Department of Critical Care Medicine, Xiang-Ya Hospital, Central South University, Changsha, Hunan Province, China.
Department of Anesthesia, Xiang-Ya Hospital, Central South University, Changsha, Hunan Province, China.
Neuroscience. 2017 Oct 11;361:34-42. doi: 10.1016/j.neuroscience.2017.08.010. Epub 2017 Aug 12.
Whether the CD38/cyclic ADP-ribose (cADPR) pathway plays a protective or detrimental role in neuroinflammation remains controversial. This study aimed to determine the role of CD38 in neuroinflammation using lipopolysaccharide (LPS)-stimulated BV2 microglial cells and co-cultured Neuro-2a (N2a) cells. In monoculture experiments, BV2 cells were divided into control, CD38 interference (CD38Ri), negative control (NC), LPS, CD38Ri+LPS, NC+LPS and 8-Br-cADPR+LPS groups. In co-culture experiments, N2a cells were co-cultured with BV2 cells for 48h. Nicotinamide adenine dinucleotide (NAD), cADPR and intracellular Ca levels and CD38 expression increased significantly in LPS-stimulated BV2 cells. CD38 knockdown or 8-Br-cADPR treatment significantly reduced NAD, cADPR and intracellular Ca levels. CD38 knockdown increased iNOS and NO levels in BV2 cells without LPS treatment; however, CD38 knockdown or 8-Br-cADPR treatment reduced iNOS and NO levels in BV2 cells with LPS treatment. CD38 knockdown increased the ratio of TUNEL-positive cells and cleaved Caspase 3/Caspase 3 ratio, and decreased the Bcl-2/Bax ratio in BV2 cells without LPS treatment; however, CD38 knockdown reduced the TUNEL positivity in BV2 cells with LPS treatment. CD38 knockdown or 8-Br-cADPR inhibited TNF-α, IL-6 (interleukin-6) and IL-1β levels in LPS-stimulated BV2 cells. Co-culture with CD38 knockdown or 8-Br-cADPR-treated BV2 cells did not influence apoptosis or iNOS expression in N2a cells. In conclusion, our results indicate that blocking the CD38/cADPR pathway reduces intracellular Ca, NO and the secretion of proinflammatory cytokines. CD38 knockdown exerted a detrimental effect in apoptosis and NO production in normal microglia, but played a protective role in apoptosis and NO production in LPS-stimulated microglia.
CD38/环 ADP-核糖（cADPR）通路在神经炎症中是发挥保护作用还是损伤作用仍存在争议。本研究旨在通过脂多糖（LPS）刺激的 BV2 小胶质细胞和共培养的 Neuro-2a（N2a）细胞来确定 CD38 在神经炎症中的作用。在单核培养实验中，BV2 细胞被分为对照组、CD38 干扰（CD38Ri）组、阴性对照组（NC）、LPS 组、CD38Ri+LPS 组、NC+LPS 组和 8-Br-cADPR+LPS 组。在共培养实验中，N2a 细胞与 BV2 细胞共培养 48h。LPS 刺激的 BV2 细胞中 NAD、cADPR 和细胞内 Ca 水平以及 CD38 表达显著增加。CD38 敲低或 8-Br-cADPR 处理显著降低 NAD、cADPR 和细胞内 Ca 水平。CD38 敲低在无 LPS 处理的情况下增加了 BV2 细胞中的 iNOS 和 NO 水平；然而，CD38 敲低或 8-Br-cADPR 处理降低了 LPS 处理的 BV2 细胞中的 iNOS 和 NO 水平。CD38 敲低增加了无 LPS 处理的 BV2 细胞中 TUNEL 阳性细胞的比例和裂解 Caspase 3/Caspase 3 比值，降低了 BV2 细胞中的 Bcl-2/Bax 比值；然而，CD38 敲低降低了 LPS 处理的 BV2 细胞中的 TUNEL 阳性率。CD38 敲低或 8-Br-cADPR 抑制了 LPS 刺激的 BV2 细胞中 TNF-α、IL-6（白细胞介素-6）和 IL-1β水平。与 CD38 敲低或 8-Br-cADPR 处理的 BV2 细胞共培养并未影响 N2a 细胞中的细胞凋亡或 iNOS 表达。总之，我们的结果表明，阻断 CD38/cADPR 通路可降低细胞内 Ca、NO 和促炎细胞因子的分泌。CD38 敲低在正常小胶质细胞中的细胞凋亡和 NO 产生中产生有害作用，但在 LPS 刺激的小胶质细胞中的细胞凋亡和 NO 产生中发挥保护作用。
