Study On The Effects Of Parecoxib Reducing Oxidative Stress Injury Induced By Hydrogen Peroxide In Rat Astrocytes And Its Mechanisms

Postoperative cognitive dysfunction (POCD) that occurred within several days after surgery and anesthesia of the consciousness, cognition, orientation, thinking, memory and sleep disorder, often occurs in the elderly. It can lead to higher mortality, delayed postoperative recovery, increased complications, prolonged hospitalization days and the increases of medical expenses, even influence the quality of life after patients discharge. The exact cause is still not very clear and this brings some difficulties in clinical prevention and treatment. Existing researches indicate that the occurrence of POCD is related to a variety of factors such as operation factors, age factors, narcotic drugs, pre-existing cerebral, cardiac, vascular disease, low education level and the postoperative complications et al. In addition, the oxidative stress in the brain, particularly in the hippocampus may be involved in the pathogenesis of POCD.Oxidative stress is thought to be involved in the pathologic state of acute brain cell damage process and neuropathology changes of many degenerative diseases such as Alzheimer’s disease. The potential molecular mechanisms of oxidative stress related cell damage have still not been fully elucidated. Currently researchers pay more and more attention to POCD. How to predict and detect POCD patients as early as possible, and to give prevention and treatment timely is the key to improve the patients quality of life. To find new therapeutic targets in order to improve the prognosis of patients will become the focus of the present study.Astrocytes (AS), one of the predominant types of glial cells, are widely distributed between neurons. They not only provide energy and metabolic support, but also play an important role in the formation and maintenance of the blood-brain barrier and regulating synaptic plasticity etc.Astrocytes that function as both supportive and metabolic cells for the brain, are key cells in the central nervous system (CNS) involved in the maintenance of the extracellular environment and in the stabilization of cell-cell communications under physiological and pathological conditions.Astrocytes provide mechanical and metabolic support for neurons. Many functions have been attributed to astrocytes including ion homeostasis, regulation of extracellular glutamate concentration, synthesis of neurotrophic factors, contribution to the CNS immune system and finally, involvement in the physiological antioxidant defense of the brain.Parecoxib, a nonsteriodial anti-inflammatory drug (NSAID), selectively blocks off the action of cyclooxygenase-2 (COX-2) enzyme has been of interest on postoperative pain. Recently, Parecoxib has been shown to have neuroprotective properties. But hitherto poorly studied and the effects of parecoxib on cultured rat primary astrocytes induced by hydrogen peroxide (H2O2) remain largely unknown. If effective, parecoxib can be used both as an analgesic and ameliorate the effects of POCD.In this study, primary cultured astrocytes of rat were given strong oxidant H2O2 treatment to cause oxidative damage and to set up oxidative stress model. Based on this, the effect of parecoxib on H2O2-induced astrocytes protection and its protective mechanisms were studied. This paper was divided into two parts:the first part discussed the primary culture of rat astrocytes and the establish of astrocytes oxidative stress model; the second part confirmed the effects of parecoxib reducing oxidative stress injury induce by hydrogen peroxide in rat astrocytes and its mechanisms.Part I The primary culture of rat astrocytes and the establish of astrocytes oxidative stress modelObjective:To explore the dissociation, the purity and the culturing method of rat primary astrocytes in vitro. Hydrogen peroxide was added to cause oxidative stress injures to astrocytes. Astrocytes oxidative stress model was established to further study oxidative stress and to provide a theoretical basis for screening effective antioxidant neuroprotective agents.Methods:Primary culture and continuous cell culture of rat hippocampal astrocytes in vitro. Under sterile environment, the hippocampuses were obtained from SD rats born in 12-24 hours, dissected and prepared cell suspension by digestion with the use of trypsin and mechanical digestion, then transferred to culture flasks without any stratum. Astrocytes were cultured in condition of 37℃,5%CO2 and the culture medium was DMEM/F12. When the cells were confluent by 80%～90% on days 9 to 12, they were purified with orbital shaker. The oligodentrocytes and microglial cells were reduced with exfoliated cells suspension was abandoned. The primary culture was passaged when it was confluent. The purity of the primary astrocyte cultures was determined by immunostaining using an antibody against an astrocyte-specific marker, glial fibrillary acidic protein (GFAP). Methyl thiazolyl tetrazolium (MTT) assay was routinely used to assess cell metabolic activity after 100μmol/L H2O2 was added to astrocytes for 24 h. Morphology of astrocytes was observed under inverted microscope. Intracellular reactive oxygen species (ROS) were measured by using an oxidation-sensitive fluorescence probe which was dichlorodihydrofluorescein diacetate (DCFH-DA). Cell apoptosis assays were performed by flow cytometry.Results:The purified rat astrocytes with typical shapes were obtained by orbital shaker in constant temperature and passage. The number of GFAP positive cells of the first generation astrocytes after primary cultured was more than 85%. The number of GFAP positive cells of the second generation astrocytes was more than 90 %. The astrocytes were used for experiments from fourth to seventh generation and the number of GFAP positive cells was more than 98%. MTT results showed that exposure to H2O2 led to loss of cell viability. Morphology of astrocytes under inverted microscope changed significantly and many cells lost structural integrity. Some cells died floating in the normal level. DCF fluorescence increased when treated with H2O2 and ROS level increased. Cell apoptosis assays performed by flow cytometry found that the apoptosis rate of H2O2 treatment group was obviously increased.Conclusions:This experiment successfully cultured the original generation of rat astrocytes in vitro, purified and identified. H2O2 was added to cause oxidative stress injures to astrocytes and astrocytes oxidative stress model was established to further observe the protective effect of drugs on oxidative stress in astrocytes.Part Ⅱ Study on the effects of parecoxib reducing oxidative stress injury induce by hydrogen peroxide in rat astrocytes and its mechanismsAim:To investigate the effects of parecoxib on cultured rat primary astrocytes induced by H2O2 and to provide a theoretical basis for the clinical cerebral protective effect of parecoxib.Methods:80μmol/L and 160μmol/L parecoxib pretreated rat primary astrocytes for 24 h, then parecoxib was maintained and 100μmol/L H2O2 was added for 24 h. MTT was used to assay was routinely used to assess metabolic activity. Morphology of astrocytes was observed under inverted microscope. Intracellular ROS were measured by flow cytometry. Cell apoptosis assays were performed by flow cytometry. Immunocytochemistry was used to examine the expression of AQP4 protein. Reverse transcription polymerase chain reaction was used to observe the expression of the gene expressions of Bax, Bcl-2 mRNA. Western blot was used to observe the expression of BDNF protein.Results:MTT results showed that in parecoxib pretreatment group, absorbance value significantly increased and cell growth inhibition phenomenon obviously improved compared with the H2O2 group. Whlie there were not significant changes between control group and parecoxib pretreatment groups. The morphology of astrocytes almost maintained basic forms. A lot of protuberances of astrocytes still existed. The integrity of cells still existed. Results of flow cytometry indicated that parecoxib was able to prevent the increase of intracellular ROS levels. The apoptosis decreased gradually. H2O2 group indicated significant differences from control group. Immunohistochemical results showed that the protein level of AQP4 was significantly increased in H2O2 group and the protein expressions of AQP4 in 80 μmol/L and 160μmol/L parecoxib pretreatment group were notably decreased. RT-PCR results showed that the expression of bax mRNA was significantly increased and the expression of bcl-2 mRNA was significantly decreased in H2O2 group. The expression of bax mRNA in 160μmol/L parecoxib was decreased and the expression of bcl-2 was increased compared to H2O2 group. Western blot results indicated that the expression of BDNF protein in H2O2 group was significantly decreased compared with control group. After parecoxib pretreatment, the expressions of BDNF protein were increased compared to H2O2 group.Conclusions:Parecoxib can protect astrocytes against oxidative damage induced by H2O2. Its mechanism may be related to reducing cellular ROS level, decreasing the apoptosis rate and restraining the expressions of AQP4, bax and increases the expressions of bcl-2 and BDNF. This perhaps indicates that parecoxib can play its neuroprotective effect.