Mechanism Of Gandou Decoction Regulating NLRP3 Inflammasome By TLR4 Pathwayand Inhibiting Neuroinflammation In A Model Of Wilson’s Disease

Wilson’s disease（WD）is a neurogenetic disorder caused by mutations in the ATP7B gene that leads to impaired copper metabolism and consequently to multi-system symptoms such as hepatic,neurological and psychiatric symptoms.WD is one of the few treatable neurological genetic disorders.When patients with neurological WD develop significant cognitive and behavioral dysfunction,conventional clinical copper chelation therapy is less effective,and some patients even experience exacerbation of clinical symptoms,which seriously affects the treatment and prognosis of WD patients,which is also the focus and difficulty of long-term WD treatment.Since the 1970s,the research team began to discuss WD from the aspects of syndrome differentiation and treatment of traditional Chinese medicine,and considered that WD was caused by copper toxin cohesion,dampness and heat of liver and gallbladder.Gandou decoction was used to treat WD patients through its role of heat-clearing and detoxification,purging viscera and promoting dampness,and achieved remarkable clinical effect.Previous studies have found that Gandou decoction can reduce oxidative stress injury of WD neurons,inhibit neuronal apoptosis,and improve high copper-induced neuronal degeneration and necrosis.Studies have shown that inflammation exists in WD neurons.Previous studies of our group found that nod-like receptor family,pyrin domain-containing protein3（NLRP3）plays an important role in the pathological process of WD neuronal injury.Inhibition of NLRP3 inflammatory body activation can significantly reduce neuronal injury caused by neuroinflammation in WD model TX mice and significantly improve their behavioral abnormalities.Therefore,the inflammatory injury induced by copper accumulation plays an important role in the process of brain injury in patients with WD.Recently,it has been found that intracellular copper accumulation can activate NLRP3 inflammatory bodies through toll-like receptor 4（TLR4）pathway,which can induce and promote neuronal inflammatory injury.Based on the regulation of NLRP3 inflammatory bodies by TLR4 pathway,this study will explore the intervention effect of Gandou decoction on neuroinflammatory injury induced by high copper,in order to provide theoretical basis for Gandou decoction in the treatment of patients with cerebral WD,and provide new treatment and research ideas for the treatment of neurogenetic diseases with traditional Chinese medicine.Objective:1.To observe the protective effect of Gandou decoction on neuronal injury induced by high copper in a model of WD.2.To observe the inhibitory effects of Gandou decoction on the activation of microglia,the activation of NLRP3 inflammatory bodies and the release of inflammatory factors induced by high copper.3.To observe the regulatory effect of Gandou decoction on TLR4 pathway,and to explore the possible mechanism of Gandou decoction protecting neurons in WD.Methods:1.Preparation of traditional Chinese medicine Gandou decoction（GDD）and rat serum containing Gandou decoction.2.In vivo experiment,DL mice were used as control group,TX mice were randomly divided into model group and model+Gandou decoction group,normal group and model group were perfused with normal saline,and model+Gandou decoction group were treated with Gandou decoction.3.Barnes maze test,Open field test and Elevated plus maze test were used to detect the action and cognitive function of mice in each group.4.Hematoxylin-eosin（HE）staining was used to detect the neuronal damage in the brain tissue of mice in each group.5.The expression levels of NLRP3,ASC and Caspase-1 m RNA in brain tissue of mice in each group were detected by RT-q PCR.6.Microglia（BV-2）were cultured in vitro and randomly divided into normal group,model group（Cu Cl₂）,model+5%Gandou decoction serum group,model+10%Gandou decoction serum group,model+15%Gandou decoction serum group.7.BV-2 cells and hippocampal neurons（HT-22）were conditionally cultured.Conditionally cultured neurons were randomly divided into normal group,Cu Cl₂-induced model group,model+5%Gandou decoction serum group,model+10%Gandou decoction serum group,model+15%Gandou decoction serum group.8.The survival rate of conditioned neurons in each group was detected by CCK8method.9.Detection of lactate dehydrogenase（LDH）leakage rate of conditioned neurons in each group by biochemical-enzyme labeling method.10.The expressions of Malondialdehyde（MDA）,Hydrogen peroxide（H₂O₂）,Superoxide dismutase（SOD）,Reduced glutathione（GSH）and Oxidized glutathione（GSSG）were detected by biochemical-enzyme labeling method.11.Detection of Reactive oxygen species（ROS）activity in conditioned culture cells by flow cytometry.12.The activation level of microglia in each group was observed by fluorescence inverted microscope.13.Elisa method was used to detect the expression of Interleukin-1β（IL-1β）,Interleukin-18,IL-18（interleukin-18）,Tumor necrotic factor-α（TNF-α）and inducible nitric oxide synthase（i NOS）in microglia.14.The levels of NLRP3 inflammatory bodies and TLR4 pathway-related proteins in mice and microglia were detected by Western Blot.Results:Part I ResearchIn vivo experiment:The results of Barnes maze showed that compared with the normal group,the latency of TX mice to find the hole was significantly increased（P<0.01）,and the treatment of Gandou decoction could significantly shorten the latency of TX mice entering the target hole（P<0.01）.The results of elevated cross maze test showed that the percentage and duration of open arm entry in TX mice were significantly lower than those in normal group（P<0.05）,while the percentage and duration of open arm in Gandou decoction group were higher than those in TX group（P<0.05）.The results of open field test showed that compared with the normal group,the time spent in the centre zone,total distance,rearing times and climbing times of TX mice were decreased（P<0.05）,while the time spent in the wall zone and immobility time of TX mice were increased（P<0.05）.The time spent in the centre zone,total distance,rearing times and climbing times in Gandou decoction group were higher than those in TX group（P<0.05）,and Gandou decoction could shorten the time spent in the wall zone and immobility time of TX mice（P<0.05）.Gandou decoction could improve the behavioral and cognitive dysfunction of TX mice.The results of HE staining showed that the morphology of neurons and glial cells in DL mice was normal,the peripheral nerve fibers were compact,and didn’t find obvious abnormality.In TX mice,most neurons in the cortex and striatum were necrotic,pyknosis and deep staining,unclear nuclear-cytoplasmic boundaries,significantly enhanced basophilia,vacuolation in the nucleus,loose peripheral nerve fibers,decreased eosinophilia,sparse arrangement of hippocampal pyramidal cells,necrosis of pyramidal cells,nuclear pyknosis or dissolution.The nuclear and cytoplasmic boundaries of a large number of cerebellar Purkinje cells were unclear,some of the nuclei were vacuolated or irregular,the neurons and glial cells in the molecular layer and white matter were pyknotic and deeply stained,and the nuclear and cytoplasmic boundaries were unclear.Basophilic enhancement.In the Gandou decoction group,the morphology of brain cells was regular and arranged neatly,the nucleus of nerve cells was large and round,the cytoplasm was rich,light blue or uniform,the nucleolus was clear,the layers and cell lines were clear,and the structure of each layer of cerebellum was clear.a small amount of microglia proliferation can be seen in the local granular layer.In vitro experiment:The results of CCK8 method showed that under the condition of co-culture of microglia and hippocampal neurons,10μM Cu Cl₂could significantly reduce the survival rate of hippocampal neurons compared with the normal group（P<0.01）,and 5%,10%and 15%Gandou decoction serum could significantly increase the viability of co-cultured cells（P<0.05）.The results of LDH detection showed that the activity of LDH in the supernatant of co-cultured cells in the model group was significantly higher than that in the normal group（P<0.01）,and the serum containing Gandou decoction could reduce the LDH activity of conditioned neurons induced by high copper（P<0.01）.The results of oxidative stress test showed that compared with the normal group of conditioned culture,the activity of ROS,the levels of MDA and H₂O₂in the model group were increased（P<0.01）,while the activity of SOD and the ratio of GSH/GSSG in the model group were lower than those in the normal group（P<0.01）.Compared with the model group,the activity of ROS,the levels of MDA and H₂O₂in the Gandou decoction serum group were lower than those in the model group（P<0.05）,while the activity of SOD and the ratio of GSH/GSSG in the Gandou decoction serum group were significantly higher than those in the model group（P<0.05）.Part II ResearchIn vivo experiment:Gandou decoction could significantly reduce the expression of NLRP3,ASC and Caspase-1 m RNA in various regions of brain tissue of TX mice（P<0.01）.The results of Western Blot showed that compared with the normal group,the expression of NLRP3,Caspase-1,IL-1βand ASC in the cortex,hippocampus,striatum and cerebellum of TX mice increased,and oral administration of Gandou decoction inhibited the expression of NLRP3 inflammatory body protein in various regions of the brain of TX mice（P<0.05）.In vitro experiment:The observation of fluorescence inverted microscope showed that compared with the normal group,the surface processes of microglia in the model group gradually contracted,and showed an activated state of larger cell body,shorter processes and round cell morphology.the serum containing Gandou decoction could significantly reduce the activation level of microglia in the model group.The results of Elisa detection showed that the expressions of IL-1β,IL-18,TNF-αand i NOS in microglia in the model group were significantly higher than those in the normal group（P<0.01）.Compared with the model group,Gandou decoction serum could reduce the contents of IL-1β,IL-18,TNF-αand i NOS in a dose-dependent manner（P<0.01）.The results of Western Blot showed that compared with the normal group,the expression of NLRP3inflammatory body related protein in microglia in the model group increased（P<0.01）,and the serum containing Gandou decoction decreased the expression of NLRP3inflammatory body protein in the model group（P<0.01）.Part III Research In vivo experiment:The results of Western Blot showed that compared with the normal group,the expressions of TLR4,Nuclear factor-κB（NF-κB）p65 and Phosphorylated-nuclear factorκB inhibitor proteinα（P-IκBα）in cerebral cortex,hippocampus,striatum and cerebellum of TX mice were significantly up-regulated（P<0.01）,and oral administration of Gandou decoction could reduce the expression of TLR4,NF-κB/p65 and P-IκBαprotein（P<0.05）.In vitro experiment:The results of Western Blot showed that compared with the normal group,the expression of TLR4,NF-κB/p65 and P-IκBαprotein in the microglia of the model group was significantly up-regulated（P<0.05）.Compared with the model group,the serum containing Gandou decoction could down-regulate the expression of TLR4,NF-κB/p65 and P-IκBαprotein（P<0.05）.Conclusion:1.Gandou decoction can improve the abnormal behavior of TX mice,reduce the neuronal damage induced by high copper,and reduce the level of cytotoxicity and oxidative stress.2.Gandou decoction can inhibit the activation of microglia and NLRP3 inflammatory bodies,and reduce the release of inflammatory cytokines.3.Gandou decoction can inhibit TLR4 signal pathway to regulate the activation of NLRP3 inflammatory bodies and reduce inflammatory response.