| Objective:To observe the effect of Six Ingredient Rehmannia Pill on rabbit intervertebral disc degeneration model in vitro extracellular matrix components, to investigate the effect of Six Ingredient Rehmannia Pill Treatment of intervertebral disc degeneration.Method:20 June old New Zealand rabbits of clean grade were selected, according to the Haschtmann method to establish the culture system of the whole intervertebral disc in vitro. Air embolism and the animals were sacrificed, under sterile conditions, the median approach or the abdominal anterior approach taken with endplate l1-l6 disc 100, try to remove the endplates attached to the bone and ligament, to retain the integrity of the cartilage endplate, continuous with 0.9% Na Cl solution, povidone iodine solution and containing 55 mmol / L sodium citrate, 50 ? g / ml gentamycin Hanks solution rinse. Disc samples were placed in 6-well plates, placed in the drum, drum speed- 10 R h-1), to containing 5% fetal bovine serum, 50 ? g / ml gentamycin, 20 mmol / L citric acid sodium / F12 medium, is placed at 37 DEG C, 5% CO2, 100% relative humidity temperature box overnight. Were randomly divided into blank control group, TNF alpha group, p38-JNK/SAPK blocking group, Liuwei Dihuang Pill serum group, TNF alpha + containing serum group of Liuwei Dihuang pill, will stay the night after the specimens were inoculated in 6-well plates, added 8ml containing 10% fetal bovine serum, 50 ? g / ml gentamycin, 25 ? g / ml vitamin C in DMEM / F12 medium, culture is placed at 37 DEG C, 5% CO2, 100% relative humidity temperature box, every two days for liquid once. TNF alpha were cultured in the liquid and the other containing 10 NG / u l TNF alpha 2 m 1; p38-JNK blocking group and the other containing p38 MAPK specific inhibitor SB203580, JNK specific blocking agent sp600125 20 mu mol / L, 10 UL, 10 NG / u l TNF alpha 2 m 1, Liuwei Dihuang pills group, serum culture medium and the other containing 10% serum Liuwei Dihuang Wan; TNF alpha + containing Liuwei Dihuang Wan Group, the serum containing 10 NG / u l TNF alpha 2 m 1, Liuwei Dihuang Pill 10% serum. The measured respectively in second, 4, 8 training, 14 days to collect samples.Results:The TNF alpha group compared with the control group, ratios of TNF alpha group protein polysaccharide expression and m RNA content, glycosaminoglycan content of polysaccharide, chondroitin sulfate and keratan sulfate, hyaluronic acid content, type II collagen expression and m RNA content was significantly lower than that of control group, the content of type I collagen and m RNA expression is obviously higher than that in control group, P<0.05, the differences have statistical significance, indicating that the rabbit intervertebral disc in vitro degeneration model was established successfully.TNF alpha + containing Liuwei Dihuang Wan serum group and TNF alpha group compared, TNF alpha + containing Liuwei Dihuang pills group, the serum protein polysaccharide expression and m RNA content, glycosaminoglycan content of polysaccharide, chondroitin sulfate and keratan sulfate ratio, content of hyaluronic acid(HA), type II collagen expression and m RNA content was significantly higher than that in the TNF group; the expression of type I collagen content and m RNA was significantly lower than that of TNF alpha group, P<0.05, the differences have statistical significance, indicating that the drug containing serum of Liuwei Dihuang Pill intervention can delay the effect of in vitro intervertebral disc degeneration.Polysaccharide TNF alpha + containing Liuwei Dihuang Wan serum group and p38-JNK group compared, TNF alpha + containing Liuwei Dihuang pills group, the serum protein expression and m RNA content, glycosaminoglycan content of polysaccharide, chondroitin sulfate and keratan sulfate ratio, content of hyaluronic acid(HA), type II collagen expression and m RNA was significantly lower than that in group p38-JNK; type I collagen content and m RNA expression was significantly higher than that of p38-JNK group, P<0.05, the difference has statistical significance, that p38-JNK pathway exists, and than TNF alpha + containing Liuwei Dihuang Wan serum group delay in vitro intervertebral disc degeneration and the effect is more obvious.TNF alpha + containing Liuwei Dihuang Wan serum group specimens, 2 days and 4 days, 4 days and 8 days, 8 days and 14 TX comparison, P < 0.05, the difference was statistically significant, the ratio of pigment protein rabbit polysaccharide expression and m RNA content, glycosaminoglycan content of polysaccharide, chondroitin sulfate and keratan sulfate, hyaluronic acid content, type II collagen expression and m RNA content was gradually decreased, the content of type I collagen and m RNA expression of content increased gradually, but compared with TNF alpha group, the trend towards smaller, indicating that Liuwei Dihuang pill can effectively delay the degeneration of intervertebral disc.TNF alpha group, 2 days and 4 days, 4 days and 8 days, 8 days and 14 TX comparison, P<0.05, the difference was statistically significant, the ratio of pigment protein rabbit polysaccharide expression and m RNA content, glycosaminoglycan content of polysaccharide, chondroitin sulfate and keratan sulfate, hyaluronic acid content, type II collagen expression and m RNA content was gradually decreased, the content of type I collagen and m RNA expression of content increased gradually, also shows that the intervertebral disc degeneration model was successfully established. Six taste glutinous rehmannia pill group compared with the control group c P was 0.05, the difference was not statistically significant.Conclusion : In the model in vitro of intervertebral disc degeneration, with prolongation of culture time, endplate cells gradually reduce the number, endplate ultrastructure progressive damage, glycosaminoglycan polysaccharide content, chondroitin sulfate / keratan sulfate ratio, hyaluronic acid content, polysaccharide and protein and m RNA expression of type II collagen and m RNA expression decreased gradually, and type I collagen m RNA expression increased gradually, Liuwei Dihuang Pill medicated serum can delay the changes, but the difference was not significant(P>0.05),In the culture medium added with TNF alpha significantly up-regulated and type I collagen m RNA expression, downregulation of glycosaminoglycan polysaccharide content, chondroitin sulfate and keratan sulfate ratio, hyaluronic acid content, protein polysaccharide and m RNA and type II collagen and m RNA expression, Liuwei Dihuang Pill medicated serum can part against TNF alpha caused the change. |
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