| BACKGROUNDSepsis is defined as the presence (probable or documented) of infection together with systemic manifestations of infection. Sepsis associated encephalopathy (SAE) is a diffuse brain dysfunction that occurs secondary to sepsis without overt center nervous system (CNS) infection, structural abnormality or other types of encephalopathy (for example, hepatic or renal or pulmonary encephalopathy), as detected by clinical or standard laboratory tests. SAE is the most common form of encephalopathy in the intensive care units and might appear before other systemic features of sepsis are obvious. SAE has a spectrum of degrees of severity, ranging from delirium to deep coma and causes increased morbidity and mortality but has limited therapeutic options. Furthermore, sepsis induced brain dysfunction could influence the function of autonomic nervous system and neuroendocrine system. There is no specific treatment for SAE and the outcome of patients relies on prompt and appropriate treatment of sepsis as a whole at this moment.Although the molecular mechanisms underlying the pathogenesis of SAE is not clear, mitochondrial dysfunction was shown to be involved in this process. While mitochondrion is the site of electron transport chain and oxidative phosphorylation system providing cellular energy in the form of adenosine triphosphate (ATP), it is also linked to other many important cellular functions such as pyruvate and fatty acid oxidation, nitrogen metabolism, heme biosynthesis etc. Furthermore, mitochondria are also involved in cell signal transduction, cell apoptosis, calcium metabolism and generation of important biological active molecules such as oxygen free radicals. Normal mitochondrial function is essential in maintaining cell survival and normal cell function, so mitochondrial dysfunction maybe closely related with many human diseases. Experimental studies have demonstrated that sepsis could cause mitochondrial uncoupling, cytochromes depletion, decreased mitochondrial membrane potential and cytochrome C oxidase activity in mice brain. Another experimental study has found increased apoptosis of neuron cells in the septic rats’ hippocampus, choroid and other areas with higher permeability of blood-brain barrier. All these evidences indicate that the functions of mitochondria are impaired in the septic brain.Mitochondrial biogenesis are controlled by both nuclear and mitochondrial transcriptional regulators. Mitochondrial biogenesis involves coordination of expression, import, and assembly of mitochondrial proteins from nuclear and mitochondrial genomes and regulation of mitochondrial content and morphology. Mitochondrial biogenesis plays a pivotal role in maintaining mitochondrial function and mass, to meet the physiological needs in eukaryotic cells. Mitochondrial biogenesis in the brain is also regulated by nuclear and mitochondrial transcriptional regulators, including nuclear respiratory factor1(NRF-1) and nuclear respiratory factor2(NRF-2, has two subunits, i.e. NRF-2a and NRF-2p) which control expression of nuclear genes encoding mitochondrial proteins; and mitochondrial transcription factor A (TEAM) which drives transcription and replication of mitochondrial DNA (mtDNA). The expression of NRF-1, NRF-2and TFAM are regulated by peroxisome proliferator activator receptor gamma coactivator-1α (PGC-la), which is the master regulator of the mitochondrial biogenesis.Enhanced mitochondrial biogenesis could maintain mitochondrial function and cellular energy state, thus contributing to tissue and organ repair during diseases. Conversely, it would accelerate the deterioration of mitochondrial function. Recent studies have shown that mitochondrial biogenesis is altered in a number of brain diseases such as Alzheimer’s disease, Huntington’s disease and chronic seizures, which mitochondrial dysfunction is one of their pathogenesis. To date it is not clear how mitochondrial biogenesis changes in septic brain.As the largest number of macroglial cells in the mammalian central nervous system, astrocytes participate in the normal physiological activities and pathological mechanisms in the central nervous system through their complex biological functions. The findings from our previous study indicate that in a rat model of sepsis, the astrocytes mitochondria damaged after a nonlethal intraperitoneal injection of lipopolysaccharide (LPS) is restored to nomal after24hours. We investigated the changes of astrocytes mitochondrial biogenesis in sepsis and we assume that astrocytes mitochondrial biogenesis is increased to meet the high-energy demand and to restore the mitochondrial structure.OBJECTIVEWe designed the study to explore the changes of astrocytes mitochondrial biogenesis under the experimental sepsis condition. We try to provide experimental evidence for further pathophysiological study of SAE. METHODS1. Primary cultures of cerebral cortical astrocytes were prepared from1-day-old newborn Sprague Dawley (SD) rat brains. The purity of the cultures was determined by immunocytochemistry using cell-type specific antibodies, i.e., anti-glial fibrillary acidic protein (anti-GFAP) for astrocytes and anti-ionized calcium binding adapter molecule1(anti-ba-1) for microglia.2. The primary cultures of rats’cerebral cortical astrocytes were treated with culture medium containing lipopolysaccharide (LPS,50ng/ml, from Escherichia coli0111:B4) and interferon-y (IFN-y,200U/ml) to mimic the effects of septic insult on astrocytes in vitro according to previous study. Assay of tumor necrosis factor-a (TNF-a), interleukin-6(IL-6) and nitric oxide (NO) levels in the culture medium and determination of the reactive oxygen species (ROS) content in the astrocytes were used to confirm the process of sepsis.3. Grouping:there were4groups in the experiment, i.e.0hour group (control group),6hours group (sepsis group, treated with50ng/ml LPS and200U/ml IFN-y),12hours group (sepsis group, treated with50ng/ml LPS and200U/ml IFN-y) and24hours group (sepsis group, treated with50ng/ml LPS and200U/ml IFN-y).4. Evaluation of the rats’ cerebral cortical astrocytes mitochondrial ultrastructure and function in each group.4.1Production of rats’ cerebral cortical astrocytes ultrathin sections and observation of the ultrathin sections through transmission electron microscope.4.2A grading scale of0-4depending on the degree of mitochondrial morphologic damage was as follows:grade0, normal mitochondria; grade1, early swelling mitochondria as manifested by separation of cristae and clearing of matrix density; grade2, more marked swelling than grade1; grade3, massive swelling with architectural disruption; grade4, findings in grade3plus rupture on inner and outer mitochondrial membranes.4.3Intercellular adenosine triphosphate (ATP) measurements5. Determination of the expression of rats’ cerebral cortical astrocytes mitochondrial biogenesis regulators5.1Evaluation of the mRNA expression of rats’ cerebral cortical astrocytes mitochondrial biogenesis regulators in each group by real-time polymerase chain reaction (RT-PCR).5.2Evaluation of the protein expression of rats’ cerebral cortical astrocytes mitochondrial biogenesis regulators in each group by western blot.5.3Evaluation of the nuclear protein expression of PGC-la in each group by western blot.6. Evaluation of the TFAM-mitochondrial DNA (TFAM-mtDNA) binding activity in each group by electrophoretic mobility shift assay (EMSA).7. Evaluation of rats’cerebral cortical astrocytes mitochondrial DNA relative content in each group by RT-PCR.8. Evaluation of rats’ cerebral cortical astrocytes mitochondria volume density in each group by "point count grids".9. Evaluation of protein expression of mitochondrial DNA-encoded proteins----cytochrome C oxidase1(C0X1) and cytochrome C oxidase2(COX2) in each group by western blot.10. Evaluation of protein expression of mitochondrial fusion and fission proteins----optic atrophy1gene protein (OPA1) and dynamin-related protein1(DRP1) in each group by western blot.11. Statistical analysis:results are presented as mean±standard deviation (x±s) with multiple replications in each experiment. Analysis of variance, one-way ANOVA or Welch test was used. The Least-significant Difference (LSD) or Dunnett’s T3test was used to evaluate differences between means, with a P-value less than0.05being considered significant.RESULTS1. More than95%of the primary cultured cells expressed abundant GFAP conformed to be astrocytes with broad cytoplasmic veils and stellate processes. Rare (<5%) cells showed iba-1positive representing microglia.2. After exposing to the LPS and IFN-y, the levels of TNF-a and IL-6in the cultured medium of3hours (186.79±40.28pg/ml;54.55±4.08pg/ml),6hours (872.93±82.47pg/ml;49.55±3.72pg/ml),12hours (705.30±124.95pg/ml;53.77±3.87pg/ml) and24hours groups (810.93±73.43pg/ml;48.51±2.93pg/ml) were significantly increased as compared with0hour group (17.59±5.77pg/ml;35.801.87pg/ml; P<0.05). The expression of intracellular ROS and NO in the culture medium of0hour group (11.44±1.50μM;2.42±0.28μM) were both very low, whereas their levels were significantly increased following3hours (16.53±3.02μM;3.31±0.46μM; P<0.05) exposure to LPS and IFN-y, and kept rising to24hours (39.40±2.16μM;13.39±0.92μM; P<0.05).3. The ultrastructure and function of rats’ cerebral cortical astrocytes mitochondria3.1The proportion of mild injured mitochondria (grade1) in the6hours group (2.97±0.92) were significantly higher than other groups (P<0.05), which inflected the mild and reversible sepsis induced injury of mitochondrial ultrastructure in the6hours group. There were no significant differences among the proportions of normal and moderate injured mitochondria (grade0and grade2) in the total mitochondria that detected among the4groups. We did not find severely injured mitochondria (grade4and5) in the study. 3.2The intracelllular ATP levels of6hours(1.48±0.05nmol/mg protein),12hours(1.80±0.12nmol/mg protein) and24hours(1.79±0.14nmol/mg protein) groups were dramatically increased as compared with0hour group(0.93士0.15nmol/mg protein;P<0.05).4.The expression of rats’ cerebral cortical astrocytes mitochondrial biogenesis regulators4.1The mRNA expressions of PGC-1α(1.77±0.06;1.91±0.04),NRF-1(1.68±0.21;2.77±0.63),NRF-2α(2.33±0.09;2.52±0.25),NRF-2p(1.41±0.18;1.72±0.11)and TFAM(1.54±0.15;1.58±0.10)in rats’ cerebral cortical astrocytes of6hours and12hours groups were significantly increased as compared with O hour group(0.86±0.10;0.96±0.09;0.93±0.09;0.92±0.09;0.91±0.12;P<0.05).But to the24hours group,only the TFAM mRNA expression(1.20±0.19)was significantly elevated as compared with0hour group(P<0.05).4.2The protein expressions of PGC-1α(0.91±0.10;0.92±0.16),NRF-1(1.00±0.23;0.99±0.31),NRF-2α(1.34±0.03;1.41±0.20),NRF-2β(0.89±0.04;1.00±0.04) and TFAM(1.84±0.19;1.78±0.08) in rats’ cerebral cortical astrocytes of6hours and12hours groups were significantly increased as compared with0hour group(0.52±0.11;0.40±0.13;1.06±0.08;0.70±O.14;0.76±0.12；P<0.05).But to the24hours group,only the TFAM protein expression(1.35±0.23)was significantly elevated as compared with O hour group(P<0.05).4.3The protein levels of PGC-1αin the nucleus of6hours(0.63±0.05) and12hours groups(0.60±0.03) were also elevated significantly as compared with0hour group(0.42±0.06;P<0.05), and returned to normal level in24hours group (0.40±0.04).5.The specificity of the shited band was confirmed by anti-body super shift. The binding activities of TFAM to mtDNA of6hours,12hours and24hours groups were not obviously changed as compared with0hour group.6. The rats’ cerebral cortical astrocytes mitochondrial DNA relative contents of12hours (1.25±0.17) and24hours groups (1.33±0.24) were increased about40%and50%separately as compared with0hour group (0.90±0.15; P<0.05).7. The rats’ cerebral cortical astrocytes mitochondrial volume density was dramatically increased in24hours groups (0.09±0.00) as compared with0hour group (0.06±0.01; P<0.05).8. The protein levels of COX1(0.86±0.06;1.09±0.14) and COX2(1.26±0.17;1.41±0.16) of6hours and12hours groups were elevated significantly as compared with0hour group (0.51±0.04;0.73±0.09; P<0.05), and returned to normal level in24hours group (0.58±0.06;0.83±0.07).9. The protein levels of OPA1(1.21±0.17;1.34±0.06) and DRP1(1.04±0.05;1.05±0.05) of6hours and12hours groups were elevated significantly as compared with0hour group (0.66±0.06;0.58±0.04; P<0.05), and returned to normal level in24hours group (0.73±0.05;0.65±0.05).CONCLUSIONS1. LPS and IFN-y co-cultured with rats’ cerebral cortical astrocytes could induce septic insult (uncontrolled inflammation and oxidative stress) on astrocytes.2. Under the experimental septic conditions, the rats’ cerebral cortical astrocytes mitochondrial ultrastructures are mild injured, which are manifested as early swelling, the separation of cristae and the clearing of matrix density3. Under the experimental septic conditions, mitochondrial biogenesis of rats’ cerebral cortical astrocytes is aroused to meet the high-energy demand and to promote mitochondria recovery.4. Under the experimental septic conditions, the expressions of rats’ cerebral cortical astrocytes mitochondrial fusion and fission proteins are increased to meet the aroused mitochondrial biogenesis and to ensure proper organization of the mitochondrial network during biogenesis. |
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