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Title: [Mechanism of p38 mitogen activated protein kinase signaling pathway on promoting the hypertrophy of human lumbar ligamentum flavum via transforming growth factor β ₁/connective tissue growth factor].
Author: Lu C, Liu Z, Zhang H, Duan Y, Cao Y.
Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2019 Jun 15; 33(6):730-735. PubMed ID: 31198002.
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. 目的: 探讨 p38 丝裂原活化蛋白激酶（mitogen activated protein kinase，MAPK）通路在 TGF-β ₁/结缔组织生长因子（connective tissue growth factor，CTGF）调控人腰椎黄韧带增生肥厚中的作用机制。. 方法: 取腰椎间盘突出髓核摘除术中获得的黄韧带组织，采用胶原酶预消化组织块培养法分离培养黄韧带细胞。分别用细胞外调节蛋白激酶通路阻断剂 PD98059、c-Jun 氨基末端激酶通路阻断剂 SP600125、p38 通路阻断剂 SB203580 处理黄韧带细胞，实时荧光定量PCR（real-time fluorescence quantitative PCR，qRT-PCR）检测 CTGF、Ⅰ型胶原和Ⅲ型胶原 mRNA 相对表达量。然后取黄韧带细胞分为 A、B、C、D 组，分别以小干扰 RNA（small interfering RNA，siRNA）、p38 siRNA、siRNA+3 ng/mL TGF-β ₁、p38 siRNA+3 ng/mL TGF-β ₁ 转染细胞，转染 24 h 后行免疫荧光染色观察 p38 和磷酸化 p38（phosphorylation p38，p-p38）表达，qRT-PCR 检测各组 CTGF、Ⅰ型胶原和Ⅲ型胶原 mRNA 相对表达量，Western blot 检测各组 CTGF 蛋白表达。. 结果: p38 通路阻断剂 SB203580 可明显降低黄韧带细胞 CTGF、Ⅰ型胶原和Ⅲ型胶原 mRNA 相对表达量（ P<0.05）。转染 24 h 后，免疫荧光染色显示 A、C、D 组细胞呈阳性反应，有 p38、p-p38 表达，且 C、D 组强于 A 组；B 组细胞呈阴性反应，无 p38、p-p38 表达。与 A 组相比，B 组 CTGF、Ⅰ型胶原和Ⅲ型胶原 mRNA 相对表达量以及 CTGF 蛋白相对表达量显著减少，C、D 组显著增加，C 组较 D 组进一步增加，差异均有统计学意义（ P<0.05）。. 结论: p38 MAPK 通路介导 TGF-β ₁/CTGF 表达，在人腰椎黄韧带细胞增生肥厚过程中具有重要作用。.

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