| Purpose and significanceCervical spondylosis, is neck intervertebral discs, bone and cartilage and ligament degenerative change happened, this change stimulation or oppression of the spinal cord, around nerve root, the blood vessel, soft tissue, sympathetic nerve, etc, this organization caused a group of syndrome of discomfort.。Epidemiological data shows:Shanghai, foshan, lanzhou, the incidence of cervical spondylosis, respectively; 13.70% 10.78%,17.35%, Cadres, technicians, accounting personnel 78.83%, respectively 74.21% incidence 58.70%, including work, nervous and long-term volt case 59.75% 14.9% of; Wuhan have symptoms of cervical spondylosis of primary and middle school students as much as 30%. Along with the work and life style changes, such as long-term working at his desk, computer and the widespread use of air conditioning, the popularity of private cars, the people and suffering from the wind cold dampness neck flexion, causing the opportunity to increase the prevalence of cervical spondylosis rising, and onset ages have younger trend.Cervical disc degeneration is considered the emergence and development of cervical spondylosis basic reasons. Degenerative disc appear capacity reduction, proteoglycan aquifer down the content, collagen type conversions, a large release inflammatory mediators. The main biomechanics function disc height, is to maintain the vertebral clearance against compression force. Cervical spondylosis patients imaging examination showed the vertebral clearance narrowed changes. Many factors involved in the disc degeneration pathological process. Among them, the disc cell nutrition decrease supply is the basic factors of cervical disc degeneration. Nerlich think disc degeneration of the most key factor is the nutrition supply reduced. VEGF is a highly specific mitotic original, it in embiyogeny and wound healing process start the formation of blood vessels. VEGF also is a kind of effective vascular permeability inducing factors, VEGF expression and organization of density and capillaries is closely related to the number of new blood vessels. VEGF by many kinds of normal cells can secrete. Vascular endothelial cells in does not normally express VEGF, but when the endothelial cells, when placed in hypoxic conditions inside cells mRNA VEGF was increased, indicating the level obviously not only through the paracrine way to remove VEGF in the role of endothelial cells, and there are secreted oneself mechanism involved. Degenerative disc of intravascular blood stasis caused pressure increases, vascular compression, circulation suffocate, local hypoxia, thus inducing VEGF expression.The water channel protein main function is to transport water. According to AQPs for water and other solute permeability, Ishibashi is divide AQPs family into two and family AQP0, AQP1, AQP2:AQP4, AQP5, AQP6, AQP8 for a family, AQP7 AQP3, Abigail, AQP9,10, belong to other. Disc is a rich moisture organs, including the moisture content of nucleus pulposus about 80%. And a degenerative disc for water content characteristic expression that drop in MPI, T2 weighted signal weakening for performance. Miyanoto think the nucleus pulposus shrinks or disappear is disc degeneration pathological index. Water metabolism in maintaining the normal physiology function discs plays an important role. Water as the main nutrients in one of the role of osmotic pressure, the next, access, and maintain discs organization of cartilage endplate hydration state. Nutrients and the permeability of fiber ring and endplate is closely related to the hydration degree, the more the higher permeability aquifer, permeate the faster. Water, as a kind of nutrients to other nutrient absorption have important role. We investigated water metabolism for understanding the disc degeneration of tumor-initiating factors have important meaning.Professor Chan famous Chinese medicine compound created a "Jingzhui Shu", through 20 years of clinical use was satisfied with the effect. Through early animal experiments and clinical trials, our team found that "Jingzhui shu" to improve microcirculation and reduce platelet aggregation, reduce blood viscosity, increased vascular wall flexibility, expansion vessel, can significantly improve nerve root cervical spondylosis Pain, numbness, stiffness, chills, muscle atrophy unable symptoms.We compared the intervertebral disc height and water content, so that the relationship between the treatment of cervical disc degeneration shu particles of the way. Through comparing the before and after the treatment of cervical disc degeneration degree, disc height, disc moisture content and water 1,3 and ventilation protein vascular endothelial growth factor, further clarify the changing relations between the jingzhuishu Granule prevent cervical disc degeneration mechanism. We studied the regulation effects of puerarin AQP1,3, further discussing compound effective composition on disc degeneration role.Method and content1,Rats of cervical disc degeneration was established3-month-old male SPF SD rat (200-220)g were used, in which conersion rat was in anesthesia through 10% Chloral hydrate(3.5ml/kg) 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 of 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. For 3 days after operation penicillin was given by intramuscular injection, inject 8ten thousand u a day. 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,X-ray observationsRespectively in 4 weeks and 8 weeks,3% of pentobarbital sodium 40mg·kg-1 intra-abdominal injection, rats after anesthesia was filmed cervical lateral a piece, strictly mastering the drug dose and anesthesia to X-ray after filming the time to control muscle tension the disc height influence degree. Calculation C5-6 disc height index (DHI).3,Pathological observationKilling 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.Specimens 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:1 Level:Normal disc, annulus and nucleus arrangement rules, the growth of cartilage., endplate cartilage and articular 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.4,Disc moisture detectionApply the vacuum freeze-drying water content determination of intervertebral disc. Will large rats were controlled, including C4-death after five vertebral asked plate used in water content determination. Take full disc tissue; divest cartilage tissue around, electronic scales weighing record (wet weight). Disc in Vacuum dryer 80 degrees below zero freeze-drying 24 hours after weighing (dry weight),1 week weighing record again. Disc water content recorded calculate formula is as follows: disc moisture content=wet weight-dry weight. According to the above formula animal intervertebral discs groups water content.5,Western blotting detection disc AQP1,3 expressionWe put the C4-5 disc cracking. The expression of AQP1,AQP3 andβ-actin was detected by way of Western blotting. We evaluated the expression of protein by analyzing the gray scale, and calculated the ratio of the value of gray scale of the protein with the value of gray scale ofβ-actin.6,Western blotting detection disc VEGF expressionWe put the C4-5 disc cracking. The expression of VEGF andβ-actin was detected by way of Western blotting. We evaluated the expression of protein by analyzing the gray scale, and calculated the ratio of the value of gray scale of the protein with the value of gray scale ofβ-actin.7,Statistical methodsApply the statistical soft system of Spss 16.0 for windows to analyze the materials, the measurement data indicated by the mean value and standard deviation. Each rat treatment 4 weeks and eight weeks disc water content, disc height index, AQP1,AQP3 and VEGF, using Independent sample t-test, different between treatment group compares the single factor analysis of variance (ANOVA), variance when using multiple comparison together-t method inspection; LSD Variance not neat, the use of approximate variance analysis, multiple comparison Welch method using Dunnett T3 method inspection. Each rat degeneration level by level on the material level of a statistical description by frequency data, using multiple independent sample non-parameter test Kruskal-Wallis method, P< 0.05 for with a statistical significance.ResultAll rats without a loss and the experimental results are incorporated into the analysis, no significant weight loss. Wound healing well, no autophagy bite and lick, no purulent secretions.1. The influence of jingzhuishu Granule in disc high index4 weeks group compared with 8 weeks, disc height index difference was significant (F=5.518, P=0.026), A statistically significant differences between groups (F=60.276, P=0.000), Groups with the interaction between before and after treatment effect (F=4.445, P=0.001)。Two independent sample t-test shows, Each groups disc height index are significant differences (t=10.787, P=0.000; t=7.640, P=0.000; t=11.532, P=0.000; t=15.278, P=0.000; t=9.751, P=0.000), except false group (t=0.793, P=0.454) Multiple comparisons between groups, Drug use 4 weeks later, compared with model group, each group is can increase disc height index significance(F=130.722, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Drug use 8 weeks later, compared with model group, each group is can increase disc height index significance(F=121.730, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst.Hence, Compared with model group, each drug intervention group all can increase the height of the disc, and exist time and index of dose-response, including cervical curative effect of particle high doses is best.2. The influence of jingzhuishu Granule in pathological sectionBy pathological visible, false group cervical intervertebral tide mark clearly visible, fiber ring structure is clear, not seen nucleus pulposus obviously shrinks, calcification articular cartilage is thinner, calcification subchondral distribution has large vessels,4 weeks group and eight weeks group no obvious difference. Model rats, cervical intervertebral appear degenerative change, fiber ring board layer structure disorder, arrangement, mild irregular corrugation of nucleus pulposus or smaller, a few prominent, the nucleus pulposus mild calcified layer and the articular cartilage were thickening calcified layer. Eight weeks than 4 weeks group of a marked change. Each drug intervention group:rat disc form a model group got obvious improvement. Puerarin extract each dose rats, disc form close to the control group, light-microscopy morphology intact, fiber circle permutation nucleus pulposus comparative rules, comparative full, tide mark visible, endplate congestion conditions improve within blood vessels, vascular shoots more than model group, including eight weeks than 4 weeks group of a marked change.4 weeks after treatment, degenerative level difference between groups was statistically significant (χ2=11.781, P=0.038); Disc degeneration level respectively 15.00.25.94.28.69.35.62.22.56.19.19, according to the average rank times we concluded, the high dose degeneration is lightest, jingfuk group is secondly serious, model group is the most serious.8 weeks after treatment, degenerative level difference between groups was statistically significant (χ2=11.992, P=0.035); Disc degeneration level respectively 13.25.26.94.29.76.34.06,22.44.20.56, according to the average rank times we concluded, the high dose degeneration is lightest, jingfuk group is secondly serious, model group is the most serious.3. The influence of jingzhuishu Granule in Disc water content4 weeks group compared with 8 weeks, Disc water content difference was significant (F=10.518, P=0.002), A statistically significant differences between groups (F=220.777, P=0.000), Groups with the interaction between before and after treatment effect (F=6.694, P=0.000)。Two independent sample t-test shows, Each groups Disc water content is significant differences (t=3.342, P=0.005; t=2.300, P=0.037; t=2.750, P=0.016; t=5.889, P=0.000; t=4.022, P=0.001), except false group (t=1.226, P=0.241)Multiple comparisons between groups, Drug use 4 weeks later, compared with model group, each group is can increase Disc water content significance(F=113.590, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Drug use 8 weeks later, compared with model group, each group is can increase Disc water content significance(F=113.119, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Hence, Compared with model group, each drug intervention group all can increase the Disc water content, and exist time and index of dose-response, including cervical curative effect of particle high doses is best.4. The influence of jingzhuishu Granule in AQP14 weeks group compared with 8 weeks, AQP1 difference was significant (F=70.653, P=0.000), A statistically significant differences between groups (F=102.600, P=0.000), Groups with the interaction between before and after treatment effect (F-476.093, P=0.000)。Two independent sample t-test shows, Each groups AQP1 is significant differences (t=7.499, P=0.000; t=12.506, P=0.000; t=6.154, P=0.004; t=9.396, P=0.001; t=9.232, P=0.001), except false group (t=1.603, P=0.184)Multiple comparisons between groups, Drug use 4 weeks later, compared with model group, each group is can increase AQP1 significance(F=366.466, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Drug use 8 weeks later, compared with model group, each group is can increase AQP1 significance(F=358.012, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Hence, Compared with model group, each drug intervention group all can increase the AQP1, and exist time and index of dose-response, including cervical curative effect of particle high doses is best.5. The influence of jingzhuishu Granule in AQP34 weeks group compared with 8 weeks, AQP3 difference was significant (F=25.427, P=0.000), A statistically significant differences between groups (F=665.591, P=0.000), Groups with the interaction between before and after treatment effect (F=26.073, P=0.000)。Two independent sample t-test shows, Each groups AQP3 is significant differences (t=6.997, P=0.002; t=10.394, P=0.000; t=13.794, P=0.000; t=5.155, P=0.007; t=9.307, P=0.001), except false group (t=2.435, P=0.720) Multiple comparisons between groups, Drug use 4 weeks later, compared with model group, each group is can increase AQP3 significance(F=566.358, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Drug use 8 weeks later, compared with model group, each group is can increase AQP3 significance(F=495.975, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Hence, Compared with model group, each drug intervention group all can increase the AQP3, and exist time and index of dose-response, including cervical curative effect of particle high doses is best.6. The influence of jingzhuishu Granule in VEGF4 weeks group compared with 8 weeks, VEGF difference was significant (F=18.345, P=0.000), A statistically significant differences between groups (F=590.710, P=0.000), Groups with the interaction between before and after treatment effect (F=29.853, P=0.000)。Two independent sample t-test shows, Each groups VEGF is significant differences (t=O12.811, P=0.000; t=4.657, P=0.010; t=6.388, P=0.003; t=7.953, P=0.001; t=5.443, P=0.006), except false group (t=2.923, P=0.056)Multiple comparisons between groups, Drug use 4 weeks later, compared with model group, each group is can increase VEGF significance(F=143.668, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Drug use 8 weeks later, compared with model group, each group is can increase AQP3 significance(F=374.502, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Hence, Compared with model group, each drug intervention group all can increase the VEGF, and exist time and index of dose-response, including cervical curative effect of particle high doses is best.7. The influence of puerarin in Disc water content One-way ANOVA shows, Each groups Disc water content is significant differences (F=321.260, P=0.000).Multiple comparisons between groups, Drug use 8 weeks later, compared with model group, each group is can increase Disc water content significance(P=0.000,0.000,0.013,0.003). high and middle puerarin group compared with celebrex group show the significant differences(P=0.000,0.011).high and middle puerarin group compared with low group show the significant differences (P=0.000,0.013). low puerarin group compared with celebrex group show no significant differences (P=0.998)Hence, Compared with model group, each drug intervention group all can increase the Disc water content, and exist dose-response, including cervical curative effect of particle high doses is best,.high and middle puerarin group more effect than celebrex group.8. The influence of puerarin in AQP1One-way ANOVA shows, Each groups AQP1 is significant differences (F=123.205, P=0.000).Multiple comparisons between groups, Drug use 8 weeks later, compared with model group, each group is can increase AQP1 significance(P=0.001,0.001,0.015,0.026). high and middle puerarin group compared with celebrex group show the significant differences (P=0.004,0.007).high and middle puerarin group compared with low group show the significant differences (P=0.001,0.019). low puerarin group compared with celebrex group show no significant differences (P=0.166)Hence, Compared with model group, each drug intervention group all can increase the AQP1, and exist dose-response, including cervical curative effect of particle high doses is best,.high and middle puerarin group more effect than celebrex group.9. The influence of puerarin in AQP3 One-way ANOVA shows, Each groups AQP3 is significant differences (F=548.956, P=0.000).Multiple comparisons between groups, Drug use 8 weeks later, compared with model group, each group is can increase AQP3 significance(P=0.000,0.000,0.005,0.046). high and middle puerarin group compared with celebrex group show the significant differences (P=0.000,0.000).high and middle puerarin group compared with low group show the significant differences (P=0.000,0.001). low puerarin group compared with celebrex group show no significant differences (P=0.233)Hence, Compared with model group, each drug intervention group all can increase the AQP3, and exist dose-response, including cervical curative effect of particle high doses is best,.high and middle puerarin group more effect than celebrex group.Conclusion1. jingzhuishu Granule can significantly delay cervical rats of the cervical intervertebral disc degeneration level, this trend has certain concentration-response and aging relations;2. jingzhuishu Granule can prevent cervical disc height lower water content and slow, and then delay reduced the cervical intervertebral disc degeneration in rats, and this trend has certain concentration-response and aging relations;3. jingzhuishu Granule can adjust cervical AQP1,3 and VEGF expression, this may be slow disc degeneration of one reason;4. puerarin can adjust cervical AQP1,3 expression, this may be the mechanism of jingzhuishu Granule treatment cervical disc degeneration. |
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