| BackgroundDiscogenic low back pain(DLBP)is a common clinical degenerative disease of lumbar spine,which is mostly caused by intervertebral disc degeneration(IDD),with"low back pain" as the main clinical symptom,and generally without nerve tissue compression.In recent years,with the progress of science and technology,especially the wide application of magnetic resonance imaging technology,people have made great progress in the diagnosis and treatment of DLBP.At present,the clinical treatment of DLBP is mainly western medicine and surgery.Although the conservative treatment is diverse and effective in the short term,it lacks significant specificity,and only stays at the level of symptomatic treatment,without intervention on the diseased disc itself,resulting in a high recurrence rate.Traditional Chinese medicine believes that DLBP belongs to "low back pain",which has its unique advantages and rich clinical experience in the treatment of such diseases.Through small sample clinical observation,we found that the application of Bushen Huoxue Fang(BSHXF)from the ancient Qing'e pill in the treatment of DLBP can significantly reduce the pain of patients,improve the lumbar function and quality of life,with high satisfaction.However,we still lack high-level evidence-based clinical research,and the evaluation index is only limited to subjective indicators,and the drug safety has not been verified;at the same time,the specific regulatory mechanism of DLBP is still lack of basic research to explain.Therefore,a parallel,double-blind,randomized,placebo-controlled trial design was first applied in this study,and the accepted subjective score combined with objective evaluation method was further adopted,so as to more comprehensively evaluate the clinical efficacy and safety of BSHXF in the treatment of DLBP.Secondly,he staining,Masson staining and immunohistochemical staining were used to observe the effect of the drug on the histological structure of rabbit degenerative intervertebral disc.Finally,MTT,immunofluorescence,Western blotting(WB)and ?-galactosidase staining were used to observe the effects of the drugs on the expression of extracellular matrix(ECM)related proteins induced by IL-1? or H2O2 in human nucleus pulposus.The purpose of this study is to explore the mechanism of BSHXF in regulating the activity and function of nucleus pulposus cells(NPCs),delaying or reversing the IDD,and to provide a new idea for the mechanism research of BSHXF in delaying IDD.Part one:Clinical efficacy and safety of Bushen Huoxue Fang in the treatment of discogenic low back painObjective:To verify the clinical efficacy and safety of BSHXF in the treatment of DLBP,and to provide high-level evidence-based medicine for further clinical application.Methods:According to the inclusion and exclusion criteria,a total of 70 patients with DLBP were included.A parallel,double-blind,randomized,placebo-controlled trial design was used,in which 35 cases in the treatment group and 35 cases in the control group were treated with lumbar traction as the basic therapy.The treatment group was treated with BSHXF,while the control group was treated with placebo.The total course of treatment was 3 weeks and the follow-up period was 3 months.The VAS score,ODI score,muscle and soft tissue tension,lumbar tenderness,lumbar range of motion,laboratory examination,adverse reactions and other indicators of the two groups were compared and observed at 6 time points,7,14,21 days after treatment,and 1 month and 3 months after the end of the treatment,and the relevant clinical trial data were recorded and analyzed.Results:Two groups of patients in gender(P=0.208),age(P=0.363),lesion segment(P=0.771),course of disease(P=0.781).The outcome evaluation indexes were as follows:Regarding the VAS scores,the post-treatment and follow-up groups were significantly lower than the pre-treatment group(P<0.05),and the treatment group was significantly lower than the control group from two weeks of post-treatment to the end of the follow-up(P<0.05);The ODI scores of all patients with DLBP after infusion were significantly lower than the baseline(P<0.05),but there were no significant differences among the two groups at three months follow-up(P>0.05);Moreover,the lumbar spine mobility,tenderness,and muscle tone scores of the patients in the treatment group were significantly higher than those in the control group(P<0.05).The clinical efficacy of the treatment after one,two and three weeks was 47.06%,61.76%and 70.59%,respectively,showing a better trend than the control group.Conclusion:Bushen Huoxue Fang has good clinical effect on patients with discogenic low back pain,can significantly reduce the degree of pain,improve dysfunction,and has high security.Part two:Effect of Bushen Huoxue Fang on rabbit intervertebral disc degeneration modelObjective:To observe the effect of BSHXF on the process of IDD by different doses of drugs to intervene the rabbit IDD model.Methods:Forty healthy rabbits were randomly divided into 5 groups:control group,model group,low-dose group,medium dose group and high-dose group,respectively.He staining and Masson staining were used to observe the effect of the drug on the morphological structure of intervertebral disc,and immunohistochemical staining was used to detect the protein expression levels of MMP3 and VEGF.The related animal experimental data were recorded and analyzed.Results:(1)HE staining:a large number of NPCs were found in normal intervertebral disc tissue,and the arrangement of ECM was uniform.After modeling,the annulus fibrosus was loose and disordered,some of them were broken,the number of cells decreased,the fingerprint like changes were prominent,and the cracks increased.After drug intervention,the number of NPCs was increased,ECM was more regular,and annulus fibrosus fracture was reduced in the middle and high dose groups;(2)Masson staining:the NPCs of normal intervertebral disc were dark red or brown,the cytoplasm was red,and the boundary between nucleus pulposus and annulus fibrosus was clear.After modeling,cracks appeared in the inner layer of annulus fibrosus,NPCs arranged disorderly,cells degenerated and apoptotic,and nucleus pulposus tissue fibrosis gradually;(3)Immunohistochemical staining:the positive staining rates of MMP3 and VEGF in the model group were significantly higher than those in the control group(P<0.05),while the expressions of MMP3 and VEGF in the medium and high dose groups were significantly lower than those in the model group(P<0.05).Conclusion:Bushen Huoxue Fang can down regulate the expression levels of MMP-3 and VEGF in the degenerative intervertebral disc tissue of rabbits,and the middle dose group has the most significant effect,suggesting that it may play a role in the process of IDD.Part three:Effect of Bushen Huoxue Fang on the activity of human nucleus pulposus cells and extracellular matrixObjective:To observe the effect of Bushen Huoxue Fang on the activity and function of NPCs and the decomposition and metabolism of ECM induced by IL-1? and H2O2.Methods:the primary human nucleus pulposus cell line was cultured,and the subcultured NPCs were used for standby;the BSHXF extract was prepared according to a certain proportion,and 0,2,5,10,50,100,200 ?g/ml was added to NPCs respectively,and the effects were observed at 24 h and 48 h after intervention.The study was divided into five groups:?Control group;?IL-1? group;? H2O2 group;?IL-1?+BSHXF group;?H2O2+BSHXF group;MTT was used to detect the proliferation of NPCs;cell immunofluorescence technique was used to detect collagen?;WB was used to detect the expression of collagen ?,aggrecan,MMP-3 and ADAMTS-5;SA-?-gal staining was used to detect the senescence of NPCs;and the relevant cell experimental data were recorded and analyzed.Results:(1)BSHXF concentration detection:?BSHXF had no side effects on NPCs,and the proliferation rate of NPCs was significantly increased after treatment with 5,10,50,100 or 200?g/ml for 48 h(P<0.05).100?g/ml had the most significant effect(P<0.01).(2)MTT:?Compared with Contrl group,the proliferation ability of the other four groups decreased significantly at 24h(P<0.01);?Compared with Contrl group,the other four groups decreased significantly at 48h and 72h(P<0.01);Compared with IL-1? group,IL-1?+BSHXF group increased significantly(P<0.05);Compared with H2O2 group,H2O2+BSHXF group increased significantly(P<0.05);(3)Immunofluorescence:?Compared with Contrl group,the other four groups was significantly down regulated;?Compared with IL-1? group,IL-1?+BSHXF group was significantly up-regulated;? Compared with H2O2 group,H2O2+BSHXF group was significantly up-regulated.(4)WB:Collagen ?,Aggrecan:?Compared with Control group:the other four groups were significantly down regulated(P<0.01);?Compared with IL-1? group,IL-1?+BSHXF group was up-regulated(P<0.01);?Compared with H2O2 group,H2O2+BSHXF group was up-regulated(P<0.01).MMP-3:?Compared with control group,the other four groups were up-regulated(P<0.01);?Compared with IL-1 ? group,IL-1?+BSHXF group was down regulated(P<0.01);?Compared with H2O2 group,H2O2+BSHXF group was significantly down regulated(P<0.01).ADAMTS-5:?Compared with Control group,IL-1?group and H2O2 group were up-regulated(P<0.01),while IL-1?+BSHXF group and H2O2+BSHXF group were down regulated(P<0.01);?C ompared with IL-1? group,IL-1?+BSHXF group was down regulated(P<0.01);?Compared with H2O2 group,H2O2+BSHXF group was significantly down regulated(P<0.01).(5)SA-?-gal staining:?Compared with Contrl group,the number of senescence cells in the other four groups increased significantly(P<0.01);?Compared with IL-1? group,the number of senescence cells in IL-1?+BSHXF group decreased significantly(P<0.05);?Compared with H2O2 group,the number of senescence cells in H2O2+BSHXF group decreased significantly(P<0.01).Conclusion:(1)Bushen Huoxue Fang can improve the activity of NPCs and promote cell proliferation,and 100?g/ml is a suitable concentration for subsequent cell experiments;(2)Bushen Huoxue Fang can delay the aging of NPCs induced by IL-1?or H2O2 to a certain extent. |
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