基质Gla蛋白在脊髓损伤后纤维瘢痕形成中的作用机制

Role and mechanism of matrix Gla protein in fibrotic scar formation after spinal cord injury

摘要

目的 探讨基质Gla蛋白(matrix Gla protein, MGP)在脊髓损伤后纤维瘢痕形成中的作用机制。 方法 选取6只C57BL/6实验小鼠,采用随机分组法分为假手术组(n=3)和脊髓损伤组(n=3)。建立小鼠脊髓损伤模型,于术后第7天,取脊髓损伤组小鼠损伤中心节段和假手术组小鼠解剖学对应节段的脊髓组织,用于转录组学测序分析,筛选出差异表达基因MGP(|log₂FC|> 1且P_adj< 0.05);采用Western blotting、RT-qPCR和免疫荧光法检测敲低MGP后小鼠胚胎成纤维细胞(mouse embryonic fibroblasts, MEFs)及原代脊髓成纤维细胞纤维化的指标表达,以及转化生长因子-β/Smad(transforming growth factor-β/Smad, TGF-β/SMAD)信号通路的活性变化;采用斜板实验和巴索小鼠运动功能评分(Basso mouse scale, BMS)检测体内成纤维细胞中特异性敲低MGP对小鼠脊髓损伤后运动功能恢复的影响。 结果 转录组测序结果显示,损伤后的脊髓组织中,MGP的表达量显著高于假手术组(P<0.05)。在体外TGF-β 诱导的纤维化进程中,敲低MEFs及原代脊髓成纤维细胞中的MGP基因,使纤维连接蛋白 1、Ⅰ型胶原蛋白、α -平滑肌肌动蛋白的mRNA和蛋白表达水平,以及SMAD家族成员2(SMAD family member 2,SMAD2)和SMAD家族成员3(SMAD family member 3, SMAD3)的磷酸化水平较诱导状态显著降低(P<0.05)。成纤维细胞中特异性敲低MGP,会促进脊髓损伤后小鼠运动功能恢复(P<0.05)。 结论 MGP可能通过靶向TGF-β/SMAD信号通路调控脊髓损伤后纤维瘢痕的形成。

Abstract

Objective To investigate the role and underlying mechanism of the matrix Gla protein(MGP)in fibrous scar formation after spinal cord injury(SCI). Methods Six C57BL/6 mice were randomly assigned to the sham group(n=3)and the spinal cord injury group(n=3). A mouse model of SCI was established. On postoperative day 7, spinal cord tissue was harvested from theepicenter of the lesion of the spinal cord injury group and the anatomically corresponding segments of the sham group for transcriptome sequencing. The differentially expressed gene MGP(|log₂FC|>1 and P_adj< 0.05)were screened and validated. To investigate the role of MGP, western blotting, RT-qPCR, and immunofluorescence were used to assess the expression of fibrosis markers and the activity of the TGF-β/SMAD signaling pathway in mouse embryonic fibroblasts(MEFs)and primary spinal cord fibroblasts after MGP knockdown. Finally, the inclined plane test and Basso mouse scale(BMS)scores were used to assess the impact of fibroblast-specific MGP knockdown on motor function recovery in mice after SCI. Results The results of transcriptome sequencing showed that the expression of MGP in the spinal cord injury group was higher than in the sham group(P<0.05); During the TGF-β-induced fibrogenesis process, MGP knockdown in MEFs and primary spinal cord fibroblasts significantly reduced the expressions of mRNA and proteins of fibronectin 1, type I collagen, and α-smooth muscle actin, as well as the phosphorylation of SMAD2 and SMAD3, compared to the induced state(P<0.05). The fibroblast-specific suppression of MGP in mice significantly promoted recovery ofmotor function after SCI(P<0.05). Conclusion MGP may modulate fibrous scar formation after SCI by targeting the TGF-β/SMAD signaling pathway.

关键词

脊髓损伤 / 基质Gla蛋白 / 原代脊髓成纤维细胞 / 纤维瘢痕 / SMAD家族蛋白

Key words

Spinal cord injury / Matrix Gla protein / Primary spinal cord fibroblasts / Fibrotic scar / SMAD proteins

参考文献

[1] Ahuja CS, Wilson JR, Nori S, et al. Traumatic spinal cord injury[J]. Nat Rev Dis Primers, 2017, 3: 17018. doi: 10.1038/nrdp.2017.18

[2] McDonald JW, Sadowsky C. Spinal-cord injury[J]. Lancet, 2002, 359(9304): 417-425.

[3] Rivers CS, Fallah N, Noonan VK, et al. health conditions: effect on function, health-related quality of life, and life satisfaction after traumatic spinal cord injury. A prospective observational registry cohort study[J]. Arch Phys Med Rehabil, 2018, 99(3): 443-451.

[4] Dias DO, Göritz C. Fibrotic scarring following lesions to the central nervous system[J]. Matrix Biol, 2018, 68/69: 561-570. doi:10.1016/j.matbio.2018.02.009

[5] Soderblom C, Luo XT, Blumenthal E, et al. Perivascular fibroblasts form the fibrotic scar after contusive spinal cord injury[J]. J Neurosci, 2013, 33(34): 13882-13887. doi:10.1523/JNEUROSCI.2524-13.2013

[6] Holl D, Hau WF, Julien A, et al. Distinct origin and region-dependent contribution of stromal fibroblasts to fibrosis following traumatic injury in mice[J]. Nat Neurosci, 2024, 27(7): 1285-1298.

[7] Ayazi M, Zivkovic S, Hammel G, et al. Fibrotic scar in CNS injuries: from the cellular origins of fibroblasts to the molecular processes of fibrotic scar formation[J]. Cells, 2022, 11(15): 2371.doi: 10.3390/cells11152371

[8] Anderson MA, O'Shea TM, Burda JE, et al. Required growth facilitators propel axon regeneration across complete spinal cord injury[J]. Nature, 2018, 561(7723): 396-400.

[9] Fraser JD, Price PA. Lung, heart, and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla protein. Implications for the possible functions of matrix Gla protein and for the tissue distribution of the gamma-carboxylase[J]. J Biol Chem, 1988, 263(23): 11033-11036.

[10] Cancela L, Hsieh CL, Francke U, et al. Molecular structure, chromosome assignment, and promoter organization of the human matrix Gla protein gene[J]. J Biol Chem, 1990, 265(25): 15040-15048.

[11] Du T, Pan L, Zheng CY, et al. Matrix Gla protein(MGP), GATA3, and TRPS1: a novel diagnostic panel to determine breast origin[J]. Breast Cancer Res, 2022, 24(1): 70. doi: 10.1186/s13058-022-01569-1

[12] Shea MK, Kritchevsky SB, Hsu FC, et al. The association between vitamin K status and knee osteoarthritis features in older adults: the health, aging and body composition study[J]. Osteoarthritis Cartilage, 2015, 23(3): 370-378.

[13] Yao YC, Shahbazian A, Boström KI. Proline and gamma-carboxylated glutamate residues in matrix Gla protein are critical for binding of bone morphogenetic protein-4[J]. Circ Res, 2008, 102(9): 1065-1074.

[14] Korosec A, Frech S, Gesslbauer B, et al. Lineage identity and location within the dermis? determine the function of papillary and?reticular fibroblasts in human skin[J]. J Invest Dermatol, 2019, 139(2): 342-351.

[15] Jerala M, Remic T, Hauptman N, et al. Thrombospondin 2, matrix Gla protein and digital analysis identified distinct fibroblast populations in fibrostenosing Crohn's disease[J]. Sci Rep, 2024, 14(1): 13810. doi:10.1038/s41598-024-64672-7

[16] Hui ST, Gong LL, Swichkow C, et al. Role of matrix gla protein in transforming growth factor-β signaling and nonalcoholic steatohepatitis in mice[J]. Cell Mol Gastroenterol Hepatol, 2023, 16(6): 943-960.

[17] 王伟, 谢杰, 方坚, 等. 水通道蛋白-4在实验性脊髓损伤大鼠的表达[J]. 神经解剖学杂志, 2005, 21(2): 139-142.

[18] WANG Wei, XIE Jie, FANG Jian, et al. The effect of rats following experimental spinal cord injury upon aquaporin-4 expression[J]. Chinese Journal of Neuroanatomy, 2005, 21(2): 139-142.

[19] Zhang C, Shao Q, Zhang Y, et al. Therapeutic application of nicotinamide: as a potential target for inhibiting fibrotic scar formation following spinal cord injury[J]. CNS Neurosci Ther, 2024, 30(7): e14826. doi:10.1111/cns.14826

[20] 闫小龙, 秦英, 邵将, 等. 姜黄素通过Wnt/β-catenin信号通路调控骨形成的机制[J]. 山东大学学报(医学版), 2024, 62(10): 76-86.

[21] YAN Xiaolong, QIN Ying, SHAO Jiang, et al. Mechanism of curcumin regulating bone formation via the Wnt/β-catenin signaling pathway[J]. Journal of Shandong University(Health Sciences), 2024, 62(10): 76-86.

[22] 韩觉明, 王晖, 吴倩, 等. B4GALNT4促进肺腺癌细胞增殖、迁移和侵袭能力[J]. 山东大学学报(医学版), 2025, 63(7): 23-31.

[23] HAN Jueming, WANG Hui, WU Qian, et al. B4GALNT4 promotes proliferation, migration and invasion of lung adenocarcinoma cells[J]. Journal of Shandong University(Health Sciences), 2025, 63(7): 23-31.

[24] 赵灿斌, 邵将, 管东辉, 等. 木犀草素在炎症微环境下调控Wnt/β-catenin信号通路促进骨髓间充质干细胞成软骨分化的机制[J]. 山东大学学报(医学版), 2025, 63(7): 11-22.

[25] ZHAO Canbin, SHAO Jiang, GUAN Donghui, et al. Mechanism of luteolin regulating Wnt/β-catenin signal pathway in inflammatory microenvironment to promote chondrogenic differentiation of bone marrow mesenchymal stem cells[J]. Journal of Shandong University(Health Sciences), 2025, 63(7): 11-22.

[26] Buss A, Pech K, Kakulas BA, et al. TGF-beta1 and TGF-beta2 expression after traumatic human spinal cord injury[J]. Spinal Cord, 2008, 46(5): 364-371.

[27] Dias DO, Kim H, Holl D, et al. Reducing pericyte-derived scarring promotes recovery after spinal cord injury[J]. Cell, 2018, 173(1):153-165.

[28] Rentsch NH, Rust R. ‘Scary’ pericytes: the fibrotic scar in brain and spinal cord lesions[J]. Trends Neurosci, 2022, 45(1): 6-7.

[29] Kaesler N, Kaushik S, Frisch J, et al. Vitamin K preserves gamma-glutamyl carboxylase activity against carbamylations in uremia: implications for vascular calcification and adjunct therapies[J]. Acta Physiol, 2025, 241(5): e70040. doi:10.1111/apha.70040

[30] Zebboudj AF, Shin V, Boström K. Matrix GLA protein and BMP-2 regulate osteoinduction in calcifying vascular cells[J]. J Cell Biochem, 2003, 90(4): 756-765.

[31] Jerala M, Hauptman N, Kojc N, et al. Expression of fibrosis-related genes in liver and kidney fibrosis in comparison to inflammatory bowel diseases[J]. Cells, 2022, 11(3): 314. doi:10.3390/cells11030314

[32] Miyata KN, Nast CC, Dai T, et al. Renal matrix Gla protein expression increases progressively with CKD and predicts renal outcome[J]. Exp Mol Pathol, 2018, 105(1): 120-129.

[33] Schmidt IM, Colona MR, Kestenbaum BR, et al. Cadherin-11, Sparc-related modular calcium binding protein-2, and Pigment epithelium-derived factor are promising non-invasive biomarkers of kidney fibrosis[J]. Kidney Int, 2021, 100(3): 672-683.

[34] Booth AJ, Hadley R, Cornett AM, et al. Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation[J]. Am J Respir Crit Care Med, 2012, 186(9): 866-876.

[35] Hardie WD, Korfhagen TR, Sartor MA, et al. Genomic profile of matrix and vasculature remodeling in TGF-alpha induced pulmonary fibrosis[J]. Am J Respir Cell Mol Biol, 2007, 37(3): 309-321.

[36] 王一帆, 郭建波, 邵宝仪, 等. TGF-β1/SMAD在糖尿病肾病中的作用机制与研究进展[J]. 四川大学学报(医学版), 2023, 54(6): 1065-1073.

[37] WANG Yifan, GUO Jianbo, SHAO Baoyi, et al. The role of TGF-β1/SMAD in diabetic nephropathy: mechanisms and research development[J]. Journal of Sichuan University(Medical Sciences), 2023, 54(6): 1065-1073.

[38] 郑婉, 杨珊珊, 张娜. 半乳糖凝集素-3对低氧性肺动脉高压小鼠肺组织氧化应激和纤维化的调节作用及其机制[J]. 山西医科大学学报, 2023, 54(6): 770-777.

[39] ZHENG Wan, YANG Shanshan, ZHANG Na. Regulatory effect and mechanism of galactin-3 on oxidative stress and fibrosis in lung tissue of mice with hypoxic pulmonary hypertension[J]. Journal of Shanxi Medical University, 2023, 54(6): 770-777.

[40] 薛丽, 叶明, 王刚. 食管鳞状细胞癌中MGP基因的表达和启动子区甲基化状态及其临床意义[J]. 国际消化病杂志, 2021, 41(6): 433-439.

[41] XUE Li, YE Ming, WANG Gang. MGP gene expression and promoter methylation status in esophageal squamous cell carcinoma and its clinical significance[J]. International Journal of Digestive Diseases, 2021, 41(6): 433-439.

基金资助

国家自然科学基金面上项目(82371392)

泰山学者特聘专家计划(tstp20231257)