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. 2020 May 25;11:760. doi: 10.3389/fphar.2020.00760

Figure 4.

Figure 4

Circumdatin D prevented inflammation-induced neuronal death in microglial-neuronal co-culture and decreased acetylcholinesterase activity. (A) Primary microglia cells were stimulated with 1 μg/ml LPS in the absence or presence of circumdatin D (10, 20, and 50 μM) for measurement of NO, TNF-α, and IL-1β. NO levels were measured by Griess method. The production of TNF-α and IL-1ß were measured by ELISA. (B) Primary neuron cell was treated with or without circumdatin D (1–100 μM) in the absence or presence of 1 μg/ml LPS for 48 h by MTT assay. (C) Primary neurons were stimulated with 1 μg/ml LPS in the absence or presence circumdatin D (1–100 μM) for 48 h and AChE enzyme activity were tested by modified Ellman's spectrophotometric method. (D) Microglial–neuronal co-cultures were treated with 1 μg/ml LPS in the absence or presence of circumdatin D for 48 h, and then apoptotic neuronal cells were determined by TUNEL assay. Data were expressed as the ratio of the number of TUNEL-positive cells to the number of Hoechst-positive cells. (E) Microglial-neuronal co-cultures were treated with 1 μg/ml LPS in the absence or presence of circumdatin D for 48 h, and then crystal violet staining was performed to observe changes in morphology. Values represent the mean ± SD of three independent experiments (#compared with the control, *compared with LPS, */#P < 0.05, **/##P < 0.01, ***P < 0.001. N.S., no significant differences from the control cells).