Effect Of Serum Containing Qing-er Prescription On Pressure Degeneration Model Of In Vitro Culture Rabbit Spinal Motion Segment

Background:Degeneration of the intervertebral disc to be linked with lower back pain,Chinese traditional medicine emphasizes that the early treatment and prevent before disease onset,which has unique advantages on curing the difficult and chronic diseases as lumbar intervertebral disc degeneration, among TCM therapies the tonifying kidney and promoting blood circulation is one of the most commonly used method for treatment of degenerative disc disease, applying of the Qing-er prescription which on behalf of the therapy to treating of related diseases has obtained satisfactory curative effect in clinic.At present,although there are a lot of researchs about the degeneration of the in tervertebral disc, it is not clear on mechanism of biological and mechanical wa y of comprehensive leading to degeneration,and there is a close relationship bet ween biomechanical and intervertebral disc degeneration,but the mechanical loadi ng is also the fundamental factor to stimulate cell metabolic activity, so in order to better prevention and treatment of intervertebral disc degeneration, the relatio n and effect between biomechanics and intervertebral disc be needed more in-dep th understanding;On the other hand, there are many complex factors about tradit ional Chinese medicine compositions and mechanism, Combined with the factors about in vivo metabolism and environmental difference of the object of study,it is difficult to full and in-depth to clarify its mechanism, so it is our challenge to eliminate theses various interference factors,and to clarify the mechanism of d egeneration and effect of TCM on treating of related diseases.Therefore, to con struct an ideal experimental model has an important significance to research the pathogenesis and clinical intervention of intervertebral disc degeneration.In vitro culture of the whole intervertebral disc organ,which can be used to study the related mechanism of metabolism and degeneration of the intervertebral disc under controlled conditions,provides a experimental platform to observe the responses of intervertebral disc on external stimuli and the histological changes of normal and degenerative intervertebral disc.On these basis, the related studies cultured of the spinal motion segment model in vitro,which on the one hand making the model be more close to the in vivo environment, on the other hand offering the fixed force point for loading device,provides the conditions to control and research of influences of biomechanics on intervertebral disc.This study will combined the spinal motion segment model with biomechanics to observe the relationship between biomechanical and intervertebral disc, and to provide a better experimental platform to research the mechanisms of traditional Chinese medicine for prevention and treatment of intervertebral disc degeneration.Objective:1.To observe the clinical curative effect of Qing’e prescription treatment of discogenic low back pain;2.Comparing the changes of nucleus pulposus after in vitro culture of rabbit whole intervertebral disc and spinal motion segment, to provide the conditions for the study of the influence of the biomechanics of intervertebral disc;3.By establishing of pressure degeneration model of in vitro culture rabbit spinal motion to study the influence of static pressure load on the intervertebral disc,and to provide conditions for further study of traditional Chinese medicine for prevention and treatment of intervertebral disc degeneration;4.Observing the effect of serum containing Qing-er prescription on pressure degeneration model of in vitro culture rabbit spinal motion segment and Preliminary investigating the mechanism, to provide the basis for tonifying kidney and promoting blood circulation for the prevention and treatment of degenerative disc disease.Method:1.Using a prospective design method> 43 cases of discogenic low back pain taking Qing-er prescription,1 agent/d, oral in morning and evening, continuous 2 weeks, then the VAS and ODI scores were recorded at pretherapy, first week of treatment, second week of treatment and follow-up after one month, while observing of the clinical symptoms, and judging the curative effect.2.The 50 intervertebral discs and the spinal motion segments were harvested respectively under aseptic conditions from 21 New Zealand white rabbits which were randomly divided into organ group with 8 and segment group with 13,and these specimens were maintained in organ culture with hyperosmotic media (410mOsm/kg),then 10 discs of the two groups were observed respectively by HE staining,proteoglycan content staining, immunohistochemistry of collagen type II and cells viability of nucleus pulposus before culture and on 3,7,14,21 days after culture.3.The 28 spinal motion segments were harvested respectively under aseptic conditions from 14 New Zealand white rabbits which were randomly divided into control group and pressure group with 7 in each one,and the control group cultured in 6-well plate, while the pressure group in the device of Spinal motion segment in vitro loading and cultivating,the two groups’ specimens were maintained in organ culture with hyperosmotic media and 10%drug free serum,then 7 discs of the two groups were observed respectively by HE staining,proteoglycan content staining, immunohistochemistry of collagen type Ⅱ and Real-time PCR testing of nucleus pulposus before culture and on 3,7,14days after culture.4.The 28 spinal motion segments were harvested respectively under aseptic conditions from 14 New Zealand white rabbits which were randomly divided into pressure group and medicated group with 7 in each one.and the two groups cultured in the device of Spinal motion segment in vitro loading and cultivating, the two groups’ specimens were maintained in organ culture with hyperosmotic media which the medicated group and pressure group respectively containing 10% Qing-er prescription containing serum and 10% drug free serum,then 7 discs of the two groups were observed respectively by HE staining,proteoglycan content staining, immunohistochemistry of collagen type Ⅱ and Real-time PCR testing of nucleus pulposus before culture and on 3,7,14days after culture.Results:1.The clinical efficiency is 86.04% of first week of treatment among 43 patient, which is 90.69% in two week of treatment and follow-up. There was a significant difference about VAS and ODI scores between pretherapy with post-treatment and follow-up(P<0.001), while the scores of second week of treatment and follow-up were better than first week of treatment(P<0.05), but there were no statistical significance between the second weeks of treatment and follow-up(P>0.05).2. HE showed the intervertebral disc tissue structure remained intact after culture of 21 days organ group and 14days segment group, but there was severely degenerated of 21 days segment group; PAS/AB staining of proteoglycan of nucleus pulposus showed that there were not decrease of tinting strength of two groups, but the strength weakened slightly of two groups at 14days, and the tinting strength became weaker at 21 days segment group, the change is more obvious than the organ group; The intensity value of type II collagen immunohistochemical staining in the nucleus pulposus were not changed significantly within 21 days organ group and 14days segment group(P>0.05), but the staining of segment group at 21 days became shallower, there was significant difference compared with before each time points and organ group on 21days(P<0.05); The intensity value of fluorescence staining of nucleus pulposus cells was not changed significantly within 7days of two groups(P>0.05), the intensity value decreased slightly at 21 days organ group and 14days segment group, but there were no significant difference compared with before time points(P>0.05),however at 21 days the intensity of segment group decreased as cells viability of Nucleus pulposus decreased, and there was a significant difference compared with before each time points and organ group on 21days(P<0.05).3.HE showed the intervertebral disc tissue structure remained intact after culture of 14days of control group, but the morphological integrity to be gradually destructed of pressure group after 3days; PAS/AB staining of proteoglycan of nucleus pulposus showed that the tinting strength to be reduced from 7days of control group, while to pressure group the tinting strength significantly decreased from 3days, and the tinting uneven distribution; The intensity value of type Ⅱ collagen immunohistochemical staining in the nucleus pulposus were not changed significantly within control group(P>0.05), but pressure group has a Obvious reaction at 3days,there are significant difference compared with the pre-culture and control group (P<0.05), and the reaction obviously reduced at 7days,comparing with the control group has a difference (P<0.05),and there is no significant difference between 14days and 7d(P>0.05).but a significant difference with the control group (P<0.01); Real time-PCR testing of Agg gene expression showed that compared with the pre-culture not significant change in control group at 3days(P>0.05), but the expression decreased at 7d and 14d,and has obvious difference with pre-culture,while to pressure group the expression significantly decreased from 3days (P＜0.05).7d and 14d further decreased,and having a significant difference with the control group (P<0.01); Expression of COL2al gene showed that compared with the pre-culture which reduced in control group after culture (P＜0.05), but the level was stable during the culture(P>0.05), the expression of the pressure group is up-regulated at 3d (P<0.05), having a significant difference with the control group (P<0.01),but which obviously reduced at 7days (P＜0.05),there are significant difference compared with the pre-culture and control group (P＜0.05), and 14d and 7d had no obvious difference,but compared with the control group had a significant difference (P＜0.01).4.HE showed the the morphological integrity of intervertebral disc tissue to be gradually destructed of pressure group after 3days, while to the medicated group the tissue structure can be remained intact after culture of 7days,and destructed at 14d as pressure group; PAS/AB staining of proteoglycan of nucleus pulposus showed that the tinting strength of pressure group significantly decreased from 3days,and decreased more obviously after 7d, the tinting uneven distribution, while to the medicated group the tinting partly decreased at 3d,and 7dand 14d were also significantly weakened, indicating that the proteoglycan content decreased; The intensity value of type II collagen immunohistochemical staining in the nucleus pulposus showed both groups have a obvious reaction at 3days (P<0.05), and medicated group was more significant(P<0.05), however the reaction of both groups were all obviously reduced at 7days(P<0.05), but reaction of medicated group stronger than pressure group(P<0.05),and at 14d the responses were weaker of two groups,and having no significant difference between them; Real time-PCR testing of Agg gene expression showed that compared with the pre-culture the expression significantly decreased of both groups from 3days (P<0.05),but medicated group much more obvious than pressure group(P<0.01), both groups at 7d and14d are further decreased (P<0.05),and there was no difference between them (P>0.05); Expression of COL2a1 gene showed that compared with the pre-culture which be up-regulated of two groups at 3d(P<0.05), and medicated group more significant(P<0. 01), however the expression level obviously decreased at 7days of both groups(P<0.05), but the medicated group still higher than the pressure group (P<0.05), at 14d the responses were weaker of two groups,and having no significant difference between them (P>0.05)Conclusion:● Qing’e prescription can significantly improve the clinical symptoms of patients of discogenic low back pain, and the curative effect being stable during follow-up period.● It is not obviously degenerated of the discs of organ group cultured within 21 days and segment group cultured within 14days,but there was significant degeneration of the intervertebral disc of segment group after cultured 21 days, so the rabbit spinal motion segment can only be used on research about the influence of the biomechanics of intervertebral disc as a vitro experimental model within 14days.● Static compression load within the physiological range can partly stimulate the metabolic function of intervertebral disc in short time,but the persistent static compression load will lead to the degeneration of intervertebral disc.● In short period (14d),the serum containing Qing-er prescription could not obviously improve the degeneration of the intervertebral disc caused by the persistent static compression load, but in the short term (7d) which can delay the degeneration of intervertebral disc to a certain extent, providing the basis for tonifying kidney and promoting blood circulation for the prevention and treatment of degenerative disc disease.● The pressure degeneration model of in vitro culture rabbit spinal motion segment can be used as a vitro experimental model to study the relationship between biomechanical and intervertebral disc degeneration,and to research the mechanisms of traditional Chinese medicine for prevention and treatment of intervertebral disc degeneration.