The role and mechanism of high mobility group box-1 protein in the degeneration of lumbar cartilage endplate in rats

English Keywords:High mobility group box-1 protein  Extracellular regulated protein kinases signaling pathway  Cartilage endplate  Rat

English Abstract:

  【Abstract】 Objectives: To explore the effect and mechanism of high mobility group box-1 protein(HMGB1) on the degeneration of lumbar cartilage endplate in rats. Methods: Four-week-old SD rats were sacrificed and the lumbar cartilage endplates were taken out under the microscope. The supernatant was extracted after digestion. After culture, isolation and purification, immunofluorescence staining was used to detect type Ⅱ collagen and　cartilage endplate cell　(CEC). Different concentrations of FBS were used to culture CEC for 24h and 48h respectively. MTT assay was used to detect cell viability. The expressions of MMP-3, vascular endothelial growth factor and interleukin were detected at 3, 6, 12 and 24 hours after adding human HMGB1 to the third generation CEC. The expression of extracellular signal-regulated kinase phosphorylation was detected by Western blot at 0, 5, 10, 30, 60 and 12０ｍｉｎ after adding 100ng/ml HMGB１ to CEC. CEC was divided into six groups: control group, HMGB1(100ng/ml) group, FPSMZ１(HMGB１ inhibitor) group, HMGB1+ FPSMZ１ group, U0126(ERK inhibitor) group and HMGB1+U0126 group. The expressions of MMP-3, VEGF and ERK signal transduction pathway phosphorylation and Elisa IL-10 were detected by Western blot. All results were repeated independently for three experiments, and their mean + standard deviation was calculated. Results: The expression of type Ⅱ collagen antibody was positive in CEC cells, and the best time and concentration for proliferation of CEC was 48 hours on 10%FBS. HMGB1 could induce phosphorylation of ERK signal transduction pathway, which gradually increased from the beginning to 10min and reached its peak at 10min. HMGB1 up-regulated the expressions of MMP-3(P=0.039) and VEGF(P=0.042), but down-regulated the expression of IL-10(P=0.025). The expression of three factors was the same as that of gene. Compared with HMGB group, phosphorylation of ERK signal transduction pathway was significantly reduced in HMGB１+FPSMZ１ group. After ERK signal transduction pathway inhibitor(U0126) added to CEC, the expressions of MMP-3(P=0.041) and VEGF(P=0.042) decreased significantly, while the expression of IL-10(P=0.004) increased significantly. After adding RAGE inhibitor(FPSMZ１) to CEC, the phosphorylation of ERK signal transduction pathway by HMGB1 decreased significantly(P=0.031). Conclusions: HMGB1 can increase the expression of MMP-3 and VEGF in rat lumbar CEC and decrease the expression of IL-10 possibly by ERK signal transduction pathway.