The Protective Effect Of Stem Cells-conditioned Media On Endoplasmic Reticulum Stress Of Retinal Ganglion Cells

Objectives:1. To study the neuroprotective effect of human umbilical cord mesenchymal stem cells-conditioned media (hUC-MSCs-CM) on the retinal ganglion cells which were under endoplasmic reticulum stress.2. To investigate the possible mechanism of the neuroprotective effect of hUC-MSCs-CM, by detecting and analyzing the change of endoplasmic reticulum stress markers.Methods:RGC-5cells were plated at a density of1×105cells/well or2×105cells/well in12-well or6-well culture plates, and were divided by control group, tunicamycin group and treatment group. The control group was sub-divided by normal control group (ND) in which RGC-5were added by completed media, and hUC-MSCs-CM group (NM) in which RGC-5were added by completed media and hUC-MSCs-CM. In tunicamycin group RGC-5were immersed by tunicamycin and completed media after48-hour culture. The tunicamycin group was sub-divided by1μg/mL group(1D),2μg/mL group(2D) and4μg/mL group(4D) according to the tunicamycin concentration. In treatment group RGC-5were added by different concentration of tunicamycin and hUC-MSCs-CM at same volumes of completed media. The treatment group was sub-divided by1μg/mL group(1M),2μg/mL group(2M) and4μg/mL group(4M) according to the tunicamycin concentration. Twenty-four hours after the addition of tunicamycin, apoptosis rate was measured by propidium iodide (PI) and Hoechst33342double staining. At the corresponding time points after the addition of tunicamycin, levels of PERK、XBP1、ATF6、 CHOP、BDNF were analyzed by semiquantitative real time PCR and immunoblot.Results:1. After adding the same concentrations of tunicamycin, the apoptosis rate of RGC-5in treatment group was significantly lower than tunicamycin group(P<0.05). Moreover with the increased concentrations of tunicamycin, the apoptosis rate of RGC-5was corresponding increased.2. At time point of9hour and12hour,the transcription level of ATF6、XBP1、CHOP in tunicamycin group were down-regulated compared with treatment group. At time point of9hour the transcription level of ATF6between two groups had significantly difference(P<0.05)..On the contrary, the transcription level of PERK between two groups had little difference.3. The transcription level of PERK、ATF6、CHOP、XBP1in4M group were down-regulated successively comparing with the4D group, while the difference is the greatest one at time point of12hour. The level of transcription and protein expression of XBP1in treatment group were significantly lower than the tunicamycin group(P<0.05).Conclusions:1. Tunicamycin could result in RGC-5undergoing endoplasmic reticulum stress.2. hUC-MSCs-CM had neuroprotective effects on RGC-5against endoplasmic reticulum stress which was induced by tunicamycin.3. At time point of9hour, hUC-MSCs-CM played a protective role through ATF6pathway to RGC-5which were undergoing unfolded protein response after being treated with tunicamycin.4. At time point of12hour, hUC-MSCs-CM play a protective role through XBP1pathway to RGC-5which were apoptosis after being treated with tunicamycin.