ATG7-mediated autophagy protects human gingival myofibroblasts from irradiation-induced apoptosis

IF 2.7 3区医学 Q1 DENTISTRY, ORAL SURGERY & MEDICINE

Journal of Oral Pathology & Medicine Pub Date : 2023-10-24 DOI:10.1111/jop.13490

Xiumei Zhuang, Xiaoxuan Lin, Ruogu Xu, Zhengchuan Zhang, Bin Zhou, Feilong Deng

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引用次数: 0

Abstract

Background

Apoptosis resistance of myofibroblasts is critical in pathology of irradiation-induced fibrosis and osteoradionecrosis of the jaw (ORNJ). However, molecular mechanism of apoptosis resistance induced by irradiation in oral myofibroblasts remains largely obscure.

Methods

Matched ORNJ fibroblasts and normal fibroblasts pairs from gingival were primarily cultured, and myofibroblast markers of α-SMA and FAP were evaluated by qRT-PCR and western blot. CCK8 assay and flow cytometric analysis were performed to investigate the cell viability and apoptosis under irradiation treatment. Autophagy-related protein LC3 and ATG7, and punctate distribution of LC3 localization were further detected. After inhibition of autophagy with inhibitor CQ and 3-MA, as well as transfected ATG7-siRNA, cell viability and apoptosis of ORNJ and normal fibroblasts were further assessed.

Results

Compared with normal fibroblasts, ORNJ fibroblasts exhibited significantly higher α-SMA and FAP expression, increased cell, viability and decreased apoptosis under irradiation treatment. LC3-II and ATG7 were up-regulated in ORNJ fibroblasts with irradiation stimulation. After inhibition of irradiation-induced autophagic flux with lysosome inhibitor CQ, LC3-II protein was accumulated and punctate distribution of LC3 localization was increased in ORNJ fibroblasts. Moreover, autophagy inhibitor CQ and 3-MA enhanced the irradiation-induced apoptosis but inhibited viability of ORNJ fibroblasts. Silencing ATG7 with siRNA could obviously weaken irradiation-induced LC3-II expression, and promoted irradiation-induced apoptosis of ORNJ fibroblasts. After knockdown of ATG7, finally, p-AKT(Ser473) and p-mTOR(Ser2448) levels of ORNJ fibroblasts were significantly increased under irradiation.

Conclusion

Compared with normal fibroblasts, human gingival myofibroblasts are resistant to irradiation-induced apoptosis via autophagy activation. Silencing ATG7 may evidently inhibit activation of autophagy, and promote apoptosis of gingival myofibroblasts via Akt/mTOR pathway.

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atg7介导的自噬保护人类牙龈肌成纤维细胞免受辐射诱导的凋亡。

背景：肌成纤维细胞的细胞凋亡抵抗在照射诱导的颌骨纤维化和放射性骨坏死（ORNJ）的病理学中至关重要。然而，辐射诱导口腔肌成纤维细胞凋亡抵抗的分子机制仍不清楚。方法：对匹配的ORNJ成纤维细胞和正常牙龈成纤维细胞对进行初步培养，并通过qRT-PCR和蛋白质印迹法评估肌成纤维细胞标志物α-SMA和FAP。采用CCK8法和流式细胞仪分析细胞在辐照处理下的活力和凋亡。进一步检测自噬相关蛋白LC3和ATG7，以及LC3定位的点状分布。在用抑制剂CQ和3-MA以及转染的ATG7 siRNA抑制自噬后，进一步评估ORNJ和正常成纤维细胞的细胞活力和凋亡。结果：与正常成纤维细胞相比，ORNJ成纤维细胞在辐照处理下表现出明显更高的α-SMA和FAP表达，增加了细胞活力，减少了细胞凋亡。LC3-II和ATG7在辐射刺激下在ORNJ成纤维细胞中上调。在用溶酶体抑制剂CQ抑制辐射诱导的自噬流量后，在ORNJ成纤维细胞中积累了LC3-II蛋白，并且LC3定位的点状分布增加。此外，自噬抑制剂CQ和3-MA增强了辐射诱导的细胞凋亡，但抑制了ORNJ成纤维细胞的活力。用siRNA沉默ATG7可以明显减弱辐射诱导的LC3-II表达，并促进辐射诱导的ORNJ成纤维细胞凋亡。ATG7敲低后，最终，ORNJ成纤维细胞的p-AKT（Ser473）和p-mTOR（Ser2448）水平在照射下显著增加。结论：与正常成纤维细胞相比，人牙龈肌成纤维细胞通过自噬激活抵抗辐照诱导的细胞凋亡。沉默ATG7可能通过Akt/mTOR途径明显抑制自噬激活，并促进牙龈肌成纤维细胞凋亡。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Oral Pathology & Medicine 医学-病理学

CiteScore

5.90

自引率

6.10%

发文量

121

审稿时长

4-8 weeks

期刊介绍： The aim of the Journal of Oral Pathology & Medicine is to publish manuscripts of high scientific quality representing original clinical, diagnostic or experimental work in oral pathology and oral medicine. Papers advancing the science or practice of these disciplines will be welcomed, especially those which bring new knowledge and observations from the application of techniques within the spheres of light and electron microscopy, tissue and organ culture, immunology, histochemistry and immunocytochemistry, microbiology, genetics and biochemistry.