| Objective: To observe Liu Wei Di Huang Wan in vitro in rabbit intervertebral disc degeneration model affect the MAPK signaling cascade, Liu Wei Di Huang Wan discuss prevention of disc degeneration effect.Methods: The serum containing drug was prepared: 6-month-old New Zealand white rabbits of clean grade 5, between body weight 2.0-3.2kg, Hunan Medical University Animal Center, Liu Wei Di Huang Wan Ning suspension oral pill, according to human and animal surface area ratio calculation animal equivalent dose administered in the control group fed with normal saline. Twice daily for 7 days, the natural feeding, breeding environment standard. In the last four hours after gavage by cardiac puncture for blood collection, centrifugation, filtration, inactivation standby. Select 6-month-old New Zealand rabbits of clean grade 20, the establishment of in vitro culture system as a whole disc Haschtmann reference method, the animals were sacrificed by air embolism after under sterile conditions before using the midline approach or intraperitoneal approach taken with the endplate of L1-L6 disc 100, try to remove the bone and ligaments attached to the end plate, preserving the integrity of endplate cartilage, continuous with 0.9% Na Cl solution, and a solution of povidone-sulfonamide containing 55 mmol / L sodium citrate, 50 μg / m L gentamicin Hanks solution rinse. Sample disc is placed in six-well plates, on a drum(drum rotation speed 10 r ? h-1), containing 5% fetal bovine serum, 50 μg / m L gentamycin, 20 mmol / L sodium citrate the DMEM / F12 culture, placed in 37 ℃, 5% CO2, 100% relative humidity incubator overnight. Were randomly divided into control group, TNF-α group, p38-JNK / SAPK blocking group, Liu Wei Di Huang Wan serum, TNF-α + Liuweidihuang pill containing serum group, the specimens were inoculated overnight in six-well plates, add 8ml DMEM with 10% fetal bovine serum, 50μg / m L gentamicin, 25μg / m L of vitamin C / F12, placed in 37 ℃, 5% CO2, 100% relative humidity incubator culture, medium was changed once every two days. TNF-α group another culture medium containing 10 ng / μl TNF-α2μl; p38-JNK blocker group and the other containing p38 MAPK specific inhibitor SB203580, JNK specific inhibitor SP600125 each 20μmol / L each 10μl, 10 ng / μl TNF-α2μl, Liu Wei Di Huang Wan serum culture medium and the other containing Liu Wei Di Huang Wan serum 10%; TNF-α + Liuweidihuang pill containing serum containing 10 ng / μl TNF-α2μl, Liu Wei Di Huang Wan serum 10%. 2,4,8,14 were cultured first day collection of specimens tested.Results: In the first 2d, 4d, 8d, 14 Tian: 1.TNF-α group and the blank group, there was significant difference P <0.05, m RNA content of TNF-α group JNK pathway and P38 protein expression were significantly higher than the control group, in the light microscope fracture annulus structure significantly increased cell nucleus morphology changes significantly. Description has entered the disc degeneration process, successful model. 2.P38.JNK + TNF-α blockers compared with TNF-α + Liu Wei Di Huang Wan serum group, P <0.05, P38.JNK blockers + m RNA content of TNF-α group P38 and JNK pathways, protein expression TNF-α were significantly lower than group + Liuweidihuang pill group. TNF-α + Liu Wei Di Huang Wan serum group and TNF-α group, P <0.05, significant difference. TNF-α + m RNA content Liuweidihuang pill serum JNK pathway and P38 protein expression were significantly lower than TNF-α group, can be explained Liu Wei Di Huang Wan can to some extent block JNK pathway activation, delay disc degeneration. Different time points, m RNA content JNK 1.TNF-α group and P38 pathways, protein expression in 2d and 4d, 4d and 8d, 8d and 14 d has a significant difference compared to P <0.05, the content has been an upward trend, a significant increase in fracture annulus structure light microscope, morphological changes of nucleus pulposus cells significantly. It may indicate successful model. 2.TNF-α + m RNA content Liuweidihuang pill serum JNK pathway and P38 protein expression in 2d and 4d, 4d compared with 8d, 8d and 14 d significant difference P <0.05, the content of the expression of an upward trend, but rise significantly lower than TNF-α group, also shows that Liu Wei Di Huang Wan can to some extent block JNK pathway activation, delay disc degeneration.Conclusions: Liu Wei Di Huang Wan possible through a certain degree of inhibition of rabbit disc cells JNK, gene and protein expression P38 MAPK, reduce inflammation disc cells, delaying the process of disc degeneration, disc cells play a protective role. |
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