Protective Effects Of Ulinastatin On Spinal Cord Injury Through Regulating AMPK/NLRP3 Pathway

Objectives: Spinal cord injury(SCI)is a serious injury to the central nervous system(CNS),which is divided into primary injury and secondary injury,the latter of which is closely associated with a violent inflammatory response.A large number of literatures have reported that AMPK and NLRP3 inflammasome signaling pathways play an important role in the inflammatory response after SCI.Microglia are the immune cells of CNS,and the neuroinflammatory response after SCI is mainly regulated by microglia.Ulinastatin(ULI)is a protease inhibitor,which can remove free radicals and inhibit the release of inflammatory factors in the body by inhibiting the activity of various enzymes.In addition,ulinastatin has been reported to have significant neuroprotective effects.The main purposes of this study are: 1)to analyze the difference of gene expression at different times in the secondary stage following SCI by bioinformatic analyses,and to screen the main signal pathways after SCI;2)To investigate the effects of ulinastatin on AMPK and NLRP3 inflammasome signaling pathway by using immortalized microglia cell line(BV-2 cells).3)To investigate whether ulinastatin can improve SCI in rats and verify the effect of ulinastatin on AMPK and NLRP3 signal pathways in Sprague-Dawley(SD)rat model;4)to collect evidences on NLRP3 and SCI by a bibliometric analysis approach.Methods: 1)Download gene chip data from Gene Expression Omnibus(GEO)database,and divide the samples into sham operation group(Control group),one week following SCI group(Week1 group),two weeks following SCI group(Week2 group),four weeks following SCI group(Week4 group)and eight weeks following SCI group(Week8 group).The data were analyzed by using limma package in R software,and the differential genes between four SCI groups and the Control groups were obtained.GO(gene ontology)function analysis was carried out on the differential genes obtained by most genes group analysis with Cluster Profiler package,and pathway enrichment analysis was carried out by using KEGG database.The PPI network was constructed in the STRING database,and the top 35 key genes were screened out with the help of Cyton CA plug-in of Cytoscape software(version 3.9.1).2)Using cell counting test to detect the effects of different concentrations of ulinastatin on the proliferation activity of BV-2 cells,and determine the appropriate drug intervention concentration for subsequent experiments.The experiment was divided into five groups: control group,model group and low,medium and high dose ulinastatin group.BV-2 cells were treated with 500 ng/ml Lipopolysaccharides(LPS)and 1mm adenosine triphosphate(ATP)to prepare the activation model of inflammatory corpuscles of NLRP3.The effect of ulinastatin on the morphology of BV-2 cells was observed by phase contrast microscope.Enzyme-linked immunosorbent assay(ELISA)and Polymerase Chain Reaction(Polymerase Chain Reaction)were used.The protein secretion and gene expression levels of IL-1β,IL-6 and TNF-α in cell culture supernatant and cell lysate were detected by PCR.Western blot(WB)was used to detect the expression levels of key proteins of AMPK and NLRP3 inflammatory corpuscles in cell lysates of each group,and the possible mechanism of ulinastatin regulating AMPK and NLRP3 inflammatory corpuscles signaling pathway was verified by AMPK inhibitors.3)SD rats were randomly divided into three groups: Sham operation group(Sham group);Spinal cord injury group(SCI+Veh group);Spinal cord injury+ulinastatin group(SCI+ULI group).Rat spinal cord injury model was established by improved Allen’s method.The water content of spinal cord of rats in each group was measured on the third day after operation,and the changes of motor function of rats in each group were evaluated by BBB scoring method.The state of motor neurons in the anterior horn of spinal cord was observed by Nissl staining,and the histopathological changes of spinal cord were observed by Hematoxylin-Eosin(HE)staining,so as to clarify the effects of ulinastatin on rat behavior and spinal cord histopathological changes.The expression levels of inflammatory factors IL-1β,IL-6 and TNF-α in each group were detected by ELISA,and the expression levels of NLRP3 inflammatory corpuscle related protein and AMPK signaling pathway related protein in spinal cord tissue of rats in each group were detected by WB.4)Search the English literature about the relationship between NLRP3 and SCI from 2013 to 2023 in the Web of Science core collection database.Using bibliometric analysis tools,such as VOSviewer,Cite Space and R language,the global trends and research hotspots in NLRP3 and SCI were analyzed.Results: 1)One week after SCI,the most differentially expressed genes were up-regulated.Functional enrichment analysis showed that genes were mainly enriched in immune and inflammatory response pathways.PPI network analysis showed that AKT1,TNF,IL-1β,IL-6 and Caspase-3 were the key genes.2)The results of CCK-8 showed that ulinastatin≤5000 U/ml had no significant inhibitory effect on the proliferation of BV-2 cells.In subsequent experiments,ulinastatin concentrations of 100,1000 and 5000 U/ml were selected to represent the intervention cells with low,medium and high drug concentrations respectively.After LPS+ATP treatment,the cell body of BV-2 cells became larger and the peripheral prominents decreased,and the morphological changes of cells in LPS+ATP+ULI group partially recovered.ELISA and PCR results showed that the expression levels of IL-1β,IL-6 and TNF-α in cell supernatance and BV-2 cells in LPS+ATP group were significantly higher than those in Control group,while the expression levels of IL-1β,IL-6 and TNF-α in LPS+ATP+ULI group were significantly decreased(P < 0.01).The effect was dose-dependent.WB results showed that the expression of p-AMPK in LPS+ATP group was significantly decreased(P<0.01),the expression level of p-m TOR was significantly increased(P<0.01),and the expression level of NLRP3 inflammasome-related protein was significantly increased(P<0.001).In LPS+ATP+ULI group,the AMPK signaling pathway was activated,the expression level of p-AMPK was increased(P<0.01),the expression level of p-m TOR was decreased(P<0.01),and the expression level of key proteins in NLRP3 signaling pathway was significantly decreased(P < 0.01).After pretreatment with compound C(CC),the LPS+ATP+ULI+CC group showed significantly increased NLRP3 inflammasome related protein expression(P<0.01)and IL-1β,IL-6 and TNF-α m RNA expression in cell lysates(P<0.01)compared with LPS+ATP+ULI group.3)BBB score results showed that the postoperative scores of rats in the SCI+Veh group and SCI+ULI group were both 0,and from the 7th day after surgery,the BBB score of rats in the SCI+ULI group was significantly higher than that in the SCI+Veh group(P<0.05).Compared with SCI+Veh group,the spinal cord tissue water content in SCI+ULI group was significantly reduced(P<0.05),and HE and Nissl staining of spinal cord tissue sections showed that the spinal cord tissue edema,cell necrosis and the number of neuron cells in SCI+ULI group were significantly improved.ELISA results showed that the expressions of inflammatory factors L-1β,IL-6 and TNF-α in SCI+ULI group were significantly lower than those in SCI+Veh group(P<0.01).WB results showed that P-AMPK in the spinal cord of rats in SCI+ULI group was significantly higher than that in SCI+Veh group(P<0.01),and the expression levels of major proteins in m TOR and NLRP3 inflamome signaling pathway were significantly lower than those in SCI+Veh group(P<0.01).4)In the part of bibliometrics,we analyzed 218 eligible literatures,including 174 articles and 44 reviews.In the past 10 years,the number of publications about NLRP3 and spinal cord injury has increased rapidly,especially in the past five years.Most of the research on this subject comes from China(n=156,71.56%),followed by the United States(n=24,11.01%)and Iran(n=16,7.34%).China scholars have made great contributions to related research.In addition,in the study of the relationship between NLRP3 and spinal cord injury,Zhejiang University published the most papers(n=18).Beyer Cordian in Germany and Zende Edeladib in Iran are the scholars with the highest average citation value.Conclusion: 1)Bioinformatics analysis results suggest that NF-κB,NLRP3 and other signal pathways play an important role in spinal cord injury.2)Ulinastatin can activate AMPK signaling pathway,inhibit NLRP3 signaling pathway and improve inflammatory response in BV-2 cells.3)Ulinastatin can inhibit the activation of NLRP3 inflammatory corpuscles by regulating AMPK/NLRP3 signaling pathway,and alleviate the neuroinflammatory reaction after SCI.4)In recent ten years,there are more and more researches on NLRP3 in spinal cord injury.By consulting the published literature,it is found that NLRP3 is very important in the pathogenesis of spinal cord injury.