乳酸通过 MCT1-mTOR 信号通路通过自噬抑制人牙周膜干细胞的成骨分化。

Lactate inhibits osteogenic differentiation of human periodontal ligament stem cells via autophagy through the MCT1-mTOR signaling pathway.

机构信息

Jiangsu Province Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, People's Republic of China; Department of Periodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, People's Republic of China; Jiangsu Province Engineering Research Center of Stomatological Translational Medicine, Nanjing, People's Republic of China.

出版信息

Bone. 2022 Sep;162:116444. doi: 10.1016/j.bone.2022.116444. Epub 2022 May 16.

DOI:10.1016/j.bone.2022.116444

PMID:35589065

Abstract

BACKGROUND

Periodontal ligament stem cells (PDLSCs) play a crucial role in periodontal bone regeneration. Lactate used to be considered a waste product of glucose metabolism. In recent years, a few pieces of evidence revealed its roles in regulating the osteogenic differentiation of stem cells, but the standpoints were controversial. This study aims to investigate the effects and the mechanisms of lactate on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs).

METHODS

The hPDLSCs were treated with different concentrations of lactic acid and lactate to differentiate the effects of the acidic PH and ion lactate. Proliferation and cytotoxicity were measured by Cell Counting Kit-8 (CCK8) assay and Live/Dead assay. The osteogenic differentiation of hPDLSCs was analyzed by alizarin red staining, alkaline phosphatase (ALP) staining, and then osteogenic proteins and genes were measured by western blot and reverse transcription-quantitative PCR (qRT-PCR). To investigate the potential signaling pathways, MCT1 inhibitor, G-protein inhibitors, and rapamycin were used, and then autophagy-related proteins and osteogenic proteins were measured by western blot.

RESULTS

The inhibition of lactic acid on the osteogenic differentiation of hPDLSCs was more significant than lactate at the same concentration. Lactate inhibited the expression of ALP which can be rescued by Gα inhibitor. Alizarin red staining, the protein expression levels of osteocalcin (OCN), osteoprotegerin (OPN), osterix (OSX), and beclin1, LC3-II/LC3-I were decreased by lactate and partly rescued by MCT1 inhibitor. Rapamycin restored the protein expression levels of beclin1, LC3-II/LC3-I and OCN, OPN, OSX under the high lactate conditions.

CONCLUSIONS

Lactate inhibits the expression of ALP via Gα subunit signaling, and inhibits mineralized nodules formation and the expression of osteogenic-related proteins via reducing autophagy through the MCT1-mTOR signaling pathway.

摘要

背景

牙周膜干细胞（PDLSCs）在牙周骨再生中起着至关重要的作用。乳酸过去曾被认为是葡萄糖代谢的废物产物。近年来，有一些证据表明它在调节干细胞的成骨分化方面发挥作用，但观点存在争议。本研究旨在探讨乳酸对人牙周膜干细胞（hPDLSCs）成骨分化的影响及其机制。

方法

用不同浓度的乳酸处理 hPDLSCs，以分化酸性 PH 和离子乳酸的作用。通过细胞计数试剂盒-8（CCK8）测定法和活/死测定法测定细胞增殖和细胞毒性。通过茜素红染色、碱性磷酸酶（ALP）染色分析 hPDLSCs 的成骨分化，然后通过 Western blot 和逆转录-定量 PCR（qRT-PCR）测定成骨蛋白和基因。为了研究潜在的信号通路，使用了 MCT1 抑制剂、G 蛋白抑制剂和雷帕霉素，然后通过 Western blot 测定自噬相关蛋白和成骨蛋白。

结果

与相同浓度的乳酸相比，抑制乳酸对 hPDLSCs 成骨分化的作用更为显著。乳酸抑制 ALP 的表达，Gα 抑制剂可挽救。茜素红染色、骨钙素（OCN）、骨保护素（OPN）、成骨特异性转录因子 2（OSX）和 beclin1、LC3-II/LC3-I 的蛋白表达水平降低由乳酸引起，并部分由 MCT1 抑制剂挽救。雷帕霉素在高乳酸条件下恢复了 beclin1、LC3-II/LC3-I 和 OCN、OPN、OSX 的蛋白表达水平。