The Mechanism Of NLRP3 Inflammasome In Neurodegeneration In Rats With Trigeminal Neuralgia

Objective:Relevant clinical studies have found that patients with chronic pain are at higher risk for neurodegeneration?such as dementia?;previous research by our team also found that trigeminal neuralgia can cause neurodegeneration in rats,but the detailed mechanism is still not clear.IL-1?is a classic pro-inflammatory cytokine,which can induce the expression of related pro-inflammatory mediators.It is at the center of the inflammatory response and is involved in the development of neuropathic pain.NLRP3inflammasome is consist of NOD-like receptor 3?NLRP3?,apoptosis-related punctate-like protein?ASC?,and cysteine protease 1 precursor?pro-caspase-1?.After the formation of NLRP3 inflammasome,it mediates the self-activation of pro-caspase-1 to caspase-1,which leads to the maturation and activation of IL-1?and IL-18,exerting immune inflammatory effects.It is speculated that NLRP3 inflammasome may be related to neuropathic pain.In addition,NLRP3 inflammasome can participate in the regulation of the neuroinflammatory response of Alzheimer's disease?AD?by regulating inflammatory factor activity and glial cell phenotype,which plays a vital role in the development of AD.It is hypothesized that neuropathic pain?NP?may induce neuroinflammation by activating NLRP3 inflammasome,leading to the occurrence of neurodegeneration.To verify this hypothesis,this experiment was designed.This experiment intends to observe the effects of TN on neurodegenerative changes in a rat model,and to explore the role of NLRP3inflammasome in it,in order to provide a basis for the prevention of neurodegenerative changes in TN patients in clinical work.Methods:1.Modeling and grouping:Thirty adult male SD rats were randomly divided into three groups after 1 week of adaptive training:sham operation group?Sham group?,surgery group?ION-CCI group?and NLRP3 inhibitor group?MCC950 group?,ten rats of each group.The Sham group served as a control and exposed only the infraorbital nerve without ligation.In the ION-CCI group,a right infraorbital nerve chronic constriction ring?ION-CCI?was used to establish a TN model.MCC950?5mg/kg?was injected intraperitoneally at 1,4,7,10,13,16,19,22,25,28 days after operation in MCC950 group.The other two groups were injected intraperitoneally with the same volume of saline at the same time.2.Mechanical pain threshold?MWT?detection:The Von Frey Filament tester was used to detect the mechanical pain threshold of all rats before,3rd,7th,14th,and 28th day after modeling.3.Morris water maze test:Before the modeling,and at 14th and 28th day after the modeling,all rats were subjected to a water maze test,namely MWM1,MWM 2,MWM 3.Observe the changes in the escape latency,the number of times to cross the platform,and the percentage of the target quadrant time in the three groups of rats.4.Detection of neurodegeneration-related indicators:Rats were sacrificed after the completion of MWM 3.A?1-42 protein,arginase1?Arg1?,and inducible nitric oxide?iNOS?in the prefrontal cortex and hippocampus were detected by enzyme-linked immunosorbent assay?ELISA?.Western blot was used to detect the expression of Tau protein in prefrontal cortex and hippocampus,and the phosphorylation level of Tau protein at Ser404 site.5.Detection of cell cycle regulatory protein:Western blot was used to detect the expression of P21 protein in the prefrontal cortex and hippocampus.6.Detection of proteins related to NLRP3 inflammasome:The expression of NLRP3 in the prefrontal cortex and hippocampus were detected by immunofluorescence and Western blot.The expression of NF-?B?P-NF-?B?IL-1?were detected by Western blot.Results:1.Rat models:A total of 4 rats in the three groups died after anesthesia or modeling.Four rats were re-selected and randomly added to each group.A total of 30 rats were included in this study.2.Changes of MWT in three groups:There was no significant difference in MWT at different time points in the sham group.?P?>?0.05?.Compared with the Sham group,the pain threshold of the ION-CCI group and the MCC950 group decreased at the 3^rd,7^th,14^th,28^thh day after operation?P?<?0.05?.Compared with the ION-CCI group,the pain threshold of the MCC950 group increased at the 7^th,14^th,28^thh day after operation?P?<?0.05?.3.Morris water maze test of three groups:There was no statistical difference in the escape latency,the crossing platform number,and the time percentage in the target quadrant in the three groups of MWM 1?P?>?0.05?.The time percentage in the target quadrant of ION-CCI group was reduced compared with the Sham group of MWM 1?P?<?0.05?.In the MWM 3,The escape latency of the ION-CCI group was longer than the Sham group?P?<?0.05?,and the crossing platform number and the time percentage in the target quadrant were reduced compared to the Sham group?P?<?0.05?.The escape latency of the MCC950 group was shorter than the ION-CCI group?P?<?0.05?,and the time percentage in the target quadrant was increased compared to the ION-CCI group?P?<?0.05?.4.The level of proteins related to neurodegeneration in three groups:Compared with the sham group,the expression of A?1-42,iNOS,^Ser404P-Tau protein in the prefrontal cortex and hippocampus increased in the ION-CCI group?P?<?0.05?,and the expression of Arg1 decreased?P?<?0.05?;the expression of A?1-42 and iNOS in prefrontal cortex and hippocampus increased in the MCC950 group?P?<?0.05?,and the expression of Arg1decreased?P?<?0.05?,the expression of ^Ser404P-Tau was not significantly changed?P>0.05?.Compared with the ION-CCI group,the expression of A?1-42 protein and iNOS in the prefrontal cortex and hippocampus of the MCC950 group decreased?P?<?0.05?,the expression of Arg1 increased?P?<?0.05?,and the expression of ^Ser404P-Tau protein in the prefrontal cortex decreased?P?<?0.05?.There was no significant difference in the level of total Tau protein in three groups?P?>?0.05?.5.The level of cell cycle regulatory protein in three groups:Compared with the Sham group,the expression of P21 protein in the prefrontal cortex and hippocampus increased in the ION-CCI group?P?<?0.05?,while the expression of P21 protein in the prefrontal cortex and hippocampus in the MCC950 group did not change significantly?P>0.05?.Compared with the ION-CCI group,the expression of P21 protein in the prefrontal cortex and hippocampus decreased in the MCC950 group?P?<?0.05?.6.The level of proteins related to NLRP3 inflammasome in three groups:Compared with the Sham group,the expression of NLRP3,P-NF-?B,and IL-1?in the prefrontal cortex and hippocampus increased in the ION-CCI group?P?<?0.05?,and the expression of NF-?B in the hippocampus increased?P?<?0.05?.Besides,there were no significant changes in the expression of NLRP3,NF-?B,P-NF-?B,and IL-1?in the prefrontal cortex and hippocampus of the MCC950 group?P>0.05?.Compared with the ION-CCI group,the expressions of NLRP3 and P-NF-?B in the prefrontal cortex and hippocampus decreased in the MCC950 group?P?<?0.05?,and the expressions of IL-1?in the hippocampus decreased?P?<?0.05?.Conclusion:1.Trigeminal neuralgia rats may exhibit neurodegenerative features,including progressive decline in learning and memory,increased expression of A?1-42,iNOS,Ser404P-Tau protein in the prefrontal cortex and hippocampus,and decreased expression of Arg1.2.The expression of P21 protein in prefrontal cortex and hippocampus of trigeminal neuralgia rats was increased,and the cell cycle of neuronal was disrupted.3.Trigeminal neuralgia can activate NLRP3 inflammasome,leading to increased expression of NLRP3,P-NF-?B,and IL-1?in the prefrontal cortex and hippocampus of rats,and a reduction of the mechanical pain threshold.4.Activation of NLRP3 inflammasome may mediate the occurrence and development of trigeminal neuralgia in rats and the neurodegenerative changes caused by it.The mechanism may be related to the accompanying neuroinflammation and neuronal cell cycle disorders.