| Background:Pain,as the fifth vital sign,is attracting more and more clinical attention.Effectively relieving or eradicating the pain of patients naturally becomes a keypoint.Lumbago and leg pain is one of the most widespread symptoms,mostly associated with disc herniation,many researchers have found that radiculitis is one of the causes of stubborn root pain.Studying and exploring the pathogenesis of radiculitis and the intrinsic mechanism of ozone therapy can provide new theoretical basis and possible target for clinical diagnosis and treatment.Radiculitis is characterized by inflammation of the dorsal root ganglion and nerve root following perineuritis or inflammatory infiltration.Patients with radicular disease showed abnormal electrophysiology,slow nerve conduction and extensive axonal injury.The causes and mechanisms of radiculitis are summarized in the following aspects.(1)nerve is subjected to mechanical compression.A large number of studies have shown that radiculitis originates from nerve compression.The nerve root has no protective sheath of connective tissue and is sensitive to mechanical compression caused by root canal lesions.Compression produces direct mechanical effects on the nerve root and other effects by impairing the blood supply and nutrition of the nerve root.Clinically,radicular pain occurs in lumbar disc herniation,which compresses the nerve roots and exposes them to chemical abnormalities caused by the breakage of nucleus pulposus and annulus fibrosus of nerve roots,such as abnormalities in tissue nutrition and oxygen supply due to microcirculation and venous occlusion-related diseases,including accumulation of metabolites,leading to the pathology process of radiculitis.Therefore,improving microcirculation,providing specific nutrition,reducing the accumulation of inflammatory chemicals in nerve roots,inhibiting the inflammatory response of nerve roots,can also improve radiculitis.(2)the hypothesis of chemical mediate.Mast cells appear in the adventitia and intima of the injured area as well as in the nerve capsule,releasing inflammatory mediators such as histamine,which stimulate nerve roots,resulting in the exudation of a large number of inflammatory proteins in the nerve roots,increasing intraneural pressure,causing local ischemia and electrolyte disorders,resulting in various symptoms,causing the pain of the zone.Other substances,such as neuropeptides,play an important role in pain perception.These pain-causing substances directly stimulate pain and reduce pain threshold.(3)autoimmune hypothesis of radicular inflammation.Some scholars believe that with the aging or degeneration of the intervertebral disc,matrix degrading enzymes in the nucleus pulposus increase,so that glycoprotein and connexin cleave into highly heterogeneous molecules,leaking out in degenerative annulus fissures,resulting in autoimmune response.Compression in mechanical compression theory,inflammatory stimulation in chemical radiculitis theory,and immune response in autoimmune theory are mutually reinforcing.Mechanical compression can increase the permeability of local blood vessels,destroy the vascular-nerve barrier.Edema in the nerve,increasing the hydraulic pressure in the nerve,further blocking the blood supply,abnormal neurotransmitter release and inflammation,so that the nerve root is stimulated to produce symptoms.At the same time,inflammatory cells and fibroblasts infiltrate the nerve roots to form fibrous scars,which in turn aggravate the compression of the nerve roots.Autophagy,which actually means eating yourself,is considered to be a very important role of eukaryotic species in regulating the metabolism of intracellular substances.Autophagy can be divided into three types:microautophagy,macroautophagy and chaperone-mediated autophagy(CMA).Autophagic cell death,also known as type ? programmed cell death,is a unique life phenomenon in eukaryotic cells,which maintains the balance of protein metabolism and homeostasis of cellular environment.When autophagic cell death occurs,chromatin does not coagulate and Caspase does not participate in this process.However,a large number of autophages with bilayer membrane structure appear in the cytoplasm.Autophages encapsulate cytoplasmic contents and damaged organelles,and then fuse with lysosomes to form monolayer membrane autophagic lysosomes.With the help of lysosomal hydrolytic enzymes,autophagic lyvsosomes decompose the contents and the degraded substances can be reused by cells.Autophagy is strictly regulated by an evolutionarily conserved set of autophagy associated genes(ATG).Excessive or inadequate intracellular autophagy can lead to diseases,such as tumors,aging and neurodegenerative diseases.Apoptosis,also known as type ? programmed cell death,refers to the process of cell death triggered by in vivo and in vitro factors,which is one of the forms of programmed cell death(PCD),and the growth and development of life,eliminating damage and aging.Cells,as well as the prevention of cancer cells have a close relationship,is a basic biological phenomenon of cells.The body clears senile or excess cells by apoptosis under physiological conditions,and apoptosis is also induced under pathological conditions.Apoptosis includes the involvement of caspase protease hydrolases,chromatin condensation,cell shrinkage,DNA degradation and fragmentation.and apoptotic body formation.In this process,cell contents usually do not leak.so it will not cause inflammatory reaction.When cells are stimulated by glucocorticoids,heat,radiation,nutritional deficiency,viral infection,hypoxia and increased intracellular calcium concentration,apoptosis signals are released.Apoptosis is mainly triggered by mitochondrial pathway or internal pathway,death receptor mediated pathway or external pathway,endoplasmic reticulum pathway and non-caspase dependent pathway.A large number of studies have shown that there is a complex crosstalk between autophagy and apoptosis pathway,both of which have a common stimulator and regulatory protein.There are several possible relationships between apoptosis and autophagy in the process of cell death:apoptosis and autophagy synergistically promote cell death,both of which may be activated simultaneously or successively,and co-regulate cell death:apoptosis and autophagy antagonize each other by accelerating the clearance of damaged organelles or protein aggregates.In addition,macromolecules can be recycled to protect cells from apoptosis;autophagy promotes the appearance of apoptosis-related features,but autophagy is not a direct cause of cell death.Many regulatory elements are involved in the interaction between apoptosis and autophagy,including Beclin 1 and Bcl-2 family protein p53.It is unclear whether neuronal apoptosis and autophagy exist during the development of radiculitis.Ozone is a colorless,pungent odour gas which is also unstable,difficult to store and have strong oxidization.It will convert into oxygen when kept in the normal temperature and pressure conditions for 40-50 minutes.The use of ozone likes a coin with two sides,studies have shown that serious side effects on organisms could occur if exposed to inhalation of ozone for long time,while small doses of ozone can effectively improve blood antioxidant capacity.It is well known that medical ozone has many biological functions,such as sterilization,antiviral,anti-inflammatory,analgesic and immunomodulation.It has been used in the treatment of lumbar disc herniation,pain,trauma and refractory ulcers,stroke and viral hepatitis,and has good effect.Up to now,the mechanisms of ozone therapy have been studied,including the dehydration and reduction of nucleus pulposus by appropriate concentration of ozone,which can alleviate the compression of intervertebral disc to a certain extent;medical ozone can stimulate the release of anti-inflammatory factors IL-10 and fibroblast growth factor-beta 1(TGF-beta 1),which can inhibit the overproduction of apoptotic protein and IL-1 beta.It can improve local oxygen Ssupply and reduce inflammatoiy reaction.However,few studies have explored at the intrinsic mechanism of low concentration ozone in the treatment of radiculitis based on autophag)y and apoptosis theory.In view of this,the paper intends to establish a rat model of chemical radiculitis,observing the expression of autophagy and apoptosis in spinal cord tissue of rats with chemical radiculitis,observing the therapeutic effect of low concentration medical ozone on radiculitis,and explore the possible mechanism of ozone treatment of radiculitis from the perspective of autophagy and apoptosis.Part ? Presence of autophagy and apoptosis of spinal cords of radiculitis ratsObjective:Radiculitis is a refractory disease and has become a medical problem to be solved urgently.The pathogenesis mechanism remains unclear.In this study,we aimed tp explore apoptosis and autophagy of spinal cords in radiculitis.Methods:36 healthy Wistar rats weighted 250-300g were randomly divided into two groups,18 rats in each group:The model group and the sham group.For the model group,radiculitis was induced by Freund's adjuvant.Hemostasis oxidized cellulose containing 50ul complete Freund's adjuvant CFA was placed on the right intervertebral foramen of L4/5 and L5 dorsal root ganglion,causing chemical radiculitis.The sham group,with hemostasis cellulose containing 50ul saline was placed.At 8h,24h,and 72 h after successful modeling,the spinal cord tissues were collected from 3 rats in each group,and the L5 dorsal root ganglion were retained.The expression of LC3 ?/NeuN was determined by double labeling immunofluorescence.Caspase3 expression was determined by immunohistochemistry,Beclinl,LC3B,PDE2A,NF-?B p65 mRNA and protein levels in the tissues were measured by RT-PCR and Western blotting,respectively.The data were statistically analyzed for significant tests.Results:1.Transmission electron microscopy:many autophagysomes including abnormal structures(such as double membrane)and many organelles were found at 8 h,24 h,and 72 h after operation.The number of autophagysomes in the ozone treatment group was decreased even to that of the sham group.2.Double labeling immunofluorescence was used to detect the expression of LC3 ?/NeuN:There were many LC3B staining areas in the spinal cords of the model group.In the sham group,there was almost no LC3B staining in the spinal cords.Similarly,there were many NeuN staining areas in the spinal cords of the model group.There were few NeuN staining areas in the sham group.3.The expression of Caspase3 was determined by immunohistochemistry.There were many caspase 3 positive staining cells in 8h,24 h,and 72 h of spinal cords in the model group,more than those in the sham group.4.RT-PCR method was used to determine the levels of Beclinl,LC3B,PDE2A and NF-?B p65 mRNA in tissues.First,the standard reagent and method were used to verify the quantitative fluorescence PCR detection system.The results showed that the correlation coefficient between the experimental system and the theoretical value reached 0.9978,which was close to 1,indicating that the experimental system could be used to accurately determine the mRNA levels of the candidate genes.The RT-PCR system was used to determine LC3B,Beclin 1,PDE2A and NF-?B p65 mRNA levels.The results showed that:compared with the sham group,the Beclin 1 and LC3B mRNA levels of 8h,24 h,and 72 h in model group were increased significantly.compared with the sham group,the PDE2A and NF-?B p65 mRNA level of 72 h in the model group was increased significantly.5.The protein levels of Beclin 1,LC3B,PDE2A and NF-?B p65 in the tissues were determined by Western blotting:compared with the sham group,the levels of Beclin 1 and LC3B protein of 8h,24 h,and 72 h in the model group were increased significantly.Compared with the sham group,the PDE2A and NF-?B p65 protein level of 72 h in model group was increased significantly.Conclusion:A rat model of chemical radiculitis was successfully established,where characteristic autophagy structure occured in spinal cords and the expression of autophagy biomarkers(LC3B and Beclin 1)and apoptosis biomarkers cleaved Caspase 3 were significantly increased.PDE2A and NF-?B p65 expression was increased significantly in the radiculitis group.These data suggested the presence of autophagy and apoptosis of spinal cords in radiculitis rats,providing a clue and a target for developing therapeutic approaches.Part ? Medical ozone influences autophagy and apoptosis in spinal cords of rats with radiculitisObjective:Radiculitis is a refractory disease and has become a medical problem to be solved urgently.Although many rehabilitation and oral medication have been used in the treatment of radiculitis,the long-term effect is relatively poor.Ozone has anti-inflammatory,analgesic,and immunomodulation effects.Therefore,we speculate that ozone can be used in the treatment of radiculitis.In this study,we explored the effects of ozone on radiculitis and its related mechanisms.Methods:54 healthy Wistar rats weighted 250-300g were randomly divided into three groups,18 rats in each group:The group A was the the model group,radiculitis was induced by Freund's adjuvant.Hemostasis oxidized cellulose containing 50ul complete Freund's adjuvant CFA was placed on L4/5 intervertebral foramen and L5 dorsal root ganglion,causing radiculitis.The group B was the ozone treatment group,after successful modeling,50 ?l 30?g/ml 03 epidural injection was performed for 3 consecutive days.The group C was the control group,with hemostasis cellulose containing 50ul saline was placed.After successful modeling,50 ?l filtered air epidural injection for the group A and C was performed for 3 consecutive days.At 8h,24h,and 72 h after successful modeling,the spinal cord tissues were collected from 3 rats in each group,and the L4/5 nerve roots and dorsal root ganglion were retained.The expression of LC3 ?/NeuN was determined by double labeling immunofluorescence.Caspase3 expression was determined by immunohistochemistry.Beclinl,LC3B,PDE2A,NF-?B p65 mRNA and protein levels in the tissues were measured by RT-PCR and Western blotting,respectively.The data were statistically analyzed for significant tests.Results:1.Transmission electron microscopy:many autophagysomes including abnormal structures(such as double membrane)and many organelles were found in the model group at 8 h,24 h,and 72 h after operation.In the ozone treatment group,the number of autophagysomes was decreased significantly,compared with the model group.The difference was statistically significant.The number of autophagysomes in the ozone treatment group was decreased even to that of the control group.2.Double labeling immunofluorescence was used to detect the expression of LC3 ?/NeuN:There were many LC3B staining areas in the model group.Compared with the model group.the area of LC3B staining in ozone treatment group was decreased significantly.In the control group,there was almost no LC3B staining in the cytoplasm.Similarly,there were many NeuN staining areas in the spinal cord of the model group.Compared with the model group,the area of NeuN staining in ozone treatment group decreased significantly.In the control group,there were few NeuN staining areas in the spinal cord.3.The expression of Caspase3 was determined by immunohistochemistry.There were many caspase 3 positive staining cells at 8h,24 h,and 72 h of spinal cord in the model group.Compared with the model group,the number of positive caspase 3 staining cells in the ozone treatment group was decreased significantly,and almost reached the level of that in the control rats.4.RT-PCR method was used to determine the levels of Beclinl,LC3B,PDE2A and NF-?B p65 mRNA in tissues.The results showed that the Beclin 1 and LC3B mRNA levels at 8h,24 h,and 72 h in model group were increased significantly,compared with the control group.Compared with the model group,the Beclin 1 and LC3B mRNA level at 8h,24 h,and 72 h in the ozone treatment group were decreased significantly,but they were still significantly higher than those in the control group.The PDE2A and NF-?B p65 mRNA levels at 72 h in the model group was increased significantly,compared with the control group.Compared with the model group,the PDE2A and NF-?B p65 mRNA levels at 72 h in the ozone treatment group was decreased significantly,but it was still significantly higher than that in the control group.5.The protein levels of Beclinl,LC3B,PDE2A and NF-?B p65 in the tissues were determined by Western blotting:the Beclin 1 and LC3B protein levels at 8h,24 h,and 72 h in model group were increased significantly,compared with the control group.Compared with the model group,the Beclin 1 and LC3B protein levels at 8h,24 h,and 72 h in the ozone treatment group were decreased significantly,but they were still significantly higher than those in the control group.The PDE2A and NF-?B p65 protein levels at 72 h in the model group was increased significantly,compared with the control group.Compared with the model group,the PDE2A and NF-?B p65 protein levels at 72 h in the ozone treatment group was decreased significantly,but it was still significantly higher than that in the control group.Conclusion:A rat model of chemical radiculitis was successfully established.Ozone treatment at the therapeutic concentration(30?g/ml)can significantly inhibit the occurrence of characteristic autophagy structure inspinal cords and the expression of autophagy biomarkers(LC3B and Beclin 1)and apoptosis biomarkers cleaved Caspase 3 in inspinal cords.Ozone inhibited the expression of PDE2A and NF-kappaB p65 in rat models of radiculitis.These data suggested that ozone at therapeutic concentration can inhibit autophagy,reduce cell apoptosis and therefore can be used to treat radiculitis. |
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