| Objective and significanceCervical Spondylosis generally refers to clinical signs and symptoms caused by the diseased neck of the spine.The present international inclined to clinical signs and symptoms of spinal cord,nerves and veins damaged caused by Cervical Intervertebral Disc Degeneration and Secondary Intervertebral Joint Degeneration.The common symptoms of Cervical Spondylosis are cervicodynia,dizziness,limb numbness and weakness,and it can be combined with sphincter disturbances seriously.Cervical Spondylosis is a common and frequently-occurring disease to Middle-aged and Old-aged. The incidence of Cervical Spondylosis in northeast and central north of China are obviously higher than the areas to the south of the Yangtze River.Many reasons can expedite proceedings of neck,lumbar spine disease.For example:working at a desk and using computer for a long time,driving,sleeping on a high pile of pillows or soft bed.It can make people keep a posture for a long time and fatigue of muscle and ligament.Besides the change of diet and the gain weight can also expedite proceedings of the disease.the task of treatment Cervical Spondylosis is heavy and the road is long.So far Cervical Spondylosis has no ideal therapy method.physical therapy,anti-inflammatory and analgesic and neck support are common measures in modern times. Spondylodesis is the main treatment method in surgical therapy. Furthermore,we are exploring the Allergenic nucleus pulpous tissue grafts in Animal Research and artificial disc implantation.The effect of non-steroid hormone is not definite,and it can stir up heavy gastrointestinal reaction.The operation treatment has great risk and greater financial commitments,so the patient is difficult to accept it.Traditional Chinese medicine has unique advantages on treating this disease,it has a long history and its effectiveness is definite.lt has made great contributions to the treatment of Cervical Spondylosis.Especially in recent years Chinese medicine has been made remarkable advances in treating Cervical Spondylosis.Much compound medicine has better stable curative effect and the price is low.The research of mechanism of Chinese medicine action is limited to microcirculation,hemodynamic, oxygen free radical,mediators of inflammation,and immunity etc.But with reference to using Chinese medicine to restrain Cervical Intervertebral Disc Degeneration and the occuring and developing of Cervical Spondylosis has been poorly studied.This article overviewed the research of the effect of compound medicine on Cervical Spondylosis,to explore the mechanism of compound medicine action.Utilizing animal model to copy disease is an important method to research and explore pathogenesis and the cause of disease.The function which results from bone and ligament to maintain the joint balance and stability is known as Hydrostatic equilibrium,the function which result from muscle is known as Dynamic balance.The normal physiological functioning and stability of cervical region can achieve Dynamic balance dependent on the action of muscle of making adjustments any time after Hydrostatic equilibrium.We can make rats model with Cervical Spondylosis of Dynamic balance and Hydrostatic equilibrium imbalance dependent on destroying the Dynamic balance and Hydrostatic equilibrium system of rats' neck.The mechanism of degeneration intervertebral disc is complex.Researchse showed that degeneration intervertebral disc' main manifestations are the change of the component of cell and extracellular matrix(ECM),and the latter is the immediate cause of losing mechanical characteristics of intervertebral disc. Normally,the ECM is in the balance between constructive metabolism and catabolism.If this balance is destroyed,moisture content and flexible of nucleus pulpous can be lost,Biomechanical of intervertebral disc should be degenerated,and degeneration intervertebral disc can be appeared.Matrix metallopteinase-3(MMP-3)also called Matrix stomelysis-1.It makes a key impotent in degradation of ECM.It not only can degrade proteoglycan and glycoprotein,but also disintegrate variety collagen.Besides,It can make an indirect impotent in degradation of cartilage matrix dependent on activating other inactive MMP.Transforming growth factor-β1 (TGF-β1) is a cell adjusts Growth Factor,can chemo attractant,inducing cartilage and adjust the compound of ECM in cartilage. TGF-β1 has plural bio-effect.It is one of the main Growth Factor to adjust the proliferation of mesenchymal stem cells(MSCs) and differentiation of chondro-genic induction.It restraints biologic activity of mediators of inflammation,and make the Allergenic cell grafts more safety.It can promote the compound of proteoglycan and collagenⅡ.Overall,the degeneration intervertebral disc is closely related to the degradation of ECM,TGF-β1 can promote the compound of ECM;MMP-3 can promote the degradation of ECM.After Experimental and Clinical Research in Early Period,we use jingzhuishu Granule that is commonly-used in clinical to treat degeneration intervertebral disc and compare the difference of TGF-β1 and MMP-3 in each group,to explore the effect way of jingzhuishu Granule.Method and content1. Rat degeneration cervical intervertebral disc model140 3-month-old male SPF SD rat (300±20)g were used,in which 140 rats was in anesthesia through 10% Chloral hydrate(3.5ml·kg-1) was given by intraperitoneal injection.Then shearing rats'sheep,iodophors disinfect aseptic technique.These rats were given a median longitudinal incision about 2-2.5cm long on the back vof neck. When the skin is incised,separating each layer muscle sufficiently,severing splenius cervicis,muscle longissimuscapitis,longissimus cervicis and longissimus atlantis at the transverse,excising costocervicalis and trachelo-occipitalis completely. Then excising ligamenta supraspinale and ligamenta interspinalia of C2-C7 in proper order,suturing peau after arresting bleeding completely.Coating some erythromycin ointment on the incision to prevention of infection after surgery.For 3 days after operation penicillin was given by intramuscular injection,inject 8ten thousand u a day. 16 rats in false operation group were given a median incision about 2-2.5 cm long on the back of neck,suturing peau after arresting bleeding,and the stitches shed off of itself. After operation,taking these rats into cage and let them play by themselves.The models complete after 12 weeks.2. Animal groupingThe models complete after 3 month.110 rats survived in operation group,80 rats were randomly chosen and divided into High doses group of jingzhuishu Granule(group C),medium doses group of jingzhuishu Granule(group D),low doses group of jingzhuishu Granule(group E)and Jingfukang Granule group (group F), model group (group B),Each group has 16 rats.16 rats in false operation group (group A) had no died.6 big groups in all and the 6 groups were divided into giving medicine for 4 weeks and 8 weeks,each group has 8 rats.3. Intragastric administrationWe compute the drug overdosage which was given to rats in each group by intragastric administration according to the medicinal preparations calculation method in science of laboratory animal.The rats received daily drug=the people with normal weight(60kg) received daily drug×0.018X5.So the rats received daily 0.7g·kg-1. d-1 in the low dose group of jingzhuishu Granule,1.4g·kg-1·d-1 in the medium dose group of jingzhuishu Granule,2.8g·kg-1·d-1 in the high dose group of jingzhuishu Granule,0.7g·kg-1·d-1 in the Jingfukang Granule group.And the rats received the same normal saline in the model and false operation groups.4. Draw materials and Preparation of specimensKilling the rats by excessive loss of blood in a short temper after 4 and 8 weeks respectively,taking the whole cervical vertebra range from basilar part of occipital bone to Second dorsal vertebra immediately.Putting the intervertebral disc and interfacing centrums of C5-6 of each rat and fixing them in 10% liquor formalin,to detect morphology and observe their degeneration situation. Cutting the intervertebral disc of C6-7 off at the interface between the end-plate osteochondritis and the centrums under the operative microscope with a magnification of 4 times. Washing the blood stains off by normal saline,0.5ml Phosphate buffer (PH7.4) is added to intervertebral disc tissue(20mg),and put them into refrigerator(-80℃).Detect them with ELISA method.5. Observation by paraffin sectionSpecimens for light microscopy in 10% neutral formaldehyde,15%EDTA decalcified for 2 weeks,With the median sagittal cervical incision,Dehydrated with alcohol gradients,Xylene,Embedded with paraffin blocks,Slices with a slicer,HE staining,Observed under the microscope,Observed nucleus,annulus,endplate and vertebral degeneration.Scores on the disc with Grading standards of Miyamoto: 1Level:Normal disc, annulus and nucleus arrangement rules, the growth of cartilage. endplate cartilage and artiular cartilage is divided into layers, tidemark clear. 2Level:Anular lamellar structure disappeared,proliferation ofcartilage endplate. 3Level:Nucleus shrink or disappear. 4Level:Fissures of the Anular. 5Level:Herniated Disc or Osteophyte formation.6. Detecting the contents of TGF-β1 and MMP-3 with ELISA method.Carry procedure on according to the instruction of Kit strictly.7. Statistical methodsDegree of intervertebral disc degeneration in each group was compared with rank sum test.The contents of TGF-β1 and MMP-3 among groups in 4 and 8 weeks of treatment was compared with one-way analysis of variance,and the two-two comparisons among the means were done by LSD method,significant level a =0.05;Independent-Samples T Test was compared between 4 and 8 weeks of treatment,significant levelα=0.05.Applying the statistical soft system of Spss 16.0 for windows to analyze the materials.Result1. Degree of intervertebral disc degeneration in each groupRats were excised the superficial and intermediate muscle on the back of neck,and were destroyed the Hydrostatic equilibrium system of neck.It can cause degeneration cervical intervertebral disc.Compared with each group(A, B, C, D, E, F group), the degree of disc degeneration there was a significant difference (P<0.05). group B was significantly degeneration, the treatment groups (C, D, E, F group) followed.2. The result of ELISA method of rats' intervertebral disc in each group2.1 The concentration of MMP-3 of cervical intervertebral disc in each group: Compared with each group, the concentration of MMP-3 has a significant difference (F=87.517. P=0.000). multiple comparison method with the results of SNK, the concentration of MMP-3 of group A,C,D,E,F Significantly lower than group B, group D,E,F Significantly lower than group C, group D and no significant difference in group F, the concentration of MMP-3 were significantly different between the time (F=6.581, P=0.012),8 weeks of treatment group, the concentrations of MMP-3 were significantly higher than the 4 weeks group.2.2 The concentration of TGF-β1 of cervical intervertebral disc in each group: Compared with each group, the concentration of TGF-β1 has a significant difference (F=87.990, P=0.000). multiple comparison method with the results of SNK, the concentration of TGF-β1 of group A,C,D,E,F Significantly higher than group B, group D,E,F Significantly higher than group C, the concentration of MMP-3 were no significantly different between the time (F=0.174, P=0.678)Conclusion1. Jingzhuishu Granule and Jingfukang Granule have significant curative effect on degeneration cervical intervertebral disc.2. We can manufacture a Cervical Spondylosis animal model which is likely induced by head drop for a long time (that is the factor of Cervical Spondylosis pothogenesy) by destroy the Dynamic balance and Hydrostatic equilibrium system of animal.3. The mechanism of relieving degeneration intervertebral disc by jingzhuishu Granule may be related with the following factor that can influence ECM: (1)It can decrease the compound of MMP-3 in intervertebral disc.(2)It can promote the compound of TGF-β1 in intervertebral disc.4. With the medication regimen of jingzhuishu Granule, the treatment of cervical disc degeneration of the effect may be more significant. |
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