Exploration Of The Mechanism For Wilson Disease With Cognitive Impairment And Effectiveness Of Huatan Quyu Recipe Based On Higher-order Cognitive Networksand Classical Pyroptosis

PURPOSE Wilson’s disease(WD)is a potentially treatable hereditary disorder of copper metabolism,mainly caused by pure or heterozygous mutations in the ATP7 B gene encoding the p-type ATPase,resulting in excessive copper into the circulation,which triggering pathological copper deposition in tissues,especially brain tissue.Cognitive impairment is a common neurological symptom that is receiving increasing attention.Rapid and accurate identification of diagnostic and effective clinical interventions are of great practical importance for the prognostic quality of survival of WD patients.This study aims to provide evidence for the identification and diagnosis of cognitive impairment in WD and the effective intervention of the Huatan Quyu recipe Gandouling through clinical studies and basic research.METHODS This study consisted of three parts.(1)In the clinical study,group-level ICA-based functional network connectivity analysis was used to explore the differences in intra-and inter-network functional connectivity of high-order cognitive networks between WD patients with cognitive impairment and normal control subjects;to detect differences in MMSE scale scores,peripheral serum IL-6,IL-17,TNF-α and classical pyroptosis indicators NLRP3,GSDMD,IL-1β,Caspase-1,IL-18;the results of functional connectivity of resting brain networks were correlated with cognitive scales,inflammatory factors and classical pyroptosis indicators to explore the neuroimaging markers for the identification and diagnosis of cognitive impairment in WD;To detect the changes of MMSE scale,peripheral serum IL-6,IL-17,TNF-α and classical pyroptosis indicators NLRP3,GSDMD,IL-1β and Caspase-1,IL-18 in WD patients with cognitive impairment after the intervention of Huatan Quyu recipe Gandouling to verify its clinical efficacy.(2)In animal experiments,we used the classic copper-poisoned rat as a model of WD and tested learning memory using the water maze experiment;HE staining and transmission electron microscopy to observe hippocampal tissue morphology and cell damage;immunohistochemistry to observe microglial activation in each group;the levels of NLRP3,GSDMD,Caspase-1,ASC and CD68 were detected by Western blot;the protein expression levels of SOD,GSH,MDA,IL-1β,IL-18,TNF-α and IL-6were detected by ELISA;the m RNA expression levels of NLRP3,GSDMD and Caspase-1 were detected by q RT-PCR.To explore the mechanism of amelioration of inflammation and classical pyroptosis pathway of WD cognitive impairment in hippocampal tissues of copper-loaded rats by Huatan Quyu recipe Gandouling.(3)In cellular experiments,we set up the NLRP3-specific inhibitor MCC950 group to regulate the activation of microglia inflammation,and detected the cellular activity by CCK-8 assay;immunofluorescence double-staining assay to detect the activation of M1/M2 type microglia;Western blot assay to detect the activation of NLRP3,ASC,Caspase-1,GADMD,IL-1β,IL-18;q RT-PCR to detect the m RNA expression levels of NLRP3,GADMD and Caspase-1 in each group.To explore the mechanism by which the Huatan Quyu recipe Gandouling regulates microglial NLRP3 inflammatory vesicle activation,mediating microglial activation and classical pyroptosis pathway amelioration,with a view to validating the clinical findings from a molecular biological perspective.RESULTS(1)In the clinical study,the WD group showed significant differences(P< 0.01)in the indices of MMSE,classical pyroptosis indicators Caspase-1,GSDMD,NLRP3,IL-1β,IL-18 and inflammatory factors IL-6,IL-17,and TNF-α;The results of resting-state functional brain network analysis showed that the brain network of cognitive impairment in WD patients was characterized by high connectivity in the DMN with activation of the right anterior cingulate and paracingulate gyrus,right insula,left nucleus accumbens,and left medial superior frontal gyrus;low connectivity in the VN,SN,SMN,and CEN with reduced functional connectivity in the right cuneus,right perisylvian cortex,bilateral middle frontal gyrus,left superior temporal gyrus,right precentral gyrus,right central gyri,right postcentral gyrus,bilateral angular gyrus and parts of the left middle temporal gyrus;The results of correlation analysis showed that there was a negative correlation between NLRP3 and VN＿SMN,Caspase-1 and SN＿VN and VN＿SMN(P< 0.05),a positive correlation between GSDMD and SN＿DMN and DMN＿SMN in WD patients with cognitive impairment(P<0.05),and a positive correlation between GSDMD and SN＿DMN and DMN＿SMN in WD patients with cognitive impairment(P< 0.05).After the intervention of Huatan Quyu recipe Gandouling,the MMSE scale scores of the patient group in the WD group increased significantly(P< 0.01);Caspase-1,GSDMD,NLRP3,IL-1β,IL-18 indices and the levels of inflammatory factors IL-6,IL-17 and TNF-α indices decreased significantly(P< 0.01).(2)In vitro experiments,the water maze experiment confirmed that copper-loaded SD rats had a significant decrease in learning and memory ability;HE staining of hippocampal tissue showed significant tissue and cell damage;transmission electron microscopy revealed significant cell pores and burnt vesicles,indicating significant cell burns in the hippocampal tissue of copper-loaded SD rats;The copper ion content of the hippocampal tissue was significantly increased;the levels of SOD and GSH were significantly decreased compared to other groups;immunohistochemistry showed significant microglial activation;WB,ELISA and q RT-PCR experiments showed a significant increase in the level of toxic products MDA.The results of WB,ELISA and q RT-PCR showed that the levels of inflammatory factors L-1β,IL-18,TNF-α,IL-6 and classical pyroptosis indicators NLRP3,GSDMD and Caspase-1 were significantly increased in hippocampal tissues of copper-loaded rats.Compared with the model group,the efficacy of the medium-dose Gandouling group on the learning and memory ability,hippocampal tissue structure and cellular and classical pyroptosis of rats was more obvious than other drug intervention groups;in addition,the SOD and GSH of the medium-dose Gandouling group were significantly increased(P< 0.01),while copper ion,MDA,IL-1β,IL-18,TNF-α,IL-6,NLRP3,GSDMD,Caspase-1 and other indicators were significantly reduced(P< 0.01).(3)In vitro experiments,CCK-8 experiments showed that the number of microglia in the model group was significantly reduced and cell activity was significantly decreased,while the number of cells in the MCC950 group was significantly increased compared with the model group;Immunofluorescence double staining experiments showed M1 type microglia were significantly activated in the model group compared with the normal control group;in WB experiments,we found that the protein levels of NLRP3-inflammatory vesicle complex were significantly increased in the model group compared with the control group,and the levels of inflammatory factors and classical pyroptosis indicators,including NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18,were significantly increased.q RT-PCR results showed that the m RNA levels of NLRP3,GADMD and caspase-1 also showed statistically significant differences.Conclusions(1)The structural characteristics of the brain network in WD patients with cognitive impairment are the hyperactivated brain area of the right anterior cingulate and paracingulate gyrus,the right insula,the left nucleus accumbens and the left medial superior frontal gyrus in the DMN,and the low connectivity of VN,SN,SMN and CEN is accompanied by reduced functional connectivity in the right cuneus,right perirhinal cortex,bilateral middle frontal gyrus,left superior temporal gyrus,right precentral gyrus,right central sulcus,right postcentral gyrus,bilateral angular gyrus,and parts of the left middle temporal gyrus.(2)The resting state brain network analysis results of WD cognitive impairment patients may have excessive internal orienting cognition,excessive deconstruction of visual images and increased activity of the biased "task negative network",as well as undercorrection of incoming information,goal-directed reduced activity in the "task-active network" of abnormal action.(3)High levels of serum NLRP3,caspase-1 and GSDMD in combination with high DMN connectivity and low VN,SMN and SN connectivity in WD patients with cognitive impairment may be a characteristic manifestation of peripheral serum and neuroimaging in patients with WD with cognitive impairment.(4)Huatan Quyu recipe Gandouling can reduce M1 microglial polarisation,inhibit microglial activation,scavenge superoxide radicals,reduce NLRP3 inflammatory vesicle release,decrease Caspase-1 expression,inhibit GSDMD shear and inflammatory factor IL-1β/IL-18 infiltration,mediate inflammatory response and the microglia classical pyroptosis pathway,promote hippocampal tissue protection and repair,and thus improve cognition.