Formulated Chinese Medicine Shaoyao Gancao Tang Reduces Tau Aggregation and Exerts Neuroprotection through Anti-Oxidation and Anti-Inflammation

Figure 6

Neuroprotection of SG-Tang on ∆K280 tau_RD-DsRed SH-SY5Y cells from BV-2 conditioned medium-induced cell death. (a) Experiment flow chart for cell viability assay. ∆K280 tau_RD-DsRed SH-SY5Y cells were plated in media with/without retinoic acid (± RA, 10 μM) on day 1 and pretreated with SG-Tang the next day for 8 h, followed by doxycycline addition (Dox, 1 μg/ml) to induce misfolded tau expression. On day 3, DMEM-F12 media was mixed with BV-2 CM and cell viability was assessed by MTT assay on day 5. (b) Cell viability assay (, − Dox vs. + Dox; ^&, + Dox/CM vs. + Dox/CM/SG-Tang-treated cells) (). (c) Experiment flow chart for LDH release, neurite outgrowth, and tau phosphorylation assays. RA (10 μM, present in cultures throughout) differentiated ∆K280 tau_RD-DsRed SH-SY5Y cells were pretreated with SG-Tang (200 μg/ml) on day 2 for 8 h, followed by inducing ∆K280 tau_RD-DsRed expression (+ Dox, 1 μg/ml). On day 3, DMEM-F12 was mixed with BV-2 CM and added to the cells. After five days, media were collected for LDH release examination. In addition, cells were examined for neurite outgrowth and tau phosphorylation. (d) LDH assay (, − Dox vs. + Dox; ^###, + Dox vs. +Dox/CM; ^&, + Dox/CM vs. + Dox/CM/SG-Tang treated cells) (). (e) Neurite outgrowth assay (). To normalize, the relative neurite outgrowth of uninduced cells is set as 100%. , − Dox vs. + Dox; ^###, + Dox vs. + Dox/CM; ^&&, + Dox/CM vs. + Dox/CM/SG-Tang-treated cells. (f) Representative microscopy images of differentiated ΔK280 tau_RD-DsRed SH-SY5Y cells uninduced (− Dox), induced (+ Dox), inflamed (+ Dox/CM), or treated with SG-Tang (+ Dox/CM/SG-Tang). Neurites were stained with TUBB3 (green) antibody. Nuclei were detected using DAPI (blue). Upper row, merged TUBB3 and DAPI signals; lower row, images of the neurites and the body of a cell being outlined by the same color for outgrowth quantification. (g) Western blot analysis of total and phosphorylated (Ser202, Thr231, and Ser396) tau (normalized to GAPDH internal control, ). , − Dox vs. + Dox; ^&, + Dox/CM vs. + Dox/CM/SG-Tang-treated cells.

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