The Expressions Of Nrf2/ARE Pathway In PC12 Cells Treated With Low-Dose LPS And Its Protective Effects To Neurons In Spinal Cord Injury

Spinal cord injury (SCI) is a kind of harmful illness to human health and its incidence increased substantially with the development of social economy. The reasons for function deficit in spinal cord injury are mainly due to the contact information disruptions between spinal cord neurons'axons and their targets, and the neurons'apoptosis or pathological death. The key method to improve SCI patient outcomes is protecting neurons, promoting the axonal regeneration. The secondary injury becomes the focus of research due to its reversible and regulatory characteristics. However, the secondary injury mechanisms are various and complicated. At present, the oxidative stress mechanism becomes a hot research point for its clear mechanism and the link with others.Inflammation response exists in the central nervous system injury, and it's reported that the inflammatory response in spinal cord injury is more serious than traumatic brain injury. On the one hand the inflammatory response could aggravate the injuries in central nervous systems, on the other hand it could promote the damage repair. Nuclear factor E2 p45 related factor 2 (Nrf2) is a new transcription factor. Under varied stimulation, Nrf2 could combine with antioxidant response element (ARE) and induce a series of protective molecules to express. Recent studies showed that Nrf2 / ARE pathway played an important role in controlling inflammation immune reaction, and might be a critical regulatory factor in natural immune response and body survival. Combined with clinical practice, we hypothesized that the inflammatory response in spinal cord injury might activate Nrf2 / ARE pathway, and moderate inflammatory response protected neurons improving the prognosis of function, in which Nrf2 /ARE pathway might play a major role.In this research, we stimulated PC12 cells with low-dose LPS to establish inflammation-stimulated cell model of the neurons in vitro. We investigated the effects of low-dose LPS on Nrf2 / ARE pathway in PC12 cells and the protective functions for PC12 cells in reperfusion injury. Then we constructed Nrf2 recombinant adenovirus vector to examine the effects of Nrf2 overexpression on cell cycle, anti-inflammation, anti-necrosis/anti-apoptosis for PC12 cells in vitro. We constructed rat model of spinal cord injury in vivo to investigate the effects on neurons'Nrf2/ARE pathway, nerve cell apoptosis, and the prognostic function in rats after transfected the Nrf2 recombinant adenovirus. This study included three parts: PART ONE: THE EFFECT ON NRF2/ARE PATHWAY IN PC12 CELLS TREATED WITH LOW-DOSE LPS AND ITS PROTECTIVE MECHANISMS IN REPERFUSION INJURYObjective To explore the influence of low-dose LPS on Nrf2 and its downstream regulatory factors HO - 1, NQO1,γ- GCS in PC12 cells, and the protective role of Nrf2 /ARE in reperfusion injury for PC12 cells preconditioned with low-dose LPS.Methods The safety doses of LPS were established by MTT method. With Real-time PCR, Western-blot, immune fluorescence, the expressions of Nrf2 mRNA, total Nrf2 protein, Nrf2 distribution in cytoplasm&nucleus were detected in PC12 cells treated with lose-dose LPS. The expressions of HO-1, NQO1,γ-GCS were detected by western-blot, too. The reperfusion injury model of PC12 cells was established. The number of apoptotic cells pre-treated with low-dose LPS during ischemia was assessed with Hoechst33258 dyeing at 24h after reperfusion. ROS was tested with Flow Cytometry at the end of ischemia and 6h after reperfusion. LDH release rate was detected by enzymatic methed at 24h after reperfusion. The expressions of Nrf2 in nucleus and HO-1, NQO1,γ- GCS in whole cell were tested at the end of ischemia.Results No more than 0.1μg/μl LPS were safe for PC12 cells(P> 0.05 ). The expressions of Nrf2 mRNA, total Nrf2 protein increased at 6h (P< 0.05 ), reaching peak at 24h(P< 0.01 ). Nrf2 protein reduced in cytoplasm(P< 0.01) meanwhile raised (P< 0.05) in nucleus at 1h. The immune-fluorescence showed the Nrf2 expression more clearly at 3h than 1h. Intracellular HO-1, NQO1,γ-GCS expressions increased and reached peak at 24h accordingly. In reperfusion model, the number of apoptotic cells significantly reduced at 24h after reperfusion, especially in the 0.05μg/μl group(vs LPS-free P< 0.05) . The release rate of LDH reduced accordingly. At the end of ischemia, ROS expression in PC12 cells increased with LPS dosage up, and at 6h after reperfusion ROS first decreased then increased with LPS dosage up, in which the 0.025μg/μl group was most obviously decreased(vs LPS-free P< 0.05 ). The expressions of Nrf2 in nucleus and HO-1, NQO1,γ-GCS in whole cells at the end of ischemia increased with LPS dosage up, among which the group of 0.05μg/μl LPS was the most obvious.Conclusions Low-dose LPS could promote Nrf2 transcription, translation, and the transfer from cytoplasm to nucleus in PC12 cells,to activate Nrf2 / ARE pathway. Pre-treatment with Low-dose LPS protected PC12 cells from reperfusion injury, in which Nrf2/ARE might play a major role.PART TWO: CONSTRUCTION OF NRF2 RECOMBINANT ADENOVIRUS AND THE INFLUENCE ON PC12 CELLSObjective To construct Nrf2 recombinant adenovirus vector, and investigate its influence on PC12 cells. Methods The Nrf2 recombinant adenovirus vectors were re-combinated with adenovirus shuttle plasmids pAV-MCMV-GFP-Nrf2 and adenovirus auxiliary packaging plasmids pBHG loxΔE1, 3 Cre in HEK293 cells. RT - PCR was used to identify the purpose gene expression. The recombinant adenovirus vectors were aplificated, purificated and qualificated. The expressions of Nrf2, HO-1, NQO1,γ-GCS were detected in PC12 cells transfected by Nrf2 recombinant adenovirus. The effects of Nrf2 recombinant adenovirus on proliferation and resistance to LPS were observed in PC12 cells under the microscope. The cell cycle of PC12 cells with Nrf2 overexpression was detected by Flow Cytometry. The expressions of IL-1β,Caspase-3 were detected by Western-blot in PC12 cells treated with LPS.Results Nrf2 recombinant adenovirus vectors have been successfully recombinated. The result of PCR showed that Nrf2 recombinant adenovirus had the same purpose gene with shuttle plasmid pAV-MCMV- GFP-Nrf2. High-titer virus liquid could be gained by amplification and purification, and the titer was 7.9 x 1010pfu/ml. Nrf2 recombinant adenovirus could promote expressions of Nrf2 (cytoplasm/nucleus), HO - 1, NQO1,γ- GCS in PC12 cells, inhibit PC12 cell proliferation, resist the toxic injury of LPS, and reduce IL - 1β, Caspase - 3 expressions.Conclusions Nrf2 recombinant adenovirus has been successfully recombinated. Nrf2 recombinant adenovirus could increase the expression of Nrf2/ARE, inhibit PC12 cells proliferation, and enhance PC12 cell abilities of anti-apoptotic/anti-necrosis and anti-inflammation.PART THREE: THE PROTECTIVE EFFECTS OF NRF2 ON NEURONS IN RATS WITH SPINAL CORD INJURYObjective To detect the protective effects of Nrf2 overexpression on neurons in spinal cord injury of rats.Methods 160 Sprague-Dawley rats were randomly divided into 4 groups: Group A (sham group), Group B (SCI group), Group C (Ad-Nrf2 group), Group D (Ad-GFP group). The rats'movement function was observed after operation, and BBB scores were recorded. The spinal cord specimens were assessed. The frozen longitudinal spinal cord slices were prepared to observe the fluorescence intensity and distribution under the fluorescence microscope. The morphological changes, edema, inflammatory response at the injury part of spinal cord tissues were investigated in transverse slices staining with HE dyeing. The expressions of Nrf2, HO-1, NQO1,γ- GCS and GAP-43 were detected by immunohistochemical methods in tissue transverse slices. The nerve cells'apoptosis was assessed with TUNEL method in tissue transverse slices.Results The movement functions of Group C were obviously improved after postoperative 2nd week(vs B, D P<0.05). At 4th week,the spinal cord specimens of Group C were kept relatively well. The strong green fluorescence in Group C could be observed at postoperative 1st day in frozen slices under the fluorescence microscope, which was mainly distributed in the injection parts. At postoperative 3rd day, the green fluorescent intensity was the strongest, even in the injury center. Then green fluorescence weakened gradually. AT postoperative 4th week, a few green fluorescent expressions could still be seen. HE showed the inflammatory response was lighter at postoperative 2nd -4th week in Group C,the neurons had been more reserved. The positive cell count of Nrf2,NQO1, HO-1,γ- GCS increased obviously in Group C. The expression peak delayed to postoperative 3rd day (vs B, D P< 0.01) . The GAP-43 positive cells increased, which expressed mostly at postoperative 7th day (vs B, D P< 0.01) . TUNEL results showed the apoptotic cells were almost in the injury area. At postoperative 7th day, the number reached peak, and apoptotic cells were obviously reduced in Group C (vs B, D P< 0.01) .Conclusions Nrf2 recombinant adenoviruses injecting locally to the injury parts of spinal cord could promoted Nrf2 / ARE patheway expression in rat spinal cords, reduced local apoptosis and inflammation, protected neurons, promoted axonal regeneration and improved rats'function recovery.