Study On The Mechanism Of Sanbi Decoction In Modulating The Inflammatory Response To Retard Intervertebral Disc Degeneration

Background:The incidence of spinal degenerative diseases(SDD)caused by intervertebral disc degeneration(IVDD)is increasing year by year.It is the main cause of spinal pain,radicular pain and even disability.The development of IVDD involves a number of mechanisms,among which,the inflammatory response mechanism is the more researched and has been proved to have an important role in the development of this disease.Pryoptosis has been found to be an effective therapeutic target for IVDD,and significant therapeutic effects can be achieved by inhibiting pyroptosis.Clinical interventions for IVDD include conservative analgesic medications or discectomy surgery.However,the effectiveness and longevity of these strategies remain unsatisfactory.Sanbi decoction(SBD)has been used for thousands of years in the treatment of SDD,but the specific mechanism is unclear due to the complexity of its components.In this study,we firstly applied bibliometric methods to analyze the research on inflammatory mechanisms in IVDD.Secondly,we further validated the important role of pyroptosis in promoting the development of IVDD through clinical samples.Than,we used transcriptomics methods to systematically explore the potential targets and pathways of SBD for the treatment of NPCs degeneration.Subsequently,we clarified through cell and animal experiments that SBD can treat IVDD by inhibiting inflammatory factors and apoptosis,promoting ECM remodeling,and other mechanisms.This study combines literature analysis,transcriptomics methods,cellular animal experiments,molecular biology research and others to explore the inflammatory mechanism and the important role of pyroptosis in IVDD,and further evaluate the pharmacological mechanism of SBD.This can provide a basis and reference for basic clinical research on TCM compound therapy for SDD.Part Ⅰ:Bibliometric analysisObjective:The bibliometric analysis of the research on inflammatory mechanisms in IVDD elucidates the current status,hotspots and possible future trends of the research,which can provide reference for researchers in this field.Methods:In this study,we searched the Web of Science Core Collection(WOSCC)database from 1 January,2001 to 7 November,2023.We analyzed and visualized the content using software packages such as Citespace,Vosviewer,and Bibliometrix.Results:This study found that the number of annual publications has been increasing year by year.In terms of journal research,Spine not only has the highest publication volume,but also has significantly higher total and average citations than other journals.In terms of country analysis,China has the highest publication volume,while the USA has the highest citation volume and total link strength.Institutional analysis shows that Sun Yat sen University has the highest publication volume and total link strength,while Thomas Jefferson University has the highest total citation volume.The author’s analysis shows that Ohtori,S has the highest publication volume,Risbud,MV has the highest citation,and Inoue,G has the highest total link strength.These authors have all made important contributions to the development of this field.The results of citation and co-citation analysis indicate that these highly cited literature are mainly classic studies and explorations of the inflammatory mechanisms in the pathogenesis of IVDD.The results of keyword analysis indicate that the current research hotspot is still further exploration of mechanisms and treatment research.Pyroptosis,necroptosis,autophagy,ferroptosis,oxidative stress,bacterial infection,CirRNA,and other mechanisms may be the focus of research in recent years.In addition to the treatment of IVDD with traditional monomers,drugs and compounds,the research in recent years tends to stem cell therapy,exosomes,hydrogels,scaffolds and others.Conclusions:The research on the inflammatory mechanism of IVDD has received widespread attention from industry professionals,and the number of publications is still increasing year by year.The current hot topic is still further exploration of the mechanism and research on treatment methods.Part Ⅱ:Clinical observationsObjective:The aim of the present study was to explore the correlation between IVDD and pyroptosis through clinical research.Methods:Fifty patients who underwent PELD in our hospital due to LDH were included.According to the rating criteria of MRI,the patients were divided into mild degeneration group(grade Ⅱ and Ⅲ)and severe degeneration group(grade Ⅳ and Ⅴ).The pain and function of patients were scored preoperatively,and the correlation between them and the grade of IVDD was searched.The differences of pyroptosis and NF-κB-related proteins between different degrees of degeneration were detected by WB assay.Results:The results of correlation analysis showed that there was a significant positive correlation between the degree of IVDD degeneration with VAS and ODI scores(P<0.05).WB results showed that the protein expressions of NLRP3,C-caspase1,GSDMD-N and NF-κB signaling pathways in the severe degeneration group were significantly higher than those in the mild degeneration group(P<0.05).Conclusions:When the degree of IVDD is aggravated,the pain in the lumbar and legs and the limitation of lumbar function may be more obvious.The higher the degree of IVD degeneration,the higher the expression of pyroptosis and NF-κB signaling pathway related proteins in NP tissue.Part Ⅲ:Transcriptomics researchObjective:Evaluate the pharmacological mechanism of SBD in the treatment of IVDD using transcriptomics methods.Methods:Finding the effective concentration of IL-1β to establish a cell degeneration model through CCK8 experiment.Validate the model through QPCR to evaluate its effectiveness.Detect the effect of SBD on the activity of RNPCs through CCK8.Detecting the proliferation effect of SBD on RNPCs through EDU.Evaluate the difference in gene expression between the model group and the SBD group through volcano and heat maps.Through GO and KEGG enrichment analysis search for possible pathways of action of SBD.Analyze the important gene set of the detection pathway through GSEA analysis.Results:When the concentration of IL-1β reaches lOng/ml,intervening in RNPCs for 24 hours can establish an effective degenerative model.IL-1β can significantly increase the mRNA expression of IL-1β,MMP13,and ADAMTS4 in the extracellular matrix(ECM),while reducing the mRNA expression of Collagen II and Aggrecan.The transcriptome sequencing results showed that compared with the model group,the SBD group upregulated 503 genes and downregulated 726 genes.We conducted data mining and enrichment analysis on differentially expressed genes and found that SBD mainly inhibits inflammatory targets when intervening in degenerated NPCs.The KEGG enrichment analysis results indicate that the top ranked pathways are TNF signaling pathway,IL-17 signaling pathway,PI3K-AKT signaling pathway,NF-κB signaling pathway,NLRP signaling pathway and others.We further confirmed the important role of the NF-κB signaling pathway and NOD like receptor signaling pathway in this study through GSEA analysis,indicating that SBD can alleviate the degeneration of IL-1βinduced NPCs by inhibiting these two pathways.Conclusions:IL-1β intervention can serve as an effective method for establishing degeneration of NPCs.The inhibition of RNPCs degeneration by SBD may be related to reducing inflammatory response,and the NF-κB signaling pathway and NOD like receptor signaling pathway may play important roles in it.Part Ⅳ:Cell experimentsObjective:Exploring the pharmacological mechanism of SBD in treating IVDD through cell experiments.Methods:Establishing an inflammatory degeneration model by intervening with IL-1β in NPCs,CCK8 detecting cell viability,EDU detecting cell proliferation,TUNEL method and cell apoptosis and necrosis detection kit detecting cell apoptosis and necrosis,ELISA detecting inflammatory factors,SA-β-Gal staining was used to detect cell aging,ROS detection kit was used to detect reactive oxygen species,protein expression was detected through immunofluorescence and Western blotting,and gene expression was detected through QPCR.Results:TUNEL,WB,qPCR and ELISA results showed that SBD could effectively inhibit cell apoptosis.The results of WB and qPCR showed that SBD could inhibit the degradation of ECM.WB,qPCR,immunofluorescence and ELISA results showed that SBD could inhibit pyroptosis.The results of WB and qPCR showed that SBD could inhibit the activation of NF-κB signaling pathway.ELISA results showed that SBD could reduce the expression of inflammatory factors.SA-β-Gal staining results showed that SBD could inhibit cell senescence;ROS detection showed that SND could reduce the expression of ROS and inhibit cellular oxidative stress.The results of apoptosis and necrosis detection showed that SBD could inhibit apoptosis and necrosis of cells.Conclusions:SBD can inhibit cell apoptosis,regulate ECM metabolic balance,inhibit inflammation and pyroptosis,which may be related to the inhibition of NF-κB signaling pathway.In addition,SBD can also regulate cell senescence,oxidative stress and cell necrosis.Part Ⅴ:Animal experimentsObjective:Exploring the efficacy and safety of SBD in the treatment of IVDD through animal experiments.Methods:The IVDD model of rats was established by intervertebral disc puncture.The effectiveness of the model was evaluated by X-ray and MRI.After successful modeling,the drug group was given different concentrations of water extract of SBD by gavage,and the control and model group were given the same amount of distilled water by gavage.After 4 weeks,we performed imaging evaluation on the rats,and then euthanized the rats and took the serum,liver and kidney tissues and IVD tissues for subsequent experiments.We evaluated the safety of SBD by liver and kidney weighing,functional detection and tissue staining,detected serum inflammatory factors by ELISA kit,and evaluated the efficacy of SBD by X-ray detection,MRI examination,immunohistochemistry,Western blotting,qPCR,HE staining and SO/FG staining.Results:The results of X-ray and MRI showed that the IVDD model could be successfully established by puncturing the rat caudal IVD.Liver and kidney function and HE staining showed that SBD did not have toxic effects on liver and kidney at a suitable concentration.The imaging results showed that SBD could significantly alleviate the decrease of NP water content and IVD height caused by puncture.He staining and SO/FG staining showed that SBD could restore the degenerated IVD tissue structure.Immunohistochemical(IHC)results showed that SBD could increase the expression of collagen II and reduce the expression of MMP13 in degenerated IVD tissues.ELISA results showed that SBD could reduce the expression of TNFα,IL-1β,IL-6 inflammatory factors in rat serum.WB and qPCR results showed that SBD could promote ECM remodeling,inhibit its degradation,and inhibit pyroptosis to alleviate IVDD.Conclusions:SBD can remodel the metabolic balance of ECM and inhibit IVDD by inhibiting inflammatory reaction and pyroptosis without obvious toxic and side effects.