Hippocampus transcriptome profiling
Gene expression profiles of the hippocampus were determined by RNA- seq and compared between S, M, E, and E + L groups. After removing unknown bases (N) > 5%, low-quality sequences and reads that were too short, 20.71, 41.44, 20.32 and 20.25 Gb of clean bases were obtained from 152.85, 305.58, 149.33 and 147.57 Mb of raw data for the S, M, E and E + L groups, respectively. The Q30 ≥ 88.03% in all cases, indicating good data quality (Table 1 and Figure S1). The ratio of matched reads in the sequence alignment exceeded 91.78%. In all matched reads, the uniquely mapped reads are more than 86.19%. These results indicate that the sequencing depth was sufficient for analyzing differential gene expression between groups. The threshold for differentially expressed genes (DEGs) was defined asัlog 2FCำ⩾1 and Q value⩽0.001. The heat map (EXP > 1) revealed differences in the gene expression profiles of the M and S groups (Fig. 3a) indicating that AMI was associated with significant changes in gene expression in the hippocampus. EA also altered transcript levels, as evidenced by the DEGs between the E and M groups. Additionally, the gene expression profilesof the E and E + L groups differed, suggesting involvement of the LC in mediating the effects of EA in the hippocampus of AMI rats. There were 703 DEGs between the S and M groups, including 391that were up-regulated and 312 that were down-regulated (Table S1). Between the M and E groups, there were 567 DEGs, including 312 and 255 that were up and down-regulated, respectively (Table S2). Of the 473 DEGs between the E group and E + L groups, 141 were up-regulated, and 332 were down-regulated (Fig. 3(b) and Table S3).The Venn diagrams revealed 67common DEGs between M vs S and E vs M, and 254 DEGs common to E vs M and E + L vs E (Fig. 3(c,d)).
Table 1
Summary of RNA sequencing results.
Group | Sample | Raw reads | Clean reads | Q30 (%) | Clean read ratio | Total mapped(%) | Uniquely mapped(%) |
S | S1 | 50946206 | 45891112 | 88.41 | 90.08 | 91.92 | 86.57 |
| S2 | 50945566 | 46006896 | 88.7 | 90.31 | 91.85 | 86.62 |
| S3 | 50945984 | 46231830 | 88.56 | 90.75 | 91.78 | 86.19 |
M | M1 | 49189326 | 44740580 | 88.21 | 90.96 | 92.14 | 87.22 |
| M2 | 52585260 | 47687462 | 91.12 | 90.69 | 92.92 | 87.76 |
| M3 | 50945510 | 46098634 | 88.66 | 90.49 | 92.05 | 87.03 |
| M4 | 50945754 | 46079410 | 88.8 | 90.45 | 92.15 | 86.91 |
| M5 | 50945670 | 45660720 | 88.03 | 89.63 | 91.65 | 86.5 |
| M6 | 50945572 | 46060494 | 88.14 | 90.41 | 92.13 | 87.01 |
E | E1 | 50945274 | 45632592 | 88.55 | 89.57 | 91.83 | 86.99 |
| E2 | 49188076 | 44788752 | 88.97 | 91.06 | 92.08 | 86.97 |
| E3 | 49188242 | 45073502 | 89.23 | 91.63 | 92.39 | 87.5 |
E + L | E + L1 | 49188236 | 45059832 | 88.93 | 91.61 | 92.15 | 87.27 |
| E + L2 | 49188160 | 44845222 | 88.93 | 91.17 | 92.08 | 87.01 |
| E + L3 | 49188216 | 45064528 | 89.05 | 91.62 | 92.39 | 87.46 |
Gene Ontology (GO) analysis
GO term enrichment was evaluated to categorize biological processes associated with the identified DEGs (Fig. 4). In the S vs M comparison (Table S1), the DEGs were mostly related to cell adhesion(Col6a5, Itga10, LOC102553715, LOC108348159, Col15a1, Gpnmb, Pcdhb2, LOC108349816, Dscam, Glycam1, Glycam1, Tnr, LOC100910275, Pcdhgb5, LOC108353166, Pcdhgb6, Fbln7, Cdhr1, Prkce, Cx3cr1, Cdh6, Wisp3, Col5a1, LOC103693608, Thbs1, LOC108348121, Pcdhgb4, Dgcr2, Pcdhb21, Pdzd2, Dpp4, Pcdh1, Pcdhgc5 and Cdh4) and ionotropic glutamate receptor signaling pathway(Grid2, Cdk5r1, Grik3, Gria1, Camk2a, Grin3a and Grin2a). In the M vs E comparison (Table S2), the DEGs were primarily associated with ion transport (Scn5a, Best3, Slc5a5, Scn11a, Slc22a2, Slc22a8, Kcnj13, Slco1a5, Atp2b4, Atp2b4, Kcnq1, Lcn2, Kcnf1, Slc24a1, Gabrq, Atp4a, Slc40a1, Hfe, Chrna4, Fxyd6, Tspo, Clic3, Steap1, Slc4a5, Scara5, Trpv4, Gabre, Slc5a11, Slc17a6, Slc38a4, Slc4a2, Cftr, Slc5a7, Kcne2 and Clic6) and neuropeptide signaling (Adcyap1, Nmur2, Crhr1, Tac1, Sstr1, Pnoc, Npsr1, Ntsr1, Ecel1, Penk, Grp, Prlhr, Glp1r, Nmbr). The DEGs between E + L and E groups were mainly related tocilium movement (Ccdc151, Ccdc114, Pih1d3, LOC108349819, Rsph4a, Dnah11, LOC103689994, Hydin and Cfap100) and iontransport (Scn5a, Best3, Slc5a5, Chrna5, Slc22a8, Kcnj13, Slco1a5, Atp2b4, Kcnq1, Grin3b, Kcnf1, Gabrq, Atp4a, Slc40a1, Hfe, Chrna4, Fxyd6, Tspo, Steap1, Slc4a5, Scara5, Trpv4, Slc17a6, Slc38a4, Slc4a2, Cftr, Kcnk16, Kcne2 and Clic6) (Table S3).
Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis
The functions of DEGs were also investigated by KEGG pathway enrichment analysis (Fig. 3a-d). Of the 67 DEGs at the intersection between M vs S and E vs M(Fig. 5a,b and Table S4) and 254 DEGs common to E vs M and E + L vs E( Fig. 5c,d and Table S5). most were associated with metabolism, including glycerolipid (LOC100911615), glycerophospholipid (Pla2g4f), Arachidonic acid (Cbr1, Pla2g4f) as well as with nervous system function including glutamatergic synapses (Pla2g4f) and long-term potentiation (Pla2g4f). cholinergic (LOC102547029, Slc18a3 and Gng4), serotonergic (LOC102547029, LOC100912642 and Gng4) and GABAergic (LOC102547029, Gng4 and Gabre) and retrograde endocannabinoid signaling (LOC102547029, Gng4 andGabre) (Figure(5b)). Other KEGG pathways that were represented among the DEGs were related tothe immune system functions, including helper T cell 17(Th17) differentiation (RT1-Bb, RT1-Da, RT1-Db1, RT1-Ba), Th1 and Th2 cell differentiation(RT1-Bb, RT1-Da, RT1-Db1 and RT1-Ba) (Fig. 5c) and tometabolism, including ether lipid (Pla2g5, Enpp2 and Pla2g4f) and arachidonic acid (Ephx2, Pla2g5, Cbr1 and Pla2g4f) (Fig. 5d).