Rotenone, a mitochondrial complex I inhibitor used to model Parkinsonian cellular stress, was applied at 10 nM for 24 hours to human SH-SY5Y neuroblastoma cells and murine BV2 microglial cells. Rotenone exposure produced concentration-dependent reductions in mitochondrial metabolic activity (MTT) and reductions in cell confluence measured by live-cell imaging. At the 10 nM experimental concentration selected for subsequent assays, both cell lines demonstrated clear rotenone-associated cytotoxic stress.
Under the chosen treatment conditions, rotenone decreased total superoxide dismutase (SOD) activity and increased markers of oxidative stress and inflammation. Culture supernatant interleukin-6 (IL-6) levels were elevated after rotenone exposure, consistent with inflammatory activation. Intracellular lipid peroxidation, quantified as malondialdehyde (MDA), was also increased following rotenone treatment, reflecting enhanced oxidative damage.
To evaluate protective pharmacological effects against rotenone-induced mitochondrial stress, the investigators pretreated cells immediately before rotenone exposure with ginsenosides Rg1, Rg2 (protopanaxatriol-type), and Rd (protopanaxadiol-type) at 20 µM. Rotenone (10 nM) was added into the same ginsenoside-containing medium and cells were co-incubated for 24 hours in complete growth medium with 10% FBS. Vehicle controls contained equivalent DMSO (≤0.1% v/v). This protocol was intended to assess prevention of rotenone-induced changes rather than treatment after established injury.
Intracellular Ca2+ dynamics were assessed using Fura-2 AM ratiometric fluorescence. After the 24-hour co-treatment period, cells were detached, suspended in a standard extracellular solution, and loaded with Fura-2 AM (2.5 µM). Store-operated Ca2+ entry (SOCE) was induced by depleting endoplasmic reticulum stores with BHQ (30 µM) followed by re-addition of extracellular Ca2+ (1.5 mM). Fluorescence was recorded at 340/380 nm excitation and 510 nm emission, and quantitative Ca2+ influx analysis used the area under the curve (AUC) for the re-addition phase. Calculations applied the Fura-2 dissociation constant and standard calibration parameters reported in the methods.
To characterize pathway components contributing to rotenone-associated Ca2+ influx and ginsenoside effects, inhibitors were applied during the Ca2+ influx phase: FIPI (PLD inhibitor, 500 nM), nifedipine (L-type Ca2+ channel blocker, 10 µM), and H-89 (PKA inhibitor, 10 µM). Nifedipine and H-89 were also combined to explore PKA-related modulation of LTCC activity. These pharmacological manipulations were used to infer pathway sensitivity rather than to identify direct molecular binding targets.
Rotenone increased intracellular Ca2+ influx in both SH-SY5Y and BV2 cells under the SOCE protocol. Pretreatment with each ginsenoside (Rg1, Rg2, Rd at 20 µM) attenuated the rotenone-induced increases in intracellular Ca2+ and reduced MDA levels, indicating antioxidative and Ca2+-modulatory effects across cell types. However, the pharmacological sensitivity profiles differed among ginsenosides. Responses in both cell types were sensitive to PLD inhibition with FIPI, implicating PLD-associated signaling in rotenone-evoked and ginsenoside-modulated Ca2+ influx. The PPD-type ginsenoside Rd exhibited broader sensitivity: its Ca2+-modulating effects involved PLD-, LTCC-, and PKA-associated components. By contrast, the PPT-type ginsenosides Rg1 and Rg2 produced predominantly PLD-associated response patterns with less evidence of LTCC- or PKA-associated modulation.
Although PLD-associated pharmacology was common to both SH-SY5Y neuronal and BV2 microglial cells, distinctions emerged in LTCC-related sensitivity. SH-SY5Y neuronal cell Ca2+ responses showed additional sensitivity to LTCC blockade with nifedipine, consistent with a role for LTCCs in neuronal Ca2+ entry under oxidative stress. In BV2 microglial cells, LTCC-associated pharmacological responses were weaker, indicating a smaller contribution of LTCCs to rotenone-evoked Ca2+ influx in this cell type. These differences underscore cell type–dependent mechanisms by which ginsenosides modulate Ca2+ dysregulation.
In rotenone-induced mitochondrial oxidative stress models using SH-SY5Y neurons and BV2 microglia, pre-treatment with structurally distinct ginsenosides (Rg1, Rg2, Rd) attenuated intracellular Ca2+ overload and lipid peroxidation. The pharmacological dissection implicates PLD-associated pathways as a shared modulatory axis for ginsenoside effects in both cell types, while LTCC and PKA components contribute more prominently to Rd-associated responses and to neuronal Ca2+ influx. The study provides comparative pharmacological profiles rather than identification of direct molecular targets, and suggests that ginsenoside structural class influences the breadth of Ca2+-regulatory mechanisms engaged under mitochondrial oxidative stress conditions. Future work would be required to translate these pathway-level findings to in vivo models and to clarify direct molecular interactions; such details were not reported within this manuscript.