Effects of homocysteine on intracellular ROS and mitochondrial membrane potential of BMSCs.

<p>(a) Intracellular ROS level was measured in BMSCs treated with homocysteine 30, 100 and 300 µM for 24 h by DCFH-DA staining. The ROS level was gradually increased with the increase of homocysteine concentration. (b) Homocysteine induced an obvious depolarization of mitochondrial membrane potential in BMSCs apoptosis by JC-1 staining.</p

Homocysteine induced apoptotic cellular changes of BMSCs.

<p>(a) AO/EB double staining demonstrated the effects of difference concentrations of homocysteine on the apoptosis of BMSCs. BMSCs were incubated with homocysteine for 24 h. (b) Hoeschest33342 staining detected the changes in the nucleus of BMSCs after treatment with homocysteine 30, 100 and 300 µM (scale bar, 20 µm). (c, e) The inhibitory effects of homocysteine on BMSCs were determined by Live/Dead staining. (d, f) TUNEL was used to determine the effects of homocysteine on BMSCs apoptosis (n = 3 independent experiments). * p<0.05 versus Control.</p

Effects of difference concentrations of homocysteine on the morphological appearance and cellular viability of BMSCs.

<p>(a) The morphology of cultured BMSCs was observed after treatment with homocysteine 30, 100, 300 and 1000 µM for 24 h. Homocysteine caused aberrant morphological appearance of BMSCs. (b) Homocysteine significantly decreased the cellular viability of BMSCs in a concentration-dependent manner. * p<0.05 versus Control.</p

JNK signal is involved in the apoptosis of BMSCs induced by homocysteine.

<p>JNK specific inhibitor effectively attenuated the apoptosis induced by homocysteine 300 µM in BMSCs. However, p38 and ERK specific inhibitors did not affect homocysteine-induced apoptotic morphological changes in BMSCs.</p

Effects of homocysteine on MAPKs and apoptotic proteins expression in BMSCs.

<p>(a) The total and phosphorylated JNK, p38 and ERK1/2 protein was detected by western blotting in BMSCs after treatment with homocysteine at the time point of 0, 2, 4, 8, 12 and 24 h. Homocysteine effectively activated phosphorylated JNK expression after treatment with homocysteine. But homocysteine did not increase the expression of total JNK protein in BMSCs. (b) Influences of homocysteine on the expression of Bcl-2, caspase-3, cleaved caspase-3, and p-p53 proteins in BMSCs. n = 3 independent experiments.</p

Increased ROS is required for homocysteine-induced apoptosis of BMSCs.

<p>(a) DMTU and NAC attenuated the increase of ROS level by homocysteine in BMSCs. (b) Effects of DMTU and NAC on the apoptotic appearance of BMSCs. The inhibition of ROS with DMTU and NAC abolished the apoptosis of BMSCs induced by homocysteine. (c) Homocysteine-induced depolarization of mitochondrial membrane potential was also effectively reserved by DMTU and NAC in BMSCs.</p