Inhibitory Effect Of Hydrogen On The Neuronal Apoptosis Induced By Spinal Cord Ischemia-reperfusion Injury

Background and objective Patients with SCI (spinal cord injury) often leads to paralysis, and even cause death. Because there is currently no effective treatments, increasing of patients with spinal cord injury has become a huge economic burden in the world. According to the investigation in America, on the cost of treatment and nursing in patients with spinal cord injury was more than $7 billion a year, and the average age of the patients was 31.7 years old (15 to 25 as the peak of onset age). It also need long-term treatment and nursing care laterly. Therefore, the study of treatment on spinal cord injury has an important social effect.The pathological process of spinal cord injury include primary injury and secondary injury. The apoptosis caused by primary damage cannot be repaired. The secondary injury induced by the primary injury. Extracellular toxins, free radicals and inflammatory mediators caused by Ischemia reperfusion injury and inflammation which lead to nerve cell necrosis and apoptosis are the pathology of secondary spinal cord injury.The study found that hydrogen has a function to neutralize free radicals. Due to its electrically neutral and it is easy to penetrate the cell membrane, generating water after oxidation, and not induce metabolic disorders, Hydrogen can effectively improve the heart, brain, et al and other important organs of ischemia-reperfusion injury. The treatment of Hydrogen on spinal cord ischemia-reperfusion injury are rare. We researched the protective effect of Hydrogen and found that animals neural function improved significantly in experiment group compared with control group. The apoptotic cells were decreased obviously in experiment group. We thought that the hydrogen could suppress the generation of oxidation product and the results were consistent with the related studies.In addition to the function of the reduction of free radicals, Hydrogen may also inhibit neuronal apoptosis in other ways. Existing literature are not specifically research on the mechanism of hydrogen as a kind of effective treatment on spinal cord injury. As a potential trentment, the mechanism of inhibition on neuronal apoptosis research is necessary.Currently, studies on hydrogen against apoptosis are rare. Zhang found that hydr- ogen can increase the expression of SOD (superoxide dismutase) and decrease the expression of ROS (reactive oxygen species). In 2014, Zhao Y found that hydrogen-rich saline may decrease cell apoptosis and attenuate myocardial reperfusion injury through inhibiting ERS (endoplasmic reticulum stress). The mechanism may be associated with decreasing the expression of GRP78, caspase-12 and Bax and increasing the expression of Bcl-2 in myocardial tissue. Chen ZF found that H2S can increase the expression of SOD and reduce the expression of ROS by adjusting ERS such as anti-inflammatory and antioxidant. In 2016, Li CH found that hydrogen sulfide prevented OGD/R(oxygen and glucose deprivation/restoration)-induced apoptosis by suppressing the phosphorylation of p38 and secretion of IL-6 in PC12 cells. H2S ingredients are same as hydrogen. We speculate that hydrogen could inhibit the ERS to prevent neuronal apoptosis and the hydrogen may provide a new treatment for spinal cord ischemia-reperfusion injury.PC12 cells were cloned from pheochromocytoma of adrenal medulla in rat and have the same function as neuroendocrine cells. PC 12 cells are often used as the passage cells and used to study the physiological-biochemical fuctions of neurons in vitro. In 2015, Vavilis T found that OGD could induced apoptosis by regulating UPR (unfolder protein response) on endoplasmic reticulum in PC 12 cells. For researching the protective mechanism of hydrogen and simulating the pathophysiological process of spinal cord ischemia reperfusion injury, we used OGSD/R (oxygen-glucose-serum deprivation/ restoration) model to induce PC 12 cells apoptosis. In our study, we established the stable OGSD/R model and thus made sure the reperfusion time window to provide the theory and experimental evidence for hydrogen. And to verify whether hydrogen can inhibit the PC 12 cells apoptosis through apoptosis pathway which related to ERS.Methods1. Subculture and identification of PC 12 cellsPCI2 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. After recovery, adherence and multiplication, the third generation of cells were used to experiment. The morphology of PC12 cells were photographed by invert microscope.2. GroupingIn Part 1, PC 12 cells were divided into normal group (no disposal) and experimental group (OGSD 12h/R 0-6h).In Part 2, PC 12 cells were divided into normal group (no disposal), control group (OGSD/R) and H2 group (pretreatment with hydrogen at the time of restoration).3. OGSD/R modelPC 12 cells were put into low-glucose DEME without serum. Then put the culture plate into culture bottle with three gas (94% N2+5% CO2,1% O2). The PC 12 cells were immigrated into high-glucose DEME after 12h. The apoptosis and related protein were detected in every group.4. Hydrogen-culture medium preparationHydrogen was dissolved into culture medium using high pressure ventilation device (0.6mmol/L). The hydrogen-culture medium was stored in aluminium containers.To maintain its saturation concentration, Hydrogen was added every 3 days and restored in refrigerator(4℃).5. Immunofluorescence assayIn Part 1, DAPI staining was performed to test the cells apoptosis at OGSD 12h/R 0-6h and take photos by microscopic to observe the cells apoptosis.In Part 2, DAPI staining was performed to test the cells apoptosis at OGSD 12h/R 1h in every group and to calculate the relative rate of apoptosis.6. Cell viability assayThe relative viability of PC12 cells were tested with CCK8 assay kit at every time.7. Flow cytometric methodThe relative rate of apoptosis was detected and compared through flow cytometry.8. ROS level detectionH2DCF-DA probe was applicated to detect ROS level in each group and collected the relevant data by flow cytometry.9. Apoptosis proteins assayThe expression of PERK-eEF2a-ATF4, caspase-3, caspase-12 and CHOP/GADD 153 were detected with western-blot.Results1. PC 12 cells grew neurite like the sympathetic neurons. The size of cells increased, which connected with each other into a mesh.2. In order to observe the change of apoptosis, DAPI staining was performed to test the cont group and OGSD 12h/R 0-6h group. We found that there were almost no apoptosis in control group (cont) and the apoptosis cells were increased in OGSD. The apoptosis and injury were further increased in OGSD/R. And the most serious injury at OGSD 12h/R 1h. which same to the cck8 assay and flow cytometric assay.3. The expression of caspase-3 and caspase-12 were lower in cont group and then increased in OGSD. The expression of caspase-3 and caspase-12 were further increased in OGSD/R. The expression of caspase-12 were reached the peak at OGSD 12h/R 1h and the caspase-3 were reached the peak at OGSD 12h/R 2h. The tendency of two apoptosis protein were consistent.4. Treament with hydrogen inhibited cell apoptosis that induced by OGSD/R. DAPI staining and CCK8 assay results reminder that the apoptosis in experiment group with hydrogen were lower than that in control group. We used western-blot to detect the expression of cleaved-caspase-3. caspase-12, CHOP/GADD153 and p-PERK-p-eIF2a-ATF4. The result showed that the expression of cleaved-caspase-3, caspase-12, CHOP/GADD153 and p-PERK-p-eIF2a-ATF4 were lower in normal group than that in control group and H2 group. The expression of cleaved-caspase-3, caspase-12, CHOP/GADD153 and p-PERK-p-eIF2a-ATF4 were lower in H2 group than that in control group. There were significant different between control group and H2 group.ConclusionsOGSD could induce the apoptosis of PCl2 cells and further injury followed by restoration and the injury showed time-dependent. Pretreatment with Hydrogen suppressed the expression of apoptosis protein associated with ERS and have the protective effect on neurons which damaged by spinal cord ischemia-reperfusion injury.