p38丝裂原活化蛋白激酶通路介导TGF-β_1/结缔组织生长因子调控人腰椎黄韧带增生肥厚的机制研究
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摘要
目的探讨p38丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)通路在TGF-β1/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-β1、p38 siRNA+3 ng/mL TGF-β1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。
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 Nterminal 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(pp38); the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ in each group were detected by qRTPCR; 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.
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 Nterminal 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(pp38); the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ in each group were detected by qRTPCR; 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.
引文
1 Binder DK, Schmidt MH, Weinstein PR. Lumbar spinal stenosis.Semin Neurol, 2002, 22(2):157-166.
2 Gu J, Liu X, Wang QX, et al. Angiotensin II increases CTGF expression via MAPKs/TGF-(3i/TRAF6 pathway in atrial fibroblasts. Exp Cell Res, 2012, 318(16):2105-2115.
3 Maezawa Y, Baba H, Uchida K, et al. Ligamentum flavum hematoma in the thoracic spine. Clin Imaging, 2001, 25(4):265-267.
4 Behm B, Babilas P, Landthaler M, et al. Cytokines, chemokines and growth factors in wound healing. J Eur Acad Dermatol Venereol,2012,26(7):812-820.
5 Zhong ZM, Zha DS, Xiao WD, et al. Hypertrophy of ligamentum flavum in lumbar spine stenosis associated with the increased expression of connective tissue growth factor. J Orthop Res, 2011,29(10):1592-1597.
6 Amudong A, Muheremu A, Abudourexiti T. Hypertrophy of the ligamentum flavum and expression of transforming growth factor beta. J Int Med Res, 2017, 45(6):2036-2041.
7方清清,李志忠,周建,等.信号分子p38参与低频脉冲电磁场促进成骨细胞矿化成熟的实验研究.中国修复重建外科杂志,2016, 30(10):1238-1243.
8 New L, Han J. The p38 MAP kinase pathway and its biological function. Trends Cardiovasc Med, 1998, 8(5):220-228.
9 Hale KK, Trollinger D, Rihanek M, et al. Differential expression and activation of p38 mitogen-activated protein kinase alpha, beta,gamma, and delta in inflammatory cell lineages. J Immunol, 1999,162(7):4246-4252.
10 Specchia N, Pagnotta A, Gigante A, et al. Characterization of cultured human ligamentum flavum cells in lumbar spine stenosis.J Orthop Res, 2001, 19(2):294-300.
11 Zhong ZM, Chen JT. Phenotypic characterization of ligamentum flavum cells from patients with ossification of ligamentum flavum.Yonsei Med J, 2009, 50(3):375-379.
12 Zhang M, Fraser D, Phillips A. ERK, p38, and Smad signaling pathways differentially regulate transforming growth factor-betal autoinduction in proximal tubular epithelial cells. Am J Pathol,2006,169(4):1282-1293.
13 Holmes A, Abraham DJ, Sa S, et al. CTGF and SMADs,maintenance of scleroderma phenotype is independent of SMAD signaling. J Biol Chem, 2001,276(14):10594-10601.
14 Du QC, Zhang DZ, Chen XJ, et al. The effect of p38MAPK on cyclic stretch in human facial hypertrophic scar fibroblastdifferentiation. PLoS One, 2013, 8(10):e75635.
15 Morales MG, Vazquez Y, Acuna MJ, et al. AngiotensinⅡ-induced pro-fibrotic effects require p38MAPK activity and transforming growth factor beta 1 expression in skeletal muscle cells. Int J Biochem Cell Biol, 2012,44(11):1993-2002.
16 Park JB, Chang H, Lee JK. Quantitative analysis of transforming growth factor-beta 1 in ligamentum flavum of lumbar spinal stenosis and disc herniation. Spine(Phila Pa 1976), 2001, 26(21):E492-E495.
17 Han J, Lee JD, Bibbs L, et al. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science, 1994, 265(5173):808-811.
18 Jiang Y, Gram H, Zhao M, et al. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38delta. J Biol Chem, 1997, 272(48):30122-30128.
19 Mavropoulos A, Orfanidou T, Liaskos C, et al. p38 MAPK signaling in pemphigus:implications for skin autoimmunity.Autoimmune Dis, 2013,2013:728529.
20 Whitmarsh AJ. A central role for p38 MAPK in the early transcriptional response to stress. BMC Biol, 2010, 8:47.
2 Gu J, Liu X, Wang QX, et al. Angiotensin II increases CTGF expression via MAPKs/TGF-(3i/TRAF6 pathway in atrial fibroblasts. Exp Cell Res, 2012, 318(16):2105-2115.
3 Maezawa Y, Baba H, Uchida K, et al. Ligamentum flavum hematoma in the thoracic spine. Clin Imaging, 2001, 25(4):265-267.
4 Behm B, Babilas P, Landthaler M, et al. Cytokines, chemokines and growth factors in wound healing. J Eur Acad Dermatol Venereol,2012,26(7):812-820.
5 Zhong ZM, Zha DS, Xiao WD, et al. Hypertrophy of ligamentum flavum in lumbar spine stenosis associated with the increased expression of connective tissue growth factor. J Orthop Res, 2011,29(10):1592-1597.
6 Amudong A, Muheremu A, Abudourexiti T. Hypertrophy of the ligamentum flavum and expression of transforming growth factor beta. J Int Med Res, 2017, 45(6):2036-2041.
7方清清,李志忠,周建,等.信号分子p38参与低频脉冲电磁场促进成骨细胞矿化成熟的实验研究.中国修复重建外科杂志,2016, 30(10):1238-1243.
8 New L, Han J. The p38 MAP kinase pathway and its biological function. Trends Cardiovasc Med, 1998, 8(5):220-228.
9 Hale KK, Trollinger D, Rihanek M, et al. Differential expression and activation of p38 mitogen-activated protein kinase alpha, beta,gamma, and delta in inflammatory cell lineages. J Immunol, 1999,162(7):4246-4252.
10 Specchia N, Pagnotta A, Gigante A, et al. Characterization of cultured human ligamentum flavum cells in lumbar spine stenosis.J Orthop Res, 2001, 19(2):294-300.
11 Zhong ZM, Chen JT. Phenotypic characterization of ligamentum flavum cells from patients with ossification of ligamentum flavum.Yonsei Med J, 2009, 50(3):375-379.
12 Zhang M, Fraser D, Phillips A. ERK, p38, and Smad signaling pathways differentially regulate transforming growth factor-betal autoinduction in proximal tubular epithelial cells. Am J Pathol,2006,169(4):1282-1293.
13 Holmes A, Abraham DJ, Sa S, et al. CTGF and SMADs,maintenance of scleroderma phenotype is independent of SMAD signaling. J Biol Chem, 2001,276(14):10594-10601.
14 Du QC, Zhang DZ, Chen XJ, et al. The effect of p38MAPK on cyclic stretch in human facial hypertrophic scar fibroblastdifferentiation. PLoS One, 2013, 8(10):e75635.
15 Morales MG, Vazquez Y, Acuna MJ, et al. AngiotensinⅡ-induced pro-fibrotic effects require p38MAPK activity and transforming growth factor beta 1 expression in skeletal muscle cells. Int J Biochem Cell Biol, 2012,44(11):1993-2002.
16 Park JB, Chang H, Lee JK. Quantitative analysis of transforming growth factor-beta 1 in ligamentum flavum of lumbar spinal stenosis and disc herniation. Spine(Phila Pa 1976), 2001, 26(21):E492-E495.
17 Han J, Lee JD, Bibbs L, et al. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science, 1994, 265(5173):808-811.
18 Jiang Y, Gram H, Zhao M, et al. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38delta. J Biol Chem, 1997, 272(48):30122-30128.
19 Mavropoulos A, Orfanidou T, Liaskos C, et al. p38 MAPK signaling in pemphigus:implications for skin autoimmunity.Autoimmune Dis, 2013,2013:728529.
20 Whitmarsh AJ. A central role for p38 MAPK in the early transcriptional response to stress. BMC Biol, 2010, 8:47.