Research Paper Volume 13, Issue 11 pp 15620—15637

Formulated Chinese medicine Shaoyao Gancao Tang reduces NLRP1 and NLRP3 in Alzheimer’s disease cell and mouse models for neuroprotection and cognitive improvement

Effects of SG-Tang on Aβ aggregation, ROS, and neurite outgrowth in Aβ-GFP-expressing cells. (A) Experimental flow chart of Aβ-GFP SH-SY5Y cells. On day 1, cells were plated with retinoic acid (RA, 10 μM) added to the culture medium. On day 2, curcumin or SG-Tang was added to the cells for 8 h, followed by inducing Aβ-GFP expression with doxycycline (Dox, 5 μg/ml) for 6 days. On day 8, GFP fluorescence, cell number, ROS, Aβ-GFP RNA and neurite outgrowth were measured. (B) Assessment of GFP fluorescence and cell number with curcumin (1.2–5 μM) or SG-Tang (1–100 μg/ml) treatment (n = 3). The relative GFP fluorescence/cell number of untreated cells (Untr.) was normalized as 100%. (C) ROS assay with curcumin (1.2–5 μM) or SG-Tang (1–100 μg/ml) treatment (n = 3). The relative ROS of uninduced cells (Dox-) was normalized (100%). (D) Measurement of Aβ-GFP RNA levels in cells treated with 5 μM curcumin and 100 μg/ml SG-Tang by real-time PCR (n = 3). (E) Fluorescence microscopy images of differentiated Aβ-GFP SH-SY5Y cells uninduced (Dox-), untreated (Dox+) or treated with curcumin (5 μM) or SG-Tang (100 μg/ml). Neurite outgrowth and cell number were measured after TUBB3 (yellow) staining (n = 3). Nuclei were counterstained with DAPI (blue). The relative cell number of uninduced cells was normalized as 100%. P values: comparisons between induced (Dox+) vs. uninduced (Dox-) cells (###: P P P P t test; C–E: ROS, Aβ-GFP RNA and neurite outgrowth: one-way ANOVA with a post hoc Tukey test).

Figure 1. Effects of SG-Tang on Aβ aggregation, ROS, and neurite outgrowth in Aβ-GFP-expressing cells. (A) Experimental flow chart of Aβ-GFP SH-SY5Y cells. On day 1, cells were plated with retinoic acid (RA, 10 μM) added to the culture medium. On day 2, curcumin or SG-Tang was added to the cells for 8 h, followed by inducing Aβ-GFP expression with doxycycline (Dox, 5 μg/ml) for 6 days. On day 8, GFP fluorescence, cell number, ROS, Aβ-GFP RNA and neurite outgrowth were measured. (B) Assessment of GFP fluorescence and cell number with curcumin (1.2–5 μM) or SG-Tang (1–100 μg/ml) treatment (n = 3). The relative GFP fluorescence/cell number of untreated cells (Untr.) was normalized as 100%. (C) ROS assay with curcumin (1.2–5 μM) or SG-Tang (1–100 μg/ml) treatment (n = 3). The relative ROS of uninduced cells (Dox-) was normalized (100%). (D) Measurement of Aβ-GFP RNA levels in cells treated with 5 μM curcumin and 100 μg/ml SG-Tang by real-time PCR (n = 3). (E) Fluorescence microscopy images of differentiated Aβ-GFP SH-SY5Y cells uninduced (Dox-), untreated (Dox+) or treated with curcumin (5 μM) or SG-Tang (100 μg/ml). Neurite outgrowth and cell number were measured after TUBB3 (yellow) staining (n = 3). Nuclei were counterstained with DAPI (blue). The relative cell number of uninduced cells was normalized as 100%. P values: comparisons between induced (Dox+) vs. uninduced (Dox-) cells (###: P < 0.001), or treated (Dox+/curcumin or SG-Tang) vs. untreated (Dox+) cells (*: P < 0.05, **: P < 0.01, ***: P < 0.001). (B: GFP fluorescence and cell number: two-tailed Student’s t test; C–E: ROS, Aβ-GFP RNA and neurite outgrowth: one-way ANOVA with a post hoc Tukey test).