| Objective:Aging model rats were used as the research object to observe the behavioral changes of rats after inhaling 3.2%sevoflurane for 6h,The pathological morphology of prefrontal cortex and microglial activation of NLRP3、ASC、caspase-1and IL-1βprotein relative expression,as well as serum IL-1βcontent were observed.To investigate the effect of neuroinflammation on sevoflurane-induced cognitive dysfunction in aging rats.Method:SPF male SD rats（N=48）aged 4 months were random Ly divided into four groups:Control group（Ctrl）;Negative control,NLRP3 inflammasome specific inhibitor MCC950 group（MCC950）;Sevoflurane group（Sev）and MCC950intervention group（Sev+MCC950）,every group n=12.（1）Before the experiment,all rats were treated with d-galactose（125mg/kg）subcutaneously on the head and neck for 42 consecutive days to establish the aging rat model.（2）Morris water maze experiment was conducted after the model was established,Including positioning voyage（5d）Space exploration trial（1d）.（3）After the water maze experiment,the Ctrl group and the MCC950 group inhaled carrier gas（O2and air 1:1）for 6h,and the Sev group and the Sev+MCC950 group inhaled 3.2%seflurane+carrier gas for 6h.After the completion of the inhalation,the Ctrl group and the Sev group intraperitoneal injection of 1m LNS at 30min,24h and 48h,respectively.MCC950 group and Sev+MCC950 group were intraperitoneally injected with MCC950（1m L,concentration 10mg/kg）at 30min,24h and 48h,respectively.（4）Morris Water Maze test was performed again in each group to detect learning and memory ability.（5）Prefrontal cortex was collected after pentobarbital anesthesia:（1）HE staining was used to observe the nucleus and tissue structure of nerve unit;（2）DNA fracture of nerve cells was observed by TUNEL apoptosis staining;（3）Iba-1expression was detected by immunohistochemistry to determine the activation status of microglia;（4）Western Blot was used to detect the relative expression of NLRP3ASC Caspase-1 and IL-1β.（5）Serum IL-1βinflammatory levels were determined by ELISA.Results:1.Morris Water Maze test:All the rats in each group could swim normally in the adaptive training,and the congenital stupid rats were excluded.In the positioning navigation test,with the increase of training days,the escape incubation period of the rats in each group showed a trend of gradually shortening and stability,which was significantly shortened on the second day compared with the first day（P<0.05）,during the next 3 days of experiment,the rats in each group were significantly shorter than the second day,and there was no significant difference after statistical analysis（P>0.05）In addition,we compared the swimming distance and swimming speed of rats in each group（P>0.05）.In the space exploration experiment,compared with Ctrl group,Sev group rats traversed the original platform less times（P<0.05）,while there was no significant change in MCC950 group and Sev+MCC950 group（P<0.05）2.HE staining:Compared with Ctrl group,the neurons in the prefrontal cortex of the rats in the MCC950 group were round or conical,and the nucleus was located in the center of the nucleus,without obvious neuron damage or inflammatory cell infiltration.In the Sev group,the volume of some neurons in the prefrontal cortex of the rats was reduced,and the nucleus of neurons was pyrotic and hyperchromatic,and the neurons were obviously damaged.Compared with the Sev group,neurons in the prefrontal cortex of rats in the Sev+MCC950M group were closely arranged,and most of the neurons were round or conical in shape,and the damage degree of neurons was reduced.3.TUNEL apoptosis staining:Compared with Ctrl group,a large number of TUNEL positive cells were expressed in the prefrontal cortex of rats in Sev group（P<0.05）,and showed that neurons were small on the surface,and cytoplasm was concentrated and dark brown.Compared with the Sev group,the expression of TUNEL positive cells in the prefrontal cortex of rats in the Sev+MCC950M group was significantly decreased,and some damaged neurons were relieved to varying degrees,with statistically significant differences（P<0.05）4.Immunohistochemical staining:Compared with Ctrl group,the expression of Iba-1protein in the prefrontal cortex of Sev group was significantly up-regulated,and the difference was statistically significant（P<0.05）,compared with Sev group,iba-1protein expression in Sev+MCC950 group was significantly decreased,and the difference was statistically significant（P<0.05）.5.Protein expression of each group by Western Blot:Compared with Ctrl group,the protein expressions of NLRP3,ASC,Caspase-1 and IL-1βin prefrontal cortex of rats in Sev group were significantly up-regulated,and the differences were statistically significant（P<0.05）,there was no significant difference in ASC protein expression among all groups（P<0.05）;Compared with Sev group,the expressions of Iba-1,NLRP3,Caspase-1,IL-1βand protein were significantly decreased in Sev+MCC950 group（P<0.05）.6.Serum IL-1βinflammatory level was detected by ELISA:The expression level of IL-1βin Sev group was significantly higher than that in Ctrl group,and the difference was statistically significant（P<0.05）;Compared with Sev group,the expression level of IL-1βin serum of Sev+MCC950 group was significantly decreased,and the difference was statistically significant（P<0.05）.Conclusion:Sevoflurane can reduce the learning and memory ability of aging rats,and damage neurons in the prefrontal cortex,enhance the activation of microglia,and activation of NLRP3 inflammasome assembly in prefrontal cortex induces the release of pro-inflammatory cytokine IL-1βthrough activation of Caspase-1,up regulate the expression of neuroinflammation-related proteins.Objective: By observing the number of Nissl corpuscles in prefrontal cortex of aging model rat 、 the relative and transcriptional expression of NFκB and NLRP3 protein,and the level of serum IL-1β inflammatory factor,to investigate the role of NFκB in NLRP3 inflammatory body in cognitive dysfunction induced by sevoflurane induced neuroinflammation in rats.Methods: 36 male aging model SD rats were divided into three groups according to random number table method:Control group（Ctrl）;Sevoflurane group（Sev）;NFκB inhibitor pyrrolidine dimercaptan PDTC group（PDTC）,every group n=12.The Ctrl group inhaled carrier gas for 6h,and the Sev group inhaled 3.2% seflurane+carrier gas for 6h.After the completion of the inhalation,the Ctrl group and the Sev group intraperitoneal injection of 1m L NS at 30 min,24h and 48 h,respectively.PDTC group were intraperitoneally injected with PDTC（1m L,concentration 100mg/kg）at 30 min,24h and 48 h,respectively.（2）Prefrontal cortex was collected after pentobarbital anesthesia:（1）The number of Nissl corpuscles was observed by Nissl staining;（2）Western Blot was used to detect the relative expression of NFκB P65 and NLRP3;（3）NFκB and NLRP3 transcription were detected by RT-PCR.Results: 1.Nissl staining: Compared with Ctrl group,the number of prefrontal lobe nirosii in Sev group was significantly decreased（P < 0.05）,compared with Sev group,the number of prefrontal cortex in PDTC group was increased（P < 0.05）.2.Western Blot of NFκB P65 and NLRP3 protein expression in prefrontal cortex and cytoplasm of rats: Compared with Ctrl group,the protein expression of NFκB P65 and NLRP3 in the prefrontal cortex of Sev group was significantly up-regulated（P <0.05）,compared with Sev group,nuclear NFκB P65 and NLRP3 protein expression in PDTC group was significantly decreased（P < 0.05）;There was no significant difference in NFκB P65 protein expression among all groups（P < 0.05）.3.Transcriptional expression of NFκB P65 and NLRP3 in rat prefrontal cortex detected by RT-PCR: Compared with Ctrl group,the transcription of NFκB P65 and NLRP3 in the prefrontal cortex of Sev group were significantly up-regulated（P <0.05）,compared with Sev group,the transcription expression of NFκB P65 and NLRP3 in PDTC group was significantly decreased（P < 0.05）.Conclusions: Sevoflurane enhances nuclear NFκB P65 translocation and mediates IL-1β activation induced by NLRP3 inflammasome pathway,which is involved in neuroinflammation in the prefrontal cortex of rats.Objective: The neuronal morphology and mitochondrial autophagy related LC3 B SQSTM1/ P62 protein in the prefrontal cortex of rats were observed,and the role of mitochondrial autophagy in neuroinflammation induced by sevoflurane in aging model rats,and the transcription expression of NF-KB、NLRP3、Caspase-1 and IL-1β,as well as the level of serum IL-1β inflammatory factors,to investigate the role of mitochondrial autophagy in neuroinflammation in sevoflurane induced aging model rats.Methods: 48 male aging model SD rats were divided into three groups according to random number table method:Control group（Ctrl）;Autophagy inhibitor 3-methyl adenine intervention group（3-MA）;Sevoflurane group（Sev）;Autophagy agonist rapamycin intervention group（Rapa）,every group n=12.The Ctrl group inhaled carrier gas for 6h,and the Sev group、3-MA group and Rapa group inhaled 3.2%seflurane+carrier gas for 6h.After the completion of the inhalation,the Ctrl group and the Sev group intraperitoneal injection of 1m L NS at 30 min,24h and 48 h,respectively.3-MA group and Rapa group were intraperitoneally injected with3-MA（1m L,concentration 15mg/kg）and rapamycin（1m L,concentration 2.0 mg/kg）at 30 min,24h and 48 h,respectively.（2）Prefrontal cortex was collected after pentobarbital anesthesia:（1）The morphology of neurons and autophagy of mitochondria were observed by transmission electron microscopy;（2）Western Blot was used to detect the relative expression of LC3 B and SQSTM1/p62;（3）NFκB、NLRP3、Caspase-1 and IL-1β transcription were detected by RT-PCR.（3）Serum IL-1β inflammatory cytokines were detected by ELISA.Results: 1.Transmission electron microscopy analysis revealed the formation of mitochondrial phagosomes in the prefrontal cortex of rats in each group.Based on TEM,we observed that sevoflurane induced mitochondrial structural damage,compared with healthy organelles in the control group,sevoflurane group showed more severe cell damage In addition,we found that in the prefrontal cortex of sevoflurane rats,the structure of mitochondrial matrix and mitochondrial crest was disturbed with alternating light and dark nuclear membrane contraction,and mitochondrial autophagy was inhibited.In addition,other organelles were blurred and difficult to identify However,rapamycin improved mitochondrial destruction in the prefrontal cortex,showing a significant increase in the number of mitochondrial phagosomes and mitochondrial lysosomes,and a significant reduction in organelle damage.However,the neuronal protective effect of rapamycin on increased mitochondrial autophagy was eliminated in the 3-MA intervention group.2.Western blot analysis of protein expression in each group: Compared with Ctrl group,LC3BII/LC3 BI protein expression in the prefrontal cortex of rats in Sev group or 3-MA group was significantly decreased,and the difference was statistically significant（P < 0.05）;The expression of P62 protein in prefrontal cortex of Rapa group was significantly decreased compared with Sev or 3-MA group（P < 0.05）.3.RT-PCR: Compared with Ctrl group,the expression of NLRP3 caspase-1 and transcription in prefrontal cortex of rats in Sev group or 3-MA group were significantly up-regulated（P < 0.05）,NLRP3 caspase-1 and transcription in Rapa group were significantly decreased compared with Sev group or 3-MA group（P <0.05）Conclusion: Sevoflurane inhibits mitochondrial autophagy in the prefrontal cortex of aging rats,and activates the expression of key molecules in NFκB and NLRP3 inflammasome pathways,as well as the production of IL-1β,aggravating neuroinflammation. |
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