| Background:At present,the treatment of spinal cord injury has still been a worldwide problem.A variety of nerve regeneration therapies in the field of basic research have shown great prospects for spinal cord injury,but there is still no effective strategy for regulating nerve repair in clinical treatment.Traditional Chinese medicine has accumulated effective experience in the treatment of spinal cord injury.The varieties of traditional Chinese medicine monomers and compounds have been proved to promote nerve regeneration and induce neuroprotection.In recent years,interdisciplinary cooperation has become an important method to break through the bottleneck of spinal cord injury research.Loading drugs into tissue engineering materials for the treatment of spinal cord injury may be an effective methods to solve this problem.Objective:1 Through clinical trial research,the study aims to evaluate the changes of early hematological indexes in patients with spinal cord injury for exploring their correlation with the severity of the disease,and to provide more objective evaluation strategy for the preliminary evaluation of spinal cord injury.2 Through network pharmacology method and in vivo experiments,this research preliminarily explores the feasibility of tetramethylpyrazine in repairing spinal cord injury.At the same time,we will explains the potential mechanism of tetramethylpyrazine in regulating microcirculation after spinal cord injury,and provides a theoretical basis for subsequent experiments on animals.3 this study is to build conductive hydrogel with loading tetramethylpyrazine,which is explored for the effectiveness of nerve repair,and reveal its targeting mechanism.According to the survey,we hope to provide a new way for spinal cord injury treatment.Methods:1 The research adopted multicenter and cross-sectional method.The subjects with acute spinal cord injury(grade A-D)were included as the experimental group,and the subjects with spinal fracture and no neurological symptoms were included as the control group.The blood samples were taken from all subjects within 8 h after injury,and the ASIA score or damage classification evaluation were performed within 24 h after injury.By comparing the changes of microcirculation,nerve injury,and other indicators in the serum of the two subjects,statistically analyzing the differences in measurement data,we explored the correlation between serological indicators and the severity of the disease to identify biomarkers with potential value.Finally we expect to provide new strategy for the early evaluation and treatment of acute spinal cord injury.2 We collected the target information of tetramethylpyrazine by using a variety of drug data platforms and literature retrieval methods and gathered the target information of spinal cord injury through the GeneCards,OMIM,TTD,Pharmgkb,DrugBank and other databases.The Cytoscape software and string platform were used to build protein interaction(PPI)network from dates taking the intersection of drugs and disease targets,which obtain the core target of tetramethylpyrazine in the treatment of spinal cord injury.We also applicated R language to analyze the intersection target of disease-drugs.Meantime,the Autodocktools software and PyMOL software were used to verify the molecular docking between the core target and the main active components.According to the screening results of the NT,the animal model of transection spinal cord injury was further used to dynamically observe and verify its targeting mechanism from the aspects of behavior,pathology,protein,and gene expression.3 A composite material of methacrylic acid and polypyrrole conductive powder was constructed by one-step method.Tetramethylpyrazine was loaded onto conductive hydrogel by physical blending to construct a new Material,which was tested by in vitro dialysis,has shown the sustained-release properties.At the same time,the material was implanted into the animal model of transection spinal cord injury to verity the biological safety and the feasibility of repairing spinal cord injury,which were evaluated based on the HE,Masson,Nissl,Immunofluorescence,Immunohistochemical staining,and Western blotting methods.4 There were four observation groups,namely sham operation group,model group,conductive hydrogel group,and conductive hydrogel with tetramethylpyrazine group.The hemorheological indexes,pathological staining and microcirculation reconstruction related proteins were evaluated at different periods by the animal model,which initially revealed the mechanism of material for spinal cord injury repair.Results:1 Clinical study(1)There were 10 patients in the experimental group.This group had 7 males and 3 females,and the age ranged from 27 to 68 years,with an average age of(51.5 ± 13.11)years.Among the ASIA damage grades,there were 3 cases of Grade A,2 cases of grade B,1 case of grade C and 4 cases of grade D.Meanwhile,There were 10 patients in the control group.This group had 5 males and 5 females,and the average age was(42.1± 17.97)years.There was no significant difference in BMI,heart rate,systolic and diastolic blood pressure at admission and injured segments between the two groups(P>0.05),and the baseline was comparable.(2)The comparison of hematological indexes between the two groups:the hematocrit increased significantly with significant statistical difference(P<0.05),and there was no significant difference in ESR between the two groups(P>0.05).There was no significant difference in other indexes of coagulation function except D-Dimer(P<0.05).The expression of ET-1 in the experimental group was higher than that in the control group(P<0.05).The expression of VACM-1 in the experimental group was significantly higher than that in the control group(P<0.01).The expression of serum NSE and GFAP increased compared with the control group,and the difference was statistically significant(P<0.05).(3)The correlation analysis between serological indexes and ASIA neurological function scores in patients with spinal cord injury was evaluated.There was no correlation between VCAM-1 expression and the sensory function score or motor function score(P>0.05).The ET-1(r=-0.887,P=0.001),NSE(r=-0.963,P=0.000)and GFAP(r=-0.887,P=0.001)indexes were negatively correlated with motor sensory function score(P<0.05).2 Experimental study Ⅰ(1)By querying temps,herb,and other databases and literature retrieval,We screened 40 targets of this drug,6631 human targets closely related to spinal cord injury,and 36 common targets of the drugs-diseases,which involving 38 common pathways.The PPI network analysis showed that F2,VEGFA,TNF,CXCL8,ELANE,MPO,and CYP19A1 genes might be the potential core targets of tetramethylpyrazine for the treatment of spinal cord injury.Further screening of GO analysis and KEGG analysis surveys,it is found that the regulation of microcirculation after spinal cord injury for tetramethylpyrazine mainly focused on endothelial cell proliferation,migration,and angiogenesis.Meantime,the target molecules closely related to this biological process were HIF-1α,VEGFA,HMOX1,TNF.We also found that HIF1α/VEGF/VEGFR2 signaling pathway could be a greater link to angiogenesis after SCI.(2)Neurobehavioral Evaluation:comparised with SCI group,the scores of BBB and inclined plate in TMP group were significantly different(P<0.05).The improvement of hindlimb motor function in the TMP group was better than that in the SCI group from the third day after injury.(3)The Pathological staining,Western blot,and PCR results of the injured area:immunofluorescence results showed that a large number of marked endothelial cells appeared around the injured area in the TMP group on the 7th day of injury.At the same time,the number of angiogenesis and microvessel density around the injury area were better than those in the SCI group at different times after injury.In addition,the TMP group decreased HIF-1α gene expression at different times after injury,On the 7th day after the injury,which showed a gradual downward trend,and its expression was lower than that in the SCI group.There was significant difference between the groups(P<0.05).The expressions of the VEGF and VEGFR2 gene in the TMP group were up-regulated at different times after injury,which was statistically different from that in the SCI group(P<0.05).The expression trend of HIF-1α,VEGF,and VEGFR2 mRNA was consistent with that of Western blot in The SCI group and TMP group.3 Experimental Study Ⅱ(1)The drug delivery rate of conductive hydrogel with tetramethylpyrazine was detected in vitro.The results showed that TMP had the largest absorption at 295nm in the UV region,and there was a certain sustained-release effect on the TGTP compared with the standard release curve.(2)We also evaluated the effectiveness of the TGTP in biosecurity and nerve regeneration by animal experiment.In 28th days after injury,the TGTP showed that the hydrogel was tightly connected with the spinal cord,and formed an integral part of the spinal cord.Meanwhile,There was no scar tissue around the hydrogel.In addition,the material did not degrade completely in the 28th day after implantation.The He staining of the spinal cord,liver,spleen,and kidney,even the Immunohistochemical staining of the injured area,showed that the implants had good biocompatibility.The Masson,Nissl,NF200-labeled fluorescence staining and protein quantitative analysis showed that it could effectively inhibit the disordered growth of glial scar,inhibit neuronal apoptosis,and promote nerve repair.4 Experimental study Ⅲ(1)There were four observation groups,namely sham operation group,model group,conductive hydrogel group,and conductive hydrogel with tetramethylpyrazine group.The BBB scores and the oblique plate score of the GTP and TGTP groups were compared with the SCI group respectively,and their differences were significantly different from that of the SCI group(P<0.001).After seventh days,the TGTP group had better improvement in hindlimb motor function than that in the GTP group.(2)The hemorheology of rats after SCI showed that the whole blood viscosity and plasma viscosity of rats in the TGTP group were lower than those of the SCI group at different time periods(P<0.05).(3)Immunofluorescence results showed that the TGTP group found a large number of CD31-labeled endothelial cells around the lesion area on the seventh day after injury.At the same time,the number of angiogenesis and micro vessel density around the injured area were better than that of the SCI group and the GTP group at different time points after injury.In addition,the TGTP group increased the expression of VEGF/VEGFR2,PDGF-B/PDGFR-β,and Ang-1/Tie2 genes after injury,which were significantly different from that of the SCI group(P<0.05),and were better than that of the GTP group.Conclusion:1 After acute spinal cord injury,there were a series of pathological changes in the circulatory system,such as coagulation dysfunction and increase of blood viscosity.The expression of ET-1,NSE,and GFAP genes in circulating blood was correlated with the severity of spinal cord injury.That could be potential biomarkers to evaluate the severity of spinal cord injury.2 Tetramethylpyrazine had a multi-target effect,which might improve the hypoxic environment in the injured area after spinal cord injury and regulate the VEGF/VEGFR2 pathway,which was not independent of the HIF-1α gene expression,to promote angiogenesis and affect the recovery of nerve function.3 The conductive hydrogel with tetramethylpyrazine,which had well physical character and biological safety,could intervention the VEGF/VEGFR2,PDGF-B/PDFGR-β,and Ang1/Tie2 signaling pathways to regulate the reconstruction of microcirculation after spinal cord injury and promote nerve repair. |
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