Carbonyl Stress On Mesenchymal Stem Cells And Protective Effects Of Ginsenoside Rg1

Objective:Reactive carbonyl species (RCS) are toxic compounds, and researchers are deeply concerned with their roles in the process of senescence. It has been shown that changes in the biological function of mesenchymal stem cells (MSCs) are involved in the aging. Little is known about effects of RCS on MSCs so far. This study was conducted to examine the effects of malondialdehyde (MDA), used as a representative RCS, on the proliferation, apoptosis and differentiation of mouse bone MSCs in vitro to gain a mechanism underlying carbonyl stress in these cells associated with the senescence. The anti-carbonyl stress activities of ginsenoside Rgl were also evaluated in the cultured MSCs to provide experimental evidences for its use in the treatment of age-related diseases.Methods:The MDA treated and control murine bone marrow primary MSC colony formation rates were assayed. After subculture, we obtained MSC passage purified cells. The cells were treated by different concentration of MDA, or pretreated by Rg1for24h and then treated by MDA. Then the cells were assayed:①using MTT to measure cell viability;②the DNA synthesis rates were detected by BrdU;③using flow cytometry, Hochest33258stain and TUNEL to analysis apoptosis rates;④calcium nodule formation were detected by alizarin red stainingand Von Kossa's stain;⑤the determination of alkaline phosphatase activity were assayed by phosphate on the nitrobenzene;⑥Oil red O staining were performed to observe the intracellular lipid droplets;⑦the mRNA expression of ALP,Runx2/Cbfal,PPARγ2, bcl-2,Bax,caspase-3were detected by Q-RT-PCR;⑧using Western blotting to detected protein expression and phosphorylation p38MAPK and JNK;⑨The JNK protein distribution in cell were assayed by immunofluorescence and laser scanning confocal microscopy.Results:(1) The depression of MDA on MSCs polificationAmong0.01-1mmol/L MDA culture system, with the increase of MDA concentration, the colony formation rates of cultured primary MSC, MTT absorbed value of MSC passage purified cells and BrdU incorporation percentage of positive cells were decreased consistently and showed dosage-dependent. Between0.1mmol/L and1.0mmol/L concentration, the indicators had significant difference.(2) The apoptosis of MSCs by MDA treatmentWith MDA concentration increased, the apoptosis rates were raised. Meanwhile, the mRNA expression of bcl-2and Box were decreased and rised, respectively. Each indicators showed significat difference among the groups with dosage-dependent. (3) The inhibition of osteogenic and induction adipogenic of by MDAAfter osteogenic inductive, both the0.1mmol/L and1mmol/L MDA concentration groups showed decreased calcium nodule formation, alkaline phosphatase activity, mRNA expression of ALP and Runx2/Cbfal compared with control. Conversely, after adipogenic-inductive, compared with control, the0.1mmol/L and1.0mmol/L MDA concentration groups displayed increased lipid droplets and PPARy2mRNA expression. The0.01mmol/L concentration MDA group showed higher lipid droplets and PPARy2mRNA expression than control but the difference showed no significant.(4) Effects of Malondialdehyde on p38MAPKand JNK signal transduction pathwaysThe protein expression and phosphorylation p38MAPK and JNK were increased, and then which were decreased in a time-dependent manner by0.1mmol/L MDA treatment compared with control. In MDA group, the distribution of JNK was transformed from cytoplasm to nucleus.(5) The anti-carbonyl stress activities of ginsenoside Rg1After pretreated with10mg/L,50mg/L or100mg/L Rgl for24h, all the groups showed increased colony formation rates compared with control. While50mg/L and100mg/L Rgl had greater effect on colony formation rate compared with10mg/L and showed significant difference. Compared with control, after pretreated with Rg1had higher MTT absorbed value, BrdU incorporation percentage of positive cells, bcl-2mRNA and protein expression. Meanwihle the apoptosis rates, mRNA and protein expression of bax and caspase-3were decreased with dosage-dependent.The Rg1pretreated for24h showed increased calcium nodule formation, ALP activities, and mRNA expression of ALP and Cbfal compared with control. In the adipogenic-inductive groups, Rg1pretreated showed decreased lipid droplets and PPARγ mRNA expression. The indicators of osteo-inductive and adipogenic-inductive showed dosage-dependent with concentration of Rg1.Conclusion:MDA induces carbonyl stress in MSCs in vitro, suggesting that MSC bio-toxic effects of carbonyl compounds on these cells contribute to the aging process, and may be associated with some age-related diseases. Ginsenoside Rg1has a cell-protective effect against the carbonyl stress in cultured MSCs, providing experimental data for ginseng and its active ingredients used for the prevention and treatment of senile diseases related to MSCs.