Ghosh Nilabh, Bregere Catherine, Bustos Pia, Guzman Raphael
Department of Biomedicine, University Hospital Basel, Basel, Switzerland.
Department of Neurosurgery, University Hospital Basel, Basel, Switzerland.
CNS Neurol Disord Drug Targets. 2023;22(5):761-773. doi: 10.2174/1871527321666220414094149.
Twenty million Americans suffer from peripheral nerve injury (PNI) and approximately $150 billion is spent annually in the United States for the treatment of nerve injuries. Moreover, 50,000 cases of PNI repairs are performed annually in the United States, with even less than 42% experiencing satisfactory sensory recovery. Available therapies control painful symptoms but do not treat axonal degeneration or neuronal cell death. Peripheral nerve fibrosis (PNF) associated with chronic inflammation, perineural adhesions, and scarring is often reported in patients with nerve injury. Unfortunately, post-surgical adhesions and fibrosis often lead to aberrated wound healing and impairment of nerve functions. Various treatment strategies have been attempted, including the use of grafts and biomaterials; however, few appear promising.
L-Alanyl-L-Glutamine (L-Ala-L-Gln) was reported to protect the lung from sepsisinduced injury and play an immunomodulatory role in stress and fibrosis. This study aimed to examine the potential anti-fibrotic effects of L-Ala-L-Gln in an in vitro model of neural fibrosis.
Primary fibroblasts isolated from rat sciatic nerve were exposed to chronic (48 h) and episodic (2 h) hypoxic conditions. Cultures were then treated for 48 h with various concentrations of L-Ala-L-Gln (0, 1, 10, and 100 mM). The expression of hypoxic and pro-fibrotic markers in the different culture conditions was assessed by immunocytochemistry and western blot analyses. Quantitative phosphor-proteomic profiling was performed to investigate mechanistically the impact of L-Ala- L-Gln on collagen biosynthesis and hypoxia-driven tissue fibrosis in vitro.
In protein expression assays, L-Ala-L-Gln significantly reduced markers related to the cellular response to hypoxia, in particular HIF-1 signaling. L-Ala-L-Gln also significantly reduced the expression of pro-fibrotic and cell-adhesion-inducing factors. Phospho-proteomic data indicated that L-Ala-L-Gln modulates several pro-fibrotic factors and associated pathways.
Altogether, our data demonstrate that L-Ala-L-Gln efficiently suppresses hypoxiamediated fibrotic processes at different concentrations in rat primary fibroblasts. Thus, L-Ala-L-Gln presents a high potential therapeutic value as an antifibrotic pharmaceutical agent for the treatment of neural fibrosis.
2000万美国人患有周围神经损伤（PNI），美国每年用于神经损伤治疗的费用约为1500亿美元。此外，美国每年进行5万例PNI修复手术，但感觉恢复良好的患者不到42%。现有的治疗方法可控制疼痛症状，但无法治疗轴突退变或神经元细胞死亡。神经损伤患者常出现与慢性炎症、神经周粘连和瘢痕形成相关的周围神经纤维化（PNF）。不幸的是，术后粘连和纤维化常导致伤口愈合异常和神经功能受损。人们尝试了各种治疗策略，包括使用移植物和生物材料；然而，几乎没有一种看起来有前景。
据报道，L-丙氨酰-L-谷氨酰胺（L-Ala-L-Gln）可保护肺免受败血症诱导的损伤，并在应激和纤维化中发挥免疫调节作用。本研究旨在探讨L-Ala-L-Gln在神经纤维化体外模型中的潜在抗纤维化作用。
将从大鼠坐骨神经分离的原代成纤维细胞暴露于慢性（48小时）和间歇性（2小时）缺氧条件下。然后用不同浓度的L-Ala-L-Gln（0、1、10和100 mM）处理细胞培养物48小时。通过免疫细胞化学和蛋白质印迹分析评估不同培养条件下缺氧和促纤维化标志物的表达。进行定量磷酸化蛋白质组分析，以从机制上研究L-Ala-L-Gln对体外胶原生物合成和缺氧驱动的组织纤维化的影响。
在蛋白质表达分析中，L-Ala-L-Gln显著降低了与细胞对缺氧反应相关的标志物，特别是缺氧诱导因子-1（HIF-1）信号。L-Ala-L-Gln还显著降低了促纤维化和细胞粘附诱导因子的表达。磷酸化蛋白质组数据表明，L-Ala-L-Gln调节多种促纤维化因子和相关途径。
总之，我们的数据表明，L-Ala-L-Gln在不同浓度下可有效抑制大鼠原代成纤维细胞中缺氧介导的纤维化过程。因此，L-Ala-L-Gln作为一种抗纤维化药物用于治疗神经纤维化具有很高的潜在治疗价值。
