Objective To investigate the mechanism of p38 mitogen activated protein kinase (MAPK) signaling pathway in regulating the hyperplasia and hypertrophy of human lumbar ligamentum flavum via transforming growth factor β1 (TGF-β1)/connective tissue growth factor (CTGF). Methods The lumbar ligamentum flavum tissue taken from patient with lumbar intervertebral disc herniation was isolated by collagenase-predigested explant cultures. The ligamentum flavum cells were treated with the extracellular regulated protein kinase pathway blocker PD98059, c-Jun N-terminal kinase pathway blocker SP600125, and p38 pathway blocker SB203580, and then the mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescence quantitative PCR (qRT-PCR). The ligamentum flavum cells were divided into 4 groups, and transfected with small interfering RNA (siRNA), p38 siRNA, siRNA+3 ng/mL TGF-β1, and p38 siRNA+3 ng/mL TGF-β1 in groups A, B, C, and D, respectively. After 24 hours of transfection, immunofluorescence staining was performed to observe the expressions of p38 and phosphorylation p38 (p-p38); the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ in each group were detected by qRT-PCR; the protein expression of CTGF in each group was detected by Western blot. Results p38 pathway blocker SB203580 could significantly reduce the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ (P<0.05). After 24 hours of transfection, immunofluorescence staining showed positive staining with p38 and p-p38 expressions in groups A, C, and D and negative staining in group B. Compared with group A, the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ and relative protein expression of CTGF in group B decreased significantly (P<0.05), while those in groups C and D increased significantly (P<0.05); and those indicators significantly increased in group C than in group D (P<0.05). Conclusion TGF-β1/CTGF based on the p38 MAPK signaling pathway play an important role in the occurance and development of hypertrophy of human lumbar ligamentum flavum.
ObjectiveTo investigate the effect of transforming growth factor β1 (TGF-β1) induced proliferation of ligamentum flavum cells and ligamentum flavum hypertrophy and its effect on connective tissue growth factor (CTGF) expression.MethodsThe ligamentum flavum tissue in lumbar intervertebral disc herniation was extracted and the ligamentum flavum cells were isolated and cultured by collagenase pre-digestion method. Morphological observation, immunofluorescence staining observation, and MTT assay were used for cell identification. The 3rd generation ligamentum flavum cells were divided into 5 groups. The cells of groups A, B, C, and D were respectively sealed with 3 ng/mL TGF-β1, 50 ng/mL CTGF, 3 ng/mL TGF-β1+CTGF neutralizing antibody, and 50 ng/mL CTGF+CTGF neutralizing antibody. Serum free DMEM was added to group E as the control. MTT assay was used to detect the effects of TGF-β1 and CTGF on the proliferation of ligamentum flavum cells. Western blot was used to detect the expression of CTGF protein. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of collagen type Ⅰ, collagen type Ⅲ, and CTGF genes.ResultsThe morphological diversity of cultured ligamentum flavum cells showed typical phenotype of ligamentum flavum fibroblasts; all cells expressed collagen type Ⅰ and vimentin, and some cells expressed collagen type Ⅲ; MTT identification showed that with the prolongation of culture time, the absorbance (A) value of each generation of cells increased gradually, and the A value of the same generation of cells at each time point was significantly different (P<0.05), there was no significant difference in A value between the cells of each generation at the same time point (P>0.05). After cultured for 24 hours, MTT assay showed that the A value of cells in groups A and B was significantly higher than that of group E (P<0.05). After adding CTGF neutralizing antibody, the A value of cells in groups C and D decreased, but it was still higher than that of group E (P<0.05). There were also significant differences among groups A, C and groups B, D (P<0.05). Western blot analysis showed that the relative expression of CTGF protein in groups A and B was significantly higher than that in group E (P<0.05), while the relative expression of CTGF protein in groups C and D was significantly lower than that in group E (P<0.05), and the difference between groups A, C and groups B, D was also significant (P<0.05). qRT-PCR detection showed that the mRNA relative expression of CTGF, collagen type Ⅰ, and collagen type Ⅲ in group A was significantly higher than that in group E (P<0.05). After adding neutralizing antibody, the mRNA relative expression of genes in group C was inhibited and were significantly lower than that in group A, but still significantly higher than that in group E (P<0.05). The mRNA relative expressions of collagen type Ⅰ and collagen type Ⅲ in group B was significantly higher than that in group E (P<0.05), but the mRNA relative expression of CTGF was not significantly different from that in group E (P>0.05); after neutralizing antibody was added, the mRNA relative expression of collagen type Ⅰ and collagen type Ⅲ in group D was inhibited and was significantly lower than that in group B, but still significantly higher than that in group E (P<0.05); there was no significant difference in the mRNA relative expression of CTGF between group D and groups B, E (P>0.05).ConclusionTGF-β1 can promote CTGF, collagen typeⅠ, collagen type Ⅲ gene level and protein expression in ligamentum flavum cells, and TGF-β1 can synergistically promote proliferation of ligamentum flavum cells through CTGF.