Efficacy And Mechanism Of Tongluo Qubi Decoction Combined With Percutaneous Endoscopic Lumbar Discectomy In The Treatment Of Lumbar Disc Herniation

ObjectiveTo evaluate the effect of clinical path(CP)of TCM therapy combined with percutaneous endoscopic lumbar discectomy(PELD)in the treatment of lumbar disc herniation(LDH),and to evaluate the effect of Tongluo Qubi Decoction combined with See electromagnetic-navigation endoscopic spinal surgery system(SEESSYS)for LDH,and to clarify the mechanism of Tongluo Qubi Decoction in the treatment of LDH by network pharmacology and experiments.Methods(1)Experiment 1:From January 1,2020 to December 30,2020,patients who underwent PELD in Guangdong Provincial Hospital of Chinese Medicine were divided into test group and control group according to whether they were included in CP management.In the control group,the routine diagnosis and treatment plan was applied during the perioperative period,while the test group was included in the CP management and applied TCM characteristic comprehensive therapy including wrist-ankle acupuncture,moxibustion,auricular acupoint sticking,acupoint sticking,and TCM hot compress during the perioperative period of PELD.The general data of the two groups were collected,including age,gender,BMI,surgical segment,etc.Perioperative indicators included medical costs,length of hospital stay,frequency of intravenous or intramuscular analgesic,and related surgical complications and adverse events were recorded.The clinical efficacy was evaluated by Oswestry Disability Index(ODI),Visual Analogue Scale(VAS),Japanese Orthopedic Association(JOA)and TCM syndrome score,modified MacNab was used to evaluate the Excellent and good rate,readmission rate and reoperation rate were also analyzed.(2)Experiment 2:Patients with LDH who were hospitalized in our hospital and received SEESSYS treatment from January 2021 to May 2021 were selected.According to whether using Tongluo Qubi Decoction in the perioperative period,they were divided into two groups,the control group was SEESSYS group,and the test group was SEESSYS combined with Tongluo Qubi Decoction group.The two groups of patients were given Celecoxib Capsules and Methylcobalamin Tablets after operation for four consecutive weeks;on this basis,the test group was given Tongluo Qubi Decoction orally,once a day for four consecutive weeks.General observation indicators included:patient age,gender,BMI,operation time,intraoperative fluoroscopy times,and length of hospital stay,and recorded whether there were complications such as discontinuation of surgery,change of surgical plan,neurovascular injury,cerebrospinal fluid leakage,and hematoma.The ODI,short-form of McGill pain questionnaire(SF-MPQ)and TCM syndrome scores were used to evaluate the clinical efficacy,and the quality of life was evaluated by the SF-12 scale.Follow-up and scoring were performed before surgery and on the 1st day,6 months and 12 months after surgery,and the modified MacNab criteria was used to evaluate the excellent and good rate at the last follow-up.(3)Experiment 3:The targets of LDH were collected from the GeneCards and OMIM database,and the Uniprot database was used to correct the targets.Baishao,Gancao,Guizhi,Danggui,Chuanxiong,Danshen,Huangqi,Baishu,Taoren and Fulin were respectively entered into the database of traditional Chinese medicine system pharmacology and analysis platform database for retrieval,and the active ingredients were screened out.The targets corresponding to the active ingredients were exported from the TCMSP database.and then imported into the Unitprot database for gene standardization.The Venny2.1 software was used to compare the LDH target genes and the genes corresponding to the target proteins of the active ingredient compounds of Tongluo Qubi Decoction.To construct the Tongluo Qubi Decoction-disease target protein interaction network and core targets,and import the potential targets of Tongluo Qubi Decoction for LDH into the DAVID database for GO function enrichment analysis and KEGG pathway enrichment analysis.The Caspase-1 gene was obtained from the gene database GeneCards OMIM.After comparing with the target genes of Tongluo Qubi Decoction,Venny 2.1 software was used to screen out the intersection genes,and Cytoscape 3.6.1 software was used to construct a visual network for the compounds with targets in Tongluo Qubi Decoction and their corresponding targets.The target genes of Caspase-1 regulated by Tongluo Qubi Decoction were imported into STRING11.0 database for analysis,and the main target genes of Tongluo Qubi Decoction in the treatment of LDH were obtained,and the experimental verification was carried out on this basis.The medicated serum with Tongluo Qubi Decoction was prepared and human nucleus pulposus cells were cultured.Interleukin-1β(IL-1β)was used to establish intervertebral disc degeneration cell model,and CCK-8 assay was used to investigate the effect of the medicated serum of Tongluo Qubi Decoction on the viability of the cells.Cell supernatant was taken and the inflammatory index levels of IL-1βand IL-18 were detected by enzyme-linked immunosorbent assay(ELISA)kits.The expression levels of Caspase-1,GSDMD,MAPK1 and MAPK3 were detected by Western blot and qPCR.Results(1)Experiment 1:There were 162 cases in the test group,including 112 males and 50 females.The age ranged from 16 to 78 years old,with an average age of(42.94± 15.29)years.BMI ranged from 16.92 to 36.24 kg/m2,with an average of(23.96±3.61)kg/m2.And 177 cases in the control group,including 103 males and 74 females.The age ranged from 21 to 79 years old,with an average age of(51.37±16.59)years.BMI ranged from 15.43 to 37.98 kg/m2,with an average of(23.78±3.20)kg/m2.At 3 and 6 months after operation,the ODI of the test group was significantly lower than that of the control group(P<0.01),but there was no significant difference in ODI between the two groups at 12 months(P>0.05).At 3 months after operation,the back pain VAS score of the test group was lower than that of the control group(P<0.01),but there was no significant difference between the two groups at 6 months and 12 months after operation(P>0.05).After 3 months,the leg pain VAS score of the test group was lower than that of the control group(P<0.05),but there was no significant difference at 6 and 12 months after surgery(P>0.05).At 3 and 12 months after operation,the JOA in the test group was higher than that in the control group(P<0.01).At 3,6 and 12 months after operation,the TCM syndrome scores of the test group were significantly lower than those of the control group(P<0.01).The cumulative frequency of postoperative intravenous and intramuscular analgesics in the test group was 18 times,the relative frequency was 11.11%;the cumulative frequency of the control group was 39 times,the relative frequency was 22.03%.The total hospitalization days in the test group were significantly less than those in the control group(P<0.01).The medical expenses of the test group were(22259.09±2460.68)RMB,and(24316.94±3669.53)RMB in the control group(P<0.01).According to the MacNab evaluation standard,the excellent and good rate of the test group was 92.59%,while that of the control group was 88.70%(P<0.05).Among the 162 patients in the test group,41 patients were treated with the Tongluo Qubi Decoction(group A)during the perioperative period,58 patients were treated with the Taoren Siwu Decoction(group B),and 63 patients were without Chinese medicine orally(group C).At 3 months after surgery,the TCM syndrome scores of the group A was better than those of the group B and group C(P<0.05).(2)Experiment 2:A total of 54 LDH patients were included in this study,3 cases were lost to follow-up,and finally 51 cases were included in statistical analysis,including 26 patients in the test group(15 males and 11 females),mean age was 41.19±12.42 years(16-68 years),and BMI was 24.16±3.99 kg/m2(18.9-36.2 kg/m2),the course of disease was 12.23± 11.72 months(2-48 months);25 patients in the control group(14 males and 11 females),the mean age was 47.12± 11.28 years(18-65 years old),BMI was 23.37±2.56 kg/m2(19.8～29.8 kg/m2),disease duration was 8.92±5.21 months(2～22 months).The total operation time of the test group was 59.46± 16.53 min(28-76 min),of which the catheter placement time was 16.19±3.60 min(10-26 min),and the endoscopic decompression time was 40.77±12.15 min(16-55 min);while the operation time of the control group was 53.40±11.88min(35-78min),of which the catheter placement time was 14.68±3.59min(8-25min),and the endoscopic decompression time was 39.12±13.46min(18-63min).There was no significant difference between the two groups in the total operation time,catheter placement time and endoscopic decompression time(P>0.05).The number of intraoperative C-arm fluoroscopy in the test group was 3.24± 1.30 times(2-6 times),and that in the control group was 3.24± 1.30 times(2-6 times),and the difference was not statistically significant(P>0.05).The postoperative hospital stay in the test group was 1.31±0.47 days(1-2 days),and the control group was 2.12±0.83 days(1-4 days).The postoperative hospital stay in the test group was shorter than that in the control group(P<0.01).There was no significant difference in SF-MPQ between the two groups at 1 day,6 months and 12 months after operation(P>0.05).One day and 12 months after the operation,the ODI between the two groups was no significant difference(P>0.05),at 6 after operation,the ODI of the test group was significantly lower than that of the control group(P<0.05).At 6 months after operation,the PCS score of the test group was better than that of the control group(P<0.05).The PCS and MCS scores at 12 months after operation between the two groups was not statistically significant(P>0.05).One day and 12 months after the operation,the TCM syndrome scores of the two groups was no significant difference(P>0.05),while 6 months after operation,the TCM syndrome scores of the test group was significantly lower than those of control group(P<0.05).At 12 months after operation,the excellent and good rate in the test group was 92.3%,and 92%in the control group.(3)Experiment 3:Through network pharmacology analysis,it was found that the mechanism of Tongluo Qubi Decoction in the treatment of LDH was related to factors such as regulating inflammatory response and cell function.Further pathway analysis results showed that Tongluo Qubi Decoction can play a role in regulating Caspase-1 and IL-17,TNF-α,C-type lectin receptor and Th17 and other signaling pathways.Its mechanism related to pyroptosis may be related to 14 genes regulating Caspase-1,including CASP3,MAPK1,RB1,STAT3,MAPK3,MAPK14,AKT1,HSP90AA1,RELA,MYC,HIF1A,AR,CDKN1A,and JUN.In order to verify the results of GO-BP analysis in network pharmacology,cell experiments were used to verify the relevant biological processes.The results of cell viability showed that the cell viability of intervertebral disc degeneration model cells was significantly improved after the intervention of the medicated serum of Tongluo Qubi Decoction.Meanwhile,TNF-α and IL-1β were down-regulated in supernatant and the cleavage of the pyroptotic core protein Caspase-1 was down-regulated,the phosphorylation of MAPK1,MAPK3 and AKT were up-regulated,and the phosphorylation of MAPK14 was down-regulated.The expression of pyroptotic core genes Caspase-1,RELA,NLRP3 and GSDMD were significantly decreased.The above results suggest that Tongluo Qubi Decoction can reduce the pyroptosis by regulating the core genes and proteins of pyroptosis,thereby delaying the degeneration of intervertebral disc cells.ConclusionThe CP of TCM combined with PELD in the treatment of LDH can effectively improve the clinical efficacy,shorten the length of hospital stay,reduce the frequency of postoperative intravenous and intramuscular injection of analgesics,and reduce medical costs.The application of Tongluo Qubi Decoction in CP is helpful to further improve the TCM syndrome score.Tongluo Qubi Decoction can improve the postoperative curative effect of SEESSYS patients,promote rapid postoperative recovery,and improve the quality of life of patients.Tongluo Qubi Decoction can mediate the pyroptosis of intervertebral disc cells by inhibiting the inflammatory response,regulating the MAPK pathway.