Study On The Mechanism Of Endogenous Antioxidant Level In Human Mesenchymal Stem Cells Influence Their Fate

Objective This study aims to investigate the mechanisms for how endogenous antioxidant level in human umbilical cord mesenchymal stem cells(h UCMSCs) influences their cellular fate under exogenous stresses.Methods Application of lipopolysaccharide(LPS)/H2O2 co-treatment caused potent oxidative/inflammatory stress in vitro that similar to the in vivo acute liver failure situation. We also used edaravone as an antioxidant and diethyl maleate(DEM) as a pro-oxidant to pre-treat h UCMSCs. Then we examined the dynamic changes of cellular endogenous antioxidant level, cellular viability, and apoptosis at various time-points post-treatment. Firstly, apoptosis was detected by immunofluorescence staining and caspase-3/7 activity measurements; Cell viability was evaluated by MTT assay; Reactive oxygen species(ROS) staining and ratio change of GSH/GSSH were applied to show cellular endogenous antioxidant level changes after treatments. Then, the m RNA expression levels of anti-apoptotic gene Bcl-2 and pro-apoptotic gene Bax1 were quantified by quantitative PCR; The protein expression of antioxidant enzymes catalase(CAT), superoxide dismutase-1(SOD1) and the involvement of MAPK, PKC pathways were measured by Western blot and normalized by internal control β-actin; Nrf2 transcription factor activity was measured by using a commercial kit. To further investigate the underlying mechanism of modulation of stem cell apoptosis and endogenous antioxidant level, we used MAPK inhibitor PD98059 or PKC inhibitor staurosporine, as well as the si RNA of Nrf2 inhibitory protein – Keap1, to confirm the effects of MAPK-PKC-Nrf2 pathway on stem cell endogenous antioxidant level.Results(1) Compared with the control group, LPS/H2O2 group caused a decrease of cell viability(P<0.05); Pre-treatment with pro-oxidant DEM further reduced the viability of stem cells challenged by LPS/H2O2 at all time-points post-treatment(P<0.05). Compared with LPS/H2O2 group, both concentrations of edaravone significantly recovered the cell viability impaired by the challenge of LPS/H2O2(P<0.05). There was no significant change of cell viability between 10 μM and 20 μM edaravone pre-treated h UCMSCs(P>0.05). Edaravone improved h UCMSCs cell morphology damaged by LPS/H2O2 challenge while DEM further exacerbated it.(2) It was shown that compared with LPS/H2O2 group, pre-treatment with edaravone(10 μM and 20 μM) or DEM significantly decreased or further increased the apoptosis of h UCMSCs, respectively(P<0.05 or <0.001). The activity change of cellular caspase-3/7 was consistent with the change of cellular apoptotic ratio; Results of q PCR exhibited that, when compared with the control group, LPS/H2O2 challenge significantly down-regulated the m RNA level of anti-apoptotic molecule Bcl-2, while up-regulated the m RNA level of pro-apoptotic molecule Bax1 at 12, 24, 36, 48, 60, 72-hour post-treatment; Pre-treatment with both concentrations of edaravone abolished such effects. DEM pre-incubation further reduced the level of Bcl-2 and increased the level of Bax1.(3) From 24-hour post-treatment afterwards, LPS/H2O2 group showed obvious DCFH-DA positive signals in the culture medium, when compared with the control group, which was attenuated by edaravone pre-treatment but worsened by DEM pre-treatment In line with the ROS production, LPS/H2O2 challenge caused significant decrease of cellular GSH/GSSG ratio when compared with that of the control group at 12, 24, 36, 48, 60 and 72 hours post-treatment(P<0.001). Such decreases were evidently restored by edaravone(10 μM and 20 μM) but further reduced by DEM.(4) Western blotting results suggested that at 12, 24, 36, 48, 60, and 72 hours after LPS/H2O2 treatment, the protein expression levels of both CAT and SOD1 were significantly down-regulated(particularly for SOD1). Edaravone pre-treatment significantly restored their expression levels while DEM pre-incubation reduced them to lower levels(P<0.05).(5) Results from MAPK studies showed that LPS/H2O2 treatment potentiated the phosphorylation of both p38 MAPK and ERK1/2 at most time points post-treatment. In agreement with previous results, pre-treatment with edaravone or DEM significantly abolished or further strengthened the effects of LPS/H2O2 treatment, respectively. After the application MAPK inhibitor PD98059 and PKC inhibitor staurosporine, the beneficial effects of 20 μM edaravone on cell viability were partially abolished; Silence of Keap1 by specific si RNA significantly increased the Nrf2 activity, and partially reversed the detrimental effects of DEM on cell viability.Conclusions(1) Pre-treatment with antioxidant Edaravone can improved h UCMSCs viability and morphology after the oxidative/inflammatory challenge;(2) h UCMSCs pre-treatment with antioxidant Edaravone can attenuated stem cell apoptosis by down-regulated the m RNA level of anti-apoptotic molecule Bcl-2, while up-regulated the m RNA level of pro-apoptotic molecule Bax1;(3) h UCMSCs pre-treatment with antioxidant Edaravone increased endogenous antioxidant level by restored levels of endogenous antioxidant enzymes impaired by oxidative/inflammatory challenge;(4) The modulation of stem cell endogenous antioxidant level was, at least partly, through the MAPK-PKC-Nrf2 pathway.