Endoplasmic Reticulum Stress Is Involved In The Neuroprotection Of Propofol In Vitro

The endoplasmic reticulum(ER) is an essential intracellular organelle for the synthesis and maturation of biological macromolecules and maintenance of Ca2+ homeostasis of eukaryotic cell. The ER environment is highly sensitive to stresses that impair folding of ER proteins, such as ischemic insult, disturbances in calcium homeostasis, oxidative stress. These stresses reduce the protein folding capacity of the ER, which results in the accumulation and aggregation of unfolded proteins----a condition referred to as ER stress. If the ER stress is too excessive to resolve, the ER switches the signaling direction from the pro-survival pathway to the pro-apoptotic pathway. Such a stress response by the ER and induction of apoptosis are believed to be involved in various tissue injuries and diseases. Cerebral ischemia can result in ATP depletion, Ca2+ overload and free radical generation, which lead to excessive ER stress thus initiate neuronal damage. Propofol(2, 6-diisopropylphenol) is an IV anesthetic widely used for the induction and maintenance of anesthesia and and ICU sedation in clinical practice. Because of its chemical structure that is similar to endogenous antioxidants vitamin E and exogenous antioxidant butyl hydroxy toluene(BHT), Propofol was considered to have the effect of scavenging free radicals and anti-oxidant activities. Numerous studies have shown that Propofol provides neuroprotective effect, but the mechanisms remain to be fully elucidated. In this study, we aim to determine whether Propofol protects cells from ER stress-initiated neural cell apoptosis and the underlying mechanisms in vitro.Objective: To investigate whether Propofol protects neural cells from ER stress-initiated apoptosis and elucidate the underlying mechanisms.Methods: SH-SY5 Y and neuro-2a cells in vitro were stimulated by tunicamycin to induce ER stress in these cells. MTT assay was used to detect cell viability after pretreated with Propofol. Flow cytometry(FCM) was used to detect the number of apoptosis cells. Reverse transcription polymerase chain reaction(RT-PCR) and western blot(WB) were used to detect Bi P and CHOP m RNA and protein level, respectively. Intracellular co-localization of two proteins were examed by immunofluorescence. The expressions of UPR associated proteins were also detected by WB. Small interference RNAs for Bi P were synthesized from Genepharm. Co. LTD. and were transfected to neuro-2a cells to knockdown endogenous Bi P.Results: 1. Propofol protects neural cells against the damage induced by ER stress inducer tunicamycin. Firstly, we used MTT assay to evaluate the effect of propofol on cell viability with orwithout exposure to tunicamycin in SH-SY5 Y cells. Compared with the control group, 10 μM of Propofol can enhance SH-SY5 Y cell viability by 110%. Pretreatment with Propofol at the concentrations of 1, 10, 20, and 40 μM significantly increased the cell viability of SH-SY5 Y cells when subjected to tunicamycin(5 mg/ml), especially at the concentration of 10 μM, which was verified directly by DAPI/PI double staining assay. We also found that pretreatment of 10 μM Propofol decreased the number of apoptotic neuro-2a cells induced by tunicamycin(5 mg/ml) significantly by FCM analysis. 2. Propofol up-regulates Bi P expression in SH-SY5 Y cells. Bi P is usually used as a marker of ER stress. To investigate the mechanism by which Propofol protects neural cells against tunicamycin-induced ER stress, we detected the expression of Bi P after Propofol treatment. SH-SY5 Y cells were incubated with different concentrations of propofol(0.01, 0.1, 1, 10, 20, 40 and 80 μM) for 24 hrs. Propofol significantly up-regulated Bi P expression in a dose-dependent manner, but not CHOP, one of the components of the ER stress-mediated apoptosis pathway. The most effective concentration of Propofol was 10 μM. Therefore, we chose 10 μM as the suitable dosage for further study. The expression of Bi P was significantly increased from 3 hrs to 24 hrs after Propofol exposure. The relative level of Bi P was highest at the time point of 6 hrs. Immunofluorescence Microscopy analysis also indicated that treatment with Propofol up-regulated Bi P expression in SH-SY5 Y cells. 3. Propofol down-regulates tunicamycin-induced CHOP expression. We have observed that Propofol did not affect CHOP level of SH-SY5 Y cells. We were wondering whether Propofol affect the CHOP expression up-regulated by tunicamycin in SH-SY5 Y cells. To test it, we pretreated the cells with Propofol for 6 hrs before the cells were exposed to tunicamycin. After tunicamycin treatment for 6 hrs, the cells were processed for Western blot. We found that Propofol dependently inhibited tunicamycin-stimulated CHOP expression. We also used western blot to examine the effect of Propofol at 10 μM against different concentrations of tunicamycin-stimulated(2.5, 5 and 10 μg/ml) CHOP expression. The result showed that CHOP were all down-regulated significantly. To confirm the effect of propofol on the two ER stress associated proteins Bi P and CHOP, we repeated the experiment in neuro-2a cell line. The cells were treated with Propofol(10 μM) for 6 hrs before tunicamycin(5μg/ml) exposure. The result showed a decrease in CHOP m RNA and protein expression. Furthermore, Immunofluorescence analysis also showed that CHOP expression was decreased by Propofol in neuro-2a cells. Importantly, Propofol inhibited CHOP expression in the nuclei. Our data suggest that Propofol not only inhibit ER stress-induced CHOP expression, but also inhibit CHOP activation. 4. Propofol differently regulates the expressions of UPR target gens. Because Propofol exerts some different effects on Bi P and CHOP expression, we next tested which of the UPR signal axes was involved respectively. neuro-2a cells were treated with Propofol(10 μM) for 6 hrs before tunicamycin(5μg/ml), total protein were extracted for WB analysis. Our data indicated that Propofol pretreatment induced XBP-1 splicing and ATF6 activation. we also examined the phosphorylation of PERK and e IF2α. Our data indicated that tunicamycin increased both e IF2α and PERK phosphorylation, which was attenuated by Propofol pretreatment. 5. Propofol inhibits ATF4 and the cleavage of caspase-3 expression. Previous data indicates that Propofol can decrease the number of apoptotic neuro-2a cells. In order to confirm this, we then examined another pro-death factor, ATF-4, and the determinative caspase, caspase 3. Our data indicated that Propofol attenuated ATF4 expression induced by tunicamycin. Caspase-3 was significantly activated when cells were incubated with tunicamycin, but it was also decreased when pretreated with Propofol. 6. Knockdown Bi P enhances the level of ER stress. To figure out the mechanisms by which Propofol protects neural cells from ERstress-induced injury, we determined the protective effect of endogenous Bi P on ER stress-induced apoptosis by using of Bi P-si RNA in neuro-2A cells. Three strips of synthetic si RNA-Bi P were adopted to knockdown the endogenous Bi P. Twenty-four hours after transfection, total protein were extracted for WB assay. The results showed that the first si RNA-Bi P was the most efficient one in the inhibition of protein level. We then used the validness-approved si RNA-Bi P to transiently transfect neuro-2a cells. 24 hrs after transfection, cells were pretreated with Propofol(10 μM) for 6 hrs before tunicamycin treatment(5μg/ml), total protein were extracted and subjected to WB with CHOP and cleaved caspase 3 antibody respectively. In this way, we found the expression of CHOP was highly increased, the same as the cleavage of caspase-3, and pretreatment of Propofol couldn’t inhibit it. 7. Knockdown Bi P alleviates the neuroprotective effect of propofol. In order to further determine whether Propofol-induced Bi P overexpression is play a vital role in anti-apoptosis, we transiently transfected si RNA-Bi P to neuro-2a cells, added Propofol to cultural medium, then treated with or without tunicamycin. Compared to the control, MTT assay indicated that Propofol had no effect on cell viability. Tunicamycin-induced apoptosis rate measured by flow cytometry showed no significant difference with or without Propofol pretreatment, suggesting neuroprotective effect of Propofol is alleviated after knockdown Bi P.Conclusions: Our findings demonstrated that clinical concentration of propofol can protect cells from ER stress induced apoptosis through PERK/e IFα/ATF4/CHOP pathway, which may be associated with the increased expression of Bi P.