| BackgroundLumbar spinal stenosis(LSS)refers to the absolute or relative stenosis of the lumbar spinal canal due to various congenital or acquired factors,which stimulates the compression of the cauda equina or spinal nerve root and causes extensive pathological changes in the nerve tissue,resulting in a series of neurological dysfunctions.disease.Although LSS is divided into congenital and acquired,degenerative lumbar spinal stenosis(DLSS)is the most common in clinical practice.As the second largest intraspinal disease after lumbar intervertebral disc herniation,DLSS has a population incidence rate of about 3.9%to 11%,and the prevalence rate of people over 60 years old can be as high as 19.4%.increasing trend with age.Against the background of my country’s aging population,the disease is increasingly threatening the quality of life and physical and mental health of the middle-aged and elderly population,and also causes a great economic burden to the society.Therefore,how to take effective treatment measures in the development of the disease and improve the clinical symptoms of patients has become an urgent need for clinicians and patients.Conservative treatment is the first choice for the vast majority of DLSS patients,including rest,drug therapy,and traditional Chinese medicine.Among them,Chinese medicine therapy has gradually become an important method for conservative treatment of DLSS in my country.This study is guided by "Yang transforms qi and yin forms" in "Huangdi Neijing",combined with the dialectical thinking of "pi shen qi deficiency and blood stasis together" of Qing Dynasty physician Chen Shiduo in the treatment of low back pain.The above-mentioned chemical therapy is used to treat DLSS(pi shen qi deficiency and blood stasis together),aiming to verify the clinical efficacy and safety of Zhuanyao Decoction in the treatment of DLSS.However,the current research on the treatment of DLSS with traditional Chinese medicine is still at the clinical level,and there is a lack of mutual confirmation between clinical and basic research,which also seriously restricts the further promotion and application of traditional Chinese medicine in the clinical treatment of DLSS.Therefore,this study will first provide reliable clinical evidence for the treatment of DLSS with Zhuanyao Decoction from a clinical perspective,and then explore the molecular mechanism of Zhuanyao Decoction in the treatment of DLSS through animal experiments on the basis of clinical efficacy.Mechanistic studies on the prevention and treatment of DLSS provide scientific evidence at the molecular level.Research ContentPart I:A case series study of Zhuanyao decoction in the treatment of degenerative lumbar spinal stenosisOBJECTIVE:To observe the clinical efficacy and safety of Zhuanyao Decoction in the treatment of DLSS,and to analyze the relevant factors affecting the clinical efficacy of Zhuanyao Decoction in the treatment of DLSS.METHODS:From October 2019 to November 2021,a total of 116 patients with DLSS(pi shen qi deficiency and blood stasis together)who met the criteria of appetite and excretion were enrolled in the Spine Clinic of Wangjing Hospital of Chinese Medicine.All patients were treated with Zhuanyao Decoction orally for two consecutive courses of treatment.The general conditions of all patients were collected before treatment,including sociodemographic information,condition information and imaging information;at the same time,the Oswestry disability index(ODI),pain visual analog scales(VAS),TCM syndrome score,intermittent claudication,frequency of lower extremity cramps and coldness of lower extremities,etc.,and adverse reactions during treatment were recorded.The patients after treatment were divided into treatment effective group and treatment ineffective group according to their clinical efficacy,and the related factors that may affect the treatment of DLSS(pi shen qi deficiency and blood stasis together)with Zhuanyao Decoction were analyzed by binary logistic regression.RESULTS:During treatment,12 patients dropped out,and 104 patients completed treatment and follow-up.(1)General conditions before enrollment,including gender,age,height,weight,course of disease,chief complaint,degree of intermittent claudication,frequency of lower extremity cramps,degree of coldness of lower extremities,imaging information,and ODI score,VAS score,TCM syndrome score.(2)Analysis of clinical efficacy and safety:after treatment and during the follow-up period,the ODI score,VAS score and TCM syndrome score were significantly different from those before treatment(P<0.05);the patients had intermittent claudication after treatment There were statistically significant differences in the degree,frequency of cramps of lower extremities and degree of coldness of lower extremities compared with those before treatment(P<0.05).The rates were 75.96%and 72.12%,respectively;no obvious adverse reactions occurred in all patients during the treatment.(3)Analysis of relevant factors affecting the curative effect:Through binary logistic regression analysis,the age,BMI,ODI score before treatment,VAS score before treatment,and TCM syndrome score before treatment had no significant effect on the curative effect of Zhuanyao Decoction in the treatment of DLSS(P>0.05),while the patient’s disease course,the number of segments of lumbar spinal stenosis,and the degree of intermittent claudication were the related factors affecting the efficacy of Zhuanyao Decoction in the treatment of DLSS(P<0.05).CONCLUSIONS:Zhuanyao Decoction has good clinical efficacy and safety in the treatment of DLSS(pi shen qi deficiency and blood stasis together),and can effectively improve lumbar and leg pain,lumbar spine function,traditional Chinese medicine syndrome,intermittent claudication,and lower limb cramps in patients with DLSS.and lower extremity symptoms.In addition,the analysis showed that the longer the course of disease,the more segments of lumbar spinal stenosis and the more severe the degree of intermittent claudication,the worse the effect of Zhuanyao Decoction treatment.Part II:Establishment and mechanism exploration of a rat model of lumbar spinal stenosisOBJECTIVE:To establish a stable and reliable chronic compression LSS rat model,and to explore the pathogenesis of LSS from the histopathological level.METHODS:64 SD rats were randomly divided into a sham operation group and a model group,with 32 rats in each group.The establishment method of model group rats:Two 4.0mm×1.0mm×1.0mm silica gel blocks were placed in the sublamina and epidural space of L5 and L6 respectively.In the sham operation group,the L5/L6 and L6/S1 intervertebral spaces were exposed,and the ligamentum flavum was punctured,but no silicone block was inserted.(1)Magnetic resonance imaging of the lumbar spine:On the 3rd day after modeling,the position of the silicone block in the spinal canal was observed by Magnetic Resonance Imaging(MRI).(2)Behavioral evaluation:1 day before surgery,1 day,3rd day,1st week,2nd week,4th week,8th week,and 12th week after operation,the two groups of rats were subjected to rotarod test.,Electronic VonFrey plantar pain measurement and determination of plantar thermal radiation pain threshold.(3)Histopathological observation:16 rats were sacrificed at the 2nd,4th,8th,and 12th week after modeling,and the cauda equina nerve of L5-L6 segment and the dorsal root ganglia on both sides of the corresponding segment were taken out.The structural changes of cauda equina nerve were observed by luxol fast blue,(LFB)and transmission electron microscopy,the apoptosis level in DRG was observed by TUNEL fluorescence detection,and the microtubule structure changes of DRG were observed by transmission electron microscopy.RESULTS:The rats in the sham-operated group and the model group survived after the operation.(1)Imaging observation:From the T2-weighted image of MRI,it was observed that the positions of all rat glue blocks were aligned with the L5 and L6 segments,and were located in the epidural space without destroying the dural sac.(2)Behavioral evaluation:Before operation,there were no significant differences in the walking time on the rotarod,the force and duration of mechanical tingling of the plantar,and the thermal radiation pain threshold of the plantar between the two groups(P>0.05).There were no significant differences in the walking time of the rats in the sham-operated group at each time point after the operation,the force and time of the mechanical tingling of the plantar,and the thermal radiation pain threshold of the plantar compared with those before the operation(P>0.05)..From the first day after operation,the time of walking on the rotator,the force and time of mechanical tingling of the plantar,and the thermal radiation pain threshold of the plantar of the model group showed a decreasing trend until the 8th and 12th day after the operation.week to reach a steady state.There were significant statistical differences in the time of walking on the rotator,the force and duration of mechanical tingling of the plantar,and the thermal radiation pain threshold of the plantar between the two groups at each observation point after operation(P<0.05).(3)Histopathological results:①There was no significant difference in the degree of axonal myelination between the time points in the sham-operated group(P>0.05),while the degree of axonal myelination in the model group gradually decreased with the extension of the compression time.The degree of myelination reached a minimum and then remained at a stable level.In addition,the degree of myelination in the model group was significantly lower than that in the sham operation group at the 8th and 12th weeks after operation(P<0.05).At the 8th week after modeling,the cauda equina nerve of the model group was observed to have obvious demyelination changes by transmission electron microscope,while the structure of the cauda equina in the sham operation group was basically normal.②The proportion of apoptosis in DRG of rats in sham operation group had no significant difference between observation time points(P>0.05);while the proportion of apoptotic cells in DRG of rats in model group showed a gradual upward trend,and the 8th week after operation Significantly higher than the 4th week after operation(P<0.05);while there was no significant difference between the 12th and 8th week after operation(P>0.05);in addition,at the 8th and 12th week after operation The ratio of apoptosis in DRG of Zhou model group was significantly higher than that of sham operation group(P<0.05).On the 8th week after modeling,the DRG neurons in the model group were significantly damaged by transmission electron microscopy,while the DRG structure in the sham-operated group was basically normal.CONCLUSIONS:The modeling method adopted this time can form a chronic LSS rat model with stable behavioral and pathological changes in the 8th week after surgery.4 This model can produce symptoms such as intermittent claudication and lower limb sensory dysfunction similar to clinical patients,and can be used as an ideal model to study the pathogenesis of LSS.At the same time demyelination of cauda equina and DRG neuron apoptosis may be the pathogenesis of LSS.Part III:The mechanism of action of Zhuanyao decoction in the treatment of lumbar spinal stenosisOBJECTIVE:Taking the LSS model rats as the carrier,the pharmacodynamic effect of Zhuanyao Decoction in the treatment of LSS was clarified,and the mechanism of the treatment of LSS was further explored from the cauda equina and DRG levels.METHODS:Ninety SD tats were randomly divided into a sham operation group(Sham),a model group(Model),a low-dose group of Zhuanyao Decoction(ZYT-L),a medium-dose group of Zhuanyao Decoction(ZYT-M),and a high-dose group of Zhuanyao Decoction(ZYT-M).Dose group(ZYT-H),15 in each group.At the 8th week after modeling,the rats in each group of Zhuanyao decoction were gavaged continuously for 8 weeks,and the Sham group and the Model group were gavaged with the same volume of distilled water.One day before the start of gavage and one week,two weeks,four weeks,and eight weeks after gavage,the two groups of rats were subjected to rotarod test,electronic Von Frey plantar pain measurement and plantar thermal radiation pain threshold measurement,respectively.Rat cauda equina nerve and DRG were collected after 8 weeks of intragastric administration,and were detected by LFB staining,immunohistochemical staining,TUNEL fluorescence detection,immunofluorescence staining,transmission electron microscopy,ELISA detection,and Western Blot(WB)detection.To evaluate the effect of the prescription and explore its mechanism of action.RESULTS:(1)Behavioral evaluation:One day before gavage,there was no significant difference in the walking time on the rod,the force and time of plantar mechanical stinging pain,and the thermal radiation pain threshold of the rats in the Sham group at each time point(P>0.05).There was no significant difference in the walking time on the rotarod,the mechanical stinging force and time of the plantar,and the thermal radiation pain threshold between the ZYT group and the ZYT dose groups(P>0.05),but the model group and the ZYT doses showed no significant differences(P>0.05).The walking time of the rats in the group was shorter than that in the sham-operated group(P<0.05).There were significant differences in the walking time on the rotarod,the force and time of plantar mechanical tingling,and the thermal radiation pain threshold of the ZYT-H group compared with the Model group after 2,4,and 8 weeks of gavage(P<0.05).The walking time of the ZYT-M group was significantly different from that of the Model group after 2 weeks of intragastric administration(P<0.05).There were statistical differences in pain force and time compared with the Model group(P<0.05).After 8 weeks of gavage,the walking time on the rotarod,the plantar mechanical stinging force and time,and the plantar thermal radiation pain threshold were significantly different from those in the Model group.There was statistical difference between groups(P<0.05).The walking time of the rats in the ZYT-L group was significantly different from that in the Model group after 4 and 8 weeks of gavage(P<0.05).There was a statistical difference between the Model group(P<0.05).(2)Cerebrospinal fluid ELISA test:①The expression level of VEGFA in the cerebrospinal fluid of the rats in the Sham group was lower than that in the other groups(P<0.05),and the expression level of VEGFA in the cerebrospinal fluid of the rats in each dose group of Zhuanyaotang was higher than that in the Model group(P<0.05).<0.05),the expression levels of VEGFA in the cerebrospinal fluid of the ZYT-M group and the ZYT-H group were higher than those in the ZYT-L group(P<0.05),and the expression level of VEGFA in the cerebrospinal fluid of the ZYT-H group was higher than that of the ZYT-L group(P<0.05).Group M.②The expression levels of TNF-α and IL-6 in the cerebrospinal fluid of the rats in the Sham group were lower than those in the other groups(P<0.05).In the Model group(P<0.05),the expression levels of TNF-α and IL-6 in the cerebrospinal fluid of the ZYT-M group and the ZYT-H group were lower than those of the ZYT-L group(P<0.05).The expression levels of TNF-α and IL-6 in ZYT-M group were lower than those in ZYT-M group(P<0.05).(3)Detection of cauda equina nerve tissue:①LFB staining:The degree of myelination of the cauda equina nerve in the Sham group was higher than that in other groups(P<0.05).Group L(P<0.05).②Immunohistochemistry:The expression of CD34 protein in the cauda equina of rats in the Model group and Zhuanyaotang groups was higher than that in the Sham group(P<0.05),and the expression of CD34 protein in the cauda equina of rats in the ZYT groups The amount was higher than the Model group(P<0.05).③WB detection:The protein expression levels of p-VEGFR2 and p-AKT/AKT in the cauda equina of rats in each group after Zhuanyaotang intervention were higher than those in the Sham group(P<0.05).The protein expression levels of p-VEGFR2 and p-AKT/AKT in the middle group were higher than those in the Model group(P<0.05).④Transmission electron microscopy:The myelin structure of the cauda equina nerve axons of the rats in the Sham group was basically normal;a large number of axons in the cauda equina nerve fibers of the rats in the Model group showed discrete myelin lamellae,and some of the myelin sheaths had sub-axonal edema.The axon was compressed,the mitochondria in the axoplasm were swollen,and the nuclear membrane of the Schwann cell nucleus was depressed;the axonal myelin lamellae were less scattered in the cauda equina nerve fibers of the ZYT-H group,and there was no obvious edema under the axonal membrane.There was no obvious swelling of mitochondria in plasma.(4)DRG tissue detection:①TUNEL fluorescence detection:There were fewer apoptotic cells in the DRG tissue of the rats in the Sham group,while a large number of apoptotic cells appeared in the DRG tissue of the rats in the Model group;after the intervention of different doses of ZYT,ZYT-H Apoptosis of DRG tissue in group rats was significantly inhibited.②WB detection:After Zhuanyaotang intervention,the expression levels of p-IKKα/β and p-P65/P65 in DRG of rats in Model group were higher than those in Sham group(P<0.05),while those in ZYT-M group and ZYT-H group were higher than those in ZYT-M group and ZYT-H group.The expression levels of p-IKKα/β and p-P65/P65 in mouse and horse DRG were lower than those in Sham group(P<0.05).After Zhuanyaotang intervention,the expression level of BCL2/BAX in the DRG of the Model group was lower than that of the Sham group(P<0.05),while the expression level of BCL2/BAX in the horse DRG of the ZYT-H group was higher than that of the Sham group(P<0.05).P<0.05),the expression level of cleaved-Caspase3 in the DRG of the Model group was higher than that of the Sham group(P<0.05),while the expression level of cleaved-Caspase3 in the horse DRG of the ZYT-H group was lower than that of the Sham group(P<0.05).0.05).③Transmission electron microscopy:the structure of nerve cells in the DRG of the rats in the Sham group was normal;while the nerve cells in the DRG of the rats in the Model group had condensed chromatin in the nucleus,depression of the nuclear membrane,increased lipofuscin in the cytoplasm,and a large amount of lipofuscin in the cytoplasm.Mitochondria were swollen and even vacuolized;there was a small amount of lipofuscin in the cytoplasm of neurons in the DRG of the cauda equina of rats in the ZYT-H group,and some mitochondria in the cytoplasm were swollen or cristae destroyed.CONCLUSIONS:Zhuanyao Decoction can effectively improve lower limb motor and sensory dysfunction in LSS model rats.Its therapeutic mechanism may be to promote angiogenesis of the cauda equina in LSS model rats by regulating the VEGF/VEGFR2/AKT pathway,thereby reducing or reversing the secondary damage caused by compression to the cauda equina.Apoptosis of DRG cells in model rats may be the main reason for improving neurogenic pain symptoms. |
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