DTI Imaging Characteristics Of Yishen Yangsui Prescription In The Treatment Of Cervical Spondylotic Myelopathy And Its Mechanism Based On RNA-se

Objectives:(1)To investigate the correlation between DTI imaging features and neurological changes in patients with spinal cord cervical spondylosis treated with Yishen Yangsui Formula,and to provide imaging support for the treatment of spinal cord cervical spondylosis with Yishen Yangsui Formula.(2)To use RNA-Seq technology to screen out the core targets and key pathways of Yishen Yangsui Formula for the treatment of spinal cord cervical spondylosis,and to explore the potential mechanism of action of Yishen Yangsui Formula for the treatment of spinal cord cervical spondylosis.METHODS:(1)A clinical study of Yishen Yangsui Formula based on magnetic resonance DTI imaging technique for the treatment of CSM.Thirty patients with CSM who met the criteria and 10 healthy subjects were included as normal controls.The patient group was given the Yishen Yangsui Formula for a period of 2 months,and the cervical spinal cord neurological function score was performed by the cervical JOA score before enrollment and after 2 months of treatment,respectively,and conventional cervical MRI as well as cervical MRI DTI scans were given to record the imaging data.And 10 healthy subjects were given conventional cervical MRI as well as cervical MRI DTI scans,respectively,and recorded image data as healthy controls.Image processing was performed using a Siemens Sygno workstation as well as NEURO3D software to generate color-coded,FA,and ADC maps,and ROI outlining was performed manually for a total of five selected discs to obtain the values of FA,ADC,λ1,λ2,λ3,and MD.The primary efficacy indicators were the values of the cervical medullary DTI imaging parameters ADC,FA,λ1,λ2,and λ3 and the mean diffusion rate MD values.The secondary efficacy indexes were the cervical JOA score and the improvement rate of JOA score.SPSS 25.0 software was used for statistical analysis.One-way ANOVA was used for continuous variables.Correlation data before and after treatment were tested by paired-samples t-test.correlation test between DTI-related parameters and JOA scores was performed by Pearson’s correlation coefficient,and differences were considered statistically significant at P<0.05.The ROC curve was plotted,and the area under the curve was calculated to measure the diagnostic efficacy of each quantitative index to identify CSM,and the cut-off value was determined.The difference was considered statistically significant at P<0.05.(2)To explore the neuroprotective mechanism of Yishen Yangsui Formula on rats with CSM based on RNA-seq technique.Sixty rats were randomly divided into blank group,sham-operated group,model group and Chinese medicine group,15 rats in each group,in which the blank group was not intervened and fed with normal diet;the sham-operated group was given saline by gavage;the model group was given saline by gavage;the Chinese medicine group was given Yishen Yangsui Formula by gavage.The CSM model of rats was established,and the dose of 9.1 g/kg/d was administered once a day for 8 weeks after modeling.The sham-operated group and the model group were gavaged with equal amount(1 mL/100 g)of saline.At the end of the drug administration period,the spinal cords of the corresponding stressed segments were taken and frozen in liquid nitrogen for transcriptome sequencing analysis.After quality control,extraction of total RNA,library construction,and finally up-sequencing,protein gene expression was quantitatively analyzed and differential genes were screened according to |log2FC|>1,P<0.05.The screened differential genes were subjected to GO functional annotation,KEGG pathway analysis,and construction of PPI protein interaction network,and the data were statistically analyzed using SPSS 25.0 software.The measurement data were expressed as mean ±standard deviation(x±s),and t-test was used for comparison between groups.P<0.05 was considered as a statistically significant difference.(3)To validate the effects of the formula on Gpnmb,Cd68 and Caspase1 mRNA and protein expression in CSM rats.BBB score was used for model evaluation and determination of spinal cord function in rats,transmission electron microscopy was used to observe the ultrastructure of spinal cord tissues,and RT-PCR was used to detect the mRNA expression of Gpnmb,Cd68 and Caspase-1 in rat spinal cord tissues.The protein expression of Gpnmb,Cd68 and Caspase-1 was detected by Western Blot.SPSS 25.0 software was used for statistical analysis of the data.Experimental data were expressed as mean ± standard deviation(x±s),and t-test was used for comparison between groups.P<0.05 was considered statistically significant difference.RESULTS:(1)FA,ADC and λ1 values were statistically significantly different between segments in the CSM group(FA:F=5.278,P=0.001;ADC:F=4.127,P=0.005;λ1:F=3.329,P=0.015),while the differences in mean dispersion rate MD were not statistically significant between segments(F=0.8,P=0.54);FA,ADC,λ1 and MD values of the cervical medulla maximal compression level(MCL)and C2/3 segment before treatment in the CSM group were statistically significantly different(FA:t=3.916,P=0.001;ADC:t=-3.025,P=0.007;λ1:t=-2.797,P=0.012;MD:t=1.723,P=0.031);FA and λ1 values were statistically significantly different between segments in the CSM group after treatment(FA:F=2.738,P=0.04;λ1:F=2.857,P=0.046),and FA and λ1 values were statistically significantly different between the cervical medullary MCL and C2/3 segment in the CSM group after treatment(FA:t=2.180(FA:t=2.180,P=0.031;λ1:t=-2.412,P=0.042);DTI-related parameters of different segments of the cervical medulla before and after treatment in the CSM group were analyzed,and the results showed statistically significant differences in FA,ADC,λ1 and MD values after treatment compared with those before treatment(FA:t=-6.938,P=0.000;ADC:t=3.340,P=0.003;λ1=3.744,P=0.001;MD:t=-0.783,P=0.041);statistically significant differences after taking the ratio of the MCL and C2/3 segment(FA:t=-6.938,P=0.000;ADC:t=0.279,P=0.000);Pearson’s correlation coefficient showed a positive correlation between FA values of the MCL and JOA and JOA improvement rates,with statistically significant differences(JOA:r=0.635,P=0.003;JOA improvement rate:r=0.338,P=0.025);ADC values were negatively correlated with JOA and JOA improvement rates,with differences(JOA:r=-0.289,P=0.037;JOA improvement rate:r=-0.164,P=0.041).There was a positive correlation between FA values after taking the ratio of the MCL and C2/3 segment with JOA and JOA improvement rate,and the difference was statistically significant(JOA:r=0.417,P=0.000;JOA improvement rate:r=0.341,P=0.045);there was a negative correlation between ADC values and JOA and JOA improvement rate,and the difference was statistically significant(JOA:r=-1.161,P=0.023;JOA improvement rate:r=-0.283,P=0.000).Linear correlation analysis was used to analyze the relationship between DTI parameters of the MCL of the cervical medulla and JOA scores in patients with CSM,and ROC working curves were plotted,which showed that the area under the curve of the FA value of the MCL for diagnosing CSM was 0.714(95%CI:0.445-0.984),and the diagnostic efficacy was highest when the diagnostic threshold was 0.464 The area under the curve for diagnosing CSM with ADC value was 0.625(95%CI:0.332-0.918),and the diagnostic efficacy was highest when the diagnostic threshold was 0.339,with a sensitivity of 0.714 and a specificity of 0.625.(2)Differential expression analysis was performed for the sham-operated and model groups,and the model and Chinese medicine groups,respectively.303 differentially expressed genes(including 153 up-regulated genes and 150 down-regulated genes)were screened in the differentially expressed genes of the sham-operated and model groups,and 341 differentially expressed genes(including 130 upregulated Among the differentially expressed genes in the model group and the Chinese medicine group,a total of 341 differentially expressed genes(130 up-regulated genes and 211 down-regulated genes)were screened,and the differentially expressed genes in the shamoperated group and the model group,and the differentially expressed genes in the model group and the Chinese medicine group were intersected to obtain 117 co-expressed genes.The top 3 pathway entries were listed according to the significance of P-value value,and their pathways were mainly significantly associated with CREB signaling pathway,neuroactive ligandreceptor interaction,PPAR signaling pathway,heterotrimeric g protein signaling pathway,ErbB signaling pathway,and cell adhesion molecules.(3)Transmission electron microscopy observed the morphology of spinal cord tissue with vacuole-like changes within the laminae structure and intracellularly,with broken and missing mitochondrial cristae.the spinal cord myelin structure in the Chinese medicine group was arranged more regularly and less loosely than that in the model group,with less destruction of mitochondrial morphology and more organelles.the RT-PCR results showed that,compared with the sham-operated group,the spinal cord tissues of rats in the model group showed significantly higher Gpnmb,Cd68,and Caspase1 mRNA expression levels were significantly higher in the model group compared with the sham-operated group(P<0.05).Compared with the model group,the expression levels of Gpnmb,Cd68 and Caspase-1 mRNA in the spinal cord tissues of rats in the Chinese medicine group were significantly lower(P<0.05);Western Blot results showed that the expression levels of Gpnmb,Cd68 and Caspase-1 protein in the spinal cord tissues of rats in the model group were significantly higher compared with the sham-operated group(P<0.05).Compared with the model group,the levels of Gpnmb,Cd68 and Caspase-1 in the spinal cord tissues of rats in the Chinese medicine group were significantly lower(P<0.05).Conclusions:(1)This study found that DTI imaging parameters FA value and ADC value have important value for early diagnosis of spinal cord injury in spinal cord type cervical spondylosis.FA value and ADC value correlate with the severity of spinal cord injury,the more severe the spinal cord injury,the lower the FA and the higher the ADC value,and the measurement of ADC value and FA has a suggestive effect on predicting the prognosis of spinal cord injury.In this study,we found that FA values increased and ADC values decreased in patients with CSM after treatment with the Yishen Yangsui Formula,and DTI imaging technology can provide imaging support for the treatment of CSM with the Yishen Yangsui Formula.(2)Gpnmb,Cd68 and Caspase-1 are the key targets of the treatment of CSM with Yikenyangmyong formula.The mechanism of action of Yishen Yangsui Formula for the treatment of CSM may be achieved by acting on these targets,which involves the molecular activities of neurons and cytosomes and many biological processes such as neurological regulation,prominent conduction and secretory regulation.signaling pathway,neuroactive ligand-receptor interaction,and PPAR signaling pathway,and acts through mediating apoptosis,scorch death,inflammatory response,oxidative stress,etc.(3)Yishen Yangsui Formula can reduce the release of inflammatory factors and regulate the scorching pathway by inhibiting the expression of Gpnmb,Cd68 and Caspase-1,which in turn reduces the inflammatory response in spinal cord tissues and plays a role in protecting the spinal cord and promoting recovery from spinal cord injury.