3.1. Identification of differentially expressed upstream cytokines in SNs and DRGs following peripheral nerve injury
Previously, the expression patterns of genes in SNs (13) and DRGs (16) at multiple time points after rat SN crush injury were determined and a global view of genetic changes following peripheral nerve injury was obtained. Considering the essential roles of cytokines in tissue remodeling and organ regeneration, IPA bioinformatic analysis was applied to screen upstream cytokines of differentially expressed genes in SNs and DRGs after nerve crush injury. The expression levels of genes coding for these upstream cytokines were further examined and differentially expressed upstream cytokines in SNs and DRGs at 1 day, 4 days, and 7 days after nerve injury were recognized (Table S1).
Table S1. List of differentially expressed upstream cytokines in SNs and DRGs at 1 day, 4 days, and 7 days after rat SN crush injury. Green color indicated down-regulated upstream cytokines while red color indicated up-regulated upstream cytokines.
SN
|
1d
|
4d
|
7d
|
Gene
|
Log Ratio
|
FDR
|
Gene
|
Log Ratio
|
FDR
|
Gene
|
Log Ratio
|
FDR
|
Ccl12
|
14.800
|
0.000
|
Ccl12
|
12.409
|
0.000
|
Ccl12
|
11.630
|
0.000
|
Cxcl2
|
12.446
|
0.000
|
Cxcl5
|
9.615
|
0.000
|
Cxcl2
|
9.887
|
0.000
|
Cxcl3
|
11.164
|
0.000
|
Cxcl2
|
9.041
|
0.000
|
Xcl1
|
9.833
|
0.000
|
Il10
|
10.552
|
0.000
|
Cxcl3
|
8.656
|
0.000
|
Cxcl5
|
9.552
|
0.000
|
Tnfsf14
|
9.445
|
0.000
|
Tnfsf14
|
8.654
|
0.000
|
Cxcl3
|
9.189
|
0.000
|
Il12b
|
7.123
|
0.007
|
Il10
|
8.317
|
0.000
|
Il17c
|
8.328
|
0.001
|
Il1rn
|
6.515
|
0.000
|
Il12b
|
7.491
|
0.001
|
Il12b
|
7.287
|
0.003
|
Csf2
|
6.509
|
0.243
|
Cd40lg
|
7.118
|
0.020
|
Il10
|
7.172
|
0.029
|
Ccl2
|
6.353
|
0.000
|
Ifng
|
6.695
|
0.146
|
Tnfsf14
|
6.772
|
0.059
|
Il1b
|
6.263
|
0.000
|
Il1a
|
6.565
|
0.000
|
Il2
|
6.317
|
0.120
|
Il1a
|
5.728
|
0.000
|
Csf2
|
5.361
|
0.531
|
Ifng
|
6.228
|
0.244
|
Ebi3
|
5.203
|
0.000
|
Ebi3
|
5.052
|
0.000
|
Il1a
|
5.438
|
0.000
|
Il36b
|
5.197
|
0.000
|
Il1rn
|
4.870
|
0.000
|
Wnt3a
|
5.218
|
0.029
|
Ifnb1
|
5.177
|
0.481
|
Osm
|
4.488
|
0.000
|
Il1rn
|
5.205
|
0.000
|
Ifna4
|
5.139
|
0.480
|
Il36a
|
4.383
|
0.530
|
Il17f
|
4.911
|
0.241
|
Cxcl1
|
5.127
|
0.000
|
Ccl2
|
4.041
|
0.000
|
Wnt7a
|
4.643
|
0.059
|
Il11
|
4.997
|
0.000
|
Lif
|
3.971
|
0.000
|
Ebi3
|
4.258
|
0.000
|
Il6
|
4.961
|
0.000
|
Crh
|
3.921
|
0.531
|
Tnfsf11
|
3.650
|
0.000
|
Osm
|
4.939
|
0.000
|
Tnfsf11
|
3.906
|
0.000
|
Slurp1
|
3.650
|
0.003
|
Ccl3
|
4.799
|
0.000
|
Il36b
|
3.828
|
0.000
|
Ccl3
|
3.494
|
0.000
|
Il2
|
4.762
|
0.478
|
Il6
|
3.658
|
0.000
|
Il1b
|
3.375
|
0.000
|
Tnf
|
4.377
|
0.000
|
Il1b
|
3.496
|
0.000
|
Osm
|
3.306
|
0.000
|
Prl
|
4.305
|
0.479
|
Pf4
|
3.458
|
0.000
|
Cd70
|
2.775
|
0.061
|
Il17f
|
3.940
|
0.478
|
Il18
|
3.320
|
0.000
|
Ccl2
|
2.620
|
0.000
|
Wnt3a
|
3.926
|
0.242
|
Ccl22
|
3.222
|
0.000
|
Lif
|
2.468
|
0.000
|
Lif
|
3.667
|
0.000
|
Ccl3
|
3.158
|
0.000
|
Pf4
|
2.463
|
0.000
|
Ccl6
|
3.458
|
0.000
|
Cxcl10
|
3.070
|
0.000
|
Cxcl10
|
2.428
|
0.000
|
Pf4
|
3.436
|
0.000
|
Faslg
|
2.658
|
0.000
|
Il36b
|
2.360
|
0.030
|
Cxcl10
|
3.109
|
0.000
|
Cd70
|
2.658
|
0.081
|
Ccl4
|
2.241
|
0.000
|
Il18
|
3.093
|
0.000
|
Tnf
|
2.643
|
0.000
|
Tnf
|
2.109
|
0.000
|
Timp1
|
3.087
|
0.000
|
Il11
|
2.188
|
0.000
|
Cxcl14
|
2.099
|
0.000
|
Cd70
|
2.542
|
0.110
|
Ccl6
|
2.173
|
0.000
|
Il18
|
2.037
|
0.000
|
Lta
|
2.307
|
0.002
|
Ccl5
|
2.073
|
0.000
|
Il6
|
1.945
|
0.000
|
Tnfsf11
|
2.027
|
0.012
|
Cxcl1
|
2.052
|
0.000
|
Ccl22
|
1.891
|
0.000
|
Il17a
|
1.805
|
0.338
|
Epo
|
1.880
|
0.122
|
Ccl28
|
1.775
|
0.152
|
Vav3
|
1.796
|
0.000
|
Tnfsf13
|
1.874
|
0.000
|
Il11
|
1.775
|
0.000
|
Ccl22
|
1.307
|
0.000
|
Timp1
|
1.663
|
0.000
|
Ccl5
|
1.759
|
0.000
|
Tnfsf13
|
1.221
|
0.000
|
Vav3
|
1.556
|
0.000
|
Faslg
|
1.422
|
0.000
|
Il33
|
1.201
|
0.000
|
Tnfsf13b
|
1.183
|
0.000
|
Ccl6
|
1.412
|
0.000
|
Aimp1
|
1.111
|
0.000
|
Spp1
|
1.089
|
0.000
|
Tnfsf13
|
1.282
|
0.000
|
Nampt
|
1.063
|
0.000
|
Il7
|
1.073
|
0.002
|
Vav3
|
1.204
|
0.000
|
Il21
|
-1.121
|
0.014
|
Wnt4
|
-1.223
|
0.022
|
Il17a
|
1.191
|
0.570
|
Cntf
|
-1.289
|
0.000
|
Tnfsf10
|
-1.490
|
0.000
|
Scgb1a1
|
1.191
|
0.213
|
Tnfsf10
|
-1.749
|
0.000
|
Ctf1
|
-1.844
|
0.000
|
Il9
|
1.191
|
0.570
|
Tnfsf15
|
-2.474
|
0.000
|
Il16
|
-2.032
|
0.000
|
Dkk3
|
1.183
|
0.000
|
Il9
|
-4.936
|
0.622
|
Tnfsf15
|
-2.097
|
0.000
|
Cxcl1
|
1.170
|
0.000
|
|
|
|
Wnt1
|
-2.249
|
0.040
|
Fam3b
|
1.039
|
0.236
|
|
|
|
Il21
|
-2.590
|
0.000
|
Timp1
|
1.013
|
0.000
|
|
|
|
Cntf
|
-2.722
|
0.000
|
Mif
|
-1.046
|
0.000
|
|
|
|
Il12a
|
-2.802
|
0.000
|
Il16
|
-1.171
|
0.000
|
|
|
|
Il17a
|
-3.913
|
0.574
|
Tnfsf15
|
-1.748
|
0.000
|
|
|
|
Csf3
|
-5.724
|
0.095
|
Cntf
|
-2.480
|
0.000
|
|
|
|
|
|
|
Ccl19
|
-2.482
|
0.000
|
|
|
|
|
|
|
Ccl21
|
-3.616
|
0.000
|
DRG
|
1d
|
4d
|
7d
|
Gene
|
Log Ratio
|
FDR
|
Gene
|
Log Ratio
|
FDR
|
Gene
|
Log Ratio
|
FDR
|
Ccl1
|
7.672
|
0.052
|
Il5
|
8.584
|
0.005
|
Prlh
|
8.197
|
0.090
|
Ifna2
|
6.993
|
0.097
|
Prlh
|
8.247
|
0.074
|
Il24
|
7.583
|
0.000
|
Il6
|
6.832
|
0.000
|
Cd40lg
|
7.196
|
0.019
|
Ifna4
|
7.019
|
0.091
|
Prl
|
5.767
|
0.178
|
Il22
|
7.147
|
0.075
|
Il5
|
6.534
|
0.308
|
Il24
|
4.839
|
0.000
|
Ifna4
|
7.069
|
0.075
|
Il6
|
6.316
|
0.000
|
Wnt3a
|
4.803
|
0.097
|
Il24
|
6.951
|
0.000
|
Ifnb1
|
6.058
|
0.311
|
Il1a
|
3.991
|
0.000
|
Il6
|
6.395
|
0.000
|
Il9
|
6.037
|
0.311
|
Ctf2
|
2.669
|
0.103
|
Il12b
|
4.247
|
0.524
|
Prl
|
5.185
|
0.307
|
Cxcl14
|
1.256
|
0.000
|
Il1a
|
3.430
|
0.000
|
Il22
|
5.097
|
0.559
|
Ifnk
|
1.183
|
0.021
|
Wnt3a
|
2.857
|
0.524
|
Csf3
|
4.824
|
0.311
|
Tnfsf14
|
1.084
|
0.509
|
Ccl22
|
1.800
|
0.000
|
Wnt3a
|
4.391
|
0.169
|
Il11
|
1.010
|
0.121
|
Ccl2
|
1.557
|
0.000
|
Il1a
|
3.123
|
0.000
|
Il12a
|
-1.086
|
0.314
|
Csf1
|
1.481
|
0.000
|
Il12a
|
1.775
|
0.001
|
Tnfsf10
|
-1.385
|
0.000
|
Cxcl14
|
1.479
|
0.000
|
Il36rn
|
1.672
|
0.220
|
Il17b
|
-1.501
|
0.305
|
Cd70
|
1.459
|
0.295
|
Ccl22
|
1.672
|
0.001
|
Ccl19
|
-1.501
|
0.003
|
Ccl11
|
1.450
|
0.000
|
Csf1
|
1.528
|
0.000
|
Ccl5
|
-2.153
|
0.000
|
Slurp1
|
1.275
|
0.029
|
Cd70
|
1.409
|
0.333
|
Cxcl10
|
-2.365
|
0.000
|
Lta
|
1.137
|
0.450
|
Ccl2
|
1.202
|
0.000
|
Tnfsf11
|
-2.376
|
0.036
|
Il17c
|
1.137
|
0.450
|
Il1b
|
1.198
|
0.000
|
Cxcl2
|
-3.724
|
0.002
|
Tnf
|
1.000
|
0.002
|
Il11
|
1.158
|
0.060
|
Wnt1
|
-3.882
|
0.369
|
Tnfsf10
|
-1.060
|
0.000
|
Cxcl5
|
1.087
|
0.493
|
Epo
|
-4.641
|
0.371
|
Crh
|
-1.267
|
0.000
|
Ctf2
|
1.087
|
0.654
|
Il17a
|
-4.927
|
0.371
|
Ccl5
|
-1.737
|
0.001
|
Ccl11
|
1.065
|
0.001
|
Il10
|
-5.673
|
0.368
|
Cxcl10
|
-3.079
|
0.000
|
Ifnk
|
1.035
|
0.049
|
Csf2
|
-6.302
|
0.372
|
Epo
|
-4.641
|
0.356
|
Il1rn
|
1.010
|
0.000
|
|
|
|
Csf2
|
-6.302
|
0.357
|
Tnfsf10
|
-1.059
|
0.000
|
|
|
|
Il17b
|
-7.245
|
0.035
|
Il21
|
-1.291
|
0.134
|
|
|
|
Cxcl2
|
-7.790
|
0.000
|
Ccl3
|
-1.372
|
0.033
|
|
|
|
|
|
|
Tnfsf11
|
-2.372
|
0.034
|
|
|
|
|
|
|
Cxcl10
|
-3.337
|
0.000
|
|
|
|
|
|
|
Cxcl2
|
-3.720
|
0.002
|
|
|
|
|
|
|
Epo
|
-4.641
|
0.357
|
|
|
|
|
|
|
Il17a
|
-4.927
|
0.357
|
|
|
|
|
|
|
Csf2
|
-6.302
|
0.358
|
3.2. Demonstration of the expression patterns of upstream cytokines in SNs and DRGs following peripheral nerve injury
To identify the dynamic changes of critical cytokines after peripheral nerve injury, SNs and DRGs intersection cytokines were further studied. A total of 27 cytokines were differentially expressed in both SNs and DRGs at 1 day, 4 days, or 7 days after nerve injury. The expression levels of these cytokines were investigated and displayed in heatmaps (Figure 2). Some cytokines showed similar expression trends in both SNs and DRGs. For example, tumor necrosis factor ligand superfamily member 10 (Tnfsf10) was down-regulated in both SNs and DRGs after nerve injury, CD40 ligand (Cd40lg) was up-regulated in both SNs and DRGs at 4 days after nerve injury, and interleukin-9 (Il9) was up-regulated in both SNs and DRGs at 7 days after nerve injury. Some cytokines, such as Il1rn and C-C motif chemokine ligand 2 (Ccl2), exhibited higher expression changes in SNs as compared with DRGs.
The expression patterns of representative cytokines revealed by sequencing assay were further validated by quantitative PCR experiments. Independent sciatic nerve crush injury experiments were performed in rats for the collection of SNs and DRGs and the conduction of PCR experiments. Cxcl10, a cytokine whose mRNA expressions were up-regulated in SNs but down-regulated in DRGs and I11rn according to sequencing data, as well as Il1rn, a cytokine whose mRNA expressions were up-regulated in both SNs and DRGs according to sequencing data, were selected for PCR validation. Outcomes from PCR experiments demonstrated that the mRNA levels of cytokine Cxc10 were increased in SNs (Figure 3A) but decreased in DRGs (Figure 3B) following nerve injury. The relative abundances of gene coding for Il1rn were up-regulated in both SNs (Figure 3C) and DRGs (Figure 3D). These outcomes were consistent with the expression trends determined by sequencing data (shown in red lines), indicating that sequencing data were of high accuracy.
3.3. Identification of significantly involved signaling pathways of differentially expressed upstream cytokines following peripheral nerve injury
Bioinformatic analyses were performed to evaluate significantly involved signaling pathways of differentially expressed upstream cytokines in SNs and DRGs after nerve injury. Activated signaling pathways that were related to nerve regeneration in up-regulated cytokines and down-regulated cytokines in SNs and DRGs were separately explored (Figure 4). Cytokine-cytokine receptor interaction and chemokine signaling were most strongly enriched signaling pathways. Other significantly enriched signaling pathways included Toll-like receptor signaling, TNF signaling, NOD-like receptor signaling, NF-κB signaling, and JAK-STAT signaling. And these signaling pathways were most robustly involved in up-regulated upstream cytokines in SNs.
3.4. Identification of significantly involved GO biological process categories and gene function regulatory networks of differentially expressed upstream cytokines following peripheral nerve injury
Critical nerve regeneration-related biological processes occurred after sciatic nerve crush injury were further discovered by categorizing differentially expressed upstream cytokines to GO terms. Inflammatory response and immune response were the most significantly involved biological processes and were also most strongly involved in up-regulated upstream cytokines in SNs (Figure 5). Some other inflammatory response and immune response-related biological processes, such as neutrophil chemotaxis, monocyte chemotaxis, cellular response to interleukin-1, also exhibited low p-values, indicating the significance of inflammation and immune responses.
To further reveal the intrinsic link among gene function, we performed a GO analysis on the differentially expressed cytokines in both SNs and DRGs at the same time point, and constructed gene function regulatory networks (GO-Tree) for the significant GO terms (p-value<0.05). The analysis showed that inflammation (Figure 6A) and immune responses (Figure 6B) were induced after peripheral nerve injury. The inflammation-centered network showed that both acute and chronic inflammatory responses were activated after nerve repair. The chemotaxis, migration, and extravasation of various types of cells, including lymphocytes, macrophages, and monocytes, contributed to activated inflammatory response (Figure 6A). The immune-centered network showed that many biological processes related with phenotype modulation of immune cells, such as the activation and proliferation of T cells, B cells, and natural killer cells, were significantly participated in the generated network. It indicated the critical roles of immune cells in nerve repair and regeneration (Figure 6B).