaRa biological engineering company (Da Lian, China) and Roche (Foster City, United States), respectively.

Animals and Treatment

Adult male wistar rats aged 6 to 7 weeks and weighing 180–220 g were obtained from Changchun Yisi Experimental Animal Technology Co., Ltd. (Changchun, China). Animals were housed three per cage under specific pathogen-free conditions with temperature of 22–25 °C, 12 h light/dark cycle, and free access to water and food. One week later, the rats were randomly divided into the Q808 group (Q808) and vehicle control group (control). In the Q808 group, 30 mg/kg Q808 was administered to the rats via oral gavage daily for 28 d. The control group received the solvents of Q808. Q808 drug solutions were freshly prepared with Tween-80 and CMC-Na as description before.¹⁶⁾ Twenty-four hours after the last administration, all of the rats were sacrificed and the hippocampus region was dissected. The study was carried out in compliance with the ARRIVE guidelines and approved by the Animal Care and Use Committee of the College of Basic Medical Sciences in Jilin University (No. 2020-31).

RNA Preparation

Total RNA was extracted from the dissected hippocampal tissue sample (about 100 mg) using TRIzol kit (Invitrogen, United States). Subsequently, RNA concentration was evaluated using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States). The 260/280 ratio between 1.8 and 2.2 for samples were considered acceptable. RNA quality was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, United States). Then, 1-2 µg of total RNA was used for generating the sequencing library as described before.¹⁶

RNA Sequencing

The libraries were sequenced at three samples per lane on a BGI T7 platform. By using Cufflinks program,⁶¹⁾ the gene expression levels in fragments per kilobase of transcript per million fragments mapped (FPKM) was calculated. HTSeq-count was used to obtain the read counts of each gene.⁶²⁾ DESeq (2012) R package was used to analyze DEGs. Genes were considered to be differentially expressed between two groups was set at p value <0.05, and log 2 of the fold-changes was filtered at >2 or <0.5.

Gene Functions and Pathways

In order to perform the functional annotation of DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses are used. In the GO analysis, the annotations were obtained from Gene Ontology (www.geneontology.org/) and NCBI (www.ncbi.nlm.nih.gov/), and the biological process (BP), cellular component (CC), and molecular function (MF) of DEGs was evaluated.⁶³⁾ For KEGG pathway analysis, the KEGG database (www.genome.jp/kegg/) was used, and the pathways which DEGs are involved in were identified.⁶⁴⁾

Protein–Protein Interaction (PPI) Network

The 51 DEGs were used to construct the PPI Network. By using the STRING database, the interactions between proteins were provided. Subsequently, Cytoscape (versio3.8.0) was used to visualized the PPI network.

Validation by qRT-PCR

RNA was extracted as described in RNA preparation section. Subsequently, the Prime ScriptTM RT Master Mix was used to synthesize first-strand cDNA and SYBR Green Master Mix was used for qRT-PCR. Reactions were performed on the QuantStudio Real-Time PCR System. The following PCR cycling parameters were used: denaturation stage at 95 °C for 10 min, followed by 60 cycles of 95 °C for 15 s and 59 °C for 30 s. The relative mRNA expression was calculated using the 2^−△△CT method and samples were normalized to Gapdh. Primers (Table 4) were designed using Primer-BLAST.

Statistical Analysis

Data analysis were carried out using GraphPad Prism 7 software. The experiment was performed with at least three biological replicates and data were expressed as mean ± standard error (S.E.M.). Unpaired t-test was performed to analyze differences between two groups. An adjusted p-value <0.05 was considered to be statistically significant.

Acknowledgments

This research was funded by the Department of Science and Technology of Jilin Province (Grant number: 20210101013JC).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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