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.