EXPERIMENTAL AND THERAPEUTIC MEDICINE 13: 1303-1312, 2017

Identification of associated with the effect of inflammation on the neurotransmission of vascular smooth muscle cell

SHUJIE GAN1, SHENLONG QIU1, YIWEN FENG1, YANPING ZHANG1, QIN QIAN1, ZHONG WAN1 and JINGDONG TANG2

1Department of Vascular Surgery, The First People's Hospital, Shanghai Jiao Tong University, Shanghai 200080; 2Department of Vascular Surgery, Shanghai Pudong Hospital, Fudan University, Pudong Medical Center, Shanghai 201399, P.R. China

Received August 11, 2015; Accepted December 9, 2016

DOI: 10.3892/etm.2017.4138

Abstract. Vascular smooth muscle cell (VSMC) accumula- for DEGs enriched in pathways associated with neurological tion and hypertrophy are common in vascular disorders, and diseases. In the PPI network for ADRA1D, CACNA1S and inflammation has a crucial role in the development of these the DEGs interacting with them, prohibitin (PHB), oxytocin diseases. To investigate the effect of inflammation on the neuro- receptor (OXTR), collapsin response mediator 1 transmission of VSMC, bioinformatic analysis was performed, (CRMP1) and dihydropyrimidinase like 2 (DPYSL2) had following next generation sequencing. Genes of lipopolysac- interaction relationships with both ADRA1D and CACNA1S. charide (LPS)‑treated A7r5 cells and phosphate‑buffered To conclude, the present study revealed that NDUFV2, PHB, saline (PBS)‑treated A7r5 cells were sequenced via next OXTR, CRMP1 and DPYSL2 may have key roles in the effect generation sequencing, and each assay was repeated three of inflammation on neurotransmission of VSMC. times. Differentially expressed genes (DEGs) were obtained using the NOISeq package in R. Subsequently, their potential Introduction functions were predicted by functional and pathway enrichment analyses using the Database for Annotation, Visualization and As a highly specialized cell in mature animals, the vascular Integrated Discovery online tool. Interaction relationships of smooth muscle cell (VSMC) has a principal function of contrac- the enriched in pathways associated with neurological tion; however, production of matrix components of the blood diseases, the proteins which had interaction relationships vessel wall and proliferation becomes the primary function of with adrenoceptor α 1D (ADRA1D) or calcium voltage‑gated VSMCs during vasculogenesis (1). Abnormal contraction of channel subunit α1 S (CACNA1S), separately, were obtained SMC is a major incentive of vasospasm of the cerebral and from STRING, and protein‑protein interaction (PPI) networks coronary arteries, as well as hypertension (2). VSMC accu- were constructed using Cytoscape software. A total of mulation and hypertrophy are common in vascular disorders, 2,038 DEGs, including 1,094 upregulated and 944 down- such as atherosclerosis, hypertension, restenosis (3,4) and regulated genes in the LPS treatment group were identified inflammation, which can be induced by hypoxia and has when compared with the control group. Enrichment analyses crucial roles in the development of these diseases (5,6). Thus, showed that NADH:Ubiquinone Core Subunit there is an urgency to elucidate the effect of inflammation on V2 (NDUFV2) was involved in several neurological diseases, the neurotransmission of VSMCs. including oxidative phosphorylation, Alzheimer's disease, Several pharmacological agents are capable of inducing Parkinson's disease and Huntington's disease. Furthermore, inflammation. In human aortic smooth muscle cells, lipopoly- NDUFV2 (degree, 20) had a higher degree in the PPI network saccharide (LPS) promotes the production of nitric oxide (NO) and Toll‑like receptor 4 (TLR4) expression, inducing inflam- matory responses (4). A previous study demonstrated that propranolol has a negative chronotropic effect on the expres- sion levels of pro‑inflammatory cytokines after myocardial Correspondence to: Dr Jingdong Tang, Department of Vascular infarction (MI) in rats (7). In rat aorta, β‑adrenoceptors were Surgery, Shanghai Pudong Hospital, Fudan University, Pudong overstimulated by the agonist, isoproterenol, which resulted Medical Center, 2800 Gongwei Road, Pudong New Area, in an increase of vascular inflammatory mediators, such as Shanghai 201399, P.R. China β κ κ E‑mail: [email protected] interleukin (IL)‑1 , IL‑6 and nuclear factor B (NF‑ B) (8). At a concentration of 20 µg/ml, the non‑selective β‑adrenergic Key words: vascular smooth muscle cell, inflammation, receptor agonist, propranolol, was revealed to suppress cell next generation sequencing, differentially expressed genes, growth of infantile hemangioma endothelial cells (IHECs) protein‑protein interaction network in vitro once the proliferation stage of IHECs has been affected for between 72 and 96 h, whereas isoproterenol yielded the opposite results (9). 1304 GAN et al: EFFECT OF INFLAMMATION ON THE NEUROTRANSMISSION OF VSMC

Previous reports have been conducted to survey the effect Cell hypoxia treatment. Cells were inoculated with 0.5% FBS of inflammation on VSMC. As an adipocytokine, extracel- in confocal plates (4x105 cells/plate). After being cultivated lular pre‑B cell colony‑enhancing factor/nicotinamide for 24 h, the 0.5% FBS medium was replaced with 10% FBS phosphoribosyltransferase/visfatin (ePBEF/NAMPT/visfatin) and cells were treated with cobalt dichloride (200 µmol/l) for functions as a direct contributor to vascular inflammation via 24 h. Subsequently, cells were induced by LPS (100 µg/ml) its NAMPT activity (10). Through regulating vascular cell for 30 min; however, cells in the control group received PBS. activation and inflammatory cell recruitment, the adhesion After cell digestion by pancreatin (Gibco; Thermo Fisher protein, cluster differentiation (CD) 44, has an important Scientific, Inc.) for 5 min, the mixture was centrifuged at room role in the development of atherosclerotic diseases (11). By temperature at 157 x g for 5 min. Then, cells were washed inhibiting the activation of hypoxia‑inducible factor‑1α, three times with DMEM at room temperature and further transcription factors, NF‑κB and activator protein‑1 (AP‑1), centrifuged at room temperature at 157 x g for 5 min. The and 3‑hydroxy‑3‑methylglutaryl‑coenzyme A (HMG‑CoA) supernatant was discarded and A7r5 cells were preserved in reductase inhibitors have anti‑proliferative and anti‑inflam- TRIzol (Invitrogen; Thermo fisher Scientific, Inc.) at ‑80˚C. matory effects on human endothelial and vascular smooth Each assay was performed in triplicate. muscle cells; thus, statins can be used to treat atheroscle- rosis (12). RNA isolation and RNA‑sequence library construction. In the present study, next generation sequencing was Total RNA of the LPS treatment group and the control conducted to obtain sequence data. Differentially expressed group were extracted using a TRIzol total RNA extraction genes (DEGs) between the LPS treatment group and the control kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to group were screened and their functions were predicted by the manufacturer's instructions. The integrity and purity of enrichment analyses. Moreover, a protein‑protein interaction total RNA was detected using 2% agarose gel electropho- (PPI) network was constructed to investigate the interaction resis and a spectrophotometer (Merinton Instrument, Ltd., relationships between these DEGs. Beijing, China), respectively. The RNA‑sequence library was constructed using methods described in a previous study (13). Materials and methods Subsequently, DNA cluster amplification was performed and high‑throughput sequencing was conducted for the library, Cell cultivation. Rat VSMC cell line A7r5, which was using Illumina Hiseq 2000 100PE (Illumina Inc., San Diego, purchased from Shanghai research Biotechnology CA, USA). After the raw data was obtained, sequences Co., Ltd. (Shanghai, China), was cultivated in Dulbecco's containing an adaptor, >50% low quality bases and >3% modified Eagle medium (DMEM) (Gibco; Thermo Fisher unknown bases were filtered out. Scientific, Inc., Waltham, MA, USA) and maintained at 37˚C using an incubator (Thermo Fisher Scientific, Inc). DEGs screening. Following filtering, the sequences were DMEM was discarded, and A7r5 cells were digested with mapped to the rat genome (rn5), using bowtie1 in TopHat pancreatin (Gibco; Thermo Fisher Scientific, Inc) for 5 min. software (version 2.1.0, accessible at http://ccb.jhu.edu/soft- Subsequently, 6‑fold DMEM was added to terminate diges- ware/tophat/index.shtml) (14). The maximum read mismatch tion and cells were centrifuged at room temperature with number was set at 2, and the parameter ‘max‑multihits’ was set at 157 x g for 5 min, and the supernatant and resuspension was 1 (15). And the other parameters were set to defaults. Combining discarded. A7r5 cells were cultured in new culture flasks with annotation information of rn5 in ensemble, expres- in a 5% CO2 incubator at 37˚C (Thermo Fisher Scientific, sion of each sample was annotated using Cufflinks software Inc.). Using the frozen stock solution made of 10% dimethyl (version 2.2.1, accessible at http://cole‑trapnell‑lab.github.io/cuff- sulfoxide (DMSO; Sigma‑Aldrich; Merck Millipore, Darm- links/) (16) under the default parameter values. The NOISeq stadt, Germany) and 90% fetal bovine serum (FBS) (Gibco; package (version 2.18.0, accessible at http://www.bioconductor. Thermo Fisher Scientific, Inc.), A7r5 cells were resuspended org/packages/release/bioc/html/NOISeq.html) (17) in R was at a density of 5x106 cells/ml. Following stewing at 4˚C for used to screen the DEGs between the LPS treatment group and 10 min, cells were cryopreserved at ‑20˚C for 2 h and stored the control group. The probability of a being DEGs (q) was at ‑80˚C overnight. set to 0.99.

Calcium detection. A7r5 cells were inoculated into confocal Functional and pathway enrichment analysis. plates (Wohong Biotechnology Co., Ltd., Shanghai, China) (GO), which consists of three categories, including biological (2x104 cells/plate) and cultivated overnight. Cells were washed process (BP), molecular function (MF) and cellular compo- with phosphate‑buffered saline (PBS) twice with 100 µl PBS nent (CC), is used to generate the vocabulary that can be containing 5 µmol/l Fluo‑4/AM and lucifugally incubated at applied to all eukaryotes (18). As a database, Kyoto Encyclo-

37˚C in a 5% CO2 incubator for 45 min. Subsequently, cells pedia of Genes and Genomes (KEGG) includes information were washed with PBS twice again and induced by LPS of known genes and their biochemical functionalities (19). (100 µg/ml) for 30 min. In the control groups, PBS was used Using the Database for Annotation, Visualization, and instead of LPS. Under confocal laser scanning microscope, Integrated Discovery (DAVID) online tool (20), GO and A7r5 cells were observed and images were captured before and KEGG pathway enrichment analyses were conducted for after treatment with isoprenaline (10 µmol/l, Melone pharma- the upregulated and downregulated genes between the LPS ceutical, Co., Ltd., Dalian, China) or propranolol (10 µmol/l, treatment and control groups, respectively. P<0.01 was used Melone pharmaceutical, Co., Ltd.). as the cut‑off criterion. EXPERIMENTAL AND THERAPEUTIC MEDICINE 13: 1303-1312, 2017 1305

Figure 1. Calcium signals in A7r5 cells observed under confocal laser scanning microscope. (A) Calcium signals in A7r5 cells in the PBS group before treatment with isoprenaline (10 mol/l) or propranolol (10 mol/l). (B) Calcium signals in A7r5 cells of the PBS group after isoprenaline treatment. (C) Calcium signals in A7r5 cells in the PBS group after being treated with propranolol. (D) Calcium signals in A7r5 cells in the LPS group before being treated with isoprenaline (10 mol/l) or propranolol (10 mol/l). (E) Calcium signals in A7r5 cells in the LPS group after isoprenaline treatment. (F) Calcium signals in A7r5 cells in the LPS group after propranolol treatment. PBS, phosphate‑buffered saline; LPS, lipopolysaccharide.

PPI network construction. Using STRING online software (version 10.0, accessible at http://www.string‑db.org/) (21), interaction relationships of the proteins encoded by the DEGs were searched, and the required confidence (combined score) >0.1 was used as the cut‑off criterion. Subsequently, PPI network was visualized using Cytoscape (version 3.2.0, accessible at http://www.cytoscape.org/) (22). Proteins in the network were named as nodes and the degree of a node was equal to the number of nodes interacted with it. Moreover, the nodes with degrees higher than 20 were defined as hub nodes.

Results

Calcium detection. In the PBS control group, A7r5 cells prior to treatment with isoprenaline or propranolol are indicated in Fig. 1A. In A7r5 cells treated with isoprenaline, the concen- tration of calcium decreased (Fig. 1B); however, the level of Figure 2. Protein‑protein interaction network for differentially expressed calcium in propranolol‑treated A7r5 cells increased (Fig. 1C). genes enriched in pathways associated with neurological diseases. Red The LPS‑treated group (Fig. 1D‑F) exhibited a reduced circles and green circles represent up‑regulated and down‑regulated genes, respectively. calcium signal prior to treatment with isoprenaline or propran- olol when compared with the PBS control group (Fig. 1D). In the A7r5 cells that were treated with isoprenaline, the concen- tration of calcium decreased (Fig. 1E). However, the level of homeostasis (P=4.81E‑04), mitogen‑activated protein kinase calcium in the A7r5 cells treated with propranolol had no kinase kinase cascade (P=7.85E‑04) and nucleoside mono- change when compared with the LPS group cells treated with phosphate metabolic process (P=8.39E‑04). The enriched isoprenaline (Fig. 1F). functions in the CC category included condensed chromo- some kinetochore (P=1.33E‑04), , centromeric DEGs analysis. Compared with the control PBS group, a total region (P=1.63E‑04) and plasma membrane (P=2.96E‑04). of 2,038 DEGs, including 1,094 upregulated and 944 down- Enriched functions in the MF category included hydrolase regulated genes were identified in the LPS‑treated group. activity, acting on carbon‑nitrogen (but not peptide) bonds in These findings indicated that the number of upregulated genes linear amidines (P=1.40E‑04), quaternary ammonium group was markedly higher in comparison to the number of down- binding (P=8.28E‑04) and phosphatidylcholine binding regulated genes. (P=9.81E‑04). The enriched KEGG pathways for upregulated genes Functional and pathway enrichment analysis. The enriched are also listed in Table I, including purine metabolism GO functions for upregulated genes are listed in Table I. The (P=3.13E‑04), neuroactive ligand‑receptor interaction enriched functions in the BP category included chemical (P=0.003996) and pyrimidine metabolism (P=0.007245). 1306 GAN et al: EFFECT OF INFLAMMATION ON THE NEUROTRANSMISSION OF VSMC P‑value 8.39E‑04 4.81E‑04 8.54E‑04 8.82E‑04 2.96E‑04 7.85E‑04 1.33E‑04 5.98E‑04 1.63E‑04 , , ,

,

, , , , , , , , , , , , , , ,

,

, , Stc1 Muc20, , Ncr3 Apoe Golph3 Ska2 , Pfkm , , , Rheb , Pkd2l1 , Gck Cldn6 , , Cd200r1 , , Myd88 Igsf11 , Mecom , , Oscp1 , Slc1a6 Slc38a4 Kl Cacnb4 Ppat , , , , Tgfbr1 Apoe , , , Faim2 , , Mal Proc Fabp4 , ,

Bub1 Cntf , , Pdyn , Apoa2 , , Slc6a7 Gpr4 Pfkm , , Oxtr Apoe , Ehd3 Wnt7b , Acy3 F2 , Itgam Scn10a , , , , , Otoa , Cacna1h F7 Sh3gl2 , , Ncr1 Cdh20 , , Gucy2c , , Cckbr , Rt1‑m10‑1 , , Lyn Ampd1 Best2 Abcg5 , , , , Cd19 Cdh5 Ppp2cb , , , Ngfr Nab1 Htr3a Pth2r P2rx7 Sctr Apoa2 , , , , , Car4 , Vpreb2 , Ubac1 Myh7 Pfkm , , , , , Avpr1b Slc43a1 Gnaz , Ppat , , Epas1

Aqp2 , , Cckbr , Guca2b P2rx7 Gpr1 , , , , Blm Vav3 Cenpa Cftr Aqp2 , , P2ry10 , Pgm1 Avpi1 , Trem1 , , , , Npffr1 , Tie1 Actn2 , Lilrb3l , , , Gip Fech Zw10 Zw10 , Slc38a1 Cacnb4 , C7 Chrng , , Slc7a1 , , , Sod2 , , , Gucy2c , Cacnb4 Aqp3 Tpo , Cacnb4 , , , Grm6 Gip Pard6a , Ptprr Aqp2 Sh2b2 Mad2l1 , Avpr1b , Fech Rt1‑m1‑4 , , Stc1 , , Slc7a10 , , , , Mapk10 , Ppp1r9a Pkp3 , Mis12 Mis12 , , , , Oxtr Nostrin Smad1 Mtnr1a , Cd300lf , Avp , , , , Taar1 Avp Gck Cspg5 Ncf4 Nab1 , , Pde3b Guca2b , Oxtr , Chrng , , , , , , Tgm3 Nab1 Pde3b , Cdca8 , , F2 , Gucy2g Pmf1 Pmf1 , Mal , , , Abcg5 Smad1 Wnt7b Cmklr1 , Mme , , , , , Cntf Pde3b Gene symbol F2 , , Mc5r , Lax1 Cd52 Spred1 , Nppc Nppc , , Slc5a11 , Scn2b , , Sash3 Guca2b , Cd22 , Iyd , , Htr6 , Guca2b , Grik2 Zp3 Vpreb2 Ska2 Ska2 , , , Spc25 , , , , Itpkb , Zap70 , P2rx7 , , Il1b Syt9 Grik2 , F10 Pgm1 , Abcg5 , C7 Taf12 , , , Cxcr6 , , Olr1469 , , Pdzd2 Chrna10 Entpd8 Entpd8 , Eif2b2 , Loc690948 Oxtr , , Agtrap Cenpf Cenpf , , , Col6a2 Eif2b2 Noxo1 Chrnb1 Slc9a4 , , , Mis12 , , , , , , Rasgrf2 Kctd6 , , Entpd8 , Slc9a4 P2rx7 Rps17 Slc9a4 Abl1 , , , Cp Rab3a Il1b , Aldob Perp Kcnq4 , Map3k1 Alox15 , , Chrna2 , , , , , , Cish , Nuf2 Nuf2 Krt1 , Adcy9 Adcy9 Fgf7 , , , Epas1 , , , Glp2r , Cckbr Chrng Muc20 Cenpi , , , , , Cp Kctd7 Folr1 Egflam , , , , Grik2 Lax1 Uts2r , Hnf1a Cp Galr2 , Rasd2 Gjb6 , Alox15 Alox15 Syn2 , , , Bub1 Bub1 Lyn , , Il1b Tyms Tyms , , , , , , Ddah1 Chrnb1 , , , Tinf2 Gpr149 , , Hnf1a , Slc23a1 Pmf1 Slc26a3 , , , , , Tgfbr1 , Fgf7 , Bbs1 Gabrr3 Slc6a19 Adss Adss , , , Fabp4 , , Gorasp1 Zmynd19 Apoa5 Rxfp1 , Gabrb2 Uts2r Tmed1 , , , , Cenpa Cenpa Apoa5 Glp2r Gja1 , Zw10 Chrnb1 Map3k2 , , Sh2b2 , , , , , Lppr4 Slc27a5 , , , Slc34a2 Ambp Mapk10 , Cenpf Rab9a , , , , , , , Cckbr Uts2 , , C7 Slc1a6 Sod2 Uts2r Ret Mdfic Gucy2g Ampd1 Gucy2g Uts2 Galr2 Slc34a2 Best2 Avpr1b Spc25 Nuf2 Tigd5 Rab9a Galr2 Slc34a2 Cav2 Hcrtr1 Slc6a13 S100g Susd2 Ptger2 Stom Ptafr Pex19 Gjb3 Gng10 Spc25

43 18 12 10 56 9 14 159 9 Gene no. Description metabolic process biosynthetic process kinetochore centromeric region Chemical homeostasis MAPKKK cascade Nucleoside monophosphate Nucleoside monophosphate Homeostatic process Condensed chromosome Chromosome/centromeric Plasma membrane Condensed chromosome/

Term GO:0048878 GO:0000165 GO:0009123 GO:0009124 GO:0042592 GO:0000777 GO:0000775 GO:0005886 GO:0000779

Table Table I. Enriched GO functions and Kyoto Encyclopedia of Genes saline control group. phosphate buffered and Genomes pathways for upregulated genes in the lipopolysaccharide treatment group compared with the Category BP BP BP BP BP CC CC CC region CC EXPERIMENTAL AND THERAPEUTIC MEDICINE 13: 1303-1312, 2017 1307

The enriched GO functions for downregulated genes are presented Table II. The enriched functions in the BP

P‑value category included oxidation reduction (P=1.19E‑05), 1.69E‑03 2.19E‑03 1.40E‑04 8.28E‑04 9.81E‑04 6.78E‑04 metabolic process (P=0.001298) and erythrocyte homeo- stasis (P=0.0013). Enriched functions in the CC category

, , included mitochondrion (P=6.87E‑07), organelle membrane (P=6.03E‑06) and mitochondrial part (P=1.67E‑05). The enriched functions in the MF category included nicotinamide Slc43a1 Slc38a1

, , adenine dinucleotide or nicotinamide adenine dinucleotide hydride binding (P=3.63E‑04), protein homodimerization Zw10 , activity (P=4.08E‑04) and iron ion binding (P=4.97E‑04). Slc38a1 Slc7a10 , , The enriched KEGG pathways for downregulated genes are also listed in Table II, including oxidative phosphorylation Mis12 , (P=2.49E‑04), Alzheimer's disease (P=0.00105), Parkinson's Slc7a10 Slc22a4 , , disease (P=0.00299), Huntington's disease (P=0.00397) and Pmf1 , proteasome (P=0.008983). Furthermore, NADH dehydroge- nase ubiquinone flavoprotein 2 (NDUFV2) was revealed to be Ska2 , Slc6a13 Slc6a13

, , involved in several neurological diseases, including oxidative phosphorylation, Alzheimer's disease, Parkinson's disease and Cenpf Allc ,

, Huntington's disease. Slc1a6 Slc1a6

, , Gene symbol Nuf2

, PPI network analysis. Pyrimidine metabolism, oxidative Padi1 , Acpp , phosphorylation, Alzheimer's disease and Parkinson's disease Slc7a1 Slc7a1 , ,

Bub1 were all metabolic pathways associated with neurological , Pctp Pctp , ,

Ddah1 diseases. DEGs enriched in these pathways and adrenergic , Slc6a7 Slc6a7

, , receptor genes α1d (ADRA1D) were used to construct a PPI Cenpa , Aldob Aldob

, , network. The PPI network consisted of 39 nodes and 183 inter- Agmat , actions (Fig. 2). ATP synthase, mitochondrial F1 complex,

Slc7a15 Slc7a15 Slc6a19 O subunit (ATP5O; degree, 22) and NDUFV2 (degree, 20) , , , Apoa5 Apoa5 Mad2l1 , , ,

Padi4 were hub nodes in the PPI network. , With the required confidence threshold of >0, the DEGs

Spc25 Arg1 Apoa2 Apoa2 Slc38a4 Slc6a19 Slc38a4 Slc43a1 which had interaction relationships with ADRA1D and voltage‑dependent L‑type calcium channel subunit α‑1S (ACNA1S) were indicated in Fig. 3A and B, respectively. DEGs which had interaction relationships with ADRA1D and

11 10 6 5 4 10 CACNA1S were merged in Fig. 4. In the PPI network, prohib-

Gene no. itin (PHB), oxytocin receptor (OXTR), collapsin response mediator protein 1 (CRMP1) and dihydropyrimidinase‑like 2b (DPYSL2) exhibited an interaction relationship with both ADRA1D and CACNA1S.

Discussion

The present study indicated that calcium signals in A7r5 cells treated with LPS were weaker when compared with

Description PBS‑treated cells (the control group). It has been reported that calcium oxalate crystals are able to trigger inflamma- tion through regulating IL‑1β (23). Calcium pyrophosphate carbon‑nitrogen (but not peptide) bonds, in linear group binding transporter activity transporter activity Kinetochore acting on Hydrolase activity, Quaternary ammonium Phosphatidylcholine binding transmembrane Amine transmembrane has been revealed to induce a novel acute inflammation, pleurisy (23). Thus, LPS induced inflammation in A7r5 cells. In the present study, a total of 2,038 DEGs, including 1,094 upregulated and 944 downregulated genes, were identified

in the LPS‑treated and control groups. Enrichment analyses

Term indicated that NDUFV2 was involved in several neurological diseases, including oxidative phosphorylation, Alzheimer's GO:0000776 GO:0016813 GO:0050997 GO:0031210 GO:0015171 GO:0005275 disease, Parkinson's disease and Huntington's disease. Further- more, NDUFV2 (degree, 20) exhibited a higher degree in the PPI network for DEGs enriched in pathways associated with neurological diseases. NDUFV2, which is located on chromo- some 18p11.31‑p11.2, has been named as a causative gene Table I. Continued. Table Category CC MF MF amidines MF MF MF molecular function; CC, cellular component; MAPKKK, mitogen‑activated protein kinase kinase. biological process; MF, GO, gene ontology; BP, 1308 GAN et al: EFFECT OF INFLAMMATION ON THE NEUROTRANSMISSION OF VSMC P‑value 1.19E‑05 6.03E ‑ 06 1.30E ‑ 03 , , ,

, 1.67E ‑ 05 , , , , , , 6.87E ‑ 07 1.63E ‑ 03

, , , , , , , , , , , , Dhrs4 Idh3b

Bnip3 Acss1 , Ccdc58 Vdac2 , ,

, , , Mrpl34 , Slc25a29 Mcart1 , Cox5b , Clpx , Me1 Mrpl22 Mthfs Hagh ,

, , , Ndufb9 , , , Hmox1 Ucp2 , Scd , , , Ndufa6 Fig4 Ndufa9 Fam110b , , , , Serinc1 Morc1 , , Oxct1 Afap1l1 Wwox , Hagh Tufm Aldh3a1 Mthfd1l , , , , , Mtx2 , Armc1 Gstm1 Cyp2e1 Cox5a , Ucp3 Timm17a Scd Mcart1 , , , , , , , , Maoa , Ndufs4 Slc25a23 Pptc7 , , , Grhpr Ndufa9 , , Gng5 Slc11a1 Gclm Mdh1 , Tyrp1 , , , Gstm1 Bbox1 Atad1 Hagh Sdhd , , , Maoa , , , Mrps7 Cd1d1 , Slc25a24 , Fads1 , Ndufv2 Ndufb9 , Mthfd1l , Fam136a Cyp2e1 Rgd1566320 , Ero1lb , , , , , Cat , , Gatc Phb Stx8 , Hsd17b4 , Slc25a24 Cyp2e1 Comtd1 1.30E ‑ 03 Clpx , , Slc25a4 , , , , Afap1l1 , Idh3a , Sdhd , Mtx2 Hibadh Ndufv2 Anpep Mthfd1l , Fdft1 , Lypla1 , Idh3a , Myg1 , , , , Ndufs4 , , Mb 1.76E ‑ 03 Ndufa9 Atp5o , , , , Loc685351 Cacng4 , , Sdhd , Timm17a Idh3a Hspe1 , , Ndufb5 , Acaa2 Ndufa6 , Faah , Aldh9a1 Vdac2 Pdp2 , Pcyox1l , , , Adh5 Prdx1 , Idh3a , Bnip3 , , , , Mdh1 Mdh1 Cisd1 Prdx1 , , Map1lc3a , Cox4i2 Adh5 , Hibadh , Cyp4f18 Hibadh , , Atp6v1g1 , , , Fgfbp3 , , Pdp2 Cox4i2 Tufm , , Oxct1 Ndufv2 , Hpd Vdac2 Slc25a29 , , , , Tlr3 , Ucp2 , Pebp1 , Ndfip2 Mrpl34 Klf1 , Ndufa9 Urod , , , Ndufb5 Cox5b , , Idh3a Aco1 Acss1 , , , Ndufb9 , , Hibadh Gclm , , Bbox1 Gene symbol , Slc2a4 Adhfe1 , , Lepre1 , Cmc1 , , Ucp2 Cat , Rb1 , Gfod2 , , Hsd17b7 Qprt Slc25a4 Mcart1 Lemd3 , , Trappc3 Idh3b , Dhrs4 Rnaset2 , , , , , , Ido1 Vps18 Mtx2 Loc685351 , , Gclm , Slc25a23 , Cyp4f18 Cox5b , , , , Prdx1 Cisd1 Hibadh Tspo Mrps16 Ndufaf1 Tyrp1 , , , , Slc25a4 , , , , Tcea1 Hk3 Sdhd Hspe1 , , , , Gars Dusp26 Cyp17a1 Idh3b , Srd5a2 Acaa2 , Ido1 , , Gstt1 , Pebp1 , Soat2 , , , , Ucp3 Ido1 Mdh1 Ldha , , Prdx1 , Adhfe1 , , Atp5e , Timm17a , Cox5a , Ndufs4 Tmem186 Bcl6 Bcmo1 Kcnab1 , , Cat , , , Atp6v1g2 , Rpusd4 Slc25a24 , , Adi1 Atp5o , Nudt9 Ireb2 , Gars Rgd1562758 Ucp3 , , , , , , , Hmox1 Gstt1 Qprt Idh3b , , Qprt , , , Atp5e Scamp1 Zfyve28 , , Me1 Cox5a , , , Ldha Ndufb5 Hagh Hsd17b7 Rer1 Phb Clstn3 Ctu1 , Uqcrc2 Mipep , , , , , , Cyp17a1 Dyrk3 , Pebp1 Ero1lb , , , , , , Idh3a Acaa2 Tpi1 , , , Sdhd Ireb2 Idh3b Alox12b Cox4i2 , Idh3b , , Hpd , , , , Slc25a29 Hsd17b4 Chchd10 , Clpx Mdh1 , , , Mipep , Uqcrc2 Ido1 Atp5e Maoa Rnf180 Acox2 , Sdhd Ireb2 , Cisd1 , Bnip3 Hspa5 Fdft1 , , , , , , , , , , Cat Tpi1 Acss1 Rgd1303003 Mrps16 , , Slc35a5 Aco1 Aco1 , , Mrps36 Coro1a , Ldha , , , , , Maoa , , Loc688320 Aldh9a1 Hsd17b6 Cyp4v3 Ldha Mthfs Hmox1 Ldha Acss1 Tpi1 Mrps36 Lemd3 Ndufs4 Rgd1309676 Ndufa12 Tshz3 Mthfs Ndufa6 Cyp17a1 Uqcrc2 Spink5 Atp5o Sacm1l Drd4 Gp1ba Gjb2 Tufm Afap1l1 Slc25a23 Phb Ndufv2 Uqcrc2

51 20 9 17 8 94 73 46 Gene number

Description Cofactor metabolic process Erythrocyte homeostasis Coenzyme metabolic process Nicotinamide nucleotide metabolic process Mitochondrion membrane Organelle Mitochondrial part Oxidation reduction

Term GO:0055114 GO:0051186 GO:0034101 GO:0006732 GO:0046496 GO:0005739 GO:0031090 GO:0044429

BP BP BP BP CC CC CC Table II. Enriched GO functions and Kyoto Encyclopedia of Genes and Genomes pathways for the down‑regulated genes intreatmentcomparedthelipopolysaccharidethegroup with genes thedown‑regulated for pathways andGenomes Genes of Encyclopedia Enriched II. GOfunctions and Kyoto Table phosphate‑buffered saline group. control Category BP EXPERIMENTAL AND THERAPEUTIC MEDICINE 13: 1303-1312, 2017 1309 P‑value 3.63E‑04 3.48E‑05 1.27E ‑ 03 4.08E‑04 4.97E‑04 8.40E‑04 4.90E‑04

, , , ,

, ,

,

, , , , Hpd

, , Cat , , Tbx1

,

,

, ,

, , , Zfp438 Cat Cyp2e1 Mdh1 Lepre1 , Ndufs4 , Acvr2b , , Ido1 ,

, Slc2a4 , , Cyp4f18 , , Rpl37 Zdhhc9 Zfp786 , Rgd1562758

, , , Trpm8 Gstm1 , , Ndufa6 Cyp2e1 Alox12b , Cyp17a1 , , , Grhpr Tpi1 , , Pde3a Adh5 Cox5b Zcchc24 , , Alox12b Cr2 Rnf180 Aldh9a1 , , Rnf112 , , , , Cyp17a1 , , Parp1 Phb Bbox1 Tgfb1 , , , Rnf166 Rnf8 , , , Spire2 , Hmox1 , Maoa , Adi1 Hagh Cox5a Camkk1 , Dpep3 , , , Bsn , Uqcrc2 Trim47l , , , Cox5b Idh3a Cyp2e1 Mdh1 Ndufv2 Loc685351 Ndufa9 , , , , , , , Mthfd1l Isl1 Slc11a1 Hsp90aa1 , Cisd1 , Aco1 , , , Tut1 , , Ctsl1 Rgd1562758 Klf1 , Zhx1 , , , Ido1 Ndufv2 Tyrp1 Afap1l1 , , Setmar Cat , , , Cryl1 Csrp2 , Idh3b Cox5a Ido1 , , , , , Prdx1 Cryba4 Actc1 , , Abo Slc25a4 , , , Gclm Morc1 , Slc11a1 , , Parp1 Sdhd Cda , , , Nudt18 Adh5 , Idh3a Idh3b Loc680200 Bcmo1 , Cyp4v3 , , , , Ndufb9 Hmox1 Sytl1 , , , , Cyp4v3 , Cat , Parp1 Eif2b3 Acaa2 Sdcbp2 , , , , , Mb Gstm5 Dnase1l2 Eno2 Ucp2 Topors , Bbox1 , , , Gene symbol , , , Acp5 Ireb2 Maoa Ireb2 , , , Vps29 , Nenf Prdx1 , , Pebp1 , Hibadh , , Pdx1 Cpb1 Fads1 , Atp5o , , Cpa1 , Ndufv2 Ucp3 , Timm17a Scd , Anpep Prkch Mipep , Prdx1 , , Me1 , , , , Gtf2a2 , , Bcl6 Aldh9a1 , Fads1 , Chn2 , Ndufa9 Asns , , , Grhpr , Rgd1562758 Clpx Adam17 Cbfa2t3 Asns , , , , Prkar1b Hdc Ndufv2 Rbm20 , Prkci , , , , , Ero1lb Osgep Scd Cox5a , , Lemd3 Ndufb5 , , , , Add2 Tcea1 Slc25a29 Ace2 , , , , Ldha Adamts4 , Bnip3 , , Idh3b Cpa1 , Adh5 , Blmh Rgd1309534 , Aco1 Anpep , , , ,

Aco1 Mcart1 Cep57 Ndufv2 Cryl1 Zbtb7c Zfyve28 , , , , Qprt Rnf208 , , , Atp5e , , Mipep Rnf168 , , Nudt9 , , Soat2 Pias1 Adh5 Mep1a , , , Mvd Adh5 Apobec4 , Aspg Mb , , , , , Slc25a23 Cisd1 , Mdh1 Sdhd , Dtx4 Cryl1 Vdac2 , , Cda , Faah Idh3a Tufm , Rnf187 Zdhhc4 , Nr1i2 Acp5 , , , , , , Pitpnm2 , , Ppm1e Mvd , , Tcea3 , Sdhd , Hpd Tesk1 , Ldha Ldha Acox2 Pdp2 Eno2 , , , , , , , Me1 Hdc Fuca1 Uqcrc2 Slc25a24 Cox4i2 Me1 Aldh9a1 Tyrp1 Slc11a1 Coro1a Bcmo1 Nenf Cyp4f18 Gda Ighmbp2 Clpx Fbxo43 Lepre1 Rnf185 Zfp365 Myrip Cpn1 Galnt13 Rbak Me1 Hibadh

31 27 13 28 26 106 21 Gene number

Description Soluble fraction Mitochondrial inner membrane NAD or NADH binding Protein Homodimerization Iron ion binding metal Transition ion binding Coenzyme binding

Term GO:0005625 GO:0005743 GO:0051287 GO:0042803 GO:0005506 GO:0046914 GO:0050662 Table II. Continued. II. Table Category CC CC MF MF MF activity MF MF molecular function; CC, cellular component; NAD, nicotinamide adenine dinucleotide. biological process; MF, GO, gene ontology; BP, 1310 GAN et al: EFFECT OF INFLAMMATION ON THE NEUROTRANSMISSION OF VSMC

Figure 3. PPI network for ADRA1D/CACNA1S and the DEGs that interact with them. (A) PPI network for ADRA1D and the DEGs that were demonstrated to interact with them. (B) PPI network for CACNA1S and the DEGs interacted with them. Red circles and green circles represent upregulated and downregulated genes, respectively. DEGs, differentially expressed genes; PPI, protein‑protein interaction; ADRA1D, adrenoceptor α 1D; CACNA1S, calcium voltage‑gated channel subunit α1 S.

Figure 4. Protein‑protein interaction network for ADRA1D/CACNA1S and the differentially expressed genes interact with them. Red circles and green circles represent upregulated and downregulated genes, respectively. ADRA1D, adrenoceptor α 1D; CACNA1S, calcium voltage‑gated channel subunit α1 S.

in neurological diseases, such as , Parkinson's oxidative and excitotoxic stress, and may serve as target for disease, and (24,25). A human disease cell designing agents to control neuronal death in brain injury, such as model has revealed that the injury of mitochondrial localiza- cerebral ischemia (27). In cardiomyocytes, overexpressed PHB tion of NDUFV2 is related to the pathogenesis of early‑onset contributes to the maintenance of the mitochondrial membrane hypertrophic cardiomyopathy and (26). potential and improves cell survival during hypoxia (28). It has Therefore, the expression of NDUFV2 may be involved in the been speculated that the function of PHB in protecting against effect of inflammation on neurotransmission of VSMC. oxidative and hypoxic stress may be correlated with its role in In the PPI network for ADRA1D, CACNA1S and the DEGs mediating the enzyme, cytochrome interacting with these components, PHB, OXTR, CRMP1 and C oxidase (29). A previous report has implicated oxytocin DPYSL2 were revealed to have interaction relationships with (OXT) in inflammatory processes (30). As G‑protein coupled both ADRA1D and CACNA1S. PHB, a member of the Band‑7 receptors, oxytocin receptors (OXTRs) are regulated by family of proteins, has a neuro‑damaging role following G‑proteins, which stimulate the phosphatidylinositol‑calcium EXPERIMENTAL AND THERAPEUTIC MEDICINE 13: 1303-1312, 2017 1311 secondary messenger system (31). In addition, OXTRs are 8. Davel AP, Fukuda LE, De Sá LL, Munhoz CD, Scavone C, Sanz‑Rosa D, Cachofeiro V, Lahera V and Rossoni LV: widely distributed in the central nervous system and mediate Effects of isoproterenol treatment for 7 days on inflammatory various behaviors (32), such as social and recognition, mediators in the rat aorta. Am J Physiol Heart Circ Physiol 295: responses to stress and anxiety, sexual and maternal behaviors, H211‑H219, 2008. 9. Zhu Y, Tuerxun A, Hui Y and Abliz P: Effects of propranolol and and bonding (33). These may indicate that the expression levels isoproterenol on infantile hemangioma endothelial cells in vitro. of PHB and OXTR are correlated with the effect of inflamma- Exp Ther Med 8: 647‑651, 2014. tion on neurotransmission of VSMC. 10. Romacho T, Azcutia V, Vázquez‑Bella M, Matesanz N, Cercas E, Nevado J, Carraro R, Rodríguez‑Mañas L, Sánchez‑Ferrer CF CRMP1 is affiliated with a cytoplasmic family of proteins and Peiró C: Extracellular PBEF/NAMPT/visfatin activates and is involved in the development of the central nervous pro‑inflammatory signalling in human vascular smooth muscle system (34). CRMP1, which may be disturbed by Speedy A1 cells through nicotinamide phosphoribosyltransferase activity. Diabetologia 52: 2455‑2463, 2009. (Spy1) from interacting with actin, has a role in the collapse 11. Cuff CA, Kothapalli D, Azonobi I, Chun S, Zhang Y, Belkin R, and regeneration of growth cones after sciatic nerve crush (35). Yeh C, Secreto A, Assoian RK, Rader DJ and Puré E: The Previous studies have identified that within hypertrophic adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation. J Clin cells of a brain that has suffered a stroke, overexpression of Invest 108: 1031, 2001. SPNA2 and DPYSL2 (also known as CRMP2) was revealed 12. Dichtl W, Dulak J, Frick M, Alber HF, Schwarzacher SP, to be correlated with neurite outgrowth and plasticity, which Ares MP, Nilsson J, Pachinger O and Weidinger F: HMG‑CoA reductase inhibitors regulate inflammatory transcription factors suggests an early activation of neuronal regeneration, repair in human endothelial and vascular smooth muscle cells. Arterio- and development (36,37). 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