Glycyrrhizin Attenuates Isoflurane-induced Cognitive Deficits In Neonatal Rats Via Inhibition Of CX3CL1/CX3CR1 Signaling Pathway

Part one Glycyrrhizin Attenuates Isoflurane-induced Cognitive Deficits in Neonatal RatsObjective:To investigate the effects of glycyrrhizin on isoflurane-induced cognitive deficits in neonatal rats.Methods:Neonatal rats (PND7) were randomly divided into four groups:control group (CON), isoflurane group (ISO), glycyrrhizin group (Gly) and isoflurane+glycyrrhizin group (ISO+Gly). Rats in control group and glycyrrhizin group were intraperitoneally injected with saline or glycyrrhizin followed by control gas exposure for 4 hours. Rats in isoflurane group and glycyrrhizin+isoflurane group were intraperitoneally injected with saline or glycyrrhizin followed by isoflurane (1.8%) exposure for 4 hours. Arterial blood was sampled at the end of isoflurane anesthesia by obtaining a single sample (100?l) via cardiac puncture. Hippocampus samples were dissociated 0 h,6 h,12 h and 48 h after control gas or isoflurane exposure. Western blotting was conducted to detect the protein levels of HMGB1 in hippocampus. Morris Water Maze was performed at P31 to evaluate spatial learning and memory.Results:There was no difference in systemic parameters including pH, PaCO2, PaO2, glucose and SaO2 among four groups. Western blot analysis showed the protein levels of HMGB1 in the hippocampus of PND7 rats increased after 4 hours of isoflurane (1.8%) exposure (P<0.05), and peaked at 12 h after isoflurane exposure. Comparing to the rats in isoflurane group, the treatment of glycyrrhizin attenuated the increase of HMGB 1 in the hippocampus of the rats induced by early exposure to isoflurane (P<0.05). The results of Morris water maze task showed that isoflurane-exposed rats displayed cognitive impairment as indicated by prolonged escape latency (P<0.01), longer traveled distance and decreased platform crossings (P<0.01). However, the application of glycyrrhizin attenuated the cognitive deficit induced by isoflurane exposure, as indicated by decreased escape latency (P<0.01), shorter traveled distance and increased platform crossings (P<0.01).Conclusion:Isoflurane exposure induced the increased expression of cytoplasmic HMGB1 in the hippocampus of neonatal rats. Glycyrrhizin treatment attenuated the increased expression of cytoplasmic HMGB1 in the hippocampus, and prevented the cognitive decline induced by neonatal exposure to isoflurane.Part two Glycyrrhizin Attenuates Isoflurane-induced Synaptic Dysfunction and Apoptosis in the Hippocampus of Neonatal RatsObjective:To investigate the effects of glycyrrhizin on isoflurane-induced synaptic dysfunction and apoptosis in the hippocampus of neonatal rats.Methods:Neonatal rats (PND7) were randomly divided into four groups:(1) control group (CON):rats were intraperitoneally injected with 100?l of saline followed by control gas exposure for 4 h; (2) isoflurane group (ISO):rats were intraperitoneally injected with 100?l of saline followed by 1.8% isoflurane exposure for 4 h; (3) glycyrrhizin group (Gly):rats were intraperitoneally injected with 20 mg/kg glycyrrhizin (100?l) followed by control gas exposure for 4 h; (4) isoflurane+glycyrrhizin group (ISO+Gly):rats were intraperitoneally injected with 20 mg/kg of glycyrrhizin (100?l) followed by 1.8% isoflurane exposure for 4 h. Hippocampus were dissociated at 12 h after isoflurane exposure and detected by Western blot analysis. ELISA was used to detect the changes of pro-inflammationary cytokines (IL-1? and TNF-a) in the hippocampus.Results:Western blot analysis revealed that glycyrrhizin effectively attenuated the increase of cleaved (active) caspase-3 and pro-apoptotic protein Bax in the hippocampus of the rats exposure to isoflurane (P<0.05), and compared to isoflurane group, glycyrrhizin reversed the decline of Bcl-2, PSD-95 and SNAP-25 induced by isoflurane exposure (P<0.05). In addition, glycyrrhizin attenuated the increase of inflammation cytokine IL-1? and TNF-ainduced by isoflurane exposure (P<0.05). Moreover, western blot analysis showed nuclear NF-?B p65 increased along with the decrease of cytoplasmic NF-?B and I?B-?, whereas these changes could be attenuated by glycyrrhizin (P<0.05).Conclusion:Glycyrrhizin treatment mitigated isoflurane-induced apoptosis, neuroinflammation and the decreased synthesis of synaptic protein in the hippocampus.Part three The neuroprotective effects of glycyrrhizin against isoflurane-induced apoptosis and inflammation is caused by the rescue of CX3CL1/CX3CR1 signaling pathwayObjective:To investigate the involvement of CX3CL1/CX3CR1 signaling pathway in the neuroprotective effects of glycyrrhizin against isoflurane-induced apoptosis and inflammation.Methods:?in vivo study:Neonatal rats (PND7) were randomly divided into the following groups:(1) control group (CON):rats were intraperitoneally injected with 100?l of saline followed by control gas exposure for 4 h; (2) isoflurane group (ISO):rats were intraperitoneally injected with 100?l of saline followed by 1.8% isoflurane exposure for 4 h; (3) isoflurane+ glycyrrhizin group (ISO+Gly):rats were intraperitoneally injected with 10,20 or 30 mg/kg of glycyrrhizin (100?l) followed by 1.8% isoflurane exposure for 4 h. Hippocampus were dissociated at 6h,12 h and 48h after isoflurane exposure and protein levels of CX3CL1 and CX3CR1 in CON and ISO group rats were detected by Western blot analysis. Another Western blotting was conducted to detect the protein levels of CX3CL1 in CON?ISO and ISO+10mg/kg Gly, ISO+20mg/kg, ISO+30mg/kg Gly group.?in vitro study:Cultured hippocampal neurons were randomly divided into four groups:(1) control group (CON):neurons were exposed to control gas (air) exposure for 4 h; (2) isoflurane group (ISO):neurons were exposed to 1.8% isoflurane exposure for 4 h; (3) glycyrrhizin group (Gly):neurons were incubated with 10?M of glycyrrhizin followed by control gas exposure for 4 h; (4) isoflurane+glycyrrhizin group (ISO+Gly):neurons were incubated with 10?M of glycyrrhizin followed by 1.8% isoflurane exposure for 4 h; (5) isoflurane+ glycyrrhizin+anti-CX3CR1 group (ISO+Gly+anti-CX3CR1):neurons were incubated with 10?M of glycyrrhizin and 1?g/ml anti-CX3CR1 antibody followed by 1.8% isoflurane exposure for 4 h. Medium were collected at 2 h after isoflurane exposure and the changes of pro-inflammationary cytokines (IL-1? and TNF-a) and CX3CL1 were detected by ELISA, protein levels of HMGB1 and phosphorylation Akt were detected by Western blot analysis. Apoptosis of neuron was detected by TUNEL.Results:?Western blot analysis showed the protein levels of CX3CL1 in the hippocampus of PND7 rats decreased after 4 hours of isoflurane (1.8%) exposure (P<0.05), and reached the minimum at 12 h after isoflurane exposure, whereas the protein levels of CX3CR1 were not affected by isoflurane exposure (P>0.05).?The decrease in hippocampal CX3CL1 protein expressions were attenuated by the treatment of 10,20 and 30 mg/kg of glycyrrhizin (P<0.01 or P<0.05).?The in vitro study showed that the treatment of glycyrrhizin alleviated elevated apoptosis and the decreased CX3CL1 release of as well as the increased release of IL-1? and TNF-a induced by isoflurane exposure (P<0.01 or P<0.05). However, these effects of glycyrrhizin were reversed by the combined treatment of anti-CX3CR1 antibody (P<0.01).Conclusion:The neuroprotective effects of glycyrrhizin against isoflurane-induced apoptosis and inflammation may be attributed to the rescue of CX3CL1/CX3CR1 signaling pathway...