Transcriptomic Analysis of Menstrual Blood-Derived Stem Cells Transplantation Combined with Exercise Training in Promoting Spinal Cord Injury Recovery in Rats

doi:10.12300/j.issn.1674-5817.2024.031

Abstract

Abstract:

Objective To explore the potential therapeutic targets and molecular mechanisms of menstrual blood-derived stem cells (MenSCs) transplantation combined with exercise training in promoting recovery in rats with spinal cord injury (SCI) through transcriptome sequencing analysis. Methods Female SD rats aged two months were selected and a SCI model was established by a hemisection at the tenth thoracic vertebra (T10). The rats were then divided into two groups: the Cell and Treadmill Training (CTMT) group, which received MenSCs transplantation and treadmill training after SCI, and the SCI group (control), with 12 rats in each group. One week after modeling, the CTMT group received a microinjection of 1×10⁵ MenSCs at the injury site, followed by two weeks of weight-supported aerobic exercise training. Spinal cord tissue from the injury site was selected for transcriptome sequencing, and mRNA expression data from both the SCI and CTMT groups were analyzed. Differential gene expression, GO (Gene Ontology) functional enrichment, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment, and protein-protein interaction (PPI) network analyses were performed. Motor function recovery was assessed using the Basso, Beattie, and Bresnahan (BBB) score, while histopathological changes at the injury site were evaluated through hematoxylin-eosin (HE) staining. Real-time fluorescent quantitative PCR and Western blotting were used to verify the expression of differentially expressed genes. Results Transcriptome sequencing analysis showed 247 upregulated genes and 174 downregulated genes in the CTMT group compared to the SCI group. Notably, genes such as Bdnf, Hmox1, Sd4, Mmp3, and Cd163 were significantly upregulated [|log2(FoldChange)|≥0.66, P<0.05]. KEGG pathway enrichment analysis and GO functional enrichment analysis indicated that these differentially expressed genes were mainly involved in growth and development, metabolic reactions, and immune-inflammatory processes, such as axon growth and the electron transport chain. The Bdnf gene was notably enriched in the PI3K-Akt signaling pathway. The BBB score showed that MenSCs transplantation combined with exercise training significantly improved the motor function of SCI rats. HE staining revealed that pathological changes at the injury site were significantly reduced in the treatment group. Furthermore, real-time quantitative PCR and Western blotting confirmed that brain-derived neurotrophic factor (BDNF) mRNA and protein expression levels in the CTMT group were significantly higher than those in the SCI group (P<0.001). Conclusion The combined exercise training with MenSCs effectively promotes the recovery of motor function in SCI rats by upregulating BDNF expression, providing a novel strategy for SCI treatment.

Key words: Spinal cord injury, Transcriptome sequencing, Menstrual blood-derived stem cells, Exercise training, Brain-derived neurotrophic factor, Rats

CLC Number:

R744

QI Longju,CHEN Shiyuan,LIAO Zehua,et al. Transcriptomic Analysis of Menstrual Blood-Derived Stem Cells Transplantation Combined with Exercise Training in Promoting Spinal Cord Injury Recovery in Rats[J]. Laboratory Animal and Comparative Medicine, 2024, 44(5): 531-542. DOI: 10.12300/j.issn.1674-5817.2024.031.

Figures/Tables 7

Figure 1 Distribution of gene expression and principal component analysis of spinal cord tissue samples in the two groups of rats
Note：A showed the distribution of sample expression levels (FPKM, fragments per kilobase of exon model per million mapped fragments); in the figure, the median, interquartile range and extreme values of the samples in SCI (spinal cord injury, as the control, n=3) group and the CTMT (the transplantation of menstrual blood-derived stem cells combined with treadmill training after SCI surgery, n=3) group were relatively close, which indicated that the sequencing results were more homogeneous within the group and the sequencing data quality was high. B showed the principal components analysis (PC1, principal component 1; PC2, principal component 2), and the samples from the same group were clustered together with close proximity, and the differences between samples mainly arose from intergroup differences.

Table 1 Significantly up-regulated and down-regulated genes in spinal cord tissues of spinal cord injury rats after transplantation of menstrual blood-derived stem cells combined with exercise training

表达上调基因 Up-regulated gene	log2(差异倍数) log2 (FoldChange)	t值 t value	P值 P value	表达下调基因 Down-regulated gene	log2(差异倍数) log2 (FoldChange)	t值 t value	P值 P value
Psme3	2.542 363 895	2.672 366 157	0.040 381 267	Ptpdc1	-0.941 169 616	-2.823 151 331	0.033 418 310
Fgd2	1.870 409 798	5.277 683 881	0.002 476 179	RT1-N2	-0.961 937 408	-3.508 946 559	0.014 746 432
Sdc4	1.591 559 898	2.530 079 050	0.048 412 378	Cd79al	-1.007 349 648	-3.292 831 274	0.018 934 378
Cthrc1	1.259 963 833	3.177 793 692	0.021 693 595	Six4	-1.071 889 332	-15.482 205 520	0.000 009 930
RGD1302996	1.205 943 515	12.811 980 370	0.000 027 300	Matn2	-1.137 472 728	-3.927 529 073	0.009 277 287
Rabepk	1.201 118 420	2.962 815 910	0.028 125 989	Clec4a3	-1.273 541 156	-2.512 270 495	0.049 532 946
Bdnf	1.141 863 432	3.525 110 815	0.001 447 754	AABR07051551.2	-1.308 692 094	-2.825 531 707	0.033 319 465
Pkig	1.036 827 364	6.255 767 217	0.001 095 780	Cmtm3	-1.324 596 882	-5.371 453 966	0.002 279 345
AABR07065925.1	0.970 924 295	6.151 591 228	0.001 189 396	Dpy19l3	-1.623 739 520	-3.419 084 313	0.016 347 174
Ehmt1	0.944 670 184	6.578 534 619	0.000 855 702	Cd74	-2.034 312 055	-2.712 776 259	0.038 372 308
AABR07062152.1	0.939 321 669	6.344 499 009	0.001 022 741	Cxcl13	-2.697 259 120	-3.378 916 162	0.017 124 825
Ctbp1	0.898 198 041	5.961 511 123	0.001 385 227	RGD1563231	-3.151 695 504	-2.603 665 774	0.044 063 014
Akr1b10	0.859 136 478	3.868 507 287	0.009 887 528	Jchain	-3.390 915 739	-2.827 200 269	0.033 250 368
Ssh1	0.818 982 469	5.813 203 431	0.001 564 263	Ighm	-3.673 986 054	-3.369 414 954	0.017 314 754
Piga	0.785 438 853	3.303 450 635	0.018 700 031	AABR07060872.1	-5.052 108 514	-3.355 872 330	0.017 589 546

Figure 2 Volcano plot and clustered heatmap of differentially expressed genes in spinal cord tissues between the two groups of rats
Note： A, in volcano plot, horizontal axis indicated the fold change in the gene expression, the vertical axis indicated the statistical significance of differential gene expression profile, the red and blue dots indicated the upregulated and downregulated genes, respectively, and the gray dots indicated the non-DEGs; B, in clustered heatmap, the horizontal axis indicated the sample names, the vertical axis indicated gene names, the pink indicated highly expressed genes, and the blue indicated low expressed genes. SCI was the control group (spinal cord injury, as the control, n=3), and CTMT was the cell and treadmill training group (the transplantation of menstrual blood-derived stem cells combined with treadmill training after SCI surgery, n=3).

Figure 3 GO enrichment and KEGG pathway enrichment analyses of differentially expressed genes in spinal cord tissues of the two groups of rats
Note：A-C indicated the 3 analysis ontologies for Gene Ontology (GO) enrichment analysis, including molecular function (A), cellular components (B) and biological processes (C), respectively. D represented the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The X-axis represented the fold enrichment, the Y-axis represented the enrichment items, and the bubble size indicated the number of genes enriched on each item. The larger the bubble, the greater the number of enriched genes. The color corresponded to P-value. The darker the red color, the more significant the difference.

Table 2 Results of KEGG pathway enrichment analysis of differentially expressed genes in spinal cord tissues of the two groups of rats

信号通路名 Term of signal pathway	计数 Count	富集系数 Gene ratio	P值 P value	基因名 Genes
Rap1 signaling pathway	10	2.506 265 664	0.010 546 665	Angpt1, Rassf5、Vegfb、Lpar1、Rapgef6、Efna4、Met、Bcar1、Rap1gap、Adcy6
Fluid shear stress and atherosclerosis	8	2.005 012 531	0.014 625 259	Prkaa1、Mef2c、Sdc4、Gsta4、Il1r2、Gstt3、Hmox1
PI3K-Akt signaling pathway	13	3.258 145 363	0.014 714 234	Prkaa1、Itga4、Angpt1、Bdnf、 Lama4、Lpar1、Vegfb、Ppp2r3b、Efna4、Gng7、Spp1、Col9a3、Met
ECM-receptor interaction	6	1.503 759 398	0.016 252 600	Sdc4、Itga4、Rgd1565355、Lama4、Spp1、Col9a3
Cytokine-cytokine receptor interaction	11	2.756 892 231	0.016 582 561	Cxcl10、Cntf、Il25、Tnfsf15、Tnfrsf9、Il1r2、Il21r、Ccr7、Ltb、Cxcl13、Ccl17
IL-17 signaling pathway	6	1.503 759 398	0.020 227 194	Cxcl10、Il25、Traf3、Mmp3、Lcn2、Ccl17
Pathways in cancer	17	4.260 651 629	0.020 758 687	Ctbp1、Lama4、Lpar1、Vegfb、Gstt3、Adcy6、Dll4、Gsta4、Peg12、Traf3、Gng7、Rassf5、Ccdc6、Hmox1、E2f3、Met
Chemical carcinogenesis- DNA adducts	5	1.253 132 832	0.040 451 734	Cyp3a9、Hpgds、Gsta4、Gstt3
Cholesterol metabolism	4	1.002 506 266	0.053 168 933	Rgd1565355、Apoh、Apoc1、Apoc3
Focal adhesion	8	2.005 012 531	0.054 767 437	Itga4、Lama4、Vegfb、Spp1、Col9a3、 Pip5k1c、Met、Bcar1
Metabolic pathway	36	9.022 556 391	0.068 256 357	Mtmr2、Gldc、Mtmr14、Pde3b、Gstt3、Hexd、Ehmt1、Adcy6、Abo、Selenbp1、Cyp3a9、Hpgds、Neu4、Spp1、Hmox1、St3gal6、Ca8、Pip5k1c、Csgalnact1、Car1、Uros、Qrsl1、 Coq7、Sirt3、Nt5c3b、Akr1b10、 Cyp2u1、Gsta4、Akr1c15、P4ha3、Piga、Cat、Acot1、Idnk
Mineral absorption	4	1.002 506 266	0.072 932 376	Atp7b、Slc40a1、Hmox1、Slc39a4
Toll-like receptor signaling pathway	5	1.253 132 832	0.074 076 217	Cxcl10、Irf3、Traf3、Spp1、Tlr3
Human papillomavirus infection	11	2.756 892 231	0.079 092 324	Irf3、Itga4、Traf3、Rt1-N2、Lama4、Spp1、Col9a3、Ppp2r3b、Rt1-T24-1、Tlr3、Llgl1
Glycosphingolipid biosynthesis	3	0.751 879 699	0.081 761 102	St3gal6、Abo

Table 2 Results of KEGG pathway enrichment analysis of differentially expressed genes in spinal cord tissues of the two groups of rats

信号通路名 Term of signal pathway	计数 Count	富集系数 Gene ratio	P值 P value	基因名 Genes
Rap1 signaling pathway	10	2.506 265 664	0.010 546 665	Angpt1, Rassf5、Vegfb、Lpar1、Rapgef6、Efna4、Met、Bcar1、Rap1gap、Adcy6
Fluid shear stress and atherosclerosis	8	2.005 012 531	0.014 625 259	Prkaa1、Mef2c、Sdc4、Gsta4、Il1r2、Gstt3、Hmox1
PI3K-Akt signaling pathway	13	3.258 145 363	0.014 714 234	Prkaa1、Itga4、Angpt1、Bdnf、 Lama4、Lpar1、Vegfb、Ppp2r3b、Efna4、Gng7、Spp1、Col9a3、Met
ECM-receptor interaction	6	1.503 759 398	0.016 252 600	Sdc4、Itga4、Rgd1565355、Lama4、Spp1、Col9a3
Cytokine-cytokine receptor interaction	11	2.756 892 231	0.016 582 561	Cxcl10、Cntf、Il25、Tnfsf15、Tnfrsf9、Il1r2、Il21r、Ccr7、Ltb、Cxcl13、Ccl17
IL-17 signaling pathway	6	1.503 759 398	0.020 227 194	Cxcl10、Il25、Traf3、Mmp3、Lcn2、Ccl17
Pathways in cancer	17	4.260 651 629	0.020 758 687	Ctbp1、Lama4、Lpar1、Vegfb、Gstt3、Adcy6、Dll4、Gsta4、Peg12、Traf3、Gng7、Rassf5、Ccdc6、Hmox1、E2f3、Met
Chemical carcinogenesis- DNA adducts	5	1.253 132 832	0.040 451 734	Cyp3a9、Hpgds、Gsta4、Gstt3
Cholesterol metabolism	4	1.002 506 266	0.053 168 933	Rgd1565355、Apoh、Apoc1、Apoc3
Focal adhesion	8	2.005 012 531	0.054 767 437	Itga4、Lama4、Vegfb、Spp1、Col9a3、 Pip5k1c、Met、Bcar1
Metabolic pathway	36	9.022 556 391	0.068 256 357	Mtmr2、Gldc、Mtmr14、Pde3b、Gstt3、Hexd、Ehmt1、Adcy6、Abo、Selenbp1、Cyp3a9、Hpgds、Neu4、Spp1、Hmox1、St3gal6、Ca8、Pip5k1c、Csgalnact1、Car1、Uros、Qrsl1、 Coq7、Sirt3、Nt5c3b、Akr1b10、 Cyp2u1、Gsta4、Akr1c15、P4ha3、Piga、Cat、Acot1、Idnk
Mineral absorption	4	1.002 506 266	0.072 932 376	Atp7b、Slc40a1、Hmox1、Slc39a4
Toll-like receptor signaling pathway	5	1.253 132 832	0.074 076 217	Cxcl10、Irf3、Traf3、Spp1、Tlr3
Human papillomavirus infection	11	2.756 892 231	0.079 092 324	Irf3、Itga4、Traf3、Rt1-N2、Lama4、Spp1、Col9a3、Ppp2r3b、Rt1-T24-1、Tlr3、Llgl1
Glycosphingolipid biosynthesis	3	0.751 879 699	0.081 761 102	St3gal6、Abo

Figure 4 Protein-protein interaction network of differentially expressed genes in spinal cord tissues of the two groups of rats
Note：The size of the dots represented the node and degree, the node with more connections had a larger size; the red color in the dots represented the up-regulation of gene expression in this node, and the blue color represented the down-regulation of gene expression.

Figure 5 Menstrual blood-derived stem cells transplantation combined with treadmill training promotes motor function recovery and BDNF expression in spinal cord injury rats
Note：A showed the local structural pathological changes of the spinal cord injury in the SCI group (spinal cord injury, as the control, n=3) and the CTMT group (the transplantation of menstrual blood-derived stem cells combined with treadmill training after SCI surgery, n=3), observed by hematoxylin-eosin staining (magnified 40 times). B, statistical results of the lesion area in Figure A (^***P<0.001, n=3). C, BBB motor score, a higher score indicated a better recovery of motor function. D, Real-time fluorescence quantitative PCR was performed to detect the changes in the transcriptional level of Bdnf gene in the injured spinal cord in rats of SCI and CTMT groups (^***P<0.001, n=5). E, Western blotting analysis was performed to detect the changes in the protein level of BDNF in SCI and CTMT groups (^***P<0.001, n=5).

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