Results
GS enhances MRC and ATP production in cardiomyocytes and neurons
To explore the molecular mechanism of GS on extensive cell protection, we first investigated the effects of GS on mitochondrial function by examining OCR, as a direct measure of mitochondrial respiration (Diepart, Verrax, Calderon, Feron, Jordan & Gallez, 2010). Oxygen consumption probes were used to assess the effect of GS on the rate of basal oxygen consumption in a variety of cell lines, including H9c2, primary neonatal cardiomyocytes, differentiated PC12, primary cortical neurons, L6, C2C12, HUVECs, BMSCs, osteoblast, 16HBE, and THP-1 cells. As expected, GS pretreatment for 48 h increased basal oxygen consumption by 2.0-fold in H9c2 cells, 2.8-fold in PC12 cells, 1.5-fold in neurons (Fig. 1A and Supplementary Fig. 3A) , 1.6-fold in C2C12 cells, and 1.5-fold in L6 cells( Supplementary Fig. 3B) . Meanwhile, we found that GS had no effect on basal OCR in HUVECs, BMSCs, osteoblast, 16HBE, and THP-1 cells (Fig. 1B,Supplementary Fig. 3B-3F) . To further assess the potential function of GS in mitochondrial oxidative capacity, we conducted a mitochondrial stress test in H9c2, PC12, and HUVECs. The pretreatment of GS at 5 μg/mL for 48 h led to increases in basal OCR, MRC, and SRC in H9c2 and PC12 cells(Fig. 1C and Fig. 1D) . The similar results for mitochondrial oxidative capacity were detected by Luxcel oxygen consumption probes in H9c2 and PC12 cells (Supplementary Fig. 4A-4B) . In HUVECs, GS pretreatment had no effects on basal OCR, MRC, and SRC(Supplementary Fig. 4C) . Furthermore, intracellular ATP content was examined in H9c2, PC12, and primary neurons after GS pretreatment. As shown in Figure 1E , GS pretreatment significantly increased ATP production in cardiomyocytes and neurons in the same manner as OCR. LC-MS analysis showed that GS at 5 μg/mL promoted relative ATP intensity and the ratio of ATP/ADP in H9c2 cells (Fig. 1F) . In HUVECs, GS slightly increased ATP content and had no effect on ATP/ADP ratio(Supplementary Fig. 4D) . To further identify the time-dependent effect of GS on energy production, the levels of basal OCR and ATP in H9c2 cells at different time points were measured after GS pretreatment for 48 h. The results in Figure 1G and 1H demonstrated that GS pretreatment for 6 h enhanced basal OCR and ATP content at concentrations of 5 and 10 μg/mL GS. In particular, basal OCR was gradually increased by GS pretreatment in time- and dose-dependent manners. Collectively, GS pretreatment enhanced MRC and ATP production in aerobic respiration- dominated cardiomyocytes and neurons, and had no obvious effect on anaerobic respiration- dominated other cell lines such as HUVECs and BMSCs.