Changhuai Lu1, Zhijun Liu1, Hongbo Zhang1, Yang Duan2, Yanlin Cao3. 1. Department of Spine Disease Area of Orthopedics and Traumatology, No.1 Traditional Chinese Medicine Hospital of Changde, Changde Hospital Affiliated to Hunan University of Traditional Chinese Medicine, Changde Hunan, 415000, P.R.China. 2. Department of Spinal Surgery, Zhujiang Hospital of Southern Medical University, Guangzhou Guangdong, 510282, P.R.China.duanyang2011@163.com. 3. Department of Spinal Surgery, Zhujiang Hospital of Southern Medical University, Guangzhou Guangdong, 510282, P.R.China.
Abstract
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 β 1 (TGF-β 1)/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-β 1, and p38 siRNA+3 ng/mL TGF-β 1 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-β 1/CTGF based on the p38 MAPK signaling pathway play an important role in the occurance and development of hypertrophy of human lumbar ligamentum flavum.
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 β 1 (TGF-β 1)/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-β 1, and p38 siRNA+3 ng/mL TGF-β 1 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-β 1/CTGF based on the p38 MAPK signaling pathway play an important role in the occurance and development of hypertrophy of human lumbar ligamentum flavum.