Gao Shumei, Zhou Qiao, Jin Hui, Shi Naiqi, Wang Xiaoyu, Zhang Li, Yan Meijuan
The Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, China.
School of Chemistry and Molecular Biosciences, the University of Queensland, Brisbane, Queensland, Australia.
Ann Transl Med. 2021 Sep;9(17):1377. doi: 10.21037/atm-21-730.
Pyrroloquinoline quinone (PQQ) is involved in various physiological and biochemical processes, including antioxidant, cell proliferation, and mitochondrial formation. It plays a vital role in protecting neurons. However, the effect of PQQ on microglia, an inflammatory cell of the central nervous system (CNS), is still unclear. This study aimed to investigate the biological role and neuroprotective mechanism of PQQ in HAPI microglial cells exposed to lipopolysaccharide (LPS).
Western blot (WB) was used to detect apoptosis and autophagy-related molecules Bax, Bcl2, active-caspase-3, caspase-3, LC3, lysosomal associated membrane protein 2 (LAMP2), AKT, tumor necrosis factor receptor (TNFR) 1, and TNFR2 expression. The phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor LY294002 was used to block the Akt pathway. WB detected the effects of PI3K on autophagy and TNFR1 and TNFR2 expression. The localization of active-caspase-3, caspase-3, LC3, LAMP2, TNFR1, and TNFR2 in cells was observed by immunofluorescence staining. The effect of PQQ on the cell cycle was examined by flow cytometry. We used 5-Ethynyl-2'-deoxyuridine (EdU) assay to detect cell proliferation. The migration ability of cells under different conditions was detected by scratch test and Transwell assay.
Our results showed that there were different effects on the apoptosis-related molecules Bcl2/Bax and active-caspase-3/caspase in HAPI microglial cells treated with PQQ at different times. PQQ had no significant effect on the LC3b/a ratio in the early stage, which was upregulated in the later stage. The expression of LAMP2 was significantly increased in both early and late stages after PQQ treatment. At the same time, we found that PQQ can reverse the translocation of LAMP2 from the cytoplasm to the nucleus in LPS-induced HAPI microglia. After PQQ treatment, TNFR1 was significantly decreased, but TNFR2 increased in LPS-induced HAPI microglia. It may be that PQQ works through the PI3K/Akt signaling pathway to up-regulate LC3, LAMP2, and TNFR1 and down-regulate TNFR2 in LPS-induced HAPI microglia. However, PQQ has little effect on LPS-induced proliferation, cell cycle, and migration of HAPI microglia.
In LPS-induced HAPI microglia, PQQ reduces the apoptosis level and increases that of autophagy. In addition, PQQ changes the distribution of LAMP2 in the cytoplasm and nucleus, which is regulated through the PI3K/Akt signaling pathway.
吡咯喹啉醌（PQQ）参与多种生理和生化过程，包括抗氧化、细胞增殖和线粒体形成。它在保护神经元方面起着至关重要的作用。然而，PQQ对小胶质细胞（中枢神经系统（CNS）的一种炎症细胞）的影响仍不清楚。本研究旨在探讨PQQ在暴露于脂多糖（LPS）的HAPI小胶质细胞中的生物学作用和神经保护机制。
采用蛋白质免疫印迹法（WB）检测凋亡和自噬相关分子Bax、Bcl2、活化的半胱天冬酶-3、半胱天冬酶-3、微管相关蛋白轻链3（LC3）、溶酶体相关膜蛋白2（LAMP2）、蛋白激酶B（AKT）、肿瘤坏死因子受体（TNFR）1和TNFR2的表达。使用磷脂酰肌醇3-激酶（PI3K）/Akt抑制剂LY294002阻断Akt通路。WB检测PI3K对自噬以及TNFR1和TNFR2表达的影响。通过免疫荧光染色观察活化的半胱天冬酶-3、半胱天冬酶-3、LC3、LAMP2、TNFR1和TNFR2在细胞中的定位。采用流式细胞术检测PQQ对细胞周期的影响。我们使用5-乙炔基-2'-脱氧尿苷（EdU）检测法检测细胞增殖。通过划痕试验和Transwell检测法检测不同条件下细胞的迁移能力。
我们的结果表明，不同时间用PQQ处理的HAPI小胶质细胞中，凋亡相关分子Bcl2/Bax和活化的半胱天冬酶-3/半胱天冬酶-3受到不同影响。PQQ在早期对LC3b/a比值无显著影响，后期上调。PQQ处理后早期和晚期LAMP2的表达均显著增加。同时，我们发现PQQ可以逆转LPS诱导的HAPI小胶质细胞中LAMP2从细胞质向细胞核的转位。PQQ处理后，LPS诱导的HAPI小胶质细胞中TNFR1显著降低，但TNFR2增加。可能是PQQ通过PI3K/Akt信号通路在LPS诱导的HAPI小胶质细胞中上调LC3、LAMP2和TNFR1并下调TNFR2。然而，PQQ对LPS诱导的HAPI小胶质细胞的增殖、细胞周期和迁移影响较小。
在LPS诱导的HAPI小胶质细胞中，PQQ降低凋亡水平并增加自噬水平。此外，PQQ改变LAMP2在细胞质和细胞核中的分布，这是通过PI3K/Akt信号通路调节的。
