The Role Of Signaling Pathway In Postoperative Cognitive Dysfunction And Lung Ischemia Reperfusion Injury And Its Regulatory Mechanisms

Section 1:The role of Toll-like receptor mediated central nervous inflammatory reaction in postoperative cognitive dysfunctionObjective: To investigate the role of the toll-like receptor mediated inflammatory response in the brain in postoperative cognitive dysfunction caused by aseptic surgical trauma.Methods: Six-eight weeks male CD-1 mice were randomly assigned to two groups(n=6): control and surgery group(right carotid artery exposure) in the first experiment, the total and membrance proteins of TLR2, TLR4, TLR9 in bilateral cerebral cortex and hippocampus at 6 hours after surgery were assessed using Western blot. The mice were assigned to: control group, CU-CPT22 group, surgery group, surgery plus CU-CPT22 group in the second experiment. Fifteen mice per group were used for the learning and memory tests(Barnes maze and fear conditioning) that were started one week after the surgery. Six mice per group were used for the bilateral cerebral cortex and hippocampal harvest at 12 h after surgery for ELISA of IL-1β and IL-6. Six mice per group were used to harvest at 6 h after surgery for immunofluorescent staining of TLR2 with Neu N, GFAP, and Iba-1.Results: In the first experiment, compared with control group, the expressions of total and membrane proteins of TLR2, TLR4 in cortex and hippocampus were significantly increased in surgery group(P<0.05), but the expressions of TLR9 were not affected(P>0.05). In the second experiment, during the Barnes maze test, training sessions had a main effect on the time to identify the target hole, surgery also had a significant effect on long term test. This surgical effect was abolished by CU-CPT22(P>0.05). Mice in the surgery group had decreased freezing behavior in context-related fear conditioning test when compared with control animals(P>0.05). This decrease was attenuated by CU-CPT22(P>0.05). However, the tone-related freezing behavior was not affected by any experimental conditions(P>0.05). Surgery significantly increased the expressions of IL-1β and IL-6, the increase was attenuated by CUCPT22(P>0.05). The results of immunofluorescence showed that the expressions of TLR2 were increased after surgery, TLR2 can express on Neu N and part of Iba-1.Conclusion: TLR2 and TLR4 mediated inflammatory response in the brain seems to play a important role in the procedure of postoperative cognitive dysfunction caused by aseptic surgical trauma. TLR2 mainly express on Neu N and Iba-1 in brain after surgery.Section 2:The role of HMGB1 in the activation of TLR2 in brain after surgery and its regulatory mechanismsObjective: To investigate the role of HMGB1 in the activation of TLR2 in brain after surgery and its regulatory mechanisms.Methods: Six-eight weeks male CD-1 mice were randomly assigned to two groups(n=10): control and surgery group(right carotid artery exposure) in the first experiment, the nuclear and Cytoplasmic proteins of HMGB1 in bilateral cerebral cortex and hippocampus at 6 hours after surgery were assessed using Western blot. The mice were assigned to: control group, Glycyrrhizin group, surgery group, surgery plus Glycyrrhizin group in the second experiment. Six mice per group were used to harvest at 6 h after surgery for Western blot of TLR2 and HMBG1. Four mice per group were used for hippocampal harvest at 6 h after surgery for Chromatin immunoprecitation.Results: In the first experiment, compared with control group, the expressions of nuclear proteins of HMGB1 in cortex and hippocampus were significantly increased in surgery group(P<0.05), but the expressions of Cytoplasmic proteins of HMGB1 were not affected(P>0.05). In the second experiment, Surgery significantly increased the expressions of HMGB1 in nuclear proteins and TLR2 in total and membrane proteins, the increase was attenuated by Glycyrrhizin. The results of Chromatin immunoprecitation showed that the DNA expressions of the promoter of TLR2 in-283 bp ~-71 bp were increased after surgery(P<0.05).Conclusion: The expressions of HMGB1 in the brain play a important role in the activation of TLR2 caused by aseptic surgical trauma. HMGB1 can increase the activity of the promoter of TLR2 in-283 bp ~-71 bp after surgery.Section 3:The role of HMGB1/TLR2 signaling pathway in lung ischemiareperfusion injuryObjective: To investigate the role of HMGB1/TLR2 signaling pathway in lung ischemia-reperfusion injury.Methods: Six-eight weeks male BALB/C mice were randomly assigned to three groups(n=6): Control, Sham and Ischemia/Reperfusion group(I/R) in the first experiment, and were randomly assigned to four groups(n=6): Control, Glycyrrhizin, I/R and I/R plus Glycyrrhizin in the second experiment. The lung tissue damage and score were assessed using HE stain and wet weight /dry weight(W/D). The total proteins of TLR2 and the nuclear and Cytoplasmic proteins of HMGB1 in lung tissue were assessed using Western blot. Six mice per group were used for ELISA of IL-1β and IL-6. Six mice per group were used for lung tissue harvest at 6 h after I/R for Chromatin immunoprecitation.Results: In the first experiment, lung tissue in control group had no pathological change, lung interval broadened and a small amount of inflammatory cells infiltrated in Sham group, lung tissue in I/R group had significantly damaged, alveolar edema and hemorrhage, a large number of inflammatory cells infiltration. The lung tissue damage score were 1.6±0.3 in Control group, 12.4±1.3 in Sham group and 28.5±2.1 in I/R group. Compared with Control and Sham group, W/D in I/R group had significantly increased(P>0.05). Ischemia/Reperfusion significantly increased the expressions of HMGB1, TLR2, IL-1β and IL-6. The lung tissue injury and the expressions of HMGB1, TLR2, IL-1β and IL-6 caused by Ischemia/Reperfusion were attenuated by Glycyrrhizin(P>0.05). HMGB1 can not increase the activity of the promoter of TLR2 in-283 bp ~-71 bp after Ischemia/Reperfusion(P>0.05).Conclusion: TLR2 and HMGB1 mediated inflammatory response in the lung tissue seems to play a important role in the procedure of lung ischemiareperfusion injury. Glycyrrhizin can attenuate the expressions of HMGB1, TLR2, IL-1β, IL-6, and can alleviate the lung tissue injury caused by Ischemia/Reperfusion.