| BackgroundRadicular cervical spondylosis(CR)is the most common type of cervical spondylosis.Cervical radiculopathy pain is a typical symptom of CR,and its incidence ws significantly higher than that of motor dysfunction.Traditional Chinese Medicine(TCM)thought this disease belongs to "arthralgia syndrome",because of "qi and blood stagnation,stop for a long time" produces "meridian obstruction,become general pain"clinical manifestations.TCM was a common conservative measure for the treatment of cervical spondylotic radiculopathy.Proprietary Chinese medicine was a branch of herbal medicine based on modern technology and widely used in clinical practice.As a proprietary Chinese medicine for the treatment of CR,Jingtong granule had the effect of promoting blood circulation,removing blood stasis and relieving pain,and had good clinical efficacy and safety.However,there was still a lack of expert consensus based on evidence-based evidence to guide the clinical application of this drug.Meanwhile,the pathogenesis of CR and the therapeutic mechanism of Jingtong granule need to be further explored.These problems seriously restricted the promotion and application of Jingtong granule in the clinical treatment of CR.Therefore,we systematically combed the existing literature and carried out relevant evidence-based research based on systematic evaluation and GRADE classification,in order to find existing research problems and provide ideas for future research.Meanwhile,high-throughput proteomics and molecular biology techniques were used to explore the pathogenesis of the disease in the central and peripheral nerves,and to study the intervention mechanism of Jingtong granule,aiming to provide molecular scientific evidence for the prevention and treatment of CR with TCM.Research ContentsPart One:Study on expert consensus of Jingtong granule in the treatment of cervical spondylotic radiculopathy based on GRADE classification systemObjective:Based on evidence-based medicine,the clinical expert consensus of Jingtong granule was formulated to guide the clinical application of Jingtong granule in CR.Methods:Based on the evidence-based methodology,this consensus adopted the internationally recognized clinical guideline formulation process.Firstly,the consensus clinical problems were determined through the combing of instructions,expert interviews,online questionnaire survey and expert consensus voting,and then the standardized literature retrieval strategy,inclusion criteria and exclusion criteria are formulated.The clinical research of Jingtong granule in the treatment of CR was retrieved and described according to the Picos principle,Secondly,based on the grade evidence classification system,the Review Manager 5.4.1 software was used for data synthesis and bias risk assessment.The GradePro tool formed the evidence summary table.After the working group formed the recommended opinions and expert consensus items,the final clinical expert consensus content was determined by voting through the expert nominal group method.Results:8 clinical problems were confirmed,and 5 recommendations and 3 expert consensus recommendations were formed,namely,cervical tong granule alone or combined with other therapies had good efficacy for CR,Jingtong granule could be used alone,and the recommendation was low-level evidence and strongly recommendation.Jingtong granule could be combined with Western medicine,and the recommendation was very low evidence and strong recommendation;Jingtong granule can be combined with external medication,and the recommendation is very low evidence and strong recommendation.Jingtong granule could joint non-drug therapy,recommendations for low evidence and strongly recommend Jingtong granule could be used alone treatment of postoperative patients with CR,recommendations for very low evidence and strongly recommendation.The expert consensus item is that Jingtong granule could be used for related patients with qi stagnation and blood stasis syndrome.Two weeks as a course of treatment,1-3 courses can be used or extended as appropriate.Jingtong granule had good clinical safety.Conclusion:Jingtong granule was effective in the treatment of cervical spondylotic radiculopathy,which was recommended for clinical use.Part Two:Study on the pathogenesis of CR based on proteomics and WGCNAObjective:Explore and verify the similarities and differences of the pathogenesis of CR in the peripheral and central nerves through high-throughput proteomics.Methods:The rat model of CR was established by chronic dorsal root ganglion compression.After completing the behavioral evaluation of mechanical hyperalgesia,thermal hyperalgesia and spontaneous pain,the materials of dorsal root ganglion,spinal cord and cerebrospinal fluid were obtained.Nissl staining was used to evaluate the number of normal neurons and TUNEL staining was used to evaluate the apoptosis of neurons.TMT labeled quantitative proteomics was used to detect the dorsal root ganglion and spinal cord of rats.Combined with conventional differential analysis and WGCNA weighted co expression network analysis,the protein data were deeply mined,and the biological function was analyzed from multiple perspectives such as go,KEGG and GSEA.Western blot(WB),ELISA,double immunofluorescence staining and transmission electron microscopy were used to verify and explore the key proteins and key pathogenesis.Results:The pain related sensory dysfunction in the model group gradually worsened after operation,and the model group showed obvious tingling hypersensitivity on the first day after operation.Compared with the force threshold of plantar mechanical tingling in the model group and the sham operation group,there were significant statistical differences from the first day to the 14th day after operation(P<0.05).Compared with the sham operation group,there was no significant difference in the plantar thermal radiation pain threshold between the two groups from the first day to the third day after operation(P>0.05),and there was significant difference in the plantar thermal radiation pain threshold from the seventh day to the 14th day after operation(P<0.05).There was significant difference in the number of spontaneous foot licking between the model group and the sham operation group at each observation time point from the first day to the 14th day after operation(P<0.05).Nissl staining showed that there was obvious injury in the nerve tissue of the model group.The number of normal neurons in the sham operation group(40.75±7.676)was significantly higher than that in the model group(28.50 ± 5.916)(P<0.05);The number of normal neurons in the sham operation group(63.50 ± 11.96)was significantly higher than that in the model group(45.75±2.217).TUNEL staining showed that the number of apoptotic cells in the model group increased significantly.In the spinal dorsal horn,the number of apoptotic cells in the sham operation group was 23.38±3.68,and the number of apoptotic cells in the model group was 30.96±3.41.There was significant difference between the two groups(P<0.05);In dorsal root ganglion,the number of apoptotic cells in sham operation group was 21.49 ± 5.88 and that in model group was 33.77±3.89.There was significant difference between the two groups(P<0.05).TMT labeled quantitative mass spectrometry showed that the distribution of detection results was in line with the theoretical situation.Conventional data difference analysis combined with WGCNA found that the potential key molecules shared by the two tissues after modeling surgery intervention were 2 up-regulated key molecules(RPN2 and HMGB1)and 5 down-regulated key molecules(GDAP1,MTX2,DNAJA3,HSPA4L and SEPT3).Biological function analysis suggests that oxidative stress and inflammatory immune response are related to the disease.Western blot finally verified that the down-regulated GDAP1 protein and up-regulated HMGB1 protein had significant changes in both tissues.Double immunofluorescence further confirmed that there were differences in the expression of GDAP1 or HMGB1 in neurons of dorsal root ganglion and spinal cord.In the study of antioxidant capacity and mitochondrial morphological changes,it was found that SOD2 and catalase in dorsal root ganglion and spinal cord were detected by WB.It was found that catalase protein in model group was significantly lower than that in sham operation group(P<0.05);In the spinal cord,the expression of SOD2 protein in the model group was significantly lower than that in the sham operation group(P<0.05).We observed the spinal cord and dorsal root ganglion under transmission electron microscope.It was found that in the model group,the number of mitochondria in dorsal root ganglion and spinal cord neurons increased and decreased,the mitochondrial cristae disappeared,and even white vacuoles appeared,the cells appeared edema,and a few cells swelled and ruptured;Demyelination could be seen in axons in ganglia.In the study of inflammation related pathways,Western blot showed that the degree of ERK1/2 phosphorylation activation in the model group was significantly higher than that in the sham operation group(P<0.05);The expression of NFKB1 in the model group was significantly higher than that in the sham operation group(P<0.05).The results in spinal cord tissue were similar to those in dorsal root ganglion.The degree of ERK1/2 phosphorylation activation in model group was also significantly higher than that in sham operation group(P<0.05);The expression of NFKB1 in the model group was significantly higher than that in the sham operation group(P<0.05).The cerebrospinal fluid of rats was detected by ELISA.The study found that TNF-α and IL-1β in cerebrospinal fluid of model group were higher than those in the sham operation group,and the difference was statistically significant(P<0.05).Conclusion:The key proteins GDAP1 and HMGB1 in the two tissues,and the decreased antioxidant stress ability and increased inflammatory response regulated by the key proteins,may be the key pathogenesis shared by the peripheral(dorsal root ganglion)and central(spinal cord)of pathological pain of CR.Part Three:Study on the mechanism of Jingtong granule in the treatment of CRObjective:To explore whether Jingtong granule can regulate the key proteins GDAP1 and HMGB1 in the pathogenesis of cervical spondylotic radiculopathy,reduce neuronal mitochondrial damage and improve the inflammatory microenvironment.Methods:Seventy-two female SD rats were selected and numbered successively,and divided into Sham group,Model group,jTKL-L group,JTKL-M group,JTKL-H group and IBU group by random number table method.Each group of 12,3 days after the building began to fill the stomach,a total of lavage after 2 weeks to be put to death all the rats,cerebrospinal fluid,dorsal root ganglion,and spinal cord.The rats were evaluated for mechanical hyperalgesia,thermal hyperalgesia and spontaneous pain.Western Blot detection,RT-PCR detection,ELISA detection,transmission electron microscopy and other techniques were used to explore and analyze the regulation of Necking Tong granules on the protein and mRNA expressions of key molecules GDAP1 and HMGB1,and to observe the changes of neuron mitochondrial injury and inflammatory microenvironment.Results:There were statistically significant differences between JTKL-H group and Model group on 10,14 and 17 days after surgery(P<0.05),and there were statistically significant differences between JTKL-M group and Model group on 10,14 and 17 days after surgery(P<0.05).There were statistically significant differences between JTKL-L group and Model group on 14 and 17 days after surgery(P<0.05),and there were statistically significant differences between IBU group and Model group on 14 and 17 days after surgery(P<0.05).There was statistical difference in the plantar heat radiation pain threshold between JTKL-H group and Model group on day 10,14 and 17 after surgery(P<0.05),and there was statistical difference in the plantar heat radiation pain threshold between JTKL-M group and Model group on day 14 and 17 after surgery(P<0.05).There was no statistical difference between JTKL-L group and Model group in plantar heat radiation pain threshold on the 14th and 17th day after surgery(P>0.05),and there was statistical difference between IBU group and Model group on the 17th day after surgery(P<0.05).There was statistical difference in the licking times of JTKL-H group on day 10,14 and 17 after surgery compared with Model group(P<0.05),and there was statistical difference in the licking times of JTKL-M group on day 14 and 17 after surgery compared with Model group(P<0.05).There was no statistical difference in the licking times of JTKL-L group on the 14th and 17th day after surgery compared with Model group(P>0.05),and there was statistical difference in the licking times of IBU group on the 17th day after surgery compared with Model group(P<0.05).The results of GDAP1 protein expression showed that the P value of One-way ANOVA was 0.001 for each group.The expression values of Sham group were significantly higher than those of the other five groups(P<0.05).The expression level of JTKL-M group was higher than that of Model group(P<0.05).The results of HMGB1 protein expression showed that the P value of one-way anOVA in each group was less than 0.0001.The expression level of Sham group was lower than that of Model group and IBU group(P<0.05).The expression value of Model group was higher than that of JTKL-L,JTKL-M and JTKL-H groups(P<0.05);The expression value of JTKL-L group was higher than that of JTKL-M and JTKL-H groups(P<0.05).The expression value of JTKL-M group was lower than that of IBU group(P<0.05).JTKL-H group was lower than IBU group(P<0.05).The mRNA expressions of GDAP1 and HMGB1 in dorsal root ganglion tissues of rats were detected by PCR 14 days after gavage.The One-way ANOVA of GDAP1 mRNA expression in the 6 groups was P=0.001;The expression level in Model group was significantly lower than that in Sham,JTKL-M and JTKL-H groups(P<0.05).The P value of HMGB1 mRNA expression in the 6 groups was 0.0054 by One-way ANOVA.The Model group was higher than JTKL-M and JTKL-H(P<0.05).The results of transmission electron microscopy showed that the cell structure of JTKL-H group was basically the same as that of JTKL-M group,and the mitochondrial structure was basically normal and the cell damage was relatively light.The expression levels of TNF-α and IL-1β in cerebrospinal fluid of rats were determined by ELISA;The expression level in Model group was significantly higher than that in Sham,JTKL-L,JTKL-M,JTKL-H and IBU groups(P<0.05).In addition,there was statistical difference between IBU group and JTKL-L group(P<0.05).One-way ANOVA of IL1β expression in 6 groups showed P value=0.0001.The expression level of Sham group was lower than that of Model,JTKL-L and IBU groups(P<0.05).Model group was higher than JTKL-M and JTKL-H(P<0.05).Conclusion:In the rat model of CR,the high concentration of Jingtong granule was found to significantly reduce mechanical pain allergy,thermal hyperalgesia and spontaneous pain.At the same time,Jingtong granule could increase the expression of GDAP1 protein and mRNA in dorsal root ganglion,reduce the expression of HMGB1 protein and mRNA,inhibited the secretion of inflammatory factors,reduced the mitochondrial structure damage of neuronal cells in dorsal root ganglion,and protected neuronal cell damage. |
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