Construction Of Conductive Hydrogels Carrying Ligustrazine Sustained-release Microparticles And Study Of Their Mechanism Of Repairing Spinal Cord Injur

Background:The difficulty of nerve repair after spinal cord injury is a worldwide clinical problem.On the one hand,early diagnosis and evaluation methods for acute spinal cord injury are simple,which limits early clinical accurate treatment.On the other hand,the imbalance of the microenvironment after spinal cord injury leads to limited nerve regeneration.Traditional Chinese medicine(TCM)has accumulated rich experience in the treatment of spinal cord injury,and many TCM components have been proved to maintain microenvironmental homeostasis and promote nerve regeneration.Local drug-loaded sustained-release technology using tissue engineering materials is a research hotspot at present.It can not only play the role of "bridge"connection of tissue engineering materials,but also enable sustained sustained-release of neurotropic drugs locally,improve the bioavailability of drugs,and is expected to become an important means of spinal cord injury repair.Objective:1 An observational control study was conducted to explore the changes of serum oxidative stress indexes in patients with spinal cord injury in the early stage,and to evaluate the correlation between them and the severity of spinal cord injury,providing a new objective evaluation index for the rapid and accurate early diagnosis and treatment of spinal cord injury.2 The conductive hydrogel carrying tetramethylpyrazine sustained-release particles(GMPT)was constructed,and the physicochemical characterization and biocompatibility of the novel drug-carrying sustained-release composite material were clarified,providing a new idea for the treatment of spinal cord injury by traditional Chinese medicine.3 Based on PI3K/AKT signaling pathway,the targeted mechanism of GMPT hydrogel in regulating oxidative stress and improving microcirculation disturbance after spinal cord injury was investigated,providing preliminary theoretical basis for clinical transformation and application of this novel composite material.Methods:1 In a retrospective case-control study,patients with acute spinal cord injury were enrolled as the experimental group and patients with simple spinal fracture and no neurological dysfunction as the control group.Whole blood and serum were extracted from all patients within 8h after injury.Meanwhile,ASIA impairment grade and ASIA neurological function score were evaluated and analyzed.The expression levels of SOD,MDA,GSH and NO in serum of the two groups were evaluated by biochemical detection,and the differences in serum oxidative stress indexes between the two groups were analyzed by comparison between the two groups.To further explore the correlation between serum oxidative stress indexes and disease severity of patients in the experimental group,screen out biomarkers with potential application value,and provide a new objective evaluation index for the early diagnosis,treatment and evaluation of spinal cord injury.2 GMPT hydrogel was constructed by UV physical crosslinking method,and its physicochemical characterization was determined by measuring its micro-morphology,composition,mechanics,electrochemistry and drug slow release.In vitro and in vivo experiments were conducted to verify its biocompatibility and degradation characteristics,providing technical support for its therapeutic effect and mechanism exploration in the followup treatment of spinal cord injury.3 GMPT hydrogel was implanted into the injured site in rats with complete spinal cord transection to observe the effect and mechanism.Five observation groups were set up,including the Sham operation group,Model group,Tetramethylpyrazine group,GMP hydrogel group and GMPT hydrogel group.The expressions of oxidative stress and vascular endothelial cell apoptosis related indicators were detected at different time points by histamathology and molecular biology techniques,and the mechanism of repairing spinal cord injury by GMPT hydrogel was preliminatively revealed.Results:1 Clinical study(1)A total of 32 patients were included in the experimental group,including 22 males and 10 females,with an average age of(46.84 ± 15.06)years old.ASIA damage classification was as follows:6 cases in grade A,7 cases in Grade B,6 cases in grade C and 13 cases in grade D.A total of 33 patients were included in the control group,including 15 males and 18 females,with an average age of(51.06±13.51)years.There were no significant differences between the two groups in average heart rate,blood pressure and injured segments(P>0.05).(2)Comparison of serum oxidative stress indexes between the two groups:the average expressions of SOD and GSH in the experimental group were significantly higher than those in the control group,and there were significant differences between the two groups(P<0.001);The mean expression levels of MDA and NO in the experimental group were significantly lower than those in the control group,and there were statistical differences between the two groups(P<0.01).(3)Correlation analysis of serum oxidative stress indexes and severity in patients with spinal cord injury:Serum SOD and GSH were positively correlated with ASIA lesion grade(P<0.001),MDA was negatively correlated with AISA damage classification(P<0.001),NO had no significant correlation with ASIA damage classification(P>0.05);There was no significant correlation between serum SOD,NO and ASIA sensory function score(P>0.05),GSH was positively correlated with ASIA sensory function score(P<0.001),MDA was negatively correlated with ASIA sensory function score(P<0.001);Serum SOD and GSH were positively correlated with ASIA motor function score(P<0.05),MDA was negatively correlated with ASIA motor function score(P<0.001),NO had no significant correlation with the ASIA exercise function score(P>0.05).2 Material study(1)The physical and chemical characterization of GMPT hydrogel showed that GMPT hydrogel had good three-dimensional network structure,which provided space for the growth of microvessels and nerve axons after spinal cord injury.The maximum absorption peak of tetramethylpyrazine was located at 295nm.Tetramethylpyrazine was continuously released in GMPT hydrogel,and the cumulative release amount reached 80.3%on the 14th day.GMPT hydrogel has mechanical properties that can match the spinal cord tissue.The introduction of conductive polypyrrole significantly improves its conductivity,which is conducive to the remodeling of electrical microenvironment after spinal cord injury.(2)The results of cytocompatibility experiment showed that neural stem cells co-cultured with GMPT hydrogel did not die in large numbers,indicating that the material had no obvious toxicity and good cytocompatibility.The hemolysis rate of GMPT hydrogel was 1.18%,which met the international standard for biomedical materials.Histocompatibility results showed that after GMPT hydrogel implantation in rats with spinal cord injury,inflammatory indexes such as C-reactive protein and erythrocyte deposition in blood were not significantly increased.Immunohistochemical results showed that GMPT hydrogel could reduce the expressions of proinflammatory factor NF-κB and TNF-α,and increase the expression of anti-inflammatory factor IL-10.HE staining showed no obvious inflammatory cell infiltration in major organs.It indicated that GMPT hydrogel had good biocompatibility.(3)GM,GMP and GMPT hydrogels were buried subcutaneously in rats to observe the degradation of materials in vivo.Macroscopic and HE staining were performed at different time points,and it was found that the hydrogel degraded a lot on day 56 after subcutaneous implantation,and conductive particles still existed in part.The thickness of skin inflammatory layer in GMPT group was lower than that in other hydrogel groups,but there was no significant statistical difference(P>0.05).3 Vivo study(1)BBB score showed that BBB score of GMPT group was significantly higher than that of model group since the 7th day after modeling(P<0.01).The results of the modified Rivlin slant plate experiment were basically consistent with the results of BBB score.The slant plate angle of GMPT group was higher than that of model group at all time points after surgery,and the data between the two groups were statistically different on day 28 after surgery(P>0.05).(2)The results of HE staining of spinal cord tissue showed that the implants in GMPT group were closely combined with tissues on both sides of the broken end,some tissue cavities were still accompanied,the range of inflammatory cell infiltration was less,and the distance between normal tissues on both sides was significantly shorter than those other groups(P<0.05).Masson staining showed that GMPT hydrogel could effectively reduce the area of collagen fiber tissue and significantly reduce CVF value compared with model group(P<0.001).Nissl staining showed that the number of residual neurons in gray matter of GMPT group increased significantly compared with model group(P<0.05).LFB staining showed that some myelin structures were intact in the spinal cord tissue of GMPT group,and the proportion of positive staining area was also significantly increased in comparison model group(P<0.01).(3)The results of spinal cord ELISA showed that SOD and GSH expression levels in GMPT group were significantly increased compared with model group on day 7 after modeling(P<0.001),ROS and MDA were significantly decreased(P<0.001).On day 28 after modeling,SOD and GSH expressions in GMPT group were significantly higher than those in model group(P<0.001),ROS expression was significantly decreased compared with model group(P<0.001),the expression level of MDA was slightly lower than that of model group(P<0.01).(4)Immunohistochemical staining showed that the average optical density(AOD)of ZO-1 in the model group was not significantly different from that in the GMPT group on the 7th day after modeling(P>0.05),the AOD of Occludin was significantly lower than that of GMPT group(P<0.001).The AOD of ZO-1 and Occludin in GMPT group increased significantly compared with that in model group at 28 days after modeling(P<0.001).Immunofluorescence staining showed that on day 7 after modeling,a large number of CD31 labeled vascular endothelial cells could be seen in GMPT group,and the number of apoptosis was significantly less than that in model group.On day 28 after modeling,the number of apoptosis of vascular endothelial cells decreased significantly in all groups,but no obvious apoptosis was observed in GMPT group.Meanwhile,the positive expression of NF200 in GMPT group was also significantly higher than that in model group.(5)Western Blot analysis showed that the expression levels of PI3K and Caspase3 in GMPT group were significantly lower than those in model group on day 7 after modeling(P<0.05),the protein expressions of p-PI3K,p-AKT and Bcl-2 were significantly increased(P<0.05),Bax protein expression level was lower than model group,but there was no statistical difference(P>0.05).On day 28 after modeling,the expressions of PI3K and Bax protein in GMPT group were significantly decreased compared with model group(P<0.05),Bcl-2 protein expression was significantly increased(P<0.05),there were no significant differences in protein indexes between the other two groups(P>0.05).Conclusions:1 After acute spinal cord injury,oxidative stress occurred in the body,serum MDA,NO and other peroxide products increased significantly,and antioxidant indexes SOD and GSH decreased.The expression levels of SOD,MDA and GSH serological indexes were correlated with the severity of spinal cord injury,and could be used as biomarkers to evaluate the severity of spinal cord injury.2 The GMPT hydrogels possess excellent mechanical,electrochemical and drug sustainedrelease properties,as well as excellent biocompatibility and degradability.As a local sustained release carrier of tetramethylpyrazine,it can meet the necessary conditions for spinal cord injury repair and has certain application potential.3 The GMPT hydrogels can inhibit oxidative stress after spinal cord injury by regulating PI3K/AKT signaling pathway,reduce the apoptosis of vascular endothelial cells,protect the structure integrity and permeability of blood-spinal barrier,improve microcirculation disorders,and promote nerve regeneration and functional recovery after spinal cord injury.