Establishment of high content co-culture screening for anti-inflammatory drugs in Alzheimer's disease

Abstract

Objectives: Inflammation in Alzheimer's disease occurs as a result of microglial activation that initiates a pro-inflammatory signalling cascade, leading to neurite retraction and neuronal cell death. The aims of the study were: (1) to design a high content assay in which co-culture of a neuron-like cell line with activated microglia or macrophage cells will allow quantitation of neurite retraction and neuronal cell death; (2) to measure the ability of Indonesian herbal extracts to reduce microglial/macrophage activation for identification of the most potent down-regulator of inflammation. Methods: A murine neuroblastoma cell line (Neuro2A) that stably expresses enhanced-green fluorescent protein (eGFP) was directly co-cultured with mouse microglia (M4T4 and C8B4) or macrophage (RAW 264.7) cells. Microglia or macrophages in the co-culture system were activated with lipopolysaccharide (LPS) and interferon gamma (IFN ) in a dose dependent manner up to 10 g/ml of LPS and 10U/ml of IFN . Neurite retraction was quantitated using HCA-vision software (CSIRO). Cell viability, nitrite concentration and TNF concentrations were used as measures of neuronal toxicity, oxidative stress and inflammation, respectively. The macrophage cells were treated with water, ethanol, and hexane extracts of Phyllanthus niruri, Andrographis paniculata, and Tinospora crispa in a dose-dependent manner, serially diluted from 2,500 g/mL. Results: Microglia C8B4 killed Neuro2A-eGFP in control without LPS and IFN activation. Therefore, the co-culture system of microglia C8B4 and Neuro2A-eGFP could not be established. M4T.4, regardless having reasonable doubling time and ability to be cultured together with Neuro2A-eGFP, cannot be activated with LPS and IFN . Therefore, M4T.4 microglia were not suitable for co-culture system with Neuro2A-eGFP. Activated macrophage RAW 264.7 induced Neuro2A-eGFP cell death dose dependently, resulting in a 72.5% reduction of Neuro2A-eGFP cell number at the highest concentration of LPS and IFN in the co-culture system. In the co-culture system, inflammatory activation decreased the average neurite length by 85% and reduced the average number of neurites by 80% when compared to control. TNF and nitrite in the co-culture system were increased dosedependently with respect to LPS and IFN activation. The concentration of NO increased to 37.4 M which was significantly higher than the 3.4 M of seen inthe control. TNF upregulation was observed up to 47.4ng/mL compared to 0.3ng/mL of the control. Tinospora crispa oven dried hexane extract that has potentially lower IC50 for nitric oxide, 4.6 3.1 g/mL. Phyllanthus niruri sun dried water extract was among the most potent reducer of nitrite with IC50 of 49.5 11.4 g/mL. Andrographis paniculata oven dried water extract and Tinospora crispa sun dried water extract were able to down-regulate nitrite production with IC50 of 79.0 21.9 g/mL and 359.3 144.7 g/mL, respectively. For TNF down regulation, Phyllanthus niruri oven dried water extract was the most potent due to an IC50 value of 10.67 1.0g/mL, while Andrographis paniculata had IC50 of 133.798.1g/mL. Coffea liberica Luwak water extract exhibited best TNF down-regulation with IC50 for TNF at 11.91.1g/mL and also showed high potency as NO down-regulator, resulting IC50 value for NO at 288.039.2g/mL. Conclusion: In comparison to previous work with microglia N-11 and Neuro2A-eGFP coculture macrophage, RAW 264.7 and Neuro2A-eGFP co-culture required higher concentration of LPS and IFN to reduce Neuro2A-eGFP viability upon LPS and IFN activation. Therefore, macrophage RAW 264.7 and Neuro2A-eGFP co-culture was suitable for high content screening assay of anti-inflammatory and neuroprotective drug. Indonesian herbal extracts show anti-inflammatory activity resulting Phyllanthus niruri and Coffea liberica Luwak as the best candidate.

Date of Award	2012
Original language	English