| Spinal cord injury (SCI) is one of the most common and devastating injuries observed in spine and neurosurgery departments. It can cause permanent disabilities such as paralysis and loss of movement or sensation, and bring heavy burden to society and family. Many studies demonstrated that apoptosis would aggravate the secondary injury to the spinal cord and reduced apoptosis can improve the function recovery following SCI.MicroRNAs (miRNAs or miRs) are endogenous, non-coding, single-stranded RNAs consisting of approximately22nucleotides. They can regulate hundreds of gene expression via RNA-induced silencing complexes (RISC), targeting them to mRNAs where they inhibit translation or direct destructive cleavage. Recently, some studies have demonstrated that miR-21was upregulated following several types of CNS injuries. In many cell types and diseases models, miR-21has been shown to be a strong anti-apoptotic factor.(TMP) is an active biomonomer extracted from traditional Chinese Herbal Medicine. In the previous studies, we had demonstrated TMP treatment had anti-apoptosis in SCI, but the mechanism underlying the anti-apoptosis effects of TMP in spinal cord injury still not clearly. So in the present study, we explored the anti-apoptosis of miR-21in SCI and to gain a deeper insight into the mechanism underlying the anti-apoptosis effects of TMP in spinal cord injury. Chapter one:The microRNA Profiling of Spinal Cord Injury in Rats and the Bioinformatics AnalysisObjectiveTo explore the microRNA profiling of spinal cord injury in rat, and the altered expression of miRNAs may contribute be involved in SCI secondary injury.Materials and methods(1) Animal grouping:12adult male SD rats (180-220g) were random devided into4group (n=3):SCI-ld, SCI-3d, Sham-ld and Sham-3d group. The rats in SCI-ld and SCI-3d group were subjected to the modified Allen’s weight drop impacting to induce the contusive spinal cord injury, and the rats in both Sham group were subjected to laminectomy only.(2) Total RNA extraction:Total RNA from10mm-long spinal cord segments containing the injury epicenter was extracted with TRIzol (Invitrogen).(3) MicroRNA array analysis:After having passed RNA quantity measurement using the NanoDrop1000, the samples were analysised using the miRCURYTM LNA Array (v.16.0).(4) Valited the the microarray results:Real-time qRT-PCR was used to valited the the microarray results.(5) Bioinformatics analysis:Mirbase, Miranda, Mirdbwere and KEGG data base were used to analysis the miRNA target genes and signal pathways.Results(1) Our microarray analysis found22miRNAs dysregulated after injury:9miRNAs increased expression and5decreased expression at1day,3 miRNAs increased expression and5decreased expression at3days post-SCI (>1.5fold change, p<0.05).(2) Real-time qRT-PCR:miR-21was up-regulated at3day post-injury, and didn’t had significat difference at1day post-injury between the two group (SCI-1d/Sham-1d)(P<0.01); miR-126was down-regulated at3day post-injury, and didn’t had significat difference at1day post-injury between the two group (SCI-1d/Sham-1d)(P<0.05); miR-24was down-regulated at3day post-injury, and didn’t had significat difference at1day post-injury between the two group (SCI-1d/Sham-1d)(P<0.01); let-7b was up-regulated at1day post-injury (P<0.05), and didn’t had significat difference at3day post-injury between the two group (SCI-3d/Sham-3d).(3) Bioinformatics analysis:miR-21has22target genes, miR-126has17target genes, miR-24has19target genes, let-7b has7target genes; these target genes were involved in Valine, leucine and isoleucine degradation, Butanoate metabolism, Endocytosis, Propanoate metabolism, Fatty acid metabolism, Tryptophan metabolism, Lysine degradation, Wnt signaling pathway, NOD-like receptor signaling pathway and Chemokine signaling pathway.Conclusion(1) The results of our study showed that SCI changes the expression levels of many miRNAs.(2) Many target genes of the dysregulated expression miRNAs were involved in many signaling pathways which may contribute to SCI secondary injury. Chapter Two:Anti-Apoptosis Effect of microRNA-21in Control of Cell Death after Contusion Spinal Cord Injury in RatsObjectiveIn many cell types and animal models, miroRNA-21(miR-21) had been shown to be a strong anti-apoptosis factor. The present study aimed to explore the effect of miR-21in spinal cord injury in a rat model.Materials and methodsA total of56adult male SD rats (180-220g) were subjected to the modified Allen’s weight drop impacting to induce the contusive spinal cord injury, then random divided into2groups (n=28):Antagomir-21group and Negative control group. In the Antagomir-21group, rats were treated intrathecally with Antagomir-21(1μL/h,20nmol/ml). In the Negative control group, rats were treated intrathecally with Antagomir-21Negative control (1μl/h,20nmol/mL) for3days. At the scheduled time points, rats in both groups were euthanized with an overdose of10%chloral hydrate (10mg/kg). Subsequently, the spinal cord was exposed, and a10-mm long segment of the spinal cord centered at the injury epicenter was harvested. The time of euthanasia was determined according to the different parameters measured:motor function score (BBB) and Lesion identification by cresyl violet staining=28days after SCI (n=4/group); western blot analysis for FasL, PDCD4and PTEN=1,3days after SCI (n=4/group/time point); quantitative real-time PCR analysis of miR-21=1,3days after SCI (n=4/group/time point); Immunohistochemistry staining, TUNEL staining and In situ hybridization=1,3days after SCI (n=4/group/time point). All data was analyzed with SPSS17.0software. T test for the comparison between control and experimental groups. P<0.05was interpreted as significant.Results(1) Behavioral analysis:A spontaneous functional recovery after SCI in the negative control group and antagomir-21group was observed. The rats in negative control group improved more rapidly. The statistical analysis showed a significantly greater increase in the BBB score from Day14to Day28in the negative control group than in the antagomir-21group (Day14, P<0.05; Day21and28, P<0.01).(2) Histologic evaluation:Compared with the negative control group, antagomir-21-treated rats had significantly larger lesion volume and smaller spared tissue.(3) miR-21expression affected by antagomir-21:miR-21was mainly located in the cytoplasm of neuron, and partly located in the cytoplasm of glial cells and oligodendrocyte. Compared with the negative control group, the number of miR-21positive cells was significantly lower in antagomir-21group at1and3days post-injury (P<0.05). qRT-PCR analysis reveal that a significant reduction in expression of miR-21was observed in the spinal cords of antagomir-21group compared with negative control group at1and3days post-injury (P<0.01).(4) Effect of antagomir-21on apopsis:Antagomir-21resulted in a marked increase in the number of TUNEL positive cells when compared with that of the negative control group at Day3(P<0.05). And the number of TUNEL positive cells didn’t have significant difference between the two group.(5) Effects of antagomir-21on the expression of miR-21target genes in spinal cord: 1) FasL:Compared to the negative control, treatment with antagomir-21significantly increased FasL expression at day3(P<0.05), but the expression of FasL was similar in both groups at day1.2) PTEN:PTEN was mainly located in the cytoplasm of neuron, and partly located in the cytoplasm of glial cells and oligodendrocyte. Compared with the negative control group, the number of PTEN positive cells was significantly higher in antagomir-21group at3days post-injury (P<0.05), and was similar in both group at1day post-injury. Western bolt analysis revealed that compared with the negative control group, the expression of PTEN protein was significantly higher in antagomir-21group at3days post-injury (P<0.05), and was similar in both group at1day post-injury.3) PDCD4:PDCD4was mainly located in the cytoplasm of neuron, and partly located in the cytoplasm of glial cells and oligodendrocyte.The number of PTEN positive cells was similar in both group at1and3days post-injury, which was in agreement with western blot data.Conclusion(1) We have demonstrated that miR-21exhibited an anti-apoptosis effect in SCI;(2) The anti-apoptosis effect of miR-21in SCI may partly by regulated the expression of its pro-apoptosis target genes-FasL and PTEN. Chapter Three:Tetramethylpyrazine Attenuates Apoptosis through Modulating microRNA-21after Contusion Spinal Cord Injury in RatsObjectiveIn the previous study, tetramethylpyrazine (TMP) has been proved to have protective effects against SCI. The present study aimed to gain a deeper insight into the mechanism underlying the anti-apoptosis effects of TMP in spinal cord injury.Materials and methodsA total of52adult male SD rats (180-220g) were subjected to the modified Allen’s weight drop impacting to induce the contusive spinal cord injury, then random divided into2groups (n=26):TMP group and Control group. In the TMP group, rats were treated intraperitoneal with TMP (200mg/kg). In the Control group, rats were treated intraperitoneal with Saline (200mg/kg) for3days. At the scheduled time points, rats in both groups were euthanized with an overdose of10%chloral hydrate (10mg/kg). Subsequently, the spinal cord was exposed, and a10-mm long segment of the spinal cord centered at the injury epicenter was harvested. The time of euthanasia was determined according to the different parameters measured:motor function score (BBB) and Lesion identification by Cresyl violet staining=28days after SCI (n=4/group); western blot analysis for FasL, PDCD4and PTEN=1,3days after SCI (n=3/group/time point); quantitative real-time PCR analysis of miR-21=1,3days after SCI (n=4/group/time point); Immunohistochemistry staining, TUNEL staining and In situ hybridization=1,3days after SCI (n=4/group/time point). All data was analyzed with SPSS17.0software. T test for the comparison between control and experimental groups. P<0.05was interpreted as significant.Results(1) Behavioral analysis:A spontaneous functional recovery after SCI in the both groups was observed. The rats in The TMP group improved more rapidly. The statistical analysis showed a significantly greater increase in the BBB score from Day14to Day28in TMP group than in Control group (7and14day, P<0.05;.21and28day, P<0.01).(2) Histologic evaluation:Compared with the Control group, TMP-treated rats had significantly smaller lesion volume and larger spared tissue.(3) miR-21expression affected by TMP:miR-21was mainly located in the cytoplasm of neuron, and partly located in the cytoplasm of glial cells and oligodendrocyte. Compared with the Control group, the number of miR-21positive cells was significantly higher in TMP group at1and3days post-injury (Day1, P<0.05;.Day3, P<0.01). qRT-PCR analysis reveal that a significant increase in expression of miR-21was observed in the spinal cords of TMP group compared with Saline group at1and3days post-injury (Day1, P<0.05;.Day3, P<0.01).(4) Effect of TMP on apopsis:TMP resulted in a marked reduction in the number of TUNEL positive cells when compared with that of the Saline group at Day1and Day3(Day1, P<0.05;.Day3, P<0.01).(5) Effects of TMP on the expression of miR-21target genes in spinal cord:1) FasL:Compared to the Saline group, treatment with TMP significantly reduced FasL expression at day3(P<0.05), but the expression of FasL was similar in both groups at day1.2) PTEN:PTEN was mainly located in the cytoplasm of neuron, and partly located in the cytoplasm of glial cells and oligodendrocyte. Compared with the Saline group, the number of PTEN positive cells was significantly lower in TMP group at3days post-injury (P<0.01), and was similar in both group at1day post-injury. Western bolt analysis revealed that compared with the Saline group, the expression of PTEN protein was significantly lower in TMP group at3days post-injury, and was similar in both group at1day post-injury (P<0.01).3) PDCD4:PDCD4was mainly located in the cytoplasm of neuron, and partly located in the cytoplasm of glial cells and oligodendrocyte. Compared with the Saline group, the number of PDCD4positive cells was significantly lower in TMP group at3days post-injury (P<0.01), and was similar in both group at1day post-injury. Western bolt analysis revealed that compared with the Saline group, the expression of PDCD4protein was significantly lower in TMP group at3days post-injury, and was similar in both group at1day post-injury (P<0.05).Conclusion(1) In the present study, we found that the administration of TMP improved the functional recovery, reduced apoptosis cell death.(2) The anti-apoptosis effect was based on the increasing miR-21expression and inhibiting the expression of apoptosis-related genes after SCI. |
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