酰胺生物碱哌啶嗪对成骨前体细胞凋亡、自噬和分化的影响。

Effects of the amide alkaloid piperyline on apoptosis, autophagy, and differentiation of pre-osteoblasts.

机构信息

Department of Oral and Maxillofacial Pathology, School of Dentistry, Kyung HeeUniversity,26, Kyungheedae-ro, Dongdaemun-gu,Seoul02453, South Korea.

National Development Institute of Korean Medicine, Gyeongsan38540, South Korea.

出版信息

Phytomedicine. 2020 Dec;79:153347. doi: 10.1016/j.phymed.2020.153347. Epub 2020 Sep 17.

DOI:10.1016/j.phymed.2020.153347

PMID:32992084

Abstract

BACKGROUND

Amide alkaloidsare typical constituents in plants of the Piperaceae family. Most of the pharmacological properties of Piper nigrum L. are attributed to the major amide alkaloid, piperine. Piperyline (PIPE) is a further amide alkaloid that has been isolated from P. nigrum.

HYPOTHESIS/PURPOSE: This study was performed to examine the biological effects of PIPE on pre-osteoblasts and elucidate the underlying mechanisms.

STUDY DESIGN

We investigated the effects of PIPE in MC3T3E-1 cells, which are widely used for studying osteoblast behavior in in vitro cell systems.

METHODS

We evaluated cell viability based on the MTT assay, apoptosis by TUNEL staining, adhesion and migration by cell adhesion and migration assays, and osteoblast differentiation by alkaline phosphatase activity and staining. Western blot and immunocytochemical analyses were used to investigate cell signaling pathways.

RESULTS

We found that at concentrations ranging from 1 to 30 μM, PIPE inhibited cell growth and induced apoptosis in pre-osteoblasts, which was accompanied by the upregulation of apoptotic proteins but downregulation of anti-apoptotic proteins. In contrast, PIPE had no appreciable effect on the autophagy pathway. Nevertheless, PIPE reduced cell adhesion and migration via the inactivation of non-receptor tyrosine kinase (Src)/focal adhesion kinase (FAK) and mitogen-activated protein kinases, and also promoted the downregulation of matrix metalloproteinase 2 and 9 levels. Furthermore, at concentrations of 10 and 30 μM, PIPE suppressed osteoblast differentiation, as indicated by reductions in alkaline phosphatase staining and activity. In addition, PIPE reduced the protein levels of phospho-Smad1/5/8 and runt-related transcription factor 2, and the mRNA levels of osteopontin, alkaline phosphatase, and osteocalcin.

CONCLUSION

The findings of this study indicate that PIPE has biological effects associated with cell adhesion, migration, proliferation, and osteoblast differentiation, and suggest a potential role for this alkaloid in the treatment of bone diseases.

摘要

背景

酰胺生物碱是胡椒科植物的典型成分。黑胡椒的大部分药理特性归因于主要酰胺生物碱胡椒碱。哌啶（PIPE）是从黑胡椒中分离出来的另一种酰胺生物碱。

假设/目的：本研究旨在研究 PIPE 对成骨前体细胞的生物学效应，并阐明其潜在机制。

研究设计

我们研究了 PIPE 在 MC3T3E-1 细胞中的作用，MC3T3E-1 细胞广泛用于研究体外细胞系统中成骨细胞的行为。

方法

我们通过 MTT 测定法评估细胞活力、通过 TUNEL 染色评估细胞凋亡、通过细胞黏附和迁移测定法评估细胞黏附和迁移、通过碱性磷酸酶活性和染色评估成骨细胞分化。Western blot 和免疫细胞化学分析用于研究细胞信号通路。

结果

我们发现，在 1 至 30 μM 的浓度范围内，PIPE 抑制成骨前体细胞的生长并诱导其凋亡，这伴随着凋亡蛋白的上调和抗凋亡蛋白的下调。相比之下，PIPE 对自噬途径没有明显影响。然而，PIPE 通过失活非受体酪氨酸激酶（Src）/黏着斑激酶（FAK）和丝裂原活化蛋白激酶，减少细胞黏附和迁移，并促进基质金属蛋白酶 2 和 9 水平的下调。此外，在 10 和 30 μM 的浓度下，PIPE 抑制成骨细胞分化，表现为碱性磷酸酶染色和活性降低。此外，PIPE 降低了磷酸化 Smad1/5/8 和 runt 相关转录因子 2 的蛋白水平，以及骨桥蛋白、碱性磷酸酶和骨钙素的 mRNA 水平。