| BackgroundWith the arrival of the global aging trend,various system-related degenerative diseases have seriously affected human health.Among them,the adverse effects of degenerative diseases of the spine on people's daily work and life have also become increasingly prominent with age.Intervertebral disc degeneration is an important pathogenic source of cervical spondylosis and lumbar spondylosis.According to statistics,there are 1 billion people suffering from cervical and lumbar diseases in the world.About 70%of people suffer from low back pain at least once in a lifetime for various reasons.This study aims to explore the role of traditional Chinese medicine in the prevention and treatment of lumbar degenerative diseases.At the same time,it uses experimental methods to explain the mechanism of action of traditional Chinese medicine and further tap the essence of the motherland medicine.In addition,the use of modern medical methods,continue to explore suitable for lumbar degeneration-related orthopedic surgery.Play the characteristics and advantages of the two kinds of medicine to jointly contribute to the prevention and treatment of lumbar degenerative diseases.Objective1 Through the systematic analysis of the relevant clinical literatures on the treatment of lumbar degenerative diseases by Chinese herbal,more Chinese herbal to be studied.At the same time,the most closely related factors and signaling pathways for disc degeneration diseases were retrieved and quantified,and they were combined through experimental verification to explore the efficacy and mechanism of Chinese herbal compound preparations in the treatment of lumbar intervertebral disc degeneration.Methods and materials Experiment 1:(1)The relevant literatures on randomized controlled clinical trials of Chinese herbal compound preparations versus western medicine for the treatment of lumbar disc herniation were searched via PubMed database,the CBM database,the CNKI database,and Wanfang database from January 1,2000 to January 1,2018.(WanFang Database),VIP Chinese(VIP)literature and then conducted Meta-analysis using Review Manager 5.3 software.The overall clinical efficacy,VAS score,JOA score,and the sub-group analysis were used to screen the Chinese herbal.(2)Searching for articles on disc degeneration and related signaling pathways published in the PubMed database,Medline database,Embase database,and web of science database between January 1,2008 and January 1,2018,through computer searches.Experiment 2:Rat lumbar intervertebral disc degeneration model was established and evaluated by three different modeling methods.Thirty-two experimental rats were randomly divided into four groups and the rats were divided into sham operation group,puncture model group,excision model group and excision+ puncture model group.There were 8 rats in each block.Firstly,according to the "three-column theory" of the spine,a degeneration model of lumbar disc instability is manufactured.Secondly,a puncture model is manufactured according to the annulus fibrosus puncture method.Finally,a combination of the two methods is used to create a "combine model.Evaluating three different "model-building"methods through imaging,behavioral and histological methods.Experiment 3:By using Bushen Zhuangjin Tang Granules to intervene rats with lumbar disc degeneration induced by resection.108 rats were randomly divided into 4w,8w,and 12w three groups,and each group consisted of 36 rats.Each group of rats was divided into sham operation group,model group,positive control group,low dose group of traditional Chinese medicine,medium dose group of traditional Chinese medicine and high dose group of traditional Chinese medicine.L4-5 and L5-6 intervertebral discs and dorsal root ganglion(DRG)were obtained at 4w,8w,and 12w after drug intervention,respectively.The behaviors,histology,Elisa method,and Western blotting were performed.Evaluate drug effectiveness and explore drug action mechanisms.Second,drug safety was evaluated by testing liver and kidney function.Experiment 4:Explore the mechanism of Bushen Zhuangjin Decoction granules in delaying the hindpaw's hyperalgesia and decreasing strength in rats.Each group of rat DRG was stained by immunohistochemistry to detect the expression of CGRP.At the same time,the key genes and signaling pathways in dorsal root ganglion were detected by bioinformatics,and the experimental methods were used to verify the drug.ResultsExperiment 1:(1)In terms of overall clinical efficacy,a total of 33 studies reported a clinical randomized controlled study comparing Chinese herbal with western medicine preparations for the treatment of lumbar disc herniation.A total of 3660 patients were included,of which 1879 were in the traditional Chinese medicine group and 1763 in the western medicine group.The results of the analysis showed that the efficacy of the Chinese herbal compound preparation group was better than that of the Western medicine preparation group,and the difference between the two groups was statistically significant[RR = 1.13;95%Cl:(1.09,1.17),P<0.00001].In VAS scores,a total of 10 studies reported VAS scores in both groups.A total of 1100 patients were included,of which 552 were in the Chinese herbal compound group and 548 were in the western medicine group.The analysis showed that the improvement of the VAS score in the Chinese herbal compound preparation group was better than that in the Western medicine preparation group.The difference between the two groups was statistically significant[SMD = 0.72;95%CI:(0.36,1.07),P<0.00001].In terms of JOA scores,a total of 6 studies reported VAS scores in two groups.A total of 593 patients were included,including 298 Chinese herbal compound preparations and 295 Western medicine preparations.The analysis results showed that the scores of JOA in the Chinese herbal compound preparation group improved better than the western medicine preparation group,and the difference between the two groups was statistically significant[SMD = 0.89;95%CI:(0.25,1.53),P = 0.007].In the subgroup analysis,a total of 4 studies reported the clinical study of Duhuo Parasitoid Soup and Bushenzhuangjin Decoction.Among them,244 people were included in the Duhuo Decoction,of which 122 were in the Chinese medicine compound preparation group and 122 in the Western medicine preparation group.The Bushen Zhuangjin Decoction granule contained a total of 182 people,including 91 cases of traditional Chinese medicine compound preparation group and 91 cases of western medicine preparation group.The results of the analysis showed that the clinical efficacy of the Duhuo Jisheng Decoction group was better than that of the western medicine group.The difference between the two groups was statistically significant[RR 1 1.17;95%CI:(1.06,1.29),P = 0.002].The clinical curative effect of Bushenzhuangjin Decoction group was better than that of Western medicine preparation group.The difference between the two groups was statistically significant[RR = 1.30;95%CI:(1.02,1.66),P = 0.04].(2)In the analysis of signal pathway related studies,we found that there are a total of 27 signal pathways included in the scope of the study,of which the TOP 10 signal pathways are:first,ERK signaling pathway;second,NF-?B signaling pathway;3rd place,Apoptosis signalling pathway,4th place,P38MAPK signalling pathway,5th place,PI3K/AKT signaling pathway,6th place,JNK signaling pathway,7th place,Wnt/?-catenin signaling pathway,8th place,P53 signalling pathway;9th,Smad signaling pathway;10th,mTOR signaling pathway.The highest IF included in the literature was 8.402,which was reported by Chen WH from the Chinese Taipei Medical University in the Biomaterials journal on the ERK signaling pathway;the lowest IF was 0.964,which was published by Xie M from Tongji University in Wuhan,China.Report on the ERK signaling pathway in Huazhong Univ Sci Technolog Med Sci.In the TOP 10 pathway,the annual trends of the signal pathways increase and change.Among them,the three signaling pathways of NF-?B signaling pathway,P38MAPK signalling pathway,and ERK signaling pathway were the most studied in 2017,with 24 articles,17 articles,and 13 articles respectively.In the analysis of preference trends,we found that P38MAPK signalling pathway,ERK signaling pathway,JNK signaling pathway,NF-?B signaling pathway,PI3K/AKT signaling pathway,and Apoptosis signalling pathway accounted for a relatively large number of published journals.Experiment 2:(1)The detection of mechanical pain threshold and sloping plate test showed that all three modeling methods can cause the pain sensitivity and strength of hindlimbs of model rats to decrease,in which the efficacy was puncture+resection method>puncture method>resection method.(2)MRI magnetic resonance imaging showed that all three methods can cause lumbar intervertebral disc degeneration in rats.Among them,the lumbar intervertebral disc degeneration induced by puncture and resection was the highest,followed by puncture and finally excision.(3)The modified histological staining of safranin O showed that all three methods could cause disc degeneration.Among them,the puncture + resection method had the most severe damage to the intervertebral disc,followed by the acupuncture method and finally the resection method.(4)The DRG immunofluorescence test showed that the CGRP expression induced by the puncture + resection method was the most,followed by the acupuncture method,and finally the resection method.Experiment 3:(1)The detection of mechanical pain threshold and sloping plate test showed that the sensitivity of rat hindlimb pain from low to high was as follows:sham operation group = high dose group of traditional Chinese medicine<positive control group<medium dose of traditional Chinese medicine group<Dose group = model group.Similarly,the hindlimb strength of rats was sham operation group = high dose group of traditional Chinese medicines>positive control group>medium dose group of traditional Chinese medicines>low dose group of traditional Chinese medicines = model group(2)histological staining of intervertebral discs suggested that the degree of degeneration of intervertebral discs was Light to heavy were as follows:sham group>high dose group>positive control group>medium dose group>low dose group = model group.The levels of TNF-a,IL-1? and IL-6 in serum were detected by ELISA.The results showed that for TNF-a,the inhibitory capacity was high dose group of traditional Chinese medicine = medium dose group of traditional Chinese medicine>positive control group>low dose group of traditional Chinese medicine;For IL-1(3,its inhibitory ability was in descending order:Chinese medicine high dose group =medium Chinese medicine dose group>positive control group>Chinese medicine low dose group;for IL-6,its inhibitory ability was high dose group of traditional Chinese medicine>medium dose group of traditional Chinese medicine = traditional Chinese medicine Low dose group = positive control group.Western blotting results showed that the Chinese herbal compound preparation could inhibit the phosphorylation of P65 and P38.The expression level of P65 phosphorylation from high to low was model group>low dose group of traditional Chinese medicine = positive control group>high dose group of traditional Chinese medicine=middle dose group of traditional Chinese medicine = sham group.The expression level of P3 8 phosphorylation from high to low was model group = low dose group of traditional Chinese medicines>positive control group>sham operation group = high dose group of traditional Chinese medicine = middle dose group of traditional Chinese medicine.In the detection of liver and kidney function,it was not found that the drug can cause symptoms of liver and kidney damage in rats.Experiment 4:From bioinformatics,we found that DRG changes in pathological conditions produced a number of abnormally up-regulated genes,of which 204 up-regulated genes and 33 down-regulated genes.In the GO analysis of genes,up-regulated genes are mainly clustered in:inflammatory response,mononuclear,lympho-neutrophil chemotaxis,glial differentiation,and neurite regulation.The down-regulated genes were mainly clustered in:transmembrane transport of ion channels,neuronal differentiation,and maintenance of intracellular homeostasis.In the signal pathway analysis process,four signal pathways are involved,which are apoptosis signaling pathway involved in apoptosis,senescence signaling pathway related to aging,MAPK signaling pathway,and p53 signaling pathway.Seven genes including Cdknla,Gadd45a,Gadd45g,Igfbp3,Pmaip1,Serpinb5 and Serpinel were enriched in p53 signaling pathway;8 genes including Casp6,Csf2rb2,Gadd45a,Gadd45g,Itprl,Jun,Pmaipl and Tubalc were enriched in Apoptosis signaling.Eight genes of Cdknla,Gadd45a,Gadd45g,Igfbp3,IL6,Itprl,Rras2,and Serpine1 were enriched in the Cellular senescence signaling pathway;12 core genes,Ccl12,Ppplrl5a,Cdknla,Jun,Atf3,IL6,were further screened and screened.Nts,Dusp1,Ccl7,Csfl,Gadd45a,Serpinel.The most important factors in the protein interaction network are IL-6 and Jun,and both genes are closely related to the MAKP signaling pathway.The detection of CGRP in DRG of rats We found that the expression of CGRP from low to high was as follows:sham operation group>positive control group = high dose group of traditional Chinese medicine>medium dose group of traditional Chinese medicine>low dose group of traditional Chinese medicine>model group.In the mechanism discovery,both c-Jun and P38MAPK showed a certain gradient trend.The c-Jun expression levels from high to low were as follows:model group = low-dose Chinese medicine group>positive control group = high-dose Chinese medicine group = traditional Chinese medicine Dosage group = Chinese medicine low dose group = sham group.The expression levels of p-P38MAPK and p-P38/P38 from high to low were as follows:model group = low dose group of traditional Chinese medicine>positive control group = high dose group of traditional Chinese medicine = middle dose group of traditional Chinese medicine =low dose group of traditional Chinese medicine = sham group.ConclusionsExperiment 1:(1)In the treatment of lumbar intervertebral disc herniation,the overall clinical effect of oral administration of Chinese herbal is better than that of oral administration of western medicine.(2)To adopt Bushen Zhuangjin Decoction granule as a follow-up experimental research intervention method.(3)NF-?B signaling pathway and P38MAPK signalling pathway are the hot signal pathways related to intervertebral disc degeneration in recent years.Most studies have confirmed that they play a key role in the process of intervertebral disc degeneration.(4)To choose NF-?B signaling pathway and P38MAPK signalling pathway as futher studies.Experiment 2:(1)Application of puncture method,resection method,puncture + resection method can all cause degeneration of intervertebral disc.The degree of degeneration is puncture + resection group>puncture group>resection group.(2)Compared with puncture and puncture+resection method,the change of intervertebral disc retraction induced by resection method conforms to the general law of degeneration,and it has advantages in follow-up studies.(3)All three methods can induce hyperalgesia and hindlimb strength in rats.The strengths were puncture + resection group>puncture group>resection group.(4)The puncture method,the resection method,and the degeneration of the intervertebral disc induced by the puncture+resection method can all cause changes in the expression of CGRP in the corresponding DRG.The CGRP expression levels from high to low were puncture +resection group>puncture group>resection group.Experiment 3:(1)Bushen Zhuangjin Decoction granule can improve the hyperalgesia and strength of the hind limbs of lumbar intervertebral disc degeneration rats(2)Bushen Zhuangjin Decoction granule can inhibit the expression of inflammatory cytokines TNF-a,IL-1? and IL-6,and down-regulate the phosphorylation of NF-?B and MAPK in P65 and P38 proteins to delay lumbar intervertebral disc degeneration.Experiment 4:(1)In the dorsal root ganglion changes after peripheral nerve injury,we found 12 genes,including Ccl12,Ppplrl5a,Cdknla,Jun,Atf3,IL-6,Nts,Duspl,Ccl7,Csfl,Gadd45a,and Serpinel.Among the larger weights are Jun and IL-6.(2)MAPK,Apoptosis,Senescence,and P53 signal pathways play an important role in the changes of dorsal root ganglia after peripheral nerve injury.(3)Bushen Zhuangjin Decoction granule can reduce the expression of CGRP and relieve the pain symptoms of rats by inhibiting inflammatory factors and further down-regulate the expression of two key proteins c-Jun and P38 in MAPK signaling pathway. |
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