Protective Effect Of Melatonin In Infectious Brain Injury Of Rats Induced By Pneumolysin

Acute bacterial meningitis is serious life-threatening infection with high mortality and morbidity. Streptococcus pneumoniae is common pathogen. There are about600,000people who suffer from streptococcus pneumoniae meningitis in the word every year. The fatality rate is up to34%.50%of survivors suffer from neuropsychological, hearing and other obstacles. As the antibiotics have been developed in recent decades, the prognosis has been improved. But antibiotics can not prevent the inflammation that induced by toxic substances relased after bacteria dissolved. Pneumolysin (PLY) is an important virulence factor of Streptococcus pneumoniae. It is released after the bacteria dissolved and has a variety of functions. It can cause severe brain damage by triggering inflammation, inducing apoptosis, damaging neurons directly. So it is particularly important to study the pathogenesis of PLY caused infectious brain injury and to find effective measures combined with antibiotics.Melatonin (MT) is indole neuroendocrine substances secreted by the pineal gland with high affinity of the central nervous system. It can cross the blood-brain barrier. Melatonin’s receptor expresses on membrane of neurons and glial cell. And the biological effects are generated by binding with specific receptor. It has antioxidant, anti-inflammatory, anti-apoptosis, and gene regulatory fuction. Melatonin has protective and conservative function on a variety of neurological diseases such as Alzheimer’s disease, epilepsy, hypoxia-ischemia brain damage in neonatal rat. But there are few studies in infectious brain damage.Apoptosis refers to the spontaneous death process after the body’s normal cells received physiological or pathological stimulation. It is closely related to the sequela of the nervous system. Apoptosis-inducing factor (AIF) is a conservative class of flavoprotein. It presents in the mitochondrial outer membrane gap. After the outside apoptotic signaling stimulated, AIF can be transferred to the nucleus, induce nuclear chromatin condensation and non-dependent cysteine aspartic enzyme DNA fragmentation. It mediated apoptosis of brain cells and play the important role in infectious brain damage.The high mobility group protein B1(HMGB1) is a kind of highly conserved non-histone proteins in cells. It was first discovered in the brain and widely distributed in the body. It is in the nucleus mainly. It’s biological function is involved in DNA repair and reorganization of nucleosome stability, cell differentiation, and steroid hormone regulation. Furthermore, it has cytokine-like functions. It is an important clinical inflammatory mediators. No paper is reported about the study of HMGB1with Streptococcus pneumoniae meningitis at present.Therefore, this study will monitor brain cell apoptosis, the expression of AIF and HMGB1based on the establishment of pneumolysin induced infectious brain injury. And explore the protective mechanism of MT on the infectious brain injury from both apoptosis and inflammatory responses.Part Ⅰ Establishment of infectious brain injury model of rats induced by pneumolysinObjectiveTo establish the infectious brain injury model of infant rats induced by pneumolysin.Methods56infant rats were randomly divided into2groups:Normal saline group(NS group, n=28),0.2mL nomal saline was applied for each rat by the left internal carotid; pneumolysin group(PLY groups, n=28),0.2mL(7μg)PLY was injected by the left internal carotid. According to different observation time points, each group was divided into2subgroups:24h and48h. There were14infant rats in every subgroup. At different time point, rats were decapitated. Brain tissue samples were prepared for the study. The Evan’s blue(EB) content of brain tissue was measured by the formamide method. The brain water content(BWC) was record by measuring both wet and dry weight. The levels of glial fibrillary acidic protein (GFAP) protein and neuron specific enolase (NSE) protein were studied by immunohistochemistry.ResultsSignificant change was observed in PLY group. Histopathological change:it could be found that the clearance around vessels became broader, inflammatory cells chemotacted and infiltrated. Gliacytes and neurons became swelled and vacuolar degenerated in PLY group. It was more serious at24h than48h. These changes were not discovered in NS group. In PLY group, at24h, the levels of brain EB, BWC, NSE and GFAP protein were higher than that at48h(P<0.05). Compared to NS group, at different time point, the levels of brain EB, BWC, NSE and GFAP protein were higher in PLY group. The difference was significant(P<0.01).ConclusionAfter applied PLY into infant rats’left internal carotid, brain tissue pathological changed significantly; the levels of brain EB, BWC, NSE and GFAP protein were increased. It was more serious at24h than48h. This suggested the infectious brain injury model could be established steadily and reliably. Part Ⅱ Intervention of melatonin to the apoptosis of nervous cells on rats brain injury induced by pneumolysinObjectiveTo explore the intervention of melatonin to the apoptosis of nervous cells on infectious brain injury model of rats induced by pneumolysin. And the mechanism was also studied. Methods84infant rats were randomly divided into3groups:NS group(n=28),0.2mL nomal saline was applied to each rat via the left internal carotid; PLY group(n=28),0.2mL(7μg)PLY was injected via the left internal carotid; MT group(n=28),0.1%MT10mg/kg was injected via intraperitoneal injection before15min PLY injection. According to different observation time points, each group was divided into2subgroups:24h and48h. There were14infant rats in every subgroup. At different time point, rats were decapitated. Brain tissue samples were prepared for the study. The EB content of brain tissue was measured by the formamide method. BWC was record by measuring both wet and dry weight. The levels of GFAP protein and NSE protein were detected by immunohistochemistry. The expression of AIF protein was detected by the same method. The apoptosis of nervous cells was detected by terminadeoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) technique.ResultsHistopathological change:it could be found that the clearance around vessels became broader, inflammatory cells chemotacted and infiltrated. Gliacytes and neurons became swelled and vacuolar degenerated in PLY group. These changes were not discovered in NS group. In MT group, these changes were not as serious as PLY group. Compared with NS group, in PLY group, the levels of brain BWC, EB content, NSE and GFAP protein, AIF were higher at24h and became lower at48h. The number of cell apoptosis were higher at24h and reached to the highest at48h. The difference was significant. In MT group, at each time point, the levels of EB, BWC, GFAP, NSE, AIF and the number of apoptosis in the brain tissue were lower than that in PLY group. Significant difference was observed. In MT group, the brain EB content and the levels of NSE, GFAP, AIF and the number of apoptosis in the brain tissue were higher than NS group at each time point. Significant difference was observed. In MT group, the BWC content was higher than NS group at24h(P<0.05). But at48h time point, there was no siginificant difference. ConclusionApoptosis played a role in the formation and development of infectious brain injury of rats and AIF was involved in neurocyte apoptosis. MT had the protective effect to the infectious brain injury, and the mechanism maybe that melatonin can inhibit the expression of AIF and reduce the apoptosis of nervous cells. Part Ⅲ Intervention of melatonin on the expression of highmobility group box1in rats brain injury induced by pneumolysinObjectiveTo explore the expression of high mobility group box1and the intervention of melatonin on the expression of HMGB1in rats brain injury induced by pneumolysin.Methods84infant rats were divided into3groups randomly:NS group(n=28),0.2mL nomal saline was applied to each rat via left internal carotid; PLY group(n=28),0.2mL(7μg)PLY was injected by left internal carotid; MT group(n=28),0.1%MT10mg/kg was injected via intraperitoneal injection before15min PLY injection. According to different observation time points, each group was divided into2subgroups:24h and48h. There were14infant rats in every subgroup. At different time point, rats were decapitated. Brain tissue samples were prepared for the study. EB content of brain tissue was measured by the formamide method. BWC was record by measuring both wet and dry weight. The levels of GFAP protein and NSE protein were detected by immunohistochemistry. The level of HMGB1protein was detected by the same method. RT-PCR technology was applied to detect the level of HMGB1mRNA. ResultsHistopathological change:it could be found that the clearance around vessels became broader, inflammatory cells chemotacted and infiltrated. Gliacytes and neurons became swelled and vacuolar degenerated in PLY group. These changes were not discovered in NS group. In MT group, these changes were not as serious as PLY group. Compared with NS group, the levels of brain EB, BWC content, NSE and GFAP protein, HMGB1protein and HMGB1mRNA were all higher in PLY group at different time point. Significant difference was observed. In MT group, at each time point, the EB, BWC content and the levels of NSE, GFAP, HMGB1protein and HMGB1mRNA in the brain tissue were lower than PLY group. There was significant difference. In MT group, EB content and the levels of NSE, GFAP, HMGB1protein in the brain tissue were higher than NS group at each time point. There was significant difference. In MT group, the BWC content and HMGB1mRNA were higher than NS group at24h(P<0.05). But at48h time point, there was no siginificant difference.ConclusionHMGB1seemed to act as a mediator in the process of brain injury induced by PLY. Melatonin could reduce the expression of HMGB1and HMGB1mRNA, and reduce inflammation of the brain.