AGEs Induce SH-SY5Y Cells Apoptosis Via Oxidative Stress And Endoplasmic Reticulum Stress

Background Alzheimer’s disease is one of the frequently encountered disease, it seriously affected physical and mental health of humen, and it has brought a heavy burden to patients family and social. Recent studies have pointed out that T2DM closely associated with the occurrence and development of AD. Luchsinger investigated1262elderly people and the results demonstrate that1/3of dementia related to diabetes. Relevant epidemiological survey found that the risk of DM patients suffer from AD was2times of nondiabetic. Mechnisms about DM caused occurrence of AD related to abnormal insulin signal transduction, hypercholesterolemia and advanced glycation end products produced in non-enzymetic glycosylation. Advanced glycation end products (AGEs) are structurally diverse irreversible polymer to which the amino sugar and protein deposits through a series of complex non-enzymatic reaction (Maillard response). AGEs played an important part in diabetic retinopathy, arteriosclerosis and diabetic nephropathy and other target organ damage.There were accumulation of AGEs in senile plaque and neurofibrillary tangles in AD patients. Takeuchi and Maczurek’s research indicated that AGEs played an important part in cognitive dysfunction resulted from DM and AD, AGEs is one of the risk factors cause cognitive decline of elderly people. AGEs may promote protein crosslinking and accumulation, inhibit the functional proteins, stimulate the generation of reactive oxygen species (ROS) directly caused biological damage. AGEs can also bind to the receptors for AGEs (RAGE), further to play indirect toxic effects through a series of signal transduction pathways.Pathological characteristic of AD is the formation of senile plaques, neurofibrillary tangles and neuronal and synaptic loss in specific brain regions. An significant pathologic feature of AD is progressive and specific neurons loss, and apoptosis holds a prominent place in cells death. Apoptosis is programmed cell death, and it has been known that there are3pathways can result cell apoptosis, including death receptors pathway, mitochondria pathway and endoplasmic reticulum stress (ERS)-mediated apoptosis. Recent studies found that ERS-mediated apoptosis play an important role in occurrence of many diseases such as diabetes and AD et al. Our previous studies indicated AGEs participated in abnormal metabolism of APP and production of Ap, ER is the main place which produced Aβ1-42and play an important part in metabolism of APP. Dysfunction of protein folding in ER, destructed calcium balance and abnormal oxidation reaction both can trigger ERS. It is not very explicitly that whether AGEs lead to apoptosis.through triggering oxidative stress and ERS and whether it involved in occurrence and development of AD. SH-SY5Y cell is closely similar to normal nerve cell in morphology and physiological functions. Therefore, we design this subject and choose SH-SY5Y as research object. We use AGE-bovine serum albumin to treat SH-SY5Y cells, and respectively pretreat cells with RAGE-antibody, alpha lipoic acid (ALA) and diphenyleneiodonium (DPI), in order to explore whether AGEs cause cell apoptosis through triggering oxidative stress and ERS, reveal the possible mechanism of AGEs involved in the occurrence and development of AD.Objective To investigate the effect of AGEs on SH-SY5Y cells apoptosis and the role of oxidative stress and ERS in this process, further to explore the possible mechanism of AGEs cause to AD.Methods As object of this study, the cultured SH-SY5Y cells were treated with different concentrations of AGE-BSA for48h, or treated with AGE-BSA(200ug/ml) for different times, cells apoptosis detected by flow cytometry (FCM) to determine the best concentration and time of AGE-BSA.Divide SH-SY5Y cells into six groups randomly, the normal control group, the BSA group, the AGE-BSA group, the AGE-BSA+anti-RAGE-Ab group, the AGE-BSA+Alpha Lipoic acid(ALA) group, the AGE-BSA+Diphenyleneiodonium (DPI) group. To detect the cells apoptosis by FCM and Hoechst staining after treatment with AGE-BSA and other drugs for48h; The level of ROS evaluated by the2’,7’-dichlorofluorescein diacetate (DCFH-DA) method. The expression of GRP78、p-eIF2α、Caspase-12was analysed by western blotting and immunofluorescence after treatment with AGE-BSA for0-48hours. Then select the peak time of each protein, and use western blotting to estimate the expression of GRP78、p-eIF2α、Caspase-12in different groups.Results1. Cell apoptosis rate measured by FCM1.1Apoptosis rate of normal control group was(2.23±0.08)%, after treatment with50,100.200,300μg/mL AGE-BSA for48hours the rate rised to(2.98±0.67)%,(8.23±0.42)%,(16.8±1.27)%,(19.6±2.89)%, after treatment with200μg/mL AGE-BSA for24hours, the rate was (7.54±1.08)%, it was (10.75±1.19)%for36hours and it was (16.8±1.27)%for48hours. Therefore, AGE-BSA can induce apoptosis, moreover the growth of apoptosis rate was in a time and concentration-dependent manner.1.2After pretreatment with RAGE-Ab, ALA and DPI respectively, the apoptosis rates have declined to (5.18±0.16)%,(5.94±0.10)%,(7.26±1.22)%, the decline rate were69.1%、64.6%、56.8%, the differences were significant compared with AGE-BSA group(P<0.05).There was no significant difference between NC group and BSA group.2. Apoptosis nucleus were detected by Hoechst33258fluorescent staining.Cells of different groups stained with Hoechst33258were observed under fluorescence microscope, the nucleus appeared uniformly dispersed of blue fluorescence, and there are no pyknoticnucleus. There were bright blue apoptosis-like nucleus in AGE-BSA group cells, and nuclear fragmentation appears. Fluorescence intensity was significantly enhanced than the normal cells. The fluorescence intensity of RAGE-Ab group、ALA group and DPI group were lower and apoptotic cells were significantly reduced than AGE-BSA group.3. The level of ROS in SH-SY5Y was detected by DCFH-DA3.1There was a small amount of ROS in cells without any AGE-BSA, and after treatment with AGE-BSA for12h, we detected the level of ROS has increased significantly, it was4times what the Oh group. The level of ROS was increased constantly along with treatment time lasting, and the differences were significant (P<0.01).3.2The ROS level of AGE-BSA group was6.95times what the NC group was(P <0.01) and there was no significant difference between NC group and BSA group. The ROS level of RAGE-Ab group, ALA group and DPI group were significantly decreased than AGE-BSA group, the decline rate were56.2%.46.5%,54.3%respectively (P<0.01). The results indicated that AGEs promoted the generation of ROS via the pathway of AGEs-RAGE-NADPH oxidase.4. Expression of GRP78, p-eIF2α, Caspase-12protein detected by immunofluorescence and western blotting4.1Estimated by immunofluorescence and western blotting, treatment with AGEs for0,12,24,48hours, the increased levels of GRP78and p-eEF2α in a time-dependent manner, with peak at24h. The Caspase-12showed the highest level at48h.4.2The levels of GRP78and p-eIF2α were analyzed in the lysates treated with200μg/mL AGE-BSA for24h, the two proteins in AGE-BSA group were respectively3.56times and6.39times what the NC group was, and which could be significantly blocked by the pretreatments of RAGE-Ab, ALA or DPI. The decline rate of GRP78were respectively39.5%,14.1%,19.3%, and that of p-eIF2α were28%,41%,33%, the differences were significant (P<0.05or P<0.01). The level of Caspase-12was measured in the lysates treated with200 μg/mL ASA-BSA for48h and apoptotic initiator marker Caspase-12were similar between the control and BSA groups, however, the level of Caspase-12in AGE-BSA group increased significantly compared with the NC group and BSA group (P<0.01). After pretreatment with RAGE-Ab, ALA or DPI, the level of Caspase-12have fallen by35.9%.43.7%,50.5%respectively(P<0.01).Conclusion1. AGEs could promote the production of ROS through binding to RAGE, further to activate NADPH oxidase.2. AGEs could trigger ERS through stimulating the generation of ROS, and further to cause nerve cells apoptosis, it may be a mechanism of AGEs cause to AD.3. Blocking the binding of AGEs-RAGE, decreasing ROS by ALA or inhibiting NADPH oxidase activity can efficiently prevent oxidative stress and ERS, consequently reduced cell death. It may provide a new method for the treatment of AD.