Influences Of Hypoxia On Autophagic Cell Death In Microglia Induced By Hypoxia

Spinal cord injury(SCI) in a common injury in clinic in the department of orthopeadics,which is usually caused by spinal fractures. As phagocytic cells of central nervous system(CNS), microglia have been proposed as primary component in the innate immune response and play an important role in maintaining CNS homeostasis. Microglia can be activated during various phases of normal tissue repair and participate in the various pathological conditions in CNS. Anoxemia is a key problem accounting for the tissue destruction following Spinal cord injury(SCI). Hypoxia-inducible factor 1-alpha(HIF-1α)is thought to be able to protect hypoxic cells from apoptosis or necrosis under ischemic and anoxic conditions. However, numerous studies have shown that hypoxia upregulates HIF-1α expression leading to cell death of microglia. Our study investigated the variation of HIF-1α and the exact mechanism of autophagic cell death through HIF-1α in the microglia induced by hypoxia.In the current study, we showed that hypoxia induced HIF-1α expression and autophagy cell death in microglia. Elevated autophagy reduced cell death at the initial stage via restraining the functions of autophagy-related genes(LC3II, Beclin-1) and modulating inflammatory cytokines expression(TNF-α, IL-1β). However, overexpression of HIF-1α induced cell death and autophagy cell death in microglia. Moreover, the influences of the HIF-1α inhibitors(2ME2) and HIF-1α siRNA on the microglia death and autophagy in vitro have also been researched.Part I The cultivation of spinal microglia and the establishment of hypoxia stress modelThe spinal microglia was isolated by Percoll density gradient centrifugation as described previously. Lumbar enlargements spinal cord of 1-day old postnatal mice were digested with 0.1% trypsin. Spinal cords were ground briefly in 4 mL ice-cold Hanks,balanced salt solution and filtered through a nylon mesh to remove astrocytes and undigested tissue. The cells were seeded into a flasks coated with poly-L-lysine and fed with DMEM(Hyclone, USA) containing 10% fetal bovine serum FBS(Gibco, USA).Culture media were refreshed twice per week for 2 weeks. Microglia were detached bygentle shaking with 120 rpm for 3 h, then resuspended in fresh DMEM supplemented with10% FBS and plated at a final density of 5×105 cells/mL on a 6-well culture plate after centrifugation at 800×g for 10 min. The cell purity was determined by immunohistochemical staining using microglia specific antibody CD11 b. The microglia cultures used were ? 95% pure.For hypoxia treatment, the culture medium was changed to fresh medium for routine culture, and carried out appropriate treatment before the cells were exposed to hypoxia in a chamber, filled with a gas mixture of 1% O2, 5% CO2, 94% N2 for the scheduled time.In order to elucidate the roles of hypoxia in microglia cells death, the primary microglia cells were treated with the indicated time. Cell viability was detected by CCK-8assay, which is considered an effective method for detecting cell death. There were time-dependent decreases in relative cell viability after hypoxia treatment. Similar results of cell mortality were detected in flow cytometryPart II Expression of inflammatory factors and hypoxia inducible factor in spinal cord microglia cells under hypoxic stressHypoxic stimulus signal could be detected by Microglia.mictoglia can activate the corresponding signal pathway under hypoxia environment. Protein expression levels of HIF-1α with microglia cells by western blot. Microglia cells were harvested at the indicated time, washed three times with ice-cold PBS, and total protein and nuclear protein were prepared with RIPA buffer(Beyotime, China), After protein concentrations determined by the BCA protein assay, equal amounts of protein were separated on 10 %SDS-polyacrylamide minigels and transferred to nitrocellulose membranes. After blocking the membranes using 5% skim milk for 60 min, we incubated the membranes with primary rabbit anti-HIF-1α Ab at a concentration of 1:200 overnight at 4 °C. Subsequently, the membranes were washed three times for 10 min each with TBST and then incubated with the corresponding secondary antibodies for 2 h at room temperature. Blots were again washed three times for 10 min each in TBST and scanned with the Bio-RAD Western blot system(Bio-RAD, USA). Background intensity was subtracted from each sample and then normalized to the Actin loading control.Quantitative RT-PCR was performed to measure mRNA levels of HIF-1α, Total RNA was extracted using Trizol reagent(Ambion, USA),and the reverse-transcription reactions were performed using an TaqMan Micro RNA Reverse Transcription Kit(Takara, Japan). Real-time PCR was performed using a standard SYBR Green PCR kit(Takara, Japan) in a 7900 Real-time PCR(Applied Biosystems, CA,USA) according to the instructions from the respective manufacturer. Supernatants were collected from cell cultures at different time points after being induced with various stimuli.Secreted cytokines TNF-α, and IL-1β in the supernatants were measured by ELISA kits as recommended according to the manufacturer’s instruction(eBioscience, California, USA).Absorbance at 405 nm was measured on microplate reader(Bio-RAD iMark Microplate Reader). A 405 was converted to protein concentrations(pg/mL) calculated from the standard curve.We found, under hypoxia, HIF-1α displays a significantly high level of expression and heterodimerizes with HIF-1β to form HIF-1 after translocating into the nucleus. Hypoxia significantly increased HIF-1α mRNA. The highest expression HIF-1α appeared at 6hs,and similar results were observed in western blot assay.These data indicate that hypoxia induces expression of HIF-1α in microglia. This effect can be observed obviously in groups induced by hypoxia after 6hs especially.The results of ELISA kits showed that hypoxia markedly increased the protein levels of IL-1β and TNF-α. IL-1β can reach the highest concentration over short periods after hypoxia-induced microglia cells.Part III Study on the expression of autophagy-related genes(LC3II, Beclin-1)in spinal cord microglia cells under hypoxic stressAccumulating reports suggested that hypoxia could induce autophagy in the microglia,which is associated with SCI. Beclin-1 and light-chain-3(LC-3) are characteristic markers protein of autophagy. The expression of LC3-II and Beclin-1 were investigated, which is considered an accurate indicator of autophagy in cells.Protein expression levels of Beclin-1,LC3-II with microglia cells by western blot. Ultrastructural features of autophagy in microglia cells were studied by Transmission electron microscopy(TEM). Briefly, the microglial cells were harvested after hypoxia treatment, washed in PBS, fixed with 1% of glutaraldehyde in 0.1 M cacodylate buffer for 2 h, postfixed with 1% OsO4 for 1.5 hrs in the same buffer. The samples were then washed again, dehydrated, and embedded in 812 embedding medium(SPI, USA). Thin sections were cut with a diamond knife on a Ultra45°(Daitome, Japan) and examined with a TECNAI G2 20 TWIN(FEI, USA) transmission electron microscope. Microglia cells were seeded on coverslip in 24-well plates and treatmented with hypoxia and other molecules. Immunohistochemical staining of LC3-II was performed using the coverslip to investigate alterations of the expression of LC3-II after hypoxia. Briefly, the cells were fixed with 4 % paraformaldehyde, permeabilized with0.3 % Triton X-100, and blocked with 5 % goat serum for 40 min, and incubated with rabbit anti- LC3-II antibodies(1:100) diluted in PBS overnight at 4 °C. Subsequently, cells were stained with the TRITC-conjugated anti-rabbit IgG secondary antibody(1:100) for 2h at room temperature, secondary antibody(1:500) for 1 h at room temperature. The cells were counterstained with DAPI for 5 min after being rinsed with PBS. The images were captured by fluorescence microscope(Olympus, Japan).We found, hypoxia markedly increased the protein levels of LC3-II after hypoxia-induced microglia cells.The protein level of LC3-II and Beclin-1 reached their peak 3hs in cells treated with hypoxia compared to control cells. Ultrastructural changes in hypoxia-treated microglia. Autophagosomes could be observed in hypoxia-treated microglia by using TEM. The protein accumulation of LC3-II was significantly increased in the model group by LC3-II immunofluorescenc.Part IV Effects of Hypoxia-Inducible Factor 1α on Autophagic Cell Death in Microglia Induced by HypoxiaTo determine whether hypoxia-induced autophagy promotes microglia survival or death, and expound the relative mechanism of action, we exposed the microglia to HIF-1αinhibitors 2ME2 and silenced HIF-1α with siRNAs targeting HIF-1α(100 nM each) for 6 h.The protein and mRNA levels of the target was evaluated by western blot and qRT-PCR assays. Simultaneously, we evaluated the subsequent cell death and inflammation induced by hypoxia treatment with the pretreatment of HIF-1α inhibitors, HIF-1α siRNAs or not.We exposed the cells to the inhibitors 2ME2. Microglia cells were seeded in 6-well plates and pretreated with 3 μM 2ME2 for 6 hours before treatment with hypoxia.Supernatants from cell cultures were collected and assayed for cytokines secretion, and cell pellets were used for RNA isolation and qRT-PCR analysis. For transfection with small interfering RNA(siR-NA), cells were plated in 6-well plates at 5×105 cells/well. Plated cells were grown in DMEM with 10% FBS overnight, and then transfected with 50 nM HIF-1α siRNAs(Ribobio, Guangzhou, China) using the transfection reagent Lipofectamine 2000(Invitrogen, California, USA) according to the manufacturer’s protocol, 24 h prior to treatment with drugs.The results showed that HIF-1α inhibitors and HIF-1α silencing could successfully decrease hypoxia-induced HIF-1α expression. The expression of LC3-II and Beclin-1 were significantly repressed in microglia cells receiving transfection with siRNAs of HIF-1α.Further studies showed that HIF-1α being inhibited or HIF-1α knockdown decreasedhypoxia-induced cell viability respectively, and this effect can be associated with the changes in expression of inflammatory cytokines in microglia cells. Production of IL-1β,TNF-α, was significantly increased in microglia cells receiving the inhibitors 2ME2 intervention and transfection with siRNAs of HIF-1α. These data suggested that HIF-1αdecreased the cell death through controlling the overexpression of inflammatory cytokines in microglia cells. Attributed the results, HIF-1α can mediate autophagic cell death in microglia thouth regulation of autophagy-related gene expression and cytokine release.In conclusion, our research data indicate that suitable expression of HIF-1α could ameliorate autophagic cell death of microglia associated with hypoxia, and provide novel therapeutic interventions for SCI associated with microglia activation.