Clinical And Mechanistic Studies Of Electroacupuncture And Spinal Manipulation Therapy For Intervertebral Disc Degeneration

ObjectiveThe study aims to examine the clinical effectiveness of electroacupuncture(EA)and spinal manipulation therapy(SMT)for intervertebral disc degeneration(IVDD).Using Single-cell RNA sequencing(scRNA-seq)and Bulk RNA sequencing(Bulk RNA-seq)to predict whether ferroptosis contributes to IVDD.In animal experiments,EA combined with SMT was demonstrated to prevent IVDD,and ferroptosis was used to investigate the mechanism.Methods1.In accordance with PRISMA guidelines,the protocol has been registered with PROSPERO,and all RCT studies of SMT and EA for low back pain(LBP)have been searched in several databases.The purpose of this study was to evaluate both short-and longterm pain,functional improvement,and adverse events.Research bias was assessed using the Cochrane risk of bias tool,and inclusion and exclusion criteria were defined.2.Patients were recruited according to inclusion and exclusion criteria,and different treatment protocols were implemented for each group.At each observation point,we evaluated the improvement in VAS and ODI scores and the incidence of adverse events in different groups.3.To examine the effect of ferroptosis on the nucleus pulposus(NP)in IVDD,we downloaded the scRNA-seq and Bulk RNA-seq datasets from the GEO official website database and performed quality control,cell annotation,differential analysis,enrichment analysis,intercellular communication analysis,and pseudotime analysis on the included data.4.Following the establishment of a rat model of IVDD in which sham operations were performed as a control group,EA,SMT,and EA combined with SMT were administered.Following 30 days of intervention,the height and volume of the IVD were measured by xray imaging,and the degree of the nucleus pulposus in IVD was assessed by HE staining and safranin O-fast green staining.The mRNA levels of relevant indicators were determined by PCR,the protein expression levels were determined by Western-blotting,and the expression levels were determined by ELISA quantitative analysis.Results1.In the meta-analysis,9 RCT studies with a total of 714 patients were included.Based on moderate-quality evidence,the overall result is that SMT is more effective than acupuncture in relieving pain;whereas there is no statistical difference between SMT and acupuncture for improving function.In a subgroup analysis,SMT was found to reduce pain more than acupuncture at 2 and 12 months,and based on low-quality evidence,the same result was also found at 3 months.Based on a subgroup analysis of function,low-quality evidence suggests that SMT may provide better functional improvement at 4 months than acupuncture.Based on a sensitivity analysis,the above results did not significantly change.In addition,no serious adverse events were reported in any of the included studies.2.Based on the overall VAS multiple comparisons,EA combined with SMT was found to be more effective than EA and SMT in improving pain and function.Based on the overall ODI multiple comparisons,EA combined with SMT leads to better results in the reduction of pain and function compared with EA,while SMT leads to better results in the improvement of function compared with EA.No serious adverse events occurred in any of the three groups during the treatment and follow-up period.There are no serious adverse events associated with EA and SMT.3.The present part analyses the expression of ferroptosis genes in different NP cells to identify potential targets for IVDD through single-cell omics analysis.In the development of IVDD,ferroptosis marker genes and suppressor genes play important roles.There was a higher expression of ferroptosis suppressor genes in healthy NP compared to degenerative NP,this indicates that the ferroptosis suppressor genes are depleted in degenerative NP.Also,oxidative stress genes were analyzed in IVD,and it was found that more cells in the degenerated IVD had oxidative stress reactions when compared with healthy NP.In the single-cell pseudotime analysis,it was found that the differentiation process of NP cells was in the order of initial nucleus pulposus cells,fibrous nucleus pulposus cells,fibrochondrocyte-like nucleus pulposus cells,and fibrochondrocyte-like nucleus pulposus cells.According to intercellular communication analysis,Homeostatic chondrocytes and Stromal cells exhibited a stronger interaction and higher signal intensity.4.Using differential analysis of multiple Bulk RNA-seq datasets,WGCNA,and LASSO analysis,ferroptosis-related genes were finally screened out.Then,the correlation between hub genes was evaluated,and PPI interaction network analysis was conducted in the String database.Additionally,patients with low expression of ferroptosis-suppressor genes had a higher infiltration of M2 macrophages as analyzed by immune cell infiltration.While M1 macrophages primarily promote inflammation,M2 macrophages are primarily antiinflammatory and play a vital role in promoting tissue regeneration.5.The IVDD model was constructed,and the height and volume of the IVD were measured by x-ray imaging.In this model.HE staining and safranin O-fast green staining were used to determine the degree of IVDD.The mRNA and protein levels of NRF2,HO-1,GPX4,and SLC7A11 in each group were determined using PCR and Western blotting.According to the results,EA combined with SMT significantly increased the expression levels of NRF2,HO-1,GPX4,and SLC7A11 compared with the model group.Conclusion1.Acupuncture and SMT are effective treatments for relieving LBP and improving function.As compared to acupuncture,SMT is more effective in relieving LBP;however,in terms of improving function,there is no statistical difference between the two methods.Based on the results of the subgroup analysis of function,SMT may provide better functional improvement than acupuncture;however,sensitivity analysis did not significantly alter the results above.Finally,acupuncture and SMT rarely cause serious adverse effects.2.According to the overall comparison,the Combined group is more effective than both the EA and SMT groups at reducing pain and improving function in patients;the SMT group may be more effective at improving function in patients.The three groups had no serious adverse events during the treatment and follow-up periods.According to the findings of the safety evaluation,EA and SMT are safe,and there are no serious adverse reactions reported.3.The ferroptosis suppressor genes are thought to play a dominant role in intervertebral disc degeneration,and its depletion may contribute to the IVDD.Also,more NP cells in the IVDD were exposed to oxidative stress.In the single-cell pseudotime analysis,it was found that the differentiation process of NP cells was in the order of initial nucleus pulposus cells,fibrous nucleus pulposus cells,fibrochondrocyte-like nucleus pulposus cells,and fibrochondrocyte-like nucleus pulposus cells.A significant number of ferroptosis-related genes were differentially expressed in the bulk RNA-seq datasets of IVDD,and most of the differential signaling pathways were related to ferroptosis,oxidative stress,glutathione synthesis and metabolism,etc.GPX4,SLC40A1,MGST1,FTH1 and other target molecules were also screened.In patients with low ferroptosis-suppressor genes expression,M2 macrophages was increased;these macrophages mainly play an anti-inflammatory role and are important in tissue repair;this indicates that M2 macrophages play an important role in patients with low ferroptosis-suppressor genes expression.4.EA combined with SMT may prevent and treat IVDD primarily by inhibiting oxidative stress;on the other hand,by up-regulating the ferroptosis genes to inhibit ferroptosis in NP cells.According to the results of this study,EA combined with SMT may delay IVDD by up-regulating ferroptosis-suppressor genes,reducing oxidative stress in NP cells,and inhibiting ROS.Additionally,EA and SMT may have a synergistic effect of inhibiting ferroptosis,which then enhances the effect of inhibiting ferroptosis of NP cells in the IVD.