人黄韧带细胞骨化发生过程中的自噬

The role of autophagy in ossification of the human ligamentum flavum

背景:黄韧带骨化发生的病理机制尚不清楚,临床上无有效的药物或非手术治疗的方法。目前研究发现,骨 桥蛋白与自噬在成骨过程中均发挥重要作用,二者在黄韧带骨化中的作用尚不清楚。 目的:通过对骨桥蛋白和自噬在黄韧带骨化发生机制的研究,尝试找出药物治疗的潜在作用靶点。 方法:①黄韧带骨化病、胸椎骨折或单纯腰椎间盘突出症患者行后路全椎板切除减压获取黄韧带,将标本分 为骨化组和非骨化组,每组各取 8 例标本,通过免疫组化染色观察骨桥蛋白、骨钙素及自噬指标 Beclin-1、 LC3、P62 的表达;②通过组织块贴壁法进行黄韧带细胞的分离培养,并用不同质量浓度的骨桥蛋白干预不 同时间来构建体外黄韧带细胞骨化模型;③非骨化组黄韧带细胞用不同浓度的自噬抑制剂 3-甲基腺嘌呤干 预后,再用 100 μg/L 骨桥蛋白诱导,应用 Western blot 检测骨化指标碱性磷酸酶、骨钙素的表达变化;④ 用 100 μg/L 骨桥蛋白干预非骨化黄韧带细胞,并在 0,15,30,60,120 min 终止骨化诱导过程,应用 Western blot 检测 MAPK 信号通路中重要分子 ERK1/2、JNK、P38 的磷酸化情况;⑤非骨化组黄韧带细胞 用 ERK1/2 特异的磷酸化阻滞剂 U0126 阻断 ERK1/2 通路磷酸化后,再用 100 μg/L 骨桥蛋白诱导,应用 Western blot 检测碱性磷酸酶以及骨钙素的表达变化。 结果与结论:①骨化黄韧带和非骨化黄韧带组织骨钙素、骨桥蛋白的免疫组化均呈阳性表达;骨化黄韧带组织 中 Beclin-1 呈阳性表达,而 LC3 及 P62 未见明显阳性结果;非黄韧带骨化组织中 Beclin-1、LC3、P62 均呈 阳性表达;②与非骨化组比较,骨化组黄韧带细胞中碱性磷酸酶和骨钙素的表达增加;骨桥蛋白可诱导黄韧带 骨化,骨桥蛋白的作用具有浓度相关性和时间相关性;③自噬强弱与骨化程度呈负相关,即自噬越明显,骨化 作用越弱;④骨桥蛋白能使 MAPK 信号通路磷酸化,并具有一定的时间相关性;抑制 MAPK 磷酸化过程后, 骨桥蛋白仍然能够诱导黄韧带细胞的骨化;⑤结果表明,黄韧带骨化发生过程中,信号通路上 ERK1/2、骨桥 蛋白、骨钙素、碱性磷酸酶分子的上下游关系为:ERK1/2→骨桥蛋白→骨钙素/碱性磷酸酶。

BACKGROUND: Pathological mechanism of ossification of the ligamentum flavum is unclear. There is no effective drug or non-surgical treatment in clinical practice. Current studies have found that osteopontin and autophagy play an important role in the process of osteogenesis, but their role in ossification of the ligamentum flavum has not been elucidated. OBJECTIVE: To seek for the potential target of drug therapy by exploring the mechanism of ossification of the ligamentum flavum. METHODS: (1) Surgical specimens of the ligamentum flavum were taken from patients with ossification of the ligamentum flavum, thoracic vertebrae or simple lumbar disc herniation undergoing posterior total laminectomy and decompression. These specimens were divided into two groups: an ossification group and a non-ossification group. Eight specimens from each group were collected. Osteopontin, osteocalcin and autophagy indexes Beclin-1, LC3 and P62 were stained by immunohistochemistry. (2) The ligamentum flavum cells were isolated and cultured by adherence method. The third generation cells were treated with osteopontin at different concentrations for different time to construct an in vitro model of ligamentum flavum ossification. (3) Autophagy inhibitor 3-methyladenine with different concentrations was used to intervene with non-ossified ligamentum flavum cells, followed by induction with 100 μg/L osteopontin. Western blot assay was used to detect the expression of alkaline phosphatase, osteocalcin. (4) Non-ossified ligamentum flavum cells were induced with 100 μg/L osteopontin, and the induction was terminated at 0, 15, 30, 60, and 120 minutes, respectively. The phosphorylation of ERK1/2, JNK and P38, which are important molecules in the MAPK signaling pathway, was detected by western blot. (5) Finally, after inhibition by ERK1/2 phosphorylation blocker U0126, the expression of alkaline phosphatase and osteocalcin was detected by western blot after induction with 100 μg/L steopontin. RESULTS AND CONCLUSION: (1) Immunohistochemical staining of osteopontin and osteocalcin in ossified and non-ossified ligamentum flavum was positive. In the ossified ligamentum flavum, Beclin-1 was positive, but LC3 and P62 were not. Beclin-1, LC3 and P62 were all positive in the non-ossified ligamentum flavum. (2) The expression of alkaline phosphatase and osteocalcin in the ossified ligamentum flavum cells was higher than that in the non-ossified ligamentum flavum cells. Osteopontin could induce ossification of the ligamentum flavum in a concentration- and time-dependent manner. (3) The degree of ossification was negatively correlated with the degree of autophagy, that is, the more obvious autophagy was, the weaker ossification was. (4) Osteopontin could phosphorylate the MAPK signaling pathway in a time-dependent manner. After inhibiting the phosphorylation of MAPK, osteopontin could still induce the ossification of ligamentum flavum cells. To conclude, in the process of ligamentum flavum ossification, the upstream and downstream relationships of ERK1/2, osteopontin, alkaline phosphatase and osteocalcin molecules in signaling pathway are ERK1/2→osteopontin→osteocalcin /alkaline phosphatase.